Discovery of Pyrrolidine Sulfonamides as Selective and Orally

Article Cite This: J. Med. Chem. 2018, 61, 9738−9755

pubs.acs.org/jmc

Discovery of Pyrrolidine Sulfonamides as Selective and Orally Bioavailable Antagonists of Transient Receptor Potential Vanilloid‑4 (TRPV4)

Downloaded via KAOHSIUNG MEDICAL UNIV on November 10, 2018 at 15:12:20 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Edward J. Brnardic,*,† Guosen Ye,† Carl Brooks,† Carla Donatelli,† Linda Barton,† Jeff McAtee,† Robert M. Sanchez,‡ Arthur Shu,‡ Karl Erhard,§ Lamont Terrell,† Grazyna Graczyk-Millbrandt,§ Yanan He,§ Melissa H. Costell,† David J. Behm,† Theresa Roethke,† Patrick Stoy,† Dennis A. Holt,† and Brian G. Lawhorn† †

Heart Failure Discovery Performance Unit, ‡Flexible Discovery Unit, and §Platform Technology and Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, United States S Supporting Information *

ABSTRACT: A novel series of pyrrolidine sulfonamide transient receptor potential vanilloid-4 (TRPV4) antagonists was developed by modification of a previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon binding to the target protein. The new template was initially discovered as a minor regio-isomeric side product formed during routine structure−activity relationship (SAR) studies, and further optimization resulted in highly potent compounds with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for identifying new heart failure medicines.

■

INTRODUCTION Heart failure (HF) is an incurable, progressive disease that affects over 6 million people in America and significantly erodes quality of life.1 A common symptom of heart failure is pulmonary congestion, which leads to shortness of breath, a decrease in exercise capacity, and an increase in heart failure rates and hospital visits.2,3 In HF, the decreased ability of the left ventricle to pump blood into the peripheral circulatory system (indicated by a reduced ejection fraction or left ventricular dilation) results in an accumulation of blood in the pulmonary veins, which produces an increase in blood pressure within these veins. Sustained high pressure in the pulmonary veins causes fluid to flow from the pulmonary circulation into the surrounding alveoli, which are responsible for transfer of oxygen to the bloodstream. Current therapies4,5 such as beta blockers, ACE inhibitors, angiotensin receptor blockers, and diuretics are unable to adequately treat HF-induced pulmonary © 2018 American Chemical Society

edema, and thus, HF remains a significant area of unmet medical need. Transient receptor potential vanilloid-4 (TRPV4), a member of the transient receptor potential (TRP) ligand-gated ion channel family that regulates Ca2+/Na+ influx into cells,6 is expressed at high levels in the pulmonary vascular endothelial cells,7,8 which form a critical part of the alveolar septal barrier. TRPV4 is activated by a variety of stimuli including warm temperatures, hypotonicity, physical cell stress/pressure, and pharmacological activators.8−13 Activation of TRPV4 in response to increased pulmonary venous pressure causes endothelial cell contraction and detachment. This reduces the integrity of the alveolar septal barrier and enhances permeability, which results in pulmonary edema and impairReceived: August 28, 2018 Published: October 10, 2018 9738

DOI: 10.1021/acs.jmedchem.8b01317 J. Med. Chem. 2018, 61, 9738−9755

Journal of Medicinal Chemistry

Article

ment of gas exchange.14,15 Recently, TRPV4 antagonists such as GSK2193874,16 and a Piperidine-Benzimidazole GSK TRPV4 antagonist17 (Figure 1) among others,18,19 have

Scheme 1

Figure 1. TRPV4 antagonists that have demonstrated efficacy in the prevention of lung edema.

demonstrated the prevention and resolution of pulmonary edema in in vivo models, suggesting a potential clinical benefit of inhibiting TRPV4 function in the treatment of acute or chronic heart failure associated lung congestion.20 Herein, we describe the discovery and optimization of a novel series of pyrrolidine sulfonamides as TRPV4 antagonists.

■

substituent resulted in a 50-fold loss in potency highlighting its critical importance for TRPV4 binding. Modification of the central ring size from the 6-membered piperidine (1) and the 4-membered azetidine (2) to the unreported pyrrolidine was accomplished via a separate route (Scheme 2). Wittig reaction of ketone 8 with methyl triphenyl phosphonium bromide in the presence of potassium tertbutoxide afforded olefin 9. Bromination with NBS in the presence of water gave an equal mix of bromo-hydroxy regio isomers that were subsequently cyclized using sodium hydride to give epoxide 10. The epoxide was then opened with 3fluoro-4-cyano-phenol (4) to give intermediate 11, which was deprotected under acidic conditions and sulfonylated with 2,4dichlorobenzenesulfonyl chloride (6) to give hydroxy pyrrolidine 12. The novel pyrrolidine core (12, IC50 = 150 nM) maintained potency relative to the previously reported piperidine (1, IC50 = 130 nM) and azetidine (2, IC50 = 50 nM) while offering potential SAR advantages due to its asymmetry and synthetic accessibility. Additionally, the pyrrolidine core has reduced conformational flexibility compared to the piperidine while allowing for more substitution patterns as compared to the azetidine core. We were eager to explore the SAR around the pyrrolidine core and initially we sought to re-evaluate the position of the hydroxyl substituent to determine its optimal placement for a possible interaction with TRPV4 (Scheme 3). The methylene hydroxy pyrrolidine intermediate 13, which was initially synthesized according to literature precedent,23 was protected as the TBS-ether before hydroborating the olefin using 9-BBN and sodium perborate to give cis- and transhydroxymethyl isomers 14 and 15. The cis- and trans-isomers were separated by silica gel column chromatography, and the stereochemistry of 14 and 15 was later assigned based on inference from analytical data on final products. Etherification of hydroxymethyl isomers 14 and 15 was accomplished by

RESULTS AND DISCUSSION Piperidine 1 and azetidine 2 (Figure 2) were previously reported21 to be potent antagonists of TRPV4 and their activity was confirmed in our own TRPV4 assay (IC50 = 130 nM and 50 nM, respectively).22

Figure 2. Azetidine and piperidine TRPV4 leads.

The literature reported structure−activity relationship (SAR) study was limited, and the few analogs that were reported varied only in the substitution pattern on both aromatic rings.21 Therefore, we initiated an investigation to expand the SAR of this promising template by focusing on modifications of the central core including changes in the central ring size and substituents. To assess the value of the hydroxy substituent, the des-hydroxy analog of 1 was synthesized (Scheme 1). Alkylation of 3-fluoro-4-cyano-phenol (4) with N-Boc-4-bromomethylpiperidine (3) gave aryl ether (5), which was deprotected under acidic conditions and then treated with 2,4-dicholorobenzenesulfonyl chloride (6) to give sulfonamide 7 (IC50 = 6300 nM). The removal of the hydroxy 9739



Article

Scheme 2

Scheme 3

treatment with mesylate 1624 under basic conditions to give 17 and 20, respectively. The synthesis was completed via Boc deprotection using TFA, followed by sulfonylation with 2,4dichlorobenzenesulfonyl chloride to give sulfonamides 18 and 21, and finally silyl deprotection using TBAF to afford racemic mixtures of cis-isomer 19, and trans-isomer 22. The stereochemistry of 19 and 22 was assigned by the observation of NOE between the pyrrolidine C3 proton and the pyrrolidine C4 proton in compound 19, and the observation of NOE

between the pyrrolidine C3 proton and the aryl ether methylene protons in 22. Shifting the hydroxyl group of 12 to the neighboring position to give cis- and trans-diastereomers (19 IC50 = 1500 nM and 22 IC50 = 1000 nM) resulted in a moderate loss in potency (6−10-fold). Compounds 19 and 22 were, however, 4−6-times more potent than the des-hydroxy piperidine 7 (IC50 = 6300 nM), which indicated a significant benefit of the 4-hydroxy substituent. We hypothesized there may be two separate hydroxy binding interactions with TRPV4 that are 9740



Article

Scheme 4

pyrrolidine to the exocyclic methylene protons, and C4 of the pyrrolidine to the exocyclic methylene protons. COSY correlations observed between the C4 proton and the secondary hydroxy, the C4 proton and a C5 proton, and between the two C2 protons confirmed the assignment. The stereochemistry of 26 was assigned based on NOE observations showing both the secondary hydroxy and tertiary hydroxy protons are near the pyrrolidine C2-Ha and C5-Hb protons, which indicated that the two hydroxy groups occupy the same face of the ring. Likewise, the exocyclic methylene and the C4 methine share NOE with both the C2-Hb and C5Ha protons. The structure of 27 was determined using 2-D NMR studies (gCOSY45, gHMQC, gHMBC, and 13C gaspe spectra). The atom connectivity of 27 was assigned based on the observation of an HMBC correlation from C1 of the aryl ether to the pyrrolidine C3 proton, which indicated that 27 was a regioisomer of 26 with connection of the benzonitrile occurring through the secondary center of the pyrrolidine rather than through the primary hydroxyl. A COSY correlation between the exocyclic methylene protons and the primary OH proton was observed that confirmed this assignment. The stereochemistry of 27 was assigned by the observation of NOE between the methine proton at the C3 position of the pyrrolidine and the exocyclic methylene protons, and the

engaged as suggested by compounds 12, and 19 or 22, and thus, we explored these possible interactions by simultaneously incorporating two hydroxyl substituents on the central ring (26, Scheme 4). Synthesis of the cis-diol 26 (Scheme 4) began with epoxidation of allylic alcohol 13, which resulted in the isolation of a single epoxide diastereomer (23), which was assigned the cis- orientation.25 Presumably, the hydroxyl group coordinates with m-CPBA and directs the epoxidation to the same face of the pyrrolidine ring. Epoxide 23 was ring opened with 3-fluoro-4-cyanophenol (4) under basic conditions and isolation of the major reaction product 24, followed by deprotection and sulfonylation with 6, gave sulfonamide 26. Interestingly, an unknown minor side product 25 with identical molecular weight as 24 was also isolated from the epoxide ring opening in 8% yield. The minor isomer was elaborated to the final product 27 to facilitate its structural assignment and confirm the assignment of 26. Following deprotection and sulfonylation, 25 was converted into 27 which by MS and 1H NMR appeared to be an isomer of 26. The structure of 26 was determined using 1H and 13C GASPE spectra and 2-D NMR data (gCOSY, gHMQC, gHMBC). The atom connectivity of 26 was assigned based on the observation of HMBC correlations from C1 of the aryl ether to the exocylclic methylene protons, C3 of the 9741



Article

absence of NOE from the C3 pyrrolidine methine proton to both the OH protons, providing evidence that 27 was the cisdiastereomer (Figure 3). On the basis of the structures of

Scheme 5

Figure 3. Mechanism of formation of 25.

advantage of the ether-linked pyrrolidine series, but also it indicated that 27 may not present an optimal arrangement of hydroxy groups for TRPV4 binding. To evaluate the effects of removing the tertiary hydroxyl of 27, two hydroxymethyl diastereomers (Scheme 6) were

starting material and the product obtained, we propose that 25 is formed from 24 under the reaction conditions via a Smiles rearrangement26−29 (Figure 3) where an intramolecular displacement at the aromatic ring is initiated by the nucleophilic secondary hydroxide and the reaction proceeds through a 6-membered transition state. When evaluated for activity against TRPV4, compound 26 (IC 50 = 150 nM) showed equivalent activity to its monohydroxy analog 12 (IC50 = 150 nM), which indicated the secondary hydroxyl offers no additional benefit in this template. Thus, the role of the secondary hydroxyl in 19 and 22 may be similar to that of the tertiary hydroxyl of 12, with the C3 placement being preferred for TRPV4 recognition. Remarkably, when compound 27 was tested against TRPV4 it displayed similar activity (IC50 = 100 nM) as its regioisomer 26 despite lacking the preferred C3-OH. We suspected that the potency of 27 may arise from its reduced conformational flexibility resulting from the removal of the methylene linker through direct linkage of the phenol to the pyrrolidine ring. Given its unique activity and novel structure, 27 was selected as a lead for further SAR studies. We hypothesized that the substituents of 27 may produce conformational bias on the pyrrolidine template30,31 and also suspected the hydroxy substituents could be involved in specific interactions with TRPV4. To understand the critical features of 27 required for TRPV4 binding, we aimed to deconstruct the pyrrolidine diol core of 27 with particular intent on understanding the role of each hydroxyl group. Recognizing that the stereochemical array of substituents found serendipitously in 27 would likely be important for TRPV4 recogntion but may not be optimal, another key element of our strategy was the systematic evaluation of the stereochemical presentation of each substituent. Thus, we designed synthetic routes that would allow us to evaluate all of the possible stereoisomers of a complete series of monohydroxy and des-hydroxy pyrrolidine analogs of 27. Synthesis of the des-hydroxy pyrrolidine analog (Scheme 5) was accomplished by first treating the commercially available hydroxy pyrrolidine 28 with 2,4-dichlorobenzenesulfonyl chloride (6) under basic conditions. The resulting sulfonamide 29 was condensed with 3-fluoro-4-cyanophenol (4) under Mitsunobu conditions to form 30. Surprisingly, the des-hydroxy pyrrolidine 30 (IC50 = 320 nM) showed only a three-fold loss in potency versus 27 (IC50 = 100 nM) compared to the 50-fold loss observed upon removal of the hydroxy group from the piperidine series (cf 1 and 7). Not only did this result highlight the inherent entropic

Scheme 6

synthesized. Condensation of hydroxy pyrrolidine 13 with 3fluoro-4-cyanophenol (4) under Mitsunobu conditions, followed by hydroboration/oxidation of olefin 31 using 9BBN and sodium perborate gave both the minor trans- and major cis-hydroxymethyl pyrrolidines 32 and 33.32 Boc removal followed by sulfonylation with 2,4-dichlorobenzenesulfonyl chloride (6) afforded compounds 34 and 35.33 While the trans-hydroxymethyl isomer 34 (IC50 = 200 nM) showed little improvement in potency compared to the deshydroxy compound 30 (IC50 = 320 nM), the cis-hydroxymethyl diastereomer 35 (IC50 = 50 nM) showed a six-fold 9742



