Download - (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

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Page 1: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US009453009B2

(ΐ2) United States PatentLiu et al

(ΐο) Patent No US 9453009 Β2(45) Date of Patent Sep 27 2016

(54) SYNTHESIS OF DEUTERATED MORPHOLINE DERIVATIVES

(71) Applicant Concert Pharmaceuticals IncLexington ΜΑ (US)

(72) Inventors Julie F Liu Lexington ΜΑ (US)Xuejun Tang Ellicott City MD (US) Scott L Harbeson Cambridge ΜΑ (US) Craig Ε Masse Cambridge ΜΑ (US)

(73) Assignee Concert Pharmaceuticals IncLexington ΜΑ (US)

( ) Notice Subject to any disclaimer the term of thispatent is extended or adjusted under 35 USC 154(b) by 0 days

(21) Appl No 14566244(22) Filed Dec 10 2014(65) Prior Publication Data

US 20150099714 Al Apr 9 2015

Related US Application Data

(63) Continuation of application No 13678093 filed on Nov 15 2012 now abandoned which is a continuation of application No 12456507 filed on Jun 17 2009 now Pat No 8354557

(60) Provisional application No 61132284 filed on Jun 17 2008

(51) Int ClC07D 41310 (200601)C07D 295135 (200601)C07D 295033 (200601)C07C 21512 (200601)C07D 29502 (200601)C07D 29506 (200601)C07D 29512 (200601)C07D 41314 (200601)C07D 26306 (200601)Α61Κ 315377 (200601)Α61Κ 4506 (200601)

(52) US ClCPC C07D 41310 (201301) Α61Κ 315377

(201301) Α61Κ 4506 (201301) C07C 21512 (201301) C07D 26306 (201301)

C07D 29502 (201301) C07D 295033 (201301) C07D 29506 (201301) C07D

29512 (201301) C07D 295135 (201301) C07D 41314 (201301) C07B 220005

(201301)(58) Field of Classification Search

CPC C07D 41310See application file for complete search history

(56) References Cited

US PATENT DOCUMENTS

6221335623915262553046277985644071066030087517990752813178552048354557

200500041182005003803220070082929200701976952008010312220080139563200801465732009009342220130281456

Β1 42001Β1 52001Β1 72001Β1 82001Β1 82002Β1 82003Β2 42009Β2 52009Β2 122010Β2 12013Al 12005Al 22005Al 42007Al 82007Al 52008Al 62008Al 62008Al 42009Al 102013

FosterGordeev et al Hester Jr et al Gadwood et al Keinan et al Ando et alIto et al Persichetti et al Tung Lui et alJilaniAllison et al Gant et al Potyen et al VeltriTung et alGant et alTung et alLiu et al

5142368

FOREIGN PATENT DOCUMENTS

GB 708523 A 51954WO 9526325 Α2 101995WO 9710223 Al 31997WO 0005231 Al 22000WO 2007118651 Al 102007

(Continued)

OTHER PUBLICATIONS

Le Cam (ldquoCamrdquo) Chemica Scripta (1971) 1(2) ρ 65-8Baillie Thomas A ldquoThe Use of Stable Isotopes in Pharmacological Researchrdquo Pharmacological Reviews vol 33 No 2 ρρ 81-132 1981Browne Thomas R ldquoStable Isotope Techniques in Early Drug Development An Economic Evaluationrdquo J Clin Pharmacol vol 38 ρρ 213-220 1998Cherrah et al ldquoStudy of Deuterium Isotope Effects on Protein Binding by Gas ChromatographyMass Spectrometry Caffeine and Deuterated Isotopomersrdquo Biomedical and Environmental Mass Spectrometry vol 14 ρρ 653-657 1987Dyck et al ldquoEffects of Deuterium Substitution on the Catabolism of -Phenylethylamine An In Vivo Studyrdquo Journal ofNeurochemistry vol 46 No 2 ρρ 399-404 1986

(Continued)

Primary Examiner mdash Yong Chu(74) Attorney Agent or Firm mdash McCarter amp English LLP Steven G Davis Emily Dertz

(57) ABSTRACTThe present invention is directed to a process for preparing a 2266-d4-morpholine derivative represented by Structural Formula (I)

D

D D

R4

(I)

3931177 A 4914232 A 5688792 A 5880118 A

11976 Coates et al 41990 Lai

111997 Barbachyn et al 31999 Barbachyn et al

or a salt thereof

5 Claims 8 Drawing Sheets

US009453009B2

US 9453009 Β2Page 2

(56) References Cited

FOREIGN PATENT DOCUMENTS

WO 2008070619 Α1 62008WO 2008127300 Α2 102008

OTHER PUBLICATIONSEllermann et al ldquoEffect of pentoxifylline on the ischemic rat kidney monitored by 3 IP NMR spectroscopy in vivordquo Biomed Biochim Acta vol 47 No 6 ρρ 515-521 1988Fisher et al ldquoThe complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolismrdquo Current Opinion in Drug Discovery amp Development vol 9 No 1 ρρ 101-109 2006Foster Allan Β ldquoDeuterium isotope effects in studies of drug metabolismrdquo TIPS ρρ 524-527 1984Foster Allan Β ldquoDeuterium Isotope Effects in the Metabolism of Drugs and Xenobiotics Implications for Drug Designrdquo Advances in Drug Research vol 14 ρρ 2-40 1985Gouyette et al ldquoSynthesis of Deuterium-labelled Elliptinium and its Use in Metabolic Studiesrdquo Biomedical and Environmental Mass Spectrometry vol 15 ρρ 243-247 1988Haskins NJ ldquoThe Application of Stable Isotopes in Biomedical Researchrdquo Biomedical Mass Spectrometry vol 9 No 7 ρρ 269-277 1982Honma et al ldquoLiberation of Deuterium from the Piperidine Ring during Hydroxylationrdquo Drug Metabolism and Disposition vol 15 No 4 ρρ 551-559 1987Kushner et al ldquoPharmacological uses and perspectives of heavy water and deuterated compoundsrdquo Can J Physiol Pharmacol vol 77 ρρ 79-88 1999Liu Youhua ldquoEpithelial to Mesenchymal Transition in Renal Fibrogenesis Pathologic Significance Molecular Mechanism and Therapeutic Interventionrdquo J Am Soc Nephrol vol 15 ρρ 1-12 2004Pieniaszek et al ldquoMoricizine Bioavailablity via Simultaneous Dual Stable Isotope Administration Bioequivalence Implicationsrdquo The Journal of Clinical Pharmacology vol 39 ρρ 817-825 1999 Slatter et al ldquoPharmacokinetics Metabolism and Excretion of Linezolid following an Oral Dose of [14C]Linezolid to Healthy Human Subjectsrdquo Drug Metabolism and Disposition vol 29 No 8 ρρ 1136-1145 2001Slatter et al ldquoPharmacokinetics toxicokinetics distribution metabolism and excretion of linezolid in mouse rat and dogrdquo Xenobiotica vol 32 No 10 ρρ 907-924 2002Τοηη et al ldquoSimultaneous Analysis of Diphenhydramine and a Stable Isotope Analog (2H10)Diphenhydramine Using Capillary Gas Chromatography with Mass Selective Detection in Biological Fluids from Chronically Instrumented Pregnant Ewesrdquo Biological Mass Spectrometry vol 22 ρρ 633-642 1993Wolen Robert L ldquoThe Application of Stable Isotopes to Studies of Drug Bioabailablity and Bioequivalencerdquo The Journal of Clinical Pharmacology vol 26 ρρ 419-424 1986Prescribing information for ZYVOX (Linezoid) Pharmacia amp Upjohn Company Revised Mar 2007 ρρ 1-34

International Search Report issued in PCT Application No PCT US0722516 on Oct 16 2008International Search Reportmdash(PCTUS2009003628) Date of Mailshying Nov 12 2009Adamus et al ldquoSynthesis of NN-dimethylmorpholinium chloride- 2-14Crdquo 1982 Journal of Labelled Compounds andRadiopharmaceuticals vol 19 ρρ 309-312Bataille et al ldquoEnantioselective synthesis of alpha- phenylalkanamines via intermediate addition of Grignard reagents to chiral hydrazones derived from (R)-(-)-2-amino-l-butanolrdquo 1998 Tetrahedron Asymmetry vol 9 ρρ 2181-2192Cottle et al ldquoPreparation of some C-alkylmorpholinesrdquo 1946 Journal of Organic Chemistry vol 11 ρρ 286-291Colvin et al ldquoAlkylating properties of phosphoramide mustardrdquo 1976 Cancer Research vol 36 ρρ 1121-1126Database CA [Online] Chemical Abstracts Service Columbus Ohio US Mikula et al ldquoApparatus and methods for preparing morpholinerdquo retrieved from STN Database accession No 19875056Database CA [Online] Chemical Abstracts Service Columbus Ohio US Lindberg et al ldquoMass spectrometry of pethidine and pethidine metabolites Study utilizing specifically deuterated pethidinerdquo retrieved from STN Database accession No 1974504086Enders et al ldquo(SS)-35-Dimethylmorpholine a novel C2-symmet- ric auxiliary First application in [4+2]-cycloadditions leading to 4-oxohexahydropyridazine derivativesrdquo 1994 Synthesis vol 1ρρ 66-72Hampton et al ldquoNew synthesis of morpholinerdquo Journal of the American Chemical Society 1936 vol 58 ρρ 2338-2339Lai ldquoHindered amines Part 6 3355-Tetrasubstituted-2- oxomorpholines and derivativesrdquo Synthesis 1984 vol 2 ρρ 122-123Loeppky et al ldquoThe synthesis of deuterium-labeled N-nitrosodiethanolamine and N-nitroso-2-hydroxymorpholinerdquo Journal of Labelled Compounds amp Radiopharmaceuticals 1994 vol 34 ρρ 1099-1110Loeppky et al ldquoProbing the Mechanism of the Carcinogenic Activation of N-Nitrosodiethanolamine with Deuterium Isotope Effects In Vivo Induction of DNA Single-Strand Breaks and Related in Vitro Assaysrdquo Chemical Research in Toxicology 1998 vol 11 ρρ 1556-1566Ludeman et al ldquoOxime Derivatives of the Intermediary Oncostatic Metabolites of Cyclophosphamide and Ifosfamide Synthesis and Deuterium Labeling for Applications to Metabolite Quantificationrdquo Journal of Pharpaceutical Sciences 1995 vol 84 ρρ 393-398 Patel et al ldquoMetal(IV) phosphates as solid acid catalysts for selective cyclodehydration of ln-diolsrdquo Green Chemistry 2001 vol 3 ρρ 143-145Jarowicki et al J Chem Soc Perkin Trans I 1998 ρ 4005-4037 Anderson et al N-Nitrosodiethanolamine Revisited Biomedical Mass Spectrometry 1980 7(5)205-210Cam et al Chemica Scripta (1971) 1(2)65-68

