2-(3-Fluoro-4-methylsulfonylaminophenyl)propanamides as potent transient receptor potential vanilloid 1 (TRPV1) antagonists: Structure-activity relationships of 2-amino derivatives in the N-(6-trifluoromethylpyridin-3- ylmethyl) C-region

Article

pubs.acs.org/jmc

2‑(3-Fluoro-4-methylsulfonylaminophenyl)propanamides as Potent

Transient Receptor Potential Vanilloid 1 (TRPV1) Antagonists:

Structure−Activity Relationships of 2‑Amino Derivatives in the N‑(6-

Triﬂuoromethylpyridin-3-ylmethyl) C‑Region

Myeong Seop Kim,^†HyungChul Ryu,^†Dong Wook Kang,^†Seong-Hee Cho,^†Sejin Seo,^†

Young Soo Park,^†Mi-Yeon Kim,^†Eun Joo Kwak,^†Yong Soo Kim,^†Rahul S. Bhondwe,^†Ho Shin Kim,^†

Seul-gi Park,^‡Karam Son,^‡Sun Choi,^‡Ian A. DeAndrea-Lazarus,^§Larry V. Pearce,^§Peter M. Blumberg,^§

Robert Frank,^∥Gregor Bahrenberg,^∥Hannelore Stockhausen,^∥Babette Y. Kogel,^∥Klaus Schiene,^∥

̈

Thomas Christoph,^∥and Jeewoo Lee*^,†

^†Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,

Seoul 151-742, Korea

^‡College of Pharmacy, Division of Life and Pharmaceutical Sciences, and National Core Research Center for Cell Signaling and Drug

Discovery Research, Ewha Womans University, Seoul 120-750, Korea

^§Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health,

Bethesda, Maryland 20892, United States

^∥Grunenthal Innovation, Grunenthal GmbH, D-52078 Aachen, Germany

S

* Supporting Information

ABSTRACT: A series of N-(2-amino-6-triﬂuoromethylpyridin-3-ylmethyl)-2-(3-ﬂuoro-4-

methylsulfonylaminophenyl)propanamides were designed combining previously identiﬁed

pharmacophoric elements and evaluated as hTRPV1 antagonists. The SAR analysis indicated

that speciﬁc hydrophobic interactions of the 2-amino substituents in the C-region of the

ligand were critical for high hTRPV1 binding potency. In particular, compound 49S was an

excellent TRPV1 antagonist (K_i(CAP)= 0.2 nM; IC_50(pH)= 6.3 nM) and was thus

approximately 100- and 20-fold more potent, respectively, than the parent compounds 2 and

3 for capsaicin antagonism. Furthermore, it demonstrated strong analgesic activity in the rat

neuropathic model superior to 2 with almost no side eﬀects. Compound 49S antagonized

capsaicin induced hypothermia in mice but showed TRPV1-related hyperthermia. The basis for the high potency of 49S

compared to 2 is suggested by docking analysis with our hTRPV1 homology model in which the 4-methylpiperidinyl group in

the C-region of 49S made additional hydrophobic interactions with the hydrophobic region.

