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Discovery of a Phosphoinositide 3-Kinase (PI3K) b/d Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kb Potency in a PI3KdSelective Template by Targeting Non-Conserved Asp856 Stéphane Perreault, Jayaraman Chandrasekhar, Zhi-Hua Cui, Jerry B Evarts, Jia Hao, Joshua A. Kaplan, Adam Kashishian, Kathleen S. Keegan, Thomas Kenney, David J Koditek, Latesh Lad, Eve-Irene Lepist, Mary E. McGrath, Leena Patel, Bart Phillips, Joseph Therrien, Jennifer Treiberg, Anella Yahiaoui, and Gary Phillips J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01821 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 20, 2017

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Journal of Medicinal Chemistry

Discovery of a Phosphoinositide 3-Kinase (PI3K) / Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3K Potency in a PI3K-Selective Template by Targeting Non-Conserved Asp856 Stephane Perreault,* Jayaraman Chandrasekhar, Zhi-Hua Cui, Jerry Evarts,† Jia Hao, Joshua A. Kaplan, Adam Kashishian, Kathleen S. Keegan, Thomas Kenney, David Koditek, Latesh Lad, Eve-Irene Lepist, Mary E. McGrath, Leena Patel, Bart Phillips, Joseph Therrien, Jennifer Treiberg, Anella Yahiaoui and Gary Phillips.

Gilead Sciences, Inc., 199 E Blaine Street, Seattle, Washington 98102, United States Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States

ABSTRACT Phosphoinositide 3-kinase (PI3K)  signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3K inhibitors in which PI3K potency was built in a PI3K-selective template. This work led to the discovery of a highly selective PI3K/ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

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INTRODUCTION The phosphoinositide 3-kinase (PI3K) pathway plays a role in a wide range of key cellular functions such as cell growth, proliferation and survival.1,2,3,4 Aberrant activation of the PI3K/AKT pathway has been observed with high frequency in human malignancies, suggesting a significant role in tumorigenesis, cancer progression and treatment resistance.5,6 Class I PI3Ks are heterodimers bearing regulatory and catalytic subunits, which are further subdivided into class IA and IB.7 Class IA is comprised of three isoforms of the catalytic subunit: p110, p110 and p110, while p110 is the only isoform in class IB. PI3K and  are ubiquitously expressed in all tissues, while PI3K and  are confined to leukocytes. Class I PI3Ks catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3).8 Pan inhibitors of the Class I PI3Ks, along with molecules that inhibit additional downstream targets, such as mTOR, have provided evidence that attenuating signaling through this node can yield clinical benefit in oncology.9 However, the utility of pan inhibitors has been limited by side effects, such as hyperglycemia, resulting in dose reductions that erode efficacy. Most recent efforts are devoted to target the relevant oncogenic PI3K isoform with isoform-selective inhibitors in order to circumvent off-target toxicities and to provide a larger therapeutic window.10,11,12,13,14,15,16 In many solid tumors associated with aberrant signaling of the PI3K isoform, the increased signaling through the PI3K pathway is not induced via activating mutations in PI3K, but rather by deactivation of the tumor suppressor phosphatase and tensin homolog (PTEN).17,18 The protein encoded by this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase which functions as a negative regulator of the lipid kinase activity. Preclinical studies have shown that down-regulation of PI3K in PTEN-deficient cancer cells results in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings.17 This discovery provided strong rationale for the development of PI3K-selective


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inhibitors. GSK-263677110,19 and AZD818610,20 are -weighted / inhibitors currently under clinical evaluation for the treatment of castration-resistant prostate cancer (Figure 1).21,22,23,24,25,26,27

Figure 1. PI3K inhibitors in clinical trials.

