Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40

Jan 9, 2018 - G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to pro...
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Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists Jun Shi, Zhengxiang Gu, Elizabeth Anne Jurica, Ximao Wu, Lauren E Haque, Kristin N. Williams, Andres S. Hernandez, Zhenqiu Hong, Qi Gao, Marta Dabros, Akin H. Davulcu, Arvind Mathur, Richard A. Rampulla, Arun Kumar Gupta, Ramya Jayaram, Atsu Apedo, Douglas B. Moore, Heng Liu, Lori K. Kunselman, Edward J Brady, Jason J. Wilkes, Bradley A. Zinker, Hong Cai, Yue-Zhong Shu, Qin Sun, Elizabeth A. Dierks, Kimberly A. Foster, Carrie Xu, Tao Wang, Reshma Panemangalore, Mary Ellen Cvijic, Chunshan Xie, Gary G. Cao, Min Zhou, John Krupinski, Jean M Whaley, Jeffrey A Robl, William R Ewing, and Bruce Alan Ellsworth J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00982 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Xie, Chunshan; Bristol-Myers Squibb Cao, Gary; Bristol-Myers Squibb Zhou, Min; Bristol-Myers Squibb Company, Discovery Chemistry Krupinski, John; Bristol-Myers Squibb, Diabetes Drug Discovery Whaley, Jean; Bristol-Myers Squibb Company, Discovery Chemistry Robl, Jeffrey; Bristol-Myers Squibb Company, Discovery Chemistry Ewing, William; Bristol-Myers Squibb, Discovery Chemistry Ellsworth, Bruce; Bristol-Myers Squibb, Discovery Chemistry

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Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists Jun Shi,* Zhengxiang Gu, Elizabeth Anne Jurica, Ximao Wu, Lauren E. Haque, Kristin N. Williams, Andres S. Hernandez, Zhengqiu Hong, Qi Gao, Marta Dabros, Akin H. Davulcu, Arvind Mathur, Richard A. Rampulla, Arun Kumar Gupta, Ramya Jayaram, Atsu Apedo, Douglas B. Moore, Heng Liu, Lori K. Kunselman, Edward J. Brady, Jason J. Wilkes, Bradley A. Zinker, Hong Cai, Yue-Zhong Shu, Qin Sun, Elizabeth A. Dierks, Kimberly A. Foster, Carrie Xu, Tao Wang, Reshma Panemangalore, Mary Ellen Cvijic, Chunshan Xie, Gary G. Cao, Min Zhou, John Krupinski, Jean M. Whaley, Jeffrey A. Robl, William R. Ewing, Bruce Alan Ellsworth

Research and Development, Bristol-Myers Squibb, Co., P.O. Box 4000, Princeton, New Jersey 08540-4000. KEYWORDS: GPR40, FFAR1, GPCR agonist, diabetes therapy, dual MOA, GSIS, insulin secretagogue, GLP-1 secretagogue, BMS-986118 Abstract G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to promote glucose-stimulated insulin secretion. Herein, we report our efforts to develop highly selective and potent GPR40 agonists with a dual mechanism of action, promoting both glucose-dependent insulin and incretin secretion.

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Employing strategies to increase polarity and the ratio of sp3/sp2 character of the chemotype, we identified BMS-986118 (compound 4), which showed potent and selective GPR40 agonist activity in vitro. In vivo, compound 4 demonstrated insulinotropic efficacy and GLP-1 secretory effects resulting in improved glucose control in acute animal models.

