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Use of ω‑Transaminase Enzyme chemistry In the Synthesis of a JAK2 Kinase inhibitor.

KASHIF HAIDERPTPC/2014/502

Pharmaceutical tech. Process ChemistryNIPER Hyderabad

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Contents :1.Introduction.

2.Scheme 1: Medicinal chemistry route.

3.Scheme 2: Postulated Ylide and enamine mechanism.

4.Scheme 3: Coupling and purification stages.

5.Scheme 4: Alternative approaches to the chiral amine.

6.Scheme 5: Proposed long-term manufacturing route.

7.Scheme 6: Approaches for the conversion of ketone to the chiral amine

8.Conclusion.

9.Refrences.

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3Introduction

ω-Transaminase are obtained from Chromobacterium violaceum and Arthrobacter citreus.

The ω-transaminase is an enzyme which is capable of producing chiral amines, compounds used to great extent in pharmaceuticals.

Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced.

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Advantages of ω‑Transaminase Enzyme chemistry chemical approaches often suffer from severe drawbacks

such as harsh reaction conditions, use of toxic transition metal catalysts and sometimes insufficient stereo selectivity in a single catalytic step, causing environmental issues and product enantiopurity lower than the pharmaceutical requirement.

ω-transaminase (w-TA) has attracted growing attention as a promising catalyst, provides an environment-friendly access to production of chiral amines with exquisite stereo selectivity and excellent catalytic turnover.

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5 1. ω-Transaminase enzyme chemistry provides an excellent methodology to build synthetically useful chiral amines from their corresponding ketones.

2. An application of this methodology, providing a long-term commercial manufacturing route to a JAK2 kinase inhibitor is reported.

3. Janus kinase or JAK inhibitors are a type of medication that functions by inhibiting the activity of one or more of the Janus kinase family of enzymes (JAK1, JAK2, JAK3, TYK2), thereby interfering with the JAK-STAT signaling pathway

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Mechanism of action.• Cytokines play key roles in controlling cell growth and the

immune response. Many cytokines function by binding to and activating type I and type II cytokine receptors. These receptors in turn rely on the Janus kinase (JAK) family of enzymes for signal transduction.

• Hence drugs that inhibit the activity of these Janus kinases

block cytokine signaling.

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Some approved JAK2 inhibitors 1. Roxolitinib

useful against JAK1/JAK2 for psoriasis and myelofibrosis.

Approved by the U.S.FDA in

November 2011 for myelofibrosis.

2. Tofacitinib

useful against JAK3 for psoriasis and rheumatoid arthritis.

US FDA approved in November 2012

for rheumatoid arthritis.

NN

N

N

NHN

N

N

ON

N

N

NH

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8Drugs in clinical trail.1.Baricitinib

useful against JAK1/JAK2 starting phase IIb for rheumatoid arthritis.

2.Lestrautinib

useful against JAK2, for acute myelogenous leukemia (AML).

3.Pacritinib

useful against JAK2 for relapsed lymphoma and advanced myeloid malignancies, chronic idiopathic myelofibrosis (CIMF)

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9 4. CYT387

Against JAK2 for myeloproliferative disorders.

5.Filgotinib

(GLPG0634)against JAK1 for RA and Crohn’s diseases.

6. TG101348

Against JAK2; phase 1 results for myelofibrosis .

7. GSK2586184

Against JAK 1, for psoriasis and ulcerative colitis , but discontinue in SLE .

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10API 1

The API 1 is an orally active inhibitor of the JAK2 kinase , with the potential to be a curative treatment for

1. idiopathic myelofibrosis (IMF).

2. polycythaemia rubra vera (PRV).

compound was discovered by AstraZeneca, at its Boston research site in North America.

Was subsequently developed at the Macclesfield facility in the UK.

