Bioorganic & Medicinal Chemistry Letters 23 (2013) 2606–2613

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Discovery of thiazolobenzoxepin PI3-kinase inhibitors that sparethe PI3-kinase b isoform

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.02.102

⇑ Corresponding author.E-mail address: stevents@gene.com (S.T. Staben).

Steven T. Staben a,⇑, Chudi Ndubaku a, Nicole Blaquiere a, Marcia Belvin b, Richard J. Bull c,Danette Dudley a, Kyle Edgar b, Daniel Gray a, Robert Heald c, Timothy P. Heffron a, Graham E. Jones c,Mark Jones c, Aleks Kolesnikov a, Leslie Lee b, John Lesnick d, Cristina Lewis d, Jeremy Murray e,Neville J. McLean c, Jim Nonomiya d, Alan G. Olivero a, Rachel Ord c, Jodie Pang f, Stephen Price c,Wei Wei Prior b, Lionel Rouge e, Laurent Salphati f, Deepak Sampath b, Jeff Wallin b, Lan Wang a,BinQing Wei g, Christian Weismann e, Ping Wu e

a Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United Statesb Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United Statesc Argenta Medicinal Chemistry, 8/9 Spire Green Centre, Harlow, Essex CM19 5TR, United Kingdomd Biochemical Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United Statese Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United Statesf DMPK, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United Statesg Computational Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States

a r t i c l e i n f o

Article history:Received 14 December 2012Revised 15 February 2013Accepted 25 February 2013Available online 7 March 2013

Keywords:PI3-kinaseIsoform selectivityStructure-based ligand design

a b s t r a c t

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepinclass of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was iden-tified that increased selectivity over PI3Kb. PI3Kb-sparing compound 27 (PI3Kb Ki,app/PI3Ka Ki,app = 57)demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differentialeffects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new struc-ture-based hypothesis for reducing inhibition of the PI3K b isoform while maintaining activity against a, dand c isoforms is presented.

� 2013 Elsevier Ltd. All rights reserved.

A previous report1 described our structure-based efforts to im-prove potency and metabolic stability from an HTS-derived 2-ami-do-thienobenzopyran resulting in 2-triazolyl-thienobenzoxepinssuch as 1 (Fig. 1). Further efforts on this series included modifica-tion of the tricyclic core and modulating PI3K isoform selectivity.We were interested in replacing the thiophene of compound 1for two reasons: First, a known pathway exists for thiophenes toundergo oxidative metabolism to reactive intermediates resultingin a potential to cause drug–drug interaction (DDI), liver toxicity,or idiosyncratic drug reactions;2 second, the thiophene ring ofcompound 1 was identified as a potential region for ligand modifi-cation that could decrease the overall lipophilicity of the tricycliccore through simple addition of heteroatoms (Fig. 1). Compound1 possesses only modest selectivity over PI3Kb relative to the otherPI3K isoforms (Fig. 1). Given the proposed role of PI3Kb in insulinsignaling and glucose homeostasis,3 a PI3Kb-sparing phenotype

was desired to achieve a lower-risk safety profile. Based on earlystrategies noted in the discovery of PI3Kb sparing inhibitor thieno-pyrimidine and early work on the 2-amido-thienobenzoxepincore,4 we envisaged 2-triazolyl-benzoxepin 8-substitution to pro-vide a handle on improving selectivity over PI3Kb. In addition toaltering isoform selectivity, we hoped to minimize the potentialfor amidase-mediated hydrolysis of the 8-amido compounds to im-prove our confidence in human PK prediction.5 Demonstrating thepotential for PK liabilities of 8-amido substitution, lead thi-enobenzoxepin 1 had high clearance in dog (in vivo Clp = 100 mL/min/kg); the major circulating metabolite was identified as theamide hydrolysis product.