development and therapeutic potential of TRPV1 antagonists

have been extensively reviewed.⁹⁻¹³

INTRODUCTION

■

The transient receptor potential V1 (TRPV1) receptor is a

molecular integrator of nociceptive stimuli, located predom-

inantly in primary sensory neurons.¹The receptor functions as

a ligand-gated and nonselective cation channel with high Ca²⁺

permeability, activated by endogenous agonists including

protons,²noxious heat,³inﬂammatory lipid mediators such as

anandamide,⁴and lipoxygenase products,⁵as well as by natural

products such as capsaicin (CAP)⁶and resiniferatoxin (RTX).⁷

The increase in intracellular Ca²⁺upon TRPV1 activation

causes excitation of the primary sensory neurons and the

consequent central perception of pain. TRPV1 antagonists

inhibit this transmission of nociceptive signaling from the

periphery to the CNS as well as block other pathological states

associated with this receptor. In recent years a number of

TRPV1 antagonists have been developed as novel analgesic and

antiinﬂammatory agents, particularly for the treatment of

chronic pain and inﬂammatory hyperalgesia.⁸The clinical

Previously, we identiﬁed a potent and stereospeciﬁc

antagonist, (S)-N-(4-tert-butylbenzyl)-2-(3-ﬂuoro-4-

methanesulfonylaminophenyl)propanamide (2),¹⁴which ex-

hibited better binding aﬃnity and more potent antagonism for

both rTRPV1 and hTRPV1 in CHO cells compared to the

prototype thiourea antagonist 1¹⁵(Figure 1). Its activity was

stereospeciﬁc with marked selectivity for the (S)-conﬁguration,

whereas the (S)-isomer was ∼2-fold more potent than the

racemate, and the (R)-isomer was 30- to 40-fold weaker. A

docking study of 2 with our hTRPV1 homology model

demonstrated a novel aspect of its binding to the receptor and

identiﬁed crucial hydrogen bonds between the ligand and the

receptor contributing to its stereospeciﬁc potency.¹⁴

Received: June 4, 2012

Published: September 7, 2012

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RESULTS AND DISCUSSION

Chemistry. The key intermediate of the C-region 8 was

synthesized starting from ethyl vinyl ether 5 in three steps by a

modiﬁcation of the previously report procedure,¹⁸as shown in

Scheme 1. Triﬂuoromethyl acetylation of 5 followed by

■

Scheme 1. Synthesis of 2-Chloro-3-cyano-6-

a

triﬂuoromethylpyridine

a

Reagents and conditions: (a) (CF₃CO)₂O, pyridine, CHCl₃; (b)

NCCH₂CONH₂, K₂CO₃, toluene; (c) POCl₃.

condensation with cyanoacetamide provided pyridone 7,

which was readily converted to 2-chloropyridine by POCl₃.

The syntheses of ﬁnal compounds are represented in Scheme 2.

Figure 1. Lead TRPV1 antagonists.

Scheme 2. Syntheses of 2-(3-Fluoro-4-

methylsulfonylaminophenyl)propanamide Analogues

a

To further optimize the antagonistic acitivity of the lead 2,

structural modiﬁcations were performed based on the three

principal pharmacophores (A-region, (4-methylsulfonylamino)-

phenyl; B-region, propanamide; C-region, 4-tert-butylbenzyl).

However, none of the compounds were found to be better than

2 in terms of both binding aﬃnity and antagonism to capsaicin

activation for rTRPV1 in CHO cells.^14,16

A series of N-arylcinnamides had been reported by the

Amgen group¹⁷as potent antagonists for rat−human chimeric

TRPV1 expressed in CHO cells. In that series, compounds 3

and 4 (Figure 1), which had 2-(piperidin-1-yl and morpholi-

no)-6-(triﬂuoromethyl)pyridin-3-yl moieties, respectively, ex-

hibited potent antagonism of activation of TRPV1 by capsaicin

and acid and further displayed good oral bioavailability.

Combining the 2-(3-ﬂuoro-4-methylsulfonaminophenyl)-

propanamide (the A and B regions in 2) and 2-(piperidinyl

or morpholinyl)-6-triﬂuoromethylpyridin-3-ylmethyl groups

(C-region of 3 and 4) provided the novel designed compounds

45 and 97. Their syntheses and biological evaluation indicated

that they exhibited highly potent antagonism toward both

capsaicin and pH for hTRPV1 in CHO cells, in which

compounds 45 and 97 showed 46- and 15-fold enhanced

potency in capsaicin antagonism compared to parent 2,

respectively. This preliminary result prompted us to investigate

extensively the structure−activity relationship of this template.

In this study, we investigated the structure−activity relation-

ships for 2-amino substituted analogues (12−99) of the

template N-(6-triﬂuoromethylpyridin-3-ylmethyl)-2-(3-ﬂuoro-

4-methylsulfonylaminophenyl)propanamide (Figure 2) as

hTRPV1 antagonists and we further characterized analgesic

activity in a neuropathic pain model and performed molecular

modeling with our hTRPV1 homology model for the most

potent antagonist in the series.

a

Reagents and conditions: (a) [method A] neat NR₁R₂; [method B]

NR₁R₂, Pd(OAc)₂, dppf, Na₂CO₃, toluene−THF (7:1); [method C]

NR₁R₂, K₂CO₃, 18-crown-6, CH₃CN; [method D] DBU, CH₃CN; (b)

[method A] 2 M BH₃−SMe₂in THF; [method B] NaBH₄,

NiCl₂·6H₂O (or CoCl₂·6H₂O), MeOH; [method C] H₂, 10% Pd−

C, conc HCl, MeOH; (c) EDC, HOBt, TEA, CH₃CN.

Amines were reacted with 8 to aﬀord 2-amino substituted

pyridines 9, and then their nitrile groups were reduced to yield

the corresponding primary amines 10.¹⁷The amines were

coupled with racemic or chiral 2-(3-ﬂuoro-4-

methylsulfonylaminophenyl)propanamide 11 to give the ﬁnal

compounds 12−99.