In this manuscript, we describe our efforts toward identifying potent, selective, and metabolically stable PI3K inhibitors suitable for the treatment of PTEN-deficient tumors. Our goals were threefold: potent inhibition of PI3K in PTEN-deficient PC3 prostate cancer cells (inhibition of pAKT with EC50 < 20 nM), isoform selectivity of greater than 100-fold over PI3K and PI3K in order to allow EC90 coverage of PI3K at trough concentrations without compromising isoform selectivity, and in vitro human predicted clearance of 0.2 L/h/kg or below to allow for a twice-daily dosing regimen. RESULTS AND DISCUSSION In an effort to develop isoform-selective PI3K inhibitors, we envisaged building PI3K potency from our previously disclosed PI3K-selective template, exemplified by idelalisib (1).28,29 As revealed by its X-ray crystal structure in the PI3Kδ isoform, the quinazolinone of idelalisib occupies a specificity pocket between Met752 and Trp760, a closed pocket in the apo structure of the enzyme (Figure 2a).30 This binding mode may imbue propeller shaped inhibitors such as idelalisib with improved selectivity over other lipid kinases that show reduced conformational flexibility in this region.31 This orthogonal arrangement orients the phenyl group to sit on the hydrophobic region II (H2). While many residues in the ATP pocket are conserved among all four class I PI3K isoforms, sequence alignment of the residues in the hydrophobic region II shows several variations, such as Asp856, which is unique to PI3K (Figure



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2b).32 Acccordingly, wee reasoned thaat a well-placeed hydrogen bbond donor ccould engage iin an interaction with Asp8 856 resulting in an improvement in PI3K K potency.333 Proof of conncept was achhieved througgh the introductiion of a 4-hyd droxy group on o the phenyl ring of idelallisib (Table 1, 1 vs. 2). Thiis modificatioon leads to a 1000-fold im mprovement in n PI3K potency. With ouur knowledge that the primary site of metabolism of idelalisiib is the purin ne moiety,34,355 we also evalluated our hyppothesis in a second analogous series beaaring a 4-amin no-5-cyanopy yrimidine hing ge binder andd a similar inccrease in PI3K K potency w was observed (3 vs. 4). Fro om these results, we began n a medicinal chemistry efffort toward thhe identificatioon of potent and d selective PI3Kβ/δ inhibittors.

Figure 2. a)) 2.4 Å X-ray cry ystal structure off idelalisib (1) bo ound in the PI3K K ATP bindingg site (pdb: 4XE00; coordinates available).300 Asp832 and Assn836 (highlightted in yellow) differ among the P PI3K isoforms. F For clarity, all otther residues havve been removed. Blue dashed lines show hydrrogen bond conttacts between thee inhibitor and thhe protein. b) Diifferent residues in the hydrophobicc region II amon ng class I PI3Ks.. Asn836 of PI3K K corresponds to Asp856 of PII3K.


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Table 1. Building PI3K potency in a PI3K-selective template

1 (idelalisib)

2

3

4

5

6

7500

1600 (593x)

2025

281 (400x)

82 (33x)

3490 (249x)

3700

2.7

54

0.7

2.5

14

PI3KIC50 (nM) 

18

14

0.34

0.3

0.6

3.2

PI3KIC50 (nM) 

2100

4300 (1592x)

154

187 (267x)

990 (396x)

990 (71x)

PI3KIC50 (nM)

a,b

PI3KIC50 (nM)

a a

a,b

a

The activity against each class I PI3K was evaluated in in vitro kinase assays containing 2xKm steady state concentrations of ATP (average of ≥ 2 determinations). b Numbers in parentheses represent fold selectivity over PI3K (PI3K or PI3K IC50 divided by PI3K IC50).

Phenol-containing compounds are prone to pharmacokinetic (fast glucuronidation as prelude to excretion) and/or toxicological limitations (formation of quinones as chemically reactive metabolites). To eliminate the phenol, but retain the proposed hydrogen bond interaction between the inhibitor and Asp856, a variety of moieties were prepared and tested, including, but not limited to, heterocycles, such as indoles, indazoles, benzotriazoles, pyrazoles, and acylated or sulfonylated (hetero)aryl amines such as carbamates and ureas.36,37,38,39 Our studies indicated that pyrazoles 5 (pyrazol-3-yl) and 6 (pyrazol-4-yl) appeared to be attractive starting points (Table 1). Further profiling of compounds 5 and 6 revealed very high predicted clearance in human hepatocytes (>0.95 L/hr/kg). This observation is consistent with our previously disclosed PI3K-selective series: the 4-amino-5-cyanopyrimidine hinge binder is a substrate for aldehyde oxidase-mediated metabolism leading to C2-oxidation on the pyrimidine.13,40 Despite the lower selectivity for the PI3K isoform, we first decided to investigate the pyrazol-3-yl series due to the favorable PI3K potency of 5. Alternative substitutions around the pyrimidine hinge binder were explored in an effort to mitigate the high predicted clearance of 5 (Table 2). As discovered previously, introduction of a primary C2-amine is well tolerated and prevents oxidation of the pyrimidine (7).12-13 The low predicted hepatic clearance was attractive but dampened by the concomitant increase in PI3K and PI3K potency. It is likely that the