In our efforts to replace the thiophene in the tricyclic core, weexamined a series of five-membered heterocycles that were antic-ipated to be more polar and have lower risk of generating reactivemetabolites (Table 1, bonds labeled a/b/c to denote connectivity).2b

A co-crystal structure of 1 bound to PI3Kc revealed an approxi-mately flat biaryl dihedral (thiophene sulfur and triazole 4-N at�180�, Fig. 1).6 Key to our design was to ensure the desired biaryldihedral between the five-membered heteroarene of the tricyclic

Table 1Replacement of the thiophene with other five-membered-heteroaryls

OR1

NN

NMe

Me

heta

b

c

Compound het R1 PI3Ka IC50 pAKT (S473) PC3 IC50

1 Sa b

c

CONH2 4.0 nM 155 nM

2a

N Sa b

c

CONH2 1.3 nM 180 nM2b CONHMe 3.3 nM 194 nM2c H 3.1 nM 159 nM

3a

NNa b

c

CONHMe 3.3 nM 516 nM3b H 3.6 nM ND

4 N N

a b

c

H 49 nM 2.4 lM

5aNN N

a b

c

H 140 nM ND

5b N NNa b

c

Cl 33 nM ND

6 S Na b

c

CONHMe 2.90 lM ND

7 NNa b

c

CONHMe 426 nM ND

ND = not determined.Assay conditions were previously disclosed.14

Bonds labeled a/b/c to denote connectivity.

PI3K IC50 (α/β/γ/δ)4.0 nM / 29 nM / 2.2 nM / 3.9 nM

O

S

NN

NMe

Me

O

H2N

compound 1 remove structure alertand decrease lipophilicity whilemaintaining biaryl dihedral (~180o)

improve selectivity over PI3Kβand eliminate potential forhydrolytic clearance

goals:8

2

O

SO

NMe

F

2

7

2-amido-thienobenzopyran HTS hit 2-triazolyl-thienobenzoxepin

Figure 1. Thienobenzoxepin lead compound 1 and strategy for further optimization.

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613 2607

core and the 1,5-substituted triazole was a favored small moleculeconformation. Analogs were prepared with and without 8-amidosubstitution for SAR comparison. Of the thiophene replacements

tested, thiazoles (2a–c) and imidazoles (3a, b) were best toleratedwith equipotent biochemical activity (PI3Ka IC50) and modest lossin cell potency (measured S473-pAKT in PC3 cells). Pyrazole (4)

Table 2Varied 8-substitution of the thiazolobenzoxepin core affects selectivity over PI3Kb

O

N S

NN

NMe

Me

R8

2

Compound R PI3K a/b/d/c IC50

(nM)PI3Kb fold-selectivitya

pAKT (S473) PC3 IC50b

(nM)Prolif IC50 (PC3)b

(lM)Prolif IC50 (MCF7-neo/HER2)b

2c H 3.6/5.5/3.5/3.0 1.5� 159 2.6 1.3

8 OH 2.5/20.3/3.4/8.4 8.1� 99 2.3 0.49

9c

OHOH

6.4/37.8/8.1/19.1 5.9� 364 2.0 1.0

10 NH

Me

O11/63/10.2/55 5.7� 199 3.4 1.2

11 NH

OH3.5/17.4/8.4/80 5.0� 186 3.3 2.3

12NH

<0.5/0.9/<0.5/3.5 2.8�⁄ 21 0.24 0.38

13c NH

H1.5/3.9/1.1/4.3 2.6� 24 0.14 0.21

14cNH

H4.9/5.4/3.3/36 1.1� 174 0.86 2.1

15NH

2.0/8.4/1.3/8.4 4.2� 57 0.16 0.16

16 NNH

<0.5/7.9/<0.5/0.7 34�⁄ 75 1.1 0.24

17HN

N2.0/32.4/2.6/1.5 16.2� 53 1.8 0.72

18N

N Me1.3/9.3/0.6/0.6 7.2� 83 2.0 0.41

19

HNO

14/77/7.6/11 5.5� ND 4.4 2.3

20N NH2

1.4/13/1.4/3.7 9.3� 71 2.8 0.805

ND = not determined.a PI3Kb IC50/PI3Ka IC50 unless otherwise noted (⁄ denotes PI3Kb IC50/PI3Ka Ki,app)b For detailed description of cellular pAKT and proliferation assays please see Ref. 14.c Tested as a mixture of enantiomers.

