Discovery of Pyrrolidine-Containing GPR40 Agonists

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Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode Elizabeth Anne Jurica, Ximao Wu, Kristin N. Williams, Andres S. Hernandez, David S. Nirschl, Richard A. Rampulla, Arvind Mathur, Min Zhou, Gary Cao, Chunshan Xie, Biji Jacob, Hong Cai, Tao Wang, Brian J. Murphy, Heng Liu, Carrie Xu, Lori K. Kunselman, Michael B. Hicks, Qin Sun, Dora M. Schnur, Doree Fay Sitkoff, Elizabeth A. Dierks, Atsu Apedo, Douglas B. Moore, Kimberly A. Foster, Mary Ellen Cvijic, Reshma Panemangalore, Neil A. Flynn, Brad D. Maxwell, Yang Hong, Yuan Tian, Jason J. Wilkes, Bradley A. Zinker, Jean M. Whaley, Joel C. Barrish, Jeffrey A. Robl, William R Ewing, and Bruce Alan Ellsworth J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01559 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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Journal of Medicinal Chemistry

Ewing, William; Bristol-Myers Squibb Ellsworth, Bruce; Bristol-Myers Squibb, Discovery Chemistry

ACS Paragon Plus Environment


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Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode Elizabeth A. Jurica,* Ximao Wu, Kristin N. Williams, Andres S. Hernandez, David S. Nirschl, Richard A. Rampulla, Arvind Mathur, Min Zhou, Gary Cao, Chunshan Xie, Biji Jacob, Hong Cai, Tao Wang, Brian J. Murphy, Heng Liu, Carrie Xu, Lori K. Kunselman, Michael B. Hicks, Qin Sun, Dora M. Schnur, Doree F. Sitkoff, Elizabeth A. Dierks, Atsu Apedo, Douglas B. Moore, Kimberly A. Foster, Mary Ellen Cvijic, Reshma Panemangalore, Neil A. Flynn, Brad D. Maxwell, Yang Hong, Yuan Tian, Jason J. Wilkes, Bradley A. Zinker, Jean M. Whaley, Joel C. Barrish, Jeffrey A. Robl, William R. Ewing, Bruce A. Ellsworth Research and Development, Bristol-Myers Squibb, Co., P.O. Box 5400, Princeton, New Jersey 08543-5400, United States KEYWORDS GPR40, FFAR1, GPCR, agonist, diabetes, insulin, secretagogue, glucose-dependent insulin secretion, GLP-1, glucagon-like peptide-1, binding mode, pyrrolidine, radioligand binding

ABSTRACT


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A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes.

The initial pyrrolidine hit was modified by moving the position of the

carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gscoupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

INTRODUCTION The International Diabetes Federation estimates that in 2014 more than 387 million people were living with diabetes and 4.9 million deaths were attributed to diabetic complications, making diabetes a worldwide epidemic affecting 1 in 11 adults.1 Nearly half of the population with diabetes is undiagnosed,2 but the progressive nature of the disease means that their condition will likely (rapidly) decline, affecting quality of life if left untreated. Type 2 diabetes, representing 95% of all diabetes cases, is characterized by an insufficiency of insulin action that results in high blood glucose levels.

Increased blood glucose can cause a variety of

microvascular complications such as nephropathy, retinopathy, and neuropathy as well as macrovascular complications such as heart disease and stroke in type 2 diabetes patients.3


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GPR40, also known as free fatty acid receptor 1 (FFAR1), is a nutrient-sensing Gprotein-coupled receptor (GPCR) that is primarily expressed in the β-cells of the pancreas where it mediates glucose-stimulated insulin secretion.4,5 GPR40 is commonly reported to be a Gqcoupled GPCR that is most potently activated by long-chain unsaturated fatty acids (C>10) at micromolar concentrations.

GPR40-dependent modulation of insulin secretion is glucose-

dependent, suggesting that drugs targeting this receptor could improve glycemic control with low risk of hypoglycemia6 in contrast to the clinical experience with non-glucose dependent insulin secretagogues such as sulfonylureas. Fasiglifam (1, TAK-875, Figure 1) is a GPR40 agonist that progressed to phase III clinical trials, validating this target as an approach for the treatment of type 2 diabetes. It is a partial agonist of GPR40 that promotes Gq-dependent Ca2+ accumulation in beta cells, amplifying the effect of glucose on insulin secretion.7,8 In the clinic, 1 reduced glycated hemoglobin (HbA1c), an integrated measure of glycemic control, by ~1% in type 2 diabetic patients.9 At the 50 mg dose used in phase III clinical trials, 1 primarily demonstrated improvement on fasting plasma glucose levels with increased insulin secretion observed. At this dose, there was no effect on the gut hormones GIP and GLP-1 (vide infra). The development of 1 was halted during phase III clinical trials due to concerns about liver safety but, since GPR40 is not expressed in liver, the findings are unlikely to be a direct result of GPR40 agonism.10,11 The clinical efficacy of 1 suggests that a chemically distinct GPR40 agonist with improved liver safety would be a valuable treatment for type 2 diabetes. Recently, GPR40 was shown to have three distinct ligand binding sites.12 AMG 837 (2) and AM-1638 (3) were demonstrated, via radioligand binding studies, to occupy distinct sites on GPR40 (Figure 1). Interestingly, there is cross-talk between these sites, and ligands can exhibit


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positive cooperative binding. Several compounds, including 3, were found to elicit a greater Ymax than endogenous fatty acid ligands. These compounds were labeled “superagonists” due to their superior ability to control glucose levels.13 These full activators of GPR40 show greater potency and efficacy in vivo at lower plasma exposures relative to partial agonists in an oral glucose tolerance test. GPR40 is also expressed in enteroendocrine cells where it is important for secretion of gut hormones such as glucagon-like peptide-1 (GLP-1),13 but many synthetic GPR40 agonists, including 1, were not known to increase GLP-1 secretion. AM-1638 is the first reported small molecule agonist to demonstrate GPR40-dependent GLP-1 secretion.13 The ability to promote GLP-1 secretion brings the possibility of an additional benefit of GPR40 agonism for the treatment of diabetes, since GLP-1 is known to have effects on gastric emptying in the gut and glucose-dependent insulin secretion in the pancreas. The native peptide has a short half-life in plasma,14 but mechanisms that target the preservation of GLP-1, such as DPP-4 inhibitors, and metabolically stable GLP-1 mimetics are clinically efficacious for the treatment of diabetes.15,16 By developing GPR40 agonists with this dual mechanism of action (i.e. promoting glucosedependent insulin and GLP-1 secretion), we hoped to discover compounds with greater glucose lowering capacity than 1 or other agonists that employ a single insulin secretagogue mechanism of action.


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Figure 1. Structures of GPR40 agonists. To establish a novel starting point for our medicinal chemistry program, we performed a virtual search of the Bristol-Myers Squibb compound collection. It was known in the literature that a carboxylic acid or acid mimetic was required for GPR40 agonism, and several agonists (Figure 1) included chirality at the β-position relative to the carboxylic acid.8,17-20 We searched for structures with a cyclic constraint bearing a carboxylic acid that might confer GPR40 potency and efficacy. This exploration led to the discovery of racemic compound 4 that was found to be a weak partial agonist of the human GPR40 receptor (hGPR40 EC50 = 5.0 µM). Despite its weak activity, we decided to employ compound 4 as a starting point since we felt the aniline nitrogen and the novel cyclic structure might serve to constrain the carboxylic acid in a bioactive conformation. By removing the lactam carbonyl in compound 5, the potency was improved twofold, suggesting that the lactam carbonyl was not important for agonist activity. In a search for the optimal spacing between the aryl group and the carboxylic acid, we homologated the acid moiety as found in compound 6, but this modification led to reduced GPR40 agonist activity. Since the distance between the carboxylic acid and aryl group seemed critical for GPR40 activity, we varied the placement of the acid on the ring, resulting in the discovery of (S)-7 and (R)-7 (Table 1) with submicromolar hGPR40 activity. Due to the improved potency of (S)-7


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relative to the starting point (compound 4), we focused on developing the SAR around this homoproline analog.

Figure 2. Pyrrolidine series evolution. While compound (S)-7 was an attractive starting point, we wanted to improve upon several features to improve its druggability. Initially, we wished to increase potency versus the hGPR40 receptor, but we also hoped to improve both potency and efficacy versus the mGPR40 receptor to facilitate preclinical characterization in efficacy models. Additionally, compound (S)-7 possessed weak cardiac Na+ channel antagonist activity (4.0 µM) and weak peroxisome proliferator-activated receptor gamma (PPARγ) transactivation (17 ± 10 µM, 54% Ymax), which was not desired. There is a possible synergy between the insulin-sensitizing mechanism of PPARγ agonism and the increased insulin secretion via GPR40 agonism that could lead to greater efficacy against diabetes. However, since the safety and efficacy of GPR40 agonists are not fully clinically validated, we did not want to complicate evaluation of the mechanism of action with the regulatory burdens of PPARγ activation.21


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RESULTS AND DISCUSSION Early syntheses of pyrrolidine-2-acetic acids, such as 7, were (purposefully) racemic so that both enantiomers could be separated and tested for GPR40 activity. The initial route to analogs such as 7 employed tosylate 9, the product of the ring opening of tetrahydrofuran 2acetic acid ethyl ester (8) with LHMDS. In a single step, compound 9 was reacted neat with aniline 10 at 130 ºC to both displace the tosylate and concomitantly cyclize to form pyrrolidine 11 in low to modest yields. The racemic mixture was separated by chiral supercritical fluid chromatography (SFC) to provide single enantiomers. The (S)-enantiomer was taken forward for further elaboration. When a benzyl ether was used as a protecting group for analog synthesis, the benzyl group was removed by hydrogenolysis using Pd/C. Phenol 12 was reacted with benzyl halide 13 to give ester 14, which was hydrolyzed with LiOH to give the carboxylic acid (S)-7. Scheme 1. Representative Synthesis of Pyrrolidine 2-Acetic Acid Analogs, as Depicted for Compound (S)-7a,b

a

Reagents and conditions. (a) i. LHMDS, THF; ii. TsCl, 60%; (b) 130 °C, neat, 16%; (c) chiral separation; (d) Pd/C, H2, MeOH/EtOH, 88%; (e) K2CO3, DMF, 93%; (f) LiOH, THF/water,


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44%. bSee supporting information for other compounds that were synthesized via this general route.