N NHN

HN

N NF

N NH

Cl

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11Scheme 1. Medicinal Chemistry route

N N

F

Cl

Cl

Zn,AcOH,THF

54%

N N

Cl

F

N N N N

Pd2(dba)3/DppfZn(CN)2

Zn/Zn(OAc)2

NMP N N

CN

F

1) MeMgCl,Toluene/THF2) Ac2O3) Chromotography

45%

F

NHAc 1)H2,Ru-Duphos,MeOH2)Boc2O3)HCl

F

NH2.HCl

N N

HN

Cl HN

1) (7) TEA,n-BuOH2) Chiral choromotography

N N

Cl

Cl

HN

N NH

97.5%ee

100% ee

60%22%

64%

(7)

N N

Cl

N NH

API (1)

Chiral amine (6)

(3)(2) (4)

(5)

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12 The Medicinal Chemistry route (shown in Scheme 1) was used

for the production of the first manufacturing batch (400g) in the large-scale laboratory at Macclesfield.

Production of the coupling partner 7 was relatively trivial: yields of 90% were achieved from commercially available 3-amino-5-methylpyrazole and 2,4,5-trichloropyrimidine.

The main part of the synthetic route (Scheme 1), however, presented a number of challenging problems, not the least of which was an overall yield to the API of only 2% from 2,4-dichloro-5-fluoropyrimidine (2).

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13 Limitations of Scheme 1

Water was difficult to remove during the workup and contaminated the product of the de-chlorination reaction leading to 2-chloro-5-fluoropyrimidine (3).

Large amounts of tar formation were observed that, in combination with the metal residues, made workup extremely difficult even on relatively small scale.

Despite excellent enantioselectivity, the Rh-DuPhos catalyst used for the reduction to produce the chiral amine 6, it was expensive.

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14Limitations contd…

Extremely water-soluble amine required BOC protection to enable extraction into an organic solvent. The BOC group was subsequently removed under anhydrous conditions.

Coupling of 6 and 7 (to form 1) was prone to significant and unpredictable levels of epimerization (60−90% ee), such that chiral chromatography was necessary to furnish the charily pure API 1.

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15 Scheme 2. Postulated ylide and enamine mechanisms

N N N N N NN N

N N N NH

HC

F F FF

FF

H2N H2N H3N+H3N+

H2NH2N

NEt3 +NHEt3 NEt3

+NHEt3 NEt3

x

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16Scheme 3. Coupling and purification stages

N N

Cl HN

Cl

N NH

KHCO3, t-AmOHMaleic acid

N N

HN

HN

N N

N NH

F

KOH,Water,i-PrOAc,Heptane

CO2H

CO2HN N

F

H2N

(6)

(7)

(8)

81% 83%

N N

HN

Cl

N NHHN

N N

F

(1)

+

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17 Crystatallisation as the maleate salt allows chiral purification.

No need for chromatography.

Gives also excellent processing properties.

API 1 after neutralization and extraction into isopropyl acetate (i-PrOAc) again crystallized by slow antisolvent addition of n-heptane.

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Scheme 4. Alternative approaches to the chiral amine (6)

N N

N N

H2N

F

N N

H2N

F

F

O

N N N N

X

FF

HN

O

F

N

N N

F

NO2

Grignard

Heck chemistry

Nitroethane

(6) (5) (4)

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Seemingly fundamental problems with the synthetic route therefore provoked an investigation into alternative routes tochiral amine 6 (Scheme 4).

Attempts using such methodologies as

Heck chemistry (utilizing vinyl butyl ether and hydrolysis) to give the ketone10, palladium-catalyzed insertion of nitro ethane , and building the pyrimidine ring, all suffered from low yields and required extensive chromatography.

The only seemingly viable alternative to emerge from this work centered on the Grignard addition to the cyanopyrimidine 4 to deliver ketone 10, and ultimately it was from this approach that the proposed long term manufacturing route was developed (Scheme 5).