cited by examiner

US Patent Sep 27 2016 Sheet 1 of 8 US 9453009 Β2

10000 r

Intravenous (5mgkg η=3)

FIG 1Α

Oral (5mgkg η=3)

mdashdiamsmdash- Νο4mdashm~~~-No 5mdashAmdash-No 6

8 10

CD-b

- hCM

Con

c(n

gm

L)

FIG IB

US Patent Sep 27 2016 Sheet 2 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

100000

10000

4mdash iv-5mgkg

po-5mgkg

CNJ

CO

A c

nj

Con

c(n

gm

L)

FIG 1C

US Patent Sep 2720ΐ6 sheet 3 of 8 US 9453009 Β2

FIG 2Α

Oral (5mgkg η=3)

10000 -bullmdashNo 10

Intravenous (5mgkg η=3)

CD

CO

CNJ

Con

c(n

gm

L)

FIG 2Β

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 2: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β2Page 2

(56) References Cited

FOREIGN PATENT DOCUMENTS

WO 2008070619 Α1 62008WO 2008127300 Α2 102008

OTHER PUBLICATIONSEllermann et al ldquoEffect of pentoxifylline on the ischemic rat kidney monitored by 3 IP NMR spectroscopy in vivordquo Biomed Biochim Acta vol 47 No 6 ρρ 515-521 1988Fisher et al ldquoThe complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolismrdquo Current Opinion in Drug Discovery amp Development vol 9 No 1 ρρ 101-109 2006Foster Allan Β ldquoDeuterium isotope effects in studies of drug metabolismrdquo TIPS ρρ 524-527 1984Foster Allan Β ldquoDeuterium Isotope Effects in the Metabolism of Drugs and Xenobiotics Implications for Drug Designrdquo Advances in Drug Research vol 14 ρρ 2-40 1985Gouyette et al ldquoSynthesis of Deuterium-labelled Elliptinium and its Use in Metabolic Studiesrdquo Biomedical and Environmental Mass Spectrometry vol 15 ρρ 243-247 1988Haskins NJ ldquoThe Application of Stable Isotopes in Biomedical Researchrdquo Biomedical Mass Spectrometry vol 9 No 7 ρρ 269-277 1982Honma et al ldquoLiberation of Deuterium from the Piperidine Ring during Hydroxylationrdquo Drug Metabolism and Disposition vol 15 No 4 ρρ 551-559 1987Kushner et al ldquoPharmacological uses and perspectives of heavy water and deuterated compoundsrdquo Can J Physiol Pharmacol vol 77 ρρ 79-88 1999Liu Youhua ldquoEpithelial to Mesenchymal Transition in Renal Fibrogenesis Pathologic Significance Molecular Mechanism and Therapeutic Interventionrdquo J Am Soc Nephrol vol 15 ρρ 1-12 2004Pieniaszek et al ldquoMoricizine Bioavailablity via Simultaneous Dual Stable Isotope Administration Bioequivalence Implicationsrdquo The Journal of Clinical Pharmacology vol 39 ρρ 817-825 1999 Slatter et al ldquoPharmacokinetics Metabolism and Excretion of Linezolid following an Oral Dose of [14C]Linezolid to Healthy Human Subjectsrdquo Drug Metabolism and Disposition vol 29 No 8 ρρ 1136-1145 2001Slatter et al ldquoPharmacokinetics toxicokinetics distribution metabolism and excretion of linezolid in mouse rat and dogrdquo Xenobiotica vol 32 No 10 ρρ 907-924 2002Τοηη et al ldquoSimultaneous Analysis of Diphenhydramine and a Stable Isotope Analog (2H10)Diphenhydramine Using Capillary Gas Chromatography with Mass Selective Detection in Biological Fluids from Chronically Instrumented Pregnant Ewesrdquo Biological Mass Spectrometry vol 22 ρρ 633-642 1993Wolen Robert L ldquoThe Application of Stable Isotopes to Studies of Drug Bioabailablity and Bioequivalencerdquo The Journal of Clinical Pharmacology vol 26 ρρ 419-424 1986Prescribing information for ZYVOX (Linezoid) Pharmacia amp Upjohn Company Revised Mar 2007 ρρ 1-34

International Search Report issued in PCT Application No PCT US0722516 on Oct 16 2008International Search Reportmdash(PCTUS2009003628) Date of Mailshying Nov 12 2009Adamus et al ldquoSynthesis of NN-dimethylmorpholinium chloride- 2-14Crdquo 1982 Journal of Labelled Compounds andRadiopharmaceuticals vol 19 ρρ 309-312Bataille et al ldquoEnantioselective synthesis of alpha- phenylalkanamines via intermediate addition of Grignard reagents to chiral hydrazones derived from (R)-(-)-2-amino-l-butanolrdquo 1998 Tetrahedron Asymmetry vol 9 ρρ 2181-2192Cottle et al ldquoPreparation of some C-alkylmorpholinesrdquo 1946 Journal of Organic Chemistry vol 11 ρρ 286-291Colvin et al ldquoAlkylating properties of phosphoramide mustardrdquo 1976 Cancer Research vol 36 ρρ 1121-1126Database CA [Online] Chemical Abstracts Service Columbus Ohio US Mikula et al ldquoApparatus and methods for preparing morpholinerdquo retrieved from STN Database accession No 19875056Database CA [Online] Chemical Abstracts Service Columbus Ohio US Lindberg et al ldquoMass spectrometry of pethidine and pethidine metabolites Study utilizing specifically deuterated pethidinerdquo retrieved from STN Database accession No 1974504086Enders et al ldquo(SS)-35-Dimethylmorpholine a novel C2-symmet- ric auxiliary First application in [4+2]-cycloadditions leading to 4-oxohexahydropyridazine derivativesrdquo 1994 Synthesis vol 1ρρ 66-72Hampton et al ldquoNew synthesis of morpholinerdquo Journal of the American Chemical Society 1936 vol 58 ρρ 2338-2339Lai ldquoHindered amines Part 6 3355-Tetrasubstituted-2- oxomorpholines and derivativesrdquo Synthesis 1984 vol 2 ρρ 122-123Loeppky et al ldquoThe synthesis of deuterium-labeled N-nitrosodiethanolamine and N-nitroso-2-hydroxymorpholinerdquo Journal of Labelled Compounds amp Radiopharmaceuticals 1994 vol 34 ρρ 1099-1110Loeppky et al ldquoProbing the Mechanism of the Carcinogenic Activation of N-Nitrosodiethanolamine with Deuterium Isotope Effects In Vivo Induction of DNA Single-Strand Breaks and Related in Vitro Assaysrdquo Chemical Research in Toxicology 1998 vol 11 ρρ 1556-1566Ludeman et al ldquoOxime Derivatives of the Intermediary Oncostatic Metabolites of Cyclophosphamide and Ifosfamide Synthesis and Deuterium Labeling for Applications to Metabolite Quantificationrdquo Journal of Pharpaceutical Sciences 1995 vol 84 ρρ 393-398 Patel et al ldquoMetal(IV) phosphates as solid acid catalysts for selective cyclodehydration of ln-diolsrdquo Green Chemistry 2001 vol 3 ρρ 143-145Jarowicki et al J Chem Soc Perkin Trans I 1998 ρ 4005-4037 Anderson et al N-Nitrosodiethanolamine Revisited Biomedical Mass Spectrometry 1980 7(5)205-210Cam et al Chemica Scripta (1971) 1(2)65-68

cited by examiner

US Patent Sep 27 2016 Sheet 1 of 8 US 9453009 Β2

10000 r

Intravenous (5mgkg η=3)

FIG 1Α

Oral (5mgkg η=3)

mdashdiamsmdash- Νο4mdashm~~~-No 5mdashAmdash-No 6

8 10

CD-b

- hCM

Con

c(n

gm

L)

