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The Rational Design of Selective Benzoxazepin Inhibitors of the #-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-isopropyl-1H-1,2,4-triazol-5-yl)-5,6dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326) Timothy P Heffron, Robert Andrew Heald, Chudi O Ndubaku, BinQing Wei, Martin Augustin, Steven Do, Kyle Edgar, Charlie Eigenbrot, Friedman Lori, Emanuela Gancia, Philip Jackson, Graham Jones, Aleksandr Kolesnikov, Leslie Lee, John Lesnick, Cristina Lewis, Neville McLean, Mario Mortle, Jim Nonomiya, Jodie Pang, Steve Price, Wei Wei Prior, Laurent Salphati, Steve Sideris, Steven Thomas Staben, Stefan Steinbacher, Vickie Tsui, Jeff Wallin, Deepak Sampath, and Alan G Olivero J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01483 • Publication Date (Web): 07 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

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The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3Kinase Culminating in the Identification of (S)-2-((2-(1isopropyl-1H-1,2,4-triazol-5-yl)-5,6dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9yl)oxy)propanamide (GDC-0326) Timothy P. Heffron,*† Robert A. Heald, ‡ Chudi Ndubaku, † BinQing Wei, † Martin Augistin,§ Steven Do, † Kyle Edgar, † Charles Eigenbrot, † Lori Friedman, † Emanuela Gancia, ‡ Philip S. Jackson, ‡ Graham Jones, ‡ Aleksander Kolesnikov, † Leslie B. Lee, † John D. Lesnick, † Cristina Lewis, † Neville McLean, ‡ Mario Mörtl,§ Jim Nonomiya, † Jodie Pang, † Steve Price, ‡ Wei Wei Prior, † Laurent Salphati, † Steve Sideris, † Steven T. Staben, † Stefan Steinbacher,§ Vickie Tsui, † Jeffrey Wallin, † Deepak Sampath, † Alan G. Olivero. † Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Argenta, Early Discovery Charles River, 7-9 Spire Green Centre, Flex Meadow, Harlow, Essex, CM19 5TR, United Kingdom. Proteros Biostructures GmbH, Bunsenstr. 7aD – 82152 Martinsried, Germany

†

Genentech, Inc.

‡

Argenta, Early Discovery Charles River

§

Proteros Biostructures ACS Paragon Plus Environment

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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ABSTRACT Inhibitors of the Class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy.

Despite the particular attraction of

targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other Class I isoforms and all other kinases tested.

In GDC-0032 (3, taselisib), we previously minimized inhibition of

PI3Kβ relative to the other Class I insoforms. Subsequently, we extended our efforts to identify PI3Kαspecific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a non-conserved residue. Several molecules selective for PI3Kα relative to the other Class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

Introduction Inhibition of the phosphoinositide 3-kinases (PI3Ks) has received significant attention for its potential in the treatment of several diseases. Within the PI3 kinase family, there are four Class I PI3K isoforms (α, β, δ, and γ). Of these isoforms, PI3Kα is the most commonly associated with cancers.1 PI3Kβ has been a target for preventing or treating the formation of blood clots2 and PI3Kβ selective inhibitors are also under clinical study for the treatment of phosphatase and tensin homologue (PTEN) deficient cancers.3,4 PI3Kδ and PI3Kγ are expressed almost exclusively in leukocytes and have been identified as targets primarily for inflammatory, autoimmune, and respiratory indications but with recent approval of idelalisib (a PI3Kδ inhibitor) for oncology applications.5,6,7,8,9 PI3Kα remains the most extensively studied isoform for oncology, with numerous clinical inhibitors under evaluation.10,11

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Despite the implicated importance of inhibiting PI3Kα in particular, achieving selective inhibition of this isoform has proved challenging and few reports of molecules with specificity for PI3Kα have appeared.12,13,14

Recently, and since the conclusion of our efforts described here, very notable

achievements in this area have been reported describing a successful parallel chemistry approach to the identification of highly selective PI3Kα inhibitors including the clinical inhibitor NVP-BYL719.15,16 While we have previously disclosed approaches to achieve inhibition of PI3Kα with biochemical selectivity over PI3Kβ,17,18 clinical candidates GDC-0941 (1) and GDC-0980 (2) are not selective for PI3Kα relative to the other Class I isoforms, and GDC-0032 (3, taselisib) is sparing of PI3Kβ (Figure 1).19,20,21 Figure 1. Structures of clinical candidate inhibitors of Class I PI3Ks, 1; class I PI3Ks and mTOR kinase, 2; Class I PI3Ks with selectivity for PI3Kα relative to PI3Kβ, 3; and PI3Kα isoform selective, 4.

With the advancement of 1-3 into clinical studies, we turned the attention of our program to identify PI3Kα inhibitors which display selectivity over each of the other Class I isoforms. Improved selectivity for PI3Kα relative to the other isoforms has the potential for increased tolerability in the clinical setting. The efforts leading to the discovery of the PI3Kα-specific inhibitor clinical candidate 4 (GDC-0326, Figure 1) are described herein. Results and Discussion ACS Paragon Plus Environment

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At the onset of our efforts to achieve a PI3Kα specific inhibitor, no PI3Kα crystal structures were available, with only PI3Kγ crystal structures available publicly or internally to our team. As a result, our approach relied on sequence homology to identify key residues to target that differ between PI3Kα and the other Class I isoforms to achieve selectivity.22 These initial efforts using the benzoxepin series of molecules, resulted only in selectivity over PI3Kβ and little to no selectivity for PI3Kα relative to the other isoforms.17,18,23 Our understanding and approach to achieving PI3Kα specific inhibitors changed when we gained access to two PI3Kα crystal structures at 3.0 Å resolution. The first structure, which does not contain a small molecule in the active site of the enzyme, appeared in the public domain.24 The second structure was acquired internally in complex with benzoxepin 5 (Figure 2A). Greater clarity into potential alphaspecificity determinants was provided by these PI3Kα crystal structures. In each of the two structures, Gln859 and His855 had well defined electron density and are seen to adopt consistent conformations (Figure 2B). This observation suggested that these two residues, which are uniquely Gln and His in PI3Kα and appear conformationally restrained, would be suitable to target to achieve high PI3Kα specificity.

Of these two residues, interaction with Gln859 presented the greater opportunity to

differentiate affinity between PI3Kα and the other Class I isoforms due to the possibility of achieving multiple unique hydrogen bonds with its side chain.25 In support of this, modeling suggested that the position of our benzoxepin series of molecules within the active site provided adequate vectors to contact Gln859. Figure 2. (A) Crystal structure of 5 in PI3Kα (PDB Code: 5DXH). Probable hydrogen bond between Gln859 and backbone NH of Thr856 is labeled in red. Also indicated are the corresponding residues in the other Class I PI3K isoforms. (B) Overlay of A with the public crystal structure of PI3Kα (PDB Code: 2RD0). (C) Structure, PI3Kα Kiapp and isoform selectivity of 5, the PI3K inhibitor contained within crystal structure A.


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With a goal of achieving the maximum enthalpic benefit through interaction with Gln859, we designed molecules which contained an amide or amide isostere as such a moiety could theoretically be able to form complementary hydrogen bonds with Gln859. To aid the design of molecules with functionality positioned to contact Gln859 we used two complementary approaches. First, we utilized the desktop modeling software Benchware3D® to design from the crystal structure of 5 in PI3Kα (Figure 2A). We also used Link and Grow computational approaches to identify linkers between the core benzoxazepin and functional groups (eg. amides, ureas, pyrazoles) that were optimally positioned to achieve hydrogen bonding interactions with Gln859.26

Primary amide functionality joined to the

benzoxazepin core by a two atom spacer frequently appeared in target molecules that were identified from both design approaches and these were prioritized for synthesis. We chose to begin our efforts with a 9-azabenzoxazepin core (see Scheme 1 and Table 1) to facilitate synthetic chemistry for the rapid production of analogs to evaluate our hypothesis to gain isoform selectivity. Scheme 1.


5

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H2 N

O

b

Cl

N

N

O

c

Cl

N

8

6: R= H

NH

N

N

I

R

Cl

O

d

Cl

NH2

10

9

a O

7: R= I

7 R 8

OH Cl

O

H N

N

e

O

11

O

Cl

O N

f

9

g

N

N

N N

N

N

O

12 N N

N

N

N

N

N

N

N

13: R= OMe h

.HCl

14: R= OH i 15: R= Cl

j

16: R= OTf O Cl HN

NH2

N k

N

.HCl 17

N

N 20 O l

N O N

N

Cl

18

19

(a) NIS, DMF; (b) t-BuONO2, TFA, MeOH; (c) Zn(CN)2, Pd(PPh3)4, DMF; (d) LiHMDS, THF, -78 °C; (e) 19, KHCO3, THF, H2O, reflux; (f) toluene, 130 oC; (g) NaH, DMF; (h) HBr, AcOH, 80 oC; (i) POCl3; (j) NaH, PhNTf2, NMP; (k) Formamide 130 oC; (l) i) n-BuLi, THF -15 oC, ii) 20, -70 oC. The synthetic route to 8-substituted 9-azabenzoxazepins began with regiospecific iodination of commercially available 2-amino-4-chloro-pyridine (6) with NIS. The amino group was then replaced with a methoxy substituent (8), to serve as a protecting group, via diazotization in the presence of methanol and TFA. Palladium-catalyzed cyanation followed by reaction with LiHMDS gave the fully functionalized pyridine amidine 10.

The imidazole ring was formed through reaction of

chloromethylketone 19 under optimized literature conditions.27 The chloroketone 19 itself was prepared from regiospecific lithiation of 1-isopropyl-1H-[1,2,4]triazole 18 followed by condensation reaction with chloromethyl Weinreb amide 20. 1-Isopropyl-1H-[1,2,4]triazole 18 was prepared from the reaction ACS Paragon Plus Environment

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of isopropylhydrazine with formamide at high temperature. With 11, the benzoxazepin ring was then formed by regiospecific reaction of the imidazole with ethylene carbonate followed by intramolecular SNAr.

The methoxy group was deprotected using HBr/AcOH to reveal pyridone 14, which

subsequently allowed for the formation of the fully elaborated core with a chloro (15) or triflate (16) leaving group at the 8-position.

Reaction of 14-16 under the specified conditions allowed for

introduction of the desired ether or amine respectively (Scheme 2). Scheme 2.


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Journal of Medicinal Chemistry R R'

14

O

O

R''O

a

O

O

N

H2 N

N

O

N

N

O

N

N N

N

24

b

N

N

N

N

21: R, R' = H, R''= Me

R c

22: R = H, R' = Me, R''= Et

R'

23: R, R' = Me, R''= Et

O

O

H2N

N

N

O N

N

N

14

H2 N

d

N

O

N N

O

25: R = H, R' = Me 26: R, R' = Me

N N

29

N

N N

O

N R e

15

R' N

N N

30: R = CONH2, R' = H 31: R, R' = H 32: R = H, R' = CONH2

N

N N

O

H2 N N

N N

f 27

N

N N

16 O

g H2 N

O

N N

N N

28

N

N N


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(a) alcohol, PPh3, DIAD, THF or dioxane; (b) 7 N NH3, MeOH, 50 oC; (c) i) LiOH.H2O, MeOH, H2O, 50 oC, ii) NH4Cl, HATU, DMF, Et3N; (d) i) NaH, DMF, PhNTf2, ii) L-prolinamide, 70-100 oC; (e) amine, Et3N, NMP, 150 oC; (f) i) 3,4-methoxybenzylamine, NMP, 85 oC, ii) TFA 40 oC; (g) i) CH3HNCH2CO2CH3, NMP, 85 oC, ii) 7 N NH3, MeOH, 50 oC.


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Table 1. Biochemical potency and Class I PI3K isoform selectivity for aza-benzoxazepins targeting hydrogen bonding with Gln859.

O

R N

N N N

N N Compound 13

24

O

R

PI3K Kiappa

PI3K Kiapp/ PI3K Kiapp



O

3.6 nM

12

0.9

0.9

0.9 nM

45

5.3

6.5

1.0 nM

139

8.6

25

3.1 nM

23

2.3

13

1.2 nM

13

1.9

0.8

0.2 nM

135

10

45

0.1 nM

282

27

111

3.2 nM

63

1.4

21

1.3 nM

10

0.7

0.5

30 nM

18

0.4

3.5

O

H2N O

O

25 H 2N O

O

26 H 2N 27

28

H 2N O

N

H 2N N 29 O

H 2N N 30

O

H 2N 31

N N

32 HN

O

a

All Kiapp values reported represent geometric means of a minimum of three determinations. These assays generally produced results within 2-fold of the reported mean.


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Using the synthetic route described in Schemes 1 and 2, we accessed both ether and amine substitutions of the pyridyl ring (Table 1).

Where R is methoxy in Table 1 (13), as a baseline

comparison, moderate selectivity was observed for PI3Kα over PI3Kβ, but no selectivity was achieved over PI3Kδ or PI3Kγ. By extending the methoxy group to include a primary amide designed to achieve hydrogen bonding contacts with Gln859 and Ser854 (24), improvement in PI3Kα potency, along with improvement in selectivity over each of the other isoforms was achieved. When the methylene of compound 24 was further substituted with a methyl group (25), some selectivity benefit was noted against the beta and gamma isoforms. However, a quaternary center (26) resulted in loss of potency compared to 24 and 25. In the series of aminopyridines (27-32), the baseline comparitor selected was the primary amine (27) which exhibited only modest selectivity over the β isoform. SAR suggested the importance of the primary amide for potency and isoform selectivity (28-30).

Tertiary amine 28

achieved high potency and isoform selectivity. With that understanding we also investigated pyrrolidine substitution and found that 29 (derived from the D-proline) was more potent than its enantiomer 30 and that both had greater isoform selectivity than the unsubstituted pyrrolidine 31. Changing the primary amide in 29 to the NHMe amide in 32 resulted in a significant reduction in potency, consistent with the model (Figure 3) that suggested methyl substitution would result in loss of hydrogen bonding interaction and a steric clash with Gln859. Figure 3. Docking model of 29 in PI3Kα. Compound 29 is predicted to form 3 concerted hydrogen bonds with the protein. A 2-dimensional depiction of structure 29 is shown next to the docking model.