Introduction Type 2 diabetes, representing over 85% of diabetes cases, is a progressive disease characterized by insulin resistance resulting in insufficient insulin action, eventually leading to a loss of glucose homeostasis. The rapidly growing prevalence of type 2 diabetes mellitus (T2DM) is a worldwide health concern due to its contribution towards cardiovascular co-morbidities (e.g. heart failure, stroke) and microvascular damage leading to blindness, kidney failure, foot ulcers, neuropathathic pain and more.1 According to the WHO (World Health Organization), over 300 million people worldwide were estimated to have diabetes in 2013,2 and that number is projected to rise to 629 million by 2045.3 Metformin is the first-line treatment of type 2 diabetes.4 Other glucose independent therapies for the treatment of diabetes include insulin secretagogues, such as sulfonylureas (lowering HbA1c by ~1.5%) and glinides (HbA1c lowering by ~1.5%).2,3,4 While effective in the short term, chronic use of these treatments can lead to hypoglycemia and β-cell exhaustion. These limitations often lead to disease progression, resulting in the need for polypharmacology or transition to insulin therapies.4 As an alternative mechanism to treat diabetes, incretin based therapies are appealing due to their ability to stimulate insulin in a glucose-dependent fashion (glucose–stimulated insulin secretion, GSIS). DPP4 inhibitors and GLP-1 mimetics are among these treatment options, and they are effective at lowering HbA1c with the benefit of a reduced risk for hypoglycemia.5,6 There remains potential opportunity for additional incretin-based therapies, since DPP4 inhibition exhibits modest HbA1c lowering

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(~0.7%) and GLP-1 mimetics can have some safety/tolerability limitations.4 Peroxisome proliferator-activated receptor γ (PPARγ) agonists increase insulin sensitivity and infrequently result in hypoglycemia but concerns around their side effects (significant weight gain, and possible cardiovascular and bladder cancer risks) have limited their clinical use.7 A promising new class of treatments for diabetes, SGLT-2 inhibitors, demonstrates insulin-independent glucose lowering while providing additional benefits such as moderate weight loss and bloodpressure lowering; however, modest efficacy (lowering HbA1c ~0.5-0.9%) leaves opportunity for other therapies that further reduce HbA1c without risk of hypoglycemia. Lastly, many patients progress to the use of injected insulin, which is an effective therapy to manage T2DM, but it must be carefully titrated to avoid incidence of hypoglycemia and weight gain.4 G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1 (FFAR1), is a Gq protein coupled receptor (GPCR) that is predominately expressed in pancreatic β-cells and is also found in the GI tract and brain.9,10 GPR40 is activated by free fatty acids (FFAs) such as docosahexaenoic acid (DHA) and linoleic acid (LA). In pancreatic β-cells, glucose metabolism induces the closure of ATP-sensitive K+ channels in the plasma membrane, which reduces K+ efflux and depolarizes the membrane, opening Ca2+ channels. Activation of GPR40 causes increases in intracellular Ca2+ concentrations via the inositol 1,4,5-trisphosphate (IP3) pathway, and increased Ca2+ stores leads to amplified insulin release after the glucose-induced membrane depolarization.11 Since the insulinotropic effect is glucose-dependent, GPR40 agonists have the therapeutic potential to treat diabetes with a low risk of hypoglycemia. In addition, GPR40–null mutant mice exhibit reduced GLP-1 and glucose-dependent insulintropic peptide (GIP) secretion suggesting that GPR40 may be involved in FFA-mediated incretin secretion.12 Several GPR40 agonists have been reported to stimulate both GLP-1 and GIP secretion in vivo, providing a

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second mechanism to control GSIS.13,14,15 The constellation of GPR40-mediated effects makes it an attractive target for the treatment of type-2 diabetes. Figure 1. Structures of dihydropyrazole and pyrrolidine GPR40 agonists

Recently, we disclosed potent GPR40 agonists 1 and 2 (Figure 1), featuring a pyrrolidine ring and a dihydropyrazole ring, respectively.16,17 GPR40 is a receptor that binds long-chain fatty acids, and we found that ligands with 4 rings (A-D ring, as depicted in Figure 1 structures 1 and 2) yielded optimal agonist activity. Both compounds exhibited potent GPR40 agonist activity and glucose-lowering efficacy in several animal models of diabetes, however, each demonstrated in vivo off-target liabilities that precluded their advancement: compound 1 caused hyperactivity in rats and compound 2 induced undesired blood-pressure and heart rate increases in rodents and monkeys at high doses.18 Both compounds also demonstrated PPARγ activity in vitro. It is known that PPARγ agonists improve insulin sensitization and this activity may provide additional therapeutic benefit in combination with GPR40 agonists. However, since PPARγ