FIG IB

US Patent Sep 27 2016 Sheet 2 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

100000

10000

4mdash iv-5mgkg

po-5mgkg

CNJ

CO

A c

nj

Con

c(n

gm

L)

FIG 1C

US Patent Sep 2720ΐ6 sheet 3 of 8 US 9453009 Β2

FIG 2Α

Oral (5mgkg η=3)

10000 -bullmdashNo 10

Intravenous (5mgkg η=3)

CD

CO

CNJ

Con

c(n

gm

L)

FIG 2Β

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 3: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US Patent Sep 27 2016 Sheet 1 of 8 US 9453009 Β2

10000 r

Intravenous (5mgkg η=3)

FIG 1Α

Oral (5mgkg η=3)

mdashdiamsmdash- Νο4mdashm~~~-No 5mdashAmdash-No 6

8 10

CD-b

- hCM

Con

c(n

gm

L)

FIG IB

US Patent Sep 27 2016 Sheet 2 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

100000

10000

4mdash iv-5mgkg

po-5mgkg

CNJ

CO

A c

nj

Con

c(n

gm

L)

FIG 1C

US Patent Sep 2720ΐ6 sheet 3 of 8 US 9453009 Β2

FIG 2Α

Oral (5mgkg η=3)

10000 -bullmdashNo 10

Intravenous (5mgkg η=3)

CD

CO

CNJ

Con

c(n

gm

L)

FIG 2Β

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 4: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US Patent Sep 27 2016 Sheet 2 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

100000

10000

4mdash iv-5mgkg

po-5mgkg

CNJ

CO

A c

nj

Con

c(n

gm

L)

FIG 1C

US Patent Sep 2720ΐ6 sheet 3 of 8 US 9453009 Β2

FIG 2Α

Oral (5mgkg η=3)

10000 -bullmdashNo 10

Intravenous (5mgkg η=3)

CD

CO

CNJ

Con

c(n

gm

L)

FIG 2Β

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 5: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US Patent Sep 2720ΐ6 sheet 3 of 8 US 9453009 Β2

FIG 2Α

Oral (5mgkg η=3)

10000 -bullmdashNo 10

Intravenous (5mgkg η=3)

CD

CO

CNJ

Con

c(n

gm

L)

FIG 2Β

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 6: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US Patent Sep 27 2016 Sheet 4 of 8 US 9453009 Β2

Mean plusmn SD (η=3)

t(hr)

FIG 2C

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

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25

30

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  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 7: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

Plas

ma

Con

cent

ratio

n (n

gm

l)

US Patent Sep 2720ΐ6 sheet 5 of 8 US 9453009 Β2

FIG 3

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 8: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US Patent Sep 2720ΐ6 sheet 6 of 8 US 9453009 Β2

Linezolid Compound 10

10

Time (h)

μ cm

r ο

οοοοο

Plas

ma C

once

ntra

tion (

ngm

l)

FIG 4

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 9: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

mtD

NA

enc

oded

pro

tein

nucl

ear

DN

A e

ncod

ed p

rote

in

Rat

io

US Patent Sep 27 2016 Sheet 7 of 8 US 9453009 Β2

FIG 5Α

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 10: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

mtD

NA

enc

oded

pro

tein

nu

clea

r D

NA

enc

oded

pro

tein

R

atio

US Patent Sep 27 2016 Sheet 8 of 8 US 9453009 Β2

Compound 103 uMl

FIG 5Β

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 11: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β2

SYNTHESIS OF DEUTERATEDMORPHOLINE DERIVATIVES

RELATED APPLICATION

This application is a continuation application of US patent application Ser No 13678093 filed Nov 15 2012 which is a continuation application of US patent applicashytion Ser No 12456507 filed on Jun 17 2009 which claims the benefit of US Provisional Application No 61132284 filed on Jun 17 2008 The entire teachings of the above application(s) are incorporated herein by refershyence

BACKGROUND OF THE INVENTION

In certain instances improvements in drug performance have been reported as a result of incorporating deuterium into specific sites of pharmaceutical agents Site specific incorporation of deuterium with acceptable chemical and isotopic yields can be difficult and expensive to achieve Therefore there is need to develop improved processes for making pharmaceutical agents having site-specific deutera- tion which are economical and have high chemical and isotopic yields

SUMMARY OF THE INVENTION

The present invention in one embodiment is directed to a novel process for making 2266-d4 morpholine derivatives The process comprises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

1

whereinR1 is mdashΗ mdashOH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashC(=0)NRHU mdash C(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl wherein the alkyl aryl hetshyeroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdash C(=0)NRHU mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl C^ alkoxy mdashOH C^ haloalkyl and Cjg haloalkoxy

each Rreg is independently an alkyl optionally substituted with halogen C^ alkyl C^ alkoxy mdashOH C^ haloalkyl or C1-6 haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen C^ alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2

mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRreg wherein each C^ alkyl is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C1 6 alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg( mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each C^ alkyl is optionally substishytuted with one or more groups selected from halogen C1-6 alkyl Cjg alkoxy mdashOH C^ haloalkyl and C^ haloalkoxy

Rreg is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashNO UCN mdashΝΗ mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRreg mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each CU6 alkyl substituent is optionally substituted with one or more groups selected from halogen C^g alkyl C^g alkoxy mdashOH C1-6 haloalkyl and C1-6 haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen C 6 alkyl mdashOR7 mdashC(=0)0R7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)R7 mdashC(=0)NRregRrf mdashS(0)R7 mdashS(0)2R7 mdashSR7 and mdashS02NRregRrf wherein each C^ alkyl substitushyent is optionally substituted with one or more groups selected from halogen Qg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

andR4 R4 R5 and R5 are each independently mdashΗ or C^

alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Qg haloalkyl or Cjg haloalkoxy

In another embodiment the present invention is directed to a synthetic intermediate for preparing 2266-d4 morphoshyline derivatives In an example of this embodiment the present invention is directed to a synthetic intermediate for preparing the deuterated linezolid of compound 10

2

(compound 10)

Such intermediates include the compounds of Formulas (I) (la) (lb) and (Ic) and the compounds of Formulas (II) (Ila) and (lib)

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 12: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β23 4

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above 40

In another embodiment the present invention is directed to a pharmaceutical composition comprising a pharmaceushytically acceptable carrier and the deuterated linezolid of compound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desig- 45

nated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

In another embodiment the present invention is directed to a method of treating a bacterial infection or a fungal disorder in a subject in need thereof comprising the step of 50

administering to the subject an effective amount of comshypound 10 or a pharmaceutically acceptable salt thereof wherein the deuterium enrichment at each position desigshynated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75 55

Another embodiment of the present invention is directed to use of compound 10 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a bacterial infection or a fungal disorder in a subject in need of the treatment wherein the deuterium enrichment at each 60

position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

Another embodiment of the present invention is directed to compound 10 or a pharmaceutically acceptable salt 65 thereof for use in treating a bacterial infection or a fungal disorder in a subject in need thereof wherein the deuterium

enrichment at each position designated as deuterium in compound 10 or a pharmaceutically acceptable salt thereof is at least about 75

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1A-1C depict concentration-time curve of linezolid in male rats following intravenous and oral administration of linezolid in combination with compound 10 FIG 1Α is a plot showing plasma concentration of linezolid versus time following intravenous administration of linezolid and comshypound 10 for each male rat tested FIG IB is a plot showing plasma concentration of linezolid versus time following oral administration of linezolid and compound 10 for each male rat tested FIG 1C is plot showing mean plasma concentrashytion of linezolid versus time following intravenous and oral administration of linezolid and compound 10 The No designation in FIGS 1Α and IB refer to the number given to the test rat

FIGS 2A-2C depict concentration-time curve of comshypound 10 in male rats following intravenous and oral administration of compound 10 in combination with linshyezolid FIG 2Α is a plot showing plasma concentration of compound 10 versus time following intravenous adminisshytration of compound 10 for each male rat tested and linshyezolid FIG 2Β is a plot showing plasma concentration of linezolid versus time following oral administration of comshypound 10 and linezolid for each male rat tested FIG 2C is plot showing mean plasma concentration of linezolid versus time following intravenous and oral administration of comshypound 10 and linezolid The No designation in FIGS 2Α and 2Β refer to the number given to the test rat

FIG 3 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying intravenous administration of linezolid and compound 10

FIG 4 is a plot showing mean plasma concentration of linezolid (--) and compound 10 (--) versus time followshying oral administration of linezolid and compound 10

FIGS 5Α and 5Β depict inhibition of mtDNA-encoded protein synthesis by linezolid (5Α) and compound 10 (5Β) The ratio of mtDNA encoded protein over nuclear DNA encoded protein was plotted against the concentration of tested compounds

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specishyfication

Unless otherwise stated when a position is designated specifically as ldquoHrdquo or ldquohydrogenrdquo the position is understood to have hydrogen at its natural abundance isotopic composhysition Also unless otherwise stated when a position is designated specifically as ldquoDrdquo or ldquodeuteriumrdquo the position is understood to have deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium which is 0015 (ie at least 525 incorporashytion of deuterium)

ldquoHalordquo or ldquohalogenrdquo means chloro bromo or fluoroldquoAlkylrdquo unless otherwise designated means an aliphatic

hydrocarbon group which may be straight-chain or branched having 1 to 15 carbon atoms Preferred alkyl groups have 1 to 12 carbon atoms Even more preferred alkyl groups are Cjg alkyl groups which are saturated straight-chain or branched hydrocarbons having one to six carbon atoms A ldquolower alkylrdquo group is a C1-4 alkyl group ldquoBranchedrdquo means that one or more lower alkyl groups such as methyl ethyl or propyl are attached to a linear alkyl chain Exemplary alkyl