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Several of the potent and selective 9-aza compounds from Table 1 were progressed to in vitro and in vivo metabolism and pharmacokinetics studies. In contrast to our previous experience with 8-pyrazole benzoxazepine analogs,21 clearance in rats for the PI3Kα isoform selective 9-aza benzoxepin analogs tested was high and not predicted by in vitro microsomal stability (Table 2) or hepatocyte stability (data not shown). Additionally, one of three compounds showed systemic clearance well above the average hepatic blood flow of a rat (31), suggestive of non-hepatic clearance mechanisms.28 Most concerning, none of the tested compounds had systemic clearance values under liver blood flow despite moderate stability in microsomes suggesting non-cytochrome P450 mediated mechanism(s) of clearance. Table 2. Predicted hepatic Cl from microsomal stability and rat in vivo Cl data for select 9-aza benzoxazepins.


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a

Hepatic clearance was predicted from liver microsome incubations using the “in vitro t1/2 method”.29 Male Sprague-Dawley rats were dosed intravenously with 1 mg/kg of each compound prepared in 60%PEG400/10% Ethanol.

b

When considering potential contributions to extrahepatic clearance, we were initially concerned that the primary amide common to the high isoform specificity (Table 1) was prone to hydrolysis. However, compound 31 lacked this amide and still seemed to be vulnerable to extrahepatic clearance. Additionally, metabolite identification studies of 29 in both rat and human hepatocytes did not reveal any amide hydrolysis or conjugation (data not shown). While an exact site of oxidation of 29 was not determined, we considered the pyridyl ring as potentially susceptible to non-cytochrome P450 mediated metabolism (eg aldehyde oxidase).30 In our previous experience with des-aza benzoxazepins, we had not encountered compounds with rat clearance in excess of liver blood flow and typically observed reasonable correlation with liver microsomal predictions of clearance. Therefore, we set out to synthesize analogs of the PI3Kα isoform specific compounds from Table 1 on the des-aza core (Scheme 3). Scheme 3.


13

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Journal of Medicinal Chemistry n O

N

O H2 N a

N

N

39: R = Me

N

N N

N N

N

4: R = H

b N

O

N

N

N

O

H2N

R

N

33

c

N

34: n = 1 35: n = 2 36: n = 0

O

Br

N

O

O

O

HO

d

N

O

H2N

O N

N 37

38

N

N

N N

N

N

N

O e

H2 N

O

O N N

40

N

N N

(a) i) amino acid, CuI, K3PO4, DMSO, 80-90 oC, ii) NH4Cl, EDCI, HOBt, DIPEA, DMF or NH4Cl, HATU, DMF, Et3N; (b) KOH, dioxane, water, Pd2dba3, 2-di-tertbutylphosphino-2’,4’,6’triisopropylbiphenyl, 90 oC; (c) i) t-Bu-(D)-lactate or t-Bu-(R)-2-hydroxybutyrate, PPh3, DIAD, THF, ii) TFA, CH2Cl2, iii) NH4Cl, HATU, DMF, Et3N; (d) 2-bromoacetamide, Cs2CO3, DMF; (e) i) ethyl-(L)-lactate, PPh3, DIAD, THF, ii) LiOH.H2O, MeOH, H2O, 50 oC, iii) NH4Cl, HATU, DMF, Et3N. The synthesis of target molecules began with 8-bromo benzoxazepin 33 (Scheme 3).21 Compound 33 was converted to desired anilines (34-36) via copper catalyzed coupling followed by conversion of the amino acid to the primary amide. The aryl ethers 4 and 38-40 were obtained via Mitsunobu reaction of phenol 37 which was also accessed via bromide 33. Table 3. Biochemical potency and Class I PI3K isoform selectivity for benzoxazepins targeting hydrogen bonding with Gln859.


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R

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O N N N

N N

Compound

PI3K Kiappa

R

PI3K Kiapp/ PI3K Kiapp/ PI3K Kiapp/ MCF7-neo/HER2 PC3 b PI3K Kiapp PI3K Kiapp PI3K Kiapp Proliferation EC50 Proliferation EC50

N 34

0.03 nM

186

7.4

67

0.01 M

0.15 M

0.3 nM

328

16

157

0.03 M

0.87 M

0.1 nM

69

7.3

47

0.05 M

0.23 M

0.3 nM

16

1.0

8.0

0.2 M

0.2 M

0.2 nM

133

20

51

0.1 M

2.2 M

0.5 nM

164

17

53

0.2 M

2.9 M

3.6 nM

19

1.0

1.3

1.0 M

4.6 M

O

H2N N 35

O H2N N 36 H2N O

O O

38 H2N O

O

4 H2N O

O

39 H2N O 40

O

H2N

a

All Kiapp values reported represent geometric means of a minimum of three determinations. These assays generally produced results within 2-fold of the reported mean. bCellular EC50 values represent geometric means of a minimum of two determinations and these assays generally produced results within 3-fold of the reported mean.


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A comparison of 34 (Table 3) with its corresponding analog in the 9-aza series (29, Table 1) reveals comparable and desirable potency and isoform selectivity. Additionally, the improved potency of 34 relative to 35 and 36 suggests the importance of optimally positioning the primary amide. We were curious to find that 8-ether analog 38 (Table 3) had little to no selectivity for PI3Kα over the other isoforms. Although the 2-ethanamide ether of 38 should be long enough for the hydrogen bonds with His855 and Gln859 (see for example Figure 3), conformational analysis showed that the addition of a 2methyl with an S chirality would help restrict the preferred C-O bond torsion (Figure 4B) to mimic the desired value of ~79°, as predicted by modeling to allow for hydrogen bonding interactions with Gln859 (Figure 4C). Indeed, a search of the small molecule crystallographic database (CSD) using Mogul confirmed the effect of such substitutions. Inclusion of an S-methyl or S-ethyl (4 and 39) led to significant improvments in selectivity over the other 3 Class I isoforms. We have previously reported on the apparent importance of PI3Kβ inhibition to inhibit proliferation of a cancer cell that has lost PTEN function.31 We have also previously demonstrated that compounds with enhanced PI3Kα to PI3Kβ enzyme selectivity in cell proliferation assays showed a qualitative trend for weaker effect on inhibiting proliferation in the PC3 cell line (PTEN-null) relative to inhibiting proliferation in the MCF7.1 cell line (PI3Kα mutant).17 Consistent with these previous studies, improvement in selectivity over PI3Kβ is apparent in the relative potency in MCF7-neo/HER2 and PC3 cell proliferation assays (Table 3). It is worth noting that the improved selectivity of compounds 4 and 39 compared to compound 38 came not from better potencies against PI3Kα but from decreased affinities toward the other isoforms. One rationale for this observation could be that the S-alkyl substitutent somehow insults the other isoforms, leading to the better selectivity. However, modeling suggests that the alkyl group points toward the solvent, not toward the protein, which is consistent with a lack of difference between the methyl (4) and ethyl group (39). We hypothesize that the affinities of compounds 4 and 39 toward the


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off-target isoforms were reduced due to desolvation penalties upon binding of the primary amide and the nearby protein residues, while for PI3Kα the desolvation energy is fully compensated by three, concerted hydrogen bonds formed by the amide with His855 and Gln859 (Figure 4C).

The balance between

desolvation and hydrogen bonding can also account for the reduced potency and PI3Kα selectivity of 40 (Table 3) for which the preferred torsional angle would not allow for engagement of the residues unique to PI3Kα. Figure 4. Distributions of the observed torsion angles in the CSD database where -CH2- separates an amide and an aryl ether (A) or -CHMe- separates an amide and an aryl ether (B). Query structures of Mogul searches (setting: exact structures) are shown in inset with the torsional bond colored in red. The preferred angles are labeled by the red arrows. (C) Docking model 4 in PI3Kα. 4 is predicted to form 3 concerted hydrogen bonds with the protein. A 2-dimensional depiction of 4 is shown next to the docking model. The dihedral angle along the C-O (colored in red) bond is 79°.


17

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After the conclusion of these studies, a crystal structure of 4 bound to PI3Kα was obtained (2.25Å resolution, Figure 5A, PDB Code: 5DXT). Consistent with our model (Figure 4C), when bound to PI3Kα the primary amide of compound 4 achieves three concerted hydrogen bonds with the protein as predicted (vide supra). The dihedral angle of interest is 68° and again consistent with expectations based on observed torsion angles in the CSD database. Additionally, a crystal structure of 4 bound to PI3Kδ was also obtained (2.64 Å resolution, Figure 5B). This crystal structure shows that the primary amide of compound 4 achieves 2 hydrogen bonding interactions, one with Asp832 (corresponding to His855 in PI3Kα) at an apparently suboptimal bonding angle and one with the backbone carbonyl of Ser831 (corresponding to the analogous interaction with the backbone carbonyl of Ser854 in PI3Kα) which appears non-optimal at a distance of 3.6 Å. Figure 5. Crystal structures of 4 bound to PI3Kα (A; PDB Code: 5DXT) and PI3Kδ (B; PDB Code: 5DXU).

In vitro metabolic stability and in vivo pharmacokinetic data for two des-aza core analogs are shown in Table 4. A comparison of 34 (Table 4) with its aza-core analog (29, Table 3) shows that des-aza core proline-derived analog 34 is stable in vitro and with encouraging clearance in rat, well below liver blood flow. The O-linked analog 4, however, is the most stable in human and rat liver microsomes, and there ACS Paragon Plus Environment

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is a good correlation with in vivo rat clearance. Furthermore, 4 achieves high oral bioavailability and has high unbound fraction. Table 4. In vitro metabolic stability and in vivo rat PK data for key des-aza benzoxazepins.

a

Hepatic clearance was predicted from liver microsome incubations using the “in vitro t1/2 method”.29 Male Sprague-Dawley rats were dosed intravenously with 1 mg/kg of each compound prepared in 60% PEG400/10% Ethanol. cMale Sprague-Dawley rats were dosed PO with 5 mg/kg of each compound in 0.5% methylcellulose with 0.2% Tween 80 (MCT). b

The balance of potency, selectivity and appealing ADME properties led us to profile 4 in further studies. Compound 4 was found to have consistently low clearance and high oral bioavailability across species tested, enabling significant sustained free drug levels (Table 5). Table 5. Pharmacokinetic data for 4 in preclinical species.


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Journal of Medicinal Chemistry O O

H2N

O N N N

N

4

N microsomes Clhep Species (mL/min/kg)a

POc

IV (1 mg/kg)b in vivo Cl (mL/min/kg)

Vss (L/kg)

Dose (mg/kg)

Cmax ( M)

AUC ( M.h)

F%

PPB%

Mouse

4

8

0.6

25

31

74

55

40

Rat

9

10

1.5

5

5.4

25.4

120

63

Cyno

9

13

1.5

2

1.5

4.8

72

39

Dog

10

7

2.5

2

2.1

2.4

100

38

a

Hepatic clearance was predicted from liver microsome incubations using the “in vitro t1/2 method”.29 Male Sprague-Dawley rats, female NCR nude mice, male cynomolgus monkeys or beagle dogs were dosed intravenously with 1 mg/kg of each compound prepared in 60% PEG400/10% Ethanol. c Compound 4 was administered PO at the indicated dose in 0.5% methylcellulose with 0.2% Tween 80 (MCT).

b

In addition to achieving selectivity over the other Class I isoforms, the PI3Kα specific inhibitor 4 also achieves a very high level of selectivity over other kinases. In a panel of 235 kinases evaluated at Invitrogen, only one was inhibited by >50% by 4 when tested at 1 µM (61% inhibition of the class II PI3KC2b).32 Also worth noting, 4 is not an inhibitor of cytochrome P450 enzymes tested (IC50 > 10 µM against 3A4, 2C9 1A2, 2C19, 2D6), is highly permeable in MDCK cells (Papp A to B: 13 x 10-6 cm/s) and has thermodynamic solubility of 82 µg/mL at pH 7.4. In vivo Efficacy and Pharmacodynamics


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The identification of 4 allowed us to compare this selective inhibitor of PI3Kα with 3, which is selective over the beta isoform. A head-to-head comparison of relevant data for the interpretation of in vivo efficacy is presented in Table 6. While 3 achieves higher total AUC than 4 in mice at the same dose, this is offset by the substantially higher free fraction of 4 in mice. Therefore, to compare the PI3Kα specific inhibitor 4 with the PI3Kβ-sparing inhibitor 3 we advanced both into in vivo efficacy studies in tumor xenograft models. Table 6. Potency, selectivity and mouse pharmacokinetic parameters for 3 and 4.

a

Female NCR nude mice were dosed intravenously with 1 mg/kg of each compound prepared in 60% PEG400/10% Ethanol and PO at 25 mg/kg in 0.5% methylcellulose with 0.2% Tween 80 (MCT). bAll Kiapp values reported represent geometric means of a minimum of three determinations. These assays generally produced results within 2-fold of the reported mean. cCellular EC50 values represent geometric means of a minimum of two determinations and these assays generally produced results within 3-fold of the reported mean. The in vivo efficacy of 4 was compared to the beta isoform sparing clinical PI3K inhibitor 3 in two breast cancer xenograft models that were grown in nude mice (MCF7-neo/HER2) and scid beige (KPLACS Paragon Plus Environment

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4). Daily, oral administration of 3 at a maximum tolerated dose of 25 mg/kg in the MCF7-neo/HER2 xenograft model resulted in 116% tumor growth inhibition (TGI) and 4 partial regressions (PRs) out of 10 animals treated when compared to vehicle treated mice (Figure 6A). Daily administration of 4 orally at 0.78, 1.56, 3.25, 6.25, or 12.5 mg/kg resulted in dose-dependent increase in TGI (73%, 79%, 83%, 101%, and 110%, respectively) and tumor regressions (6 PRs out of 10 animal at 6.25 and 12.5 mg/kg) when compared to vehicle treated mice (Figure 6A). At the maximum tolerated dose of 25 mg/kg, daily doses of 3 resulted in 125% TGI in the KPL-4 xenograft models with 8 PRs and 2 complete regressions (CRs) out of 10 animals treated when compared to vehicle treated mice (Figure 6B). Daily administration of compound 4 orally at 0.78, 1.56, 3.25, 6.25, or 12.5 mg/kg also resulted in dose-dependent increase in TGI (73%, 97%, 97%, 122%, and 121%, respectively) in the KPL-4 xenograft model. Notably, maximum efficacy of 4 was observed at 6.25 mg/kg in the KPL-4 model based on TGI and tumor regressions (9 PRs and 1 CR out of 10 animal treated) when compared to vehicle treated mice (Figure 6B). Doses of 4 up to 12.5 mg/kg were well tolerated based on less than 10% body weight loss (data not shown).