The route depicted in Scheme 1 was short, convergent, and it served our initial needs; but, as we found that the (S)-isomers were consistently more potent (see table in supporting information), we sought a new route that would tap the chiral pool of substituted pyrrolidines to synthesize single isomers. Pyrrolidine analogs were synthesized from substituted L-prolines that were commercially available. Aryl iodides, such as compound 16, could be synthesized by in situ formation of diazonium salts and displacement with KI (Scheme 2). From substituted prolines such as 17, the desired prolinol 18 was synthesized via transient (Boc) amine protection and reduction of the carboxylic acid followed by N-Boc deprotection. Unfortunately, direct Narylation of the homologated proline esters via Pd- and Cu-catalyzed couplings mostly led to oligomerization of the proline, resulting in poor yields of N-arylated homoproline analogs. These problems could be circumvented by coupling the reduced β-amino alcohol 19 to an aryl iodide 1622 to give alcohol 20 followed by mesylation and cyanation that, after basic hydrolysis of nitrile 21, led to homologated acid 22. Acidic (HCl) nitrile hydrolysis conditions were unacceptable as these conditions led to epimerization of the acetic group. The above methods form the basis of the synthesis of all N-aryl heterocyclic carboxylic acids reported herein. Scheme 2. Representative Synthesis of Chiral Pyrrolidine GPR40 Agonists, as Depicted for Compound 22a,b


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a

Reagents and conditions. (a) TsOH, NaNO2, KI, 74%; (b) i-BuO2CCl, NMM, THF; NaBH4, H2O, 95%; (c) HCl, dioxane, quant. (d) CuI, NaOH, i-PrOH, 90 °C, 74%; (e) MsCl, NEt3, CH2Cl2; (f) NaCN, DMSO, 50 °C, 100% (2 steps); (g) KOH, EtOH/H2O, 120 °C, 43%. b Similar compounds made using an analogous method; see supporting information. GPR40 is characterized as a Gq-coupled GPCR that promotes intracellular Ca2+ accumulation. As such, we primarily tested compounds in a Ca2+-coupled fluorescence assay, searching for full agonists that elicited high Ymax in CHO cell lines expressing either human or mouse GPR40. Later (vide infra), we found that the CHO cell line used for screening highly overexpressed hGPR40, and compounds with lower efficacy were indistinguishable from compounds with higher efficacy due to receptor reserve. For this reason, Ymax values are not reported here for human EC50 values in the CHO cell assay since all compounds produced 100% Ymax. A HEK cell line with an inducible GPR40 expression vector was employed when we wished to distinguish partial and full activators,23 but this assay system had relatively low throughput and inconsistent receptor expression across different induction experiments so it was not used for routine screening.

For rapid screening, we also employed a CHO cell line


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expressing the mouse GPR40 receptor, which seemed to be somewhat more discriminatory for intrinsic efficacy differences (Ymax) among compounds. In practice, we used mGPR40 efficacy (Ymax) as a surrogate for hGPR40 intrinsic activity and only followed up on select compounds using inducible expression systems to assign compounds as full or partial hGPR40 agonists.

Figure 3. Arbitrary nomenclature (A-C) for rings on the pyrrolidine chemotype of GPR40 agonists. Variations in the N-heterocyclic ring size were explored to determine if a five-membered ring with (S)-stereochemistry had the optimal size and stereochemistry for GPR40 agonist efficacy. For descriptive purposes, the saturated nitrogen heterocycle is referred to as the A ring, the central aromatic ring as the B ring, and the appended aryl ring as the C ring (Figure 3). The A ring was modified to explore the effect of ring size (4-7) on GPR40 activity in combination with a 2, 4-dichlorobenzyl-linked C ring.

Azetidine (S)-23 had submicromolar GPR40 agonist

activity in both the human and the mouse GPR40 cell lines but the compound proved to be chemically unstable due to beta-elimination to the open form.

Azetidine (R)-23 had no

detectable GPR40 agonist activity (at the concentrations tested). In contrast, pyrrolidine (S)-7 was equipotent versus hGPR40 but it showed reduced potency versus the mouse receptor as compared to the corresponding azetidine analog. Both individual isomers of the 6- and 7membered ring compounds (24 (enantiomer 1), 24 (enantiomer 2), 25 (enantiomer 1), and 25 (enantiomer 2)) lost significant GPR40 agonist activity (absolute stereochemistry was not


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determined) versus the 5-membered analogs (S)-7 and (R)-7. These results provided confidence that the original five-membered ring with (S)-stereochemistry, as found in (S)-7, was preferred due to good hGPR40 activity and chemical stability, so further SAR explorations employed a pyrrolidine A-ring. Table 1. Effect of A-ring Size on GPR40 Activity

hEC50 (µM)1 Compound number

Ring Size ± S.D.

4

hKi (µM)2

4

S.D.4 (Ymax) 0.44 ± 0.38

0.26 ± 0.12

(S)-23

mEC50 (µM)3 ±

n.d.

(n = 0)

(88%)

4 (R)-23

135

n.d.

>17

0.34 ± 0.20

0.891

4.5 ± 1.8 (66%)

0.88 ± 0.061

n.d.

>175

>33

n.d.

>33

(n = 0) 5 (S)-7 (n = 1) 5 (R)-7 (n = 1) 24 (enantiomer 1)

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(n = 2) 6 24 (enantiomer 2)

>17

n.d.

>33

6.45

n.d.

>16

115

n.d.

>33

(n = 2) 7 25 (enantiomer 1) (n = 3) 7 25 (enantiomer 2) (n = 3) 1

hGPR40 EC50 in Ca2+ FLIPR assay. 2Human binding Ki versus a radioligand. 3mGPR40 EC50 in Ca2+ FLIPR assay. 4mean of at least two runs. 5N = 1.

With a focus on optimizing the agonist activity of the pyrrolidine-2-acetic series, we next sought to optimize the B ring via heteroaryl replacements and pendant substituents.

This

investigation was accomplished by substitution of a nitrogen or introduction of a methyl group at the 2- and 3-positions of the aryl ring. For comparison purposes, the effect of these changes is illustrated with use of either a 2,4-dichlorobenzyl C ring or a 2-methylbenzyl C ring (Table 2). While the GPR40 activity of a 2,4-dichlorobenzyl moiety in compound (S)-7 versus 2methylbenzyl group in compound 28 are not significantly different, the 2,4-dichlorobenzyl analogs had off-target activities (Na+ channel antagonist, PXR), which led us to focus upon the 2methyl derivatives for later analogs. Both the 2- and 3-pyridine-containing analogs (compounds 26 and 29) had similar hGR40 potency to the parent phenyl analog (S)-7, but they demonstrated a significant loss of mGPR40 activity. The 2- and 3-methyl substitutions (compounds 27 and 30) were not tolerated on the B ring for either human or mouse GPR40 potency, suggesting that this group may occupy a very narrow pocket in the receptor.


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Table 2. SAR of B Ring Analogs

hEC50 (µM)1 ±

Compound

2

hKi (µM)

mEC50 (µM)3 ±

B-ring

R

(S)-7

Ph

2,4-Cl

0.34 ± 0.20

0.895

4.5 ± 1.8 (66%)

26

2-pyridyl

2,4-Cl

0.22 ± 0.047

n.d.

>33

27

2-Me-Ph

2,4-Cl

8.95

n.d.

14 (70%)5

28

Ph

2-Me

0.19 ± 0.080

n.d.

3.4 ± 2.2 (41%)

29

3-pyridyl

2-Me

0.45 ± 0.060

1.8 ± 0.29

>33

30

3-Me-Ph

2-Me

>33

n.d.

>33

number

S.D.4

S.D.4 (Ymax)

1


The linkage between the B and C rings was studied to examine the effects of length on SAR for GPR40 agonist activity (Table 3). A two atom linker, as in benzyl ether 28, showed good hGPR40 potency. Shortening the linker to a methylene resulted in similar human potency. The biaryl ether linker 32 provided the best hGPR40 agonist potency. The sulfoxide, sulfone, and carbonyl linkers (compounds 33, 35, and 36) were not tolerated, suggesting that either a change in conformation or polarity at this position was not tolerated. These results suggest that a one atom linker was preferred for GPR40 activity. The one atom linker was also beneficial because


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(S)-7 (X = CH2O) displayed undesired PPARγ transactivation (17 ± 10 µM, 54% Ymax in a Gal4 gene reporter assay); whereas, compound 31 (X = CH2) showed no PPARγ transactivation and compound 32 (X = O) showed only weak PPARγ transactivation (32 ± 19 µM, 27% Ymax). Table 3. SAR of B- and C-Ring Linker, X

Compound

1

4

2

mEC50 (µM)3 ±

X

hEC50 (µM) ± S.D.

hKi (µM)

28

OCH2

0.19 ± 0.080

n.d.

3.4 ± 2.2 (73%)

31

CH2

0.15 ± 0.075

1.3 ± 0.53

>33

32

O

0.074 ± 0.039

4.4 ± 1.8

5.6 ± 3.3 (69%)

33

S

0.57 ± 0.31

n.d.

>17

34

SO5

>33

n.d.

>33

35

SO2

>33

n.d.

>33

36

CO

4.66

>33

>33

number

S.D.4 (Ymax)

1

hGPR40 EC50 in Ca2+ FLIPR assay. 2Human binding Ki versus a radioligand. 3mGPR40 EC50 in Ca2+ FLIPR assay. 4mean of at least two runs. 5mixture of diastereomers at sulfur. 6N = 1.

Having optimized the disposition of the carboxylic acid relative to the A-B-C rings, we sought to explore C-ring substituents. Relative to the unsubstituted phenyl analog 37 (hGPR40 EC50 = 0.56 µM), a 2,4-dichloro group retained similar potency, as found in compound 38. Introduction of a methyl group at the C-ring 2-position (compound 32) resulted in almost a 10-fold


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improvement in hGPR40 agonist potency compared to the unsubstituted compound 37. The 2methyl substituent seemed optimal, since a 2-fluoro or a 2-ethoxy group resulted in potency losses (compounds 39 and 40), indicating the GPR40 receptor pocket preferred a small group at the ortho position. Larger groups, such as 2-t-Bu and 2-Ph (compounds 42 and 43) decreased potency as well, suggesting a small binding pocket. Small substituents in the 3- and 4-positions were tolerated (compounds 44-48) but they did not substantially affect potency as compared to the 2-position. However, these modifications did not lead to a significant increase in mGPR40 potency and efficacy, which was desired to enable preclinical evaluation of GPR40 pharmacology. Table 4. SAR of Substitution on C Ring

hEC50 (µM)1 ±

Compound R

S.D.

number

4

hKi (µM)2

mEC50 (µM)3 ± S.D.4 (Ymax)

37

H

0.56 ± 0.29

6.5 ± 0.33

>16

38

2,4-Cl

0.365

n.d.