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agonism may introduce increased safety risks, and the safety profile of GPR40 agonists has not been fully de-risked in clinical development, we decided that it would be prudent to develop a GPR40 selective agonist that was devoid of PPARγ activity.7 The undesired hyperactive and cardiovascular effects of compounds 1 and 218 also caused concern for advancement of these compounds, especially in a population that is prone to cardiovascular (CV) disease.12 We found that the CV liabilities of 2 were due to off-target pharmacology since hemodynamic changes were observed when compound 2 was administered to GPR40 knockout mice.17 Despite extensive in vitro profiling efforts, we were unable to identify the origin of the undesired CV effects.17 The rather high log P and associated lipophilicity of these compounds may have contributed to the promiscuity of their actions in vivo. We therefore adopted a strategy to reduce log P and fractional aromaticity of the ligands, believing that this approach would lead to compounds that matched the spatial/electronic requirements for GPR40 agonism while being less likely to match those of any unidentified targets.19,20 Herein, we describe the identification of a series of pyrrolidine and dihydropyrazole derivatives leading to compound 4, a novel GPR40 agonist with an improved potency, pharmacokinetics, glucose-lowering efficacy, and safety profile. Chemistry The synthesis of dihydropyrazole analogs is detailed in Scheme 1. The preparation of dihydropyrazole phenol intermediate 14 began with acylation of hydrazine 5 with TFAA to afford hydrazide 6. Treatment of 6 with benzenesulfonyl chloride and DIEA afforded a hydrazonoyl chloride that was subjected to a [3+2] cycloaddition with alkene 7 providing dihydropyrazole 8 with 2:1 diastereoselectivity (the stereochemistry of 8 was confirmed by Xray analysis).21 Intermediate 8 was homologated to ester 12 by a four-step sequence. First,

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intermediate 8 was reduced with NaBH4 in THF and H2O to give alcohol 9. The resulting alcohol was converted to mesylate 10 via reaction with MsCl and Et3N in DCM at room temperature. Displacement of 10 via treatment with potassium cyanide in DMSO at 40 ºC generated nitrile 11 that was subsequently hydrolyzed to the corresponding methyl ester 12 using 3.6 M HCl in MeOH at room temperature. Miyaura borylation22 of 12 afforded boronate 13 that was subjected to peroxide-mediated (H2O2) oxidation to produce key intermediate, phenol 14. Synthesis of Dring analogs (see Figure 1 for ring nomenclature) was accomplished via coupling between 4hydroxyl piperidine 15 and various aryl bromides using 1st generation S-Phos precatalyst and LiHMDS in THF at 60 °C. Tosylation of alcohols 16a-ac with TsCl and pyridine afforded the corresponding tosylates 17a-ac. Displacement of the tosyl group with dihydropyrazole phenol 14 under basic conditions generated substituted methyl esters 18a-ac. Finally, hydrolysis of esters 18a-ac furnished the desired dihydropyrazole analogs 3a and 19a-ac with different aromatic/heteroaromatic D-rings.

Scheme 1. Synthesis of dihydropyrazole D-ring analogsa A. Synthesis of dihydropyrazole A-B ring

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a

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Reagents and conditions: a) TFAA, DCM, 0 ºC to rt, 81%; b) benzenesulfonyl chloride, DIEA,

DCM, 0 ºC to rt, 90%; c) Ag2CO3, dioxane, 65 ºC, 46%; d) NaBH4, THF/H2O, rt, 94%; e) MsCl, Et3N, DCM, rt, 98%; f) KCN, DMSO, 40 ºC, 92%; g) HCl (3.6 M in MeOH), rt, 82%; h) Pd(dppf)Cl2, bis(pinacolato)diboron, KOAc, DMF, 80 ºC, 90%; i) 30% aq. H2O2, EtOAc, rt, 85%; B. Synthesis of D-ring analogsa

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Ar = 16a Ph 16b 2-Cl-Ph 16c 2-Me-Ph 16d 2-Et-Ph 16e 3-MeO-Ph 16f 4-Cl-Ph 16g 4-F-Ph 16h 3-F-4-MeO-Ph 16i 2-F-6-MeO-Ph 16j 2-F-6-Me-Ph 16k 2,6-di-Me-Ph 16l 2,6-di-Me-4-MeO-Ph 16m 2-F-5-EtO-Ph 16n 2-F-5-Me-Ph 16o 2-F-5-Et-Ph