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increased potency in all isoforms is the result of a third hydrogen bond involving the 2-amino moiety of the pyrimidine hinge binder. Additionally, compound 7, as well as closely related cyclopropyl analog 8, displays very low forward permeability as determined by a Caco-2 cell monolayer assay. Our first approach to improving the permeability of this series consisted of reducing the total polar surface area (tPSA) and increasing the lipophilicity as measured by cLogP. Substitution of the nitrile group at the C5-position of the pyrimidine by a hydrogen atom (9) contributes to a reduction in tPSA (170 Å2 to 146 Å2). Unfortunately, this modification results in a substantial loss in potency. Replacement of the nitrile group by a chlorine atom (10) is well tolerated in terms of PI3K potency. This change is also accompanied by a desirable increase in forward permeability, presumably the result of a higher cLogP and lower tPSA (146 Å2). A more lipophilic cyclopropyl group at R1 (11) leads to an even greater improvement in permeability. Moreover, compounds 10 and 11 afford better selectivity over the PI3K and PI3Kisoforms relative to the nitrile analogs 7 and 8. The predicted clearances of 10 and 11 are higher but remain in an acceptable range for further consideration. Metabolite identification studies on compound 11, after incubation with human hepatocytes, reveal the formation of an oxidative metabolite (M+16) of the pyrimidine moiety. This metabolic pathway is not observed in compounds with the electron withdrawing nitrile group at the C5-position of the pyrimidine (7 and 8). As a second approach to improve the permeability, we envisaged reducing the number of hydrogen bond donors. Replacement of the primary amine at R3 by a methyl group (12) or a chlorine atom (13) results in an increase in forward permeability at the expense of higher predicted clearances. The loss in metabolic stability can be rescued by introducing a difluoromethyl group at R3 (14). This modification is also accompanied by a substantial improvement in forward permeability. The trifluoromethyl analog 15 exhibits low PI3K potency. We believe this is the result of an inevitable electrostatic repulsion between the third fluorine and the carbonyl group of Glu846 in PI3K (vide infra).


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Table 2. Optimization of the 2-aminopyrimidine hinge binder

Compound 7

R1 CH3

8

R2

IC50 (nM)a

R3 PI3Kb

PI3K

PI3K

PI3Kb

cLogP

CLpr (L/hr/kg)c

Caco-2 (AB/BA)d

CN

NH2

23 (33x)

0.7

0.2

54 (77x)

1.48

0.06

0.05/10

CN

NH2

89 (56x)

1.6

0.6

199 (124x)

1.92

0.05

0.16/14

9

CH3

H

NH2

>10000 (>6x)

1584

317

>10000 (>6x)

1.93

ND

ND

10

CH3

Cl

NH2

302 (144x)

2.1

2.1

1247 (594x)

2.70

0.22

1.1 / 20

11

Cl

NH2

1074 (136x)

7.9

5.2

4150 (525x)

3.14

0.20

2.2/12

12

CN

CH3

45 (26x)

1.7

0.8

267 (157x)

2.57

0.40

2.0/17

13

CN

Cl

308 (59x)

5.2

5.9

1300 (250x)

2.54

0.73

1.3/24

CN

CHF2

686 (74x)

9.3

13

1131 (122x)

2.27

0.16

9.0/42

CN

CF3

>10000 (>15x)

671

309

>10000 (>15x)

2.40

ND

ND

14 15

CH3

a

The activity against each class I PI3K was evaluated in in vitro kinase assays containing 2xKm steady state concentrations of ATP (average of ≥ 2 determinations). b Numbers in parentheses represent fold selectivity over PI3K (PI3K or PI3K IC50 divided by PI3K IC50). c Determined from human hepatocytes. d 106 cm/s. ND = Value not determined.