2608 S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613

and triazole (5a, b) replacements for the thiophene were less effec-tive and had 11- to 45-fold higher IC50 against PI3Ka relative to theclosest thiazole or imidazole comparators (2c/3b). Illustrating thenecessity of appropriate biaryl dihedral, compounds 6 and 7 wereprepared and tested in our PI3Ka biochemical assay revealing lar-gely decreased activity; nitrogen–nitrogen lone pair repulsion be-tween the triazole 4-N and thiazole or pyrazole nitrogen likelypromotes 180� rotation of triazole to a non-optimal binding con-formation. The thiazolobenzoxepin core was initially chosen forfurther modification given moderately superior cell potency andeasier synthetic accessibility relative to the imidazobenzoxepincore.7

Compound 2c (8-substituent = R = H) had no selectivity overPI3Kb (PI3Kb IC50/PI3Ka IC50 = 1.5, Table 2). Given our previoussuccess,4 and the recently disclosed success of others8 in directingligands toward the lower hinge region, substitution at the 8-posi-tion of our thiazolobenzoxepin is expected to modulate isoform

selectivity. A wide variety of substitution was examined withhopes for identification of initial hints of selectivity over PI3Kb.For inhibitors where the PI3Ka IC50 was low, a PI3Ka Ki was mea-sured at lower enzyme concentration to determine the selectivityratio over PI3Kb (i.e., 12 and 16). In contrast to 2-amido-thi-enobenzoxepin compounds, most varieties of 8-substitution onthe 2-triazolyl-thiazolobenzoxepin core provided only modest iso-form selectivity (Table 2). Benzylic alcohol (8), diol (9), aminoacyl(10) and aminoalkyl (11) substitution provided ligands that had�five- to eight-fold selectivity over PI3Kb and modest anti-prolif-erative activity in both PC3 and MCF7-neo/HER2 cell lines. Moreconformationally rigid cyclic aliphatic amines (12–15) also didnot possess the PI3Kb-sparing phenotype we desired, although12, 13 and 15 had improved anti-proliferative effects in both celllines tested (proliferation IC50 <400 nM). The erosion in selectivityfor these examples was potentially caused by the noted negativeelectrostatic potential of PI3Kb in the lower hinge region of the

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613 2609

binding site which favors positively charged ligands.4 Interestingly,pyrazole and imidazole substitution at the 8-position of the thia-zolobenzoxepin core provided improved selectivity (16 and 17,34- and 16-fold selectivity, respectively). This result was not un-ique to all aromatic heterocycle substitutions, however, as alterna-tive five- and six-membered heterocycles had <10-fold selectivity.Given the initial level of selectivity for 16 (34-fold) and good cellu-lar activity and solubility of 12 (kinetic solubility = 97 lM,<400 nM IC50 in both proliferation assays), these ligands were se-lected for further modification toward improvement of isoformselectivity.

We further examined 8-(3-azetidynyl)- and 8-(4-pyrazolyl)-substituted thiazolobenzoxepins (series A and B, respectively) foreffects on selectivity over PI3Kb, cell potency, solubility and rodentclearance. Table 3 dictates a trend for b-sparing activity withincreasing bulk of substitution in both series. For example, increas-ing bulk of 1-alkyl azetidine substitution from 21 to 23 to 26 im-proves selectivity over PI3Kb from 2.4-fold to 10-fold to 30-fold.A similar trend was observed for 1-pyrazole substitution (i.e., 27vs 28/31). Larger selectivity observed with pyrazole substitutionin every direct comparison (2.3- to 13-fold improvement in foldselectivity over PI3Kb). Interestingly, the total plasma clearanceof series B was consistently lower than series A. However, in caseswhere plasma protein binding was measured, this appeared to belargely an effect of increased plasma protein binding as Clp,u valueswere generally similar in direct comparisons (i.e., 25 and 31,

Table 3SAR for 8-(3-azetidynyl)- and 8-(4-pyrazolyl) substituted thiazolobenzoxepins (series A an

O

N S

NN

NMe

Me

NRR

A

8

Compound Scaffold R PI3Ka Ki,app, PI3Kb IC50

[sel. ratio]apAKT (S473) PC3IC50

b (nM)