Article

Scheme 7

Scheme 8

nM) into its mono hydroxy substituents revealed there was no advantage in combining the cis-hydroxy of 40 (IC50 = 100 nM) and trans-hydroxymethyl of 34 (IC50 = 200 nM) suggesting that neither of the pyrrolidine C4-substituents of 27 were presented in an optimal configuration. The substantial potency enhancements (>6-fold) offered by both 35 and 38 over 30 prompted synthesis of 42, an analog that combines each of these substituents. Compound 42, the diastereomer of 27, was synthesized (Scheme 8) from intermediate 31. The Boc protecting group was removed with trifluoroacetic acid, and treatment of the unprotected pyrrolidine TFA salt with 2,4-dichlorobenzenesulfonyl chloride (6) and aqueous K2CO3 in DCM gave sulfonamide 41. Dihydroxylation of the exocyclic olefin with catalytic OsO4, using NMO as a co-oxidant, provided transpyrrolidine diol 42. The stereochemical configuration of 42 was assigned based on an NOE between the C3 pyrrolidine methine proton and the tertiary hydroxy proton, and the lack of an NOE between the C3 pyrrolidine methine proton and the exocyclic methylene protons. The dihydroxylation proceeds in excellent yield and with high diastereoselectivity (dr 99:1) with the trans-isomer presumably arising through steric bias wherein OsO4 preferentially approaches the olefin on the ring face opposite the phenoxy substituent. Notably, the

improvement, which confirmed our hypothesis that the hydroxyl groups in 27 may not be optimally placed for an interaction with TRPV4. Compound 34 was, however, twofold less potent than 27, which indicated the potential importance of the tertiary C3-pyrrolidine hydroxyl group. Next, we assessed the individual C3-hydroxy diastereomers (Scheme 7) of 27 lacking only the hydroxymethyl group, which were synthesized by ring-opening of epoxide 3634 with 3-fluoro-4-cyano phenol (4) under basic conditions to give the trans-isomer 37. Intermediate 37 was either directly deprotected and sulfonylated to give the trans-hydroxypyrrolidine sulfonamide 38, or it was inverted via esterification under Mitsunobu conditions with para-nitrobenzoic acid followed by ester hydrolysis with LiOH to give the cis-hydroxypyrrolidine 39. Compound 39 was then deprotected under acidic conditions and sulfonylated to give the cis-hydroxypyrrolidine sulfonamide 40. Similar to the C4-hydroxymethyl substituent, we found that the C4-hydroxy substituent was not optimally oriented in 27 as the cis-hydroxypyrrolidine analog 40 (IC50 = 100 nM) showed only a three-fold improvement in potency compared to the des-hydroxy pyrrolidine 30 (IC50 = 320 nM), while the transhydroxypyrrolidine diastereomer 38 (40 nM) showed an eightfold improvement. Thus, deconstructing diol 27 (IC50 = 100 9743



Article

the pyrrolidine ring and minimizes dipole repulsion of the neighboring C−O bonds.30,31 Thus, we speculate the potency gains arising from the tertiary-hydroxyl may be due to its impact on preorganizing the pyrrolidine template into the TRPV4 binding orientation. The primary hydroxyl group may reinforce this conformation through an intramolecular H-bond with the tertiary hydroxyl or may engage in a direct interaction with the TRPV4 protein. The exceptional TRPV4 antagonist activity of 42 prompted further profiling of the trans-oriented pyrrolidine diol template. Thus, 42, which had been prepared as a racemic mixture, was separated into its constituent enantiomers using chiral chromatography and the configuration of each enantiomer was assigned by ab initio vibrational circular dichroism (VCD) analysis. The TRPV4 activity was found to predominately reside in the (3S,4R)-enantiomer 51 (IC50 = 2.5 nM), with the (3R,4S)-enantiomer showing 100-fold less potency (IC50 = 250 nM). A chiral synthesis of 51 was developed and optimized to access large scale quantities for biological evaluation (Scheme 9). Beginning with commercially available N-Boc-pyrroline (43), bromination with 1,3-dibromo-5,5-dimethylhydantoin in acetonitrile/water afforded the trans-bromo-hydroxypyrrolidine 44, which was cyclized to form epoxide 36 in an aqueous solution of sodium hydroxide. Exposure of 36 to an excess of dimethylsulfonium methylide (generated from the reaction of trimethylsulfonium iodide with n-butyl lithium) produced allylic alcohol 13, which was then treated with benzoyl chloride and triethylamine to give the benzoyl ester intermediate 45.

stereochemical outcome is the inverse of that observed when epoxidizing the hydroxy olefin 13 (Scheme 4). Thus, both cisand trans-pyrrolidine isomers can be accessed in highly stereoselective fashion by selection of appropriate intermediate and reagent combinations. Gratifyingly, the SAR was additive when combining the trans-hydroxy C4-substituent (8-fold potency enhancement over des-hydroxy pyrrolidine 30) with the cis-hydroxymethyl C4-substituent (6-fold) to give diol 42, which exhibited exceptional potency (IC50 = 4 nM) representing a 80-fold improvement over the unsubstituted pyrrolidine 30. The additive SAR demonstrates that each hydroxyl group of 42 is independently responsible for potency enhancements and plays a unique role in TRPV4 recognition. Notably, the transconfigured tertiary hydroxyl group elicits a strong bias on the pucker of the pyrrolidine ring, with the trans-diaxial orientation of the C3-phenoxy and C4-hydroxyl being the favored low energy conformation based on ab initio QM calculations (Figure 4).35 This conformation maximizes gauche effects in

Figure 4. Representation of the low energy conformation of 42.

Scheme 9

9744



Article

and modulate the PK properties of 51 by optimizing both the aryl ether and the sulfonamide substituents. Evaluation of a small set of aryl ether substituent patterns revealed that the SAR for the pyrrolidine diol was analogous to that reported for piperidine 1 and the 3-fluoro, 4-nitrile substitution pattern of 51 was found to be optimal for potency and pharmacokinetics on the trans-pyrrolidine diol template (data not shown).21 The similarity in aryl ether and sulfonamide SAR between 1, 2, and 51 suggests an overlap in binding modes between the two series and confirms that the conformational rigidity and introduction of the diol are the primary drivers of the enhanced potency in the pyrrolidine diol template. We then focused our attention on the optimization of the sulfonamide moiety to further build on the SAR previously established for the piperidine and azetidine cores.21 We discovered that substituting in the 3-position consistently led to a loss in potency regardless of the electronic nature of the substituents, suggesting a possible steric component. In contrast, 2-substituents were found to enhance activity compared to the unsubstituted benzene, which suggested that the ortho-substitution may favorably bias the analogs into the TRPV4 binding conformation. Notably, 2-substituted aryl sulfonamides are expected to adopt a bisected conformation about the S−Ar bond, whereas unsubstituted aryl sulfonamides prefer a perpindicular orientation.41 Small, electron withdrawing groups offered the most potency enhancement in both the 2- and 4-position, which indicated an apparent preference for an electron-deficient aryl sulfonamide. The SAR was additive when combining substitution in the 2- and 4-positions and of particular interest was the 2-CN,4-CF3-sulfonamide 52, which balanced potency (1 nM) and lipophilicity (ChromlogD = 4.6) with moderate CL and good oral bioavailability (72%) (Table 2). An established in vivo pharmacokinetic/pharmacodynamic (PK/PD) model of TRPV4-mediated pulmonary edema was used to evaluate the effects of pyrrolidine 52 on mean arterial pressure (MAP) and lung wet weight (normalized to body weight; LW/BW) in Sprauge-Dawley rats following 10 min i.v. infusion with the TRPV4 agonist GSK1016790A at 10 μg/kg/ min.12,20,42 Prior to agonist administration, rats were pretreated with vehicle or a dose-range of pyrrolidine 52 via a regimen consisting of an i.v. bolus loading dose (0.03, 0.08, and 0.23 mg/kg) followed by a continuous 60 min i.v. infusion (0.6, 1.7, and 5.1 μg/kg/min) to provide steady-state plasma exposure. Steady-state plasma concentrations were 30 ± 9.8, 40 ± 7.1, and 90 ± 1.3 nM, respectively. In vehicle treated rats, GSK1016790A significantly decreased MAP and increased the LW/BW ratio, while treatment with pyrrolidine 52 inhibited these responses in a dose-dependent manner (Figure 5). Full inhibition of these responses was achieved using the 5.1 μg/kg/min dose, which demonstrated protection against TRPV4-mediated blood pressure and pulmonary edema effects in the rat. Notably, the observed effects in the agonist-driven PK/PD experiment correlate with prevention of lung edema in heart failure disease models including acute pressure overload and chronic myocardial infarction studies in rodents.20

Separation of the enantiomers of 45 was accomplished using Chiral HPLC and the absolute configuration of the enantiomers was assigned by ab initio VCD analysis.36 Ester hydrolysis of the (S)-enantiomer 46 using KOH in MeOH produced the (S)-enantiomer of the Boc protected hydroxyl pyrrolidine 47. Compound 47 was converted to the aryl ether 48 by reaction with 3-fluoro-4-cyanophenol (4) using Mitsunobu conditions (PS−PPh3/DIAD), which occurred with clean inversion of stereochemistry to provide the desired (R)-phenyl ether. The Boc protecting group was removed with trifluoroacetic acid to give the TFA salt of pyrrolidine 49. Treatment of the pyrrolidine 49 with 2,4-dichlorobenzenesulfonyl chloride (6) and K2CO3 gave sulfonamide 50. The synthesis was completed via dihydroxylation of the exocyclic olefin of the pyrrolidine ring with catalytic OsO4 using NMO as a co-oxidant, which provided 51. trans-Pyrrolidine diol 51 is a novel, advanced lead compound for identifying TRPV4 antagonist drug-candidates. A side-by-side assessment of the biological, physicochemical, and pharmacokinetic properties of 51 and the reported lead 1 was conducted (Table 1).37 Compound 51 exhibits a 50-fold Table 1. Profile of trans-Pyrrolidine-diol 51 versus Piperidine 1

compound

1

51

hTRPV4 IC50a ChromlogDb aq. solubility rat CLc rat MRTc rat oral Fc rat PPB, fud rat CLud

130 nM 6.3 7.5 μM 19 mL/min/kg 1.5 h 56% 1.3% 1461 mL/min/kg

2.5 nM 4.6 360 μM 58 mL/min/kg 0.9 h 35% 5.9% 966 mL/min/kg

a

hTRPV4 potency was determined in a FLIPR assay.22 bChromlogD was used to measure lipophilicity.38 cRat pharmacokinetic properties of clearance (CL) and mean resonance time (MRT) measurements were derived from i.v. leg, and oral bioavailability (oral F) measurements were derived from oral leg.39,40 dUnbound clearance (CLu) was determined based on in vitro plasma protein binding (PPB).39,40

improvement in TRPV4 activity, significantly reduced lipophilicity as measured by ChromLogD,38 and higher aqueous solubility. The pharmacokinetic properites of 1 and 51 are similar, with 51 showing a moderate clearance, MRT, and oral biovailability in rats.39,40 Importantly, 51 demonstrates excellent selectivity for TRPV4, exhibiting little or no activity against a broad panel of TRP channels (IC50 > 10 μM for TRPA1, TRPV1, TRPM2, TRPM4, TRPM8, TRPC3, TRPC4, TRPC5, TRPC6). We sought to further improve the potency

■

CONCLUSION In summary, SAR studies on a reported piperidine sulfonamide TRPV4 antagonist led to a novel series of pyrrolidine sulfonamide TRPV4 antagonists. The key pyrrolidine diol core arose serendipitously as an unexpected side-product from 9745



Article

Table 2. Profile of trans-Pyrrolidine-diol 52

value for the treatment of pulmonary edema associated with heart failure.

■

compound

52

hTRPV4 IC50a ChromlogDb aq. solubility rat CLc rat MRTc rat oral Fc rat PPB, fud rat CLud

1 nM 4.6 90 μM 82 mL/min/kg 0.8 h 72% 4.9% 1400 mL/min/kg

EXPERIMENTAL SECTION

General Experimental. Biological assays45 and Rat Pharmacokintetics46 were conducted as described. The purity of each antagonist was determined to be >95% on a Waters Acquity LCMS equipped with a Acquity UPLC BEH C18 1.7 mm, 2.1 × 50 mm2 column using a gradient of 3% to 100% MeCN, 0.1% formic acid at 1 mL/min flow rate with an Acquity PDA detector at 210 and 350 nm. Mass determinations were conducted using a Waters Acquity SQD with pos/neg switching in ESI mode. Preparative HPLC was conducted on a Waters 2525 system at a flow rate of 50 mL/min with 254 nm detection using a Sunfire C18 OBD 5 μm column (30 × 150 mm2) with a gradient of MeCN/H 2 O/0.1% TFA. Flash column chromatography was conducted on silica gel eluting with EtOAchexanes, EtOAc-heptane, or MeOH−CH2Cl2 mixtures. The following intermediates were synthesized following literature procedures: 4-((1((2,4-dichlorophenyl)sulfonyl)-4-hydroxypiperidin-4-yl)methoxy)-2fluorobenzonitrile (1),21 4-((1-((2,4-dichlorophenyl)sulfonyl)-3-hydroxyazetidin-3-yl)methoxy)-2-fluorobenzonitrile (2),23 (±) tertbutyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (13),23 4cyano-3-fluorophenyl methanesulfonate (16),24 and tert-butyl 6-oxa3-azabicyclo[3.1.0]hexane-3-carboxylate (36).34 Other required reagents and building blocks were either purchased and used as is or synthesized as described further. tert-Butyl 4-((4-cyano-3-fluorophenoxy)methyl)piperidine-1-carboxylate (5). A solution of tert-butyl 4-(bromomethyl)piperidine-1carboxylate 3 (500 mg, 1.797 mmol) and cesium carbonate (878 mg, 2.70 mmol) in DMSO (2 mL) and water (2 mL) was treated with 2fluoro-4-hydroxybenzonitrile 4 (296 mg, 2.157 mmol), and the resulting mixture was subjected to microwave irradiation for 30 min at 100 °C. The mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and concentrated to give 5 (500 mg, 83%) as a colorless oil. MS (m/z) 357.2 (M + Na+). 4-((1-((2,4-Dichlorophenyl)sulfonyl)piperidin-4-yl)methoxy)-2fluorobenzonitrile (7). A solution of 5 (500 mg, 1.5 mmol) in DCM (6 mL) was treated with a solution of 4 M HCl in dioxane (3 mL), and the reaction solution was stirred for 1 h at 23 °C and then concentrated to give a white solid. The solid material was redissolved in DCM (6 mL) and subsequently treated with triethylamine (1.04 mL, 7.48 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride 6 (551 mg, 2.24 mmol), and the resulting mixture was stirred for 3 h at 23 °C. The mixture was diluted with dicholoromethane, washed with water, washed with brine, dried over Na2SO4, filtered, concentrated, and subjected first to flash chromatography (0−10% EtOAc/ Hexanes) and then to reverse phase HPLC (MeCN/Water/0.1% TFA) to give 7 (22 mg, 3%) as a white solid: 1H NMR (400 MHz,

a

hTRPV4 potency was determined in a FLIPR assay.22 bChromlogD was used to measure lipophilicity.38 cRat pharmacokinetic properties of clearance (CL) and mean resonance time (MRT) measurements were derived from i.v. leg, and oral bioavailability (oral F) measurements were derived from oral leg.39,40 dUnbound clearance (CLu) was determined based on in vitro plasma protein binding (PPB).39,40

a routine synthetic reaction that proved to have potent TRPV4 activity. The template was optimized by identifying the preferred stereochemical presentation of the diol moiety. The enhanced potency of the pyrrolidine diol template was primarily attributed to an increase in structural rigidity (reduction of entropy) via the deletion of the aryl ether methylene linker, truncation of the piperidine ring to the pyrrolidine, and conformationl bias imparted by the diol substituents. The TRPV4 activity and oral bioavailability were further enhanced through modifications of the sulfonamide aryl substituent pattern. Robust efficacy in the in vivo PK/PD model was observed with the optimal exemplar from the series (52), and therefore, pyrrolidine sulfonamide 52 (GSK3395879) represents an optimized lead with drug-like properties from a chemical series that has potential therapeutic