Structure−Activity Relationship (SAR) Analysis. The

synthesized TRPV1 ligands were evaluated in vitro for

antagonism as measured by inhibition of activation by four

separate stimuli (capsaicin (CAP), pH, heat, and N-acetyldop-

amine (NADA)), as indicated. The assays were conducted

using a ﬂuorometric imaging plate reader (FLIPR) with human

TRPV1 heterologously expressed in Chinese hamster ovary

(CHO) cells. The results are summarized in Tables 1−5,

together with the potencies of the previously reported parent

antagonists 1−4.

The 2-unsubstituted analogue 12 was initially prepared but

was found to be a weak antagonist, indicating that a

hydrophobic substituent at the 2-position was necessary for

substantial activity. The SAR for derivatives incorporating an

amino group into the 2-position of the pyridine C-region was

investigated starting from 2-acyclic amino derivatives (Table 1).

The secondary amino derivatives, incorporating alkylamino

Figure 2. General structure of designed compounds.

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Table 1. In Vitro hTRPV1 Antagonistic Activities for 2-Acylic Amino Derivatives

13−16, arylamino 17−21, and benzylamino 22−26 groups,

showed reasonable potencies for CAP antagonism, with values

for K_i(CAP)ranging from 9.1 to 65.7 nM. Exceptions were the 4-

chloroanilino 19 and pyridinylmethylamino 24−26 analogues,

which were devoid of activity.

Tertiary acyclic amino analogues 27−32 were also examined.

The activity was enhanced as the number of carbons in the

chain increased, and the derivatives exhibited much better

potencies than the corresponding secondary amino surrogates

(for example, 13 vs 28 and 14 vs 29). In particular, the

dipropylamino analogue 29 and the dibutylamino analogue 30

displayed excellent antagonism toward both CAP and pH (for

29, K_i(CAP)= 0.2 nM, IC_50(pH)= 14.7 nM; for 30, K_i(CAP)= 0.6

nM, IC_50(pH)= 10.5 nM). The methylbutylamino analogue 31

(ﬁve carbons) showed a potency between those of 28 (four

carbons) and 29 (six carbons), suggesting that the lipophilicity

is a contributor to the activity. The methylphenylamino

analogue 32 showed a signiﬁcant increase in potency (75-fold

for CAP, 25-fold for pH) compared to the phenylamino

analogue 17, indicating that an NH in a secondary amino group

was detrimental to antagonism.

Next, we sought to evaluate the SAR for monoazacyclic rings.

The introduction of unsubstituted azacyclic rings, including 1-

pyrrolidinyl 34, 1-piperidinyl 45, 1-azepanyl 79, and 1-azocanyl

80 analogues, provided potent antagonists with subnanomolar

activities (K_i(CAP)= 0.43−1.3 nM) regardless of ring size.

In the SAR of substituted 1-pyrrolidinyl analogues (Table 2),

we restricted our syntheses to the chiral propionic acid 11S at

the condensation step because the substituted pyrrolidines were

themselves chiral. The 2-methylpyrrolidine analogue 34

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a

Table 2. In Vitro hTRPV1 Antagonistic Activities for 2-Pyrrolidinyl Derivatives

a

The asterisk (∗) indicates (S)-propanamide.

showed enhanced antagonism to CAP but markedly reduced

antagonism to pH compared to the pyrrolidine analogue 33.

The structural modiﬁcation of the 2- or 3-position of

pyrrolidine resulted in a similar SAR pattern in which the

hydrophilic substituents (35, 36, 38, 40, 41) led to the loss of

activity whereas the hydrophobic ones (37, 39, 42−44)

retained potency. The stereochemistry of the substituents did

not aﬀect the antagonism (40 vs 41 and 42 vs 43).

benzyl-1-piperidinyl analogues, 57−60, exhibited great poten-

cies, suggesting that there is a large hydrophobic pocket in

TRPV1 suﬃcient to accommodate the benzyl group.

As piperidine surrogates, the 4-substituted piperazinyl 81−96

and morpholinyl 97−99 analogues were examined (Table 4).