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 13: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β26

-continued5

groups include methyl ethyl n-propyl i-propyl n-butyl t-butyl n-pentyl 3-pentyl heptyl octyl nonyl decyl and dodecyl preferred are methyl and i-propyl

ldquoArylrdquo means an aromatic carbocyclic radical containing 6 to 10 carbon atoms Exemplary aryl groups include phenyl or naphthyl

ldquoHeteroarylrdquo means 5-12 membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon for example nitrogen oxygen or sulfur Exemplary heteroaryl groups include pyrazinyl furanyl thienyl pyridyl pyrimidinyl isoxazolyl isothiaz- olyl pyridazinyl 124-triazinyl thiadiazolyl oxadiazolyl quinolinyl and isoquinolinyl

ldquoAralkylrdquo means an aryl-alkyl group in which the aryl and alkyl components are as previously described Preferred aralkyls contain a lower alkyl moiety Exemplary aralkyl groups include benzyl and 2-phenethyl

ldquoHeteroaralkylrdquo means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described

ldquoCycloalkylrdquo means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms

ldquoHeterocycloalkylrdquo means a non-aromatic mono- or mulshyticyclic hydrocarbon ring system in which at least one of the carbon atoms in the ring system is replaced by a heteroatom for example nitrogen oxygen or sulfur Exemplary heteroshycycloalkyl groups include pyrrolidinyl piperidinyl tetrahy- dropyranyl tetrahydrofuranyl tetrahydrthiopyranyl andtet- rahydrothiofuranyl

ldquoCycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

ldquoHeteroycloalkylalkylrdquo means a group in which the cycloalkyl and alkyl components are as previously described

The term ldquocompoundrdquo when referring to a compound of this invention refers to a collection of molecules having an identical chemical structure except that there may be isoshytopic variation among the constituent atoms of the molshyecules Thus it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorposhyration of deuterium in the various synthesis steps used to prepare the compound However as set forth above the relative amount of such isotopologues in toto will be less than 499 of the compound In other embodiments the relative amount of such isotopologues in toto will be less than 475 less than 40 less than 325 less than 25 less than 175 less than 10 less than 5 less than 3 less than 1 or less than 05 of the compound

A Bronsted acid is a proton donor A Lewis acid is an electron pair acceptor Examples of Bronsted and Lewis acids are well known to the skilled artisan and are comshymercially available from a wide variety of sources

Other definitions are set forth in the table below

HPLC High performance liquid chromatographyHr HourKg Kilogram

LC Liquid chromatographyL LiterLOQ Limit of quantitationug or pg Microgrammg MilligrammL MilliliterMin MinuteMS Mass spectrometryΝΑ Not applicable

The present invention is directed to a process for preparshying 2266-d4 morpholine derivatives represented by Strucshytural Formula (I)

or a salt thereof Values and specific values for each variable in Structural Formula (I) are provided in the following paragraphs

R1 is mdashΗ OH mdashNO mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0)NRUU mdashC(=0)ORg -phthalimido mdashS02mdash R4 or a group selected from alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl aryl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocycloalkylalkyl are each indeshypendently optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRrf wherein each Cjg alkyl is optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy In one embodiment R1 is mdashΗ optionally substituted alkyl optionally substituted aryl optionally subshystituted heteroaryl optionally substituted aralkyl mdashC(=0) NRregRrf mdashC(=0)ORg or mdashS02mdashR4 In another embodishyment R1 is mdashΗ or optionally substituted benzyl wherein the benzyl is optionally substituted with one or more groups selected from halogen mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0) Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregR4 In another embodiment R1 is mdashΗ or unsubshystituted benzyl In another embodiment R1 is an alkyl group that is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg haloalkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRlsquo4 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRregRlsquo4 wherein the Cjg alkyl and Cjg haloalkyl substitutents are each further optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and C1-6 haloalkoxy

Rreg for each occurrence is independently an alkyl optionshyally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 14: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β2

R is alkyl aryl heteraryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)0RregmdashC(=0)Rreg mdashno2 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRdegC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg 5 mdashSRreg and mdashS02NRdegR7 wherein each Cjg alkyl substishytuted is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg and Rrf are each independently mdashΗ or an alkyl option- 10 ally substituted with one or more groups selected from halogen Cj6 alkyl mdashORreg mdashC(=0)0Rreg mdashC(=0)RregmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRdegRlsquo7 mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and 15 mdashS02NRdegR7 wherein each Cjg alkyl substituent is optionshyally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

Rreg is alkyl optionally substituted with one or more groups 20

selected from halogen Cjg alkyl mdashOR7 mdashC(=0)0RmdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2mdashNRregC(=0)R mdashC(=0)NRdegRlsquo7 mdashS(0)R mdashS(0)2RmdashSR7 and mdashS02NRdegRlsquo7 wherein each Cjg alkyl substitushyent is optionally substituted with one or more groups 25

selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is mdashΗ or alkyl optionally substituted with one or more 30 groups selected from halogen Cjg alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHRregmdashN(Rreg)2 mdashNRregC(=0)R mdashC(=0)NRregRreg mdashS(0)RmdashS(0)2R mdashSR7 and mdashS02NRdegRreg wherein each C^ 35 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxymdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally independently substituted with one or more 40 halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

In one embodiment Rreg is alkyl optionally substituted with one or more groups selected from halogen Cjg alkylmdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashC(=0) 45 NRregRreg and mdashS02NRdegRlsquo7 wherein each alkyl substitushyent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

In one embodiment R4 R4 R5 and R5 are all mdashΗ 50In one embodiment the process of the invention comshy

prises reacting a compound of Formula (II) with an acid to form the compound of Formula (I) or a salt thereof

7

(Reaction 1)55

D D D D

Ν an acid R4 -

ho yr yr oh

R4 R4 R5 R5

(Π)

-D

-R5 60Ν

R4 | Rdeg R1

(I)

65Typically the reaction is performed at a temperature in the

range of 100-200deg C such as 120-160deg C such as 140 to

150deg C over a time ranging from 1 to 24 hours such as between 10 and 18 hours such as between 16 and 18 hours

In one embodiment the compound of Formula (II) or a salt thereof is prepared by reacting a compound of Formula (III) with a reducing agent

8

(Reaction 2)

R

Ν

R4 R4 R5 R5

(III)

reducing agent

D D D D

HO OH

R4 R4 R5 R5

(Π)

wherein each R2 is D mdashOH or mdashO(alkyl) For example R2 may be mdashOH or mdashO(alkyl) If R2 is mdashO(alkyl) the alkyl group in mdashO(alkyl) is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashORreg mdashC(=0)ORreg mdashC(=0)Rreg mdashN02 mdashCN mdashNH2 mdashNHRreg mdashN(Rreg)2 mdashNRregC(=0)Rreg mdashC(=0)NRregRreg mdashS(0)Rreg mdashS(0)2Rreg mdashSRreg and mdashS02NRdegRreg wherein each C1-6 alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy In one embodishyment R2 is mdash0(0^3 alkyl) In another embodiment R2 is mdashOEt (Et=ethyl)

In one embodiment the process for preparing the comshypound of Formula (I) comprises(a) reacting a compound of Formula (III) with a reducing agent to form the compound of Formula (II) and (b) reacting the compound of Formula (II) with an acid to form the optionally substituted morpholine derivative of Formula (I) or a salt thereof

In another embodiment the process described above further comprises the step of removing R1 from a compound of Formula (I) when it is other than mdashΗ to form a morphoshyline derivative of Formula (la) or a salt thereof

(Reaction 3)

In a specific embodiment R1 for Reaction 3 is benzyl and the benzyl group is removed under hydrogenation condishytions In one embodiment reagents for removing R1 from the compound of Formula (I) include hydrogen gas In one embodiment hydrogenation may be performed using a metal-based catalyst More specifically a palladium-based catalyst or a platinum-based catalyst may be used Suitable palladium-based catalysts are well-known to one skilled in the art In one embodiment the palladium-based catalyst is

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 15: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β29 10

Pd(OH)2 on carbon In another embodiment the catalyst is an elemental palladium catalyst such as PdC Pdalumina or palladium black A suitable platinum-based catalysts is for example an elemental platinum catalyst such as PtC

In one embodiment for any one of compounds of Struc- 5 tural Formulas (I) (la) (II) and (III) R4 R4 R5 and R5 are all mdashΗ

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl to optionally substituted as defined above a benzyl optionally substituted as defined above mdashSQ2R or mdashC(=0)NRTU

-continued(Ic)

(Π)

In another embodiment for any one of compounds of Structural Formulas (I) (II) and (III) R1 is mdashΗ an alkyl optionally substituted as defined above benzyl or mdashS02RIn another embodiment R1 is mdashΗ benzyl or mdashS02R In a more specific embodiment R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl In an even more specific embodiment R1 is benzyl In one aspect of this more specific embodiment 2o R1 is benzyl and R4 R4rsquo R5 and R5rsquo are all mdashΗ