Thus, increased unbound

concentration of 4 at doses that were approximately 4-fold lower (6.25 mg/kg) than the maximum tolerated dose of 3 (25 mg/kg) resulted in comparable TGI in both the MCF7-neo/HER2 and KPL-4 breast cancer xenograft models. Figure 6. Dose-response curves of fitted tumor volumes in response to 3 and 4 at the doses shown against MCF7-neo/HER2 (A) and KPL-4 (B) breast cancer xenografts in mice relative to vehicle (MCT; 0.5% methycellulose/0.2% Tween-80) after daily (QD) oral (PO) dosing for 21 days. Rx=treatment period


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The relationship between pharmacokinetics and pharmacodynamics of 4 relative to 3 was also investigated in the MCF7-neo/HER2 xenograft model (Figure 7). The phosphorylation levels of Akt in MCF7-neo/HER2 tumors, as well as plasma drug concentrations of both compounds, were evaluated 1 hour after a single dose of vehicle (MCT), 3 at 25 mg/kg or 4 at 0.39, 0.78, 1.56, 3.25, 6.25, or 12.5 mg/kg. A single dose of 3 resulted in a 74% decrease of phosphorylated Akt (pAktSer473) levels while a 4-fold lower dose of 4 at 6.25 mg/kg resulted in a similar decrease in Akt phosphorylation (78%) (Figure 7).33 The comparable decreases in pAkt between these doses of 4 and 3 is likely due to similar unbound plasma drug concentrations that were achieved in mice after 1 hour of dosing (Figure 7). More importantly, these doses of 4 and 3 that potently inhibited PI3K (based on robust suppression of PI3K) were sufficient to induce tumor regressions in the MCF7-neo/HER2 xenograft model. Figure 7. Quantification of pAktSer473 levels in MCF7-neo-HER2 tumor xenografts 1 hour after a single dose of MCT vehicle (0.5% methycellulose/0.2% Tween-80), 3 at 25 mg/kg or 4 at 0.39, 0.78, 1.56, 3.25, 6.25 or 12.5 mg/kg. Phosphorylated Akt (pAktSer473) and total Akt (tAkt) levels were measured by Mesoscale Discovery assay and values are expressed as ratio of pAkt/tAkt. Error bars represent SEM for


23

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tumor xenograft samples from four different animals. Corresponding unbound plasma drug concentrations (closed diamonds) for each dose of 3 or 4 are plotted on the right y-axis.

Plasma Concentration

4

0.14

3.5

0.12

3

0.1

2.5

0.08

2

0.06

1.5

0.04

1

0.02

0.5

0

Unbound Drug [uM]

pAkt/tAkt

0.16

Ratio pAkt/tAkt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 MCT

25 3

0.39

0.78

1.56

3.125

6.25

12.5

4

An optimized synthetic route to 4 is shown in Scheme 4.

A Mitsunobu reaction employing

intermediate 37 was used for installation of the ether side chain required for isoform selectivity. While not being atom efficient, this methodology is reliably stereospecific. The amide was formed directly from ester 47 by reaction with concentrated ammonia. This route provided 4 in 19% overall yield with a longest linear sequence of 7 steps. Scheme 4.


24


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HO

F

Ph

a

O

Ph

F

Page 26 of 56

O

F

b

NH2

72%

90%

N

N

43

42

41 Ph

O

OH

F

Ph H N

c 84%

NH.HCl

O

F e

d

N

58%

70%

N

N N

N

N

44

N

45

N

N

O Ph

HO

O

O

f

N

N

N

g

N

N

N N

h

N

87%

N

37

O

O

O N

83%

N 46

O

47

4

98%

N N

N N

(a) K2CO3, BnBr, KI, acetone; (b) LiHMDS, THF, -78 oC; (c) 19, KHCO3, THF, H2O; (d) 1,3dioxolan-2-one, toluene, 100 oC; (e) NaH, DMF; (f) H2, Pd/C, EtOAc, IMS; (g) (R)-2-hydroxypropionic acid methyl ester, PPh3, DIAD, dioxane; (h) 7 N NH3, MeOH.

Conclusion Structure-based drug design targeting non-conserved residues allowed for the realization of potent benzoxazepin inhibitors of PI3Kα that are remarkably selective over the other Class I isoforms in enzymatic assays. Crystal structures of 4 in PI3Kα and PI3Kδ were solved which support the proposed basis for PI3Kα selectivity. Optimization of pharmacokinetics resulted in the identification of 4 which demonstrated comparable in vivo efficacy at reduced dose when compared to the clinical inhibitor 3. The in vitro characterization and pharmacokinetic parameters estimated in preclinical species suggest 4 would have low plasma CL in human.34 The overall profile of 4 led to its selection as a clinical development candidate (not currently in clinical trials) and allows for further study of a highly optimized specific inhibitor of the PI3Kα isoform. Experimental Section


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Chemistry. All solvents and reagents were used as obtained.

1

H NMR spectra were recorded with a

Bruker Avance DPX400 Spectrometer or a Varian Inova 400 NMR spectrometer, and referenced to tetramethyl silane. Chemical shifts are expressed as δ units using tetramethylsilane as the external standard (in NMR description, s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak). All final compounds were purified to >95% chemical purity, as assayed by HPLC (Waters Acquity UPLC column, 21 mm × 50 mm, 1.7 µm) with a gradient of 0-90% acetonitrile (containing 0.038% TFA) in 0.1% aqueous TFA, with UV detection at λ = 254 and 210 nm, and with CAD detection with an ESA Corona detector. High Pressure Liquid Chromatography - Mass Spectrometry (LC-MS) experiments to determine retention times (rt) and associated mass ions were performed using various methods which are fully described in the supporting information. Where measured, chiral purity was assessed by SFC using Chiralpak AS column, using isocratic methods of methanol with 0.1 % ammonium hydroxide/carbon dioxide. All compounds were named using Autonom 1.0 (MDL Information Systems Inc.). 4-Chloro-5-iodo-pyridin-2-ylamine (7). To a solution of 2-amino-4-chloropyridine (6) (150 g, 0.78 mol) in DMF (1.5 L) was added NIS (341 g, 1.52 mol) and the reaction mixture stirred at room temperature for 18 h before being concentrated in vacuo to 300 mL volume. The resultant residue was poured into 10% aqueous sodium thiosulfate solution (1.2 L), stirred for 15 min and the precipitate formed collected by filtration, washed with water then dried at 35 oC in vacuo to give the title compound as a pale brown solid (185 g, 62%). 1H NMR (CDCl3) δ ppm 8.33 (1 H, s), 6.68 (1 H, s), 4.52 (2 H, s). 4-Chloro-5-iodo-2-methoxy-pyridine (8). To a solution of 7 (64.2 g, 0.25 mol) in methanol (1.1 L) and TFA (93.7 mL, 1.26 mol) was added tert-butyl nitrite (150 mL, 1.26 mol) so as to maintain temperature less than 3 oC. The resultant mixture was stirred at room temperature for 1h then allowed to warm to room temperature and stirred for 16 h. The reaction was quenched by the careful addition of water then concentrated in vacuo to ¼ volume. The resultant residue was treated with water (1 L) and the precipitate formed collected by filtration and dried in vacuo at 35 oC to give the title compound (62.3


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Page 28 of 56

g, 92%). Contains 16% unidentified impurity. ¹H NMR (DMSO-d₆) δ ppm 8.56 (1 H, s), 7.20 (1 H, s), 3.86 (3 H, s). 4-Chloro-6-methoxy-nicotinonitrile (9). A suspension of 8 (30.5 g, 0.11 mol), zinc (II) cyanide (7.97 g, 68 mmol), Pd(PPh3)4 (6.56 g, 5.66 mmol) and DMF (450 mL) was degassed and then heated at 120 oC for 1h before being concentrated in vacuo. The resultant residue was treated with water then extracted with DCM, the organic extract dried (MgSO4), filtered, then concentrated in vacuo. The resultant residue was crystallized from DCM to give the title compound (10.1 g, 54%). The mother liquors were concentrated in vacuo and the residue subjected to flash chromatography (SiO2 gradient 0 to 100% ethyl acetate in cyclohexane) then crystallization from cyclohexane to give the title compound (5.16 g, 28%; 82% total). 1H NMR (CDCl3) δ ppm 8.45 (1 H, s), 6.90 (1 H, s), 4.01 (3 H, s). 4-Chloro-6-methoxy-nicotinamidine hydrochloride (10). To a solution of 9 (10.1 g, 59.7 mmol) in THF (300 mL) at -78oC was added LHMDS (65.7 mL) dropwise and the reaction mixture stirred for 30 min before allowing to warm to room temperature and stirring for a further 1h. The reaction was quenched by the addition of 1N HCl (to pH ~1) and then extracted three times with ethyl acetate. The aqueous layer was concentrated in vacuo to give a brown solid which was azeotroped with toluene to give the title compound as a tan solid. Mixture with ammonium chloride, 72% title compound by weight. (15.2 g, 83%). ¹H NMR (DMSO-d₆) δ ppm 9.68 (4 H, d, J = 15.8 Hz), 8.46 (1 H, s), 7.47 (5 H, t, J = 50.7 Hz), 7.27 (1 H, s), 3.95 (3 H, s). 4-Chloro-5-[4-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-1H-imidazol-2-yl]-2-methoxy-pyridine (11).

A

suspension of 10 (50.9 mmol) and potassium bicarbonate (20.4 g, 202.5 mmol) in THF (128 mL) and water (21 mL) was heated to reflux then treated with a solution of 19 (9.55 g, 50.9 mmol) in THF (25 mL). The reaction mixture was stirred at reflux for 24 h before being concentrated in vacuo. The resultant residue was diluted with water and extracted with ethyl acetate. The combined extracts were dried (Na2SO4), treated with charcoal (15 g), filtered and concentrated in vacuo to give a solid. The solid was triturated with 10% diethyl ether in pentane then dried in vacuo to give the title compound as a


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pale brown solid (8.74 g, 54%). LC-MS: [M+H]+= 319/321.

1

H NMR (CDCl3) δ ppm 9.03 (1 H, s),

7.89 (1 H, s), 7.83 (1 H, s), 7.26 (1 H, s) 6.88 (1 H, s), 4.01 (3 H, s), 1.58 (6 H, d, J = 6.6 Hz). 2-[2-(4-Chloro-6-methoxy-pyridin-3-yl)-4-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-imidazol-1-yl]ethanol (12). To warmed ethylene carbonate (34 g, 0.39 mol) was added 11 (8.74 g, 27.4 mmol) and the mixture heated at 130 °C for 3 h. The cooled reaction mixture was diluted with DCM then subjected to flash chromatography (Si-PPC, gradient 0-5% methanol in DCM) to give the title compound as a brown foam (7.52 g, 75%). LC-MS: [M+H]+= 363/365. 1H NMR (CDCl3) δ ppm 8.27 (1 H, s), 8.02 (1 H, s), 7.85 (1 H, s), 6.93 (1 H, s), 5.98-5.82 (1 H, m), 4.00 (5 H, m), 3.88 (2 H, t, J = 5.1 Hz), 1.51 (6 H, d, J = 6.6 Hz). 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-8-methoxy-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulene (13). A solution of 12 (7.52 g, 20.7 mmol) in DMF (100 mL) was cooled to 0 °C and treated with sodium hydride (804 mg, 20.1 mmol). The resultant mixture was stirred at 0 °C for 10 min then allowed to warm to room temperature and stirred for 72 h. Further sodium hydride (150 mg, 3.75 mmol) was added and stirring continued until no starting material remained (TLC) before the reaction was concentrated in vacuo. The residue was dissolved in ethyl acetate then washed with saturated brine (x3), dried (Na2SO4) and concentrated in vacuo. The resultant residue was triturated in pentane/diethyl ether (5:1) to give the title compound as a brown solid (5.38 g, 79%). LC-MS: [M+H]+= 327.