16p 2-F-5-iPr-Ph 16q 2-F-5-Cl-Ph 16r 2-Cl-5-F-Ph 16s 2,5-di-Cl-Ph 16t 2,5-F-Ph 16u 2-Cl-5-MeO-Ph 16v 2-Cl-5-Me-Ph 16w 2-CN-5-MeO-Ph 16x 2-Py 16y 6-F-2-Py 16z 3-Me-6-F-2-Py 16aa 3-Py 16ab 2-F-5-MeO-4-Py 16ac 2-Cl-5-MeO-4-Py

a

Reagents and conditions: j) ArBr, 1st generation S-Phos precat., LiHMDS, THF, 60 ºC, 30%70%; k) TsCl, pyridine, DMAP, rt, DCM, 55%-80%; l) 14, Cs2CO3, DMF, 60 ºC, 30%-60%; m) LiOH, H2O, THF, rt, 70%-95%. The synthesis of compounds where the C-ring (see Figure 1) is substituted is depicted in Scheme 2. In order to introduce a 3-substituted piperidine ring (Scheme 2), the synthesis began with commercially available racemic 3-fluoro-piperidin-4-one (20a) or 3-methyl-substituted piperidin-4-one

(20b).

L-Selectride-mediated

reduction

of

ketones

20a-b

(with

diastereoselectivity over 20:1) followed by TBS protection (using TBS-OTf) of the resulting cisalcohol afforded intermediates 21a-b. Hydrogenolysis of the benzyl group using 10% palladium on carbon under 1 atm H2 provided amines of structure 22a-b. Subsequent SNAr reaction of the resulting amines 22a-b with 4-bromo-5-chloro-2-methoxypyridine and K2CO3 in DMSO at 100 °C gave a racemic adduct. At this point in the synthesis, the resulting racemic alcohols were subjected to chiral separation to give chiral alcohols 23a-b. Next, displacement of the hydroxyl group in intermediates 23a-b with phenol 14 was accomplished by Mitsunobu reaction using Bu3P and ADDP in toluene at elevated temperature (50 °C). The resulting methyl esters 24a-b

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were hydrolyzed using LiOH in THF and H2O at room temperature to generate the corresponding fluoro (25) and methyl (4) analogs.

Scheme 2.a Synthesis of C-ring analogs

a

Reagents and conditions: a) L-Selectride, THF, -78 ºC, 88%-98%; b) TBS-OTf, Et3N, DCM, 0 ºC to rt, 76%-92%; c) H2 (1 atm), Pd/C, MeOH, rt, 97%-100%; d) K2CO3, 100 ºC, DMSO, TBAF, THF, rt; followed by chiral separation, 30-32%; e) Bu3P, ADDP, toluene, rt, 14, 5464%; f) LiOH, H2O, THF, rt, 78-80%.

Results and Discussion Although the pharmacological origins of blood pressure and heart rate elevations observed with compound 2 (as well as hyperactivity associated with compound 1) were unclear, we envisioned that increasing polarity and the sp3/sp2 carbon ratio of our ligands may be beneficial in reducing off-target activities.19,20 Previous SARs demonstrated that the C-ring was promiscuous for GPR40 activity,16 so we decided to focus on replacing this portion of the molecule with a series of saturated heterocycles that were designed to maintain the distance and constrained nature of

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the phenyl C-ring (Structure A). As shown in Table 1, pyrrolidine 26 (see Supplemental Information for the synthesis of pyrrolidine analogs) demonstrated moderate but encouraging hGPR40 agonist activity. Following our previously described SAR for the series,16,17 incorporation of a 4-(R)-CF3 substituent to the pyrrolidine ring led to significantly (7-fold) improved in vitro potency (e.g. 3b) in this oxygenated piperidine C-ring series. Carbon-linked analogs (e.g. 27) were less potent than the oxygen-linked analogs (e.g. 3b) which was surprising given that carbon-linked analogs were more potent when the C-ring was phenyl (structures 1 and 2). To determine if the pyrrolidine C-ring unit was optimal, pyrrolidine (28-31) analogs (generated following the same general chemical protocols as outlined in Scheme 1) were tested, but their GPR40 agonist activities were inferior to the oxygenated piperidine C-ring analog. When the C-ring piperidine was incorporated into the dihydropyrazole series (Table 2), we discovered lead 3a which exhibited not only potent hGPR40 activity but was devoid of PPARγ activity as well (3a: PPARγ EC50 > 50 µM, 1: PPARγ EC50 = 4.4 µM and 2: PPARγ EC50 = 2.6 µM ). Unfortunately the electron donating nature of the piperidine nitrogen affected the metabolic fate of the D-ring, rendering it less stable to oxidative metabolism and glutathione (GSH) addition in liver microsomes. Compound 3a also demonstrated increased (vide infra, Table 5) clearance in rodents due to demethylation of the methoxy group. We therefore set out to identify a compound with an improved PK profile and a reduced propensity for GSH adducts while maintaining the potency and selectivity profile of 3a.

