Discovery of TRPM8 Antagonist (S)-6-(((3-Fluoro-4-(trifluoromethoxy

Article Cite This: J. Med. Chem. 2018, 61, 8186−8201

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Discovery of TRPM8 Antagonist (S)‑6-(((3-Fluoro-4(trifluoromethoxy)phenyl)(3-fluoropyridin-2yl)methyl)carbamoyl)nicotinic Acid (AMG 333), a Clinical Candidate for the Treatment of Migraine Daniel B. Horne,*,†,⊥ Kaustav Biswas,† James Brown,† Michael D. Bartberger,† Jeffrey Clarine,‡ Carl D. Davis,‡ Vijay K. Gore,† Scott Harried,† Michelle Horner,§ Matthew R. Kaller,† Sonya G. Lehto,† Qingyian Liu,† Vu V. Ma,† Holger Monenschein,† Thomas T. Nguyen,† Chester C. Yuan,† Beth D. Youngblood,† Maosheng Zhang,† Wenge Zhong,† Jennifer R. Allen,† Jian Jeffrey Chen,† and Narender R. Gavva† Departments of †Amgen Discovery Research, ‡Pharmacokinetics and Drug Metabolism, and §Comparative Biology and Safety Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States S Supporting Information *

ABSTRACT: Transient-receptor-potential melastatin 8 (TRPM8), the predominant mammalian cold-temperature thermosensor, is a nonselective cation channel expressed in a subpopulation of sensory neurons in the peripheral nervous system, including nerve circuitry implicated in migraine pathogenesis: the trigeminal and pterygopalatine ganglia. Genomewide association studies have identified an association between TRPM8 and reduced risk of migraine. This disclosure focuses on medicinal-chemistry efforts to improve the druglike properties of initial leads, particularly removal of CYP3A4-induction liability and improvement of pharmacokinetic properties. A novel series of biarylmethanamide TRPM8 antagonists was developed, and a subset of leads were evaluated in preclinical toxicology studies to identify a clinical candidate with an acceptable preclinical safety profile leading to clinical candidate AMG 333, a potent and highly selective antagonist of TRPM8 that was evaluated in human clinical trials.

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INTRODUCTION The transient-receptor-potential (TRP) channels are nonselective, Ca2+-permeable ion channels, some of which function as molecular sensors. The TRP-channel family comprises 28 distinct members that are activated by a variety of stimuli, including voltage, pH, temperature (cold and heat), mechanical pressure, osmotic pressure, and endogenous and exogenous ligands. Among the TRP channels, a subset are implicated in temperature sensation and thermoregulation (TRPV1−4 are activated by heat or warmth, TRPM8 is activated by innocuous cold, and TRPA1 is activated by noxious cold). TRPM8 ion channels are activated by cool temperatures (18−23 °C) as well as exogenous ligands such as menthol and icilin.1 TRPM8 is expressed in the lung, bladder, arteries, and prostate and in key components of the peripheral nervous system, such as the dorsal root ganglia (DRG), pterygopalatine ganglia (SPG), and trigeminal ganglia (TG), which are involved in pain sensation and migraines. Potential applications of modulating TRPM8 for treatment of neuropathic2 and chemotherapy-induced cold pain,3 prostate cancer,4 and overactive- and painful-bladder syndromes5 have been described. Previous investigations from various groups have resulted in identification of several smallmolecule scaffolds that modulate TRPM8 activity.6−11 Herein we communicate our efforts targeting TRPM8 inhibition as a potential treatment for migraine. © 2018 American Chemical Society

We previously described SAR studies leading to the identification of TRPM8 inhibitors 112a and 212b (Figure 1)

Figure 1. Previously reported tetrahydroisoquinoline and tetrahydronaphthyridine TRPM8 antagonists.

and results from rat in vivo pharmacodynamic (PD) models of TRPM8 target engagement, the icilin-induced wet-dog shake (WDS), and cold-pressor test (CPT). Compound 2 was a potent inhibitor of TRPM8 (hTRPM8 IC50 = 25 nM and rTRPM8 IC50 = 115 nM), had reasonable pharmacokinetic (PK) properties in rats, and was effective in vivo in both the rat WDS and CPT models of TRPM8 inhibition. Initially, we evaluated potent TRPM8 antagonists as potential therapeutics for neuropathic pain, but in our hands, 2 and other TRPM8 Received: April 2, 2018 Published: August 27, 2018 8186

DOI: 10.1021/acs.jmedchem.8b00518 J. Med. Chem. 2018, 61, 8186−8201

Journal of Medicinal Chemistry

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antagonists were ineffective in animal models of neuropathic pain.13 However, data later emerged from genomewide association studies (GWAS) that identified an association between single-nucleotide polymorphisms (SNPs) that are intergenic to TRPM8 (one SNP located about 1000 bp 5′ to the start codon and a second SNP in the intron) and a reduced risk for migraine.14 On the basis of this new genetic data, we pivoted our existing TRPM8 preclinical program with the intent of seeking a therapeutic for migraine pain. Our lead optimization efforts described herein, seeking to improve in vitro and in vivo properties of early leads, ultimately led to the nomination of development candidate (S)-6-(((3fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)nicotinic acid (35), which was subsequently advanced into human clinical studies.

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Figure 2. Identification of potent biarylmethanamine-based TRPM8 antagonists as a starting scaffold.

RESULTS AND DISCUSSION As previously reported,12b optimization of a tetrahydroisoquinoline series of TRPM8 antagonists resulted in tetrahydronaphthyridine 2, which showed improved PK properties and demonstrated potent activity in two TRPM8-engagement PD models: (i) the icilin-induced rat WDS model, with complete inhibition at 10 mg/kg (Cu = 0.19 μM), and (ii) the rat CPT, with complete inhibition of blood-pressure increase at 10 mg/kg po (Cu = 0.49 μM). However, despite achieving plasma exposures >10-fold over the exposure levels resulting in complete inhibition of rat WDS or CPT, 2 failed to show efficacy in rodent models of neuropathic pain. In addition, we discovered that potent induction of CYP3A4 in human hepatocytes, regulated through the pregnane X receptor (PXR), was endemic throughout this series. Because CYP3A4 plays an important role in human drug metabolism, upon reinitiating our program toward a treatment for migraine, we undertook efforts to eliminate the CYP-induction potential of this series of compounds. In addition, we desired to restart our efforts with a novel chemical scaffold that might provide potentially improved properties, because fairly extensive SAR investigations had already been explored in the tetrahydroisoquinoline and tetrahydronaphthyridine series. In an earlier investigation into alternative syntheses of the tetrahydronaphthyridine core of compound 2, we discovered that one of the uncyclized intermediates in that synthesis, urea analogue 3, suffered minimal loss of potency compared with the cyclized analogue, 2 (Figure 2). We viewed analogue 3 as a novel starting scaffold we could use to potentially address the suboptimal properties of the series. At this point we also discovered that the preferred stereochemistry of the uncyclized analogues was opposite that of the tetrahydronaphthyridine series. Confirmation of stereochemistry by X-ray crystal and VCD or optical-rotation confirmed our stereochemical assignments of both series (Figure 3 and the SI). We postulate that the uncyclized series may be binding in an alternative binding conformation than the cyclized series because there is some divergence in the SARs of the series, although there are some common SARs that suggest the series share the same binding pocket. Initial SAR in the biarylmethanamide series began with substitution of the 3-Br substituent of analogue 3, seeking improvements in potency. The 3-position was tolerant of several lipophilic groups, such as F (120 nM), allyl (28 nM), CF3 (4, 51 nM), and propynyl (48 nM), and the 3-CF3 analogue was selected for further SAR exploration.

A small library of amides (52 analogues) and ureas (203 analogues) was made on the basis of the biarylmethanamine of analogue 4. A variety of aryl amides from the library were welltolerated and retained potency on TRPM8 (34 of 52, 20-fold loss of activity, but TRPM8 inhibition could be increased with incorporation of increasingly lipophilic groups at the 3- and 4positions (9−11, 13, and 14) of the pendant phenyl group. In contrast, substitution at the 2-position of the phenyl (12) and incorporation of heterocycles (19 and 20) or benzylic groups (21) were generally not well-tolerated, leading to less-potent analogues. Investigation of the A-ring demonstrated that removal or transposition of the pyridine nitrogen (22 and 23) led to losses in potency. In addition, the changes above that led to more potent compounds had minimal effects on CYP induction and in vitro stability, likely because of the inability to incorporate polarity into this sector of the molecule. It became apparent at this stage that a handle to address CYP3A4 induction and in vitro stability liabilities would need to come from modifications made elsewhere in the molecule. Because it has been demonstrated that PXR transactivation and the resulting CYP3A4-induction potential could be decreased by lowering lipophilicity or introducing polarity into the periphery of the molecule,15 we turned our attention to the C-ring of the molecule, on the basis of the previous SAR showing that it was unlikely that incorporation of polarity was going to be 8187



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Figure 3. (a) Compound 35 inhibition of icilin-induced wet-dog shakes (WDS) in a dose-dependent manner. (b) Plasma-concentration-dependent inhibition of WDS by 35. ***p < 0.05 by Dunnett’s multiple comparisons.

excellent rat PK, suitable for further profiling in the rat WDS biochemical-challenge model (Table 3). In the progression of this program, we used the rat WDS model as a measure of in vivo TRPM8 target engagement and triaged compounds on the basis of their in vivo potency. Compounds 31, 32, 33, and 35 all demonstrated robust efficacy in the rat WDS model, with plasma levels resulting in 90% inhibition of WDS (IC90,ub) corresponding to 1−10-fold coverage over the in vitro rTRPM8 IC50. Compound 35 exhibited statistically significant (p < 0.05 by Dunnett’s multiple comparisons) and dose-dependent prevention of icilin-induced rat WDS at 0.6, 1, and 3 mg/kg (Figure 3). The effect at 3 mg/kg is considered to be full inhibition (unbound plasma concentration of 120 nM). The estimated ED90 was 1.1 (0.75−1.74) mg/kg (plasma IC90 = 0.80 μM, IC90,ub = 0.024 μM). Because TRPM8 can be activated by cold in addition to icilin, we evaluated doses in the same range of effect in rat WDS in the model of cold-induced increase in blood pressure: the coldpressor-test model. Because validated rodent models of migraine did not exist, we were unable to evaluate the potential efficacy of the compounds against migraine pain preclinically; thus, the CPT was intended to be used as a measure of target engagement that could also be translated to a biomarker for human TRPM8 target engagement by evaluation of human CPT in future clinical studies. Compound 35 inhibited cold-induced increases in blood pressure in the rat CPT in a dose-dependent manner, with 1 and 3 mg/kg producing significant inhibition (p < 0.05 by Dunnett’s multiple comparisons), with an ED90 of 1.1 mg/kg (IC90 = 1.1 μM, IC90,ub = 0.032 μM; Figure 4). Compound 35 also fully blocked the cold-pressor response at 3 mg/kg at an average total plasma concentration of 3.3 μM (plasma unbound concentration of 0.10 μM). Prior to initiating rat and dog toxicology studies, preliminary investigation of potential adverse cardiovascular effects were evaluated using the ex vivo isolated-rabbit-heart (IRH) model. In the IRH model, pyridone 31 caused >20% reduction of left ventricle contractility beginning at 0.3 μM (Table 3), but 31 was inactive in patch-clamp assays against hERG, NaV1.5, and Ltype Ca channels; however, other ion channels could also be responsible for the contractility finding. The plasma-exposure ratio of the IRH study at the rat WDS IC90,ub was only 1-fold, and it was deemed a significant risk, which was confirmed upon further profiling of 31 in an anesthetized-dog CV study. Therefore, further development of pyridone 31 was discontinued. Pyridone analogues 32 and 33 demonstrated improved IRH multiples compared with that of 31 and had acceptable cardiovascular profiles in dog CV studies, whereas carboxylic