1

H NMR

(CDCl3) δ ppm 9.35 (1 H, s), 7.87 (1 H, s), 7.63 (1 H, s), 6.37 (1 H, s), 6.03-6.02 (1 H, m), 4.54-4.53 (2 H, m), 4.53-4.33 (2 H, m), 3.99 (3 H, s), 1.57 (6 H, d, J = 6.6 Hz). 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-ol (14). A solution

of

2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-8-methoxy-4,5-dihydro-6-oxa-1,3a,9-triaza-

benzo[e]azulene (1.00 g, 2.97 mmol) in acetic acid (40 mL) was treated with 48% aqueous HBr (37.7 mL) and heated at 80 °C for 5 h. The reaction mixture was concentrated in vacuo then the resultant residue was suspended in water (60 mL) and the pH was adjusted to ~6 using 5N aqueous NaOH. The precipitate formed was collected by filtration, washed with water then dried in vacuo. The resultant solid was triturated in acetone to give the title compound as a beige solid (3.58 g, 69%). LC-MS: [M+H]+= ACS Paragon Plus Environment

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Page 30 of 56

313. ¹H NMR (DMSO-d₆) δ ppm 8.42 (1 H, s), 7.90 (1 H, s), 7.83 (1 H, s), 5.84 (1 H, s), 5.78 (1 H, m), 4.71-4.30 (4 H, m), 1.45 (6 H, d, J = 6.6 Hz). 8-Chloro-2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulene (15). A mixture of 14 (200 mg, 0.64 mmol) and phosphorus(V) oxychloride (15 mL) was heated at 150 °C for 2.25 h using microwave irradiation. The reaction mixture was concentrated in vacuo and the resultant residue taken up into 10% methanol in DCM. The solution was loaded onto a NH2-cartridge eluting with 10% methanol in DCM. The eluent was concentrated in vacuo to give the title compound as an off white solid (168 mg, 79%). LC-MS: [M+H]+= 331/333. 1H NMR (DMSO-d₆) δ ppm 9.29 (1 H, s), 8.01 (1 H, s), 7.93 (1 H, d, J = 0.6 Hz), 7.24 (1H, s), 5.92-5.80 (1 H, m), 4.70-4.63 (2 H, m), 4.634.56 (2 H, m), 1.48 (6 H, d, J = 6.6 Hz). Trifluoro-methanesulfonic acid 2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9triaza-benzo[e]azulen-8-yl ester (16). To a solution of 14 (400 mg, 1.28 mmol) in N-methyl-2pyrrolidone (6 mL) was added sodium hydride (57 mg, 1.54 mmol, 65% in mineral oil) and heated at 40 °C for 1 hour. The reaction mixture was cooled to room temperature before the addition of N-phenylbis(trifluoromethanesulfonimide) (550 mg, 1.54 mmol) and the reaction stirred for 19 hours at room temperature. The reaction was quenched with water and the resultant precipitate collected by filtration and dried in vacuo to give the title compound as a pale brown solid (428 mg, 75%). 1H NMR (CDCl3) δ ppm 9.50 (1 H, s), 7.88 (1 H, d, J = 0.7 Hz), 7.70 (1 H, s), 6.84 (1 H, s), 5.99 -5.87 (1 H, m), 4.68-4.61 (2 H, m), 4.55-4.48 (2 H, m), 1.58 (6 H, d, J = 6.6 Hz). 1-Isopropyl-1H-[1,2,4]triazole (18). A solution of isopropyl hydrazine hydrochloride (17) (60 g, 0.54 mmol) in formamide (270 mL) was heated at 130 oC for 3 days. The cooled solution was diluted with saturated brine (700 mL) and extracted with ethyl acetate (4 x 1L). The combined organic extracts were dried (Na2SO4), filtered and concentrated in vacuo to give an oil. (The product isopropyl triazole is volatile so vacuum is kept to ~100 mbar and temperature to ~35 oC during concentration). The oil was subjected to distillation under reduced pressure (25 mbar, bp 85-90 oC) to give the title compound as a colourless oil (54 g, 90%). ¹H NMR (CDCl3) δ ppm 8.10 (1 H, s), 7.95 (1 H, s), 4.63-4.50 (1 H, m), ACS Paragon Plus Environment

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1.56 (6 H, d, J = 6.7 Hz). The product is contaminated with a minor amount of formamide. This does not interfere with the lithiation step but can be removed by extraction of the triazole product into cyclohexane. To a solution of 1-isopropyl-1H-

2-Chloro-1-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-ethanone (19).

[1,2,4]triazole (135 mmol, wet with cyclohexane) in THF (220 mL) at -20 oC was added n-butyllithium (54 mL, 2.5M, 135 mmol) dropwise over 10 min and then the mixture stirred and allowed to warm to -5 o

C. After 30 min 1H NMR of a quenched (MeOD) portion showed ~50% lithiation. Further n-

butyllithium (28 mL, 2.5M, 70 mmol) was added and stirring continued for 20 min at -5 to 0 oC. NMR of a quenched (MeOD) portion showed full lithiation.

1

H

The reaction mixture (pale yellow

suspension) was cooled to -70 oC and treated with 2-chloro-N-methoxy-N-methyl acetamide 20 (27.8 g, 202 mmol, solution in 60 mL THF) dropwise over 45 min and stirring continued for 1.5 h. The cold reaction mixture was added to 1M HCl (400 mL) with vigorous stirring, the mixture stirred allowed to warm to room temperature. The mixture was extracted twice with ethyl acetate (2 x 500 mL), the organic extracts dried (Na2SO4), filtered and concentrated in vacuo. The resultant oil was dissolved in pentane, cooled in dry ice and sonicated intermittently to give a crystalline solid which was filtered to give the title compound as an off-white solid (22g, 87%). ¹H NMR (CDCl3) δ ppm 7.96 (1 H, s), 5.51 (1 H, sept., J= 6.7 Hz), 1.56 (6 H, d, J = 6.7 Hz). General procedure A. To a suspension of the appropriate pyridone or phenol (1 equiv), the appropriate alcohol (1.3 equiv) and triphenylphosphine (1.5 equiv) in THF or dioxane was added dropwise DIAD (1.5 equiv) and the reaction mixture stirred at room temperature for 16 h. The reaction mixture was diluted with water and extracted with DCM. The combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo to give the crude product. 2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-yloxy]acetamide (24).

Following general procedure A using 14 and methylglycolate, then flash

chromatography (Si-PPC, gradient 0-5% methanol in DCM) gave a yellow solid (88 mg, 24%). LC-MS: [M+H]+ = 385. The intermediate ester 21 (88 mg, 0.23 mmol) was dissolved in a solution of ammonia ACS Paragon Plus Environment

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in methanol (7N, 5 mL) and the reaction mixture heated at 50 °C for 2 h, during which a white precipitate formed. The reaction mixture was concentrated in vacuo and the residue subjected to reverse phase preparative HPLC (C-18, gradient 10-90% MeCN in water, 0.1% formic acid) to give the title compound as a white solid (42 mg, 50%). LC-MS: [M+H]+ = 370. 1H NMR (d6-DMSO) δ ppm 9.07 (1H, s), 7.88 (1H, s), 7.86 (1H, s), 7.41 (1H, s), 7.18 (1H, s), 6.45 (1H, s), 5.87 (1H, sept, J = 6.7 Hz), 4.66 (2H, s), 4.60-4.44 (4H, m), 1.44 (6H, d, J = 6.7 Hz). (S)-2-((2-(1-isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydroimidazo[1,2-d]pyrido[3,4-f][1,4]oxazepin9-yl)oxy)propanamide (25). Following general procedure A using 14 and ethyl-(L)-lactate, then flash chromatography (Si-PPC, gradient 0-5% methanol in DCM) to give 22 as a yellow oil contaminated with triphenylphosphine oxide (70 mg, 29% based on NMR quantification of PPh3O). LC-MS: [M+H]+ = 413. To a solution of the intermediate ester (70 mg, 0.17 mmol) in methanol (5 mL) and water (1 mL) was added lithium hydroxide monohydrate (23 mg, 0.58 mmol) and the reaction mixture stirred at 50 °C for 1 h. The reaction mixture was concentrated in vacuo and the resultant residue azeotroped with acetonitrile (3 × 20 mL). The residue was dissolved in DMF (3 mL), HATU (220 mg, 0.58 mmol), ammonium chloride (46 mg, 0.86 mmol) and triethylamine (120 µL, 0.86 mmol) were added and the reaction mixture stirred at room temperature for 1.5 h. The reaction mixture was quenched with water then extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was subjected to reverse phase preparative HPLC (C-18, 10-90% MeCN in water 0.1% formic acid) to give the title compound as a white solid (28 mg, 43%). 63% ee (SFC, retention time = 1.01 min, opposite enantiomer at 0.56 min). LC-MS: [M+H]+ = 384. 1H NMR (d6-DMSO) δ ppm 9.05 (1H, s), 7.87 (1H, s), 7.86 (1H, s), 7.38 (1H, br s), 7.05 (1H, br s), 6.41 (1H, s), 5.87 (1H, sept, J = 6.6 Hz), 5.17 (1H, q, J = 6.8 Hz), 4.60-4.43 (4H, m), 1.50-1.34 (9H, m). 2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-yloxy]2-methyl-propionamide (26). Following general procedure A using 14 and ethyl 2-hydroxyisobutyrate, then flash chromatography (Si-PPC, gradient 0-5% methanol in DCM) gave a 23 as a yellow oil (103 mg, 40%). LC-MS: [M+H]+ = 427. To a solution of 23 (103 mg, 0.24 mmol) in methanol (5 mL) was ACS Paragon Plus Environment

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added water (0.5 mL) and lithium hydroxide monohydrate (19 mg, 0.48 mmol). The reaction mixture was heated at 50 °C for 16 h before the reaction was cooled to room temperature and the pH adjusted with HCl (1N, aq.) to give pH~4. The reaction mixture was concentrated in vacuo to give the crude product as a yellow solid. LC-MS: [M+H]+ = 399. The yellow solid (96 mg, 0.24 mmol) in DMF (3 mL) was added HATU (184 mg, 0.48 mmol), ammonium chloride (39 mg, 0.72 mmol) and triethyl amine (100 µL, 0.72 mmol) then the reaction mixture stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo and the resultant residue treated with water then extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was subjected to reverse phase preparative HPLC (C-18, 10-90% MeCN in water 0.1% formic acid) to give the title compound as a white solid (38 mg, 38%). LC-MS: [M+H]+ = 398. 1H NMR (d6-DMSO) δ ppm 8.99 (1H, s), 8.11 (1H, s), 7.87 (1H, s), 7.86 (1H, s), 7.16 (1H, s), 6.89 (1H, s), 6.37 (1H, s), 5.89 (1H, sept, J = 6.6 Hz), 4.59-4.42 (4H, m), 1.54 (6H, s), 1.44 (6H, d, J = 6.6 Hz). 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-ylamine (27). A mixture of 16 and 3,4-methoxybenzylamine in NMP was heated at 85 °C for 18 h. To the reaction mixture was added water and the resultant precipitate filtered off then dried in vacuo to give a white solid. A solution of the white solid (378 mg, 0.82 mmol) and TFA (15 mL) was heated at 40 °C for 5 h. The reaction mixture was concentrated in vacuo and the residue loaded onto an SCX-2 cartridge, washing with methanol and eluting with 2 M ammonia in methanol. The basic fractions were combined and concentrated in vacuo. The resultant residue was subjected to flash chromatography (Si-PPC, gradient 0 to 10% methanol in DCM) to give the title compound as a white solid (140 mg, 55% total). LC-MS: [M+H]+ = 312. ¹H NMR (DMSO-d6) δ ppm 8.93 (1 H, s), 7.88 (1 H, s), 7.80 (1 H, s), 6.19 (2 H, br, s), 5.98 (1 H, s), 5.90-5.88 (1 H, m), 4.49-4.43 (4 H, m), 1.47 (6 H, d, J = 6.6 Hz). 2-{[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-yl]methyl-amino}-acetamide (28). To a solution of 16 (100 mg, 0.23 mmol) in NMP (1 mL) was added methylamino-acetic acid methyl ester (51 mg, 0.50 mmol). The resultant solution was stirred for 18 h at ACS Paragon Plus Environment

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85 °C before a further addition of methylamino-acetic acid methyl ester (51 mg, 0.50 mmol) was made and the reaction stirred for 8 h at 85 °C then at room temperature for 65 h. The reaction was quenched with water and the resultant precipitate collected by filtration. The residue was subjected to flash chromatography (Si-PPC, gradient 0-8% methanol in CH2Cl2) giving a 2:1 mixture of the title compound and starting material as a white solid (38 mg, 26%). A solution of this mixture (54 mg, 0.14 mmol), THF (2 mL) and 7 N ammonia in methanol (10 mL) was stirred at 40 °C for 19 h then at 50 °C for 18 h. The reaction mixture was cooled then concentrated in vacuo and the resultant residue was subjected to flash chromatography (Si-PPC, gradient 0-10% methanol in CH2Cl2) to give 28 as a white solid (16 mg, 30%). LC-MS: [M+H]+ = 383. ¹H NMR (DMSO-d6) δ ppm 9.06 (1 H, s), 7.88 (1 H, d, J = 0.6 Hz), 7.83 (1 H, s), 7.30 (1 H, s), 6.98 (1 H, s), 6.11 (1 H, s), 5.95 (1 H, h, J = 6.6 Hz), 4.54-4.50 (2 H, m), 4.50-4.46 (2 H, m), 4.13 (2 H, s), 3.05 (3 H, s), 1.47 (6 H, d, J = 6.6 Hz). General procedure B. A suspension of the pyridone 14 in DMF or NMP was treated with sodium hydride (1.2 eq.) and the reaction mixture stirred at room temperature or 40 °C for 15 min to 1.25 h before the addition of benzenebis(trifluoromethane) sulfonamide (1.2 eq.). Stirring was continued at room temperature until complete consumption of pyridone was seen (TLC or LC-MS) then the appropriate amine added (1 to 2.5 eq.) and the reaction mixture heated at 70 to 100 °C until no further reaction was seen. The crude products were isolated by removal of solvent in vacuo, precipitation from the reaction mixture by addition of water and extraction with ethyl acetate or CH2Cl2, or by using an Isolute SCX-2 cartridge. (S)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-yl]pyrrolidine-2-carboxylic acid amide (29). Following general procedure B using 14 and L-prolinamide then flash chromatography (Si-PPC, gradient 0 to 8% methanol in DCM) followed by recrystallization from methanol gave a white solid (115 mg, 44%). 98% ee (SFC, retention time = 1.48 min, opposite enantiomer at 0.48 min). LC-MS: [M+H]+ = 409. ¹H NMR (DMSO-d₆) δ ppm 9.06 (1 H, s), 7.88 (1 H, s), 7.83 (1 H, s), 7.33 (1 H, br), 6.92 (1 H, br), 5.97-5.96 (1 H, m), 5.94 (1 H, s), 4.53-4.45 (4 H, m),