21 A OH 1.9 nM, 4.6 nM [2.4�] 37

22 A OHOH

0.69 nM, 4.0 nM [5.9�] 21

23 AOH

Me Me 0.90 nM, 8.5 nM [10�] 69

24 AO

OH 0.61 nM, 7.6 nM [12�] 67

25 A

O

Me MeNH2 0.94 nM, 13 nM [14�] 54

26 AMe Me

NH2

O

0.47 nM, 14.2 nM [30�] 89

27 B OH 0.27 nM 8.6 nM [32�] 24

28 BOH

Me Me 0.15 nM, 14 nM [92�] 47

29 B SO2Me 0.06 nM, 1.8 nM [29�] 20

30 B NH2 0.06 nM, 2.4 nM [44�] 22

31 BMe Me

NH2

O

0.11 nM 7.4 nM [68�] 9

ND = not determined.a PI3Kb IC50/PI3Ka Ki,app.b For detailed description of cellular pAKT and proliferation assays please see Ref. 14.c Clp values determined from IV dosing in Sprague Dawley rats at1 mg/kg (discrete) o

Clp,u = 1153 and 625 mL/min/kg, respectively). Based on acceptablePI3Kb selectivity, cell potency and rodent clearance values 27 and28 were selected for further close-in modification of substitutionon the triazole ring (Table 4). Methyl substitution at the 3-positionof the 1-isopropyl-1,2,4-triazole was well tolerated in terms of po-tency, selectivity and rodent clearance (32). 3-amino substitutionof the 1-isopropyl-1,2,4-triazole (33–34) and 3-oxo substitutionof the 4-isopropyl-1,2,4 triazole (35) improved cellular activity,however worsened rodent clearance. Trifluoroethyl substitutionon the triazole was also well tolerated and compound 36 is themost b-sparing inhibitor made in this series (160-fold selectivityover PI3Kb, PI3Kd IC50 <0.5 nM, PI3Kc IC50 = 1.4 nM).

As anticipated from earlier structures of benzoxepin ligands, anX-ray structure of 36 (Fig. 2A, PDB-ID: 4J6I, 160-fold selective overPI3Kb) in PI3Kc revealed positioning of the 8-(4-pyrazolyl) in thevicinity of the lower hinge region (toward C-terminal end of hinge),the oxepin oxygen maintaining a hinge-binding interaction withVAL882, the triazole nitrogen atoms within water-mediated or di-rect H-bond distance to key affinity pocket residues (TYR867 andLYS883, respectively, Fig. 2A). The carbonyl of hinge-residueVAL882 is in close-proximity to the pyrazole 3-H forming a non-classical H-bond.

The ability to gain PI3Ka over PI3Kb activity through 8-substitu-tion with appropriately charged thienobenzoxepin 8-substitutiondue to large differences in electrostatic potential at the solventfront of the different isoforms has been well described (for

d B, respectively)

O

N S

NN

NMe

Me

NN

B

8

Prolif IC50

(PC3)b (lM)Prolif IC50 (MCF7-neo/HER2)b (lM)

Kineticsolubility

Clp (mL/min/kg)c

Rat Mouse

0.40 0.52 84 lM 128 132

0.44 0.18 77 lM 57 165

1.0 0.43 35 lM 163 170

0.40 0.17 2.7 lM ND ND

0.42 0.18 57 lM 60 ND

1.2 0.44 ND 55 104

0.63 0.20 4.7 lM 5.4 9.5

0.97 0.15 1.1 lM 6.8 3.1

0.36 0.08 1.1 lM 6.1 3.2

1.6 0.50 57 lM ND ND

0.26 0.11 3.7 lM 10 13.4

r 0.5 mg/kg (cassette) and nude mice at 1 mg/kg (discrete).

Table 4SAR for 1,2,4-triazolyl substituents

O

N S

R'

RR = A

R = B

NN

OH

NN

OH

Me Me

8

2

Compound R R0 PI3Ka Ki,app, PI3Kb IC50

[sel. ratio]apAKT (S473) PC3IC50

b (nM)Prolif IC50

(PC3)b (lM)Prolif IC50 (MCF7-neo/HER2)b (lM)