Acids that are suitable for Reaction 1 are well known to one skilled in the art The acid can be a Lewis acid or a Bronsted acid Examples of suitable acid include but not limited to hydrochloric acid sulfuric acid phosphoric acid 25 trifluoroacetic acid HBF4 ZnCl2 optionally with a coshysolvent such as THF toluenesulfonic acid optionally with a co-solvent such as toluene and boron trifluoride etherate In one embodiment the acid is an aqueous acid More specifi- cally the acid is sulfuric acid Even more specifically the acid is 70 sulfuric acid

Suitable reducing agents for Reaction 2 are also well known to one skilled in the art Some examples of suitable reducing agents include but are not limited to diborane-d6 35

(B2D6) DSiCl3 Et3SiD diisobutylaluminum deuteride (DIBAL-D) LiAlD4 LiBD4 and NaBD4 In one embodishyment the reducing agent is selected from the group consistshying of LiAlD4 LiBD4 and NaBD4 More specifically the reducing agent is LiAlD4 40

The present invention is also directed to the compounds of Formulas (I) (la) (lb) and (Ic) and the synthetic intermeshydiates of Formulas (II) (Ha) and (lib)

R4 R4 R5 R5

(Ha)

or a salt thereof wherein each of R1 R4 R4 R5 and R5 are as defined above

In one embodiment R1 in any one of Structural Formulas(I) (Ic) (II) and (lib) is benzyl mdashS02-aryl or mdashS02- heteroaryl each of which is optionally substituted In one embodiment R1 in any one of Structural Formulas (I) (Ic)(II) and (lib) is mdashΗ or benzyl

As an example the present invention is directed to compounds 3 and 3a and to synthetic intermediate comshypound 2

D DD^deg^D

(I) D D and

50ΝΗ

D D Bn D D(2)

(la) HOΝ

OH

55or a salt of any of the foregoing

The process of the present invention can be used to prepare deuterated version of morpholine-containing pharshymaceutical agents Such pharmaceutical agents include but

(lb) 60 are not limited to xamoterol xamoterol fumarate myco- phenolate mofetil rocuronium rocuronium bromide moclobemide landiolol linezolid emorfazone moricizine moricizine hydrochloride timolol timolol maleate molsidshyomine gefitinib pinaverium pinaverium bromide nimora-

65 zole linsidomine momiflumate rivaroxaban aprepitant fosaprepitant radafaxine and pharmaceutical acceptable salts thereof

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 16: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β211 12

As used herein ldquomorpholine-containing pharmaceutical agentsrdquo refers to any pharmaceutical agents that contain one or more morpholine moiety The morpholine moiety can have one or more substituents on the morpholine ring

In one embodiment the process of the present invention can be used to make deuterated linezolid comprising a morpholine moiety represented by the following structural formula

Examples of deuterated linezolid of the present invention are represented by the following structural formulas

(10)

or a pharmaceutically acceptable salt of either of the foreshygoing

The deuterated linezolid 10 can be prepared according to Scheme 2 described below

One embodiment of the present invention is directed to deuterated linezolid 10 and the synthetic intermediates repshyresented by the following structural formulas

-continued(5)

(6)

(7)

(8)

(9)

g0 or a salt of any of the foregoingIn one embodiment the deuterium enrichment at each

position for any one of the compounds represented by Structural Formulas (I)-(Ic) (Il)-(IIb) (2) (3) (3a) (4)-(11) is at least about 70 at least about 75 at least about 80

65 at least about 85 at least about 90 at least about 95 at least about 97 at least about 975 at least about 990 or at least about 995 The percentage for deuterium

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 17: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β213 14

enrichment refers to mole percentage When any of these compounds are analyzed by 1H NMR the lack of a visible signal corresponding to the protons alpha to the oxygen indicates deuterium enrichment at those positions of at least 95

As used herein salts include acid salts and base salts For example acid salts of a compound of the present invention containing an amine or other basic group can be obtained by reaction of the compound with a suitable organic or inorshyganic acid resulting in anionic salt In one embodiment acid salts of the present invention are pharmaceutically acceptshyable salts Such pharmaceutically acceptable salts include but not limited to acetate benzenesulfonate benzoate bicarshybonate bitartrate bromide calcium edetate camsylate carshybonate chloride citrate dihydrochloride edetate edisylate estolate esylate fumarate glyceptate gluconate glutamate glycollylarsanilate hexylresorcinate hydrobromide hydroshychloride hydroxynaphthoate iodide isethionate lactate lactobionate malate maleate mandelate mesylate methyl- sulfate mucate napsylate nitrate pamoate pantothenate phosphatediphospate polygalacturonate salicylate stearshyate subacetate succinate sulfate tannate tartrate teoclate tosylate and triethiodide salts Non-pharmaceutically acceptable salts are also included in the present invention such as trifluoroacetic acid salt

Salts of the compounds of the present invention containshying a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base In one embodishyment base salts of the present invention are pharmaceutishycally acceptable salts Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceushytically acceptable cation which includes alkali metal salts (especially sodium and potassium) alkaline earth metal salts (especially calcium and magnesium) aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine triethyl- amine morpholine pyridine piperidine picoline dicyclo- hexylamine NN-dibenzylethylenediamine 2-hydroxyeth- ylamine bis-(2-hydroxyethyl)amine tri-(2-hydroxyethyl) amine procaine dibenzylpiperidine dehydroabietylamine NN-bisdehydroabietylamine glucamine N-methylglu- camine collidine quinine quinoline and basic amino acids such as lysine and arginine

A ldquopharmaceutically acceptable saltrdquo means any nonshytoxic salt that upon administration to a recipient is capable of providing either directly or indirectly a compound of this invention

In another embodiment a pharmaceutical composition comprising a deuterated linezolid of the present invention further comprises a second therapeutic agent The second therapeutic agent includes any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with an antimicrobial comshypound in particular in anti-microbial therapy combination therapy with other anti-microbial andor anti-inflammatory agents is envisaged Combination therapies according to the present invention thus include the administration of a deushyterated linezolid of the present invention (ie a deuterated linezolid comprising a 2266-d4 morpholinyl moiety and having a deuterium enrichment at each position designated as deuterium of at least about 70) at least one compound of formula I or la as well as optional use of other antishymicrobial agents and optional use of cyclooxygenase inhibishytors particularly selective inhibitors of cyclooxygenase-2 Other anti-microbial therapies and anti-inflammatory agents are described for instance in International Publication Nos WO 0134128 and WO 03061704 which applications are

incorporated by reference to the extent that they disclose combinations of anti-microbial and anti-inflammatory therashypies

Examples of second therapeutic agents that may be for- 5 mulated with a deuterated linezolid of this invention include

but are not limited to gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

In the pharmaceutical compositions of the invention the to compound of the present invention is present in an effective

amount As used herein the term ldquoeffective amountrdquo refers to an amount which when administered in a proper dosing regimen is sufiScient to treat (eg reduce or ameliorate the severity duration or progression of the taiget disorder

15 prevent the advancement of the taiget disorder cause the regression of the target disorder or enhance or improve the prophylactic or therapeutic efifect(s) of another therapy) the target disease or disorder

The interrelationship of dosages for animals and humans 20 (based on milligrams per meter squared of body surface) is

described in Freireich et al (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately detershymined from height and weight of the patient See eg Scientific Tables Geigy Pharmaceuticals Ardsley ΝΥ

25 1970 537 An effective amount of a compound of this invention can range from about 50 mg to about 2000 mg every 24 hours if appropriate in the form of several indishyvidual doses In one embodiment the effective amount of a compound of this invention ranges from about 250 mg to

30 about 1250 mg every 24 hours in the form of a single dosage or two separate dosages of about 125 mg to about 625 mg each given every 12 hours In another embodiment the effective amount of a compound of this invention ranges from about 750 mg to about 1250 mg every 24 hours in the

35 form of a single dosage or two separate dosages of about 375 mg to about 625 mg each given every 12 hours In still another embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 1200 mg every 24 hours in the form of a single dosage or two separate

40 dosages of about 225 mg to about 625 mg each given every 12 hours In a more specific embodiment the effective amount of a compound of this invention ranges from about 450 mg to about 750 mg every 24 hours in the form of a single dosage or two separate dosages of about 225 mg to

45 about 375 mg each given every 12 hours Other ranges of a compound of this invention that fall within or between any of the above-recited ranges are also within the scope of the invention Effective doses will also vary as recognized by those skilled in the art depending on the diseases treated the

50 severity of the disease the route of administration the sex age and general health condition of the patient excipient usage the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician

55 The milligram amounts of compounds present in the pharmaceutical compositions of the present invention and for use in the methods of the present invention represent the amount of free base compound It will be understood that the use of pharmaceutical salts of the compounds of the present

60 invention will require that the stated amounts be increased so that a mole equivalent of the free base compound is used

For pharmaceutical compositions that comprise a second therapeutic agent an effective amount of the second therashypeutic agent is between about 20 and 100 of the dosage

65 normally utilized in a monotherapy regime using just that agent Preferably an effective amount is between about 70 and 100 of the normal monotherapeutic dose The normal

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

5

10

15

20

25

30

35

40

45

50

55

60

65

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

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  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 18: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β2

monotherapeutic dosages of these second therapeutic agents are well known in the art See eg Wells et ah eds Pharmacotherapy Handbook 2nd Edition Appleton and Lange Stamford Conn (2000) PDR Pharmacopoeia Tar- ascon Pocket Pharmacopoeia 2000 Deluxe Edition Taras- con Publishing Loma Linda Calif (2000) each of which references are entirely incorporated herein by reference