tolerated in the A- or B-rings with respect to maintaining TRPM8 potency. Initial incorporation of polar groups into the periphery of the C-ring such as hydroxy (25), nitrile (26), pyridyl (27), or methoxy pyridyl (28) functionalities were not successful in reducing PXR transactivation as compared with the unsubstituted benzamide (Table 2), because these analogues all still showed potency in vitro in PXR transactivation (>90% POC at 2 μM), and this was confirmed for analogue 28 in the humanhepatocyte CYP3A4-induction assay. These changes also had only a minor effect on calculated log D (cLogD) values of the analogues. Our first breakthrough in reducing CYP induction was a change to the C-ring of the analogues that drastically lowered the cLogD values of the molecule (pyridone 29), which for the first time showed lower CYP induction (17% at 1 μM) while still maintaining potency on TRPM8 (hTRPM8 IC50 = 6 nM). We next attempted to reduce lipophilicity further by replacement of the A-ring trifluoromethyl group (at position X). We demonstrated that substitution of the trifluoromethyl group with a fluorine was tolerated, with analogue 31 reducing CYP induction significantly (6% at 1 μM) while still maintaining potency on TRPM8 (hTRPM8 = 10 nM). In contrast, attempts to reduce CYP induction by replacing the trifluoromethyl group in early urea analogue 4 with fluoro gave fluoro urea analogue 30, which resulted in a significant loss of potency and was also confirmed to still have potent activation of hPXR. Further SAR studies around the pyridone 31 scaffold identified several additional analogues, including pyridones 32 and 33. These pyridones were potent on human and rodent TRPM8, had low turnover in liver microsomes, and had reduced CYP-induction activity. Pyridones 31−33 were differentiated in subsequent in vivo PK and toxicological studies. Concurrent with the discovery of the pyridones as a C-ring subunit with desirable in vitro properties, carboxylic acid 34 emerged as a functional group that was simultaneously able to reduce CYP induction and maintain potency. Additional analogues of carboxylic acid 34 identified nicotinic acid 35, which had a slight improvement in potency with the other properties in the appropriate ranges. Substitution of the 3-fluoro of 35 with a trifluoromethyl group gave analogue 36, in which CYP induction was significantly increased. It should also be noted that the reduction in CYP-induction potential, as achieved by lowering lipophilicity with pyridone and carboxylic analogues 29−35, was also accompanied by significantly decreased intrinsic clearance in liver microsomes. Evaluation of in vivo rat PK of pyridone and carboxylic acid containing analogues demonstrated that the compounds had 8188



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Table 1. SAR Summary for Biarylmethanamides

IC50 values based on inhibition of icilin (1 μM)-induced influx of Ca2+ into human-, rat-, or mouse-TRPM8-expressing CHO cells. Each IC50 value reported represents mean IC50 values calculated from three 22-point concentration−response plots; when replicate experiments were performed, the data are reported as the mean value and standard deviation. bIn vitro microsomal stability (intrinsic clearance, CLint), measured in a highthroughput automated format in human liver microsomes (HLM) and rat liver microsomes (RLM). cExpressed as a percentage of the activation a

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Table 1. continued response with rifampicin when both compounds are used at a concentration of 1 or 10 μM dCalculated log D (cLogD) determined with the ACD/ LogD Suite.16

Table 2. Optimization of CYP-Induction Liability

a IC50 values based on inhibition of icilin (1 μM)-induced influx of Ca2+ into human-, rat-, or mouse-TRPM8-expressing CHO cells. Each IC50 value reported represents mean IC50 values calculated from three 22-point concentration−response plots; when replicate experiments were performed, the data are reported as the mean value and standard deviation. bIn vitro microsomal stability (intrinsic clearance, CLint), measured in a highthroughput automated format in human liver microsomes (HLM) and rat liver microsomes (RLM). cExpressed as a percentage of the activation response with rifampicin when both compounds are used at a concentration of 1 or 10 μM dCalculated log D (cLogD) determined with the ACD/ LogD Suite.16

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Table 3. Rat Pharmacokinetic Properties, WDS Results, and Isolated-Rabbit-Heart CV Assessments for Selected Compounds

PK studies in male Sprague−Dawley rats conducted with sampling times up to 24 h, n = 3 animals per study. Interanimal variability was ≤30%; iv studies were done in fed rats, and po studies were done in fasted rats. bDose of 2 mg/kg iv in 100% DMSO. cDose of 1 mg/kg iv in 1% Tween 80, 2% HMPC, 97% water/MSA, pH 2.2. dDose of 1 mg/kg iv in 100% DMSO. eDose of 1 mg/kg po in 1% Tween 80, 2% HMPC, 97% water/MSA, pH 2.2. fDose of 1 mg/kg po in 1% Tween 80, 2% HMPC, 97% water/NaOH, pH 9.0. gIsolated-rabbit-heart assay run with continuous buffer infusion at test-article concentrations of 0.1, 0.3, 1.0, 3.0, and 10.0 μM. NOEL is the maximum concentration at which no significant effects on CV parameters (HR, Qtc, Qrs, Jtc, and LV dpt) were observed. a

Addition of aryl Grignard reagents to the imines gave (S)diarylmethyl sulfinamides 39a−39n with good diastereoselectivity (typically >9:1 dr). It should be noted that the addition of Grignard reagents to (S)-tert-butylsulfinyl imines 38a−38c gave predominantly (S)-stereochemistry at the benzylic amine center as the major diastereomer. This stereochemistry is a reversal of the stereochemistry that is typically observed with N-tertbutylsulfinyl aromatic aldimines. A similar reversal of stereochemistry was previously reported to occur with 2-pyridylsulfinyl aldimine18 and is likely a result of chelation of the pyridine and imine nitrogens and nucleophile addition from the lesshindered top face of intermediate A. Profiling of the enantiomers of urea products 44 and 46 demonstrated a significant loss of potency for the (R)-enantiomers (Scheme 3). Finally HCl treatment of sulfinamide 39 gave amine hydrochlorides 40a− 40n. Amine intermediates 40o−40u were prepared by analogous procedures detailed in the Experimental Section. Finally, formation of analogues using biarylmethanamine 40 was accomplished with either amide-forming conditions to give enantiomerically pure amide 41 or isocyanates to give urea 42 (Scheme 2). Assignment of absolute stereochemistry of analogues was accomplished by X-ray analysis of a key chiral intermediate, bromide 43, which led to a more potent enantiomer of 3-pyridyl urea, 44 (Scheme 3). A subset of additional analogues were

acid analogue 35 showed no effect in IRH or in early in vivo dog cardiovascular safety assessments. On the basis of the above results, pyridone 32 and carboxylic acid 35 were advanced to rat and dog toxicology studies. Pyridone 32 had high safety multiples in a rat 14 day toxicology assessment; however, a finding of vasculitis was identified in a 14 day dog toxicology study at a dose of 10 mg/kg and above and resulted in a low safety multiple. Fortunately, carboxylic acid 35 (AMG 333) was well-tolerated in 14 day rat and dog toxicology studies (Figure 5) and was subsequently nominated as a clinical candidate for the treatment of migraine. In vitro toxicology profiling showed that 35 was selective over several other TRP channels (IC50 TRPV1, V3, V4 > 20 μM; TRPA1 > 40 μM) and had no hits in off-target-activity panels (CEREP, 144 targets at 10 μM, POC > 45%; Ambit kinase, 100 at 1 μM, POC > 50%). Compound 35 was well-tolerated in 28 day rat and dog preclinical safety studies and advanced to Phase 1 human clinical trials.

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CHEMISTRY A general, enantioselective route to analogues was developed using Ellman’s chiral tert-butylsulfinamide chemistry.17 Beginning with 3-substituted picolinaldehydes 37a−37c, formation of tert-butylsulfinyl imines 38a−38c was accomplished with (S)tert-butylsulfinamide and copper(II) sulfate (Scheme 1). 8191



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Figure 4. Rat cold-pressor test (CPT). (a) Increase in blood pressure during 5 min of exposure to cold; dose−response with 0.3, 1, and 3 mg/kg 35. (b) Dose-dependent inhibition of cold-induced increase in blood pressure in CPT by 35. (c) Plasma-concentration-dependent inhibition of CPT by 35. ***p < 0.05 by Dunnett’s multiple comparisons.

Scheme 1. Enantioselective Synthesis of Biarylmethanaminesa

Figure 5. Fourteen day rat and dog toxicology safety multiples for compound 35.

confirmed to contain (S)-stereochemistry using the computational-optical-rotation and VCD-spectra-analysis method described in our previous manuscript.12b

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EXPERIMENTAL SECTION

In Vitro Human- and Rat-TRPM8 Functional Assay. Chinesehamster-ovary (CHO) cells stably expressing rat TRPM8 were generated using the tetracycline-inducible T-REx expression system from Invitrogen, Inc. To enable a luminescence readout based on intracellular increases in calcium, each cell line was also cotransfected with a pcDNA3.1 plasmid containing jellyfish aequorin cDNA.2 Cells were seeded in 96-well plates 24 h before the assay, and TRPM8channel expression was induced with 0.5 μg/mL tetracycline. On the

a Reagents and conditions: (a) (S)-tert-Butylsulfinamide, CuSO4, DCM, rt. (b) ArBr, Mg, THF; 3, THF, −78 °C to rt. (c) HCl, MeOH/1,4-dioxane, rt.

day of the assay, the growth media was removed and cells were incubated with assay buffer for 2 h. Cells were then exposed to test compounds (at varying concentrations) and incubated for 2.5 min prior 8192



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Scheme 2. Amide and Urea Formationa