Figure 5. Effect of TRPV4 antagonist TRPV4 agonist-induced reduction of mean arterial pressure (MAP; A) and elevation of lung weight normalized to body weight (LW/BW; B).43,44 ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus vehicle. #p < 0.05, ##p < 0.001 versus baseline. 9746



Article

DMSO-d6) δ ppm 7.97 (d, J = 8.53 Hz, 1H), 7.93 (d, J = 2.01 Hz, 1H), 7.82 (t, J = 8.41 Hz, 1H), 7.66 (dd, J = 2.01, 8.53 Hz, 1H), 7.16 (dd, J = 2.38, 11.92 Hz, 1H), 6.96 (dd, J = 2.26, 8.78 Hz, 1H), 3.97 (d, J = 6.27 Hz, 2H), 3.75 (d, J = 12.55 Hz, 2H), 2.73−2.84 (m, 2H), 1.87−1.99 (m, 1H), 1.76−1.85 (m, 2H), 1.29 (dq, J = 4.02, 12.30 Hz, 2H). MS (m/z) 443.1 (M + H+). tert-Butyl 3-methylenepyrrolidine-1-carboxylate (9). A solution of bromo(methyl)triphenylphosphorane (19.3 g, 54.0 mmol) in diethyl ether (150 mL) was treated with KOt-Bu (6.06 g, 54.0 mmol), and the mixture was stirred at 25 °C for 1 h before being treated with a solution of tert-butyl 3-oxopyrrolidine-1-carboxylate 8 (10.0 g, 54.0 mmol) in dry diethyl ether (100 mL), and the resulting mixture was stirred overnight at 25 °C. The mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed three times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give 9 (5.2 g, 58%): 1H NMR (400 MHz, CHLOROFORM-d) δ 4.98 (td, J = 1.94, 7.15 Hz, 2H), 3.94 (s, 2H), 3.48 (t, J = 7.40 Hz, 2H), 2.57 (t, J = 6.78 Hz, 2H), 1.49 (s, 9H). MS (m/z) 205.9 (M + Na+). (±) tert-Butyl 1-oxa-5-azaspiro[2.4]heptane-5-carboxylate (10). A solution of 9 (1.00 g, 5.46 mmol) in DMSO (10 mL) was cooled to 0 °C and treated with 1-bromopyrrolidine-2,5-dione (1.94 g, 10.9 mmol) and water (0.197 mL, 10.9 mmol). The mixture was warmed to 25 °C and stirred overnight. The reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed three times with water, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give a mixture of tert-butyl 3-(bromomethyl)-3hydroxypyrrolidine-1-carboxylate and tert-butyl 3-bromo-3(hydroxymethyl)pyrrolidine-1-carboxylate regioisomers (800 mg, 58%). MS (m/z) 223.9 (M + H+ − t-Bu). The hydroxy bromo regioisomers (800 mg, 2.86 mmol) were dissolved in THF (20 mL) and cooled to 0 °C before being treated with sodium hydride (156 mg of a 60% dispersion in mineral oil, 3.89 mmol). The mixture was warmed to 25 °C and stirred overnight and then quenched with the addition of aqueous saturated ammonium chloride solution. The mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed three times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−60% EtOAc/hexanes) to give 10 (380 mg, 74%): 1H NMR (400 MHz, CHLOROFORM-d) δ 3.63 (m, 3H), 3.30 (d, J = 11.80 Hz, 1H), 2.96 (s, 2H), 2.16−2.40 (m, 1H), 1.77−1.93 (m, 1H), 1.49 (s, 9H). (±) tert-Butyl 3-((4-cyano-3-fluorophenoxy)methyl)-3-hydroxypyrrolidine-1-carboxylate (11). A solution of 10 (5.10 g, 25.6 mmol) and 2-fluoro-4-hydroxybenzonitrile (4) (4.21 g, 30.7 mmol) in isopropanol (150 mL), DMSO (30 mL), and water (30 mL) was treated with cesium carbonate (10.0 g, 30.7 mmol), and the mixture was heated overnight at 60 °C. The volatile solvents were removed under reduced pressure, and the mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed three times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−70% EtOAc/hexanes) to give 11 (4.3 g, 50%): 1H NMR (400 MHz, DMSO-d6) δ 7.78−7.93 (m, 1H), 7.20 (dd, J = 2.38, 11.92 Hz, 1H), 7.01 (dd, J = 2.26, 8.78 Hz, 1H), 5.27 (s, 1H), 4.09 (d, J = 2.76 Hz, 2H), 3.15−3.53 (m, 4H), 1.89−2.09 (m, 1H), 1.74−1.87 (m, 1H), 1.40 (d, J = 3.01 Hz, 9H). MS (m/z) 280.9 (M + H+ − t-Bu). (±) 4-((1-((2,4-Dichlorophenyl)sulfonyl)-3-hydroxypyrrolidin-3yl)methoxy)-2-fluorobenzonitrile (12). A solution of 11 (280 mg, 0.832 mmol) in DCM (20 mL) was treated with TFA (1.5 mL, 19.5 mmol), and the solution was stirred for 2 h at 25 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM/hexanes several times to give the trifluoroacetic acid salt of 2-fluoro-4-((3-hydroxypyrrolidin-3yl)methoxy)benzonitrile (290 mg, 100%): 1H NMR (400 MHz, DMSO-d6) δ 8.87−9.34 (m, 2H), 7.87 (t, J = 8.28 Hz, 1H), 7.21 (dd, J = 2.01, 11.80 Hz, 1H), 7.02 (dd, J = 1.76, 8.78 Hz, 1H), 5.77 (s,

1H), 4.10−4.28 (m, 2H), 3.36 (dd, J = 5.40, 8.66 Hz, 2H), 3.09−3.26 (m, 2H), 1.89−2.15 (m, 2H). MS (m/z) 237.0 (M + H+). A solution of the trifluoroacetic acid salt of 2-fluoro-4-((3hydroxypyrrolidin-3-yl)methoxy)benzonitrile (100 mg, 0.286 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (70.3 mg, 0.286 mmol) in DCM (10 mL) was treated with triethylamine (0.100 mL, 0.716 mmol), and the reaction mixture was stirred overnight at 25 °C. The mixture was diluted with water and extracted three times with DCM. The combined organic extracts were passed through a phase separator, concentrated, and subjected to flash chromatography (0− 70% EtOAc/hexanes) to give 12 (120 mg, 89%): 1H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 8.53 Hz, 1H), 7.89 (d, J = 2.01 Hz, 1H), 7.84 (t, J = 8.28 Hz, 1H), 7.64 (dd, J = 1.76, 8.53 Hz, 1H), 7.09−7.19 (m, 1H), 6.96 (dd, J = 1.88, 8.66 Hz, 1H), 5.45 (s, 1H), 3.99−4.16 (m, 2H), 3.54 (dd, J = 5.14, 8.66 Hz, 2H), 3.46 (d, J = 10.29 Hz, 1H), 3.28−3.37 (m, 3H), 1.98−2.10 (m, 1H), 1.87−1.96 (m, 1H). MS (m/z) 444.8 (M + H+). (±) tert-Butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (13). A suspension of trimethylsulfonium iodide (762 g, 3740 mmol) in THF (2500 mL) at −10 °C was treated with nBuLi (1380 mL, 3460 mmol) over 45 min while maintaining an internal temperature between −5 and −10 °C. After being stirred for an additional 30 min, the mixture was treated with a solution of tert-butyl 6-oxa-3azabicyclo[3.1.0]hexane-3-carboxylate 36 (173 g, 934 mmol) in THF (500 mL) over 20 min before being warmed to 23 °C and stirred overnight. The mixture was diluted with TBME (2 L) and washed with water (2 L). The aqueous was extracted once more with 1 L of TBME, and the combined orgainc layers were washed with brine, dried over magnesium sulfate, filtered, and concentrated to give 13 (158 g, 85%) as a yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 5.36 (d, J = 5.27 Hz, 1H), 5.13 (br. s., 1H), 5.06 (br. s., 1H), 4.44 (d, J = 4.77 Hz, 1H), 3.76−3.99 (m, 2H), 3.49−3.59 (m, 1H), 2.94−3.07 (m, 1H), 1.40 (s, 9H). ( ±) ci s - ter t-B u t yl 3- ( (t ertbu tyldimethylsilyl)oxy)-4(hydroxymethyl)pyrrolidine-1-carboxylate (14) and (±) trans-tertButyl 3-((tert-butyldimethylsilyl)oxy)-4-(hydroxymethyl)pyrrolidine-1-carboxylate (15). A solution of tert-butyl 3-hydroxy4-methylenepyrrolidine-1-carboxylate 13 (6.8 g, 34.1 mmol) in DMF (100 mL) was treated with TBSCl (6.17 g, 41.0 mmol) followed by imidazole (4.65 g, 68.3 mmol), and the mixture was stirred overnight at 23 °C. The reaction mixture was diluted with water, and the product was extracted three times with EtOAc. The combined organic layers were washed three times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−60% EtOAc/hexanes) to give (±) tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-methylenepyrrolidine-1-carboxylate (9.4 g, 88%) as a light yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 5.09 (br. s., 2H), 4.53− 4.74 (m, 1H), 3.77−3.96 (m, 2H), 3.51−3.69 (m, 1H), 2.87−3.11 (m, 1H), 1.40 (s, 9H), 1.24 (br. s., 1H), 0.73−0.96 (m, 9H), 0.09 (s, 6H). A 0.5 M solution of 9-borabicyclo[3.3.1]nonane in THF (15 mL, 7.5 mmol) was treated with a solution of (±) tert-butyl 3-((tertbutyldimethylsilyl)oxy)-4-methylenepyrrolidine-1-carboxylate (2 g, 6.4 mmol) in THF (2 mL), and the mixture was stirred for 1 h before being diluted with water (5 mL). The reaction mixture was then treated with sodium perborate tetrahydrate (9.8 g, 64 mmol), and the mixture was vigorously stirred for 1 h. The suspension was diluted with saturated aq. NaHCO3 solution, and the aqueous phase was extracted three times with EtOAc. The combined organic layers dried over with Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−50% EtOAc/hexanes) to give 14 (520 mg, 23%) and 15 (170 mg, 8%) as colorless oils. 14: 1H NMR (400 MHz, CHLOROFORM-d) δ 4.45 (d, J = 3.26 Hz, 1H), 3.82−3.95 (m, 1H), 3.69−3.81 (m, 1H), 3.40−3.61 (m, 2H), 3.33 (d, J = 8.78 Hz, 2H), 2.17−2.42 (m, 2H), 1.48 (s, 9H), 0.92 (s, 9H), 0.13 (d, J = 4.52 Hz, 6H). MS (m/z) 276.1 (M + H+ − t-Bu). 15: 1H NMR (400 MHz, CHLOROFORM-d) δ 4.10−4.29 (m, 1H), 3.51−3.75 (m, 4H), 3.08−3.25 (m, 2H), 2.20−2.41 (m, 1H), 9747