These results prompted us to further investigate different quinazolinone and R1 substitutions in the 2,6diamino-5-chloropyrimidine and the 2-amino-5-cyano-4-(difluoromethyl)pyrimidine hinge binder series. While exploring alternate substitutions on the quinazolinone ring, we found that minor modifications have substantial impact on potency and selectivity (Table 3). Substituents such as 5-fluoro (16a-b), 6fluoro (17a-b), and 8-fluoro (18a-b) all result in a loss of PI3K potency. Interestingly, the 6,8difluoroquinazolinone motif found in 19a-b results in a 2-fold increase in PI3K potency relative to the monofluorinated analogs 17a-b and 18a-b. Potency and isoform selectivity can be further improved by replacing the 8-fluoro with an 8-chloro (20a-b). Combining the 8-chloro-6-fluoroquinazolinone with various R1 moieties (21a-b and 22a-b) results in a loss of potency. Nonetheless, the biochemical profile of compound 21b remains promising based on potency and isoform selectivity.



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Table 3. SAR results for quinazolinone ring and R1 modifications

IC50 (nM)a Core

IC50 (nM)a Compound

Compound PI3Kb

PI3K

PI3K

PI3Kb

PI3Kb

PI3K

PI3K

PI3Kb

16a

1085 (31x)

35

21

>10000 (>286x)

16b

1511 (14x)

109

51

>10000 (>92x)

17a

322 (8x)

42

9.8

>10000 (>238x)

17b

720 (6x)

121

26

7989 (66x)

18a

357 (11x)

34

12

>10000 (>294x)

18b

187 (6x)

29

9.5

4262 (147x)

19a

291 (17x)

17

3.0

4407 (259x)

19b

171 (11x)

15

6.2

2311 (154x)

20a

850 (109x)

7.8

5.3

>10000 (>1282x)

20b

284 (109x)

2.6

3.3

1277 (491x)

21a

2080 (74x)

28

15

>10000 (>357x)

21b

756 (168x)

4.5

7.2

3466 (770x)

22a

6937 (50x)

138

132

>10000 (>72x)

22b

1303 (81x)

16

13

7836 (490x)

a

The activity against each class I PI3K was evaluated in in vitro kinase assays containing 2xKm steady state concentrations of ATP (average of ≥ 2 determinations). b Numbers in parentheses represent fold selectivity over PI3K (PI3K or PI3K IC50 divided by PI3K IC50).

The improvement in PI3K potency observed with the introduction of the C2-amine on the pyrimidine hinge binder (5 vs. 7) led us to further explore the pyrazol-4-yl series exemplified by compound 6 (Table 1). However, in a head-to-head comparison between the pyrazol-3-yl series (23) and the corresponding pyrazol-4-yl series (24) (SAR pair analysis of compounds in which the only variable is the connectivity point on the pyrazole, Figure 3a), we observed that the pyrazol-4-yl leads to inferior PI3Kpotency in most cases (Figure 3b). In terms of selectivity, it appears that the combination of the pyrazol-3-yl with the 2,6-diamino-5-chloropyrimidine (green circles) leads to greater PI3K selectivity (Figure 3c). In a third analysis, we observed that the pyrazol-3-yl analog generally leads to greater PI3K selectivity when


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compared to the corresponding pyrazol-4-yl compound (Figure 3d). This last trend was independent of the hinge binder and led us to abandon the pyrazol-4-yl series.

Xn

O

Xn

NH N

N

N

R1

N HN 23 pyrazol-3-yl

N NH

O

R1

N NH2

N N

R2 3

R

HN 24 pyrazol-4-yl

NH2

N N

R2 3

R

R2 = CN and R3 = NH2 R2 = Cl and R3 = NH2 R2 = CN and R3 = CHF2

PI3K IC50 of pyrazol-3-yl (nM)

b) PI3K potency: pyrazol-3-yl vs. pyrazol-4-yl

a) SAR pair analysis: pyrazol-3-yl vs. pyrazol-4-yl

PI3K IC50 of pyrazol-4-yl (nM) c) PI3K / PI3K selectivity: pyrazol-3-yl vs. pyrazol-4-yl

d) PI3K / PI3K selectivity: pyrazol-3-yl vs. pyrazol-4-yl PI3K IC50 / PI3K IC50 of pyrazol-3-yl