The 1-piperazinyl analogue 81 proved to be a weak antagonist;

the 4-methylpiperidyl analogue 82 showed slightly better

activity. The introduction of a more lipophilic group into the

4-position of piperazine led to an improvement in the

antagonism except in the case of the 4-pyridyl analogue 92,

conﬁrming that a hydrophobic substituent at the 4-position is a

determinant for high potency. Among the morpholinyl

analogues, the 1-morpholinyl analogue 97 and the 2,6-

dimethylmorpholinyl analogue 98 exhibited potent antagonism;

the 4,4-dioxothiomorpholinyl analogue 99 showed only weak

potency reﬂecting its polar sulfonyl group.

Detailed in vitro activity of 49S, the most potent antagonist

in this study, was investigated for multiple TRPV1 activators

including capsaicin, pH, heat (45 °C), and NADA and

compared to the activity of lead 2 (Table 5). Compound 49S

showed excellent antagonism of all four TRPV1 activators and

was approximately 140- to 660-fold more potent than 2.

Selectivity of compound 49S was assessed at 10 μM against a

panel of 135 other receptors and enzymes (CEREP). Even at

this concentration, which was 4 orders of magnitude higher

than its K_ifor capsaicin, 49S was negative for all but seven

targets and gave greater than 50% inhibition for only three.

While detailed mechanistic studies were not carried out, we

conﬁrmed that 49S inhibited [³H]resiniferatoxin binding to

human TRPV1 (data not shown), as has been repeatedly

observed for structurally related TRPV1 antagonists. [³H]-

Resiniferatoxin binding provides a convenient measure for

ligand interaction at the capsaicin binding site on TRPV1. We

conclude that 49S is exerting its antagonistic activity, as fully

expected, at the capsaicin binding site rather than as a channel

blocker.

The SAR of six-membered azacyclic analogues was

investigated next (Table 3). The 1-piperidinyl analogue 45

exhibited excellent antagonism; its activity was stereospeciﬁc,

with the (S)-isomer 45S (K_i(CAP)= 0.3 nM) representing the

active conﬁguration. The potency of 45S was ∼15-fold higher

than that of the lead 3, which has the same C-region, indicating

that the 2-(3-ﬂuoro-4-methylsulfonaminophenyl)propanamide

template for the A and B regions was superior to that of the

arylcinnamide for antagonism. The tetrahydropyridinyl ana-

logue 46 was highly potent like 45. The methylpiperidinyl

derivatives 47−49 were examined, and the 4-methyl-1-

piperidinyl analogue 49 exhibited stereospeciﬁc, potent

antagonism toward both CAP and pH. The active isomer

49S was found to be the most potent antagonist in this study

with K_i(CAP)= 0.2 nM and IC_50(pH)= 6.3 nM. Its potency was

thus 100-fold and 200-fold better than the reference propamide

2 for CAP and pH antagonism, respectively. The structural

analysis comparing 2 and 49S indicated that the additional 4-

methylpiperidine moiety in 49S provided a new hydrophobic

interaction with the receptor, which could explain the enhanced

potency of 49S. The docking analysis using our hTRPV1

homology model will be described in the next section.

The impressive potency of 49 prompted us to investigate a

variety of 4-substituted piperidinyl analogues, 50−78. As

demonstrated in the SAR of the pyrrolidines, most hydrophobic

4-substituents provided signiﬁcant antagonism. Conversely,

incorporation of hydrophilic substituents, such as the 4-keto 64,

the 4-hydroxyl 65, the 4-pyrrolidinyl 75, and the 4-piperidinyl

76, caused a dramatic loss of activity. Interestingly, a series of 4-

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Table 3. In Vitro hTRPV1 Antagonistic Activities for 2-Piperidinyl Derivatives

In Vivo Activity. As part of the initial in vivo character-

ization of 49S, analgesic activity of 49S was evaluated orally in

the rat Bennett model¹⁹as a neuropathic pain model and its

activity was compared to that of parent 2 (Figure 3). The

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Table 4. In Vitro hTRPV1 Antagonistic Activities for 2-Piperazinylpyridines and 2-Morpholinylpyridines

analgesic potency of 49S demonstrated dose-dependent eﬃcacy

with ED₅₀= 0.9 mg/kg po (max 60% at 3.16 mg/kg) and was

superior to that of 2. Side eﬀects like sedation or decreased

locomotion were not observed.

Consistent with its in vitro mechanism of action, in vivo 49S

also blocked response to capsaicin (Figure 4A). The intra-

peritoneal injection of 3 mg/kg capsaicin resulted in a decrease

of body temperature as expected,^20,21with a reduction from

37.1 0.1 to 34.0 0.1 °C (15 min after capsaicin injection; p

< 0.05). By 30 min body temperature had returned to normal.