ITS, 1× PS, and 1× L-glu), and the cells were placed back in the incubator overnight. On the third day, the medium was aspirated and replaced with fresh culture medium containing the test articles dissolved in DMSO (final DMSO concentration of 0.1% in the medium); this process was repeated 24 h later for a total 48 h incubation with test article. On day 5, hepatocytes were lysed in 100 μL of 1× Quantigene 2.0 lysis mixture and stored at −80 °C prior to being assayed for CYP3A4 and β-actin mRNA contents using the Quantigene 2.0 branched DNA assay (Affymetrix) according to the manufacturer’s instructions. Plate-washing steps were performed on an Elx405 automated microplate washer (BioTek Instruments Inc.), and luminescence was analyzed on a Luminoskan Ascent microplate luminometer (Thermo Labsystems). Rat Pharmacokinetics. Fed male Sprague−Dawley rats (n = 3 per group) were dosed intravenously with test article formulated in DMSO, and fasting male Sprague−Dawley rats (n = 3 per group) were dosed by oral gavage with test compound formulated as a suspension in 5% Tween 80/Oraplus. Blood samples were taken over 16 h after dosing, with plasma prepared by centrifugation and analyzed by LC-MS/MS. Total-plasma-concentration time-course data were analyzed by noncompartmental pharmacokinetic methods. In Vivo Assays. Icilin-Induced Wet-Dog Shaking in Rats. All experimental procedures for in vivo experiments were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC). Male Sprague−Dawley rats (220−300 g, Harlan, n = 8 per treatment) were first habituated to the testing room for 30 min. Then, each rat was placed into the automated LABORAS (Metris) apparatus with a transparent Plexiglas shoebox cage for 20 min. Antagonists (31, 32, and 33 were in 2% HPMC and 1% Tween 80, pH 2.2, with MSA; 35 was in 2% HPMC and 1% Tween 80, pH 9.0, with NaOH) or vehicle control (2% HPMC and 1% Tween 80, pH 2.2, with MSA, or 2% HPMC and 1% Tween 80, pH 9.0, with NaOH) were administered po 120 min prior to administration of icilin (3.0 mg/kg in 2% HPMC and 1% Tween 80 in a injection volume of 2.5 mL/kg po), and WDS was recorded for a duration of 30 min after icilin administration. Cold-Pressor Test (CPT) in Rats. TRPM8 antagonists were evaluated in rat CPT to determine whether TRPM8 antagonists would attenuate the increase in blood pressure resulting from exposure to cold stimulation of the paws and ventral half of the body. Male Sprague− Dawley rats weighing 350−450 g were instrumented with a unilateral carotid artery cannula connected to a transducer for measuring blood pressure using a Digi-Med Blood Pressure Analyzer, Model 400. Animals were orally administrated 35 (2% HPMC, 1% Tween 80, pH 9.0, with NaOH) or vehicle control (2% HPMC, 1% Tween 80, pH 9.0, with NaOH) 120 min prior to the cold challenge and anesthetized with sodium pentobarbital at 60 mg/kg ip 20 min prior to cold. Blood pressure was recorded for 4 min for the precold baseline and for an additional 5 min during immersion of the paws and ventral half of the body in ice water (approximately 0 °C). Percent inhibition attributed to treatment with the test compound was then determined using the

a

Reagents and conditions: (a) DIPPA, HATU, DMF, R3CO2H. (b) CDI, DCM, R2NH2, then 40a−40n and DIPEA.

Scheme 3. Determination of Absolute Stereochemistry

to the addition of the agonist, 1 μM icilin (1-(2-hydroxyphenyl)-4-(3nitrophenyl)-3,6-dihydropyrimidin-2-one).3 The luminescence was measured by a charged-couple-device (CCD)-camera-based FLASH luminometer built by Amgen, Inc. Compound activity was calculated using GraphPad Prism 4.01 (GraphPad Software Inc.); the data reported represent mean IC50 values calculated from three 22-point concentration−response plots; when replicate experiments were performed, the data are reported as the mean value and standard deviation. Human- and Rat-Liver-Microsomal Stability. Liver-microsomal stability was measured at 37 °C in phosphate buffer (66.7 mM, pH 7.4). Test compounds (1 μM) were incubated with pooled human or rat liver microsomes at 0.25 mg/mL protein with or without NADPH (1 mM). After 30 min, the reaction was stopped by the addition of acetonitrile containing 0.5% formic acid and internal standard. The quenched samples were centrifuged at 1650g for 20 min. The supernatants were analyzed directly for unchanged test compound using liquid chromatography and tandem-mass-spectrometry detection (LC-MS/ MS). Intrinsic clearance was calculated on the basis of substratedisappearance rate assuming first-order elimination of compound over the 30 min incubation. CYP3A4 Induction. Cryopreserved human hepatocytes were thawed and isolated using CHRM (Lifetech) according to the manufacturer’s instructions. Hepatocytes were then resuspended with plating medium (1× DMEM supplemented with 0.1 μM dexamethasone, 10% fetal bovine serum, 1× ITS, 1× PS, and 1× L-glu) and plated on 96-well collagen I coated plates with a cell density of approximately 70 000 viable cells per well. The plates were then placed in a 37 °C incubator under an atmosphere of 95% air/5% CO2 and 90% relative humidity and allowed to attach overnight. The following day, the plating medium was removed and replaced with culture medium (1× Williams’ medium E supplemented with 0.1 μM dexamethasone, 1×

(

following formula: 1 −

cold‐evoked change in MBP cold‐evoked change in MBP postvehicle

) × 100. Plasma

was collected through an artery catheter immediately after CPT for PK analysis. Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Anhydrous solvents were obtained from Aldrich or Fisher Scientific and used directly. All reactions involving air- or moisture-sensitive reagents were performed under a N2 or Ar atmosphere. All final compounds were purified to >95% purity, as determined by LC-MS with an Agilent 1100 and HP 1100 spectrometer. Silica-gel chromatography was performed using either glass columns packed with silica gel (100−200 or 200−400 mesh, Aldrich Chemical) or prepacked silica-gel cartridges (Biotage or ISCO). 1H NMR spectra were determined with a Bruker 300 MHz or DRX 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm, δ units). Chiral purifications by preparative supercriticalfluid chromatography (SFC) were performed on an Agilent preparative SFC system using Chiralcel or Chiralpak OD-H, AD, AD-H, or AS columns. Elution was by gradient using 5−55% MeOH with 0.2−1% 8193



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diethylamine modifier in supercritical carbon dioxide over 2−5 min at 50−70 mL/min. Trifluoromethylphenyl Grignard reagents used in the synthesis of amines have been reported to undergo highly exothermic decomposition;19 appropriate precautions during generation of this reagent should be taken. Amine Intermediates. (S)-(3-Bromopyridin-2-yl)(4(trifluoromethyl)phenyl)methanamine Hydrochloride (40a). Step 1. To a solution of 3-bromopicoline (25 g, 0.145 mol) in CCl4 were added N-bromosuccinimide (51.66 g, 0.29 mol) and benzoylperoxide (2.5 g, 0.010 mol). The mixture was then gradually heated to reflux and stirred for 30 h. The reaction mixture was then cooled to rt. The succinimide was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting product was purified by flash column chromatography using silica (100−200 mesh) with 10% EtOAc in hexanes to furnish 3-bromo-2-dibromomethylpyridine (10.0 g, 0.0303 mol, 21% yield) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.70 (dd, J = 1.46, 4.53 Hz, 1H), 7.88 (dd, J = 1.46, 8.04 Hz, 1H), 7.14− 7.22 (m, 2H). Step 2. A suspension of 3-bromo-2-dibromomethyl-pyridine (10.0 g, 30.3 mmol) in morpholine (30.0 mL) was heated to 60 °C for 1 h. The reaction mixture was cooled to rt and diluted with EtOAc (200 mL). The pH was adjusted to pH 4 by adding citric acid (40.0 g). The reaction mixture was then extracted with EtOAc (3 × 200 mL), and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography using silica (100−200 mesh) with 3% EtOAc in hexane as eluent to give 3-bromo-pyridine-2-carbaldehyde, 37a (4.06 g, 21.8 mmol, 72% yield), as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 10.27 (s, 1H), 8.78 (dd, J = 1.32, 4.53 Hz, 1H), 8.06 (dd, J = 1.32, 8.18 Hz, 1H), 7.34−7.43 (m, 1H). Step 3. To 3-bromo-pyridine-2-carbaldehyde, 37a (2.03 g, 10.9 mmol), in DCM (50 mL) were added (S)-2-methylpropane-2sulfinamide (1.98 g, 16.4 mmol) and copper(II) sulfate (5.23 g, 32.7 mmol). After being stirred for 16 h at rt, the suspension was filtered through a Celite cartridge, and the cartridge rinsed with DCM. The filtrate was concentrated in vacuo, and the residue was suspended in 20% EtOAc/hexanes and again filtered through a Celite cartridge. The filtrate was adsorbed onto a plug of silica gel and chromatographed through a prepacked silica-gel column (40 g), eluted with 0 to 30% EtOAc in hexane, to provide (S)-N-((3-bromopyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide, 38a (2.14 g, 7.40 mmol, 68% yield), as a light-yellow oil. Step 4. To a stirred suspension of magnesium (2.14 g, 88 mmol) in ether (50 mL) was added 4-bromobenzotrifluoride (5.06 mL, 36.2 mmol). The stirring was continued for 4 h. (Caution! This reaction is exothermic.) The solution was transferred dropwise to a stirred −78 °C solution of (S)-N-((3-bromopyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide, 38a (5.1 g, 17.64 mmol), in THF (100 mL). The stirring was continued for another hour after the addition; then, the reaction was quenched with saturated NH4Cl, and the product was extracted with Et2O (3×), dried over MgSO4, and concentrated in vacuo. The material was purified by ISCO (0−70% EtOAc/hexanes) to give (S)-N-((S)-(3-bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)-2-methylpropane-2-sulfinamide, 39a (6.30 g, 14.5 mmol, 82% yield), as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.57 (br d, J = 4.09 Hz, 1H), 7.85 (d, J = 8.04 Hz, 1H), 7.54 (s, 4H), 7.14 (dd, J = 4.68, 7.89 Hz, 1H), 6.58 (br d, J = 7.31 Hz, 1H), 6.35 (br d, J = 7.75 Hz, 1H), 1.43 (s, 9H). Step 5. To a stirred solution of (S)-N-((S)-(3-bromopyridin-2yl)(4-(trifluoromethyl)phenyl)methyl)-2-methylpropane-2-sulfinamide, 39a (0.220 g, 0.505 mmol), in EtOH/DCM (1:1, 8 mL) was added HCl (4 M in dioxane, 1.01 mL, 4.04 mmol) at 0 °C. Stirring was continued for 1 h; then, the solution was concentrated to dryness to provide (S)-(3-bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methanamine hydrochloride, 40a, as a solid, which was used without further purification in the subsequent step. (R)-(3-Bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methanamine Hydrochloride (ent-40a). To a stirred solution of (R)N-((R)-(3-bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)-2methylpropane-2-sulfinamide (0.96 g, 2.205 mmol, prepared by a