33

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4.30 (1 H, d, J = 8.5 Hz), 3.59 (1 H, s), 3.37 (1 H, d, J = 9.9 Hz), 2.18 (1 H, m), 1.95 (3 H, m), 1.47 (6 H, dd, J = 6.6, 3.4 Hz). (R)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triazabenzo[e]azulen

8-yl]-

pyrrolidine-2-carboxylic acid amide (30). A solution of 15 (25.0 mg, 0.0756 mmol), D-prolinamide (57 mg, 0.50 mmol) and triethylamine (0.125 mL, 0.897 mmol) in NMP (1.36 mL, 14.1 mmol) was heated at 150 °C for 2 d. The reaction was filtered through Celite® rinsing with EtOAc. The filtrate was washed water followed by brine and the organic layer was dried (Na2SO4), then concentrated in vacuo to give 32. ee > 98% (SFC, retention time = 0.48 min, opposite enantiomer at 1.48 min). MS: (ESI+) 409.2 1

H NMR (500 MHz, DMSO) δ 9.10 – 9.03 (s, 1H), 7.91 – 7.86 (s, 1H), 7.86 – 7.82 (s, 1H), 7.41 – 7.31

(s, 1H), 6.99 – 6.91 (s, 1H), 6.03 – 5.90 (m, 2H), 4.57 – 4.43 (m, 4H), 4.37 – 4.24 (d, J = 9.0 Hz, 1H), 3.67 – 3.54 (t, J = 7.7 Hz, 1H), 3.45 – 3.37 (m, 1H), 2.25 – 2.11 (m, 1H), 2.03 – 1.88 (dddd, J = 13.3, 10.0, 7.0, 3.0 Hz, 3H), 1.52 – 1.43 (dd, J = 6.6, 4.5 Hz, 6H). 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-8-pyrrolidin-1-yl-4,5-dihydro-6-oxa-1,3a,9-triazabenzo[e]azulene (31). 31 was prepared similarly to 30. LC-MS: (ESI+) = 366.2. 1H NMR (DMSO) δ ppm 9.08 (1 H, s), 7.88 (1 H, s), 7.82 (1 H, s), 5.94 (1 H, dd, J = 13.6, 6.9 Hz), 4.61 – 4.32 (4 H, m), 3.40 (4 H, d, J = 6.3 Hz), 1.95 (4 H, t, J = 6.5 Hz), 1.48 (6 H, d, J = 6.6 Hz). (S)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a,9-triaza-benzo[e]azulen-8-yl]pyrrolidine-2-carboxylic acid methylamide (32). 32 was prepared similarly to 31. ee > 98% (SFC, retention time = 1.24 min, opposite enantiomer at 1.00 min). M/z 423.2, calc. 422.22. 1H NMR (DMSO) δ ppm 9.06 (1 H, s), 7.88 (1 H, s), 7.83 (1 H, s), 7.74 (1 H, d, J = 4.3 Hz), 5.95 (2 H, sept, J = 6.7 Hz), 4.58 – 4.41 (4 H, m), 4.35 (1 H, d, J = 8.5 Hz), 3.67 – 3.57 (1 H, m), 3.41 – 3.33 (1 H, m), 2.57 (3 H, d, J = 4.6 Hz), 2.15 (1 H, dd, J = 10.8, 7.7 Hz), 1.94 (3 H, d, J = 6.6 Hz), 1.47 (6 H, dd, J = 6.6, 2.4 Hz). 8-Bromo-2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulene (33) was prepared according to the procedures described in Ref 21.


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(S)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-yl]pyrrolidine-2-carboxylic acid amide (34). A mixture of 33 (375 mg, 1.0 mmol), methyl (2S)pyrrolidine-2-carboxylate (4.0 equiv., 4.0 mmol), cuprous iodide (0.20 equiv., 0.20 mmol) , N,N'dimethylethylenediamine (0.40 equiv., 0.40 mmol) and potassium phosphate tribasic (2.0 equiv., 2.0 mmol) in DMSO (6 mL) was heated at 90 °C for 16 h. The mixture was partitioned between 0.5% aqueous citric acid and ethyl acetate. The organic layer was extracted with 1 M aq. sodium carbonate and then discarded. The basic aqueous solution was neutralized by careful addition of 1 M aq. HCl and extracted with ethyl acetate. The organic extracts were washed with water, brine, dried over magnesium sulfate and concentrated in vacuum. The residue was used in the next step without further purification. Yield 140 mg. MS: 409 (M+1), 407 (M-1). HATU (1.20 equiv., 0.4114 mmol, 100 mass%) was added to a mixture of (2S)-1-[2-(2-isopropyl-1,2,4-triazol-3-yl)-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin9-yl]pyrrolidine-2-carboxamide (70 mg, 0.17 mmol), ammonium chloride (2.0 equiv., 0.69 mmol) and triethylamine (4.0 equiv., 1.37 mmol) in N,N-dimethylformamide (4 mL, 51.7 mmol). The mixture was stirred for 30 min and then concentrated in vacuo. The residue was partitioned between ethyl acetate and water and the organic extracts were washed with sat. aq. sodium bicarbonate, 0.5% aq. citric acid, water, brine, then dried over MgSO4 and concentrated. The residue was purified by reverse phase chromatography (acetonitrile gradient) yielding 70 mg (50%) of a white powder. >98% ee (SFC, retention time = 2.69 min, opposite enantiomer at 2.08 min). LC-MS: [M+H]+ = 408.2. NMR (DMSOd6) δ ppm 8.19 (1 H, d, J = 8.9 Hz), 7.87 (1 H, s), 7.77 (1 H, s), 7.40 (1 H, s), 7.04 (1 H, s), 6.36 ( 1 H, dd, J = 8.9, 2.5 Hz), 6.06 (1 H, d, J = 2.4 Hz), 5.92 (1 H, p, J = 6.6 Hz), 4.51-4.37 (4 H, m), 4.05-3.93 (1 H, m), 3.57 (1 H, t, J = 7.9 Hz), 2.22 (1 H, dd, J = 13.7, 6.3 Hz), 1.97 (3 H, tt, J = 9.4, 5.8 Hz), 1.47 (6 H, dd, J = 6.6, 3.3 Hz). (S)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8yl]piperdine-2-carboxylic acid amide (35). A mixture of 33 (1.01 g, 2.7 mmol), methyl piccolinate (3.86 g, 27.0 mmol), copper (I) iodide (0.2 g, 1.08 mmol), 2-(S)-piperidinecarboxylic acid (0.14 g, 1.08 mmol) and potassium phosphate (1.14 g, 5.4 mmol) in DMSO (10 mL) were heated at 80 °C for 24 h. 35 ACS Paragon Plus Environment

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The cooled reaction mixture was loaded onto an Isolute SCX-2 cartridge, washed with methanol and eluted with 2 M ammonia in methanol. The resultant residue was triturated with diethyl ether to give 1[2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-yl]-piperidine-2(S)-carboxylic acid (1.04 g, 87 % total). LC-MS: [M+H]+ = 423. A mixture of the intermediate acid (1.15 g, 2.73 mmol), EDCI (0.58 g, 3.0 mmol), HOBt (0.41 g, 3.0 mmol) and DIPEA (2.34 mL, 13.65 mmol) in DMF was stirred at room temperature (2 mL) for 5 min before ammonium chloride (0.44 g, 8.18 mmol) was added and the reaction mixture stirred for 18 h. The reaction mixture was concentrated in vacuo and the resultant residue triturated with water before being subjected to flash chromatography (Si-PPC, gradient 0 to 10% methanol in DCM) to give the title compound as a white solid (0.08 g, 6%). ee > 98% (SFC, retention time = 1.21 min, opposite enantiomer at 0.86 min). LC-MS: [M+H]+ = 422. ¹H NMR (DMSO-d6) δ ppm 8.18 (1 H, d, J = 9.06 Hz), 7.89 (1 H, d, J = 0.63 Hz), 7.80 (1 H, s), 7.31 (1 H, br, s), 7.01 (1 H, br, s), 6.72 (1 H, dd, J = 9.16, 2.60 Hz), 6.40 (1 H, d, J = 2.54 Hz), 5.92-5.91 (1 H, m), 4.44-4.43 (4 H, m), 4.39 (1 H, m), 3.60 (1 H, d, J = 12.44 Hz), 3.39-3.32 (1 H, m), 2.06 (1 H, d, J = 13.57 Hz), 1.75 (2 H, m), 1.58 (3 H, m), 1.48 (6 H, dd, J = 6.60, 2.59 Hz). (S)-1-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-yl]azetidine-2-carboxylic acid amide (36). Following the procedure for 35 using 33 and (L)-azetidine-2carboxylic acid gave the title compound.

ee > 98% (SFC, retention time = 1.88 min, opposite

enantiomer at 0.77 min). LC-MS: [M+H]+ = 394. 1H NMR (DMSO-d6) δ ppm 8.22 (1 H, d, J = 8.7 Hz), 7.88 (1 H, d, J = 0.6 Hz), 7.80 (1 H, s), 7.53 (1 H, s), 7.24 (1 H, s), 6.27 (1 H, dd, J = 8.8, 2.4 Hz), 5.99 (1 H, d, J = 2.3 Hz), 5.91-5.90 (1 H, m), 4.47-4.43 (4 H, m), 4.30 (1 H, dd, J = 8.8, 6.9 Hz), 3.93-3.92 (1 H, m), 3.71 (1 H, q, J = 7.8 Hz), 2.54-2.46 (1 H, m), 2.34-2.27 (1 H, m), 1.48 (3 H, d, J = 3.3 Hz), 1.46 (3 H, d, J = 3.3 Hz). 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-ol (37).

A

mixture of 33 (9.5 g, 25.4 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (855 mg, 2.0 mmol), tris(dibenzylideneacetone)dipalladium (0) (475 mg, 0.5 mmol) and potassium hydroxide (4.2 g, 76.2 mmol) were suspended in dioxane (29 mL) and water (15 mL). The suspension was degassed with ACS Paragon Plus Environment

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nitrogen and the reaction mixture heated at 90 °C for 1 h. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (20 mL). The aqueous fraction was acidified to pH~5 by addition of 1M hydrochloric acid causing a precipitate to form. The suspension was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were dried (MgSO4), concentrated in vacuo. The resultant residue was triturated with diethyl ether to give an orange solid (3.6 g, 46%). 1H NMR (DMSO-d6) δ ppm 9.90 (1 H, br s), 8.22 (1 H, d, J = 8.9 Hz), 7.88 (1 H, s), 7.82 (1 H, s), 6.61 (1 H, dd, J = 8.9, 2.6 Hz), 6.41 (1 H, d, J = 2.6 Hz), 5.90 (1 H, sept, J = 6.5 Hz), 4.49-4.39 (4 H, m), 1.47 (6 H, d, J = 6.5 Hz). 2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-yloxy]acetamide (38). To a solution of 37 (80 mg, 0.26 mmol) in DMF (3 mL) was added 2-bromoacetamide (53 mg, 0.39 mmol) and cesium carbonate (109 mg, 0.33 mmol). The reaction mixture stirred at room temperature for 16 h before being diluted with water. The mixture was extracted with ethyl acetate (3 × 10 mL) and the combined organic extracts washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was subjected to flash chromatography (Si-PPC, gradient 0-10% methanol in DCM) to give the title compound as a white solid (61 mg, 64%). LC-MS: [M+H]+ = 369. 1H NMR (d6-DMSO) δ ppm 8.28 (1 H, d, J = 9.8 Hz), 7.85 (1 H, s), 7.82 (1 H, s), 7.51 (1 H, s), 7.35 (1 H, s), 6.76 (1 H, dd, J = 9.8, 2.7 Hz), 6.54 (1 H, d, J = 2.7 Hz), 5.85 (1 H, sept, J = 6.6 Hz), 4.49-4.39 (6 H, m), 1.43 (6 H, d, J = 6.6Hz). (S)-2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8yloxy]-propionamide (4). Following general procedure A using 37 and (+)-tert-butyl D-lactate, then flash chromatography (Si-PPC, gradient 0-7% methanol in CH2Cl2) gave a yellow solid (70 mg, 62%). LC-MS: [M+H]+ = 440. To a solution of the solid (64 mg, 0.15 mmol) in CH2Cl2 (10 mL) was added TFA (0.5 mL) and the reaction mixture stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo and the residue dissolved in DMF (2 mL). HATU (130 mg, 0.34 mmol), ammonium chloride (27 mg, 0.51 mmol) and triethylamine (71µL, 0.51 mmol) were added and the reaction mixture stirred at room temperature for 1 hour. The reaction mixture was concentrated in 37 ACS Paragon Plus Environment

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vacuo, the residue dissolved in ethyl acetate (10 mL) and the mixture washed with water extracting with ethyl acetate (3 × 10 mL). The combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo. The resultant residue was subjected to flash chromatography (Si-PPC gradient 010% methanol in ethyl acetate) to give the title compound as a white solid (50 mg, 90%). LC-MS: [M+H]+ = 383. ee 97% (SFC, retention time = 0.94 min, opposite enantiomer at 0.52 min). 1H NMR (d6-DMSO) δ ppm 8.26 (1 H, d, J = 8.6 Hz), 7.81 (1 H, s), 7.85 (1 H, s), 7.51 (1 H, s), 7.21 (1 H, s), 6.72 (1 H, dd , J = 8.6, 2.6 Hz), 6.49 (1 H, d, J = 2.6 Hz), 5.85 (1 H, sept, J = 6.6 Hz), 4.62 (1 H, q, J = 6.6 Hz), 4.55-4.36 (4 H, m), 1.47-1.36 (9 H, m);

13

C NMR (d6-DMSO) δ ppm 173.5, 159.2, 156.9, 150.6,

147.6, 144.6, 131.2, 130.5, 124.0, 111.4, 111.2, 106.0, 74.0, 69.0, 50.7, 50.0, 22.8, 19.1. (S)-2-((2-(1-isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9yl)oxy)butanamide (39). Following the procedure for 4 above using 37 and (R)-2-hydroxy-butyric acid tert-butyl ester gave the title compound.

ee > 98% (SFC, retention time = 0.87 min, opposite

enantiomer at 0.46 min). LC-MS: [M+H]+ = 397. 1H NMR (d6-DMSO) δ ppm 8.26 (1 H, d, J = 8.6 Hz), 7.93 (1 H, s), 7.88 (1 H, s), 7.54 (1 H, s), 7.28 (1 H, s), 6.77 (1 H, dd , J = 8.6, 2.6 Hz), 6.56 (1 H, d, J = 2.6 Hz), 5.88 (1 H, sept, J = 6.6 Hz), 4.48 (5 H, m), 1.83 (2 H, m), 1.48 (6 H, dd, J = 6.60, 2.59 Hz), 0.97 (3 H, t, J = 7.7 Hz). (R)-2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8yloxy]-propionamide (40).