3.25 (120%)

32

2-Me

0.074 ± 0.039

4.4 ± 1.8

5.6 ± 3.3 (69%)

39

2-F

0.15 ± 0.10

1.3 ± 0.0010

6.9 ± 2.4 (50%)

40

2-OEt

1.75

n.d.

>33


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41

3-Me

0.26 ± 0.17

n.d.

2.4 ± 0.98 (83%)

42

2-t-Bu

6.55

n.d.

>17

43

2-Ph

3.5 ± 0.48

n.d.

1.3 ± 0.68 (72%)

44

3-Cl

0.68 ± 0.18

n.d.

>16

45

3-NMe2

1.3 ± 0.56

n.d.

>33

46

4-Me

0.49 ± 0.14

n.d.

>33

47

4-Cl

0.29 ± 0.089

n.d.

>33

48

4-OMe

0.52 ± 0.33

n.d.

>33

1


Having made significant changes to the linking moiety and C-ring substituent, we looked to reoptimize the pyrrolidine A ring, exploiting the synthetic intermediacy of prolinol derivatives, many of which are readily available. Introduction of a methyl group at the 2- or 5-positions dramatically reduced potency (compounds 49 and 57, Table 5). These SAR points suggest the importance of the pyrrolidine heterocycle and the aniline planarity with the aryl ring. Small substituents at the 3- and 4-positions (compounds 50 and 52) improved agonist potency against both human and mouse GPR40 but unexpectedly the 4-cis-fluoro substituent (compound 53), while enhancing hGPR40 potency, exhibited attenuated potency versus the mouse receptor. These results prompted us to synthesize the 4-cis-CF3 analog 54, providing for the first time in this series submicromolar agonist potency versus the mouse receptor. A similar result was obtained with the oxygen linker, as seen in compound 22 (first depicted in Scheme 2). In


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contrast to the 4-cis-CF3 substituent, the polar 4-cis-OMe and larger 4-cis-phenyl groups were disfavored (compounds 55 and 56). We hypothesized that one role of the 4-cis-CF3 group is to alter the conformation of the pyrrolidine ring. Indeed, an energetic exploration of the A and B rings of unsubstituted analog 32 and analog 22 reveals that the unsubstituted pyrrolidine 32 weakly prefers to have the acid group pseudo-equatorial (by 2.2 kcal/mol) while the pseudoaxial conformation of the acid group is more accessible with the 4-CF3-substituted pyrrolidine 22 (negligible energy difference for the conformations) (Figure 4). The favorable profile afforded by the 4-cis-CF3 substituent led us to utilize this moiety for future SAR explorations. With the optimized 4-cis-CF3 group on the pyrrolidine A ring, our attention was again refocused on further substitution of the phenyl C ring (Table 6). The 2-Me group in compound 22 was required for optimal potency. Substitution at the 3-, 4-, and 6-positions (compounds 60-64) led to analogs with similar potency as compound 22.

Table 5. SAR of Substitution on A Ring

hEC50 (µM)1

Compound R

X ± S.D.

number

4

2

hKi (µM)


31

H

CH2

0.15 ± 0.075

1.3 ± 0.53

>33

49

2-Me

CH2

>33

235

>33


17

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50

3-trans-Me

CH2

0.12 ± 0.065

0.31 ± 0.0050

1.9 ± 0.98 (27%)

51

3-trans-Ph

CH2

11 ± 6.1

n.d.

>33

52

4-cis-Me

CH2

0.14 ± 0.050

0.36

1.1 ± 0.75 (43%)

53

4-cis-F

CH2

0.074 ± 0.037

0.18 ± 0.067

>33

54

4-cis-CF3

CH2

0.067 ± 0.026

0.073 ± 0.010 0.71 ± 0.30 (54%)

22

4-cis-CF3

O

0.073 ± 0.047

0.035 ± 0.027 0.33 ± 0.33 (84%)

55

4-cis-OMe

O

2.3 ± 1.2

9.0 ± 3.9

0.96 ± 0.17 (76%)

56

4-cis-Ph

CH2

>17

>25

>33

57

5-cis-Me

CH2

6.25

n.d.

>33

1


Figure 4. Overlay of low energy conformations of compounds 32 and 22 A and B rings only, demonstrating the influence of a 4-cis-CF3 group on the pyrrolidine ring structure. Table 6. Effect of C-Ring Substitutions on GPR40 Activity in CF3-Containing Pyrrolidine Analogs


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hEC50 (µM)1 ±

Compound R

S.D.4

number

2

hKi (µM)


22

2-Me

0.073 ± 0.047

0.039 ± 0.027

0.33 ± 0.12 (84%)

58

2-CF3

0.31 ± 0.11

n.d.

1.3 ± 0.26 (42%)

59

2-OCF3

0.17 ± 0.067

n.d.

0.74 ± 0.35 (44%)

60

2-Me, 4-OBn

0.12 ± 0.049

0.010 ± 0.0060

1.5 ± 0.86 (34%)

61

2-Me, 4-OMe

0.055 ± 0.025

0.039 ± 0.018

0.17 ± 0.041 (41%)

62

2-Me, 3-Me

0.11 ± 0.090

0.06 ± 0.02

0.50 ± 0.42 (100%)

63

2-Me, 3-Cl

0.082 ± 0.054

0.013 ± 0.007

0.43 ± 0.11 (50%)

64

2-Me, 6-Me

0.082 ± 0.024

n.d.

0.27 ± 0.049 (36%)

1

hGPR40 EC50 in Ca2+ FLIPR assay. 2Human binding Ki versus a radioligand. 3mGPR40 EC50 in Ca2+ FLIPR assay. 4mean of at least two runs.

While we thought that improved potency was possible,24-26 we hoped that compound 22 would be of sufficient potency to blunt a glucose excursion in an intraperitoneal glucose tolerance test (iGTT) in a normal lean mouse. The compound achieved a Cmax of 4.7 µM (AUC04h

= 7.0 µM*h) when it was administered orally at 10 mg/kg in mice, but there was no reduction


19

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in plasma glucose excursion after a glucose challenge.

Even a 100 mg/kg dose was not

efficacious, suggesting that greater agonist potency and/or efficacy (Ymax) was required. We wondered if the intrinsic efficacy (Ymax = 84%) of compound 22 in the Ca2+ FLIPR assay may have been misleading due to the artificial overexpression in the CHO cell line outlined earlier. We suspected the suboptimal intrinsic efficacy may have contributed to the lack of an in vivo effect in the mouse. As such, we decided to perform an in-depth examination of its agonist activity in hGPR40-expressing HEK cells. While it appeared that all of our compounds were full agonists in the hGPR40 overexpressing CHO cell line, an inducible expression revealed partial versus full agonists. Under conditions that induced high expression of hGPR40, compound 22 appeared to be a full activator as compared to linoleic acid (Figure 5). However, when tested under conditions that induced low expression of hGPR40, we found that 22 was a partial activator of hGPR40. While a similar experiment in the mouse cell line was not performed, we surmised the lack of in vivo activity in the mouse pharmacology model was due to its partial agonism. Additionally, when tested for in vitro GLP-1 secretion in STC-1 cells, a GPR40expressing murine enteroendocrine cell line that produces GLP-1, compound 22 did not produce a response. We noticed that many GPR40 ligands, including linoleic acid/DHA, are significantly longer than compound 22 and, given the sensitivity of GPR40 to ligand length (vis-à-vis medium and short-chain fatty acids), we suspected that we may need to add substituents to the C-ring to improve agonist efficacy and/or potency. Given that the C-ring was permissive to substitution at the 3- and 4-positions, it made sense to append aryl and benzyl groups at these positions. In a similar manner, scientists at Amgen had employed changes (meta versus para) to the central linkage of their chemotype in conjunction with placement of a fluoro-methoxyphenyl terminal


20


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Page 22 of 54

substituent.17,27 This combination resulted in a more robust agonist efficacy response in their chemical series.

A 2-F, 5-OMe phenyl group was thus appended to biarylmethane and

biarylether substituents in both the unsubstituted and 4-cis-CF3-substituted (S)-pyrrolidines (e.g. compounds 31 and 22).

Appending the aryl ring to the 4-position of the phenyl C-ring

(compound 66) resulted in similar human GPR40 potency as compared with compound 31, however it exhibited surprisingly robust agonist efficacy (Ymax = 160%) at the mouse receptor with micromolar potency. In the 4-CF3-substituted pyrrolidine series, connecting a 2-F, 5-OMe phenyl group to the 3-position of the phenyl C ring resulted in a reduction of hGPR40 activity as compared to 22, although Ymax (170%) was significantly enhanced. Appending the 2-F, 5-OMe phenyl group to the 4-position of the C-ring in compound (S,S-68) resulted in reduced human and mouse potency but very potent binding. Table 7. Substitution of Aryl Groups on the Phenyl C Ring

Compound number

A

X

R3

R4

hEC50 (µM)1 ± S.D.4

hKi (µM)2


31

H

CH2

H

H

0.15 ± 0.075

1.3 ± 0.53

>33

65

H

O

H

0.31 ± 0.17

n.d.