The oral administration of 0.3 mg/kg 49S (15 min before

capsaicin injection) completely inhibited the eﬀect of capsaicin

on body temperature (Figure 4A).

Table 5. In Vitro hTRPV1 Antagonistic Activities for 2 and

49S to Multiple Activators

activator, parameter

2

49S (nM)

CAP, (f)K_i(nM)

pH, IC₅₀(nM)

28

0.2

1281

174

6.6

6.3

heat 45 °C, IC₅₀(nM)

NADA, (f)K_i(nM)

0.8

0.01

Furthermore, we could demonstrate that 49S targeted

TRPV1 in vivo. Although TRPV1 knockout mice show normal

body temperature,²²induction of modest hyperthermia is a

common acute side eﬀect of administration of TRPV1

antagonists.²³We therefore examined the eﬀect on body

temperature of administration of 49S. As illustrated in Figure 4,

oral administration of 1 mg/kg 49S in C57BL/6J wild type

mice induced a body temperature increase of about 1.3 0.1

°C 45 and 60 min after substance administration. In TRPV1

knockout mice (Figure 4B) this hyperthermic response was

completely absent.

Figure 3. Eﬀect of compound 2 (30 min after po administration) and

compound 49S (45 min after po administration) on CCI-induced cold

allodynia in rats. Data are presented as the mean SEM; (∗) p < 0.05

vs vehicle.

Molecular Modeling. Compound 49S was identiﬁed as a

highly potent antagonist showing ∼100-fold greater potency

compared to prototype 2, with this enhancement in potency

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in silico mutation and reﬁnement by energy minimization.

Using our hTRPV1 model, we then performed the ﬂexible

docking study of 2 and 49S to investigate their binding

interactions and found compound 2 ﬁtted well into the binding

site (Figure 5). The sulfonylaminobenzyl group (A-region)

occupied the deep bottom hole and was involved in a

hydrophobic interaction with Tyr511. An oxygen atom and

NH of the sulfonamide group participated in hydrogen bonding

with Ser512 and Ile564. The amide group (B-region) made a

hydrogen bond with Tyr511 and also contributed to the

appropriate positioning of the C-region for the hydrophobic

interaction. The hydrophobic 4-tert-butylbenzyl group (C-

region) was oriented toward the upper hydrophobic region of

the binding site and formed the hydrophobic interaction with

Leu547.

As we expected, compound 49S showed an excellent ﬁt to

the binding site and its 4-methylpiperidine ring in the C-region

made an additional hydrophobic interaction with hTRPV1

(Figure 6). The sulfonylaminophenyl and amide moieties (A

and B regions) made tight interactions with the binding site

residues via the hydrophobic interaction with Tyr511 and

hydrogen bonding with Tyr511, Ser512, and Ile564 as shown

for 2. The 3-triﬂuoromethyl group in the C-region, like the 4-

tert-butylbenzyl group of 2, formed a hydrophobic interaction

with Leu547. Furthermore, the 4-methylpiperidinyl group in

the C-region made an additional hydrophobic interaction with

the hydrophobic region composed of Met514 and Leu515.

That might explain why the activity of 49S was dramatically

increased, compared with that of 2.

Figure 4. Eﬀect of compound 49S on body temperature: (A) eﬀect of

49S on capsaicin-induced hypothermia; (B) eﬀect of 49S on body

temperature in wild-type and TRPV1 KO mice.

being attributable to an additional 4-methylpiperidinyl group in

49S. To clarify the basis for this enhanced activity, we used our

hTRPV1 homology model to compare the binding interactions

of 49S and 2.

CONCLUSION

■

A variety of 2-amino substituted 6-triﬂuoromethylpyridin-3-

ylmethylamines as the C-region were coupled with the well

characterized A,B-region TRPV1 antagonistic template 2-(3-

We constructed the tetramer homology model of human

TRPV1 (hTRPV1) based on our rat TRPV1 model²⁴through

Figure 5. Flexible docking of 2 in the hTRPV1 model. (A) Binding mode of 2. The key residues are marked and displayed as capped-stick with

carbon atoms in white. The helices are colored in gray, and the helices of the adjacent monomer are displayed in line ribbon. The ligand is depicted

as ball-and-stick with carbon atoms in magenta. The van der Waals surface of the ligand is presented with its lipophilic potential property. Hydrogen

bonds are shown as black dashed lines, and nonpolar hydrogens are undisplayed for clarity. (B) Surface representations of the docked ligand and

hTRPV1. The fast Connolly surface of hTRPV1 was generated by MOLCAD and colored by the lipophilic potential property. The surface of

hTRPV1 is Z-clipped and that of the ligand is in its carbon color for clarity. (C) van der Waals surface of the ligand colored by its lipophilic potential

property.