It is expected that some of the second therapeutic agents referenced above will act synergistically with the comshypounds of this invention When this occurs its will allow the effective dosage of the second therapeutic agent andor the compound of this invention to be reduced from that required in a monotherapy This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a compound of this invention synergistic improvements in efficacy improved ease of administration or use andor reduced overall expense of compound preparation or forshymulation

According to another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease that is beneficially treated by linezolid comprisshying the step of administering to said subject an effective amount of a deuterated linezolid or a pharmaceutical comshyposition of this invention Such diseases are well known in the art and include for instance the treatment or prevention of a variety of disease states typically treated by antimicroshybial therapy (eg infection fungal disorders) The deutershyated linezolid of this invention therefore have utility in the treatment of disorders including those mediated by Gramshypositive bacteria and certain Gram-negative and anaerobic bacteria

In one embodiment the invention provides a method of treating a subject suffering from or susceptible to an infecshytion caused by a bacteria selected from Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae Streptococcus pneumoniae Streptococcus pyrogenes Enterococcus faecalis Staphylococcus epidermidis Staphy- loccocus haemolyticus and Pasteurella multocida

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus an infection of the eye and tuberculosis

In another embodiment the invention provides a method of treating a subject suffering from diabetic foot infections nocardiosis endophthalmitis keratitis conjunctivitis or impetigo

In another embodiment the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder (or symptoms thereof) selected from a Gram-positive bacterial infection Vancomycin-resistant Enterococcus faecium infection nosocomial pneumonia due to Staphylococcus aureus and Streptococcus pneumoniae complicated skin and skin structure infections caused by Staphylococcus aureus Streptococcus pyogenes or Strepshytococcus agalactiae uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococshycus pyogenes and community-acquired pneumonia caused by Streptococcus pneumoniae or Staphylococcus aureus

15In another embodiment the invention provides a method

of treating a patient suffering from or susceptible to a bacterial infection comprising the step of administering to the patient in need thereof over a 24 hour period between about 450 mg and about 750 mg of a deuterated linezolid of this invention In another embodiment the patient is adminshyistered between 450 mg and 700 mg of a deuterated linezolid of the present invention

In another embodiment the above method of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with linshyezolid

In a specific embodiment the combination therapies of this invention include co-administering a deuterated linshyezolid of the present invention and a second therapeutic agent selected from gentamicin tobramycin aztreonam cefazolin ceftazidime piperacillin ciprofloxacin ofloxacin levofloxacin celecoxib and rofecoxib

Example 1

Synthesis of 2266-d4-Morpholine (3a)

16

Scheme 1 Preparation of Intermediate 3a

O Bn O

Step 1 22-(Benzylazanediyl)bis(ll-d2-ethanol) (2) To a solution of diethyl benzyliminodiacetate (1 550 g 1969 mmol) in anhydrous tetrahydrofuran (500 mL) at 0deg C was added lithium aluminum deuteride (165 g 3938 mmol Cambridge Isotopes 98 atom D) in portions with internal temperature below 10deg C After addition the reaction was stirred overnight at room temperature and then quenched sequentially with water (165 mL) 15 wt sodium hydroxshyide (165 mL) and water (495 mL) at 0deg C The suspension was stirred 2 hours at room temperature filtered over celite cake and washed with THF (400 mL) The filtrate was evaporated in vacuo to give 2 (365 g 93) as a pale yellow oil

Step 2 N-benzyl-2266-d4-morpholine (3) A solution of 2 (365 g 1834 mmol) in 70 sulfuric acid (138 mL) was heated in a sealed tube at 150deg C for 16 hours cooled to room temperature and slowly poured onto crushed ice (300 g) The resulting mixture was slowly basified to pH 9 with solid potassium carbonate and mixed with EtOAc (500 mL) The suspension was filtered over a celite cake and washed

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US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 19: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β218

-continued17

with EtOAc (400 mL) For the filtrate the two layers were split and the aqueous layer was extracted with EtOAc (2x300 mL) The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give 3 (316 g 95) as lightly tan oil The signal corresponding to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 ΜΕ1ζ instrushyment The absence of the signal indicates that less than 5 of hydrogen is present

Step 3 2266-d4-Morpholine (3a) A solution of (3 316 g) in methanol (300 mL) was shaken under hydrogen (30 psi) with Pd(OEl)2 on carbon (63 g) as catalyst until no further hydrogen was consumed The reaction mixture was filtered over a celite cake and washed with methanol (400 mL) The filtrate was evaporated at 25deg C to give 3a as a pale yellow oil in quantitative yield The signal correspondshying to the protons alpha to the oxygen was not visible in the XH NMR spectrum performed on a Varian Mercury 300 MHz instrument The absence of the signal indicates that less than 5 of hydrogen is present

Example 2

Synthesis of (S)mdashN-((3-(3-Fluoro-4-(2266-d4- morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)

acetamide (10)

7

Scheme 2 Preparation of Compound 1030

35

40

45

50

9

10

Step 1 2266-d4-4-(2-Fluoro-4-nitrophenyl)morpholine (4) To a solution of 34-difluoronitrobenzene (12) (265 g 1663 mmol) and diisopropylethylamine (76 mL 4365 mmol) in acetonitrile (350 mL) was added 3a (1746 mmol) The reaction was stirred at reflux for 16 hours then was concentrated in vacuo The crude residue was taken up with water (300 mL) The precipitate was filtered washed with

60 water (200 mL) and heptane (300 mL) and dried under vacuum at 40deg C for 5 hours to give 4 (348 g 91) as a bright yellow solid

Step 2 3-Fluoro-4-(2266-d4-morpholino)aniline (5) A solution of 4 (348 g) in ethanol (400 mL) was shaken under

65 hydrogen (30 psi) with 10 wt PdC (70 g containing 50 wt water) until no additional hydrogen was consumed (ca 3 hours) The reaction mixture was filtered over Celite and

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 20: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β219

washed with ethanol (400 mL) The filtrate was concentrated in vacuo to give 5 (267 g 85) as a white solid

Step 3 (R)-l-Chloro-3-(3-fluoro-4-(2266-d4-mor-pholino)phenylamino)propan-2-ol (6) To a solution of 5 (236 g 118 mmol) in 2-propanol (30 mL) was added (R)-(-)-epichlorohydrin (12 g 130 mmol) The reaction was stirred at reflux for 15 hours and another 024 g (26 mmol) of (R)-(-)-epichlorohydrin was added The reaction was stirred at reflux another 6 hours and the solvent was removed to give 6 as an oil that was used in the next step without further purification

Step 4 (R)-5-(Chloromethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (7) A solution of 6 (ca 118 mmol) and ll-carbonyldiimidazole (268 g 165 mmol) in dichloromethane (100 mL) was stirred overnight at room temperature and concentrated to give a crude oil containing 7

Step 5 (S)-2-((3-(3-Fluoro-4-(2266-d4-morpholino)phenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-13-dione

20Example 3

Pharmacokinetic Study in Rats

Materials and Methods1808 mg of linezolid and 1001 mg of compound lOwere

dissolved in 10 DMI 15 Ethanol 35 PG and 40 D5W independently to yield a final concentration at 10 mgmL (ρΗ-6) The combo dose was prepared by mixing both by 11 to yield a concentration of 5 mgmL for each compound (pH~6) for intravenous and oral administration The obtained solution was clear and colourless The con-

15 centrations of linezolid and compound 10 in each individual dose were confirmed by HPLC method

Male Sprague Dawley rats (body weight 170 g to 220 g) were used in this study Before the pharmacokinetic studies animals were randomly assigned to the treatment groups The treatment schedules are shown in Table 1

TABLE 1

Experimental Design

No of Male Rats Test Article

Test ArticleFormulation

Target Dose Dose LevelRoute (mgkg)

Target DoseConcentration(mgmL)

Target Dose Volume (mLkg)

3 linezolid + 10 DMI15 Ethanol IV 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

3 linezolid + 10 DMI15 Ethanol ΡΟ 5 mgkg 5 mgmL 1compound 10 35 PG40 D5W for each for each

(8) To a solution of 7 (ca 118 mmol) in DMF (50 ml) was added phthalimide potassium salt (284 g 153 mmol) The reaction mixture was heated at 100deg C for 6 hours cooled to room temperature taken up with water (100 mL) and extracted with ΜΤΒΕ (3x100 mL) The combined organic layers were washed with brine (2x200 mL) dried over sodium sulfate and concentrated in vacuo The crude solid was triturated with ΜΤΒΕ (100 mL) to give 8 (33 g 66 for 3 steps) as a white solid

Step 6 (S)-5-(Aminomethyl)-3-(3-fluoro-4-(2266-d4- morpholino)phenyl)oxazolidin-2-one (9) A solution of 8 (33 g 768 mmol) and hydrazine monohydrate (207 g 423 mmol) in methanol (40 mL) was stirred at reflux for 1 hour The reaction mixture was concentrated in vacuo taken up with water (100 mL) and extracted with dichloromethane (3x100 mL) The combine organic layers were washed with water (150 mL) dried over sodium sulfate and concentrated in vacuo to give 9 (216 g) as a tan oil in quantitative yield