3.0 ± 2.4 (83%)


21

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F

H

66

CH2

0.22 ± 0.11

0.315

1.9 ± 0.76 (160%)

H

0.073 ± 0.047

0.039 ± 0.027

0.33 ± 0.12 (84%)

H

0.505

0.0491

0.78 (170%)5

0.49 ± 0.21

0.045 ± 0.010

2.4 ± 1.3 (46%)

H O

22

CF3

O

67

CF3

O

(S,S)-68

CF3

CH2

H

H

1

hGPR40 EC50 in Ca2+ FLIPR assay. 2Human binding Ki versus a radioligand. 3mGPR40 EC50 in Ca2+ FLIPR assay. 4mean of at least two runs. 5N = 1. However, it is notable that in the earlier SAR developed for partial activators with the CF3-pyrrolidine, including compound 22, the compounds were synthesized using commercial (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid, resulting in enantiopure (>99% ee) compounds. When compound 68 was initially synthesized, it was the first CF3-pyrrolidine analog we made using CF3-prolinol that was synthesized in-house for scale-up and analog synthesis following a literature route.28 To evaluate the impact of agonist efficacy on in vivo pharmacology, we scaled up compound 68 and, upon rigorous characterization, found that 68


22


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Page 24 of 54

contained ~10-20% of its enantiomer as a result of epimerization of an intermediate in the synthesis of the 4-CF3-prolinol. The synthetic route to compound 68 begins with the oxidation of (2S,4R)-2-benzyl 1-tert-butyl 4-hydroxypyrrolidine-1,2-dicarboxylate 69 with PCC to give the carbonyl 70 (Scheme 3). The tertiary alcohol 71 was formed by reaction with CF3TMS and TBAF and it was eliminated through the use of SOCl2 and pyridine. We postulate that ester 72 was partially racemized at this step since this stereocenter controls formation of the CF3 stereocenter and no diastereomers were observed in subsequent steps. The olefin was reduced concomitantly with hydrogenolysis of the benzyl ester via H2 and Pd/C to give acid 73 possessing a cis relationship between the CF3 and the carboxylic acid functionalities. The carboxylic acid was reduced using mixed anhydride formation and NaBH4 reduction and the Boc group was removed to give prolinol salt 75. CF3Prolinol 75 was coupled to 1-bromo-4-iodobenzene followed by homologation via mesylation and NaCN displacement to provide nitrile 77. The

CD

ring

system

was

formed

via

Suzuki

coupling

of

(2-fluoro-5-

methoxyphenyl)boronic acid 78 and 4-bromo-2-methylbenzoic acid 79 in good yield.

The

carboxylic acid was reduced with LAH to give alcohol 81, which was converted to bromide 82 via reaction with MsCl and LiBr. An organozinc reagent of bromide 82 was generated using activated zinc, and this organometallic reagent was reacted with bromide 77 in good yield. Acid 68 was formed by KOH-mediated hydrolysis of the nitrile to give 68 as a scalemic mixture (as an 85:15 ratio of S,S:R,R). The enantiomers were separated by chiral SFC. Later, enantiopure prolinol was obtained following an alternative literature route29 and/or via crystallization of a diastereomeric amine salt with (1R)-( ̶ )-10-camphorsulfonic acid (see supporting information). Scheme 3. Synthesis of (S,S)-68 and 68a


23

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a

Reagents and conditions. (a) PCC, CH2Cl2, 80%; (b) CF3TMS, TBAF, 79%; (c) SOCl2, pyridine, DIPEA, Boc2O, 62%; (d) H2, Pd/C, EtOH, 90%; (e) NMM, i-BuCOCl, NaBH4, THF/water, 84%; (f) HCl/dioxane, 93%; (g) 1-bromo-4-iodobenzene, CuI, NaOH, i-PrOH, 77%; (h) MsCl, TEA, CH2Cl2; NaCN, DMSO, 87% (2 steps); (i) TBAB, Pd(Ph3P)4, Na2CO3, water, (92%); (j) LAH, THF, 89%; (k) LiBr, MsCl, TEA, THF, 89%; (l) Zn, TMS-Cl, ethylene dibromide; Pd(Ph3P)4, THF, 86%; (m) KOH, EtOH/water, 70%; (n) SFC chiral separation.

Compound 68 was initially tested in in vitro assays as a scalemic mixture since it appeared to be very potent and efficacious (high Ymax) in both human and mouse FLIPR assays. The intrinsic efficacy compound 68 was evaluated using a cell line with inducible expression of hGPR40 (Figure 5).

The inducible assay results showed that scalemic 68 exhibited full


24


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Page 26 of 54

activation of GPR40 even at low expression levels of receptor. A similar observation was made in a mouse inducible expression cell line (data not shown). The inter-species consistency of GPR40 agonism enabled preclinical characterization of 68 in mouse with improved confidence that it could model human pharmacology.

Figure 5. (A) Inducible hGPR40 assay with high expression of GPR40. (B) Inducible hGPR40 assay with low expression of GPR40. Once the enantiomers were separated, we were surprised to find that (S,S)-68 (as a single enantiomer) retained potent hGPR40 binding (0.045 µM) but (R,R)-68 was the more potent and efficacious agonist (Figure 6). Furthermore, compound (R,R)-68 occupied a binding mode that positively potentiated the radioligand used for the binding assay while (S,S)-68 displaced the radioligand (Figure 7).


25

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F

F N

(S)

N

CF 3

(S)

OMe

(S,S)-68

OMe

84

OH

O

hGPR40 = 0.49 ± 0.21 µM mGPR40 = 2.4 ± 1.3 µM (46%)

F

hGPR40 = 1.7 µM mGPR40 = 1.1 µM (140%)

F N

OMe

CF 3

(S)

O

HO

(R)

(R)

(R,R)-68 HO

O

hGPR40 = 0.11 ± 0.056 µM mGPR40 = 0.054 µM ± 0.036 (210%)

Figure 6.

N

CF 3

(R)

OMe

(S)

CF 3

(R)

85 HO

O

hGPR40 = 0.15 ± 0.11 µM mGPR40 = 0.053 ± 0.028 µM (210%)

Impact of pyrrolidine substituent stereoisomers on GPR40 binding potency and

agonist efficacy.

Figure 7. Binding assay results with compounds (S,S)-68 and (R,R)-68 (negative slope indicates radiolabel displacement; positive slope indicates increase in radiolabel binding).


26


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Page 28 of 54

In an effort to deduce which chiral center was responsible for the change in binding mode and agonist efficacy, the (2S,4R)- and (2R,4S)-(trifluoromethyl)-analogs of 68 were synthesized. Compound 84 with (2S)-stereochemistry remained a micromolar partial activator while (2R)analog 85 retained full activation and good potency, suggesting that the (R)-acetic substituent potently influenced agonist activity in combination with the 2-F,5-OMe phenyl ring (Figure 6). We were surprised to find that (R,R)-68 was the most potent analog, since earlier work in the chemotype indicated that the (S)-enantiomer was always more potent across 40 different enantiomeric pairs tested (see supporting information). It was subsequently reported in the literature that others had also observed that a change in stereochemistry in different chemotypes could result in complimentary binding modes and full agonist efficacy.13,27 Since free fatty acids are present in circulation, we studied the activation of hGPR40 by compounds (S,S)-68 and (R,R)-68 in combination with DHA, a potent and efficacious endogenous long chain fatty acid4 (Figure 8).

At sub-efficacious (3 nM) concentrations,

compound (R,R)-68 significantly enhances the agonist activity of DHA and left shifts the concentration response curve. On the other hand, (S,S)-68 does not significantly potentiate the activity of DHA in vitro. Even concentrations of (S,S)-68 that show significant agonism do not shift the DHA curve (data not shown). This data supports a change in binding mode, suggesting that compound (R,R)-68 can be a full agonist and a positive allosteric modulator of DHA’s effect on hGPR40.


27

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Figure 8. (A) Potency of DHA versus DHA + 3 nM compound (R,R)-68. (B) Potency of DHA versus DHA + 10 nM compound (S,S)-68. We evaluated the effect of compound (R,R)-68 on insulin and GLP-1 secretion in vitro. In isolated primary mouse islets, (R,R)-68 showed a dose-dependent (0.016-2.0 µM) increase in insulin secretion (Figure 9), demonstrating that (R,R)-68 promoted glucose-stimulated insulin secretion.

Compound (R,R)-68 was also quite potent in the STC-1 cell line, a mouse

enteroendocrine cell line,30,31 showing a 0.33 µM EC50 for GLP-1 secretion (Figure 10). Interestingly, enantiopure (S,S)-68 also exhibited GLP-1 secretion but with an EC50 of 3.2 µM, (10-fold less potent than its enantiomer). These findings demonstrated that these full activators show a robust dual mechanism of action as compared to partial agonists, effecting both glucosestimulated insulin secretion as well as GLP-1 secretion. However, it remained unclear if the dual mechanism of action was due to its binding mode, agonist efficacy, or another determinant.


28


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Page 30 of 54

Figure 9. In vitro insulin secretion from isolated primary mouse islets.

Figure 10. GLP-1 secretion in STC-1 cells.

While GPR40 has been traditionally known to signal through the Gq–coupled pathways, in a recent publication32 it was reported that synthetic agonists can promote GPR40-dependent cAMP accumulation via a Gs-coupled pathway.

The Gq + Gs-coupled agonists showed


29

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substantially higher in vivo glucose lowering efficacy via increased GLP-1 as compared with agonists with only Gq-coupled activity. Endogenous long chain fatty acids as well as partial activators (such as 1) only act through the Gq-coupled pathway. Partial agonists 22 and (S,S)-68 as well as full agonist (R,R)-68 were screened for Gs-coupled activity in a cAMP assay in a hGPR40-expressing HEK cell line, demonstrating that (R,R)-68 potently activated cAMP accumulation with good efficacy while (S,S)-68 showed lower potency and efficacy (Table 8). Compound 22 was inactive in the cAMP assay. These results are consistent with others’ findings that ligands that promote GPR40-dependent activation of Gq- and Gs-coupled pathways demonstrate a dual mode of action (i.e. insulin and incretin secretion).32 Table 8. cAMP Assay for GPR40 Partial and Full Agonists

1

Compound number

hEC50 (µM)1 ± S.D.2

Ymax

22

>20

5%

(S,S)-68

2.4

28%

(R,R)-68

0.21

140%

hGPR40 EC50 in Ca2+ FLIPR assay. 2mean of at least two runs.

To study the effects of intrinsic activity in vivo, the pure enantiomers (S,S)-68 and (R,R)68 were evaluated for their ability to improve glycemic control in mice. The compounds were tested in an oral glucose tolerance test in C57BL/6J mice at 0.3 or 1 mg/kg doses (Figure 11). In this assay, the compounds were administered orally 1 hour prior to an orally administered glucose bolus, and plasma glucose levels were measured at -60, -20, 0, 20, 40, 60, and 120 min timepoints. Statistically significant glucose lowering was not observed for the 0.3 mg/kg (R,R)68 or 1 mg/kg (S,S)-68 doses. Relative to vehicle, the 1 mg/kg dose of (R,R)-68 was highly


30


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Page 32 of 54

effective in reducing glucose excursion (∆AUC0-120’ = 39%), leading to further focus on this compound.