Kineticsolubility

Clp (mL/min/kg)c

Rat Mouse

27 A NN

NMe

Me Me0.27 nM, 8.6 nM [32�] 24 0.63 0.20 4.7 lM 5.4 9.5

32 A NN

NMe

Me H0.18 nM, 7.0 nM [39�] 89 1.5 0.39 2.5 lM 1.9 2.2

33 A NN

NMe

Me NH2

0.04 nM, 1.8 nM [43�] 38 0.33 0.06 1.2 lM ND ND

28 B NN

NMe

Me H0.15 nM. 14 nM [92�] 47 0.97 0.15 <1.1 lM 6.8 3.1

34 B NN

NMe

Me NH2

0.13 nM, 9.2 nM [72�] 31 0.60 0.07 <1.1 lM 55 4

35 B N NHNMe

MeO

0.18 nM, 3.9 nM [22�] 10 0.66 0.08 ND 84 22

36 B NN

NF3C 0.14 nM, 15 nM [160�] 122 1.1 0.31 <1.1 lM 10 8.2

ND = not determined.a PI3Kb IC50/PI3Ka Ki,app.b For detailed description of cellular pAKT and proliferation assays please see Ref. 14.c Clp values determined from IV dosing in Sprague Dawley rats at 1 mg/kg (discrete) or 0.5 mg/kg (cassette) and nude mice at 1 mg/kg (discrete).

2610 S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613

example, PI3Kb potency increases relative to PI3Ka potency withbasic amine substitution).4 However, it was still unclear whybulky, uncharged substitution provided good levels of selectivityfor the above-mentioned examples (including 36). In the absenceof X-ray structures for isoforms other than PI3Kc, SAR was fol-lowed without strong rationale for achieving selectivity overPI3Kb. Since initiation of this work, X-ray structures of PI3Kb9

and PI3Ka10 and PI3Kd15a have been reported. One difference ofnote is the conformation of a tryptophan residue present in all iso-forms (aTRP780, bTRP781, dTRP760, cTRP812). This is the sametryptophan residue that is thought to be a crucial for obtaining iso-form specificity for PI3Kd as the result of an ‘induced fit’ movementof adjacent residues or specific interaction by inhibitors.15 The con-formation of the indole of this TRP residue is unique in PI3Kb. Fig-ure 2B presents a different orientation of the structure of inhibitor36 bound to PI3Kc along with selected residues aligned fromPI3Ka, d and c. Figure 2C shows alignment of inhibitor 36 in thePI3Kb X-ray structure. The conformation of the indole of this TRPresidue is similar among a, d and c however a Ca–Cb rotation ofbTRP781 presents an alternate orientation (compare Fig. 2B andC). We hypothesize that differences in second shell residues pro-mote a unique orientation of this TRP in PI3Kb. First, the indole

N–H donates an H-bond to acidic residues in both PI3Ka(GLU798) and PI3Kc (GLU814) that occupy a similar region of thebinding site but are not present in PI3Kb or d. This interaction couldfavor the observed orientation of the indole in these isoforms. Inplace of GLU814, PI3Kb and d possess neutral residues with non-polar side chains (bVAL783 and dMET762 respectively) and thusthe indole can occupy other energetically favorable orientations.It is possible that the branched valine residue of PI3Kb disfavorsorientations similar to those observed in a, d and c; the Cc methylinstead approaches van der Waals distance to the indole 4/5 posi-tion (Fig. 2C). It appears from the above SAR that in this uniqueconformation the TRP sidechain is more easily insulted by stericbulk of the inhibitor. Also of note is the differential angle andatoms presented for p-stacking by the indole in PI3Ka, d and c rel-ative to b.11 This observation could partially explain why non-aro-matic substitution in the same region results in molecules withlower overall selectivity over PI3Kb (for example, series A versusseries B in Table 3).

PI3Kb-sparing inhibitor 27 had low clearance and high bioavail-ability in rodents, acceptable PK in dog and moderate predictedclearance in human (hepatocyte Clp = 12 mL/min/kg, Fig. 3A) andwas chosen for further in vitro and in vivo evaluation. Taconic nude

Figure 2. Structure analysis of the binding mode of 36. Legend: PI3Ka = magenta = PDB-ID: 3HIZ,10 PI3Kb = gray = PDB-ID: 2Y3A,9 PI3Kd = purple = PDB-ID: 2WXM,15

PI3Kc = green. (A) Compound 36 (cyan) complexed with PI3Kc (green)- PDB-ID: 4J6I. Potential polar interactions indicated with yellow dashed lines. (B) Crystal structure of36 (cyan) in PI3Kc (green) with aligned PI3K-a and -d structures. Second-shell residues that could influence the tryptophan sidechain orientation are highlighted (aASN782;dMET762; cGLU814). Potential H-bonding interactions between cGLU814 and aGLU798 and the indole N–H are indicated with yellow dashed lines. (C) Crystal structure of 36(cyan) in PI3Kc with aligned PI3Kb key residues (gray; van der Waals surfaces indicated by dots, PDB: 2Y3A). The branching Cc methyl of bVAL783 enforces a conformationalchange for bTRP781. Note the proximity of bVAL783 with the indole 4/5 position of bTRP781 and encroachment of this indole’s 6/7 positions with the 8-pyrazole andsidechain (red lines/lengths). This orientation of tryptophan indole ring is unique in PI3Kb relative to the other isoforms.