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Figure 6. Flexible docking of 49S in the hTRPV1 model: (A) binding mode of 49S; (B) surface representations of the docked ligand and hTRPV1;

(C) van dar Waals surface of the ligand colored by its lipophilic potential property. The ligand is depicted as ball-and-stick with carbon atoms in

purple. The details are the same as in Figure 5.

2-Hydroxy-6-(triﬂuoromethyl)nicotinonitrile (7). To an ice

cold solution of ethyl vinyl ether (9.58 mL, 0.1 mol) and pyridine (8.1

mL, 0.1 mmol) in CHCl₃(anhydrous 100 mL) was added

triﬂuoroacetic anhydride (21 g, 0.1 mol) at 0 °C. The reaction

mixture was gradually stirred at 0 °C to room temperature for 5 h. The

mixture was concentrated in vacuo to obtain crude (E)-4-ethoxy-1,1,1-

triﬂuorobut-3-en-2-one 6 (10.92 g, 65%) as a light yellow liquid, which

was directly used for the next step without further puriﬁcation.

A mixture of 6 (10 g, 59.5 mmol), 2-cyanoacetamide (5 g, 59.5

mmol), and K₂CO₃(10.37 g, 75 mmol) in toluene (200 mL) was

reﬂuxed under a Dean−Stark trap for 9 h. The progress of the reaction

was monitored by TLC (40% EtOAc/hexane, R_f≈ 0.2). After the

starting material disappeared, the reaction mixture was concentrated in

vacuo to give the residue which was puriﬁed by ﬂash column

chromatography on silica gel using EtOAc/hexane (1:25) as eluant to

yield compound 7 (8.6 g, 77%) as a yellow solid, mp 210−215 °C. ¹H

NMR (300 MHz, CD₃OD) δ 8.02 (d, J = 7.5 Hz, 1H), 6.97 (d, J =

7.68 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 169.27, 151.01−

149.98 (q, J = 34.3), 147.42, 127.00−118.83 (q, J = 272.4), 118.16,

109.05, 102.67; IR (KBr) 3408, 2825, 2704, 2231, 1827, 1680, 1590,

1562, 1484, 1437, 1348, 1312, 1290, 1218, 1199, 1180, 1153, 1116,

1095 cm⁻¹; LC−MS (ESI) m/z 189 (MH⁺).

ﬂuoro-4-methylsulfonylaminophenyl)propionic acid to provide

a series of propanamides for evaluation as potent TRPV1

antagonists (12−99). The analysis of structure−activity

relationship indicated that a critical feature of the 2-amino

substituents for activity was that they support a hydrophobic

interaction with the receptor. Among the compounds of the

series, compound 49S showed the best TRPV1 antagonism,

blocking the activations by capsaicin, pH, heat, and NADA. It

was selective, and it demonstrated strong analgesic activity in a

neuropathic pain model with almost no side eﬀects. Consistent

with its action in vivo being through TRPV1, compound 49S

blocked capsacin-induced hypothermia but caused modest

TRPV1-related hyperthermia in mice. The docking study with

our homology model indicated that 49S showed an excellent ﬁt

to the binding site in which the sulfonylaminobenzyl and

propanamide moieties interacted with Tyr511, Ser512, and

Ile564 as found in 2 and the 4-methylpiperidinyl group in the

C-region made an additional hydrophobic interaction with the

hydrophobic region composed of Met514 and Leu515,

resulting in its high potency.

2-Chloro-6-(triﬂuoromethyl)nicotinonitrile (8). A mixture of 7

(7.52 g, 40 mmol) and POCl₃(26 mL, 280 mmol) was reﬂuxed for 3

h. The reaction mixture was cooled and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography on silica gel

using EtOAc/hexane (1:20) as eluant to yield compound 8 (4.28 g,

52%) as a white solid, mp 37−40 °C. ¹H NMR (300 MHz, CDCl₃) δ

8.22 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H); ¹³C NMR (100

MHz, CDCl₃) δ 153.19, 151.23−150.13 (q, J = 36.8), 144.42, 123.87−

115.67 (q, J = 273.5), 119.11−119.04 (q, J = 2.7), 113.94, 113.47; IR

(KBr) 3177, 3084, 3036, 2921, 2832, 2241, 1946, 1833, 1709, 1590,

1567, 1457, 1364, 1337, 1250, 1226, 1192, 1155, 1109, 1077 cm⁻¹;

LC−MS (ESI) m/z 207 (MH⁺).