Figure 11. In vivo evaluation of (R,R)-68 and (S,S)-68 efficacy in mice. Table 9. Pharmacokinetic Profile of (R,R)-68 in Mice dose (mg/kg)

0.2

T1/2 (h)

7

Vss (L/kg)

0.1

Cl (mL/min/kg)

0.2

F (%)

83a

a

0.5 mg/kg dose Given the favorable in vivo efficacy profile and its desired dual mechanism of action,

compound (R,R)-68 was extensively profiled for ADME properties (Table 9). Compound (R,R)68 had low clearance at 0.2 mL/min/kg with a 7 hour elimination half-life and low volume of distribution (Vss = 0.1 L/kg) when administered intravenously in mice. Good bioavailability (83%) was observed. The compound exhibited high plasma protein binding with free fractions of 1.1% and 0.1% in mouse and human plasma, respectively.

Compound (R,R)-68 is


31

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metabolically stable when incubated with human, mouse, and monkey (cyno) liver microsomes with a half-life >120 minutes. A major metabolite results from demethylation of the fluoromethoxyphenyl D ring. A small amount of glutathione (GSH) adduct formation was observed in the presence of liver microsomes and NADPH, suggesting some metabolic reactivity of the compound. Due to increased risk of cardiovascular (CV) pathology in type 2 diabetic patients, compound (R,R)-68 was evaluated in CV safety studies. To evaluate the potential for undesired CV effects, (R,R)-68 was tested in a single-dose (80 mg/kg) study in male rats that were fitted with telemetry equipment for non-invasive heart rate and blood pressure measurement, and locomotor activity was also monitored. The compound had no effect on mean arterial blood pressure and heart rate, but mild transient hyperactivity was observed after administration of (R,R)-68, highlighting one concern for advancement of this compound. When (R,R)-68 was extensively counterscreened for off-target activities, it was found to have PPARγ binding (1.6 µM) and transactivation activity (EC50 = 4.4 µM, Ymax = 40%) in a Gal4 reporter assay. To further study its PPARγ activity, (R,R)-68 was incubated with 3T3L1 cells to assess effects on free glycerol levels as a marker of PPARγ-mediated adipocyte differentiation. In this assay, (R,R)-68 promoted 3T3L1 differentiation at 3 µM, further suggesting that the compound could demonstrate in vitro PPARγ-related effects.

PPARγ agonists have many

benefits in type 2 diabetes that have been clinically validated, acting to improve insulin sensitivity, and this effect could be beneficial in conjunction with the GPR40 insulin secretagogue effect to promote greater reductions in HbA1c.33 However, there are potential regulatory barriers to clinical advancement of PPARγ agonists: the FDA requires studies to evaluate the risk of adverse CV events as well as risk assessment for bladder cancers and bone


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effects.21

Page 34 of 54

As such, we did not want to complicate development of a GPR40 agonist by

introducing polypharmacology.34-37

In addition to the PPARγ activity, (R,R)-68 showed

micromolar CYP inhibition (2C8 IC50 = 2.1 µM, 2C9 IC50 = 3.8 µM, 3A4 IC50 = 1.7 µM), prompting concerns over drug-drug interactions in the clinic. Furthermore, (R,R)-68 bound to other nuclear hormone receptors (progesterone receptor and androgen receptor).

For these

reasons, compound (R,R)-68 was not advanced, but it was viewed as a good lead for further optimization due to its dual mechanism of action and favorable ADME profile.

CONCLUSIONS In summary, we report a series of novel pyrrolidine GPR40 agonists. In our early work, we found that a biaryl ether or biaryl methane linker was optimal with respect to the pyrrolidine2-acetic moiety.

Addition of a 4-cis-CF3 group to the pyrrolidine ring increased in vitro

mGPR40 potency as well as improved the human binding Ki, resulting in the identification of compound 22 as a program lead. However, upon more extensive evaluation, we found that compound 22 was a partial agonist versus both the mouse and human receptors, leading to an understanding that the overexpressed CHO cell line could not distinguish full and partial hGPR40 agonists. By optimizing the mGPR40 potency and efficacy to induce Ca2+ flux in the CHO cell line, we identified compounds that were efficacious in mice as demonstrated by a reduction in glucose excursion in an oGTT. These compounds retained full agonist efficacy versus hGPR40, providing confidence that the observed rodent activity would translate into the clinic. In one exciting breakthrough, the discovery of a minor enantiomeric impurity (R,R)-68 revealed that the two enantiomers occupied different binding sites on the receptor and that compounds binding in these different sites could enhance the binding and efficacy of the other.


33

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Inducible GPR40 expression studies demonstrated that (R,R)-68 was a full activator while (S,S)68 remained a partial agonist. In addition to its ability to promote glucose-dependent insulin secretion from islets, (R,R)-68 was also shown to stimulate GLP-1 secretion in the STC-1 assay, demonstrating a desirable dual mechanism of action. Compound (R,R)-68 was effective at lowering plasma glucose levels in mice in an oral glucose tolerance test at 1 mg/kg and also showed synergistic effects with free fatty acids in vitro. Compound (R,R)-68 was well tolerated at high doses in mice and telemetrized rats and displayed a favorable ADME profile.

In

extensive profiling, compound (R,R)-68 showed several off-target effects in vitro and in vivo (hyperactivity) and its advancement was halted for these reasons, as well as concern over irreversible metabolic conjugation with glutathione and off-target PPARγ activity. Nevertheless, (R,R)-68 served as a valuable research tool to explore the therapeutic utility of GPR40 agonists in vitro and in vivo. Further optimization will be reported in due course wherein reduced off target activity and increased in vivo potency were achieved.

EXPERIMENTAL SECTION Chemistry. All anhydrous reactions were carried out using oven-dried glassware under an atmosphere of argon or nitrogen. All reagents and solvents were obtained from commercial vendors and used without further purification unless otherwise indicated. NMR spectra (1H, 13C) were recorded on JEOL JNM-ECP500, JEOL GSX400 and Bruker 400 spectrometers. Chemical shifts were given in parts per million (ppm) downfield from internal reference tetramethylsilane standard; coupling constants (J values) were given in hertz (Hz). LC/MS data were recorded on a Shimadzu LC-10AT equipped with a SIL-10A injector, a SPD-10AV detector normally operating at 220 nm and interfaced to a Micromass ZMD mass spectrometer. LC/MS or HPLC


34


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Page 36 of 54

retention times were reported using a Phenomenex Luna C-18 4.6 mm × 50 mm column eluting with a 12 min gradient of 0-100% solvent B, where solvent A was 5:95:0.05 CH3CN-H2O-TFA and solvent B was 95:5:0.05 CH3CN-H2O-TFA. Reactions were monitored by TLC using 0.25 mm E. Merck silica gel plates (60 F254) and were visualized using UV light. All compounds were found to be >95% pure by HPLC analysis unless otherwise noted. 1-(4-Iodophenoxy)-2-methylbenzene (16). To a solution of 4-(o-tolyloxy)aniline 15 (0.50 g, 2.5 mmol) in CH3CN (10 mL) was added p-TsOH (1.4 g, 7.5 mmol). The thick suspension was cooled to 0 °C and a solution of sodium nitrite (0.35 g, 5.0 mmol) and KI (1.0 g, 6.3 mmol) in water (1.5 mL) was added gradually. The reaction mixture was stirred for 1 min at 0 °C and then warmed to rt for 1 h. To the mixture was added water (40 mL), sat. NaHCO3 (until pH = 9-10) and 2 M aq. Na2S2O3 (5 mL). The product was extracted with CH2Cl2 (3x). The combined organic layers were washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography to provide 1-(4-iodophenoxy)-2-methylbenzene (0.57 g, 1.9 mmol, 74 % yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.64 - 7.56 (m, 2H), 7.31 - 7.26 (m, 1H), 7.24 - 7.16 (m, 1H), 7.16 - 7.08 (m, 1H), 6.98 - 6.90 (m, 1H), 6.73 - 6.64 (m, 2H), 2.23 (s, 3H). (2S,4S)-tert-Butyl 2-(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1-carboxylate (18). (2S,4S)-1-(tert-Butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid 17 (purchased from NeoMPS SA) (1.8 g, 6.3 mmol) was dissolved in dry THF (26 mL) and the solution was cooled to -10 °C. 4-Methylmorpholine (0.73 mL, 6.7 mmol) and isobutyl chloroformate (0.88 mL, 6.7 mmol) were added sequentially and the mixture was stirred at -10 °C for 45 min. The mixture was filtered and added to a solution of NaBH4 (0.48 g, 13 mmol) in water (3.4 mL) cooled to 0 °C. The reaction mixture was stirred for 2 h and slowly warmed to rt. The reaction


35

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was quenched with sat. aq. NH4Cl and the product was extracted with EtOAc (3 x). The combined organic layers were washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography to provide (2S,4S)-tert-butyl 2(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1-carboxylate (1.6 g, 6.1 mmol, 95 % yield) as a colorless oil. LC-MS Anal.Calc’d for C11H18F3NO3: 269.12, found [M+H] 270.0. 1H NMR (400 MHz, CDCl3) δ 4.89 - 4.67 (m, 1H), 4.09 - 3.94 (m, 1H), 3.90 - 3.77 (m, 1H), 3.77 - 3.59 (m, 3H), 3.33 (br t, J=10.6 Hz, 1H), 2.91 - 2.77 (m, 1H), 2.36 - 2.22 (m, 1H), 1.48 (s, 9H). (2S,4S)-tert-Butyl 2-

((2S,4S)-4-(Trifluoromethyl)pyrrolidin-2-yl)methanol, HCl (19).

(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1-carboxylate (1.6 g, 6.1 mmol) was dissolved in a 4 N solution of HCl in dioxane (10 mL, 40 mmol) and stirred at rt for 1 h. The reaction mixture

was

concentrated

and

azeotroped

with

MeOH

to

give

((2S,4S)-4-

(trifluoromethyl)pyrrolidin-2-yl)methanol, HCl (1.3 g, 6.1 mmol, 100 % yield) as a colorless oil. LC-MS Anal.Calc’d for C6H10F3NO: 169.07, found [M+H] 170.0.