Article

4.52 Hz, 1H, OH). 13C NMR (126 MHz, CHLOROFORM-d) δ 164.5 (arylether C3), 163.5 (arylether C1), 139.6 (benzenesulfonamide C4), 135.4 (benzenesulfonamide C1), 134.3 (arylether C5), 133.3 (benzenesulfonamide C2), 132.8 (benzenesulfonamide C6), 131.9 (benzenesulfonamide C3), 127.4 (benzenesulfonamide C5), 114.1 (nitrile CN), 111.5 (arylether C6), 102.8 (arylether C2), 93.7 (arylether C4), 70.5 (pyrrolidine C3), 65.9 (CH2OAr), 56.4 (pyrrolidine C2), 48.0 (pyrrolidine C5), 43.7 (pyrrolidine C4). For compound 19, the 1H NMR and 13C NMR resonances were assigned based on gCOSY45, gHMQC, gHMBC, and 13C GASPE spectra, and all 2D correlations agree with the assigned structure. MS (m/z) 445.0 (M + H+). (±) trans-tert-Butyl 3-((tert-butyldimethylsilyl)oxy)-4-((4-cyano3-fluorophenoxy)methyl)pyrrolidine-1-carboxylate (20). A solution of 15 (170 mg, 0.51 mmol) and 4-cyano-3-fluorophenyl methanesulfonate 16 (130 mg, 0.62 mmol) in DMF (2.3 mL) was treated with Cs2CO3 (200 mg, 0.62 mmol), and the mixture was heated overnight at 100 °C. The reaction mixture was cooled to room temp, diluted with water (5 mL), and extracted three times with EtOAc. The combined organic layers were washed several times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give 20 (67 mg, 28%) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.46−7.68 (m, 1H), 6.59−6.91 (m, 2H), 4.29 (dd, J = 5.52, 14.56 Hz, 1H), 3.91−4.12 (m, 2H), 3.52−3.81 (m, 2H), 3.13−3.41 (m, 2H), 2.48−2.66 (m, 1H), 1.49 (s, 9H), 0.74−1.09 (m, 9H), −0.02− 0.24 (m, 6H). MS (m/z) 395.1 (M + H+ − t-Bu). (±) trans-4-((4-((tert-Butyldimethylsilyl)oxy)-1-((2,4dichlorophenyl)sulfonyl)pyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (21). A solution of 20 (81 mg, 0.18 mmol) in DCM (1 mL) was treated with trifluoroacetic acid (0.40 mL), and the mixture was stirred for 30 min at 23 °C. The reaction mixture was concentrated, diluted with water (5 mL), and adjusted to pH of 8−9 using 2 N aq. NaOH solution. The reaction mixture was extracted four times with DCM, and the combined organic layers were dried over Na2SO4, filtered, and concentrated to give (±) trans-4-((4-((tertbutyldimethylsilyl)oxy)pyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (51 mg, 77%) as a red oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.54 (t, J = 6.90 Hz, 1H), 6.66−6.89 (m, 2H), 4.22 (br. s., 1H), 3.88−4.00 (m, 2H), 3.39 (t, J = 10.67 Hz, 1H), 2.81−3.05 (m, 2H), 2.67 (dd, J = 6.15, 9.41 Hz, 1H), 2.47 (d, J = 6.78 Hz, 1H), 0.82−1.05 (m, 9H), 0.00−0.20 (m, 6H). MS (m/z) 351.1 (M + H+). A mixture of (±) trans-4-((4-((tert-butyldimethylsilyl)oxy)pyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (51 mg, 0.15 mmol) and potassium carbonate (50 mg, 0.36 mmol) in chloroform (1 mL) and water (1 mL) was treated with 2,4-dichlorobenzene-1-sulfonyl chloride 6 (55 mg, 0.22 mmol), and the reaction mixture was stirred overnight at 23 °C. The reaction mixture was diluted with saturated aqueous Na2CO3 (5 mL) and extracted three times with DCM. The combined organic layers were washed with brine, filtered through a phase separator, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give 21 (54 mg, 66%) as a colorless oil. 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.04 (d, J = 8.53 Hz, 1H), 7.51−7.67 (m, 2H), 7.40 (dd, J = 2.01, 8.53 Hz, 1H), 6.62−6.88 (m, 2H), 4.35 (q, J = 5.02 Hz, 1H), 3.90−4.08 (m, 2H), 3.68−3.82 (m, 2H), 3.46 (dd, J = 5.77, 9.79 Hz, 1H), 3.26 (dd, J = 4.64, 10.16 Hz, 1H), 2.52−2.67 (m, 1H), 0.80−0.98 (m, 9H), 0.06 (s, 6H). MS (m/z) 559.0 (M + H+). (±) trans-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxypyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (22). A solution of 21 (54 mg, 0.097 mmol) in THF (2.1 mL) was treated with TBAF (1 M in THF, 0.29 mL, 0.29 mmol) and stirred for 1 h at 23 °C. The reaction mixture was quenched with addition of saturated aq. NH4Cl solution, the volatile solvents were removed under reduced pressure, and the mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−50% EtOAc/hexanes) to give 22 (20 mg, 47%) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 8.05 (d, J = 8.53 Hz, 1H, benzenesulfonamide C6-H), 7.51−7.61 (m, 2H, benzenesulfonamide C3-H, arylether C5-H), 7.41 (dd, J = 2.01,

1.63−1.68 (m, 1H), 1.48 (s, 9H), 0.84−1.03 (m, 9H), 0.03−0.20 (m, 6H). MS (m/z) 332.2 (M + H+). (±) cis-tert-Butyl 3-((tert-butyldimethylsilyl)oxy)-4-((4-cyano-3fluorophenoxy)methyl)pyrrolidine-1-carboxylate (17). A solution of 14 (360 mg, 1.1 mmol) and 4-cyano-3-fluorophenyl methanesulfonate 16 (230 mg, 1.1 mmol) in DMF (4.9 mL) was treated with Cs2CO3 (420 mg, 1.3 mmol), and the mixture was heated overnight at 100 °C. The reaction mixture was cooled to room temp, diluted with water (5 mL), and extracted three times with EtOAc. The combined organic layers were washed several times with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give 17 (180 mg, 35%) as a white solid: 1H NMR (400 MHz, CHLOROFORM-d) δ 7.55 (t, J = 7.65 Hz, 1H), 6.65− 6.85 (m, 2H), 4.36−4.56 (m, 1H), 4.19 (dt, J = 2.64, 8.34 Hz, 1H), 3.99 (td, J = 6.15, 8.53 Hz, 1H), 3.37−3.69 (m, 3H), 3.26 (td, J = 10.13, 17.38 Hz, 1H), 2.63 (d, J = 4.27 Hz, 1H), 1.49 (s, 9H), 0.86 (s, 9H), 0.09 (s, 3H), −0.01 (d, J = 5.02 Hz, 3H). MS (m/z) 451.1 (M + H+). (±) cis-4-((4-((tert-Butyldimethylsilyl)oxy)-1-((2,4dichlorophenyl)sulfonyl)pyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (18). A solution of 17 (180 mg, 0.40 mmol) in DCM (1 mL) was treated with trifluoroacetic acid (0.40 mL) for 30 min at 23 °C. The reaction mixture was concentrated, diluted with water (5 mL), and adjusted to pH of 8−9 using 2 N aq. NaOH solution. The reaction mixture was extracted four times with DCM, and the combined organic layers were dried over Na2SO4, filtered, and concentrated to give (±) cis-4-((4-((tert-butyldimethylsilyl)oxy)pyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (120 mg, 84%) as a red oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.54 (dd, J = 7.53, 8.78 Hz, 1H), 6.60−6.83 (m, 2H), 4.35−4.50 (m, 1H), 4.19 (t, J = 8.53 Hz, 1H), 3.97 (dd, J = 6.02, 8.78 Hz, 1H), 3.13 (dd, J = 8.53, 11.04 Hz, 1H), 2.97−3.07 (m, 1H), 2.80−2.96 (m, 2H), 2.45−2.62 (m, 1H), 0.79− 0.92 (m, 9H), 0.02−0.16 (m, 3H), 0.00 (s, 3H). MS (m/z) 351.1 (M + H+). A mixture of (±) cis-4-((4-((tert-butyldimethylsilyl)oxy)pyrrolidin3-yl)methoxy)-2-fluorobenzonitrile (120 mg, 0.35 mmol) and potassium carbonate (120 mg, 0.88 mmol) in chloroform (1 mL) and water (1 mL) was treated with 2,4-dichlorobenzene-1-sulfonyl chloride 6 (130 mg, 0.54 mmol), and the reaction mixture was stirred overnight at 23 °C. The reaction mixture was diluted with saturated aqueous Na2CO3 (5 mL) and extracted three times with DCM. The combined organic layers were washed with brine, filtered through a phase separator, concentrated, and subjected to flash chromatography (0−40% EtOAc/hexanes) to give 18 (140 mg, 70%) as a colorless oil. 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.03 (d, J = 8.53 Hz, 1H), 7.49−7.63 (m, 2H), 7.39 (dd, J = 2.01, 8.53 Hz, 1H), 6.58−6.83 (m, 2H), 4.47−4.60 (m, 1H), 4.15 (t, J = 8.41 Hz, 1H), 3.97 (dd, J = 6.27, 8.78 Hz, 1H), 3.76 (dd, J = 7.78, 9.03 Hz, 1H), 3.66 (dd, J = 3.89, 10.67 Hz, 1H), 3.33−3.48 (m, 2H), 2.60−2.81 (m, 1H), 0.67− 0.89 (m, 9H), 0.03 (s, 3H), −0.02 (s, 3H). MS (m/z) 559.1 (M + H+). (±) cis-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxypyrrolidin3-yl)methoxy)-2-fluorobenzonitrile (19). A solution of 18 (140 mg, 0.26 mmol) in THF (5.5 mL) was treated with TBAF (1 M in THF, 0.78 mL, 0.78 mmol) and stirred for 1 h at 23 °C. The reaction mixture was quenched with addition of saturated aq. NH4Cl solution, the volatile solvents were removed under reduced pressure, and the mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−50% EtOAc/hexanes) to give 19 (56 mg, 46%) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 8.06 (d, J = 8.53 Hz, 1H, benzenesulfonamide C6-H), 7.50−7.64 (m, 2H, benzenesulfonamide C3-H, arylether C5-H), 7.41 (dd, J = 2.01, 8.53 Hz, 1H, benzenesulfonamide C5-H), 6.65−6.85 (m, 2H, arylether C2-H, arylether C6-H), 4.59 (d, J = 3.76 Hz, 1H, pyrrolidine C3-H), 4.28 (dd, J = 7.40, 9.16 Hz, 1H, ArOCHH), 4.08 (dd, J = 6.90, 9.16 Hz, 1H, ArOCHH), 3.78 (dd, J = 8.16, 9.41 Hz, 1H, pyrrolidine C5-H), 3.66−3.73 (m, 1H, pyrrolidine C2-H), 3.58−3.64 (m, 1H, pyrrolidine C2-H), 3.42 (t, J = 9.79 Hz, 1H, pyrrolidine C5-H), 2.71−2.85 (m, 1H, pyrrolidine C4-H), 1.89 (d, J = 9748



Article

A solution of the trifluoroacetic acid salt of (±) 4-((3,4dihydroxypyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (260 mg, 0.71 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (190 mg, 0.78 mmol) in DCM (5 mL) and water (5 mL) was treated with K2CO3 (490 mg, 3.6 mmol), and the reaction mixture was stirred overnight at 23 °C. The mixture was extracted three times with DCM, and the combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−80% EtOAc/hexanes) to give 26 (230 mg, 67%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.53 Hz, 1H), 7.86 (d, J = 2.65 Hz, 1H), 7.80−7.85 (m, 1H), 7.62 (dd, J = 2.26, 8.53 Hz, 1H), 7.11 (dd, J = 2.26, 11.80 Hz, 1H), 6.93 (dd, J = 2.26, 8.78 Hz, 1H), 5.48 (d, J = 5.77 Hz, 1H), 5.31 (s, 1H), 3.93−4.10 (m, 3H), 3.64 (dd, J = 6.65, 9.41 Hz, 1H), 3.50 (d, J = 10.29 Hz, 1H), 3.35−3.38 (m, 1H), 3.19−3.32 (m, 1H). MS (m/z) 460.8 (M + H+). (±) cis-4-((-1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxy-4(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (27). A solution of 25 (150 mg, 0.426 mmol) in DCM 5 mL was treated with trifluoroacetic acid (0.164 mL, 2.13 mmol) and stirred for 1 h at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM/ hexanes several times to give the trifluoroacetic acid salt of (±) cis-2fluoro-4-((4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (156 mg, 100%) as white solid. MS (m/z) 253.0 (M + H+). A solution of the trifluoroacetic acid salt of (±) cis-2-fluoro-4-((4hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (156 mg, 0.426 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (115 mg, 0.469 mmol) in DCM (5 mL) and water (5 mL) was treated with K2CO3 (294 mg, 2.13 mmol), and the reaction mixture was stirred overnight at 23 °C. The mixture was extracted three times with DCM, and the combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−80% EtOAc/hexanes) to give 27 (56 mg, 26%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.53 Hz, 1 H, benzenesulfonamide C6-H), 7.93 (d, J = 2.01 Hz, 1H, benzenesulfonamide C3-H), 7.79−7.84 (m, 1H, arylether C5-H), 7.62−7.68 (m, 1H, benzenesulfonamide C5-H), 7.19 (dd, J = 2.26, 11.80 Hz, 1H, arylether C6-H), 6.99 (dd, J = 2.51, 8.78 Hz, 1H, arylether C6-H), 5.38 (s, 1H, OH), 5.17−5.22 (m, 1H, CH2OH), 4.94 (t, J = 5.40 Hz, 1H, pyrrolidine C3-H), 3.89 (dd, J = 5.90, 10.42 Hz, 1H, pyrrolidine C2-H), 3.49 (d, J = 10.04 Hz, 1H, pyrrolidine C5H), 3.44 (dd, J = 4.89, 10.42 Hz, 1H, pyrrolidine C2-H), 3.37 (d, J = 5.77 Hz, 2H, CH2OH), 3.27 (d, J = 10.04 Hz, 1H, pyrrolidine C5-H). 13 C NMR (126 MHz, DMSO-d6) 164.3 (arylether C3), 163.8 (arylether C1), 138.8 (benzenesulfonamide C4), 135.4 (benzenesulfonamide C1), 135.0 (arylether C5), 133.0 (benzenesulfonamide C6), 132.8 (benzenesulfonamide C2), 132.2 (benzenesulfonamide C3), 128.4 (benzenesulfonamide C5), 114.8 (nitrile CN), 113.8 (arylether C6), 104.3 (arylether C2), 92.4 (arylether C4), 79.6 (pyrrolidine C2), 77.0 (pyrrolidine C3), 63.1 (CH2OAr), 53.5 (pyrrolidine C5), 50.6 (pyrrolidine C4). For compound 27, the 1H NMR and 13C NMR resonances were assigned based on gCOSY45, gHMQC, gHMBC, and 13C GASPE spectra, and all 2D correlations agreed with the assigned structure. MS (m/z) 460.8 (M + H+). (±) 1-((2,4-Dichlorophenyl)sulfonyl)pyrrolidin-3-ol (29). A mixture of (±) pyrrolidin-3-ol 28 (0.91 g, 10 mmol) in DCM (20 mL) and sodium carbonate (10 mL of a 10% aqueous solution, 8.2 mmol) at 0 °C was treated with 2,4-dichlorobenzene-1-sulfonyl chloride (2.0 g, 8.2 mmol), and the reaction mixture was warmed to 23 °C and stirred overnight. The mixture was diluted with dicholoromethane, washed consecutively with water, 0.1 N HCl solution, and brine, and then dried over Na2SO4, filtered, and concentrated to give 29 (2.3 g, 93%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.53 Hz, 1H), 7.92 (d, J = 2.26 Hz, 1H), 7.64 (dd, J = 2.01, 8.53 Hz, 1H), 5.09 (d, J = 3.26 Hz, 1H), 4.29 (dd, J = 2.51, 4.52 Hz, 1H), 3.37−3.44 (m, 3H), 3.19 (td, J = 1.57, 10.42 Hz, 1H), 1.93 (dtd, J = 4.52, 8.80, 13.02 Hz, 1H), 1.69−1.84 (m, 1H). MS (m/z) 296.0 (M + H+).