procedure analogous to that of (S)-enantiomer 40a) in EtOH (20 mL) at 0 °C was added HCl (4 M in dioxane, 5.51 mL, 22.1 mmol). Stirring was continued for 2 h. The mixture was concentrated to the dryness to give the title compound (730 mg, 100% yield) as a tan solid. 1H NMR (300 MHz, MeOH-d4) δ 8.76 (d, J = 4.24 Hz, 1H), 8.12 (d, J = 8.18 Hz, 1H), 7.65−7.78 (m, 4H), 7.43 (dd, J = 4.75, 8.11 Hz, 1H), 6.04 (s, 1H). (S)-(4-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40b). Step 1. To a solution of 3(trifluoromethyl)picolinaldehyde (9.80 g, 56.0 mmol) and DCM (50 mL) were added (S)-2-methylpropane-2-sulfinamide (10.3 g, 85.0 mmol) and copper(II) sulfate (35.3 g, 221 mmol). After 1.5 h at rt, the reaction was filtered through a pad of Celite-brand filter agent and rinsed with DCM. The filtrate was concentrated in vacuo to give a darkgreen oil. The crude oil was loaded onto a silica-gel column and eluted with 30% EtOAc in hexanes to give (S)-2-methyl-N-((3(trifluoromethyl)pyridin-2-yl)methylene)propane-2-sulfinamide, 38b (13.2 g, 47.5 mmol, 85% yield), as a golden oil. 1H NMR (300 MHz, CDCl3) δ 9.02 (d, J = 4.3 Hz, 1H), 8.70 (d, J = 1.3 Hz, 1H), 8.38 (d, J = 7.7 Hz, 1H), 7.79 (dd, J = 7.9, 4.8 Hz, 1H), 1.18 (s, 9H). MS (ESI positive ion) m/z: 279.1 (M+1). Step 2. Method A: To an oven-dried round-bottom flask containing magnesium (3.46 g, 143 mmol) and Et2O (120 mL) were added diisobutylaluminum hydride (0.950 mL, 0.950 mmol) and 1 mL of 1bromo-4-(trifluoromethyl)benzene (12.5 mL, 91 mmol) dropwise. The solution was stirred for ∼20 min, during which time the reaction went from clear to a brownish tint. The reaction was placed in an ice bath, and the remaining 1-bromo-4-(trifluoromethyl)benzene was added dropwise over 20 min. In a separate round-bottom flask, a solution of (S)-2methyl-N-((3-(trifluoromethyl)pyridin-2-yl)methylene)propane-2sulfinamide (38b, 13.22 g, 47.5 mmol) and THF (80 mL) was cooled to −78 °C for 10 min, and the Grignard solution was added over 30 min. After 1 h, the reaction was quenched with saturated aqueous potassium sodium tartrate (10 mL). The reaction was poured into H2O (150 mL). The entire solution was filtered through a pad of Celite-brand filter agent and rinsed with THF and EtOAc. The resulting filtrate was separated, and the organics were concentrated in vacuo to give the crude product as a dark-orange oil. The crude product was adsorbed onto a plug of silica gel and chromatographed through a Redi-Sep prepacked silica-gel column (120 g), eluted with 0−40% EtOAc in hexanes, to provide (S)-2-methyl-N-((S)-(4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)propane-2-sulfinamide, 39b (14.83 g, 34.9 mmol, 77% yield), as a golden oil. 1H NMR (600 MHz, DMSO-d6) δ 8.93 (d, J = 4.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.71−7.67 (m, 2H), 7.61−7.59 (m, 1H), 7.54 (d, J = 8.4 Hz, 2H), 6.08 (d, J = 9 Hz, 1H), 5.90 (d, J = 9 Hz, 1H), 1.20 (s, 9H). MS (ESI positive ion) m/z: 425.1 (M+H). Method B: (S)-2-Methyl-N-((3-(trifluoromethyl)pyridin-2-yl)methylene)propane-2-sulfinamide, 38b (0.493 g, 1.772 mmol), was dissolved in dry THF (10 mL) and cooled in an ice bath. 4Trifluoromethylphenylmagnesium bromide (2.0 mmol) was added, and the reaction was stirred until the starting sulfinamide had been consumed. Saturated aqueous NH4Cl (10 mL), H2O (100 mL), and EtOAc (100 mL) were added, and the phases were mixed and separated. The organic layer was dried with magnesium sulfate and evaporated to dryness under reduced pressure. Purification using silica chromatography (hexane to EtOAc gradient) gave 39b. Method C: 1-Bromo-4-(trifluoromethyl)benzene (1.2 equiv) was dissolved in dry THF (0.3 mM) and cooled in an ice bath. Isopropylmagnesium chloride−lithium chloride complex (14% solution in THF, 1.0 equiv) was added, and the mixture was stirred for 10 min. A solution of (S)-2-methyl-N-((3-(trifluoromethyl)pyridin-2yl)methylene)propane-2-sulfinamide, 38b (1 equiv), in dry THF (0.3 mM) was added, and the reaction was stirred. After 1 h, it was quenched by addition of saturated aqueous NH4Cl. H2O and EtOAc were added, and the phases were mixed and separated. The organic phase was dried with magnesium sulfate and evaporated to dryness under reduced pressure. Purification using silica chromatography (hexane to EtOAc gradient) gave 39b. Step 3. To a cooled (0 °C), stirred solution of ((S)-2-methyl-N-((S)(4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)8194



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Step 4. To a cooled (0 °C), stirred solution of (S)-N-((S)-(3-fluoro4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)-2-methylpropane-2-sulfinamide, 39d (220 g, 539 mmol), in Et2O (440 mL) was added saturated HCl in Et2O (2.2 L). Stirring was continued for 2 h at 0 °C. The progress of the reaction was monitored by TLC (100% EtOAc). After completion of the reaction, the reaction mixture was concentrated, triturated with 10% EtOH in Et2O, stirred for 5−10 min, filtered, and dried under vacuum to afford (S)-(3-fluoro-4(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methanamine hydrochloride, 40d (154 g, 452 mmol, 84% yield), as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 3H), 8.59 (d, J = 4.8 Hz, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.75 (dd, J = 11.2, 1.6 Hz, 1H), 7.68 (t, J = 9.2 Hz, 1H), 7.55−7.63 (m, 1H), 7.45 (d, J = 8.8 Hz, 1 H), 6.06 (d, J = 4.0 Hz, 1H). MS (ESI positive ion) m/z: 305.1 (M+H). MS (ESI positive ion) m/z: calcd for C13H9F5N2O, 304.1; found, 305.1 (M+H). (S)-(4-Fluorophenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40e). Following the procedures for 40a and 40b, using Method A and (4-fluorophenyl)magnesium bromide as Grignard reagent afforded 40e. MS (ESI positive ion) m/z: 271.0 (M +H) for free base. (S)-p-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40f). Following the procedures for 40a and 40b, using Method B and p-tolylmagnesium chloride as Grignard reagent afforded 40f. MS (ESI positive ion) m/z: 267.0 (M+H) for free base. (S)-m-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40g). Following the procedures for 40a and 40b, using Method B and m-tolylmagnesium chloride as Grignard reagent afforded 40g. MS (ESI positive ion) m/z: 267.0 (M+H) for free base. (S)-o-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40h). Following the procedures for 40a and 40b, using Method B and o-tolylmagnesium bromide as Grignard reagent afforded 40h. MS (ESI positive ion) m/z: 267.0 (M+H) for free base. (S)-(4-Methoxyphenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40i). Following the procedures for 40a and 40b, using Method A and 1-bromo-4-methoxybenzene as the Grignard precursor afforded 40i. MS (ESI positive ion) m/z: 266.0 (MNH3Cl). (S)-(4-Trifluoromethoxyphenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40j). Following the procedures for 40a and 40b, using Method C and 1-iodo-4-trifluoromethoxybenzene as the Grignard precursor afforded 40j. The material was carried forward to the next step without any further characterization. (S)-(3-Trifluoromethylphenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40k). Following the procedures for 40a and 40b, using Method A and 1-bromo-3-trifluoromethylbenzene as the Grignard precursor afforded 40k. MS (ESI positive ion) m/z: 321.0 (M+H) for free base. (S)-(3-Fluoro-4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40l). Following the procedures for 40a and 40b, using 4-bromo-1-fluoro-2(trifluoromethyl)benzene as the Grignard precursor afforded 40l. MS (ESI positive ion) m/z: 338.9 (M+H) for free base. (S)-(3-Fluoro-4-(trifluoromethyl)phenyl)(3-fluoropyridin-2-yl)methanamine Hydrochloride (40m). Following the procedures for 40a and 40b, using Method A and 4-bromo-2-fluoro-1(trifluoromethyl)benzene as the Grignard precursor afforded 40m. MS (ESI positive ion) m/z: 289.1 (M+H) for free base. (S)-(3-Fluoropyridin-2-yl)(4-(trifluoromethoxy)phenyl)methanamine Hydrochloride (40n). Following the procedures for 40a and 40b, using Method A and 1-bromo-4-(trifluoromethoxy)benzene as the Grignard precursor afforded 40n. MS (ESI positive ion) m/z: 287.0 (M+H) for free base. (S)-Cyclohexyl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40o). Following the procedures for 40a and 40b, using Method A and 2-bromopyridine as the Grignard precursor afforded 40o. MS (ESI positive ion) m/z: 254.0 (M+H) for free base. (R)-Thiazol-2-yl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40p). Following the procedures for 40a and 40b, using Method C and 2-bromothiazole as the Grignard precursor afforded 40p. The material was carried forward to the next step without any further characterization.

propane-2-sulfonamide, 39b (27 g, 63 mmol), in Et2O (270 mL) was added 4.0 M HCl in 1,4-dioxane (157 mL, 630 mmol, 10 equiv) at 0 °C, and the reaction mixture was stirred for 30 min at the same temperature. The reaction progress was monitored by TLC (50% EtOAc in petroleum ether). After completion of the reaction, the reaction mixture was concentrated under reduced pressure and triturated with diethyl ether to give a white solid, which was filtered and dried to give (S)-(4(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methanamine hydrochloride, 40b (14 g, 39 mmol, 70% yield), as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.26 (s, 3H), 9.08 (d, J = 4.2 Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.82−7.77 (m, 3H), 7.67 (d, J = 8.4 Hz, 2H), 5.94 (s, 1H). MS (ESI positive ion) m/z: 321.1 (M+H) for free base. (S)-(3-Fluoropyridin-2-yl)(4-(trifluoromethyl)phenyl)methanamine Hydrochloride (40c). Following the procedures for 40a and 40b, using Method C and 1-bromo-4-(trifluoromethyl)benzene as Grignard reagent afforded 40c. MS (ESI positive ion) m/z: 271.0 (M +H) for free base. (S)-(3-Fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methanamine Hydrochloride (40d). Step 1. To a stirred solution of DABCO (444.1 g, 3.965 mol) in anhydrous Et2O (7 L) at −25 °C in a 20 L four-neck round-bottom flask was added n-BuLi (2.5 M in hexane, 1.586 L, 3.965 mol). The mixture was stirred between −25 to −10 °C for 45 min and then cooled to −70 °C. To the above solution was added 3-fluoropyridine (350 g, 3.605 mol) dropwise, and the reaction was stirred between −70 to −60 °C for 1.5 h before DMF (526 mL, 7.21 mol) was added. The progress of the reaction was monitored by TLC (5% EtOAc in petroleum ether). After 1 h of stirring at −70 °C, water (1.2 L) was added, and the reaction was allowed to warm to rt. The layers were separated, and the aqueous layer was extracted with DCM (5 × 2 L). The combined organic layers were washed with brine and dried over sodium sulfate. After removal of solvent, the crude was purified by silica-gel chromatography using a gradient of EtOAc in hexane to give 3-fluoropicolinaldehyde (203 g, 1.62 mol, 45% yield) as a pale-yellow oil. 1H NMR (400 MHz, CDCl3) δ 10.21 (s, 1H), 8.63 (t, J = 2.2 Hz, 1H), 7.54−7.57 (m, 2H). MS (ESI positive ion) m/z: calcd for C6H4FNO, 125.0; found, 126.0 (M+H). Step 2. A mixture of 3-fluoropicolinaldehyde, 37c (300 g, 2.40 mol); copper(II) sulfate (572 g, 3.60 mol); and (S)-2-methylpropane-2sulfinamide (319 g, 2.64 mol) in DCM (3 L) in a 10 L three-neck round-bottom flask was stirred at rt for 3 h. The progress of the reaction was monitored by TLC (30% EtOAc in petroleum ether). After completion of the reaction, the solid was filtered, and the filtrate was concentrated under vacuum. The crude was purified by column chromatography using silica (60−120 mesh) with 20% ethyl acetate/nhexane to give (S)-N-((3-fluoropyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide, 38c (200 g, 0.877 mmol, 37% yield), as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.64 (s, 1 H), 7.55 (t, J = 8.4 Hz, 1H), 7.46 (d, J = 3.6 Hz, 1H), 1.29 (s, 9H). MS (ESI positive ion) calcd for C10H13FN2OS, 228.1; found, 155.0 (m-, o-, and t-Bu) Step 3. To a stirred suspension of magnesium turnings (78.9 g, 3.29 mol) in THF (2.7 L) was added 3-fluoro-4-trifluoromethoxy bromo benzene (566 g, 2.19 mol). Stirring was continued for 4 h. (Caution! This reaction is slightly exothermic; cool with a water bath if needed.) The solution was cannulated to a stirred solution of (S)-N-((3fluoropyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide, 38c (270 g, 1.18 mol), in THF (2.7 L) at −78 °C dropwise. Stirring was continued for 1 h. The progress of the reaction was monitored by TLC (50% EtOAc in petroleum ether). After completion of the reaction, the reaction mixture was quenched with saturated aqueous NH4Cl (2.5 L), and the solution was extracted with EtOAc (5 × 1 L). The organic layers were combined, dried over Na2SO4, concentrated, and purified by column chromatography using silica (100−200 mesh) with 25−30% EtOAc in petroleum ether as eluent to give (S)-N-((S)-(3-fluoro-4(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)-2-methylpropane-2-sulfinamide, 39d (165 g, 404 mmol, 37% yield), as a brown oil. The less polar, minor isomer was not isolated. 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 4.5 Hz, 1H), 7.18−7.39 (m, 5H), 5.92 (d, J = 5.7 Hz, 1H), 5.64 (d, J = 5.4 Hz, 1H), 1.28 (s, 9H). MS (ESI positive ion) m/z: calcd for C17H17F5N2O2S, 408.1; found, 409.1 (M+H). 8195