PI3K IC50 / PI3K IC50 of pyrazol-3-yl

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


PI3K IC50 / PI3K IC50 PI3K of pyrazol-4-yl

PI3K IC50 / PI3K IC50 of pyrazol-4-yl

Figure 3. SAR pair analysis illustrating that the pyrazol-3-yl series (23) is superior to the pyrazol-4-yl series (24) in terms of PI3K potency and PI3K selectivity. Each colored shape represents a pair of compounds 23 and 24 with identical substituents Xn, R1, R2, and R3, the only variable being the connectivity point on the pyrazole (pyrazol-3-yl vs. pyrazol-4-yl). The value on the x-axis corresponds to the pyrazol-4-yl compound 24 and the value on the y-axis is for the corresponding pyrazol-3-yl compound 23.

The binding mode of this series of inhibitors has not been characterized crystallographically in PI3K.41 However, it can be modeled in a PI3K homology model (coordinates have been included in Supporting Information) based on the crystal structure of PI3Kδ co-crystallized with recently disclosed (S)-2,4-



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diamino-6 6-((5-chloro-8 8-fluoro-4-ox xo-3-(pyridin-3-yl)-3,4-dihy hydroquinazollin-2yl)(cyclop propyl)methylamino)-pyrim midine-5-carb bonitrile (GS--9901, pdb: 55T8I).12 Comppounds such as 11 and 14 aree likely to bin nd to the ATP P binding site of the kinasee domain of PI3Kβ (Figuree 4). Similar too other prop peller shaped PI3K inhibito ors, the quinaazolinone occuupies an induuced specificity pocket betw ween Met773 an nd Trp781 off PI3Kβ. Addiitionally, mollecular modelling indicates that the hydrrogen bond doonor in the pyrazole ring can n form an eneergetically fav vorable interaaction with Asp856. The 2,6-diamino-5chloropyrrimidine of 11 1 is expected to t serve as a three-point t hiinge binder (F Figure 4a). On the other haand, the 2-amin no-5-cyano-4 4-(difluoromeethyl)pyrimidiine moiety off compound 114 is likely to bind to the hiinge with two hydrogen h bon nds (Figure 4b b). Modeling also suggestss a weakly atttractive interaaction betweeen the hydrogen of the 4-diflu uoromethyl grroup and the carbonyl c of G Glu846. Previiously discusssed compoundd 15, bearing a 4-trifluoromeethyl group on n the pyrimid dine, most likeely suffers froom a repulsivve interaction with the same Glu846 G carbo onyl.

Figure 4. Docked D poses of compound c 11 (aa) and 14 (b) in th he homology moodel of PI3K. IIn both cases, thee pyrazole-NH iis predicted to o hydrogen bond to Asp856. For clarity, all otherr residues have bbeen removed. B Blue dashed liness show predictedd hydrogen bo ond contacts betw ween the inhibittor and the protein. The PI3Kβ hhomology modell used was built ffrom the crystal structure pd db: 5T8I (coordin nates have been included in Supp porting Informattion).12


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To shed some light on this possible interaction between the pyrazole moiety and Asp856, we synthesized the corresponding isoxazole 25 (Table 4). The ring lacks the hydrogen bond donor and leads to a 250-fold drop in PI3K potency while PI3K remains equipotent. This result supports the prediction of an interaction between the hydrogen bond donating pyrazole and Asp856 in this series of PI3K inhibitors. Table 4. Pyrazol-3-yl vs. isoxazol-3-yl

10

25

PI3KIC50 (nM) a

302

3538

PI3KIC50 (nM) a

2.1

539

PI3KIC50 (nM) 

2.1

1.9

PI3KIC50 (nM) a

1247

499

a

a

The activity against each class I PI3K was evaluated in in vitro kinase assays containing 2xKm steady state concentrations of ATP (average of ≥ 2 determinations).