Following general procedure A 37 and ethyl-(L)-lactate, then flash

chromatography (Si-PPC, gradient 0-10% methanol in ethyl acetate) gave a yellow solid (315 mg, 66%). LC-MS: [M+H]+ = 412. To a solution of the yellow solid (315 mg, 0.77 mmol) in methanol (10 mL) was added water (1 mL) and lithium hydroxide monohydrate (46 mg, 1.15 mmol). The reaction mixture was heated at 50 °C for 16 h then HCl (1N, aq.) added until pH~4. The reaction mixture was concentrated in vacuo. The resultant residue was dissolved in DMF (5 mL) and HATU (583 mg, 1.53 mmol), ammonium chloride (122 mg, 2.29 mmol) and triethylamine (320 µL, 2.29 mmol) added. The reaction mixture was stirred at room temperature for 1 h before being concentrated in vacuo. The residue was dissolved in ethyl acetate (10 mL) and the mixture washed with water, extracting with ethyl acetate 38 ACS Paragon Plus Environment


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(3 × 10 mL). The combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo. The resultant residue was subjected to flash chromatography (Si-PPC, gradient 0-10% methanol in ethyl acetate) and the product was crystallised from acetonitrile to give the title compound as a white solid (75 mg, 26%). LC-MS: [M+H]+ = 383. ee 97% (SFC, retention time = 0.52 min, opposite enantiomer at 0.94 min). 1H NMR (d6-DMSO) δ ppm 8.26 (1 H, d, J = 8.6 Hz), 7.85 (1 H, s), 7.81 (1 H, s), 7.50 (1 H, s), 7.21 (1 H, s), 6.71 (1 H, dd , J = 8.6, 2.6 Hz), 6.49 (1 H, d, J = 2.6 Hz), 5.85 (1 H, sept, J = 6.6 Hz), 4.63 (1H, q, J = 6.5 Hz), 4.44 (4 H, s), 1.83 (2 H, m), 1.44-1.40 (9 H, m). 4-Benzyloxy-2-fluoro-benzonitrile (42). A solution of 4-hydroxy-2-fluoro-benzonitrile 41 (80 g, 0.58 mol), potassium carbonate (162 g, 1.17 mol) and benzyl bromide (76.4 mL, 0.64 mol) and potassium iodide (9.6 g, 0.058 mol) in acetone (600 mL) was stirred at room temperature and a significant exothermic reaction was observed. Stirring continued for 18 h without cooling. The reaction mixture was diluted with water (600 mL) and extracted with EtOAc (500 mL x 2). The combined organic extracts were washed with brine (500 mL), dried (Na2SO4), filtered and concentrated in vacuo. The resultant residue was triturated in cyclohexane (300 mL) and the crystalline solid was filtered off and washed to give the title compound (118.2 g, 90%). 1H NMR (CDCl3) δ ppm 7.51 (1 H, dd, J = 8.70, 7.48 Hz), 7.40 (5 H, m), 6.86-6.76 (2 H, m), 5.11 (2 H, s). 4-Benzyloxy-2-fluoro-benzamidine hydrochloride (43). A solution of 42 (84 g, 0.37 mol) in THF (450 mL) under an atmosphere of nitrogen, was cooled to -70 °C. The resultant suspension was treated with a solution of 1M LiHMDS in THF (440 mL, 0.44 mol) over 10 min to reach a maximum temperature of -55 °C. The reaction mixture was allowed to warm to room temperature and stirred for 3 days. The reaction mixture was poured onto ice/1M HCl mixture, the pH adjusted to ~1 by addition of 6M HCl and washed with EtOAc (500 mL). The organic layer was extracted with 1M HCl and the combined aqueous extracts washed with EtOAc (500 mL). The acid aqueous extracts were concentrated to low volume in vacuo and the resultant solid filtered off, washed with H2O and dried in vacuo to give the title compound as a pale cream crystalline solid (74.5g, 72%). 1H NMR (d6-DMSO) δ ppm 9.30 (4 H, d, J = 10.39 Hz), 7.64 (1 H, t, J = 8.59 Hz), 7.49-7.35 (5 H, m), 7.19 (1 H, dd, J = 12.88, 2.41 Hz), ACS Paragon Plus Environment

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7.06 (1 H, dd, J = 8.77, 2.41 Hz), 5.25 (2 H, s). 5-[2-(4-Benzyloxy-2-fluoro-phenyl)-1H-imidazol-4-yl]-1-isopropyl-1H-[1,2,4]triazole

A

(44).

solution of 43 (74.5 g, 0.265 mol) in THF (705 mL) was treated with potassium hydrogen carbonate (106 g, 1.06 mol) and water (150 mL). The resultant mixture was heated to reflux to give a white suspension. Whilst maintaining gentle reflux a solution of 19 (50 g, 0.265 mol) was added dropwise over 40 min. The resultant suspension gradually dissolved giving a dark red mixture that was refluxed for 18 h. The mixture was cooled to room temperature, diluted with saturated aqueous NaCl solution (500 mL) and extracted with EtOAc (500 mL). The organic extract was washed with saturated aqueous NaCl solution, dried (Na2SO4), filtered and concentrated in vacuo to give a pink solid. The solid was triturated in a mixture of MTBE/pentane (1:1 by volume, 300 mL), collected by filtration, washed with a MTBE/pentane mixture and dried in vacuo to give the title compound as a pale pink solid (84 g, 84%). 1

H NMR (d6-DMSO) δ ppm 7.95-7.93 (2 H, m), 7.76 (1 H, s), 7.44-7.42 (5 H, m), 7.12 (1 H, dd, J =

13.1, 2.5 Hz), 7.03 (1 H, dd, J = 8.7, 2.47 Hz), 5.90 (1 H, m), 5.21 (2 H, s), 1.46 (6 H, d, J = 6.6 Hz). 2-[2-(4-Benzyloxy-2-fluoro-phenyl)-4-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-imidazol-1-yl]-ethanol 45). A suspension of 44 (48 g, 0.55 mol) and 1,3-dioxolan-2-one (30 mL) in toluene (100 mL) was heated at 130 °C for 7.5 h allowing some solvent to evaporate until reaction was initiated. The cooled reaction mixture was concentrated in vacuo and the residue treated with acetonitrile (50 mL), stirred, sonicated and cooled in an ice bath. The resultant solid was collected by filtration, washed with cold acetonitrile then diethyl ether and dried in vacuo at 60 °C to give the title compound (65.2 g, 70%). 1H NMR (CDCl3) δ ppm 8.04 (1 H, s), 7.80 (1 H, d, J = 0.7 Hz), 7.43-7.41 (6 H, m), 6.89 (1 H, dd, J = 8.6, 2.5 Hz), 6.79 (1 H, dd, J = 11.7, 2.5 Hz), 5.93-5.92 (1 H, m), 5.11 (2 H, s), 4.02 (2 H, t, J = 4.9 Hz), 3.89 (2 H, t, J = 5.0 Hz), 1.49 (6 H, d, J = 6.6 Hz). LC-MS: [M+H]+ = 422. 8-Benzyloxy-2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulene (46). A solution of 45 (25.0 g, 59.3 mmol) in DMF (890 mL) was treated with sodium hydride (3.51 g, 94.89 mmol) portionwise. The resultant mixture was stirred for 23 h at room temperature and then quenched with ice and water. The precipitate was collected by filtration, washed with water and dried in ACS Paragon Plus Environment

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vacuo at 60 °C to give the title compound as an off-white solid (13.9 g, 58%). 1H NMR (CDCl3) δ ppm 8.43 (1 H, d, J = 9.0 Hz), 7.87 (1 H, s), 7.63 (1 H, s), 7.41-7.39 (5 H, m), 6.82 (1 H, dd, J = 9.0, 2.6 Hz), 6.64 (1 H, d, J = 2.6 Hz), 6.00 (1 H, m), 5.10 (2 H, s), 4.50-4.48 (2 H, m), 4.42-4.41 (2 H, m), 1.59 (6 H, d, J = 6.6 Hz). LC-MS: [M+H]+ = 402. 2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-ol (37). A solution of 46 in a mixture of EtOAc (350 mL) and IMS (150 mL), under argon atmosphere, was treated with Pd/C. The atmosphere was exchanged with hydrogen and the resultant reaction mixture was stirred at room temperature for 20 h. The hydrogen atmosphere was exchanged with argon, the reaction mixture was diluted with CH2Cl2 (~ 60 mL), filtered through a pad of Celite and washed with a mixture of DCM/IMS (9:1 by volume). The resultant solution was concentrated in vacuo to give the title compound as an off-white solid (9.42 g, 51%). More material was recovered by thorough washing of the Celite residue to provide the title compound in 83% total yield. 1H NMR (d6-DMSO) δ ppm 8.22 (1 H, d, J = 8.8 Hz), 7.89 (1 H, s), 7.82 (1 H, s), 6.61 (1 H, dd, J = 8.8, 2.4 Hz), 6.41 (1 H, d, J = 2.4 Hz), 5.95-5.86 (1 H, m), 4.45 (4 H, m), 1.47 (6 H, d, J = 6.6 Hz). LC-MS:[M+H]+ = 312. (S)-2-[2-(2-isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8-yloxy]propionic acid methyl ester (47). A suspension of 37 (6.3 g, 20.23 mmol), (R)-2-hydroxy-propionic acid methyl ester (2.51 mL, 26.3 mmol) and triphenylphosphine (7.96 g, 30.35 mmol) in dioxane (190 mL) was cooled with a water bath and then treated with diisopropylazodicarboxylate (5.98 mL, 30.35 mmol) dropwise and then stirred at room temperature for 2 hours. The resultant mixture was concentrated in vacuo, the residue was dry loaded onto silica (~120 g) and subjected to flash chromatography (Si-PPC, gradient 0-10% MeOH in MTBE) to give the title compound as an off-white foam (7.0 g, 87%). 1H NMR (d6-DMSO) δ ppm 8.31 (1 H, d, J = 9.0 Hz), 7.89 (2 H, m), 6.75 (1 H, dd, J = 9.0, 2.6 Hz), 6.51 (1 H, d, J = 2.6 Hz), 5.92-5.85 (1 H, m), 5.07 (1 H, m), 4.49 (4 H, m), 3.70 (3 H, s), 1.52 (3 H, d, J = 6.7 Hz), 1.47 (6 H, d, J = 6.6 Hz). LC-MS: [M+H]+ = 398. (S)-2-[2-(2-Isopropyl-2H-[1,2,4]triazol-3-yl)-4,5-dihydro-6-oxa-1,3a-diaza-benzo[e]azulen-8yloxy]-propionamide (4). A solution of 47 (8.84 g, 22.24 mmol) in 7 N NH3 in MeOH (150 mL) was 41 ACS Paragon Plus Environment

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stirred at room temperature for 7 h. The resultant white suspension was concentrated in vacuo and the residue triturated in diethyl ether. The solid was collected by filtration and dried in vacuo to give the title compound as an off-white solid (8.37 g, 98%). Characterization of Biochemical and Cellular Activity in vitro. Enzymatic activity of the Class I PI3K isoforms was measured using a fluorescence polarization assay that monitors formation of the product 3,4,5-inositoltriphosphate molecule (PIP3) as it competes with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidyl inositide-3phosphate product results in a decrease in fluorescence polarization signal as the labeled fluorophore is displaced from the GRP-1 protein binding site. Class I PI3K isoforms were purchased from Perkin Elmer or were expressed and purified as heterodimeric recombinant proteins. Tetramethylrhodaminelabeled PIP3 (TAMRA-PIP3), di-C8-PIP2, and PIP3 detection reagents were purchased from Echelon Biosciences. PI3K isoforms were assayed under initial rate conditions in the presence of 10 mM Tris (pH 7.5), 25 µM ATP, 9.75 µM PIP2, 5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 2% (v/v) DMSO at the following concentrations for each isoform: PI3Kα,β at 60 ng/mL; PI3Kγ at 8 ng/mL; PI3Kδ at 45 ng/mL. After assay for 30 minutes at 25 °C, reactions were terminated with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and 4.2 µg/mL GRP-1 detector protein before reading fluorescence polarization on an Envision plate reader. Apparent Ki values were determined at a fixed concentration of ATP near the measured Km for ATP for each isoenzyme, and were calculated by fitting of the dose-response curves to an equation for tight-binding competitive inhibition. All apparent Ki values represent geometric means of minimum of three determinations. These assays generally produced results within 2-fold of the reported mean. Anti-proliferative cellular assays were conducted using PC3, KPL-4, and MCF7-neo/HER2 human tumor cell lines. PC3 and KPL-4 cells were provided by ATCC and MCF7-neo/HER2 cells were provided by Genentech Research laboratories. MCF7-neo/HER2 is an in vivo selected line developed at Genentech and originally derived from the parental human MCF7 breast cancer cell line (ATCC,