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613 2611

mice bearing MCF7-neo/HER2 xenografts received a single dose of27 at 12.5, 25, 50 and 100 mg/kg. Tumor samples were collected at4-h post-dose and analyzed for pAKT (S473), pPRAS40 and pS6RP.The analysis of tumor pAKT (S473) levels (bar graph, ratio relativeto total AKT) and plasma exposure ( ) is shown in Figure 3B. A dosedependent decrease in pAKT is evident for the 12.5, 25 and 50 mg/kg doses; the 50 and 100 mg/kg doses of 27 providing comparableknockdown to pan-PI3K inhibitor GDC-0941 (37) at 150 mg/kg.12

pPRAS40 and pS6RP were also knocked down to a significant ex-tent (data not shown, 80% and 83% decrease at 100 mg/kg at 4-h,respectively).

We were interested to determine the selectivity for PI3Ka overPI3Kb for compound 27 in cells; however isoform specific cell read-outs were not readily available. We often used the difference inMCF7-neo/HER2 (PTEN WT) and PC3 (PTEN null) cell proliferationIC50 values to qualitatively define selectivity as there is a differencein the role of PI3Kb in PTEN null and PTEN WT cells (PTEN = phos-phatase and tensin homologue).13 Indeed, most compounds fromTables 2–4 that have moderate-to-high degree of selectivity versus

the PI3Kb biochemical assay show cellular selectivity for MCF7-neo/HER2 over PC3 cells. Given the PI3Kb sparing nature of 27,we determined its anti-proliferative activity in a number of PTENnull and PTEN WT cell lines to determine whether this was a con-sistent trend. The data is presented as a ratio relative to pan-PI3Kinhibitor GDC-0941 (37) in Figure 2C. Notably, there is a statisti-cally significant decrease in activity for 27 in PTEN null comparedto PTEN WT cells (mean 1.5-fold ratio of 27 IC50/37 IC50 for PTENWT vs 4.6 for PTEN null, p = 0.0085).

In summary, we identified several suitable five-membered het-erocyclic alternatives to thiophenes within a benzoxepin class ofPI3K inhibitors. Specific 8-substitution was discovered that in-creased selectivity for PI3Ka over PI3Kb. Compound 27 had a suit-able profile for advancement and displayed dose dependentknockdown of pAKT, pPRAS40 and pS6RP in a MCF7-neoHER2xenograft model and had an expected differential effect on PTENnull versus PTEN WT cancer cells. A new structure based hypothe-sis for decreasing inhibition of the PI3Kb isoform while maintain-ing activity against a, d and c isoforms was presented.

A

compound 27 :

PI3K Ki,app (α / β / γ / δ)a: 0.27 / 15 / 0.55 / 0.61 nM

mTOR IC50 > 4.3 μM; DNA-PK IC50 = 0.34 μM

Rat PK: Clpb = 5.4 mL/min/kg; F (5 mg/kg) = 83%c

Mouse PK: Clpb = 9.5 mL/min/kg; F (25 mg/kg) = 110%c

Dog PK: Clpb= 14 mL/min/kg; F (2 mg/kg) =22%c

Human hepatocyte Cl: 12 mL/min/kg

MDCK Papp (A-B): 16 cm-6/s

Solubility (pH 7.4)d: 2 - 31 µM

GDC-0941 (37):

PI3K Ki,app (α / β / γ / δ)a: 2.6 / 17 / 1.4 / 31 nM

B

C Fold EC50 increase (compound 27 / compound 37)

Compound 27 (37)