1

H NMR (400 MHz,

METHANOL-d4) δ 3.92 - 3.86 (m, J=11.9, 3.5 Hz, 1H), 3.86 - 3.77 (m, 1H), 3.70 (dd, J=11.7, 5.9 Hz, 1H), 3.61 (br d, J=2.8 Hz, 1H), 3.51 - 3.37 (m, 2H), 2.42 (dt, J=13.8, 6.9 Hz, 1H), 1.98 (ddd, J=13.2, 10.6, 10.3 Hz, 1H). ((2S,4S)-1-(4-(o-Tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)methanol (20). 1-(4Iodophenoxy)-2-methylbenzene (0.18 g, 0.59 mmol), ((2S,4S)-4-(trifluoromethyl)pyrrolidin-2yl)methanol, HCl (0.10 g, 0.49 mmol), CuI (2.3 mg, 0.012 mmol), and freshly powdered NaOH (0.059 g, 1.5 mmol) were combined in a capped vial, which was sealed and purged with argon. Isopropanol (3 mL) was added and the reaction mixture was heated to 90 °C overnight. The reaction mixture was cooled to rt and diluted with water. The product was extracted with CH2Cl2 (3x).

The combined organic layers were washed with brine, dried (MgSO4), and


36


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Page 38 of 54

concentrated. The crude product was purified by silica gel chromatography to provide ((2S,4S)1-(4-(o-tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)methanol (0.13 g, 0.36 mmol, 74 % yield) as a colorless oil. LC-MS Anal.Calc’d for C19H20F3NO2: 351.14, found [M+H] 352.0. 1H NMR (400 MHz, CDCl3) δ 7.40 (br d, J=7.1 Hz, 1H), 7.29 - 7.18 (m, 3H), 6.94 (d, J=9.1 Hz, 1H), 6.71 (d, J=9.1 Hz, 1H), 4.99 (s, 2H), 3.99 - 3.87 (m, J=7.5, 7.5, 4.8, 2.8 Hz, 1H), 3.80 (ddd, J=11.2, 4.4, 4.2 Hz, 1H), 3.69 - 3.58 (m, 2H), 3.51 (t, J=9.2 Hz, 1H), 2.38 (s, 3H), 2.37 - 2.23 (m, 2H), 1.62 (dd, J=7.7, 3.9 Hz, 1H), 1.27 (t, J=7.1 Hz, 1H). 2-((2S,4S)-1-(4-(o-Tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile

(21).

((2S,4S)-1-(4-(o-tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)methanol (0.073 g, 0.21 mmol) was dissolved in CH2Cl2 (1 mL) and the solution was cooled to 0 °C. Methanesulfonyl chloride (0.024 mL, 0.31 mmol) and TEA (0.058 mL, 0.41 mmol) were added sequentially and the reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with EtOAc and washed with 1 N HCl, sat. aq. NaHCO3, and brine. The organic layer was dried (MgSO4) and concentrated. The crude product was redissolved in DMSO (1 mL) and NaCN (0.041 g, 0.83 mmol) was added. The reaction mixture was stirred at 50 °C overnight. The reaction mixture was cooled to rt and water was added. The product was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried (MgSO4), and concentrated. The crude product was purified

by silica

gel chromatography to provide 2-((2S,4S)-1-(4-(o-

tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (0.075 g, 0.21 mmol, 100 % yield) as a colorless oil. LC-MS Anal.Calc’d for C20H19F3N2O: 360.14, found [M+H] 360.9. 1H NMR (400 MHz, CDCl3) δ 7.24 (br d, J=6.8 Hz, 1H), 7.16 - 7.09 (m, 1H), 7.06 - 6.97 (m, 1H), 6.91 (d, J=9.1 Hz, 2H), 6.80 (d, J=8.1 Hz, 1H), 6.62 (d, J=9.1 Hz, 2H), 4.20 (dddd, J=10.7, 5.7, 5.5, 3.2 Hz, 1H), 3.59 (d, J=8.3 Hz, 2H), 3.18 - 3.00 (m, 1H), 2.79 (dd, J=16.8, 3.2 Hz, 1H), 2.69


37

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(ddd, J=13.7, 9.3, 7.6 Hz, 1H), 2.47 (dd, J=16.9, 8.8 Hz, 1H), 2.28 (s, 3H), 2.22 (ddd, J=13.7, 8.0, 5.8 Hz, 1H). 2-((2S,4S)-1-(4-(o-Tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetic acid, TFA (22). 2-((2S,4S)-1-(4-(o-Tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (0.036 g, 0.099 mmol) was dissolved in EtOH (1.5 mL) in a microwave tube and 6 M aq. KOH (0.50 mL, 3.0 mmol) was added. The reaction was microwaved at 150 °C for 30 min. The reaction mixture was concentrated. The residue was acidified to pH 2 with 1 N aq. HCl and the product was extracted with EtOAc (3x).

The combined organic layers were dried (MgSO4), and

concentrated. The crude product was purified by RP-Prep. HPLC. The HPLC fractions were lyophilized to give 2-((2S,4S)-1-(4-(o-tolyloxy)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetic acid, TFA (0.021 g, 0.042 mmol, 43 % yield) as a brown solid. LC-MS Anal.Calc’d for C20H20F3NO3: 379.14, found [M+H] 379.9.

1

H NMR (400 MHz, CD3CN) δ 7.25 (br d, J=7.6

Hz, 1H), 7.13 (br t, J=7.7 Hz, 1H), 7.01 (t, J=7.3 Hz, 1H), 6.86 (d, J=9.1 Hz, 2H), 6.75 (d, J=8.1 Hz, 1H), 6.70 (d, J=9.1 Hz, 2H), 4.26 - 4.14 (m, 1H), 3.60 - 3.45 (m, 2H), 3.27 - 3.08 (m, 1H), 2.81 (dd, J=15.9, 3.0 Hz, 1H), 2.62 (ddd, J=13.5, 9.3, 7.8 Hz, 1H), 2.24 (s, 3H), 2.31 - 2.19 (m, 1H), 2.04 - 1.96 (m, 1H). Analytical HPLC: RT = 9.8 min, HI: 98.6%. (S)-2-Benzyl 1-tert-butyl 4-oxopyrrolidine-1,2-dicarboxylate (70). To a solution of (2S, 4R)-2-benzyl 1-tert-butyl 4-hydroxypyrrolidine-1,2-dicarboxylate 69 (20 g, 62 mmol) in CH2Cl2 (400 mL) was added pyridinium chlorochromate (200 g, 93 mmol). The resultant solution was stirred overnight at rt. The solution was filtered through a Celite pad and washed with sat. aq. NaHCO3 and 10% citric acid. The solution was dried (Na2SO4) and concentrated. The crude product was purified by silica gel chromatography to provide (S)-2-benzyl 1-tert-butyl 4oxopyrrolidine-1,2-dicarboxylate (16 g, 50 mmol, 80 % yield) as a colorless oil.

LC-MS


38


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Anal.Calc’d for C17H21NO5: 319.14, found [M+H-Boc] 221.0.

Page 40 of 54

1

H NMR (400 MHz, CDCl3) δ

7.34 (br d, J=3.5 Hz, 5H), 5.36 - 5.06 (m, 2H), 4.96 - 4.66 (m, 1H), 3.96 - 3.78 (m, 2H), 3.04 2.78 (m, 1H), 2.56 (br d, J=18.4 Hz, 1H), 1.51 - 1.32 (m, 9H). (2S)-2-Benzyl 1-tert-butyl 4-hydroxy-4-(trifluoromethyl) pyrrolidine-1,2-dicarboxylate (71). To a solution of (S)-2-benzyl 1-tert-butyl 4-oxopyrrolidine-1,2-dicarboxylate (20 g, 62 mmol) at 0 °C in THF (300 mL) was added (trifluoromethyl)trimethylsilane (18 g, 130 mmol) and 1 M TBAF in THF (2.2 mL, 2.2 mmol). The reaction mixture was warmed to rt and stirred overnight. Sat. aq. NH4Cl (100 mL) was added and the reaction mixture was stirred for 15 min. A 1 M solution of TBAF in THF (100 mL, 100 mmol) was added and the mixture was stirred for 2 h. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with water and brine, dried (Na2SO4), and concentrated. Purification via silica gel chromatography provided (2S)-2-benzyl 1-tert-butyl 4hydroxy-4-(trifluoromethyl)pyrrolidine-1,2-dicarboxylate (19 g, 49 mmol, 79 % yield) as colorless oil. LC-MS Anal.Calc’d for C18H22F3NO5: 389.15, found [M+H-Boc] 290.0. 1H NMR (300 MHz, CDCl3) δ 7.41 - 7.29 (m, 5H), 5.34 - 5.12 (m, 2H), 4.65 - 4.42 (d, J=9.51 Hz, 1H), 3.87 - 3.60 (m, 2H), 2.66 - 2.46 (m, 1H), 2.19 (t, J=15.3 Hz, 1H), 1.48 - 1.32 (m, 9H). (S)-2-Benzyl

1-tert-butyl

(scalemic mixture) (72).