8.53 Hz, 1H, benzenesulfonamide C5-H), 6.63−6.82 (m, 2H, arylether C2-H, arylether C6-H), 4.45 (br. s., 1H, pyrrolidine C3H), 4.02 (dd, J = 4.14, 6.40 Hz, 2H, ArOCHH), 3.72−3.87 (m, 2H, pyrrolidine C2-H, pyrrolidine C5-H), 3.47 (dd, J = 5.52, 10.04 Hz, 1H, pyrrolidine C5-H), 3.39 (dd, J = 4.27, 10.79 Hz, 1H, pyrrolidine C2-H), 2.62−2.75 (m, 1H, pyrrolidine C4-H), 2.07 (s, 1H, OH). 13C NMR (126 MHz, CHLOROFORM-d) δ 164.5 (arylether C3), 163.9 (arylether C1), 139.7 (benzenesulfonamide C4), 135.2 (benzenesulfonamide C1), 134.4 (arylether C5), 133.3 (benzenesulfonamide C2), 132.9 (benzenesulfonamide C6), 131.9 (benzenesulfonamide C3), 127.4 (benzenesulfonamide C5), 114.1 (nitrile CN), 111.5 (arylether C6), 102.9 (arylether C2), 94.0 (arylether C4), 72.3 (pyrrolidine C3), 67.7 (CH2OAr), 54.3 (pyrrolidine C2), 48.2 (pyrrolidine C5), 46.3 (pyrrolidine C4). For compound 22, the 1H NMR and 13C NMR resonances were assigned based on gCOSY45, gHMQC, gHMBC, and 13C GASPE spectra, and all 2D correlations agreed with the assigned structure. MS (m/z) 445.0 (M + H+). (±) tert-Butyl 7-hydroxy-1-oxa-5-azaspiro[2.4]heptane-5-carboxylate (23). A solution of (±) tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate 13 (5.0 g, 25.1 mmol) in DCM (70 mL) was treated with m-CPBA (11.2 g, 50.2 mmol) and stirred overnight at 23 °C. The reaction mixture was quenched with the addition of saturated aq. Na2S2O3 solution followed by careful treatment with saturated aq. NaHCO3 solution. The mixture was extracted three times with DCM, and the combined organic layers were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−50% EtOAc/hexanes) to give 23 (1.2 g, 22%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 5.13 (d, J = 5.77 Hz, 1H), 3.98−4.08 (m, 1H), 3.55 (ddd, J = 5.90, 11.04, 13.93 Hz, 1H), 3.46 (dd, J = 8.91, 11.67 Hz, 1H), 3.20−3.29 (m, 1H), 3.12−3.19 (m, 1H), 2.92−2.97 (m, 1H), 2.86 (d, J = 5.02 Hz, 1H), 1.40 (d, J = 3.76 Hz, 9H). (±) cis-tert-Butyl 3-((4-cyano-3-fluorophenoxy)methyl)-3,4-dihydroxypyrrolidine-1-carboxylate (24) and (±) cis-tert-Butyl 4-((4cyano-3-fluorophenoxy)-3-hydroxy-3-(hydroxymethyl)pyrrolidine1-carboxylate (25). A solution of 23 (1.2 g, 5.6 mmol) and 2-fluoro4-hydroxybenzonitrile (0.84 g, 6.1 mmol) in isopropanol (30 mL), DMSO (5.0 mL), and water (5.0 mL) was treated with cesium carbonate (2.2 g, 6.7 mmol), and the mixture was heated overnight at 80 °C. The volatile solvents were removed under reduced pressure and reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−70% EtOAc/hexanes) to give 24 (1.1 g, 56%) and 25 (150 mg, 7.6%) as a white solids. 24: 1H NMR (400 MHz, DMSO-d6) δ 7.83 (dd, J = 8.03, 8.78 Hz, 1H), 7.19 (dd, J = 2.26, 12.05 Hz, 1H), 7.00 (dd, J = 2.26, 8.78 Hz, 1H), 5.24 (dd, J = 5.90, 11.42 Hz, 1H), 5.06 (d, J = 7.03 Hz, 1H), 3.97−4.12 (m, 3H), 3.51 (ddd, J = 2.38, 7.28, 10.16 Hz, 1H), 3.32− 3.41 (m, 1H), 3.25−3.31 (m, 1H), 3.11 (dt, J = 7.53, 10.42 Hz, 1H), 1.39 (d, J = 4.27 Hz, 9H). MS (m/z) 296.9 (M + H+ − t-Bu). 25: 1H NMR (400 MHz, DMSO-d6) δ 7.78−7.84 (m, 1H), 7.26 (dd, J = 2.38, 11.92 Hz, 1H), 7.06 (dd, J = 2.26, 8.78 Hz, 1H), 5.19 (s, 1H), 5.15 (dt, J = 2.51, 5.65 Hz, 1H), 4.85−4.93 (m, 1H), 4.01−4.08 (m, 1H), 3.80 (dd, J = 6.27, 11.04 Hz, 1H), 3.35−3.44 (m, 2H), 3.22−3.31 (m, 1H), 3.17 (dd, J = 3.89, 11.17 Hz, 1H), 1.39 (d, J = 4.52 Hz, 9H). MS (m/z) 296.8 (M + H+ − t-Bu). (±) cis-4-((-1-((2,4-Dichlorophenyl)sulfonyl)-3,4-dihydroxypyrrolidin-3-yl)methoxy)-2-fluorobenzonitrile (26). A solution of 24 (250 mg, 0.71 mmol) in DCM 5 mL was treated with trifluoroacetic acid (0.27 mL, 3.6 mmol) and stirred for 1 h at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM/hexanes several times to give the trifluoroacetic acid salt of (±) 4-((3,4-dihydroxypyrrolidin-3yl)methoxy)-2-fluorobenzonitrile (260 mg, 100%) as white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (br s, 1H), 8.87 (br s, 1H), 7.80−7.92 (m, 1H), 7.19 (dd, J = 2.26, 11.80 Hz, 1H), 7.00 (dd, J = 2.26, 8.78 Hz, 1H), 4.17 (t, J = 7.40 Hz, 1H), 4.11 (s, 2H), 3.37−3.49 (m, 1H), 3.25−3.34 (m, 1H), 3.15−3.24 (m, 1H), 2.97−3.09 (m, 1H). MS (m/z) 253.0 (M + H+). 9749



Article

(±) 4-((1-((2,4-Dichlorophenyl)sulfonyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (30). A solution of 29 (250 mg, 0.844 mmol), triphenylphosphine (244 mg, 0.929 mmol), and 2-fluoro-4-hydroxybenzonitrile (127 mg, 0.929 mmol) in THF (2.6 mL) under a nitrogen atmosphere at 0 °C was treated with diisopropyl azodicarboxylate (181 μL, 0.929 mmol), and the reaction mixture was warmed to 23 °C and stirred overnight. The mixture was diluted with EtOAc, washed consecutively with water, 10% aqueous sodium carbonate solution, and brine, and then dried over Na2SO4, filtered, concentrated, and subjected to reverse phase HPLC (MeCN/Water/ 0.1% TFA) to give 30 (180 mg, 48%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.88−7.98 (m, 2H), 7.77−7.86 (m, 1H), 7.62 (dd, J = 2.26, 8.53 Hz, 1H), 7.07 (dd, J = 2.26, 11.80 Hz, 1H), 6.85 (dd, J = 2.38, 8.66 Hz, 1H), 5.18 (br. s., 1H), 3.60−3.70 (m, 1H), 3.40−3.59 (m, 3H), 2.26 (dtd, J = 4.39, 9.35, 14.05 Hz, 1H), 2.04− 2.17 (m, 1H). MS (m/z) 415.0 (M + H+). (±) tert-Butyl 3-(4-cyano-3-fluorophenoxy)-4-methylenepyrrolidine-1-carboxylate (31). A solution of 2-fluoro-4-hydroxybenzonitrile (4) (0.76 g, 5.5 mmol), (±) tert-butyl 3-hydroxy-4methylenepyrrolidine-1-carboxylate (13) (1.0 g, 5.0 mmol), and triphenylphosphine (2.0 g, 7.5 mmol) in THF (20 mL) at 0 °C was treated dropwise with DIAD (1.5 mL, 7.5 mmol), and the mixture was stirred for 30 min before being warmed to 23 °C and stirred overnight. The reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−60% EtOAc/hexanes) to give 31 (1.4 g, 90%) as a pale yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 7.85 (t, J = 8.41 Hz, 1H), 7.30 (dd, J = 2.26, 11.80 Hz, 1H), 7.06 (dd, J = 2.26, 8.78 Hz, 1H), 5.47 (br s, 1H), 5.43 (s, 1H), 5.34 (s, 1H), 4.03 (m, 1H), 3.87−3.98 (m, 1H), 3.73 (dt, J = 4.64, 12.23 Hz, 1H), 3.45 (dd, J = 2.51, 12.30 Hz, 1H), 1.40 (br d, J = 7.53 Hz, 9H). MS (m/z) 262.9 (M + H+ − t-Bu). (±) trans-tert-Butyl 3-(4-cyano-3-fluorophenoxy)-4(hydroxymethyl)pyrrolidine-1-carboxylate (32) and (±) cis-tertButyl 3-(4-cyano-3-fluorophenoxy)-4-(hydroxymethyl)pyrrolidine1-carboxylate (33). A solution of 31 (0.78 g, 2.4 mmol) in THF (12 mL) was treated with 9-BBN (5.9 mL, 2.9 mmol), and the mixture was stirred for 1 h at 23 °C before being treated with a solution of sodium perborate tetrahydrate (3.8 g, 24 mmol) in water (4 mL), and the resulting mixture was stirred overnight at 23 °C. The reaction mixture was treated with 10% NaHCO3 solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, concentrated, and subjected to flash chromatography (0−10% MeOH/DCM) to give 32 (50 mg, 6%) and 33 (500 mg, 61%). 32: MS (m/z) 267.0 (M + H+ − t-Bu). 33: 1H NMR (400 MHz, CHLOROFORM-d) δ 7.46−7.62 (m, 1H), 6.65−6.87 (m, 2H), 4.97 (br. s., 1H), 3.74−3.99 (m, 2H), 3.49− 3.74 (m, 3H), 3.29 (t, J = 10.79 Hz, 1H), 2.57−2.80 (m, 1H), 2.21 (br. s., 1H), 1.34−1.56 (m, 10H). MS (m/z) 267.0 (M + H+ − t-Bu). (±) trans-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (34). A solution of 32 (50 mg, 0.15 mmol) in DCM (1 mL) was treated with TFA (0.057 mL, 0.74 mmol) and stirred for 40 min at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM several times to give the trifluoroacetic acid salt of (±) trans-2-fluoro-4-((4(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (35 mg, 100%). MS (m/z) 236.9 (M + H+). A solution of the trifluoroacetic acid salt of (±) trans-2-fluoro-4((4-(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (35 mg, 0.15 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (76 mg, 0.31 mmol) in DCM (3 mL), methanol (3 mL), and water (3 mL) was treated with K2CO3 (205 mg, 1.48 mmol), and the reaction mixture was stirred for 20 min overnight at 23 °C. The mixture was extracted with DCM, and the organic layer was dried over magnesium sulfate, filtered, concentrated, and subjected to reverse phase HPLC (MeCN/ Water/0.1% TFA) to give 34 (6 mg, 10%). 1H NMR (400 MHz, CHLOROFORM-d) δ 8.03 (d, J = 8.53 Hz, 1H), 7.57 (d, J = 2.01

Hz, 1H), 7.49−7.56 (m, 1H), 7.39 (dd, J = 2.01, 8.53 Hz, 1H), 6.76 (d, J = 9.29 Hz, 2H), 4.99 (td, J = 1.69, 4.89 Hz, 1H), 3.89 (dd, J = 5.02, 11.80 Hz, 1H), 3.79 (dd, J = 5.65, 10.67 Hz, 1H), 3.60−3.71 (m, 3H), 3.39 (dd, J = 2.89, 10.16 Hz, 1H), 2.67−2.74 (m, 1H). MS (m/z) 444.9/446.9 (M + H+). (±) cis-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (35). A solution of 33 (600 mg, 1.78 mmol) in DCM (10 mL) was treated with TFA (0.69 mL, 8.9 mmol), and the mixture was stirred for 1 h at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM several times to give the trifluoroacetic acid salt of trans-(±) trans-2-fluoro-4-((4(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (625 mg, 100%) as colorless waxy solid. MS (m/z) 237.1 (M + H+). A solution of the trifluoroacetic acid salt of trans-(±) trans-2fluoro-4-((4-(hydroxymethyl)pyrrolidin-3-yl)oxy)benzonitrile (625 mg, 1.78 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride (482 mg, 1.963 mmol) in DCM (5 mL) and water (5 mL) was treated with K2CO3 (294 mg, 2.13 mmol), and the reaction mixture was stirred overnight at 23 °C. The mixture was extracted three times with DCM, and the combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−80% EtOAc/hexanes) to give 35 (500 mg, 60%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 8.78 Hz, 1H), 7.84 (d, J = 2.01 Hz, 1H), 7.79 (dd, J = 8.03, 8.78 Hz, 1H), 7.56 (dd, J = 2.01, 8.53 Hz, 1H), 7.07 (dd, J = 2.26, 11.80 Hz, 1H), 6.84 (dd, J = 2.26, 8.78 Hz, 1H), 5.08 (t, J = 3.39 Hz, 1H), 4.76 (t, J = 5.14 Hz, 1H), 3.60−3.71 (m, 2H), 3.43−3.60 (m, 3H), 3.24 (dd, J = 9.54, 10.79 Hz, 1H), 2.65 (dqd, J = 4.02, 7.45, 11.04 Hz, 1H). MS (m/z) 445.1 (M + H+). tert-Butyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (36). A solution of 44 (247 g, 928 mmol) in THF (2000 mL) was treated with 2 N solution of sodium hydroxide (835 mL, 1670 mmol) over 20 min, and the reaction mixture was stirred at 23 °C for 1 h. The mixture was extracted with TBME (2 × 1500 mL), and the combined organic layers were washed with brine, dried over magnesium sulfate, filtered, concentrated, and subjected to flash chromatography (20− 35% EtOAc/heptane) to give 36 (173 g, 96%) as a pale yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 3.82 (d, J = 12.80 Hz, 1H), 3.75 (d, J = 12.80 Hz, 1H), 3.68 (d, J = 3.26 Hz, 2H), 3.32 (dd, J = 4.77, 12.80 Hz, 2H), 1.45 (s, 9H). (±) trans-tert-Butyl 3-(4-cyano-3-fluorophenoxy)-4-hydroxypyrrolidine-1-carboxylate (37). A solution of tert-butyl 6-oxa-3azabicyclo[3.1.0]hexane-3-carboxylate 36 (4.8 g, 26 mmol) and 2fluoro-4-hydroxybenzonitrile 4 (3.9 g, 28 mmol) in isopropanol (60 mL), DMSO (10 mL), and water (10 mL) was treated with cesium carbonate (10 g, 31 mmol), and the mixture was heated overnight at 80 °C. The volatile solvents were removed under reduced pressure and reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−70% EtOAc/hexanes) to give 37 (3.2 g, 38%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.87 (t, J = 8.41 Hz, 1H), 7.27 (dd, J = 2.26, 11.80 Hz, 1H), 7.04 (dd, J = 2.26, 8.78 Hz, 1H), 4.80 (br d, J = 4.52 Hz, 1H), 4.19 (br s, 1H), 3.68 (dt, J = 4.27, 11.92 Hz, 1H), 3.34−3.49 (m, 2H), 3.27 (dd, J = 3.51, 11.54 Hz, 1H), 1.40 (d, J = 3.26 Hz, 9H). MS (m/z) 267.0 (M + H+ − tBu). (±) trans-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxypyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (38). A solution of 37 (220 mg, 0.68 mmol) in DCM (10 mL) was treated with TFA (0.26 mL, 3.4 mmol), and the mixture was stirred for 1 h at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM/hexanes several times to give the trifluoroacetic acid salt of (±) trans-2-fluoro-4-((4hydroxypyrrolidin-3-yl)oxy)benzonitrile (230 mg, 100%) as colorless waxy solid. MS (m/z) 223.2 (M + H+). A solution of the trifluoroacetic acid salt of (±) trans-2-fluoro-4((4-hydroxypyrrolidin-3-yl)oxy)benzonitrile (230 mg, 0.68 mmol), and 2,4-dichlorobenzene-1-sulfonyl chloride (180 mg, 0.75 mmol) in 9750