O

N S

NN

NMe

Me

NN

HO

N

NSN

O

N

MeO2S

NHN

PTEN Null mean fold EC50 increase: 4.6PTEN WT mean fold EC50 increase: 1.5

p=0.0085

Figure 3. Relevant in vivo and in vitro properties observed for 27 and GDC-0941 (37). (A) Key selectivity and PK data for 27 and GDC-0941 (37). aAll values are apparent Kis.bValues determined from IV dosing in Sprague Dawley rats at 1 mg/kg (discrete), nude mice at 1 mg/kg (discrete), and beagle at 1 mg/kg (discrete). cBioavailability (%F)determined on oral dosing of MCT suspension at listed dose level. dThermodynamic solubility was measured using the shake-flask method and varied depending on lot of 27.(B) Measurement of pAKT/tAKT (tAKT = total AKT, left-hand y-axis, bar graph) and plasma concentration (right hand y-axis, ) levels at 4 h post GDC-0941 (37) and 27 inTaconic nude mice implanted with MCF7-neo/HER2 xenografts. Numbers indicate percent reduction relative to MCT vehicle. ± error bars = SEM for biomarkers and plasma PK,four animals per dose. ⁄p <0.05, ⁄⁄p <0.001. (C) Ratio of cell IC50s for compound 27 versus pan-PI3K-inhibitor GDC-0941 (37) in a group of PTEN null and PTEN WT cellproliferation assays. Fold increase = proliferation IC50 for 27/proliferation IC50 for 37.

2612 S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613

Acknowledgments

The authors would like to thank Yanzhou Liu, Yutao Jiang, ChrisHamman, Mengling Wong and Michael Hayes for analytical andpurification support. We also thank the Baculovirus ExpressionGroup at Genentech for protein production.

References and notes

1. Staben, S. T.; Blaquiere, N.; Tsui, V.; Kolesnikov, A.; Do, S.; Bradley, E. K.; Dotson,J.; Goldsmith, R.; Heffron, T. P.; Lesnick, J.; Lewis, C.; Murray, J.; Nonomiya, J.;Olivero, A. G.; Pang, J.; Rouge, L.; Salphati, L.; Wei, B.; Wiesmann, C.; Wu, P.Bioorg. Med. Chem. Lett. 2013, 23, 897–901.

2. Thiophene-containing compounds, including tienilic acid (TA), suprofen,tenidap, ticlopidine, and methapyrilene exhibit various toxicities in humansand animals. Some of these toxicities have been hypothesized to result fromelectrophilic-activation of the thiophene by P450 mediated oxidation, andthiophenes are thus considered structural alerts. For examples and reviews onmetabolic activation of thiophenes see: (a) Dansette, P. M.; Amar, C.; Smith, C.;Pons, C.; Mansuy, D. Biochem. Pharmacol. 1990, 39, 911–918; (b) Dalvie, D. K.;Kalgutkar, S. A.; Khojasteh-Bakht, C. S.; Obach, S. R.; O’Donnell, P. J. Chem. Res.Toxicol. 2012, 15, 269–299; (c) Dansette, P. M.; Bertho, G.; Mansuy, D. Biochem.Biophys. Res. Commun. 2005, 338, 450–455; (d) Hu, Y.; Yang, S.; Shilliday, F. B.;Heyde, B. R.; Mandresll, K. M.; Robins, R. H.; Xie, J.; Reding, M. T.; Lai, Y.;Thompson, D. C. Drug Metab. Dispos. 2012, 38, 1522; For attempted connectionof thiophene metabolism to idiosyncratic drug reactions or specific toxicity see,for example: (e) Kalgutkar, S. A.; Didiuk, T. M. Chem. Biodivers. 2009, 6, 2115–2137; (f) Ju, C.; Uetrecht, J. P. Curr. Drug Metab. 2002, 3, 367–377; (g) Uetrecht,J. P. Drug Metab. Rev. 2006, 38, 745–753; (h) Uetrecht, J. P. Chem. Res. Toxicol.2008, 21, 84–92.

3. For a recent review including discussion of the disputed role of PI3Kb inmetabolism see the following: (a) Costa, C.; Hirsch, E. Curr. Top. Microbiol.Immunol. 2011, 346, 182; Recent reviews that discuss the isoform selectivity ofPI3K inhibitors include the following: (b) Marone, R.; Cmiljanovic, V.; Giese, B.;Wymann, M. P. Biochim. Biophys. Acta 2008, 1784, 159–185; (c) Knight, Z. A.;Shokat, K. M. Biochem. Soc. Trans. 2007, 35, 245–249; (d) Ward, S. G.; Finan, P.