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Manassas, VA). Cell lines were cultured in RPMI supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin, 10 mM HEPES and 2 mM Glutamine at 37°C under 5% CO2. MCF7-neo/HER2 and KPL-4 cells were seeded in 384-well plates in media at 1000 cells/well while PC3 cells were seeded in 384-well plates in media at 3000 cells/well, and incubated overnight prior to the addition of compounds to a final DMSO concentration of 0.5% v/v. MCF7-neo/HER2 cells were incubated for 3 and KPL-4 and and PC3 cells were incubated for 4 days, prior to the addition of CellTiter-Glo® reagent (Promega) and reading of luminescence using an Analyst plate reader. For antiproliferative assays, a cytostatic agent such as aphidicolin and a cytotoxic agent such as staurosporine were included as controls. Dose-response curves were fit to a 4-parameter equation and relative EC50’s were calculated using Assay Explorer software. All cellular EC50 values represent geometric means of a minimum of two determinations and these assays generally produced results within 3-fold of the reported mean. Plasma Protein Binding. The extent of protein binding was determined in vitro, in CD-1 mouse, Sprague-Dawley rat, cynomolgus monkey, beagle dog and human plasma (Bioreclamation, Inc., Hicksville, NY) by equilibrium dialysis using the RED Device (Thermo Fisher Scientific, Rockford, IL). Compounds were added to pooled plasma (n ≥ 3) at a total concentration of 5 µM. Plasma samples were equilibrated with phosphate-buffered saline (pH 7.4) at 37°C in 90% humidity and 5% CO2 for 4 hours. Following dialysis, compound concentration in plasma and buffer was measured by LC-MS/MS. The percent unbound compound in plasma was determined by dividing the concentration measured in the post-dialysis buffer by that measured in the post-dialysis plasma and multiplying by 100. Metabolic Stability in Liver Microsomes. The oxidative metabolism of compounds was evaluated in pooled liver microsomes (Corning, Tewksbury, MA) from CD-1 mice, Sprague-Dawley rats, cynomolgus monkeys, beagle dogs, and humans. The incubation mixture was prepared for each species in 0.1 M-potassium phosphate buffer (pH 7.4) containing 0.5 mg/mL microsomal protein, 1 mM NADPH, and 1 µM of compound. Reactions were initiated with the addition of NADPH. Samples were incubated at 37°C and aliquots were sampled at 0, 20, 40 and 60 min. Reactions were quenched with 43 ACS Paragon Plus Environment

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acetonitrile containing 0.1% formic acid and internal standard at each timepoint. Samples were centrifuged at 3000 g for 10 min. The supernatant was diluted with water (1:2 ratio) and the percentage of compound remaining was determined by LC–MS/ MS using the t = 0 peak area ratio values as 100%.35 The in vitro Clint and scaled hepatic Cl were determined as described by Obach.29 ACKNOWLEDGMENT The authors wish to thank our analytical chemistry group for compound purification and determination of purity and ee by HPLC/SFC, mass spectroscopy, and 1H NMR. We thank Krista K. Bowman, Alberto Estevez, Kyle Mortara, and Jiansheng Wu for technical assistance of protein expression and purification. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland for provision of synchrotron radiation beamtime at beamline X06SA of the SLS and would like to thank Meitian Wang and Tomizaki Takashi.

Supporting Information Available. A kinase selectivity panel for 4, a figure explaining the Link and Grow strategies used to design PI3Kα specific inhibitors, crystal structure metrics as well as a depiction of a crystal structure of a benzoxepin bound to PI3Kγ (PDB 3R7R) where residues are labeled to highlight differences between PI3Kα and the other isoforms within the active site are available as supporting information.

PDB ID Codes. The coordinates of 5 in PI3Kα have been deposited with PDB Code 5DXH. The coordinates of 4 in PI3Kα and PI3Kδ have been deposited with PDB Codes 5DXT and 5DXU respectively.

Corresponding Author. *Email: [email protected], Phone: (650) 467-3214. Fax: (650) 225-2061.

Abbreviations used. PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; HATU,

N,N,N',N'-tetramethyl-O-(7-Azabenzotiazol-1-yl)uronium

hexafluorophosphate;

NIS,

N-

iodosuccinimide; DIAD, diisopropylazodicarboxylate; Si-PPC pre-packed silica cartridge; TGI, tumor ACS Paragon Plus Environment

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growth inhibition; PR, partial regression; CR, complete regression; IMS, industrial methylated spirits; ABT, 1-aminobenzotrizole.

1

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Barry, S. T. Inhibition of PI3Kβ signaling with AZD8186 inhibits growth of PTEN-deficient breast and prostate tumors alone and in combination with docetaxel. Mol. Cancer Ther. 2015, 14, 48-58. (b) Barlaam, B.; Cosulich, S.; Degorce, S.; Fitzek, M.; Green, S.; Hancox, U.; Lambert-van der Brempt, C.; Lohmann, J.-J.; Maudet, M.; Morgentin, R.; Pasquet, M.-J.; Péru, A.; Plé, P.; Saleh, T.; Vautier, M.; Walker, M.; Ward, L.; Warin, N. Discovery of (R)-8-(1-(3,5-Difluorophenylamino)ethyl)-N,N-dimethyl2-mopholino-4-oxo-4H-chromene-6-carboxamide (AZD8186): A Potent and Selective Inhibitor of PI3Kβ and PI3Kδ for the Treatment of PTEN-Deficient Cancers. J. Med. Chem. 2015, 58, 943-962. (c) Bedard, P. L.; Davies, M. A.; Kopetz, S.; Flaherty, K. T.; Shapiro, G.; Luke, J. J.; Spreafico, A.; Wu, B.; Gomez, C.; Cartot-Cotton, S.; Mazuir, F.; Micallef, S.; Demers, B.; Juric, D. First-in-human trial of the PI3Kβ-selective inhibitor SAR260301 in patients with advanced solid tumors (NCT01673737). J. Clin. Oncol. 2015, 33, suppl; abstr 2564. (d) Arkenau, H.-T.; Mateo, J.; Lemech, C. R.; Infante, J. R.; Burris, H. A.; Bang, Y.-J.; Eder, J. P.; Herbst, R. S.; Sharma, S.; Motwani, M.; Kumar, R.; De Bono, J. S. A phase I/II, first-in-human dose-escalation study of GSK2636771 in patients (pts) with PTEN-deficient advanced tumors. J. Clin. Oncol. 2014, 32, suppl; abstr 2514. 5

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Inflammatory Responses. Biochem. Biophys. Res. Commun. 2003, 308, 764-769.

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Masinovsky, B.; Dick, K.; Sowell, C. G.; Staunton, D. E. Essential Role of Phosphoinositide 3-Kinase δ in Neutrophil Directional Movement. J. Immunol. 2003, 170, 2647-2654. (c) Fruman, D. A. Phosphoinositide 3-Kinase and its Targets in B-cell and T-cell Signaling. Curr. Opin. Immunol. 2004, 16, 314-320. (d) Okkenhaug, K.; Vanhaesbroeck, B. PI3K in Lymphocyte Development, Differentiation, and Activation. Nat. Rev. Immun. 2003, 3, 317-330. (e) Rommel, C.; Camps, M.; Ji, H. PI3Kδ and PI3Kγ: Partners in Crime in Inflammation in Rheumatoid Arthritis and Beyond? Nat. Rev. Immunol. 2007, 7, 191-201. (f) Ameriks, M. K.; Venable, J. D. Small Molecule Inhibitors of Phosphoinositide 3Kinase (PI3K) δ and γ. Curr. Top.Med. Chem. 2009, 9, 738-753. ACS Paragon Plus Environment

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(a) Ruckle, T.; Schwarz, M. K.; Rommel, C. PI3Kγ Inhibition: Towards an Aspirin for the 21st

Century? Nat. Rev. Drug Disc. 2006, 5, 903-918. (b) Barbier, D. F; Barolome, A.; Hernandez, C.; Flores, J. M.; Redondo, C.; Fernandez-Arias, C.; Camps, M.; Ruckle, T.; Schwarz, M. K.; Rodriguez, S.; Martinez, C.; Balomenos, D.; Rommel, C.; Carrera, A. C. PI3Kγ Inhibition Blocks Glomerulonephritis and Extends Lifespan in a Mouse Model of Systemic Lupus. Nat. Med. 2005, 11, 933-935. (c) Wymann, M. P.; Bjorklof, K.; Calvez, R.; Finan, P.; Thomas, M.; Trifilieff, A.; Barbier, M.; Altruda, F.; Hirsch, E.; Laffargue, M. Phosphoinositide 3-Kinase γ: A Key Modulator in Inflammation and Allergy. Biochem. Soc. Trans. 2003, 31, 275-280. (d) Hirsch, E.; Katanaev, V. L.; Garlanda, C.; Azzolino, O.; Pirola, L.; Silengo, L.; Sozzani, S.; Mantovani, A.; Altruda, F.; Wymann, M. P. Central role for G protein-coupled phosphoinositide 3-kinase γ in inflammation. Science, 2000, 287, 1049-1053. (e) Camps, M.; Rückle, T.; Ji, H.; Ardissone, V.; Rintelen, F.; Shaw, J.; Ferrandi, C.; Chabert, C.; Gillieron, C.; Francon, B.; Martin, T.; Gretener, D.; Perrin, D.; Leroy, D.; Vitte, P.-A.; Hirsch, E.; Wymann, M. P.; Cirillo, R.; Schwarz, M. K.; Rommel, C. Blockade of PI3Kγ Suppresses Joint Inflammation and Damage in Mouse Models of Rheumatoid Arthritis. Nat. Med. 2005, 11, 936943. (f) Pomel, V. Klicic, J.; Covini, D.; Church, D. D.; Shaw, J. P.; Roulin, K.; Burgat-Charvillon, F.; Valognes, D.; Camps, M.; Chabert, C.; Gillieron, C.; Francon, B.; Perrin, D.; Leroy, D.; Gretener, D.; Nichols, A.; Vitte, P. A.; Carboni, S.; Rommel, C.; Schwarz, M. K.; Rückle, T. Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase γ. J. Med. Chem. 2006, 49, 3857-3871. (g) Pereira, A. R.; Strangman, W. K.; Marion, F.; Feldberg, L.; Roll, D.; Mallon, R.; Hollander, I.; Andersen, R. J. Synthesis of Phosphatidylinositol 3-Kinase (PI3K) Inhibitory Analogues of the Sponge Meroterpenoid Liphagal. J. Med. Chem. 2010, 53, 8523-8533. (h) Leahy, J. W.; Buhr, C. A.; Johnson, H. W.; Kim, B. G.; Baik, T.; Cannoy, J.; Forsyth, T. P.; Jeong, J. W.; Lee, M. S.; Ma, S.; Noson, K.; Wang, L.; Williams, M.; Nuss, J. M.; Brooks, E.; Foster, P.; Goon, L.; Heald, N.; Holst, C.; Jaeger, C.; Lam, S.; Lougheed, J.; Nguyen, L.; Plonowski, A.; Song, J.; Stout, T.; Wu, X.; Yakes, M. F.; Yu, P.; Zhang, W.; Lamb, P.; Raeber, O. Discovery of a Novel Series of Potent and Orally 47 ACS Paragon Plus Environment

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Bioavailable Phosphoinositide 3-Kinase γ Inhibitors. J. Med. Chem. 2012 55, 5467-5482. (i) Collier, P. N.; Martinez-Botella, G.; Cornebise, M.; Cottrell, K. M.; Doran, J. D.; Griffith, J. P.; Mahajan, S.; Maltais, F.; Moody, C. S.; Huck, E. P.; Wang, T.; Aronov, A. M. Structural Basis for Isoform Selectivity in a Class of Benzothiazole Inhibitors of Phosphoinositide 3-Kinase γ. J. Med. Chem. 2015, 58, 517-521. (i) Discovery and Optimization of a Series of 2-aminothiazole-oxazoles as Potent Phosphoinositide 3-kinase γ Inhibitors. Oka, Y.; Yabuuchi, T.; Fujii, Y.; Ohtake, H.; Wakahara, S.; Matsumoto, K.; Endo, M.; Tamura, Y.; Sekiguchi, Y. Bioorg. Med. Chem. Lett. 2012, 22, 7534-7538. 7

(a) Fruman, D. A.; Rommel, C. ΠΙ3Κδelta Inhibitors in Cancer: Rationale and Serendipity Merge in

the Clinic. Cancer Discov. 2011, 7, 562-572. (b) Lannutti, B. J.; Meadows, S. A.; Herman, S. E. M.; Kashishian, A.; Steiner, B.; Johnson, A. J.; Byrd, J. C.; Tyner, J. W.; Loriaux, M. M.; Deininger, M.; Druker, B. J.; Puri, K. J.; Ulrich, R. G.; Giese, N. A. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability. Blood 2011, 117, 591-594. (c) Subramaniam, P. S.; Whye, D. W.; Efimenko, E.; Chen, J.; Tosello, V.; Keersmaeker, K. D.; Kashishian, A.; Thompson, M. A.; Castillo, M.; CordonCardo, C.; Davé, U. P.; Ferrando, A.; Lannutti, B. J.; Diacovo, T. G. Targeting Nonclassical Oncogenes for Therapy in T-ALL. Cancer Cell 2012, 21, 459-472. 8

Schmid, M. C.; Avraamides, C. J.; Dippold, H. C.; Franco, I.; Foubert, P.; Ellies, L. G.; Acevedo, L.