Article

6.27, 9.54 Hz, 1H), 3.48 (dd, J = 3.64, 11.17 Hz, 1H), 3.26 (dd, J = 6.78, 9.54 Hz, 1H). MS (m/z) 430.9 (M + H+). (±) 4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-methylenepyrrolidin3-yl)oxy)-2-fluorobenzonitrile (41). A solution of 31 (62 g, 120 mmol) in DCM (600 mL) was treated with trifluoroacetic acid (75 mL, 970 mmol), and the reaction mixture was stirred overnight at 23 °C. The solvent was removed under reduced pressure, the resulting oil was diluted with diethyl ether, and the solid precipitate was collected by filtration and dried to give the trifluoroacetic acid salt of (±) 2-fluoro-4-((4-methylenepyrrolidin-3-yl)oxy)benzonitrile (26 g, 68%) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 9.40 (br. s., 2H), 7.79−8.01 (m, 1H), 7.31 (dd, J = 2.38, 11.67 Hz, 1H), 7.06 (dd, J = 2.38, 8.66 Hz, 1H), 5.63 (s, 1H), 5.59 (d, J = 3.76 Hz, 1H), 5.51 (s, 1H), 3.98−4.06 (m, 1H), 3.85−3.93 (m, 1H), 3.50−3.66 (m, 2H). MS (m/z) 219.2 (M + H+). A solution of the trifluoroacetic acid salt of (±) 2-fluoro-4-((4-methylenepyrrolidin-3-yl)oxy)benzonitrile (26 g, 78 mmol) and 2,4-dichlorobenzene-1-sulfonyl chloride 6 (27 g, 110 mmol) in water (250 mL) and DCM (250 mL) was treated with potassium carbonate (110 g, 780 mmol), and the reaction was stirred overnight at 23 °C. The reaction mixture was diluted with DCM, and the organic layer was separated, dried over Na2SO4, filtered, and subjected to flash chromatography (0−100% EtOAc/hexanes) to give 41 (18 g, 54%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.91−8.01 (m, 1H), 7.84 (t, J = 8.28 Hz, 1H), 7.64 (dd, J = 2.13, 8.66 Hz, 1H), 7.16 (dd, J = 2.51, 11.80 Hz, 1H), 6.93 (dd, J = 2.38, 8.66 Hz, 1H), 5.44−5.53 (m, 2H), 5.39 (s, 1H), 4.20 (d, J = 14.31 Hz, 1H), 4.04 (d, J = 14.31 Hz, 1H), 3.76 (dd, J = 4.39, 11.92 Hz, 1H), 3.59 (dd, J = 1.63, 11.92 Hz, 1H). MS (m/z) 427.0 (M + H+). (±) trans-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxy-4(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (42). A solution of 41 (250 mg, 0.58 mmol) in THF (7 mL) was treated with osmium tetroxide (2.5% in tert-butanol) (0.37 mL, 0.03 mmol) followed by NMO (100 mg, 0.88 mmol), and the reaction mixture was stirred overnight at 23 °C. Note: Osmium tetraoxide is a toxic and volatile reagent and we recommend quenching the reaction with aqueous sodium thiosulfate followed by standard workup prior to chromatography, particularly on larger scale. The mixture was concentrated and the residue was subjected to flash chromatography (0−100% EtOAc/hexanes) to give 42 (260 mg, 96%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.53 Hz, 1H), 7.85 (d, J = 2.26 Hz, 1H), 7.81 (t, J = 8.28 Hz, 1H), 7.57 (dd, J = 2.01, 8.53 Hz, 1H), 7.11 (dd, J = 2.26, 11.80 Hz, 1H), 6.87 (dd, J = 2.26, 8.78 Hz, 1H), 5.54 (s, 1H), 4.86 (t, J = 5.65 Hz, 1H), 4.71 (d, J = 3.01 Hz, 1H), 3.86 (dd, J = 3.26, 11.80 Hz, 1H), 3.29−3.65 (m, 3H). MS (m/ z) 460.9 (M + H+). (±) trans-tert-Butyl 3-bromo-4-hydroxypyrrolidine-1-carboxylate (44). A slurry of 1,3-dibromo-5,5-dimethylimidazolidine-2,4dione (265 g, 927 mmol) in acetonitrile (2000 mL) and water (333 mL) was slowly treated with a solution of 43 (300 g, 1773 mmol) in acetonitrile (400 mL) while the internal reaction temperature was maintained between 5 and 10 °C before being warmed to 23 °C and stirred overnight. The reaction mixture was quenched with the addition of 5% aq. Na2S2O3 solution and extracted with t-butylmethyl ether (2 × 2L). The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, and concentrated to give 44 (247 g, 100%). 1HNMR (400 MHz, CHLOROFORM-d) δ 4.74 (s, 1H), 4.39−4.49 (m, 1H), 4.16−4.23 (m, 1H), 4.03 (dd, J = 4.39, 12.67 Hz, 1H), 3.78−3.88 (m, 1H), 3.65−3.78 (m, 1H), 3.41 (dd, J = 12.05, 18.07 Hz, 1H), 1.39−1.51 (m, 9H). (±) tert-Butyl 3-(benzoyloxy)-4-methylenepyrrolidine-1-carboxylate (45). A solution of (±) tert-butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate 13 (158 g, 793 mmol) and Et3N (170 mL, 1.19 mol) in 2-methyltetrahydrofuran (1500 mL) at 10 °C was slowly treated with benzoyl chloride (110 mL, 952 mmol) while an internal temperature was maintained between 10 and 12 °C, followed by DMAP (19.4 g, 159 mmol) and before being warmed to 23 °C and stirred overnight. The mixture was washed with water (1 L), and the organic layer was dried over MgSO4, filtered, concentrated, and subjected to flash chromatography (10−25% EtOAc/hexanes) to give 45 (210 g, 85%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ:

DCM (5 mL) and water (5 mL) was treated with K2CO3 (470 mg, 3.4 mmol), and the reaction mixture was stirred overnight at 23 °C. The mixture was extracted three times with DCM, and the combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10− 80% EtOAc/hexanes) to give 38 (250 mg, 80%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.53 Hz, 1H), 7.89 (d, J = 2.01 Hz, 1H), 7.83 (t, J = 8.41 Hz, 1H), 7.61 (dd, J = 2.01, 8.53 Hz, 1H), 7.11 (dd, J = 2.38, 11.67 Hz, 1H), 6.89 (dd, J = 2.26, 8.78 Hz, 1H), 5.77 (d, J = 3.26 Hz, 1H), 4.83 (d, J = 2.76 Hz, 1H), 4.26 (br s, 1H), 3.77 (dd, J = 3.89, 11.92 Hz, 1H), 3.61 (dd, J = 4.27, 10.79 Hz, 1H), 3.53 (d, J = 11.80 Hz, 1H), 3.35 (d, J = 10.79 Hz, 1H). MS (m/ z) 431.0 (M + H+). (±) cis-tert-Butyl 3-(4-cyano-3-fluorophenoxy)-4-hydroxypyrrolidine-1-carboxylate (39). A solution of 4-nitrobenzoic acid (148 mg, 0.887 mmol), 37 (260 mg, 0.807 mmol), and triphenylphosphine (317 mg, 1.210 mmol) in THF (5.0 mL) at 0 °C was treated with DIAD (0.235 mL, 1.21 mmol), and the mixture was stirred for 30 min before being warmed to 23 °C and stirred overnight. The reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−60% EtOAc/hexanes) to give (±) cis-tertbutyl 3-(4-cyano-3-fluorophenoxy)-4-((4-nitrobenzoyl)oxy)pyrrolidine-1-carboxylate (315 mg, 83%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 8.78 Hz, 2H), 8.01−8.10 (m, 2H), 7.81 (dd, J = 7.91, 8.66 Hz, 1H), 7.34 (dd, J = 2.13, 11.92 Hz, 1H), 7.00−7.10 (m, 1H), 5.72−5.81 (m, 1H), 5.41 (br d, J = 4.27 Hz, 1H), 3.75−3.94 (m, 2H), 3.55 (dd, J = 4.52, 11.80 Hz, 1H), 3.47 (dd, J = 5.27, 11.29 Hz, 1H), 1.42 (s, 9H). MS (m/z) 416.1 (M + H+ − tBu). A solution of (±) cis-tert-butyl 3-(4-cyano-3-fluorophenoxy)-4-((4nitrobenzoyl)oxy)pyrrolidine-1-carboxylate (310 mg, 0.67 mmol) in methanol (15 mL) and water (5.0 mL) was treated with lithium hydroxide monohydrate (28 mg, 0.67 mmol) and reaction mixture was stirred overnight at 23 °C. The volatile solvents were removed under reduced pressure and reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10−60% EtOAc/hexanes) to give 39 (160 mg, 76%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.82 (t, J = 8.41 Hz, 1H), 7.25 (dd, J = 2.51, 12.05 Hz, 1H), 7.03 (dd, J = 2.26, 8.78 Hz, 1H), 5.35 (d, J = 4.77 Hz, 1H), 4.96 (br d, J = 3.76 Hz, 1H), 4.35−4.43 (m, 1H), 3.56−3.68 (m, 1H), 3.49 (dt, J = 6.15, 10.35 Hz, 1H), 3.28−3.33 (m, 1H), 3.12−3.21 (m, 1H), 1.39 (d, J = 5.52 Hz, 9H). MS (m/z) 267.0 (M + H+ − t-Bu). (±) cis-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxypyrrolidin3-yl)oxy)-2-fluorobenzonitrile (40). A solution of 39 (160 mg, 0.50 mmol) in DCM (30 mL) was treated with TFA (0.19 mL, 2.5 mmol), and the mixture was stirred for 1 h at 23 °C. The solvent was removed under reduced pressure, and the resulting residue was redissolved and concentrated from DCM/hexanes several times to give the trifluoroacetic acid salt of (±) cis-2-fluoro-4-((4-hydroxypyrrolidin-3-yl)oxy)benzonitrile (170 mg, 100%) as colorless waxy solid. MS (m/z) 223.1 (M + H+). A solution of the trifluoroacetic acid salt of (±) cis-2-fluoro-4-((4hydroxypyrrolidin-3-yl)oxy)benzonitrile (170 mg, 0.50 mmol), and 2,4-dichlorobenzene-1-sulfonyl chloride (130 mg, 0.55 mmol) in DCM (5 mL) and water (5 mL) was treated with K2CO3 (340 mg, 2.5 mmol), and the reaction mixture was stirred overnight at 23 °C. The mixture was extracted three times with DCM, and the combined organic extracts were washed twice with brine, dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (10− 70% EtOAc/hexanes) to give 40 (190 mg, 84%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.53 Hz, 1H), 7.91 (d, J = 2.01 Hz, 1H), 7.81 (dd, J = 8.03, 8.78 Hz, 1H), 7.62 (dd, J = 2.01, 8.53 Hz, 1H), 7.13 (dd, J = 2.38, 11.92 Hz, 1H), 6.92 (dd, J = 2.38, 8.66 Hz, 1H), 5.54 (d, J = 5.27 Hz, 1H), 4.99 (q, J = 3.76 Hz, 1H), 4.42−4.51 (m, 1H), 3.73 (dd, J = 4.77, 11.04 Hz, 1H), 3.60 (dd, J = 9751