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(S)-2-Phenyl-1-(3-(trifluoromethyl)pyridin-2-yl)ethanamine Hydrochloride (40q). Following the procedures for 40a and 40b, using Method B and benzylmagnesium bromide as the Grignard precursor afforded 40q. MS (ESI positive ion) m/z: 267.0 (M+H) for free base. (S)-(4-(Trifluoromethyl)phenyl)(4-(trifluoromethyl)pyridin-3-yl)methanamine Hydrochloride (40r). Step 1. To a solution of 4(trifluoromethyl)nicotinic acid (2.11 g, 11.04 mmol) and N,Odimethylhydroxylamine hydrochloride (1.077 g, 11.04 mmol) in DCM (20 mL) were added N-ethyl-N-isopropylpropan-2-amine (3.78 mL, 22.08 mmol) and HATU (4.20 g, 11.04 mmol). The reaction was stirred at rt under N2 for 5 h. The reaction was then diluted with H2O (50 mL) and extracted with DCM (2 × 50 mL). The organic layers were combined, dried (MgSO4), and concentrated to give the amide. Purification by ISCO (80 g of SiO2, 10−50% EtOAc/hexanes) gave N-methoxy-N-methyl-4-(trifluoromethyl)nicotinamide (2.40 g, 10.25 mmol, 93% yield) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.84 (d, J = 5.12 Hz, 1H), 8.74 (s, 1H), 7.58 (d, J = 5.12 Hz, 1H), 3.44 (s, 3H), 3.38 (s, 3H). MS (ESI positive ion) m/z: 235.1 (M+H). Step 2. To a solution of N-methoxy-N-methyl-4-(trifluoromethyl)nicotinamide (1.50 g, 6.41 mmol) in THF (20 mL) at 0 °C was added diisopropylaluminum hydride (7.69 mL, 7.69 mmol, 1.0 M in hexanes). After this addition, the reaction was immersed in an ice/water bath and stirred for 1 h. Additional diisopropylaluminum hydride (7.69 mL, 7.69 mmol) was added, and the reaction was stirred 1 h at 0 °C. The reaction was quenched by slow addition of H2O (2 mL), which was followed by addition of saturated aqueous sodium potassium tartrate (100 mL). The reaction was diluted with Et2O and stirred at rt for 1 h. The organic layer was separated, and the aqueous layer was extracted with Et2O. The combined organic layers were dried (MgSO4), and the solution was concentrated to 10 mL in vacuo to give a solution of the aldehyde, which was used in the next step without further purification. To a the solution of 4-(trifluoromethyl)nicotinaldehyde, from above, were added DCM (10 mL), (R)-2-methylpropane-2-sulfinamide (1.553 g, 12.81 mmol), and copper sulfate (4.09 g, 25.6 mmol). The suspension was stirred at rt under N2 for 68 h. The suspension was then filtered through Celite-brand filter agent, and the solid was washed with DCM (2 × 20 mL). The filtrates were concentrated and purified by ISCO (40 g of SiO2, 10−50% EtOAc/hexane) to give (R)-2-methyl-N-((4(trifluoromethyl)pyridin-3-yl)methylene)propane-2-sulfinamide (800 mg, 2.87 mmol, 45% yield) as a light-yellow oil. MS (ESI positive ion) m/z: 279.0 (M+H). Step 3. Following the procedure detailed above for 40a and 40b, using Method A and 4-bromobenzotrifluoride gave 40r. MS (ESI positive ion) m/z: 320.9 (M+H) for free base. (S)-(3-Fluoro-4-(trifluoromethyl)phenyl)(2-(trifluoromethyl)phenyl)methanamine (40s). Step 1. To a solution of magnesium (0.066 g, 2.71 mmol) in THF (4 mL) was added dropwise 1-bromo-2(trifluoromethyl)benzene (0.138 mL, 1.016 mmol). The mixture was stirred at rt for 2.5 h. The solution was cooled to −78 °C, and a solution of (S)-N-(3-fluoro-4-(trifluoromethyl)benzylidene)-2-methylpropane2-sulfinamide (0.200 g, 0.677 mmol) in THF (1 mL) was added. The cooling bath was removed, and the mixture was gradually warmed to rt and stirred at rt for 2 h; then, the mixture was heated to 60 °C for 5 min. The reaction was quenched with saturated NH4Cl and extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude was purified by silica-gel chromatography (12 g, 10−50% EtOAc-hexane). (S)-N((S)-(3-fluoro-4-(trifluoromethyl)phenyl)(2-(trifluoromethyl)phenyl)methyl)-2-methylpropane-2-sulfinamide (210.6 mg, 0.477 mmol, 70% yield) was obtained as a light-yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.04 Hz, 1 H), 7.51−7.65 (m, 3 H), 7.45 (br s, 1 H), 7.26 (br s, 2 H), 6.10 (br s, 1 H), 3.72 (br s, 1 H), 1.27 (s, 9 H). MS (ESI positive ion) m/z: 338.0 (M+H). Step 2. To a solution of (S)-N-((S)-(3-fluoro-4-(trifluoromethyl)phenyl)(2-(trifluoromethyl)phenyl)methyl)-2-methylpropane-2-sulfinamide (210.6 mg, 0.477 mmol) in MeOH (2 mL) was added hydrogen chloride (1 M in Et2O, 0.954 mL, 0.954 mmol). The mixture was stirred at rt for 3 h. The mixture was concentrated in vacuo, taken up in DCM (35 mL), washed with saturated aqueous NaHCO3 twice, dried over Na2SO4, and concentrated in vacuo. The residue was purified

by silica-gel chromatography (12 g, 10−50% EtOAc/hexane) to afford (S)-(3-fluoro-4-(trifluoromethyl)phenyl)(2-(trifluoromethyl)phenyl)methanamine, 40s, as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.68 (br d, J = 6.65 Hz, 1H), 7.53 (br d, J = 6.85 Hz, 2H), 7.31−7.43 (m, 3H), 7.27−7.30 (m, 1 H), 5.68 (br s, 1H), 1.77 (br s, 2H). MS (ESI positive ion) m/z: 338.0 (M+H). (S)-Quinolin-3-yl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40t). Following the procedures for 40a and 40b, using Method C and 3-iodoquinoline as the Grignard precursor afforded 40t. MS (ESI positive ion) m/z: 304.1 (M+H) for free base. (S)-Cyclohexyl(3-(trifluoromethyl)pyridin-2-yl)methanamine Hydrochloride (40u). Following the procedures for 40a and 40b, using Method B and cyclohexylmagnesium bromide as the Grignard reagent afforded 40u. MS (ESI positive ion) m/z: 259.1 (M+H) for free base. Analogues. 1-((S)-(3-Bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)-3-((S)-1,1,1-trifluoropropan-2-yl)urea (4). A mixture of di(1H-imidazol-1-yl)methanone (0.083 g, 0.509 mmol) and (S)1,1,1-trifluoropropan-2-amine (0.058 g, 0.509 mmol) in DCM (1 mL) was stirred at rt for 1 h. The reaction mixture was added to a stirred solution of (S)-(3-bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methanamine, 40a (0.187 g, 0.463 mmol), and DIPEA (0.242 mL, 1.39 mmol) in DCM (2 mL). The resulting mixture was stirred at rt over the weekend, then concentrated, and purified by silica-gel flash chromatography (0−40% EtOAc/hexanes) to give the title compound (148 mg, 68% yield) as a white solid. 1H NMR (300 MHz, MeOH-d4) δ 8.63 (d, J = 4.4 Hz, 1 H), 8.03 (d, J = 8.0 Hz, 1 H), 7.51−7.65 (m, 4 H), 7.28 (dd, J = 8.1, 4.6 Hz, 1 H), 6.54 (s, 1 H), 4.41 (dt, J = 14.5, 7.3 Hz, 1 H), 1.27 (d, J = 7.0 Hz, 3 H) . MS (ESI positive ion) m/z: calcd for C17H14BrF6N3O, 469.0, 471.0; found 469.9, 471.9 (M+H). 1-((S)-(4-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2yl)methyl)-3-((S)-1,1,1-trifluoropropan-2-yl)urea (5). Following the procedure used for the formation of urea 4 with amine intermediate 40b and (S)-1,1,1-trifluoropropan-2-amine gave the title compound in 69% yield. 1H NMR (300 MHz, MeOH-d4) δ 8.88 (dd, J = 4.8, 0.9 Hz, 1 H), 8.17 (dd, J = 8.0, 1.0 Hz, 1 H), 7.58−7.65 (m, 2 H), 7.55 (dd, J = 8.0, 4.8 Hz, 1 H), 7.45−7.52 (m, 2 H), 6.57 (s, 1 H), 4.42 (dt, J = 14.4, 7.3 Hz, 1 H), 1.27 (d, J = 6.9 Hz, 3 H). MS (ESI positive ion) m/z: calcd for C18H14F9N3O, 459.1; found 460.0 (M+H). General Amide-Formation Procedures. To a solution of amine hydrochloride 40a−40u (50 mg, 0.156 mmol), the corresponding carboxylic acid (0.156 mmol), and DIPEA (0.080 mL, 0.468 mmol) in DCM or DMF (1 mL) at rt was added an amide-coupling reagent, such as HATU, TBTU, or EDCI (0.156 mmol, 1.0 equiv). The reaction was stirred 3 h at rt. The reaction was diluted with DMF (1 mL), filtered through a syringe filter, and then purified by preparative reverse-phase HPLC (gradient elution 10−100% MeCN/0.1% TFA in H2O). Afterward, one of the following procedures was used: (1) The product-containing fractions were then combined, and the solvent was removed by lyophilization to provide the target compound as a TFA salt. (2) The product was dissolved in MeOH (1 mL) and washed through PL-HCO3 MP-resin, and the resin was further washed with MeOH (2 × 0.4 mL). The combined filtrates were then concentrated and dried in vacuo to give the title compounds as free bases. (3) The product-containing fractions were concentrated, the solids were dissolved in DCM, and the organic layer was extracted with saturated aqueous NaHCO3, dried, and concentrated to provide the title compounds as free bases. (S)-N-((4-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2yl)methyl)quinoline-6-carboxamide (6). Following the general amidecoupling procedure, using amine hydrochloride 40b and quinoline-6carboxylic acid gave the title compound in 94% yield. 1H NMR (300 MHz, MeOH-d4) δ 9.08 (br d, J = 3.36 Hz, 1H), 8.94 (br d, J = 2.63 Hz, 1H), 8.86 (br d, J = 7.89 Hz, 1H), 8.65 (br s, 1H), 8.35 (br d, J = 8.62 Hz, 1H), 8.17 (br t, J = 8.92 Hz, 2H), 7.81−7.96 (m, 1H), 7.63 (br s, 5H), 6.98 (br s, 1H) MS (ESI positive ion) m/z: calcd for C24H15F6N3O 475.1; found, 476.0 (M+H). (S)-N-((4-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2yl)methyl)quinoline-7-carboxamide (7). Following the general amidecoupling procedure, using amine hydrochloride 40b and quinoline-7carboxylic acid gave the title compound in 69% yield. 1H NMR (400 8196