General Procedure for Amination (9). Method A (for

Compounds 13−16, 22, 27−31, 44). A mixture of 2-chloro-6-

triﬂuoromethyl-nicotinonitrile (1.0 mmol) was dissolved in amine

(10.0 mmol). The mixture was stirred for 16 h at room temperature.

The aqueous portion was extracted with EtOAc (30 mL) twice. The

combined organic extracts were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography on silica gel using EtOAc/hexane (1:15) as eluant.

EXPERIMENTAL SECTION

General. All chemical reagents were commercially available.

Melting points were determined on a Buchi melting point B-540

■

̈

apparatus and are uncorrected. Silica gel column chromatography was

performed on silica gel 60, 230−400 mesh, Merck. Nuclear magnetic

resonance (¹H NMR and ¹³C NMR) spectra were recorded on JEOL

JNM-LA 300 [300 MHz (¹H), 75 MHz (¹³C)] and Bruker Avance 400

MHz FT-NMR [400 MHz (¹H), 100 MHz (¹³C)] spectrometers.

Chemical shifts are reported in ppm units with Me₄Si as a reference

standard. Infrared (IR) spectra were recorded on a JASCO FT/IR-

4200 spectrometer. Mass spectra were recorded on a VG Trio-2 GC−

MS instrument and a 6460 Triple Quad LC−MS instrument. All ﬁnal

compounds were puriﬁed to >95% purity, as determined by high-

performance liquid chromatography (HPLC). HPLC was performed

on an Agilent 1120 Compact LC (G4288A) instrument using Agilent

TC-C18 column (4.6 mm × 250 mm, 5 μm), Agilent Eclipse Plus C18

column (4.6 mm × 250 mm, 5 μm), and Daicel Chiralcel OD-H

column (4.6 mm × 250 mm, 5 μm). Optical rotations were measured

in a JASCO DIP-2000 digital polarimeter.

8399

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Journal of Medicinal Chemistry

Article

Method B (for Compounds 17−21, 23, 32, 46). A mixture of 2-

chloro-6-triﬂuoromethylnicotinonitrile (1.0 mmol), bis-

(triphenylphosphine)palladium(II) dichloride (0.10 mmol), and

copper iodide (0.20 mmol) was dissolved in 1-methyl-2-pyrrolidinone

(10 mL). After the mixture was stirred for 10 min, alkyene and N,N-

diisopropylethylamine (2.0 mmol) were added to the mixture. The

mixture was stirred for 12 h at 90−110 °C. The mixture was ﬁltered

over Celite, and the aqueous portion was extracted with ether twice.

The combined organic extracts were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography on silica gel using EtOAc/hexane (1:15) as eluant.

Method C (for Compounds 24−26, 33−43, 54, 57−61, 73−99). A

mixture of 6-tert-butyl-2-hydroxynicotinonitrile (1.0 mmol) and 1-

bromopentane (2.0 mmol), 18-crown-6-ether (cat.), and K₂CO₃(4.0

mmol) was dissolved in CH₃CN/DMF (1:2) solution. The mixture

was reﬂuxed for 12 h and then cooled to room temperature. The

aqueous portion was extracted with EtOAc (30 mL). The organic

phase was dried over MgSO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography on silica gel

using EtOAc/hexane (1:1) as eluant.

4.37 (m, 2H), 3.69 (q, J = 6.78 Hz, 1H), 3.00 (s, 3H), 1.36 (d, J = 6.78

Hz, 3H). MS (FAB) m/z 420 (MH⁺).

N-((2-(Butylamino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)propanamide (13).

Yield 71%, white solid, mp 65−67 °C. ¹H NMR (300 MHz,

CDCl₃) δ 7.50 (dd, J = 8.4 Hz, 1H), 7.22 (d, J = 7.3 Hz, 1H), 7.15 (dd,

J = 11.2 Hz, 1H), 7.06 (d, 1H), 6.76 (d, J = 7.3 Hz, 1H), 6.51 (br s,

1H), 5.95 (br t, 1H), 5.71 (br t, 1H), 4.42−4.43 (m, 2H), 3.53−3.33

(m, 3H), 3.03 (s, 3H), 1.59−1.50 (m, 2H) 1.51 (d, J = 7.1 Hz, 3H),

1.45−1.33 (m, 2H), 0.95 (t, 3H). MS (FAB) m/z 491 (MH⁺).