Article

(d, J = 8.53 Hz, 1H), 7.50−7.63 (m, 2H), 7.41 (dd, J = 1.76, 8.53 Hz, 1H), 6.63−6.80 (m, 2H), 5.27−5.47 (m, 2H), 5.15 (br. s., 1H), 4.20− 4.29 (m, 1H), 4.09−4.16 (m, 1H), 3.92 (dd, J = 5.27, 11.29 Hz, 1H), 3.70 (dd, J = 3.51, 11.29 Hz, 1H). MS (m/z) 426.9 (M + H+). 4-(((3S,4R)-1-((2,4-Dichlorophenyl)sulfonyl)-4-hydroxy-4(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (51). A solution of 50 (250 mg, 0.585 mmol), 4-methylmorpholine 4-oxide (103 mg, 0.878 mmol), and 2.5% osmium tetroxide in t-butanol (0.297 mL, 0.029 mmol) in THF (7 mL) was stirred overnight at 23 °C. Note: Osmium tetraoxide is a toxic and volatile reagent, and we recommend quenching the reaction with aqueous sodium thiosulfate followed by standard workup prior to chromatography, particularly on larger scale. The mixture was concentrated and the residue was subjected to flash chromatography (0−100% EtOAc/hexanes) to give 51 (164 mg, 60%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.53 Hz, 1H), 7.73−7.87 (m, 2H), 7.57 (dd, J = 2.01, 8.53 Hz, 1H), 7.06−7.14 (m, 1H), 6.88 (dd, J = 2.01, 8.78 Hz, 1H), 5.50 (s, 1H), 4.82 (t, J = 5.52 Hz, 1H), 4.72 (d, J = 2.76 Hz, 1H), 3.86 (dd, J = 3.14, 11.92 Hz, 1H), 3.44−3.66 (m, 4H), 3.38 (d, J = 10.29 Hz, 1H). MS (m/z) 460.9 (M + H+). 4-(((3S,4R)-1-((2-Cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (52). A mixture of 49 (49.5 g, 149 mmol) in THF (700 mL) and NaHCO3 (350 mL of a 2 M aqueous solution, 700 mmol) was treated with 2,4-dichlorobenzene-1-sulfonyl chloride (36.5 g, 135 mmol), and the reaction mixture was stirred for 15 min at 23 °C. The organic layer was separated, washed with water and brine, dried over Na2SO4, filtered through a silica plug, and concentrated. The resulting gray solid was triturated with diethyl ether and collected by filtration to give (R)-4-((1-((2-cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4methylenepyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (53.8 g, 88%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.21− 8.35 (m, 2H), 7.81 (t, J = 8.3 Hz, 1H), 7.03 (dd, J = 2.3, 11.8 Hz, 1H), 6.81 (dd, J = 2.3, 8.8 Hz, 1H), 5.49 (s, 1H), 5.46 (d, J = 3.0 Hz, 1H), 5.39 (s, 1H), 4.16−4.25 (m, 1H), 4.06−4.13 (m, 1H), 3.76− 3.85 (m, 1H), 3.65−3.72 (m, 1H). MS (m/z) 452.0 (M + H+). A solution of (R)-4-((1-((2-cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4-methylenepyrrolidin-3-yl)oxy)-2-fluorobenzonitrile (6.18 g, 13.7 mmol), 4-methylmorpholine 4-oxide (2.41 g, 20.5 mmol), and 2.5% osmium tetroxide in t-butanol (8.6 mL, 0.69 mmol) in THF (150 mL) was stirred overnight at 23 °C. The reaction mixture was diluted with EtOAc quenched with the careful addition of 10% aqueous Na2S2O3 solution, and the mixture was stirred for 1 h. The organic layer was separated, washed with 10% aqueous NaHCO3 solution, water, and brine, and then dried over Na2SO4, filtered, concentrated, and subjected to flash chromatography (0−100% EtOAc/hexanes) to give 52 (3.7 g, 53%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.20−8.26 (m, 1H), 8.15−8.20 (m, 1H), 7.76 (t, J = 8.4 Hz, 1H), 7.01 (dd, J = 2.4, 11.7 Hz, 1H), 6.77 (dd, J = 2.3, 8.8 Hz, 1H), 5.53 (s, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.69 (d, J = 2.8 Hz, 1H), 3.87 (dd, J = 3.1, 12.2 Hz, 1H), 3.41−3.57 (m, 5H). MS (m/z) 486.1 (M + H+).

7.91−8.07 (m, 2H), 7.62−7.71 (m, 1H), 7.47−7.59 (m, 2H), 5.73 (dd, J = 2.9, 4.4 Hz, 1H), 5.42 (s, 1H), 5.35 (s, 1H), 4.05−4.16 (m, 1H), 3.92−4.02 (m, 1H), 3.73−3.85 (m, 1H), 3.50 (dd, J = 2.6, 12.4 Hz, 1H), 1.41 (br s, 9H). (S)-tert-Butyl 3-(benzoyloxy)-4-methylenepyrrolidine-1-carboxylate (46). Racemic tert-butyl 3-hydroxy-4-methylenepyrrolidine-1carboxylate 45 (800 g) was resolved in 12.5 g batches at a 10 min cycle time via preparative HPLC (Chiralpak IC, 100 × 250 mm2) eluted with heptanes/IPA (75/25) at a flow rate of 500 mL/min. The respective enantiomer fractions were combined, concentrated, and reconcentrated from Et2O to give 46 (352 g, 44%, 98% ee) as a pale yellow liquid. 1H NMR (400 MHz, CD3OD) δ: 7.99−8.06 (m, 2H), 7.60−7.67 (m, 1H), 7.47−7.54 (m, 2H), 5.80 (br s, 1H), 5.49 (br s, 1H), 5.36 (br s, 1H), 4.16−4.25 (m, 1H), 4.01−4.09 (m, 1H), 3.83 (br s, 1H), 3.64 (dd, J = 2.1, 12.4 Hz, 1H), 1.51 (s, 9H). MS (m/z) 303.9 (M + H+). (S)-tert-Butyl 3-hydroxy-4-methylenepyrrolidine-1-carboxylate (47). KOH pellets (61.4 g, 1.09 mol) were added to MeOH (200 mL), and the solution was cooled in an ice bath to reduce the temperature to 25 °C and then treated with a solution of 46 (83 g, 274 mmol) in MeOH (100 mL). The resulting mixture was stirred for 1 h at 23 °C, filtered through Celite, while rinsed with MeOH (60 mL) and MTBE (100 mL), filtered, and concentrated. The residue was dissolved in water (250 mL), extracted with MTBE (3 × 330 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated to give 47 (55.0 g, 95%) as a pale brown oil. 1H NMR (400 MHz, CD3OD) δ: 5.27 (br s, 1H), 5.14 (br s, 1H), 4.54−58 (m, 1H), 4.03−4.12 (m, 1H), 3.91− 3.99 (br m, 1H), 3.58−3.68 (m, 1H), 3.21−3.28 (1H, partially hidden by solvent peak), 1.49 (s, 9H). MS (m/z) 199.9 (M + H+). (R)-tert-Butyl 3-(4-cyano-3-fluorophenoxy)-4-methylenepyrrolidine-1-carboxylate (48). A solution of 47 (100 g, 500 mmol) in THF (1200 mL) was treated with 2-fluoro-4-hydroxybenzonitrile (83 g, 600 mmol) and trimethylphosphine (1 M in THF, 600 mL, 600 mmol). The mixture was cooled to 0 °C and treated dropwise with DEAD (40 wt % in toluene, 320 mL, 700 mmol) over a period of 1 h while the internal temperature was maintained below 8 °C. The reaction mixture continued to stir at 0 °C for 30 min before being warmed to 23 °C and stirred overnight. The reaction mixture was poured into 1 N NaOH (aq) (1 L) and stirred vigorously while a saturated solution of Na2S2O3 (aq) (50 mL) was added. The mixture was poured into a 50/50 mixture of EtOAc/hexanes and the layers separated. The organic layer was washed with 1 N NaOH (aq) (3 × 1 L), water (1 × 1 L), and brine (1 × 1 L), dried over Na2SO4, filtered, and concentrated to a mixutre of an orange oil and a white solid. The oil was triturated with hexanes:EtOAc, filtered to remove the white solid, and concentrated to give 48 (152 g, 95% yield) as an orange oil. 1 H NMR (400 MHz, DMSO-d6) δ: 7.86 (t, J = 8.3 Hz, 1H), 7.31 (dd, J = 2.3, 11.8 Hz, 1H), 7.07 (dd, J = 8.8, 2.3 Hz, 1H), 5.47 (br s, 1H), 5.43 (br s, 1H), 5.34 (s, 1H), 4.03 (m, 1H, partially hidden by solvent peak), 3.89−3.99 (m, 1H), 3.68−3.80 (m, 1H), 3.46 (dd, J = 2.3, 12.3 Hz, 1H), 1.41 (d, J = 7.8 Hz, 9H). MS (m/z) 263.2 (M + H+ − t-Bu). (R)-2-Fluoro-4-((4-methylenepyrrolidin-3-yl)oxy)benzonitrile, Trifluoroacetic Acid Salt (49). A solution of 48 (122 g, 326 mmol) in DCM (1000 mL) was treated with TFA (150 mL, 195 mmol) and stirred overnight at 23 °C. The reaction mixture was concentrated, and the resulting oil was treated with Et2O (1 L), sonicated, and allowed to stand overnight. The resulting precipitate was collected by filtration, washed with Et2O, and dried to give 49 (93.5 g, 86% yield) as a gray solid. MS (m/z) 219.2 (M + H+). (R)-4-((1-((2,4-Dichlorophenyl)sulfonyl)-4-methylenepyrrolidin3-yl)oxy)-2-fluorobenzonitrile (50). A solution of the trifluoroacetic acid salt of 49 (1.03 g, 4.72 mmol) in water (20 mL) and DCM (20 mL) was treated with 2,4-dichlorobenzene-1-sulfonyl chloride (1.74 g, 7.08 mmol) and potassium carbonate (6.52 g, 47.2 mmol), and the reaction mixture was stirred overnight at 23 °C. The organic layer was separated and the aqueous layer was extracted with DCM. The combined organic layers were washed with brine, concentrated, and subjected to flash chromatography (0−100% EtOAc/hexanes) to give 50 (1.12 g, 56%). 1H NMR (400 MHz, CHLOROFORM-d) δ 8.04

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01317.

■

SMILES representation of compounds with key data (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (484) 923-3471. ORCID

Edward J. Brnardic: 0000-0001-8304-9894 Brian G. Lawhorn: 0000-0003-4618-5314 9752



Article

Notes

(8) Delany, N. S.; Hurle, M.; Facer, P.; Alnadaf, T.; Plumpton, C.; Kinghorn, I.; See, C. G.; Costigan, M.; Anand, P.; Woolf, C. J.; Crowther, D.; Sanseau, P.; Tate, S. N. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol. Genomics 2001, 4, 165−174. (9) Güler, A. D.; Lee, H.; Iida, T.; Shimizu, I.; Tominaga, M.; Caterina, M. Heat-evoked activation of the ion channel, TRPV4. J. Neurosci. 2002, 22, 6408−6414. (10) Strotmann, R.; Harteneck, C.; Nunnenmacher, K.; Schultz, G.; Plant, T. D. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat. Cell Biol. 2000, 2, 695− 702. (11) Everaerts, W.; Nilius, B.; Owsianik, G. The vanilloid transient receptor potential channel TRPV4: From structure to disease. Prog. Biophys. Mol. Biol. 2010, 103, 2−17. (12) Willette, R. N.; Bao, W.; Nerurkar, S.; Yue, T-l.; Doe, C. P.; Stankus, G.; Turner, G. H.; Ju, H.; Thomas, H.; Fishman, C. E.; Sulpizio, A.; Behm, D. J.; Hoffman, S.; Lin, Z.; Lozinskaya, I.; Cassillas, L. N.; Lin, M.; Trout, R. E. L.; Bartholomew, J. V.; Thorneloe, K.; Lashinger, E. S. R.; Figueroa, D. J.; Marquis, R.; Xu, X. Systematic Activation of the Transient Receptor Potential Vanilloid Subtype 4 Channel causes endothelial failure and circulatory collapse: Part 2. J. Pharmacol. Exp. Ther. 2008, 326, 443. (13) Thorneloe, K. S.; Sulpizio, A. C.; Lin, Z.; Figueroa, D. J.; Clouse, A. K.; McCafferty, G. P.; Chendrimada, T. P.; Lashinger, E. S.; Gordon, E.; Evans, L.; Misajet, B. A.; Demarini, D. J.; Nation, J. H.; Casillas, L. N.; Marquis, R. W.; Votta, B. J.; Sheardown, S. A.; Xu, X.; Brooks, D. P.; Laping, N. J.; Westfall, T. D. N-((1S)-1-{[4-((2S)-2{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)- 1piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I. J. Pharmacol. Exp. Ther. 2008, 326, 432−442. (14) Jian, M. Y.; King, J. A.; Al-Mehdi, A. B.; Liedtke, W.; Townsley, M. I. High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4. Am. J. Respir. Cell Mol. Biol. 2008, 38, 386−392. (15) Alvarez, D. F.; King, J. A.; Weber, D.; Addison, E.; Liedtke, W.; Townsley, M. I. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ. Res. 2006, 99, 988−995. (16) Cheung, M.; Bao, W.; Behm, D. J.; Brooks, C. A.; Bury, M. J.; Dowdell, S. E.; Eidam, H. S.; Fox, R. M.; Goodman, K. B.; Holt, D. A.; Lee, D.; Roethke, T. J.; Willette, R. N.; Xu, X.; Ye, G.; Thorneloe, K. S. Discovery of GSK2193874: An orally active, potent, and selective blocker of Transient Receptor Potential Vanilloid 4. ACS Med. Chem. Lett. 2017, 8, 549−554. (17) Hilfiker, M. A.; Hoang, T. H.; Cornil, J.; Eidam, H. S.; Matasic, D. S.; Roethke, T. J.; Klein, M.; Thorneloe, K. S.; Cheung, M. Optimization of a novel series of TRPV4 antagonists with in vivo activity in a model of pulmonary edema. ACS Med. Chem. Lett. 2013, 4, 293−296. (18) Duncton, M. A. J. Small Molecule Agonists and Antagonists of TRPV4. In TRP Channels as Therapeutic Targets; Arpad, S., Ed.; Elsevier, 2015, pp 205−219. (19) Vincent, F.; Duncton, M. A. J. TRPV4 agonists and antagonists. Curr. Top. Med. Chem. 2011, 11, 2216−2226. (20) Thorneloe, K. S.; Cheung, M.; Bao, W.; Alsaid, H.; Lenhard, S.; Jian, M.-Y.; Costell, M.; Maniscalco-Hauk, K.; Krawiec, J. A.; Olzinski, A.; Gordon, E.; Lozinskaya, I.; Elefante, L.; Qin, P.; Matasic, D. S.; James, C.; Tunstead, J.; Donovan, B.; Kallal, L.; Waszkiewicz, A.; Vaidya, K.; Davenport, E. A.; Larkin, J.; Burgert, M.; Casillas, L. N.; Marquis, R. W.; Ye, G.; Eidam, H. S.; Goodman, K. B.; Toomey, J. R.; Roethke, T. J.; Jucker, B. M.; Schnackenberg, C. G.; Townsley, M. I.; Lepore, J. J.; Willette, R. N. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure. Sci. Transl. Med. 2012, 4, 159ra148.

The authors declare the following competing financial interest(s): Authors affiliated with GlaxoSmithKline have received compensation in the form of salary and stock.