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MHz, MeOH-d4) δ 8.94 (td, J = 1.98, 4.06 Hz, 2H), 8.52 (s, 1H), 8.39− 8.45 (m, 1H), 8.21 (dd, J = 0.98, 8.02 Hz, 1H), 7.98−8.07 (m, 2H), 7.55−7.68 (m, 6H), 6.99 (s, 1H) MS (ESI positive ion) m/z: calcd for C24H15F6N3O 475.1; found, 476.0 (M+H). (S)-N-(Phenyl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline7-carboxamide (8). Following the general amide-coupling procedure, using amine hydrochloride 40t and quinoline-7-carboxylic acid gave the title compound in 74% yield. 1H NMR (400 MHz, CDCl3) δ 8.94 (dd, J = 4.1, 1.4 Hz, 1H), 8.87 (d, J = 4.3 Hz, 1H), 8.53 (s, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.07 (dd, J = 8.4, 1.6 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.36−7.51 (m, 4H), 7.20− 7.33 (m, 3H), 6.93 (d, J = 8.0 Hz, 1H). MS (ESI positive ion) m/z: calcd for C23H16F3N3O, 407.1; found, 408.0 (M+H). (S)-N-((4-Fluorophenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (9). Following the general amide-coupling procedure, using amine hydrochloride 40e and quinoline-7-carboxylic acid gave the title compound in 27% yield. 1H NMR (400 MHz, CDCl3) δ 8.98 (d, J = 4.1 Hz, 1H), 8.89 (d, J = 4.7 Hz, 1H), 8.45−8.56 (m, 2H), 8.19 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.39−7.50 (m, 4H), 6.98 (t, J = 8.6 Hz, 2H), 6.88 (d, J = 7.8 Hz, 1H). MS (ESI positive ion) m/z: calcd for C23H15F4N3O, 425.1; found, 426.0 (M+H). (S)-N-(p-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline7-carboxamide (10). Following the general amide-coupling procedure, using amine hydrochloride 40f and quinoline-7-carboxylic acid gave the title compound in 60% yield. 1H NMR (400 MHz, CDCl3) δ 8.95 (dd, J = 4.2, 1.7 Hz, 1H), 8.87 (d, J = 4.1 Hz, 1H), 8.53 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.16 (dd, J = 8.2, 0.8 Hz, 1H), 8.07 (dd, J = 8.5, 1.7 Hz, 1H), 7.98 (dd, J = 8.0, 1.0 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 8.3, 4.2 Hz, 1H), 7.33−7.42 (m, 3H), 7.05−7.16 (m, 2H), 6.89 (d, J = 7.8 Hz, 1H), 2.28 (s, 3H). MS (ESI positive ion) m/z: calcd for C24H18F3N3O, 421.1; found, 422.0 (M+H). (S)-N-(m-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline7-carboxamide (11). Following the general amide-coupling procedure, using amine hydrochloride 40g and quinoline-7-carboxylic acid gave the title compound in 83% yield. 1H NMR (400 MHz, CDCl3) δ 8.96 (dd, J = 4.1, 1.2 Hz, 1H), 8.88 (d, J = 4.5 Hz, 1H), 8.54 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 8.08 (dd, J = 8.4, 1.4 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.46 (dd, J = 8.3, 4.2 Hz, 1H), 7.40 (dd, J = 7.8, 4.9 Hz, 1H), 7.30 (s, 1H), 7.24 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 2.31 (s, 3H). MS (ESI positive ion) m/z: calcd for C24H18F3N3O, 421.1; found, 422.0 (M+H). (S)-N-(o-Tolyl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline7-carboxamide (12). Following the general amide-coupling procedure, using amine hydrochloride 40h and quinoline-7-carboxylic acid gave the title compound in 48% yield. 1H NMR (400 MHz, CDCl3) δ 8.93− 8.98 (m, 1H), 8.87 (d, J = 4.69 Hz, 1H), 8.52 (s, 1H), 8.18 (d, J = 8.22 Hz, 1H), 8.05 (d, J = 8.61 Hz, 1H), 7.99 (br d, J = 7.82 Hz, 2H), 7.86 (d, J = 8.41 Hz, 1H), 7.46 (dd, J = 4.21, 8.31 Hz, 1H), 7.40 (dd, J = 4.89, 7.82 Hz, 1H), 7.20−7.27 (m, 1H), 7.13−7.20 (m, 2H), 7.02−7.09 (m, 1H), 6.97−7.02 (m, 1H), 2.67 (s, 3H), MS (ESI positive ion) m/z: calcd for C24H18F3N3O, 421.1; found, 422.0 (M+H). (S)-N-((4-Methoxyphenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (13). Following the general amidecoupling procedure, using amine hydrochloride 40i and quinoline-7carboxylic acid gave the title compound in 87% yield. 1H NMR (400 MHz, CDCl3) δ 8.93 (d, J = 3.3 Hz, 1H), 8.86 (d, J = 4.3 Hz, 1H), 8.46− 8.58 (m, 2H), 8.13 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.34−7.48 (m, 4H), 6.79− 6.92 (m, 3H), 3.73 (s, 3H). MS (ESI positive ion) m/z: calcd for C24H18F3N3O2, 437.1; found, 438.0 (M+H). (S)-N-((4-(Trifluoromethoxy)phenyl)(3-(trifluoromethyl)pyridin2-yl)methyl)quinoline-7-carboxamide (14). Following the general amide-coupling procedure, using amine hydrochloride 40j and quinoline-7-carboxylic acid gave the title compound in 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 4.2, 1.7 Hz, 1H), 8.87−8.92 (m, 1H), 8.49−8.56 (m, 2H), 8.21 (dd, J = 8.2, 0.8 Hz, 1H), 8.08 (dd, J = 8.5, 1.7 Hz, 1H), 8.03 (dd, J = 8.2, 0.6 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.43−7.52 (m, 2H), 7.21−7.26 (m, 1H), 7.18 (dd, J = 12.2, 2.1

Hz, 1H), 6.90 (t, J = 8.6 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H). MS (ESI positive ion) m/z: calcd for C24H15F6N3O2, 491.1; found, 492.0 (M +H). (S)-N-((3-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2yl)methyl)quinoline-7-carboxamide (15). Following the general amide-coupling procedure, using amine hydrochloride 40k and quinoline-7-carboxylic acid gave the title compound in 30% yield. 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 4.2, 1.5 Hz, 1H), 8.92 (d, J = 4.5 Hz, 1H), 8.50−8.58 (m, 2H), 8.20 (d, J = 8.2 Hz, 1H), 8.09 (dd, J = 8.4, 1.6 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.69−7.78 (m, 2H), 7.39−7.55 (m, 4H), 6.94 (d, J = 7.6 Hz, 1H). MS (ESI positive ion) m/z: calcd for C24H15F6N3O, 475.1 ; found, 476.0 (M+H). (S)-N-((3-Fluoro-4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (16). Following the general amide-coupling procedure, using amine hydrochloride 40l and quinoline-7-carboxylic acid gave the title compound in 64% yield. 1H NMR (300 MHz, MeOH-d4) δ 8.94 (dd, J = 1.53, 4.31 Hz, 2H), 8.52 (s, 1H), 8.43 (d, J = 8.33 Hz, 1H), 8.22 (d, J = 7.78 Hz, 1H), 7.99−8.08 (m, 2H), 7.58−7.70 (m, 3H), 7.38−7.45 (m, 2H), 6.96 (s, 1H) . MS (ESI positive ion) m/z: calcd for C24H14F7N3O, 493.1; found, 494.0 (M+H). (S)-N-(Quinolin-3-yl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (17). Following the general amide-coupling procedure, using amine hydrochloride 40t and quinoline-7-carboxylic acid gave the title compound in 82% yield. 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 4.3, 1.6 Hz, 1H), 8.95 (d, J = 4.5 Hz, 1H), 8.87 (dd, J = 4.1, 1.6 Hz, 1H), 8.64 (d, J = 7.8 Hz, 1H), 8.56 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.09 (dd, J = 8.6, 1.6 Hz, 1H), 8.05 (s, 1H), 8.03 (s, 1H), 7.93 (s, 1H), 7.83−7.91 (m, 2H), 7.44−7.51 (m, 2H), 7.37 (dd, J = 8.3, 4.2 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H). MS (ESI positive ion) m/z: calcd for C26H17F3N4O, 458.1; found, 459.0 (M +H). (S)-N-(Cyclohexyl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (18). Following the general amide-coupling procedure, using amine hydrochloride 40u and quinoline-7-carboxylic acid gave the title compound in 58% yield. 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 1.27, 4.01 Hz, 1H), 8.79 (d, J = 4.30 Hz, 1H), 8.48 (s, 1H), 8.19 (d, J = 8.22 Hz, 1H), 8.09 (d, J = 8.23 Hz, 1H), 7.98 (d, J = 7.82 Hz, 1H), 7.89 (d, J = 8.61 Hz, 1H), 7.43−7.55 (m, 2H), 7.35 (dd, J = 4.99, 7.73 Hz, 1H), 5.68 (br t, J = 7.92 Hz, 1H), 1.86−1.98 (m, 2H), 1.77 (br s, 1H), 1.59−1.73 (m, 2H), 1.33−1.47 (m, 1H), 1.12− 1.33 (m, 5H). MS (ESI positive ion) m/z: calcd for C23H22F3N3O, 413.2; found, 414.0 (M+H). (S)-N-(Pyridin-2-yl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (19). Following the general amide-coupling procedure, using amine hydrochloride 40o and quinoline-7-carboxylic acid gave the title compound in 51% yield. 1H NMR (400 MHz, CDCl3) δ 8.91−9.04 (m, 2H), 8.87 (d, J = 4.69 Hz, 1H), 8.72 (s, 1H), 8.56 (d, J = 4.69 Hz, 1H), 8.27 (d, J = 8.22 Hz, 1H), 8.13 (d, J = 8.41 Hz, 1H), 8.02 (d, J = 8.02 Hz, 1H), 7.91 (d, J = 8.61 Hz, 1H), 7.67−7.74 (m, 1H), 7.59 (d, J = 7.82 Hz, 1H), 7.53 (dd, J = 4.30, 8.22 Hz, 1H), 7.39 (dd, J = 4.99, 7.73 Hz, 1H), 7.18−7.23 (m, 1H), 6.98 (d, J = 7.43 Hz, 1H). MS (ESI positive ion) m/z: calcd for C22H15F3N4O, 408.1; found, 409.1 (M+H). (R)-N-(Thiazol-2-yl(3-(trifluoromethyl)pyridin-2-yl)methyl)quinoline-7-carboxamide (20). Following the general amide-coupling procedure, using amine hydrochloride 40p and quinoline-7-carboxylic acid gave the title compound in 59% yield. 1H NMR (400 MHz, CDCl3) δ 8.98 (dd, J = 1.37, 4.11 Hz, 1H), 8.88 (d, J = 4.50 Hz, 1H), 8.56−8.66 (m, 2H), 8.19 (d, J = 8.02 Hz, 1H), 8.03−8.14 (m, 2H), 7.90 (d, J = 8.41 Hz, 1H), 7.69 (d, J = 3.33 Hz, 1H), 7.47 (dd, J = 4.11, 8.22 Hz, 2H), 7.31 (d, J = 3.13 Hz, 1H), 7.25 (d, J = 8.02 Hz, 1H). MS (ESI positive ion) m/z: calcd for C20H13F3N4OS, 414.1; found, 415.0 (M +H). (S)-N-(2-Phenyl-1-(3-(trifluoromethyl)pyridin-2-yl)ethyl)quinoline-7-carboxamide (21). Following the general amide-coupling procedure, using amine hydrochloride 40q and quinoline-7-carboxylic acid gave the title compound in 71% yield. 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 1.56, 4.11 Hz, 1H), 8.74 (d, J = 4.30 Hz, 1H), 8.43 (s, 1H), 8.17 (d, J = 8.02 Hz, 1H), 7.98 (br d, J = 8.22 Hz, 2H), 7.85 8197