Compounds meeting potency and selectivity criteria were then evaluated for tumor cell activity by measuring inhibition of AKT phosphorylation in the PTEN-deficient PC3 prostate cancer cell line (Table 5). Correlation between biochemical and cellular potency is more consistent for compounds with the 2,6diamino-5-chloropyrimidine hinge binder (10, 11 and 20a), while larger shifts were observed with the 2amino-5-cyano-4-(difluoromethyl)pyrimidine (14, 20b and 21b). This observation, combined with higher predicted human clearances for compounds 20b and 21b, led us to focus on analogs with the 2,6-diamino5-chloropyrimidine hinge binder (10, 11 and 20a).



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Table 5. Activity against AKT phosphorylation in PC3 cells and predicted human hepatic clearance

a

Compound

PI3K IC50 (nM)a

PC3 EC50 (nM)a

CLpr (L/hr/kg)b

10

2.1

4.2

0.22

11

7.9

7.6

0.20

20a

7.8

20

0.17

14

9.3

45

0.16

20b

2.6

15

0.29

21b

4.5

42

0.34

Average of ≥ 2 determinations. b Determined from human hepatocytes.

We next evaluated the pharmacokinetic profiles of the most promising compounds 10, 11 and 20a. Despite moderate oral bioavailability, the in vivo rat pharmacokinetic profile of 10 was promising, exhibiting good correlation between in vitro and in vivo clearances (Table 6). Introduction of a more lipophilic cyclopropyl at R1 (11) enhances the forward permeability leading to an overall improvement in oral bioavailability. Compound 20a, bearing an 8-chloro-6-fluoroquinazolinone, leads to an oral bioavailability of 66% in rat. We attribute this result to lower observed clearance and improved forward permeability as measured by a Caco-2 cell monolayer assay. Inspired by the rat pharmacokinetic profiles of 11 and 20a, we envisaged combining the positive features of these two analogs in one compound along with the metabolically stable 2,6-diamino-5cyanopyrimidine hinge binder exemplified in compounds 7 and 8. The resulting compound (26) yields a promising amalgamation of both bio- and physicochemical properties including a significant improvement in forward permeability in comparison to 7 and 8. Encouraged by the low predicted clearance and the improved permeability, we progressed compound 26 to in vivo pharmacokinetic evaluation. In addition to poor correlation between predicted and observed clearances, oral dosing of 26 at 5 mg/kg in rat suffers from a disappointing bioavailability of 12%. Among these four compounds, 20a is the most attractive based on its combination of potency and selectivity, lower predicted and observed clearances, and bioavailability.


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Table 6. Summary of pharmacokinetic profiles in rat

10

11

20a

26

302 / 2.1 / 2.1 / 1248

1074 / 7.9 / 5.2 / 4150

850 / 7.8 / 5.3 / >10000

84 / 0.7 / 0.6 / 632

4.2

7.6

20

3.7

Caco-2 (AB / BA)

1.1 / 19.6

2.2 / 11.8

5.7 / 21.7

1.5 / 17.3

CLpr (h) (L/h/kg) c

0.22

0.20

0.17

0.05

CLpr (r) (L/h/kg)

c

1.45

1.37

0.88

0.81

CLobs (r) (L/h/kg)

d

1.43 ± 0.26

0.58 ± 0.07

0.26 ± 0.02

0.07 ± 0.00

1.29 ± 0.10

0.83 ± 0.10

0.52 ± 0.03

0.22 ± 0.01

26 ± 12

42 ± 22

66 ± 17

12 ± 5

PI3K /IC50 (nM) EC50 PC3 (nM) a b

Vss (r) (L/kg) d,e F (r) (%)

f,g

a

a

Average of ≥ 2 determinations. b 106 cm/s. c Predicted clearance in hepatocytes. d Intravenous dose of 1 mg/kg. e Volume of distribution at steady state. f Oral dose of 5 mg/kg. g Number of animals per dosing route = 3. ND = Value not determined. h = human, r = rat.

A summary of the pharmacokinetic parameters in additional preclinical species after intravenous and oral administration of 20a is presented in Table 7. The in vivo data indicate that 20a has low to intermediate total clearance (CL) in comparison to hepatic blood flow in all species evaluated. Volumes of distribution (Vss) are higher than total body water in all species with the exception of rat in which higher plasma protein binding (PPB) is observed. The apparent elimination half-life (t1/2) and mean residence time (MRT) are 2 to 3 hours in rat, 4 to 5 hours in dog and rhesus monkey, and 5 to 6 hours in cynomolgus monkey. More than half of the administered dose is orally bioavailable in rat, dog and cynomolgus monkey. The high oral bioavailability is consistent with low observed clearance. Further characterization of 20a revealed that it does not inhibit major human CYP450 enzymes (IC50 > 25 uM) or hERG (IC50 > 25 M) in binding assays. Compound 20a does not significantly interact with any protein kinases using the KINOMEscan™ (DiscoveRx) platform at a compound concentration of 10 M, validating that 20a is a potent and selective inhibitor of PI3K/δ (see Supporting Information).