M.; Manglicmot, J. R.; Song, X.; Wrasidlo, W.; Blair, S. L.; Ginsber, M. H.; Cheresh, D. A.; Hirsch, E.; Field, S. J.; Varner, J. A. Receptor Tyrosine Kinases and TLR/IL1Rs Unexpectedly Activate Myeloid Cell PI3Kγ, A Single Convergent Point Promoting Tumor Inflammation and Progression. Cancer Cell 2011, 19, 715-727. 9

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm406387.htm


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Reviews of Class I PI3K inhibitors under clinical evaluation: (a) Carnero, A. Novel Inhibitors of the

PI3K Family. Expert Opin. Invest. Drugs 2009, 18, 1265-1277. (b) Courtney, K. D.; Corcoran, R. B.; Engelman, J. A. The PI3K Pathway as a Drug Target in Human Cancer. J. Clin. Oncol. 2010, 28, 1075. (c) Hixon, M. L.; Paccagnella, L.; Millham, R.; Perez-Olle, R.; Gualberto, A. Development of Inhibitors of the IGF-1R/PI3K/Akt/mTOR Pathway. Rev. Recent Clin. Trials 2010, 5, 189-208. (d) Shuttleworth, S. J.; Silva, F. A.; Cecil, A. R.; Tomassi, C. D.; Hill, T. J.; Raynaud, F. I.; Clarke, P. A.; Workman, P. Progress in the preclinical discovery and clinical development of class I and dual class I/IV phosphoinositide 3-kinase (PI3K) inhibitors. Curr. Med. Chem. 2011, 18, 2686-2714. (e) Denny, W. A. Phosphoinositide 3-kinase α inhibitors: a patent review. Expert Opin. Ther. Pat. 2013, 23, 789-799. (f) Yap, T. A.; Bjerke, L.; Clarke, P. A.; Workman, P. Drugging PI3K in cancer: refining targets and therapeutic strategies. Curr. Opin. Pharmacol. 2015, 23, 98-107. 11

For PI3Kα isoform selective inhibitors in clinical trials see: (a) Fritsch, C.; Huang, A.; Chatenay-

Rivauday, C.; Schnell, C.; Reddy, A.; Liu, M.; Kauffmann, A.; Guthy, D.; Erdmann, D.; De Pover, A.; Furet, P.; Gao, H.; Ferretti, S.; Wang, Y.; Trappe, J.; Brachmann, S. M.; Maira, S. M.; Wilson, C.; Boehm, M.; Garcia-Echeverria, C.; Chene, P.; Wiesmann, M.; Cozens, R.; Lehar, J.; Schiegel, R.; Caravatti, G.; Hofmann, F.; Sellers, W. R. Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials. Mol. Cancer Ther. 2014, 13, 1117-1129. (b) Barlaam, B.; Cosulich, S.; Delouvrie, B.; Fitzek, M.; Germain, H.; Green, S.; Harris, C. S.; Hudson, K.; Lambert-van der Brempt, C.; Lamorlette, M.; Antoine, L. G.; Morgentin, R.; Ouvry, G.; Page, K.; Pasquet, G.; Ruston, L.; Saleh, T.; Vautier, M.; Ward, L. Discovery of AZD8835, a potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of PIK3CA-dependent cancers. Cancer Res. 2015, 75, suppl; abstr 2830. 12

For recent reviews of isoform selective PI3K inhibitors see: (a) Marone, R.; Cmiljanovic, V.; Giese,

B.; Wymann, M. P. Targeting Phosphoinositide 3-Kinase—Moving Towards Therapy. Biochim. ACS Paragon Plus Environment

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Biophys. Acta, 2008, 1784, 159-185. (b) Knight, Z. A.; Shokat, K. M. Chemically Targetting the PI3K Family. Biochem. Soc. Trans. 2007, 35, 245- 249. (c) Ward, S. G.; Finan, P. Isoform-specific Phosphoinositide 3-Kinase Inhibitors as Therapeutic Agents. Curr. Opin. Pharmacol. 2003, 3, 426-434. (d) Sundstrom, T. J.; Anderson, A. C.; Wright, D. L. Inhibitors of Phosphoinositide-3-Kinase: A Structure-Based Approach to Understanding Potency and Selectivity. Org. Biomol. Chem. 2009, 7, 840850. 13

(a) Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Ishikawa, N.; Koizumi, T.; Ohishi, T.; Yamano,

M.; Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Waterfield, M. D.; Parker, P.; Workman, P. Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110α inhibitors. Bioorg. Med. Chem. 2007, 15, 403-412. (b) Pereira, A. R.; Strangman, W. K.; Marion, F.; Feldberg, L.; Roll, D.; Mallon, R.; Hollander, I.; Andersen, R. Synthesis of Phosphatidylinositol 3Kinase (PI3K) Inhibitory Analogues of the Sponge Meroterpenoid Liphagal.

J. J. Med. Chem. 2010,

53, 8523-8533. (c) Gilbert, A. M.; Nowak, P.; Brooijmans, N.; Bursavich, M. G.; Dehnhardt, C.; Delos Santos, E.; Feldberg, L. R.; Hollander, I.; Kim, S.; Lombardi, S.; Park, K.; Venkatesan, A. M.; Mallon, R. Novel Purine and Pyrazolo[3,4-d]pyrimidine Inhibitors of PI3 Kinase-a: Hit to Lead Studies. Bioorg. Med. Chem. Lett. 2010, 20, 636-639. (d) Kendall, J. D.; Rewcastle, G. W.; Frederick, R.; Mawson, C.; Denny, W. A.; Marshall, E. S.; Baguley, B. C.; Chaussade, C.; Jackson, S. P.; Shepherd, P. R. Synthesis, biological evaluation and molecular modeling of sulfonohydrazides as selective PI3K p110α inhibitors. Bio. Org. Med. Chem. 2007, 15, 7677-7687. (e) Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.; Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.; Williams, R. L.; Shokat, K. M. A Pharmacological Map of the PI3-K Family Defines a Role for p110α in Insulin Signaling. Cell 2006, 125, 733-747. 14

Jamieson, S.; Flanagan, J. U.; Kolekar, S.; Buchanan, S.; Kendall, J. D.; Lee, W.-J.; Rewcastle, G.

W.; Denny, W. A.; Singh, R.; Dickson, J.; Baguley, B. C.; Shepherd, P. R. A drug targeting only p110α ACS Paragon Plus Environment

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can block phosphoinositide 3-kinase signaling and tumour growth in certain cell types. Biochem. J. 2011, 438, 53-62. 15

Furet, P.; Guagnano, V.; Fairhurst, R. A.; Imbach-Weese, P.; Bruce, I.; Knapp, M.; Fritsch, C.;

Blasco, F.; Blanz, J.; Aicholz, R.; Hamon, J.; Fabbro, D.; Caravatti, G. Discovery of NVP-BYL719 a Potent and Selective Phosphatidylinositol-3 Kinase Alpha Inhibitor Selected for Clinical Evaluation. Biorg. Med. Chem. Lett. 2013, 23, 3741-3748. 16

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, L.; Leblanc, C.; LeGrand, D.; McCarthy, C.; O’Connor, D.; Owen, C.; Oza, M. S.; Pilgrim, G.; Press, N. E.; Sviridenko, L.; Whitehead, L. Development of Isoform Selective PI3-Kinase Inhibitors as Pharmacological Tools for Elucidating the PI3K Pathway. Biorg. Med. Chem. Lett. 2012, 22, 5445-5450. 17

Heffron, T. P.; Wei, B.; Olivero, A.; Staben, S. T.; Tsui, V.; Do, S.; Dotson, J.; Folkes, A.;

Goldsmith, P.; Goldsmith, P.; 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. The Rational Design of PI3 Kinase Inhibitors Exhibiting Selectivity Over the PI3K-β Isoform. J. Med. Chem. 2011, 54, 7815-7833. 18

Staben, S. T.; Ndubaku, C.; Blaquiere, N.; Belvin, M.; Bull, R. J.; Dudley, D.; Edgar, K.; Gray, D.;

Heald, R.; Heffron, T. P.; Jones, G. E.; Jones, M.; Kolesnikov, A.; Lee, L.; Lesnick, J.; Lewis, C.; Murray, J.; McLean, N. J.; Nonomiya, J.; Olivero, A. G.; Ord, R.; Pang, J.; Price, S.; Prior W. W.; Rouge, L.; Salphati, L.; Sampath, D.; Wallin, J.; Wang, L.; Wei, B.; Wiesmann, C.; Wu, P. Discovery of thiazolobenzoxepin PI3-kinase inhibitors that spare the PI3-kinase β isoform. Biorog. Med. Chem. Lett. 2013, 23, 2606-2613.


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Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.; Chuckowree, I. S.;

Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes, A.; Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.; Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann, C.; Workman, P.; Zhyvoloup, P.; Zvelebil, M. J.; Shuttleworth, S. J. The Identification of 2(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2d]pyrimidine (GDC-0941) as a Potent, Selective, Orally Bioavailable Inhibitor of Class I PI3 Kinase for the Treatment of Cancer. J. Med. Chem. 2008, 51, 5522-5532. 20

Sutherlin, D. P.; Bao, L.; Berry, M.; Castanedo, G.; Chuckowree, I.; Dotson, J.; Folks, A.;

Friedman, L.; Goldsmith, R.; Gunzner, J.; Heffron, T.; Lesnick, J.; Lewis, C.; Mathieu, S.; Murray, J.; Nonomiya, J.; Pang, J.; Pegg, N.; Prior, W. W.; Rouge, L.; Salphati, L.; Sampath, D.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wei, B. Q.; Wiesmann, C.; Wu, P.; Zhu, B.-Y.; Olivero, A. Discovery of a Potent, Selective, and Orally Available Class I Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor (GDC-0980) for the Treatment of Cancer. J. Med. Chem. 2011, 54, 7579-7587. 21

Ndubaku, C. O.; Heffron, T. P.; Staben, S. T.; Baumgardner, M.; Blaquiere, N.; Bradley, E.; Bull,

R.; Do, S.; Dotson, J.; Dudley, D.; Edgar, K. A.; Friedman, L. S.; Goldsmith, R.; Heald, R. A.; Kolesnikov, A.; Lee, L.; Lewis, C.; Nannini, M.; Nonomiya, J.; Pang, J.; Price, S.; Prior, W. W.; Salphati, L.; Sideris, S.; Wallin, J. J.; Wang, L.; Wei, B.; Sampath, D.; Olivero, A. G. Discovery of 2{3-[2-(1-Isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9yl]-1H-pyrazol-1-yl}-2-methylpropanamide (GDC-0032): A β-Sparing Phosphoinositide 3-Kinase Inhibitor with High Unbound Exposure and Robust in Vivo Antitumor Activity. J. Med. Chem. 2013, 56, 4597-4610.


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A depiction of a crystal structure of a benzoxepin bound to PI3Kγ (PDB 3R7R) where residues are

labeled to highlight differences between PI3Kα and the other isoforms within the active site, can be found as Supporting Information. 23

Staben, S. T.; Siu, M.; Goldsmith, R.; Olivero, A. G.; Do, S.; Burdick, D. J.; Heffron, T. P.; Dotson,

J.; Sutherlin, D. P.; Zhu, B.-Y.; Tsui, V.; Le, H.; Lee, L.; Lesnick, J.; Lewis, C.; Murray, J. M.; Nonomiya, J.; Pang, J.; Prior, W. W.; Salphati, L.; Rouge, L.; Sampath, D.; Sideris, S.; Wiesmann, C.; Wu, P. Structure-based design of thienobenzoxepin inhibitors of PI3-kinase. Bioorg. Med. Chem. Lett. 2011, 21, 4054-4058. 24

Huang, C.-H.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.; Velculesco, V. E.; Kinzler, K. W.;

Vogelstein, B.; Gabelli, S. B.; Amzel, L. M. The Structure of Human p110α/p85α Complex Elucidates the Effects of Oncogenic PI3Kα Mutations. Science, 2007, 318, 1744-1748. 25

After the conclusion of our research efforts a report suggesting the importance of hydrogen bonding

with Gln859 to achieve PI3Kα specificity in a different series of molecules. See Refs 15, 16. 26

A figure depicting the computational Link/Grow strategy is included as Supporting Information.

27

Li, B.; Chiu, C. K.-F.; Hank, R. F.; Murry, J.; Roth, J.; Tobiassen, H. An Optimized Process for

Formation of 2,4-Disubstituted Imidazoles from Condensation of Amidines and α-Haloketones. Org. Process Res. Dev., 2002, 6, 682–683. 28

There are multiple reported values for liver blood flow of a rat. One estimate is 55.2 mL/min/kg:

Tschida S. J.; Vance-Bryan, K.; Zaske, D. E. Anti-infective agents and hepatic disease. Med. Clin. North America, 1995, 79, 895-917.


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Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones,B. C.; MacIntyre, F.; Rance, D. J.;

Wastall, P. The prediction of humanpharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 1997, 283, 46–58. 30

Pryde, D. C.; Dalvie, D.; Hu, Q.; Jones, P.; Obach, R. S.; Tran, T.-D. Aldehyde Oxidase: An

Enzyme of Emerging Importance in Drug Discovery. J. Med. Chem. 2010, 53, 8441–8460. 31

Edgar, K. A.; Wallin, J. J.; Berry, M.; Lee, L. B.; Prior, W. W.; Sampath, D.; Friedman, L. S.;

Belvin, M.

Isoform-Specific Phosphoinositide 3-Kinase Inhibitors Exert Distinct Effects in Solid

Tumors. Cancer Res. 2010, 70, 1164-1172. 32

Enzymatic IC50 values were determined by Invitrogen for 4 against the following kinases, PI3KC2b

(IC50 = 261 nM), PI3KC2a (IC50 > 10 µM), hVPS34 (IC50 = 2.84 µM). Kiapp for mTOR was determined to be 4.3 µM. A complete list of kinases tested at Invitrogen is included as Supporting Information. 33

The in vitro EC50 values for inhibition of pAKT in MCF7-neo/HER2 cells are 4 nM and 10 nM for

3 and 4 respectively. 34

Hepatic CL was predicted to be 5 mL/min/kg by microsomal incubations. Allometric scaling

projected a plasma CL of 3 mL/min/kg (based on the rule of exponents). 35

Halladay, J. S.; Wong, S.; Jaffer, S. M.; Sinhababu, A. K.; Khojasteh-Bakht, S. C. Metabolic Stability

Screen for Drug Discovery Using Cassette Analysis and Column Switching. Drug Metab. Lett. 2007, 1, 67-72.


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402x203mm (96 x 96 DPI)


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