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(d, J = 7.4 Hz, 1H), 8.92 (d, J = 3.9 Hz, 1H), 8.22−8.36 (m, 1H), 8.17 (d, J = 2.5 Hz, 1H), 7.90 (dd, J = 9.7, 2.6 Hz, 1H), 7.68−7.75 (m, J = 8.2 Hz, 2H), 7.63 (dd, J = 7.8, 4.9 Hz, 1H), 7.41−7.57 (m, J = 8.0 Hz, 2H), 6.76 (d, J = 7.4 Hz, 1H), 6.33 (d, J = 9.6 Hz, 1H). MS (ESI positive ion) m/z: calcd for C20H13F6N3O2, 441.1; found, 442.1 (M+H). 1-((S)-(3-Fluoropyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)3-((S)-1,1,1-trifluoropropan-2-yl)urea (30). To a solution of (S)-1,1,1trifluoropropan-2-amine (88 mg, 0.783 mmol) and DCM (2.5 mL) was added CDI (69 mg, 0.426 mmol). The solution was stirred for 30 min and then treated with a solution of amine hydrochloride 40c (200 mg, 0.652 mmol) and DIPEA (0.398 mL, 2.28 mmol) in DCM (1.5 mL). After 16 h, the crude reaction was adsorbed onto a Redi-Sep prepacked silica-gel column (4 g), eluted with 20−40% EtOAc in hexane, to provide the title compound (140 mg, 52.5% yield). 1H NMR (300 MHz, CDCl3) δ 8.33 (d, J = 4.7 Hz, 1 H), 7.52 (d, J = 8.5 Hz, 2 H), 7.46 (d, J = 8.3 Hz, 2 H), 7.38 (t, J = 8.3 Hz, 2 H), 7.23−7.29 (m, 1 H), 6.80 (d, J = 7.2 Hz, 1 H), 6.39 (dd, J = 7.2, 1.8 Hz, 1 H), 4.84 (d, J = 9.5 Hz, 1 H), 4.44−4.62 (m, 1 H), 1.25 (d, J = 6.9 Hz, 3 H). MS (ESI positive ion) m/z: calcd for C17H14F7N3O, 409.1; found, 410.0 (M+H). (S)-N-((3-Fluoropyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)6-oxo-1,6-dihydropyridine-3-carboxamide (31). Following the general amide-coupling procedure, using amine hydrochloride 40c and 6oxo-1,6-dihydropyridine-3-carboxylic acid gave the title compound in 64% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.01 (br s, 1H), 9.15 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 4.5 Hz, 1H), 8.19 (d, J = 2.5 Hz, 1H), 7.92 (dd, J = 9.7, 2.6 Hz, 1H), 7.77 (td, J = 9.3, 1.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 (dt, J = 8.5, 4.4 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.35 (d, J = 9.8 Hz, 1H). MS (ESI positive ion) m/z: calcd for C19H13F4N3O2, 391.1; found, 392.1 (M+H). (S)-N-((3-Fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2yl)methyl)-6-oxo-1,6-dihydropyridine-3-carboxamide (32). Following the general amide-coupling procedure, using amine hydrochloride 40d and 6-oxo-1,6-dihydropyridine-3-carboxylic acid gave the title compound in 86% yield. 1H NMR (300 MHz, MeOH-d4) δ 8.48 (d, J = 4.5 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 9.6, 2.6 Hz, 1H), 7.63 (td, J = 9.1, 1.3 Hz, 1H), 7.34−7.51 (m, 3H), 7.26−7.34 (m, 1H), 6.65 (s, 1H), 6.53 (d, J = 9.6 Hz, 1H). MS (ESI positive ion) m/z: calcd for C19H12F5N3O3, 425.1; found, 425.9 (M+H). (S)-N-((3-Fluoro-4-(trifluoromethyl)phenyl)(3-fluoropyridin-2-yl)methyl)-6-oxo-1,6-dihydropyridine-3-carboxamide (33). Following the general amide-coupling procedure, using amine hydrochloride 40m and 6-oxo-1,6-dihydropyridine-3-carboxylic acid gave the title compound in 59% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.00 (br s, 1H), 9.15 (d, J = 7.8 Hz, 1H), 8.43 (dt, J = 4.7, 1.4 Hz, 1H), 8.16 (d, J = 2.3 Hz, 1H), 7.89 (dd, J = 9.6, 2.7 Hz, 1H), 7.70−7.81 (m, 2H), 7.57 (d, J = 11.9 Hz, 1H), 7.48 (dt, J = 8.5, 4.4 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.34 (d, J = 9.6 Hz, 1H). MS (ESI positive ion) m/z: calcd for C19H12F5N3O2, 409.1; found, 410.0 (M+H). (S)-4-(((3-Fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2yl)methyl)carbamoyl)benzoic Acid (34). Following the general amidecoupling procedure, using amine hydrochloride 40d and 4(methoxycarbonyl)benzoic acid gave the intermediate ester (structure not shown) in 75% yield. To a solution of (S)-methyl 4-(((3-fluoro-4(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)benzoate (105 mg, 0.225 mmol) and THF (2 mL)/MeOH (1 mL) was added 1 M LiOH (1 mL). The solution was stirred at rt. After 16 h, the reaction was diluted with water. The aqueous solution was acidified with 1 N HC1 to a pH of 7 and extracted with EtOAc (3 × 15 mL). The combined EtOAc layers were dried over MgSO4 and concentrated in vacuo to give the title compound (99% yield) as a white solid. 1H NMR (300 MHz, MeOH-d4) δ 8.49 (d, J = 4.7 Hz, 1H), 8.06−8.15 (m, J = 8.5 Hz, 2H), 7.89−8.00 (m, J = 8.5 Hz, 2H), 7.64 (t, J = 9.1 Hz, 1H), 7.31− 7.51 (m, 4H), 6.70 (s, 1H). MS (ESI positive ion) m/z: calcd for C21H12F5N2O4, 452.1; found, 453.0 (M+H). (S)-6-(((3-Fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2yl)methyl)carbamoyl)nicotinic Acid (35). Step 1. To a solution of (S)(3-fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methanamine hydrochloride, 40d (17.0 g, 49.9 mmol), in N,Ndimethylformamide (166 mL) were added 5-(methoxycarbonyl)pyridine-2-carboxylic acid (9.94 g, 54.9 mmol), HATU (20.9 g, 54.9