Step 7 (S)mdashN-((3-(3-Fhioro-4-(2266-d4-morpholino) phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (10) To a solution of 9 (216 g 722 mmol) in toluene at room temperature was added acetic anhydride (2 mL 209 mmol) The reaction mixture was warmed at 35deg C for 5 minutes and then stirred overnight at ambient temperature The reaction mixture was cooled to 0deg C filtered washed with toluene and dried at 40deg C for 4 hours to give 10 (12 g 49) as a white solid The signal corresponding to the protons alpha to the oxygen is not visible in the 14 NMR spectrum

35 Blood samples were collected by retro-orbital at 0 (preshydose) and 0083 025 05 1 2 4 6 8 10 12 and 24 hours post-dose The plasma samples and the dose formulation were stored at -20deg C until bioanalysisSample Analysis

40 The concentrations of linezolid and compound 10 in plasma were determined using a high performance liquid chromatographymass spectrometry (F1PLCMSMS) methodLC-MSMS Apparatus

45 The LC system comprised an Agilent (Agilent Technoloshygies Inc USA) liquid chromatograph equipped with an isocratic pump (1100 series) an autosampler (1100 series) and a degasser (1100 series) Mass spectrometric analysis was performed using anAPI3000 (triple-quadrupole) instru-

50 ment from ΑΒ Inc (Canada) with an ESI interface The data acquisition and control system were created using Analyst 14 software from ABI Inc

Other equipment XW-80A Vortex mixer (Shanghai) TGL-16B high speed centrifuge (Shanghai) Millipore Aca-

55 demic Ultrapure-water generating systemInternal Standard (Quetiapine) was a gift from Shanghai

Institute of Pharmaceutical Industry Acetonitrile and methashynol (Tedia Inc USA) were F1PLC grade All other solvents and chemicals were analytical grade or better

60 LC-MSMS Conditions Chromatographic ConditionsColumn Phenomenex Gemini C6-pheny 5 pm (50

mmx46 mm)Mobile phase 01 Formic acidMethanol=1090

65 Elution rate 1000 pLmin Column temperature 25deg CInjection volume 5 pL

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

28

5

10

15

20

25

30

35

  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 21: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β221

MassScan type Positive MRM Ion source Turbo spray Ionization model ESI Nebulize gas 8 Lmin Curtain gas 8 Lmin Collision gas 4

LminIonspray voltage 4500 ν Temperature 450deg COther parameters

Data AnalysisPharmacokinetic Data Analysis

The concentrations in plasma below the limit of quanti- 5 tation (LOQ=5 ngmL) were designated as zero The pharshy

macokinetic data analysis was performed using noncompart- mental analysis modules in WinNonlin20 The

22

Drug name Qi Q3 Dell time DP (ν) FP (ν) ΕΡ (ν) CE (ν) CXP (ν)

Linezolid 33807 29627 200 ms 61 160 10 27 20

compound 10 34217 30019 200 ms 56 170 10 27 20

Quetiapine 3842 2532 200 ms 50 200 10 31 15

Preparation of Standard Stock SolutionA stock solution of linezolid and compound 10 was

prepared by dissolving the drug in methanol to yield a final 20

concentration of 200 pgmL respectively Then proper volshyume of these two solutions were transferred into one flask and diluted to the mark with methanol to make a mixture of two compounds with the same concentration of 25 pgmL 25

An aliquot of this mixture was diluted using methanol to get a series of working solutions of 25 50 250 500 2500 5000 and 25000 ngmL Seven calibration standard samples containing 5000 1000 500 100 50 10 and 5 ngmL were obtained by adding 20 pL working solution prepared above 30

into seven EppendorfF tubes containing 100 pL blank plasma QC samples were prepared by spiking 100 pL blank plasma with 20 pL working solutions of20000 4000 and 40 ngmL to yield final concentration of 4000 800 and 8

ngmL 35

Stock solution of Quetiapine (internal standard IS) was prepared by dissolving the drug in methanol to a final concentration of 200 pgmL This solution was diluted with methanol to yield a final concentration of 50 ngmLPlasma Sample Process 40

Plasma samples (01 mL) were transferred to EppendorfF tube then 20 pL methanol and 300 pL IS solution (50 ngmL) were added to it After Vortexing for 1 min and centrifuging for 5 min at 15000 rpm 5 pL of supernatant ^ was injected into LCMSMSMethod Validation Results SpecificityThe chromatographic conditions showed that the blank plasma had no interference to the test compounds and IS 50

determinationCalibration Curve

The analytical curves were constructed using seven nonshyzero standards ranging from 5 to 5000 ngmL A blank sample (matrix sample processed without internal standard) 55 was used to exclude contamination The linear regression analysis of linezolid and compound 10 were performed by plotting the peak area ratio (y) against the concentration (χ) in ngmL for linezolid or compound 10 respectively The linearity of the relationship between peak area ratio and 60

concentration were demonstrated by the correlation coeflS- cients (R) obtained for the linear regressions of linezolid and compound 10Intra-assay Accuracy

The intra-assay accuracy results (ranged from 8354 to 65 10638 for linezolid and 9400 to 11332 for comshypound 10) showed that the method is reliable

bioavailability was calculated as F()=(DoselVx AUCoraZ(0_co))(DoseoraZxAUCv(0_co))100Results and DiscussionPharmacokinetics of Linezolid after Combinatory Adminisshytration

The individual and average concentration-time data of linezolid following intravenous and oral administration linshyezolid in combination with compound 10 are listed in Table 2 and shown in FIGS 1A-1C Selected noncompartmental pharmacokinetic parameters following intravenous and oral dose are listed in Table 3

TABLE 2

Plasma Concentration of Linezolid in Male Rats Following Intravenous and Oral Administration in Combo with Compound 10

Time (hr) Plasma Concentration (ngmL)

IV-5 mgkg R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 632403 530966 598354 587241 51624025 435205 472702 474246 460718 2210805 332369 395946 404558 377624 394281 291908 263607 283548 279688 145402 136181 135043 159624 143616 138754 27833 32060 36130 32008 41496 5801 6151 6963 6305 5968 1553 2606 1675 1945 576

10 446 724 729 633 16212 0 0 0 ΝΑ ΝΑ24 0 0 0 ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 0 0 0 ΝΑ ΝΑ0083 311708 87270 82997 160658 130830025 359456 176467 109944 215289 12920705 326677 214220 113283 218060 1067491 254456 196434 116957 189282 690282 142406 172858 86003 133756 440694 38280 78974 66496 61250 208486 7020 18451 42025 22499 178508 2139 4616 14645 7133 6622

10 000 1947 4028 1992 201412 000 000 515 172 29724 000 000 000 ΝΑ ΝΑ

SD Standard deviationΝΑ Not applicable or failed to collect samples

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

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  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 22: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β224

TABLE 323

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0pgLhr

AUC^jpgLhr

MRT(0_hr

asymp) ^ℓ2ζhr

Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

IV-5 mgkg

R 1 820053 820749 128 108 0083 095 061 632403R 2 819382 820453 134 103 0083 090 061 530966R 3 895618 896661 136 099 0083 080 056 598354mean 845017 845954 133 103 0083 088 059 564660SD 43823 43914 004 005 0 008 003 47651

PO-5 mgkg

R 4 736111 736621 154 096 025 ΝΑ ΝΑ 359456 8702R 5 742944 743426 228 104 05 ΝΑ ΝΑ 214220 8782R 6 547404 548019 317 083 1 ΝΑ ΝΑ 116957 6471mean 675486 676022 233 094 058 ΝΑ ΝΑ 230211 7985SD 110975 110906 082 011 038 ΝΑ ΝΑ 122038 1312

20

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for linezolid was059plusmn003 Lhrkg which corresponded to 1782 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 25 half-life (T12) for linezolid was 103plusmn005 hr

Following an IV combo administration of linezolid and compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and 30

Αυθ(οinfin) for linezolid was 564660plusmn47651 pgL and 845954plusmn43914 hrpgL The volume of distribution at terminal phase was 088plusmn008 Lkg which corresponded to 13134 of the total body water (067 Lkg) in the rats

Following an oral combo administration of linezolid and 35 compound 10 at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax and Tmax for inezolid were 230211plusmn122038 pgL and 058plusmn038 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 676022plusmn110906 hrpgL and 094plusmn011 hr respectively The meanplusmnSD value of bioavailability for inezolid was 7985plusmn1312Pharmacokinetics of Compound 10 after Combinatory Administration

The individual and average concentration-time data of compound 10 following intravenous and oral administration compound 10 in combination with linezolid are listed in Table 4 and shown in FIGS 2A-2C Selected noncompart- mental pharmacokinetic parameters following intravenous 50

and oral dose are listed in Table 5

TABLE 4

Plasma Concentration of Compound 10 in Male Rats Following Intravenous and Oral Administration in Combo with Linezolid

Time (hr)

IV-5 mgkg

Plasma Concentration (ngmL)

R7 R8 R9 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 623985 522629 616063 587559 56370025 472924 460804 515586 483105 2877505 359016 425451 441269 408579 436451 330580 274530 298525 301212 281212 151770 145258 173663 156897 148804 31918 36842 43889 37550 60176 7089 7076 8803 7656 9938 1594 2951 2509 2351 692