Table 1. C-Ring SAR with a pyrrolidine A ring

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FLIPR hGPR40 Compound

C-ring linker

CLogP

Y a,b

EC50 (µM) 26

H

1.20, 1.60

2.49

3b

CF3

0.21±0.06

4.55

27

CF3

0.53±0.14

5.73

28

CF3

2.83

4.48

29

CF3

5.36

4.48

30

CF3

0.94

4.60

31

CF3

0.63

4.60

a

S.D.: standard deviation, provided for results of n>3 tests; other EC50’s reported as the results of each separate test occasion. b all compounds showed full activation as compared to compound 2 and DHA in this assay.(See SI for detailed information)

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Since we identified the D-ring as the primary site of metabolism of 3a (vide infra, Figure 2), emphasis was placed on exploration of this portion of the molecule, focusing first on monosubstituted D-ring analogs (Table 2). Compared to unsubstituted analog 19a, ortho-substitution on the D-ring (19b-d) resulted in improved GPR40 activity, in contrast to para-substitution (19f, 19g) which decreased activity. Both o-chloro (19b) and m-methoxy (19e) substituents exhibited a 4-fold improved activity, indicating the benefit of having a chloro-substituent at the orthoposition and a methoxy-substituent at the meta-position relative to the piperidine nitrogen attachment. A survey of di- and tri-substituted D-ring analogs revealed that 2,6- (19i-j), 3,4(19h) and 2,4,6-substituted analogs (19l) were not in the desired potency range. Interestingly, 2,5-disubstitutions (19m-w) showed a positive effect on agonist activity and this substitution pattern was deemed to be optimal in this series. Unfortunately the D-ring substituents did not adequately address metabolism concerns. However, electron deficient heterocycles were introduced in an effort to reduce the potential for D-ring GSH adduct formation. As shown in Table 2, replacing the D-ring phenyl, as in compound 19a, pyridine analogs (19y, 19z, 19ab and 19ac) showed a tendency towards improved potency. Surprisingly, the 2-chloro-5-methoxy-4-pyridine analog (19ac) maintained similar in vitro potency as the lead compound 3a despite different electronic characteristics of the D-ring. Since the piperidine C-ring seemed to provide preferred spatial placement of the B and D rings, we decided to evaluate conformational constraints in the piperidine group in an attempt to further increase potency. 3-Substituted piperidines were synthesized as shown in Table 3. The introduction of fluorine (32, 25) or/and methyl (33-35, 4) group on the C-ring provided a significant improvement in EC50 values. Methyl substitution (4) improves potency by ~2 fold as

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compared to 3a. (Analogs are counterscreened against a proprietary panel of GPCRs (including the related FFARs), NHRs, transporters, ion channels, and hERG. Compound 4 had no detectable activities below 10 µM against any counterscreen with the exception of MAO B (IC50 = 6 µM).) With several analogs in the desired potency range in hand, biotransformation studies were initiated as a means to further differentiate between preferred compounds.