(d, J = 8.41 Hz, 1H), 7.67 (br d, J = 8.41 Hz, 1H), 7.46 (dd, J = 4.30, 8.22 Hz, 1H), 7.36 (dd, J = 4.79, 7.73 Hz, 1H), 7.13−7.24 (m, 3H), 7.04−7.12 (m, 2H), 6.06−6.13 (m, 1H), 3.38 (dd, J = 5.28, 13.69 Hz, 1H), 3.13 (dd, J = 7.82, 13.69 Hz, 1H). MS (ESI positive ion) m/z: calcd for C24H18F3N3O, 421.1; found, 422.0 (M+H). (S)-N-((4-(Trifluoromethyl)phenyl)(4-(trifluoromethyl)pyridin-3yl)methyl)quinoline-7-carboxamide (22). Following the general amide-coupling procedure, using amine hydrochloride 40r and quinoline-7-carboxylic acid gave the title compound in 48% yield. 1H NMR (300 MHz, MeOH-d4) δ 9.14 (dd, J = 4.8, 1.3 Hz, 1H), 8.76− 8.92 (m, 3H), 8.61 (s, 1H), 8.17- 8.32 (m, 2H), 7.92 (dd, J = 8.4, 4.9 Hz, 1H), 7.84 (d, J = 5.1 Hz, 1H), 7.68- 7.79 (m, J = 8.2 Hz, 2H), 7.46- 7.58 (m, J = 8.0 Hz, 2H), 6.99 (s, 1H). MS (ESI positive ion) m/z: calcd for C24H15F6N3O, 475.1; found, 476.0 (M+H). (S)-N-((3-Fluoro-4-(trifluoromethyl)phenyl)(2-(trifluoromethyl)phenyl)methyl)quinoline-7-carboxamide (23). Following the general amide-coupling procedure, using amine 40s and quinoline-7-carboxylic acid gave the title compound in 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.97 (dd, J = 4.1, 1.6 Hz, 1H), 8.43 (s, 1H), 8.19- 8.26 (m, 1H), 8.07 (dd, J = 8.5, 1.7 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.60 (q, J = 7.0 Hz, 2H), 7.48- 7.54 (m, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.11−7.25 (m, 2H), 6.83−6.93 (m, 2H). MS (ESI positive ion) m/z: calcd for C25H15F7N2O, 492.1; found, 493.0 (M+H). (S)-N-((3-Fluoro-4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)benzamide (24). Following the general amidecoupling procedure, using amine hydrochloride 40l and benzoic acid gave the title compound in 50% yield. 1H NMR (300 MHz, MeOH-d4) δ 8.91 (d, J = 4.74 Hz, 1H), 8.21 (d, J = 7.80 Hz, 1H), 7.85 (d, J = 7.59 Hz, 2H), 7.41−7.69 (m, 5H), 7.37 (d, J = 3.22 Hz, 1H), 7.34 (s, 1H), 6.90 (s, 1H). MS (ESI positive ion) m/z: calcd for C21H13F7N2O, 442.1; found, 443.0 (M+H). (S)-4-Hydroxy-N-((4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)benzamide (25). Following the general amidecoupling procedure, using amine hydrochloride 40b and 4-hydroxybenzoic acid gave the title compound in 17% yield. 1H NMR (400 MHz, MeOH-d4) δ 9.15 (d, J = 4.9 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.00 (s, 2H), 7.84−7.88 (m, 2H), 7.81−7.84 (m, 1H), 7.76−7.81 (m, 2H), 7.15 (s, 1H), 7.07 (d, J = 8.6 Hz, 2H). MS (ESI positive ion) m/z: calcd for C21H14F6N2O2, 440.1; found, 441.0 (M+H). (S)-4-Cyano-N-((4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)benzamide (26). Following the general amidecoupling procedure, using amine hydrochloride 40b and 4cyanobenzoic acid gave the title compound in 71% yield. 1H NMR (400 MHz, MeOH-d4) δ 8.94 (d, J = 3.9 Hz, 1H), 8.22 (d, J = 7.2 Hz, 1H), 7.97−8.06 (m, 2H), 7.81−7.90 (m, 2H), 7.64−7.70 (m, 2H), 7.57−7.63 (m, 3H), 6.94 (s, 1H). MS (ESI positive ion) m/z: calcd for C22H13F6N3O, 449.1; found, 450.0 (M+H). (S)-N-((4-(Trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2yl)methyl)nicotinamide (27). Following the general amide-coupling procedure, using amine hydrochloride 40b and nicotinic acid gave the title compound in 77% yield. 1H NMR (400 MHz, MeOH-d4) δ 8.99− 9.05 (m, 1H), 8.94 (d, J = 4.1 Hz, 1H), 8.71 (dd, J = 4.9, 1.6 Hz, 1H), 8.26−8.33 (m, 1H), 8.22 (dd, J = 7.9, 0.9 Hz, 1H), 7.64−7.69 (m, 2H), 7.58−7.63 (m, 3H), 7.56 (ddd, J = 7.9, 5.0, 0.8 Hz, 1H), 6.96 (s, 1H). MS (ESI positive ion) m/z: calcd for C20H13F6N3O, 425.1; found, 426.0 (M+H). (S)-6-Methoxy-N-((4-(trifluoromethyl)phenyl)(3(trifluoromethyl)pyridin-2-yl)methyl)nicotinamide (28). Following the general amide-coupling procedure, using amine hydrochloride 40b and 6-methoxynicotinic acid gave the title compound in 49% yield. 1 H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 7.4 Hz, 1H), 8.91 (d, J = 4.5 Hz, 1H), 8.71 (d, J = 2.3 Hz, 1H), 8.25 (d, J = 7.4 Hz, 1H), 8.18 (dd, J = 8.7, 2.4 Hz, 1H), 7.66−7.76 (m, J = 8.2 Hz, 2H), 7.62 (dd, J = 7.9, 4.8 Hz, 1H), 7.47−7.58 (m, J = 8.0 Hz, 2H), 6.86 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 7.4 Hz, 1H), 3.89 (s, 3H). MS (ESI positive ion) m/z: calcd for C21H15F6N3O2, 455.1; found, 456.0 (M+H). (S)-6-Oxo-N-((4-(trifluoromethyl)phenyl)(3-(trifluoromethyl)pyridin-2-yl)methyl)-1,6-dihydropyridine-3-carboxamide (29). Following the general amide-coupling procedure, using amine hydrochloride 40b and 6-oxo-1,6-dihydropyridine-3-carboxylic acid gave the title compound in 36% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.12 8198



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mmol), and DIPEA (18.23 mL, 105 mmol). The resulting mixture was then stirred at rt overnight. Upon completion of the reaction, H2O (400 mL) was added, and the mixture was extracted with EtOAc (2 × 200 mL). The combined organic extracts were dried over MgSO4 and concentrated. The crude product was purified by silica-gel flash column chromatography (solid loading, 0−100% EtOAc/hexane) to give (S)methyl 6-(((3-fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2yl)methyl)carbamoyl)nicotinate (11.5 g, 24.6 mmol, 49.3% yield) as a white solid. Additional less-pure product (8.5 g, 46% purity at 254 nM) was also isolated. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (d, J = 7.4 Hz, 1 H), 9.21 (dd, J = 2.2, 0.8 Hz, 1 H), 8.58 (d, J = 4.7 Hz, 1 H), 8.52 (dd, J = 8.2, 2.2 Hz, 1 H), 8.20 (dd, J = 8.1, 0.7 Hz, 1 H), 7.84 (s, 1 H), 7.49− 7.64 (m, 3 H), 7.38 (s, 1 H), 6.59 (dd, J = 7.4, 1.4 Hz, 1 H), 3.95 (s, 3 H). MS (ESI, positive ion) m/z: calcd for C21H14F5N3O4, 467.1; found, 468.0 (M+H). Step 2. To a suspension of (S)-methyl-6-(((3-fluoro-4(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)nicotinate (11.5 g, 24.6 mmol) in MeOH (140 mL) was added lithium hydroxide hydrate (2.07 g, 49.2 mmol) in 47 mL of water. The mixture was stirred at rt for 5 min. The solid (starting material) did not dissolve well in MeOH. THF (5 mL) was added, and the mixture was stirred at rt for 1 h. The mixture was concentrated to remove THF and MeOH, and the aqueous solution was cooled to 0 °C. The pH was adjusted to 6−7 using 2 N HCl and concentrated HCl. EtOAc (300 mL) was added, and the resulting mixture was then stirred at rt for 15 min. The organic layer was collected, and the aqueous layer was extracted with EtOAc (200 mL). The combined organic extracts were dried over MgSO4 and concentrated. The solid mixture was then purified by silica-gel flash column chromatography (solid loading, 40% EtOAc/hexane, then 10% MeOH in EtOAc) to afford (S)-6-(((3-fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)nicotinic acid (7.47 g, 67%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.75 (br s, 1H), 9.85 (d, J = 7.4 Hz, 1H), 9.18 (d, J = 1.4 Hz, 1H), 8.58 (d, J = 4.7 Hz, 1H), 8.48 (dd, J = 8.1, 2.1 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 9.3 Hz, 1H), 7.50−7.61 (m, 3H), 7.36 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 7.2 Hz, 1H). MS (ESI, positive ion) m/z: calcd for C20H12F5N3O4, 453.1; found, 454.0 (M+H). (S)-1-((3-Bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)3-(pyridin-3-yl)urea (44). A solution of amine hydrochloride 40a was diluted with DCM (5 mL) and treated with DIPEA (0.883 mL, 5.05 mmol) followed by 3-pyridyl isocyanate (0.061 g, 0.505 mmol). The resulting mixture was stirred at rt overnight, concentrated, and purified by ISCO (3% MeOH/DCM) to give the title compound (0.175 g, 77% yield) as a white solid. 1H NMR (400 MHz, MeOH-d4) δ 8.65 (br d, J = 4.30 Hz, 1H), 8.52 (s, 1H), 8.12 (br d, J = 4.69 Hz, 1H), 8.04 (d, J = 8.02 Hz, 1H), 7.94 (br d, J = 8.22 Hz, 1H), 7.62 (s, 4H), 7.27−7.34 (m, 2H), 6.60 (s, 1H). MS (ESI positive ion) m/z: calcd for C19H14BrF3N4O, 450.0; found, 450.9 (M+H). (R)-1-((3-Bromopyridin-2-yl)(4-(trifluoromethyl)phenyl)methyl)3-(pyridin-3-yl)urea (46). A mixture of (R)-(3-bromopyridin-2-yl)(4(trifluoromethyl)phenyl)methanamine hydrochloride, ent-40a (0.196 g, 0.485 mmol); DIPEA (0.254 mL, 1.46 mmol); and 3-isocyanatopyridine (0.058 g, 0.485 mmol) in DCM (4 mL) was stirred at rt for 24 h. The mixture was concentrated and purified by ISCO (65% EtOAc/ hexanes) to give the title compound (175 mg, 80% yield) as a white solid. 1H NMR (300 MHz, MeOH-d4) δ 8.65 (d, J = 4.53 Hz, 1H), 8.52 (d, J = 2.05 Hz, 1H), 8.12 (d, J = 4.68 Hz, 1H), 8.04 (d, J = 8.18 Hz, 1H), 7.88−7.99 (m, 1H), 7.61 (s, 4H), 7.24−7.36 (m, 2H), 6.60 (s, 1H), 4.83 (s, 7H), 3.31−3.35 (m, 3H). MS (ESI positive ion) m/z: calcd for C19H14BrF3N4O, 450.0; found, 450.9 (M+H).

preclinical safety studies, which resulted in the advancement of 35 to Phase 1 human clinical trials.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00518. Computed and observed optical rotation data used for absolute stereochemical determination, X-ray crystal data for 48, and TRPM8 IC50’s including standard deviations (PDF) Molecular-formular strings (CSV) PDB coordinates for 48 (PDB) Accession Codes

X-ray crystal data for 48 was deposited in the Cambridge Structural Database (CCDC) under accession code 1841307.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 617-949-5134. E-mail: [email protected]. ORCID

Daniel B. Horne: 0000-0001-7760-6498 Kaustav Biswas: 0000-0001-9971-1424 Michael D. Bartberger: 0000-0002-5167-3139 Present Address ⊥

D.B.H.: Sage Therapeutics, 215 First Street, Cambridge, MA 01760, United States Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Steve Hitchcock, Randy Hungate, and Ken Wild for support of this research program. Thanks also are given to Kyung Gahm and Wes Barnhart for HPLC chiral separations, Yong-Jae Kim and David Bauer for managing the outsourced materials and scale-ups for toxicology studies, and Mike Hayashi and Josh Dekeyser for hepatocyte-induction studies. We also thank the entire TRPM8 research team (Neuroscience, Pharmaceutics, Toxicology, and Pharmacokinetics and Drug Metabolism).

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ABBREVIATIONS USED AUC0‑∞, area under the plasma-concentration−time curve from time 0 to infinity; CHRM, cryopreserved-hepatocyte-recovery medium; CL, total body clearance; cLogD, calculated log D; CPT, cold-pressor test; DIPEA, diisopropylethyl amine; DMEM, Dulbecco’s modified Eagle’s medium; DRG, dorsal root ganglia; GWAS, genomewide association studies; HLM, human liver microsomes; HMPC, hydroxymethyl propyl cellulose; ITS, insulin transferrin selenium; L-glu, L-glutamine; MSA, methanesulfonic acid; PS, penicillin/streptomycin; PXR, pregnane X receptor; RLM, rat liver microsomes; (SNPs), single-nucleotide polymorphisms; SPG, pterygopalatine ganglia; TRPM8, transient-receptor-potential melastatin type 8; TG, trigeminal ganglia; Vss, volume of distribution; WDS, wet-dog shake

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CONCLUSION Our efforts to improve suboptimal properties of our initial tetraisoquinoline urea leads led to clinical candidate 35 for migraines. Extensive optimization to reduce a PXR liability by reducing cLogD was effective in reducing the CYP3A4induction liability. Compound 35 was efficacious in rat WDS biochemical-challenge models and cold-pressor target-engagement models at equivalent exposures and well-tolerated in 8199



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Article

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