Curr. Opin. Pharmacol. 2003, 3, 426–434; (e) Sundstrom, T. J.; Anderson, A. C.;Wright, D. L. Org. Biomol. Chem. 2009, 7, 840–850.

4. Heffron, T. P.; Wei, B.; Olivero, A.; Staben, S. T.; Tsui, V.; Do, S.; Dotson, J.; Folkes,A. J.; Goldsmith, P.; Goldsmith, R.; Gunzner, J.; Lesnick, J.; Lewis, C.; Mathieu, S.;Nonomiya, J.; Shuttleworth, S.; Sutherlin, D. P.; Wan, N. C.; Wang, S.;Wiesmann, C.; Zhu, B.-Y. J. Med. Chem. 2011, 54, 7815.

5. vNoted differences in carboxylesterase and amidase activity betweenspecies: (a) Taketani, M.; Shii, M.; Ohura, K.; Ninomiya, S.; Imai, T. Life Sci.2007, 81, 924–932; Species specific amide cleavage leading to lowbioavailability in human: (b) Liu, L.; Halladay, J. S.; Shin, Y.; Wong, S.;Coraggio, H. L.; Baumgardner, M.; Le, H.; Gopaul, S.; Boggs, J.; Kuebler, P.;Davis, J. C., Jr.; Liao, C.; Lubach, J. W.; Deese, A.; Sowell, C. G.; Currie, K. S.;Young, W. B.; Khojasteh, C.; Hop, C. E. C. A.; Wong, H. Drug Metab. Dispos.2011, 39, 1840.

6. See Ref. 1: PDB-ID: 4HLE.7. (a) Syntheses of all compounds within this manuscript have been generally

described in a series of patents: Blaquiere, N.; Do, S.; Dudley, D.; Folkes, A. J.;Goldsmith, R. A.; Heald, R.; Heffron, T.; Kolesnikov, A.; Ndubaku, C.; Olivero, A.G.; Price, S.; Staben, S. T.; Wei, B. WO/2011/036284.; (b) Blaquiere, N.; Do, S.;Dudley, D.; Folkes, A. J.; Heald, R.; Heffron, T.; Jones, M.; Kolesnikov, A.;Ndubaku, C.; Olivero, A. G.; Price, S.; Staben, S. T.; Wang, L. WO/2011/036280.;(c) Do, S.; Goldsmith, R.; Heffron, T.; Kolesnikov, A.; Staben, S.; Olivero, A. G.;Siu, M.; Sutherlin, D. P.; Zhu, B.-Y.; Goldsmith, P.; Bayliss, T.; Folkes, A.; Pegg, N.WO/2009/123971.

8. Researchers from Novartis have recently disclosed the ability to generate a, cand d selective molecules through a parallel synthesis exploration of thisregion: Bruce, I.; Akhlaq, M.; Bloomfield, G. C.; Budd, E.; Cox, B.; Cuenoud, B.;Finan, P.; Gedeck, P.; Hatto, J.; Hayler, J. F.; Head, D.; Keller, T.; Kirman, T.;Leblanc, C.; Le Grand, D.; McCarthy, C.; O’Connor, D.; Owen, C.; Oza, M. S.;Pilgrim, G.; Press, N. E.; Sviridenko, L.; Whitehead, L. Bioorg. Med. Chem. Lett.2012, 22, 5445.

9. Zhang, X.; Vadas, O.; Perisic, O.; Anderson, K. E.; Clark, J.; Hawkins, P. T.;Stephens, L. R.; Williams, R. L. Mol. Cell 2011, 41, 567–578.

10. Huang, C.-H.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.; Velculescu, V. E.;Kinzler, K. W.; Vogelstein, B.; Gabelli, S. B.; Amzel, L. M. Science 2007, 318, 1744.

11. PDB analysis of p-interaction between tryptophan rings and aromatic aminoacid side chains (Phe, Tyr, His) see: Samanta, U.; Pal, D.; Chakrabarti, P. Biol.Crystallogr. 1999, D55, 1421.

12. MTD was not established.

S. T. Staben et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2606–2613 2613

13. Edgar, K. A.; Wallin, J. J.; Berry, M.; Lee, L. B.; Prior, W. W.; Sampath, D.;Friedman, L. S.; Belvin, M. Cancer Res. 2010, 70, 1164–1172.

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