■

ACKNOWLEDGMENTS We gratefully acknowledge intellectual and experimental contributions from Stan Martens, Elizabeth Davenport, Michael Fischer, Michael Klein, Kalindi Vaidya, Jeffrey Guss, and Brian Donovan for running the TRPV4 assays; Ron Ramsubhag, Dominic Pascoe, and David Cowan for synthesis of intermediates and compounds; and Doug Minnick for VCD analysis.

■

ABBREVIATIONS USED TRPV4, transient receptor potential vanilloid-4; GSK, GlaxoSmithKline; 9-BBN, 9-borabicyclo[3.3.1]nonane; GASPE, gated spin−echo; OsO4, osmium tetraoxide; NMO, N-morpholine oxide; QM, quantum mechanic; Cl, iv clearance; Vdss, volume of distribution; MRT, mean resonance time; F, oral bioavailability; PPB, plasma−protein binding; fu, unbound free fraction; LW/BW, lung weight/body weight ratio; DIAD, diisopropyl azodicarboxylate; PS−PPh3, polymer supported triphenylphosphine; THF, tetrahydrofuran; Na2SO4, sodium sulfate; K2CO3, potassium carbonate; TBME, tert-butyl methyl ether; Na2S2O3, sodium thiosulfate

■

REFERENCES

(1) Benjamin, E. J.; Blaha, M. J.; Chiuve, S. E.; Cushman, M.; Das, S. R.; de Ferranti, S. D.; Floyd, J.; Fornage, M.; Gillespie, C.; Isasi, C. R.; Jiménez, M. C.; Jordan, L. C.; Judd, S. E.; Lackland, D.; Lichtman, J. H.; Lisabeth, L.; Liu, S.; Longenecker, C. T.; Mackey, R. H.; Matsushita, K.; Mozaffarian, D.; Mussolino, M. E.; Nasir, K.; Neumar, R. W.; Palaniappan, L.; Pandey, D. K.; Thiagarajan, R. R.; Reves, M. J.; Ritchey, M.; Rodriguez, C. J.; Roth, G. A.; Rosamond, W. D.; Sasson, C.; Towfighi, A.; Tsao, C. W.; Turner, M. B.; Virani, S. S.; Voeks, J. H.; Willey, J. Z.; Wilkins, J. T.; Wu, H.; Alger, H. M.; Wong, S. S.; Munter, P.; et al. On Behalf of the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics2017 Update: A Report from the American Heart Association. Circulation 2017, 135, e31−e458. (2) Adams, K. F., Jr.; Fonarow, G. C.; Emerman, C. L.; LeJemtel, T. H.; Costanzo, M. R.; Abraham, W. T.; Berkowitz, R. L.; Galvao, M.; Horton, D. P. For the ADHERE Scientific Advisory Committee and Investigators. Characteristics and outcomes of patients hospitalized for heart failure in the United States: Rationale, design, and preliminary observations from the first 100,000 cases in the Acute Decompensated Heart Failure National Registry (ADHERE). Am. Heart J. 2005, 149, 209−216. (3) Gheorghiade, M.; Filippatos, G.; De Luca, L.; Burnett, J. Congestion in acute heart failure syndromes: An essential target of evaluation and treatment. Am. J. Med. 2006, 119, S3−S10. (4) Mudd, J. O.; Kass, D. A. Tackling heart failure in the twenty-first century. Nature 2008, 451, 919−928. (5) Kaye, D. M.; Krum, H. Drug discovery for heart failure: A new era or the end of the pipeline? Nat. Rev. Drug Discovery 2007, 6, 127− 139. (6) Voets, T.; Prenen, J.; Vriens, J.; Watanabe, H.; Janssens, A.; Wissenbach, U.; Bödding, M.; Droogmans, G.; Nilius, B. Molecular determinants of permeation through the cation channel TRPV4. J. Biol. Chem. 2002, 277, 33704−33710. (7) Wu, S.; Jian, M. Y.; Xu, Y. C.; Zhou, C.; Al-Mehdi, A. B.; Liedtke, W.; Shin, H. S.; Townsley, M. I. Ca2+ entry via alpha1G and TRPV4 channels differentially regulates surface expression of Pselectin and barrier integrity in pulmonary capillary endothelium. Am. J. Physiol. Lung Cell Mol. Physiol. 2009, 297, L650−L657. 9753



Article

(21) Skerratt, S. E.; Mills, J. E.; Mistry, J. Identification of false positives in “HTS hits to lead”: the application of Bayesian models in HTS triage to rapidly deliver a series of selective TRPV4 antagonists. MedChemComm 2013, 4, 244−251. (22) All potency values are an average of at least four measurements. The potency values for 1 and 2 were different in our TRPV4 assay (IC50 = 130 nM and 50 nM, respectively) compared to the values reported in ref 21 (IC50 = 12 nM and 4 nM, respectively), presumably due to notable differences in the assay conditions as follows. (1) In our assay, we used BHK or HEK MSRII to express human TRPV4 versus CHKOK1 cells. (2) In our assay, we incubated cells with blocker for 10 min versus 20 min. (3) In our assay we used an EC80 of TRPV4 agonist GSK634775 versus an EC75 of TRPV4 agonist GSK1016790. (23) Alcaraz, L.; Cridland, A.; Kinchin, E. Novel conversion of 1,2disubstituted cis-epoxides to one-carbon homologated allylic alcohols using dimethylsulfonium methylide. Org. Lett. 2001, 3, 4051−4053. (24) 4-Cyano-3-fluorophenyl methanesulfonate (16). A solution of 2-fluoro-4-hydroxybenzonitrile (2.0 g, 15.6 mmol) and triethylamine (2.2 mL, 16.0 mmol) in THF (20 mL) at 0 °C was treated with methanesulfonyl chloride (1.1 mL, 14.6 mmol) and stirred for 30 min at 0 °C and then overnight at 23 °C. The solvent was removed under reduced pressure to give 16 (2.86 g, 87%) as a beige solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.67−7.82 (m, 1H), 7.16−7.36 (m, 2H), 3.28 (s, 3H). MS (m/z) 216.1 (M + H+). (25) The stereochemistry of 23 was assigned as cis-stereochemistry by inference from NMR experiments of final products 26 and 27. (26) Levy, A. A.; Rains, H. C.; Smiles, S. The rearrangement of hydroxy-sulphones. Part I. J. Chem. Soc. 1931, 0, 3264−3269. (27) Tozer, B. T.; Smiles, S. A rearrangement of aryl salicylates. J. Chem. Soc. 1938, 1897−1900. (28) Shin, D. S.; Park, J. K. Characteristics of the intermediates in the cyclization reactions of heterocycle-fused[1,4]oxazine derivatives: Stepwise versus concerted. Bull. Korean Chem. Soc. 2007, 28, 2219− 2225. (29) A Payne rearrangment resulting in the migration of the epoxide is not possible with the cis-pyrrolidine since it would require invertive nucleophilic attack of the free hydroxyl group on the proximal epoxide, which is not possible since the free hydroxyl group and the epoxide oxygen are on the same face of the pyrrolidine. (30) Shoulders, M. D.; Hodges, J. A.; Raines, R. T. Reciprocity of steric and stereoelectronic effects in the collagen triple helix. J. Am. Chem. Soc. 2006, 128, 8112−8113. (31) DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines, R. T.; Markley, J. L. Collagen stability: Insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegativite substituents on prolyl ring conformations. J. Am. Chem. Soc. 2002, 124, 2497−2505. (32) The stereochemical assignments of 32 and 33 were assigned based on inference from the stereochemical assignment of 34 and 35. Compound 35 displayed a strong NOE between the C3 pyrrolidine methine proton and the C4 pyrrolidine methine proton indicating a cis- configuration. (33) The relative stereochemistry between 34 and 35 was confirmed by the observation of a strong NOE between the methine protons in compound 35, consistent with the cis- isomer. (34) Compound 36 was synthesized according to the procedure found in the following patent application: Hartman, G. D.; Flores, O. A.. Hepatitis B Antivial Agents. Patent WO2013096744 A1, June 27, 2013. (35) The calculated low E conformation is experimentally supported by VCD and IR measurements. The geometry optimization, frequency, and IR and VCD intensity calculations of the 86 conformers that resulted from the conformational search were carried out at the DFT level (b3lyp/dgdzvp2 scrf = (solvent = ethanol)) with Gaussian 09 on Linux Cluster rtpus190.1: Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.;

Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2009. (36) The (R)-enantiomer of 45 can be hydrolyzed with KOH in MeOH and then treated with benzoic acid under Mitsunobu conditions to provide more (S)-enantiomer as described in: Brnardic, E. J.; Brooks, C. A.; Lawhorn, B. G.; Ye, G.; Barton, L. S.; Budzik, B. W.; Matthews, J. J.; Patterson, J. R.; Pero, J. E.; Sanchez, R.; Sender, M. R.; Terrell, L. R.; Behm, D. J.; Thomas, J. V. TRPV4 Antagonists. Patent WO2018055524 A1, Mar 29, 2018. (37) DMPK properties are an average of measurements taken in two animals using Sprague-Dawley rats in a noncrossover design. (38) Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Getting physical in drug discovery II: the impact of chromatographic hydrophobicity measurements and aromaticity. Drug Discovery Today 2011, 16, 822−830. (39) Ward, K. W.; Smith, B. R. A comprehensive quantitative and qualitative evaluation of extrapolation of intravenous pharmacokinetics parameters from rat, dog, and monkey to humans. I. Clearance. Drug Metab. Dispos. 2004, 32, 603−611. (40) Ward, K. W.; Smith, B. R. A comprehensive quantitative and qualitative evaluation of extrapolation of intravenous pharmacokinetics parameters from rat, dog, and monkey to humans. II. Volume of distribution and mean residence time. Drug Metab. Dispos. 2004, 32, 612−619. (41) Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small molecule conformational preferences derived from crystal structure data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008, 48, 1−24. (42) Mendoza, S. A.; Fang, J.; Gutterman, D. D.; Wilcox, D. A.; Bubolz, A. H.; Li, R.; Suzuki, M.; Zhang, D. X. TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress. Am. J. Physiol. Heart Circ. Physiol. 2010, 298, H466. (43) All animal studies were conducted in accordance with the GSK Policy on the Care, Welfare, and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee at GSK. (44) Data are presented as mean ± SEM, and steady-state plasma concentrations are indicated on the graphs. For the vehicle-treated group, changes in MAP were analyzed using one-way ANOVA with Dunnett’s multiple comparisons post hoc test, where #p < 0.05 and ##p < 0.001 versus baseline. Pyrrolidine sulfonamide 52 treated groups were compared to vehicle using two-way ANOVA with Bonferroni’s multiple comparisons post hoc test. Basal LW/BW ratios were compared to vehicle using a one-tailed, unpaired t test with Welch’s correction for variance, while pyrrolidine sulfonamide 52 treated groups were compared to vehicle using one-way ANOVA with Dunnett’s multiple comparisons post hoc test. In all cases, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus vehicle. (45) TRPV4 channel activation results in an influx of divalent and monovalent cations including calcium. The resulting changes in intracellular calcium were monitored using a calcium specific fluorescent dye Fluo-4 (MDS Analytical Technologies). BHK/AC9 cells or HEK MSRII cells were transduced with BacMam virus expressing the human TRPV4 gene at a MOI of 78 were plated in a 384 well poly-D lysine coated plate (15 000 cells/well in 50 μL of culture medium containing DMEM/F12 with 15 mM HEPES, 10% FBS, 1% penicillin−streptomycin and 1% L-glutamine). Cells were incubated for 24 h at 37 °C and 5% CO2. Culture medium was then 9754



Article

aspirated using a Tecan plate-washer and replaced with 20 μL/well of dye loading buffer: HBSS, 500 μM Brilliant Black (MDS Analytical Technologies), and 2 μM Fluo-4 AM. Dye-loaded plates were then incubated in the dark at room temperature for 1−1.5 h. Ten microliters of test compounds diluted in HBSS (with 1.5 mM calcium chloride, 1.5 mM magnesium chloride, and 10 mM HEPES, pH 7.4) + 0.01% Chaps was added to each individual well of the plate, incubated for 10 min at room temperature in the dark, and then 10 μL of agonist (N-((S)-1-(((R)-1-((2-cyanophenyl)sulfonyl)-3-oxoazepan-4-yl)amino)-4-methyl-1-oxopentan-2-yl)benzo[b]thiophene-2carboxamide (see Reference 20) (hereinafter: Agonist Compound) was added to have a final concentration equals to the agonist EC80. Calcium signals were measured using FLIPRTETRA (MDS Analytical Technologies) or FLIPR384 (MDS Analytical Technologies), and the inhibition of Agonist Compound-induced calcium signal by the test compound was determined. (46) Pharmacokinetic studies were conducted on a single day in male Sprague-Dawley rats using a noncrossover design with two animals per route of administration (i.v. and p.o.). Studies were conducted in a cassette fashion with up to five compounds per cassette. At least three days prior to the start of the study, rats received surgically implanted femoral vein, femoral artery, and gastric catheters for i.v. infusion of the test molecules, blood sampling, and oral dose administration, respectively. Dose solutions were filtered prior to administration with the actual dose administered quantified. All PK parameters were calculated based on actual dosage administered to each animal. For i.v. infusion, a dose volume of 4 mL/kg in 5% DMSO, 20% Cavitron (pH 4) was administered for a target dose of 1 mg/kg; i.v. infusions were carried out over 30 min. Oral administration via gastric bolus used a 16 mL/kg dose volume in 5% DMSO, 6% Cavitron (pH 4) for a target dose of 2 mg/kg. Blood samples (110 μL) were collected into sodium heparinized tubes at predetermined time points up to 24 h following compound administration. Blood samples were centrifuged to obtain plasma, and 30 μL of plasma aliquots of each sample were either analyzed immediately or were stored at −80 °C until analyzed. Proteins were precipitated by the addition of 120 μL of acetonitrile containing an appropriate internal standard. Samples were centrifuged to remove precipitate, and aliquots of the resulting supernatant were analyzed for test compound concentrations by HPLC−MS/MS. Analytes were quantified by comparison to a standard curve prepared in identical matrix using Analyst 1.5.1 software. Pharmacokinetic parameters were calculated via noncompartmental analysis with linear-log trapezoidal calculation of area under the curve using Phoenix Winnonlin 6.1.0 software. Oral bioavailability was calculated in a noncrossover fashion for each oral study animal by the ratio of oral dose-normalized area under the curve (0−∞) to the mean i.v. dose-normalized area under the curve (0−∞).

9755


Discovery of Pyrrolidine Sulfonamides as Selective and Orally

Recommend Documents