Table 2. In Vitro Activities of substituted D-ring Analogs

FLIPR hGPR40 Compound

D-ring

CLogP EC50 (µM)a

19a

Ph

1.93

4.22

3a

2-F-5-MeO-Ph

0.11±0.04

5.39

19b

2-Cl-Ph

0.45

6.02

19c

2-Me-Ph

0.50

4.63

19d

2-Et-Ph

1.18

5.12

19e

3-MeO-Ph

0.62

4.73

19f

4-Cl-Ph

6.27

5.34

19g

4-F-Ph

2.73, 2.55

4.77

19h

3-F-4-MeO-Ph

3.62

4.95

19i

2-F-6-MeO-Ph

5.90

5.39

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19j

2-F-6-Me-Ph

0.80

6.06

19k

2,6-di-Me-Ph

1.34

5.02

19l

2,6-di-Me-4-MeO-Ph

3.06

4.49

19m

2-F-5-EtO-Ph

0.22

5.75

19n

2-F-5-Me-Ph

0.44

6.06

19o

2-F-5-Et-Ph

0.45, 0.72

6.51

19p

2-F-5-iPr-Ph

0.60

6.79

19q

2-F-5-Cl-Ph

0.51

6.16

19r

2-Cl-5-F-Ph

0.36

6.16

19s

2,5-di-Cl-Ph

0.25

6.63

19t

2,5-di-F-Ph

0.48

5.68

19u

2-Cl-5-MeO-Ph

0.10

5.87

19v

2-Cl-5-Me-Ph

0.36

6.44

19w

2-CN-5-MeO-Ph

0.68

5.10

19x

2-Pyridylb

2.61

3.20

19y

6-F-2-Pyridyl

0.39, 0.86

4.34

19z

3-Me-6-F-2-Pyridyl

0.37

4.02

19aa

3-Pyridyl

7.68

3.07

19ab

2-F-5-MeO-4-Pyridyl

0.15, 0.18

4.21

19ac

2-Cl-5-MeO-4-Pyridyl

0.11

4.27

a

S.D.: standard deviation, provided for results of n>3 tests; other EC50’s reported as the results of each separate test occasion.

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Table 3. Re-exploration of C-ring SAR

FLIPR hGPR40 Compound

C-ring

CLogP

D-ring a

EC50 (µM)

32

2-F-5-MeO-Ph

0.07±0.15

25

2-Cl-5-MeO-Pyridyl

0.18

33

2-F-5-EtO-Ph

0.15±0.08

34

2-F-5-EtO-4-Pyridyl

0.28

35

2-Cl-5-EtO-4-Pyridyl

0.25, 0.19

5.61

4.50

6.23

5.07

5.13

2-Cl-5-MeO-44

0.07±0.04 Pyridyl

4.76

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2-Cl-5-MeO-442

0.32 Pyridyl

4.76

a

S.D.: standard deviation, provided for results of n>3 tests; other EC50’s reported as the results of each separate test occasion.

Biotransformation In vitro biotransformation studies in liver microsomes on three advanced compounds (3a, 33 and 4) highlighted significant differences with respect to metabolic stability and GSH adduct formation. The proposed pathways that lead to the formation of de-alkylated and oxidized metabolites and glutathione conjugates are outlined in Figure 2. Compounds 3a, 33 and 4 underwent CYP450-mediated oxidative de-alkylation to form phenol-type metabolites (36, 37 and 38, respectively) and oxidative GSH addition forms GSH adducts (39 and 40).As shown in Table 4, compound 3a exhibited high amounts of de-methylated and GSH-conjugated metabolites both in rat liver microsomes (RLM) and human liver microsomes (HLM) while compounds 33 and 4 showed a much lower percentage of the corresponding de-alkylated/GSHadducts in both (human and mouse) microsomal preparations. We attribute the lack of GSH adducts to the introduction of an electron deficient pyridine (4) vs phenyl (3a) D-ring. Since high levels of GSH adducts are associated with an increased risk of idiosyncratic drug reactions,23 the metabolism profiles for 33 and 4 proved especially desirable. As a result of their improved metabolic profiles and potent GPR40 agonist activities, we selected these compounds for advanced in vivo evaluations. Figure 2. Formation of oxidation metabolites and related GSH conjugates

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Table 4. Metabolic fate of compounds 3a, 33 and 4 in liver microsome incubationsa Human liver microsomes

Rat liver microsomes

% parent

% D-ring

% GSH

T1/2

% parent

% D-ring

% GSH

T1/2

Compound

dealkylation

adducts

(min)

Compound

dealkylation

adducts

(min)

remaining

metabolites

remaining

metabolites

3a

42

65

4

48

27

36

3-14

23

33

93

1.5