10 000 706 953 553 49512 BLQ BLQ BLQ ΝΑ ΝΑ24 BLQ BLQ BLQ ΝΑ ΝΑ

PO-5 mgkg R10 Rll R12 Mean SD

0 BLQ BLQ BLQ ΝΑ ΝΑ0083 317706 91300 88724 165910 131465025 357765 184244 112293 218101 12619005 358670 213740 121821 231410 1194091 268305 225376 128126 207269 718222 164999 184314 101710 150341 432094 43042 86128 68257 65809 216476 8936 22910 46714 26187 191018 1339 5204 16020 7521 7610

10 790 2383 5081 2751 216912 000 000 971 324 56124 BLQ BLQ BLQ ΝΑ ΝΑ

TABLE 5

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_) AUC^^) MRT(0-(asymp) tf2z Τmax Vz CLZ Cmax FpgLhr pgLhr hr hr hr Lkg Lhrkg ggL

IV-5 mgkg

R 1 897823 897914 130 083 0083 067 056 623985R 2 863163 863433 139 100 0083 084 058 522629R 3 973659 975085 142 104 0083 077 051 616063mean 911548 912144 137 096 0083 076 055 569346SD 56513 57170 006 011 0 009 004 66068

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

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  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 23: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

US 9453009 Β22625

TABLE 5-continued

Selected Pharmacokinetics Parameters of Linezolid in Rats Following Intravenous and Oral Administration in Combo with Compound 10

AUC(0_0μgLhr

AUC^jμgLhr

MRT(0_(hr

Kgt hr Tmaxhr

VzLkg

CLZLhrkg

CmaxpgL

F

PO-5 mgkg

R 4 804961 806108 159 101 050 ΝΑ ΝΑ 358670 8825R 5 809094 809642 232 105 100 ΝΑ ΝΑ 225376 8870R 6 601961 603482 319 109 100 ΝΑ ΝΑ 128126 6599mean 738672 693938 237 105 083 ΝΑ ΝΑ 237391 8098SD 118413 336536 080 004 029 ΝΑ ΝΑ 115741 1298

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD value of systemic clearance for compound 10 was055plusmn004 Lhrkg which corresponded to 1662 of rat hepatic blood flow (331 Lhrkg) The meanplusmnSD value of 2o half-life (T12) for compound 10 was 096plusmn011 hr

Following an IV combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the meanplusmnSD values of Cmax (at 5 minutes after dosing) and Αυθ(οinfin) for compound 10 was 569346plusmn66068 pgL and 25

912144plusmn57170 hrpgL The volume of distribution at terminal phase was 076plusmn009 Lkg which corresponded to 11343 of the total body water (067 Lkg) in the rats

Following an oral combo administration of compound 10 and linezolid at a nominal dose of 5 mgkg for each the 30 meanplusmnSD values of Cmax and Tmax for compound 10 were 237391 plusmn115741 pgL and 083plusmn029 hr respectively the meanplusmnSD values of AUC^^ and half-life (T12) were 693938plusmn336536 hrpgL and 105plusmn004 hr respectively The meanplusmnSD value of bioavailability for compound 10 was 35 8098plusmn1298

CONCLUSIONS

Following combo IV injection of compound 10 with 40

linezolid the mean values of systemic clearance and half- life for linezolid were 059 Lhrkg and 103 hr respectively the mean value of Vz was 088 Lkg The mean value of bioavailability after oral administration for linezolid was 7985 45

Following combo IV injection of compound 10 with linezolid the mean values of systemic clearance and half- life for compound 10 were 055 Lhrkg and 096 hr respectively the mean value of Vz was 076 Lkg The mean value of bioavailability after oral administration for com- 50

pound 10 was 8098A graph showing the mean plasma concentration of

linezolid and compound 10 over time following intravenous injection of a combination of linezolid and compound 10 is shown in FIG 3 A graph showing the mean plasma con- 55 centration of linezolid and compound 10 over time followshying oral administration of a combination of linezolid and compound 10 is shown in FIG 4

Example 4 60

Mitochondria Toxicity Study

FlepG2 cells were seeded at 50000 cells per well in 6-well plates and grown in Fligh-Glucose DMEM in the 65 presence of 6 different concentrations of compound (100 ρΜ 10 ρΜ 1 ρΜ 100 ηΜ 10 ηΜ and 1 ηΜ) Each

concentration was tested in triplicate Cells were also grown in the corresponding DMSO concentrations present in each of the treatments (5xl0_1 5xl0_2 5xl0_3 5xl0_4 5xl0_5 5χ10-6)

The medium with the compound was changed after 72 hours of treatment and kept for further analysis

When the cells reached an average of 4 population doublings in the compound they were trypsinized centrishyfuged and washed with phosphate buffered saline The cells were solubilized in 15 laurylmaltoside (in 25 mM Flepes 100 mM NaCl pH 74) centrifuged at 25000 g for 20 minutes and supernatants kept for assay

Enzyme quantity from each well was assessed with duplishycate dipsticks Each dipstick was loaded with 2 pg of solubilized protein to determine the levels of Complex IV (a mtDNA-encoded protein) and Frataxin (a nuclear DNA- encoded protein) Extracts were then stored at -80deg C for further analysis

The amount of enzymes captured on the dipstick was determined quantitatively with a Hamamatsu Immunochro- mato Reader The absorbance signal of two dipsticks meashysuring enzyme quantity from the same well was averaged (CVlt1) and the means for each triplicate treatment were normalized by interpolation against an assay specific calishybration curve

Complex IVFrataxin Ratios were determined for each triplicate treatment from interpolated values Triplicate ratios for each treatment concentration were analyzed using a non-linear regression curve in Graph Pad (Log [inhibitor] vs response-variable slope) Results are shown in FIGS 5Α-5Β IC50 values for linezolid and compound 10 are 78 and 98 μΜ respectively

In conclusion the mitochondria toxicity for compound 10 is comparable to that of linezolid

While this invention has been particularly shown and described with references to example embodiments thereof it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

What is claimed is1 A compound represented by Formula (I)

D D

TN R4 | R~

R1

(I)

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

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  • Bibliographic data
  • Abstract
  • Description
  • Claims
  • Drawings
Page 24: (ΐ2) United States Patent - Concert Pharmaΐ2) United States Patent Liu et al. (ΐο) ... “Synthesis of Deuterium-labelled Elliptinium and ... et al., “Mass spectrometry of pethidine

27US 9453009 Β2

or a salt thereof whereinR1 is mdashOH mdashNO mdashNH2 mdashNHR mdashN(Rdeg)2

mdashC(=0)NRRrf mdash C(=0)0Rg -phthalimidomdashS02mdashR or a group selected from alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl heterocycloalkylalkyl wherein the alkyl heteroaryl aralkyl heteroaralkyl cycloalkyl heterocycloalkyl cycloalkylalkyl and heterocyshycloalkylalkyl are each independently optionally substishytuted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashno2 mdashcn mdashnh2 mdashNHR mdashN(Rdeg)2 mdashNRC (=0)R mdash C(=0)NRRrf mdashS(0)R mdashS(0)2R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

each R is independently an alkyl optionally substituted with halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl or Cjg haloalkoxy

R is alkyl aryl heteroaryl aralkyl or heteroaralkyl each of which is optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0) NRRrf mdashS(0)R mdashS(0)2R mdashSR andmdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R and Rrf are each independently mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cjg alkyl mdashOR mdashC(=0)0R mdashC(=0) R mdashno2 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)RmdashS(0)2 R mdashSR and mdashS02NRRrf wherein each Cjg alkyl is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

R is mdashΗ or alkyl optionally substituted with one or more groups selected from halogen Cx 6 alkyl mdashOR7 mdashC(=0)0R mdashC(=0)R mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R) mdashNRC(=0)R mdashC(=0)NRRrf mdashS(0)R7 mdashS(0)2R mdashSR7 andmdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH C1-6 haloalkyl and Cjg haloalkoxy

R7 is alkyl cycloalkyl heterocycloalkyl aryl or hetshyeroaryl

Rg is alkyl optionally substituted with one or more groups selected from halogen Cj 6 alkyl mdashOR7 mdashC(=0) OR7 mdashC(=0)R7 mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 mdashNRC(=0)R7 mdashC(=0)NRRrf mdashS(O) R7 mdashS(0)2R7 mdashSR7 and mdashS02NRRrf wherein each Cjg alkyl substituent is optionally substituted with one or more groups selected from halogen C1-6 alkyl C1-6 alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy and

R4 R4 R5 and R5 are each independently mdashΗ or C1-4 alkyl optionally substituted with one or more halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl or Cjg haloalkoxy

and wherein the deuterium enrichment at each position designated as deuterium is at least about 85

2 The compound of claim 1 wherein each of R4 R4 R5 and R5 is hydrogen and wherein the deuterium enrichment at each position designated as deuterium is at least about 95

3 The compound of claim 1 wherein R1 is benzyl mdashS02-aryl or mdashS02-heteroaryl

4 The compound of claim 2 wherein R1 is benzyl5 The compound of claim 1 wherein R is alkyl optionshy

ally substituted with one or more groups selected from halogen Cj6 alkyl mdashN02 mdashCN mdashNH2 mdashNHR mdashN(R)2 Uc(=0)NRRrf and mdashS02NRRrf wherein each C1-6 alkyl substituent is optionally substituted with one or more groups selected from halogen Cjg alkyl Cjg alkoxy mdashOH Cjg haloalkyl and Cjg haloalkoxy

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