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Table 7. Summary of pharmacokinetic parameters for 20a in preclinical species Parameter

PPB (% free)a 3

H CLpr (L/h/kg) CL (L/h/kg)

b

c

Vss (L/kg) d Terminal t 1/2 (h)

SpragueDawley rat

Beagle dog

Cynomolgus monkey

Rhesus monkey

0.85

1.96

3.05

3.33

0.89

0.67

0.25

0.58

0.26 ± 0.02

0.25 ± 0.11

0.24 ± 0.07

0.41 ± 0.03

0.52 ± 0.03

1.14 ± 0.36

1.50 ± 0.34

1.88 ± 0.17

2.69 ± 0.27

3.91 ± 0.23

4.96 ± 0.46

3.61 ± 0.21

MRT (h) f

1.97 ± 0.20

4.77 ± 0.56

6.28 ± 0.59

4.54 ± 0.19

F (%)

66.4 ± 17.5

90.4 ± 21.7

68.6 ± 10.5

ND

g

e

a

Plasma protein binding. b Predicted clearance in hepatocytes using 3H compound. c Intravenous dose of 1 mg/kg. d Volume of distribution at steady state. e Half-life iv. f Mean residence time iv. g Oral doses of 20a (5 mg/kg) were formulated in 5% ethanol, 50% PEG 300, and 45% water. ND = Value not determined. (Mean ± SD, n=3)

As previously discussed, in PTEN-deficient tumors, the counterbalance to constitutive PI3K activity is absent, leading to increased signaling through the PI3K/AKT pathway. The phosphorylation state of AKT has been extensively used as a downstream measure of PI3K activity.12-27 Downstream signaling effects of 20a inhibition have been characterized in a subset of PTEN-deficient tumor cell lines that show dependence on PI3K for viability: the prostate carcinomas PC3 and LNCaP, and the breast adenocarcinomas MDA-MB-415 and ZR-75-1 (Table 8). We also included the kidney carcinoma A498 cell line which has a PI3K activating mutation. Compound 20a inhibits the phosphorylation of AKT1 Ser473 in these PI3K-dependent cell lines with EC50 values ranging from 3.4 nM to 7.3 nM. Cell viability was also found to be potently inhibited by 20a with GI50 ranging from 2 to 120 nM. Table 8. Activity of 20a against AKT1 Ser473 phosphorylation in PI3K-dependent tumor cell lines Cell line PC3

a

LNCaPa MDA-MB-415 ZR-75-1 A498b a

a

a

EC50 (nM)c

GI50 (nM)

5.4

120

7.3

38

5.7

12

3.4

2

5.6

80

PTEN-deficient. b PI3K mutant. c Data determined from the ratio of Units pAKT1 Ser473 per nanogram total AKT1.

The activity of 20a was assessed in a human PTEN-deficient, androgen-responsive LNCaP xenograft tumor model. As shown previously, AKT Ser473 phosphorylation and cell viability of LNCaP tumor cells are potently inhibited by PI3Kβ inhibitor 20a (EC50 = 7.3 nM and GI50 = 38 nM). Mice engrafted with LNCaP tumor cells were treated with 20a at 15 mg/kg, 7.5 mg/kg, and 3 mg/kg twice a day for 13


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consecutiv ve days follow wing the estab blishment of the tumor (Fiigure 5). Tum mor burden exxpressed as voolume (mm3) waas measured tw wice a week throughout t th he experimentt. After the finnal dose, treattment with 200a resulted in n tumor grow wth inhibition of 73%, 65% % and 42% in the 15 mg/kgg, 7.5 mg/kg aand 3 mg/kg dose groups, reespectively (p p

PI3K - ACS Publications - American Chemical Society

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