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Scheme 5. Proposed long-term manufacturing route

N N N N N N

N NN N

F F

CN

F

O

F

H2N.HCl (7).KHCO3,t-AmOH

Maleic acid

Pd(dba)3/Dppf/ZnAcetone cyanohydrinBuOAc

MeMgCl/THF2-MeTHFAqueous HCl

1) Transaminase (from vibrio Fluvialis)Pyridoxal PhosphateS-MethylbenzylamineKH2PO4/Water/Toluene2) Boc2O/2-MeTHF3) HCl/ IPA

Cl

HN

HN

N N

F

N NH

COOH

COOH

KOH,aq EtOHN N

Cl HN

HN

N N

F

N NH

87%

80% 81%

75%70-90% soln. yield

X

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Acetone cyanohydrin as cyanide source and Butyl acetate (BuOAc) as solvent gave clean conversion of 2-chloro-5-fluoropyrimidine to product with controllable exotherm through the slow addition of the (liquid) acetone cyanohydrin.

A 70% solution yield was obtained in the laboratory. Higher solution yields of 90% were achieved using 2-bromo-5-flouropyrimidine.

Clean conversion of the cyanopyrimidine 4 to the ketone 10 was realized with relatively straightforward processing, via conventional Grignard chemistry.

This reaction was demonstrated on 20 L scale with little problem.

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22Scheme 6. Approaches for the conversion of ketone to the chiral amine

N N

O

N N

N N

N N

F

H2N

Enzyme

H2N

F

N N

F

H2N

Resolve

N N N N

F F

H2NNOR

F

H2N

(R= H,Me,Bz)

F

(6)

(6)

(6)

(10)

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With a promising route to the ketone in hand, numerous strategies were explored to convert 10 to chiral amine 6 (scheme 6 ).

Many of these processes exposed the inherent fragility of the pyrimidine ring, and the formation of oily tars was common place.

The only promising alternative identified was an enzymatic approach using transaminases, the conditions of which were by necessity very mild and provided the added attraction of a single chemical processing step.

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24 Conclusion

1. Manufacturing route of API 1 is reported via enzyme catalytic reaction.

2. w–transaminase enzyme chemistry is utilized to construct the key chiral amine coupling partner.

3. Demonstration of useful application of w-transaminase enzyme methodology

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4. Enzymatic transamination reactions are important and abundant in nature and involve the transfer of an amino group from α-amino acids to α-keto acids.

6. Recent years some notable examples have been described such as the preparation of sitagliptin by Merck.

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References (1) Deshpande, A.; Reddy, M. M.; Schade, G. O. M.; Ray, A.;Chowdary,

T. K.; Griffin, J. D.; Sattler, M. Leukemia 2012, 26 (4),708−715.

(2) Ioannidis, S.; Lamb, M. L.; Wang, T.; Almeida, L.; Block, M. H.Davies A. M.; Peng, B.; Su, M.; Zhang, H.-J.; Hoffmann, E. J. Med. Chem. 2011, 54 (1), 262−276.

(3) Liu, S.; Berry, N.; Thomson, N.; Pettman, A.; Hyder, Z.; Mo, J.;Xiao, J. J. Org. Chem. 2006, 71 (19), 7467−7470.

(4) Vogl, E. M.; Buchwald, S. L. J. Org. Chem. 2002, 67 (1), 106−111. Behrens GA, Hummel A, Padhi SK, Schatzle S, Bornscheuer UT

(5) Behrens GA, Hummel A, Padhi SK, Schatzle S, Bornscheuer UT (2011) Discovery and protein engineering of biocatalysts for organic synthesis. Adv Synth Catal 353:2191–2215

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Oxidative addition

complexation

insertionrotation

Beta hydride elimination

Reductive elimination Or deprotonation

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29 Bis diphenyl phosphine ferrocene(Dppf),

acetic acid (AcOH), tetra hydro furan,

acetic anhydride(Ac2o),

ruthenium duphos ligand(Ru-duphos),

Di-tert.-butyl di carbonate(boc2o),

N-methyl pyroolidone (NMP),

Hydroxy butanol (BuOH),

Iso propyl acetae (i-PrOAc)

Butyl Acetate (BuOAc),

Iso propyl Alcohol (IPA)