4-(trifluoromethyl)-1H-pyrrole-1,2(2H,5H)-dicarboxylate To a solution of (2S)-2-benzyl 1-tert-butyl 4-hydroxy-4-

(trifluoromethyl)pyrrolidine-1,2-dicarboxylate (10 g, 25 mmol) at 0 °C in pyridine (330 mL) was added SOCl2 (20 mL). The reaction mixture was refluxed for 1 h. The reaction mixture was quenched with water and extracted with EtOAc (3 x 200 mL). The combined organic layer was dried (MgSO4) and concentrated. The residue was taken up in CH2Cl2 (200 mL) and DIPEA (3.3 g, 25 mmol) and Boc2O (5.0 g, 25 mmol) were added. The reaction mixture was stirred at rt


39

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overnight. The reaction mixture was diluted with CH2Cl2 (200 mL) and washed with 1 N HCl (100 mL), sat. aq. NaHCO3 (100 mL), water (100 mL), and brine (50 mL), and dried over anhydrous Na2SO4. The crude material was purified by silica gel chromatography to provide (S)-2-benzyl 1-tert-butyl 4-(trifluoromethyl)-1H-pyrrole-1,2(2H,5H)-dicarboxylate (6.0 g, 16 mmol, 65 % yield) as a colorless oil. LC-MS Anal.Calc’d for C18H20F3NO4: 371.13, found [M+H-Boc] 272.0. 1H NMR (400 MHz, CDCl3) δ 7.40 - 7.31 (m, 5H), 6.26 (dt, J=15.3, 1.9 Hz, 1H), 5.29 - 5.09 (m, 3H), 4.45 - 4.30 (m, 2H), 1.49 - 1.34 (m, 9H). (2S,4S)-1-(tert-Butoxycarbonyl)-4-(trifluoromethyl)

pyrrolidine-2-carboxylic

acid

(scalemic mixture) (73). Ten percent Pd/C (5.5 g) was added to a solution of (S)-2-benzyl 1tert-butyl 4-(trifluoromethyl)-1H-pyrrole-1,2(2H, 5H)-dicarboxylate (6.0 g, 16 mmol) in EtOH (250 ml). The solution was stirred under hydrogen (1 atm) at rt overnight. The reaction mixture was filtered through Celite and concentrated. The crude material was recrystallized to give (2S,4S)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid (4.1 g, 14 mmol, 90 % yield) as a colorless solid. LC-MS Anal.Calc’d for C11H16F3NO4: 283.10, found [M+H-Boc] 184.1. 1H NMR (400 MHz, CDCl3) δ 4.55 - 4.26 (m, 1H), 3.98 - 3.79 (m, 1H), 3.54 - 3.41 (m, 1H), 3.02 - 2.89 (m, 1H), 2.67 - 2.48 (m, 1H), 2.41 - 2.13 (m, 1H), 1.52 - 1.39 (m, 9H). (2S,4S)-tert-Butyl (scalemic

mixture)

2-(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1-carboxylate (74).

To

a

solution

of

(2S,4S)-1-(tert-butoxycarbonyl)-4-

(trifluoromethyl)pyrrolidine-2-carboxylic acid (4.8 g, 20 mmol) in THF (200 mL) cooled to -10 °C was added N-methyl morpholine (2.1 g, 21 mmol) and isobutyl chloroformate (2.9 g, 21 mmol). The reaction mixture was stirred at -10 °C for 1 h. The reaction mixture was filtered through Celite washed with THF. This solution was added dropwise to a solution of NaBH4 (1.5 g, 40 mmol) in water (15 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then


40


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Page 42 of 54

warmed to rt and stirred for 1 h. The reaction was quenched with sat. aq. NH4Cl (100 mL) and the solution was stirred for 15 min. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with water and brine, dried (Na2SO4), and concentrated.

The crude material was purified by silica gel

chromatography to afford (2S,4S)-tert-butyl 2-(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1carboxylate (3.8 g, 14 mmol, 71 % yield) as colorless oil. LC-MS Anal.Calc’d for C11H18F3NO3: 269.12, found [M+H-Boc] 170.0. 1H NMR (400 MHz, CDCl3) δ 4.87 - 4.78 (m, 1H), 4.06 - 3.95 (m, 1H), 3.91 - 3.77 (m, 1H), 3.77 - 3.61 (m, 3H), 3.33 (t, J=10.4 Hz, 1H), 2.96 - 2.75 (m, 1H), 2.39 - 2.21 (m, 1H), 1.48 (s, 9H). ((2S,4S)-4-(Trifluoromethyl) pyrrolidin-2-yl)methanol hydrochloride (scalemic mixture) (75). To a solution of (2S,4S)-tert-butyl 2-(hydroxymethyl)-4-(trifluoromethyl)pyrrolidine-1carboxylate (4.4 g, 16 mmol) in dioxane (10 mL) at 0 °C, a 4 M solution of HCl in dioxane (4.9 mL, 20 mmol) was added and the reaction mixture was warmed to rt and stirred for 1 h. The reaction mixture was concentrated to give ((2S,4S)-4-(trifluoromethyl) pyrrolidin-2-yl)methanol hydrochloride (3.1 g, 15 mmol, 93 % yield) as a pale yellow oil. 1H NMR (400 MHz, DMSOd6) δ 9.99 - 9.73 (br. s, 1H), 9.26 - 8.99 (br. s, 1H), 3.75 - 3.59 (m, 3H), 3.55 - 3.41 (m, 3H), 2.50 (m, 1H), 2.35 - 2.22 (m, 1H), 1.89 - 1.67 (m, 1H). ((2S,4S)-1-(4-Bromophenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)methanol mixture) (76).

(scalemic

1-Bromo-4-iodobenzene (3. 8 g, 13 mmol), ((2S,4S)-4-(trifluoromethyl)

pyrrolidin-2-yl)methanol hydrochloride (2.3 g, 11 mmol), CuI (0.053 g, 0.28 mmol), and NaOH (1.3 g, 33 mmol) were combined in a flask, which was purged with argon. i-PrOH (33 mL) was added and the reaction mixture was refluxed for 16 h. The reaction mixture was cooled to rt and diluted with water. The product was extracted with CH2Cl2 (3x). The combined organic layers


41

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were washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography to afford ((2S,4S)-1-(4-bromophenyl)-4-(trifluoromethyl)pyrrolidin-2yl)methanol (2.8 g, 8.6 mmol, 77 % yield) as an off white solid. LC-MS Anal.Calc’d for C12H13BrF3NO: 323.01, found [M+H] 323.9, 325.9.

1

H NMR (400 MHz, CDCl3) δ 7.32 (td,

J=3.3, 9.1 Hz, 2H), 6.57 (td, J=3.3, 8.8 Hz, 2H), 4.06 - 3.97 (m, 1H), 3.83 - 3.75 (m, 1H), 3.73 3.65 (m, 1H), 3.57 (d, J=8.3 Hz, 2H), 3.08 - 2.89 (m, 1H), 2.44 (td, J=8.2, 13.4 Hz, 1H), 2.30 (ddd, J=7.1, 9.6, 13.4 Hz, 1H), 1.43 (dd, J=4.5, 7.1 Hz, 1H). 2-((2S,4S)-(1-(4-Bromophenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile

(scalemic

mixture) (77). ((2S,4S)-1-(4-Bromophenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)methanol (2.8 g, 8.6 mmol) was dissolved in CH2Cl2 (40 mL) and the solution was cooled to 0 °C. Triethylamine (2.4 mL, 17 mmol) and MsCl (1.0 mL, 13 mmol) were added sequentially and the reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with EtOAc and washed with 1 N HCl, sat. NaHCO3 (aq.), and brine.

The organic layer was dried (MgSO4) and

concentrated. The crude product was dissolved in DMSO (40 mL) and NaCN (1.7 g, 34 mmol) was added. The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was cooled to rt and quenched with water. The product was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography to afford 2-((2S,4S)-(1-(4-bromophenyl)-4(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (2.5 g, 7.5 mmol, 87 % yield) as a white solid. LCMS Anal.Calc’d for C13H12BrF3N2: 332.01, found [M+H] 333.0, 335.0.

1

H NMR (400 MHz,

CDCl3) δ 7.37 (d, J=8.8 Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.33 - 4.09 (m, 1H), 3.71 - 3.50 (m, 2H), 3.22 - 3.00 (m, 1H), 2.86 - 2.63 (m, 2H), 2.49 (dd, J=8.6, 16.7 Hz, 1H), 2.23 (ddd, J=5.7, 8.1, 13.8 Hz, 1H).


42


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2'-Fluoro-5'-methoxy-3-methylbiphenyl-4-carboxylic

acid

Page 44 of 54

(80).

2-Fluoro-5-

methoxyphenylboronic acid 78 (1.0 g, 6.1 mmol), 4-bromo-2-methylbenzoic acid 79 (1.2 g, 5.6 mmol), TBAB (1.8 g, 5.6 mmol), Pd(Ph3P)4 (0.13 g, 0.11 mmol), and Na2CO3 (2.4 g, 22 mmol) were combined in a microwave tube and degassed water (11 mL) was added. The vial was sealed and microwaved at 130 °C for 20 min.

The reaction mixture was diluted with

EtOAc/water and acidified to pH 1 with 1 N aq. HCl. The layers were separated and the aqueous layer was extracted with EtOAc (2x). The combined organic layers were dried (MgSO4) and concentrated to give 2'-fluoro-5'-methoxy-3-methylbiphenyl-4-carboxylic acid (1.3 g, 5.1 mmol, 92 % yield) as a pale yellow solid, which was used without further purification. LC-MS Anal.Calc’d for C15H13FO3: 260.08, found [M+H] 261.0. 1H NMR (500 MHz, CDCl3) δ 12.28 (br s, 1H), 8.16 (d, J=8.5 Hz, 1H), 7.53 - 7.42 (m, 2H), 7.09 (t, J=9.4 Hz, 1H), 6.97 (dd, J=6.2, 3.2 Hz, 1H), 6.88 (dt, J=9.0, 3.3 Hz, 1H), 3.84 (s, 3H), 2.74 (s, 3H). (2'-Fluoro-5'-methoxy-3-methylbiphenyl-4-yl)methanol

(81).

2'-Fluoro-5'-methoxy-3-

methylbiphenyl-4-carboxylic acid (3.1 g, 12 mmol) was dissolved in THF (50 mL) and cooled to 0 °C. LAH (0.98 g, 26 mmol) was added in several portions. The reaction mixture was warmed to rt and stirred for 1 h. The reaction mixture was recooled to 0 °C and quenched by the addition of water (0.98 mL), 15% aq. NaOH (0.98 mL), and water (2.9 mL). The reaction mixture was warmed to rt and stirred for 30 min. The solids were filtered off and the organic layer was dried (MgSO4) and concentrated. The crude product was purified by silica gel chromatography to afford (2'-fluoro-5'-methoxy-3-methylbiphenyl-4-yl)methanol (2.6 g, 10 mmol, 89 % yield) as a colorless oil. LC-MS Anal.Calc’d for C15H15FO2: 246.11, found [M+-OH] 229.0. 1H NMR (500 MHz, CDCl3) δ 7.46 - 7.42 (m, J=7.7 Hz, 1H), 7.41 - 7.35 (m, 2H), 7.07 (dd, J=9.9, 9.1 Hz, 1H),


43

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6.94 (dd, J=6.3, 3.0 Hz, 1H), 6.83 (ddd, J=8.9, 3.6, 3.4 Hz, 1H), 4.74 (s, 2H), 3.82 (s, 3H), 2.41 (s, 3H), 1.80 (s, 1H). 4'-(Bromomethyl)-2-fluoro-5-methoxy-3'-methylbiphenyl (82). Lithium bromide (1.1 g, 12 mmol) was added to dry THF (12 mL) and the reaction mixture was stirred for 10 min until the solids dissolved. The solution was cannulated into a flask containing (2'-fluoro-5'-methoxy-3methylbiphenyl-4-yl)methanol (0.30 g, 1.2 mmol) and NEt3 (0.85 mL, 6.1 mmol) was added. The reaction mixture was cooled to 0 °C and MsCl (0.24 mL, 3.1 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. The reaction mixture was diluted with hexanes and washed with water and brine. concentrated.

The organic layer was dried (MgSO4) and

The crude product was purified by silica gel chromatography to afford 4'-

(bromomethyl)-2-fluoro-5-methoxy-3'-methylbiphenyl (0.33 g, 1.1 mmol, 89 % yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.43 - 7.32 (m, 3H), 7.06 (dd, J=9.9, 9.1 Hz, 1H), 6.93 (dd, J=6.3, 3.0 Hz, 1H), 6.83 (dt, J=8.8, 3.4 Hz, 1H), 4.57 (s, 2H), 3.82 (s, 3H), 2.48 (s, 3H). 2-((2S,4S)-(1-(4-((2'-Fluoro-5'-methoxy-3-methylbiphenyl-4-yl)methyl)phenyl)-4(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (scalemic mixture) (83). To a suspension of zinc dust (0.61 g, 9.3 mmol) in anhydrous THF (3 mL) was sequentially added with stirring ethylene dibromide (0.021 mL, 0.24 mmol) and TMS-Cl (0.016 mL, 0.12 mmol) under argon. The mixture was heated with stirring at 65 °C for 20 min. A solution of 4'-(bromomethyl)-2fluoro-5-methoxy-3'-methylbiphenyl (1.9 g, 6.2 mmol) in THF (5.9 mL) was added dropwise to the zinc. The reaction mixture was stirred at 65 °C for 2 h and cooled to rt. A solution of 2((2S,4S)-(1-(4-bromophenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (1.3 g, 3.8 mmol) and Pd(Ph3P)4 (0.18 g, 0.15 mmol) in THF (3.8 mL) was cannulated into the zinc solution and the flask was rinsed with THF (1.5 mL). The reaction mixture was heated to 75 °C. After 1 h,


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the reaction mixture was cooled to rt and quenched with 1.5 M K2HPO4. The reaction mixture was diluted with EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine, dried (MgSO4), and concentrated. The crude product was purified by silica gel chromatography to afford 2-((2S,4S)(1-(4-((2'-fluoro-5'-methoxy-3-methylbiphenyl-4-yl)methyl)phenyl)-4(trifluoromethyl)pyrrolidin-2-yl)acetonitrile (1.6 g, 3.3 mmol, 86 % yield) as a yellow oil. LCMS Anal.Calc’d for C28H26F4N2O: 482.20, found [M+H] 483.2. 1H NMR (500 MHz, CDCl3) δ 7.37 - 7.29 (m, 2H), 7.15 (d, J=8.0 Hz, 1H), 7.09 (d, J=8.8 Hz, 2H), 7.05 (dd, J=8.8, 9.9 Hz, 1H), 6.94 (dd, J=3.2, 6.2 Hz, 1H), 6.81 (td, J=3.5, 9.0 Hz, 1H), 6.60 - 6.55 (m, 2H), 4.24 (ddt, J=3.0, 5.5, 8.3 Hz, 1H), 3.94 (s, 2H), 3.82 (s, 3H), 3.66 - 3.54 (m, 2H), 3.13 - 2.99 (m, 1H), 2.82 (dd, J=3.2, 16.9 Hz, 1H), 2.69 (ddd, J=7.7, 9.1, 13.8 Hz, 1H), 2.50 - 2.41 (m, 1H), 2.31 (s, 3H), 2.22 (ddd, J=5.6, 8.1, 13.8 Hz, 1H). 2-((2S,4S)-1-(4-((2'-Fluoro-5'-methoxy-3-methyl-[1,1'-biphenyl]-4-yl)methyl)phenyl)-4(trifluoromethyl)pyrrolidin-2-yl)acetic acid, HCl (S,S,-68) and 2-((2R,4R)-1-(4-((2'-fluoro5'-methoxy-3-methyl-[1,1'-biphenyl]-4-yl)methyl)phenyl)-4-(trifluoromethyl)pyrrolidin-2yl)acetic acid, HCl (R,R,-68).

2-((2S,4S)-(1-(4-((2'-Fluoro-5'-methoxy-3-methylbiphenyl-4-

yl)methyl)phenyl)-4-(trifluoromethyl)pyrrolidin-2-yl)acetonitrile

(1.1

g,

2.3

mmol)

was

dissolved in EtOH (23 mL) in a pressure vessel and a 6 M solution of KOH (7.6 mL, 45 mmol) was added. The pressure vessel was sealed and heated to 120 °C for 2 h. The reaction mixture was cooled to rt, concentrated, and redissolved in EtOAc. The solution was acidified to pH 2 with 1 N HCl (aq.) and the product was extracted with EtOAc (3x). The combined organic layers were dried (MgSO4), and concentrated. The crude product was purified by RP-Prep. HPLC. The product was redissolved in CH3CN (5 mL) and 3 N HCl (aq.) (2 mL) was added.


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The mixture was concentrated and the procedure was repeated (twice). The aqueous layer was lyophilized to give compound 68 as a scalemic mixture (light peach solid, 0.85 g, 1.4 mmol, 70 % yield).

Half (40 mg) of the mixture of isomers was separated by preparative SFC

chromatography (Chiralpak AD-H, 250 x 21 cm ID, 5 µm; 65 mL/min, 100 bar BP, 35 °C; 25% MeOH/75% CO2; 256 nm detector wavelength; 0.5 mL injection volume of 40 mg/mL solution in MeOH). The chiral purity of the samples was analyzed by analytical SFC chromatography (Chiralpak AD-H, 250 x 4.6 cm ID, 5 µm; 3.0 mL/min, 100 bar BP, 35 °C; 25% MeOH/75% CO2; 220 nm detector wavelength; 10 µL injection volume in MeOH). The product-containing fractions for each isomer were concentrated and redissolved in CH3CN (1 mL) and 3 N HCl (aq.) (0.5 mL) was added. The solutions were concentrated and the procedure was repeated (2x) to ensure salt exchange. The aqueous layers were lyophilized to give the single enantiomers. Compound (S,S,-68) (beige powder, 21 mg, Chiralpak AD-H rt: 7.496, ≥99.6% er). LC-MS Anal.Calc’d for C28H27F4NO3: 501.19, found [M+H] 502.4.

1

H NMR (400 MHz, CD3CN) δ

7.38 (s, 1H), 7.37 - 7.31 (m, 1H), 7.24 - 7.14 (m, 5H), 7.11 (dd, J=9.1, 10.4 Hz, 1H), 6.99 (dd, J=3.2, 6.4 Hz, 1H), 6.88 (td, J=3.5, 8.9 Hz, 1H), 4.28 - 4.19 (m, 1H), 4.00 (s, 2H), 3.81 (s, 3H), 3.80 - 3.73 (m, 1H), 3.65 (dd, J=9.6, 11.1 Hz, 1H), 3.47 - 3.31 (m, 1H), 2.84 (dd, J=3.8, 16.4 Hz, 1H), 2.74 - 2.59 (m, 2H), 2.30 (s, 3H), 2.14 (ddd, J=8.7, 8.9, 13.2 Hz, 1H). Analytical HPLC: RT = 12.9 min, HI: 98.7%. Compound (R,R-68) (light pink solid, 4.0 mg, Chiralpak AD-H rt: 13.278, 97.3% er). LC-MS Anal.Calc’d for C28H27F4NO3: 501.19, found [M+H] 502.4.

1

H

NMR (400 MHz, CD3CN) δ 7.72 (d, J=8.8 Hz, 2H), 7.43 - 7.31 (m, 4H), 7.21 (d, J=8.2 Hz, 1H), 7.11 (dd, J=8.8, 10.4 Hz, 1H), 6.98 (dd, J=3.0, 6.3 Hz, 1H), 6.88 (td, J=3.6, 8.8 Hz, 1H), 4.26 (tdd, J=4.9, 9.2, 11.3 Hz, 1H), 4.13 - 4.01 (m, 3H), 3.80 (s, 3H), 3.77 - 3.70 (m, 1H), 3.69 - 3.57


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(m, 1H), 3.08 (dd, J=8.8, 17.0 Hz, 1H), 2.83 (dd, J=4.4, 17.0 Hz, 1H), 2.75 (ddd, J=5.5, 7.1, 12.6 Hz, 1H), 2.36 - 2.22 (m, 4H). Analytical HPLC: RT = 12.9 min, HI: 98.5%.

ASSOCIATED CONTENT Experimental procedures for the synthesis of compounds 5-85 and methods for in vitro and in vivo biological studies may be found in the Supporting Information section. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: +1-609-818-4645. Fax: +1-609-818-3550. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the GPR40 team at both Bristol-Myers Squibb and Biocon Bristol-Myers Squibb Research Center for their efforts and dedication. We thank Tatyana Zvyaga and Chi Sum for their assistance in the manuscript preparation.


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ABBREVIATIONS GPR40, free fatty acid receptor 1; GPCR, G-protein-coupled receptor; GLP-1, glucagon-like peptide-1; GIP, gastric inhibitory polypeptide; hGPR40, human GPR40 receptor; mGPR40, mouse GPR40 receptor; SAR, structure activity relationship; CHO, Chinese hamster ovary; FLIPR, fluorescent imaging plate reader; HEK, human embryonic kidney; PPARγ, peroxisome proliferator-activated receptor gamma; EC50, half maximal effective concentration; cAMP, cyclic adenosine monophosphate; ADME, absorption, distribution, metabolism, and excretion; GSH, glutathione; CYP, cytochrome P450; AUC, area under the curve

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Graphic and Synopsis Here

Binding Assay

(R,R)-68 enhances radioligand binding

(S,S)-68 displaces radioligand (R,R)-68 (2R,4R) stereochemistry (S,S)-68 (2S,4S) stereochemistry


54

Discovery of Pyrrolidine-Containing GPR40 Agonists

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