N-((2-(Cyclohexylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

propanamide (14). Yield 93%, white solid, mp 82−84 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.51 (t, 1H), 7.22 (d, J = 5.8 Hz, 1H), 7.18 (dd,

J = 8.9, 1.5 Hz, 1H), 7.08 (d, J = 6.6 Hz, 1H), 6.74 (d, J = 5.8 Hz, 1H),

6.47 (br s, 1H), 5.84 (br d, 1H), 5.67 (br t, 1H), 4.32 (m, 2H), 3.91

(m, 1H), 3.48 (q, J = 5.7 Hz, 1H), 3.03 (s, 3H), 1.98−1.61 (m, 5H),

1.52 (d, J = 5.7 Hz, 3H), 1.42−1.07 (m, 5H). MS (FAB) m/z 517

(MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((4-

methylcyclohexyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)propanamide (15). Yield 25%, yellowish solid, mp 82 °C.

¹H NMR (600 MHz, DMSO-d₆) δ 9.50 (s, 1H), 8.57 (br t, 1H), 7.40

(dd, J = 33.18, 6.78 Hz, 1H), 7.31 (t, J = 8.28 Hz, 1H), 7.21 (d, J =

11.34 Hz, 1H), 7.12 (d, J = 8.28 Hz, 1H), 6.86 (dd, J = 18.12, 6.78 Hz,

1H), 6.30 (dd, J = 75.54, 6.78 Hz, 1H), 4.15 (m, 2H), 3.67 (m, 2H),

2.99 (s, 3H), 1.66 (m, 2H), 1.52 (m, 2H), 1.44 (m, 1H), 1.36 (d, J =

6.84 Hz, 3H), 1.17 (m, 2H), 0.98 (m, 2H), 0.88 (d, J = 6.06 Hz, 3H).

MS (FAB) m/z 531 (MH⁺).

Method D (for Compounds 45, 47−53, 55, 56, 62−72). A mixture of

2-chloro-6-triﬂuoromethylnicotinonitrile (1.0 mmol), piperidine (2.0

mmol), and DBU (2.0 mmol) was dissolved in acetonitrile (10 mL).

The mixture was stirred for 12 h at room temperature. The aqueous

portion was extracted with EtOAc (30 mL) twice. The combined

organic extracts were dried over MgSO₄, ﬁltered, and concentrated in

vacuo. The residue was puriﬁed by ﬂash column chromatography on

silica gel using EtOAc/hexane (1:15) as eluant.

General Procedure for Reduction (10). Method A (for

Compounds 17−21, 23−26, 32−35, 37−53, 57−81, 83, 85, 86, 89,

90, 93−99). To a stirred solution of nitrile (2.00 mmol) in THF

(anhydrous, 10 mL) was added 2 M BH₃·SMe₂in THF (3 mL, 3

equiv) at room temperature. After being reﬂuxed for 8 h, the mixture

was cooled to room temperature and 2 M HCl solution was added.

The mixture was then reﬂuxed for 30 min. After cooling to room

temperature, the mixture was neutralized by 2 M NaOH solution and

the aqueous portion extracted with EtOAc several times. The

combined organic layers were washed with brine, dried over MgSO₄,

and concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography on silica gel using CH₂Cl₂/MeOH (10:1) as eluant.

Method B (for Compounds 13−16, 22, 27−31, 36, 56). Nitrile (1.0

mmol) and NiCl₂·6H₂O (2.0 mmol) were dissolved in MeOH (8 mL).

Sodium borohydride (4.0 mmol) was added slowly to the mixture. The

mixture was reﬂuxed for 12 h and then cooled to room temperature.

The solution was ﬁltered through Celite and dried over MgSO₄,

ﬁltered, and concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography on silica gel using CH₂Cl₂/MeOH (10:1) as

eluant.

tert-Butyl 4-(((3-((2-(3-Fluoro-4-(methylsulfonamido)-

phenyl)propanamido)methyl)-6-(triﬂuoromethyl)pyridin-2-yl)-

amino)methyl)piperidine-1-carboxylate (16). Yield 55%, white

solid, mp 83−85 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.48 (dd, J = 8.2,

8.2 Hz, 1H), 7.25 (d, 1H), 7.16 (d, 1H), 7.06 (d, 1H), 6.77 (d, J = 7.3

Hz, 1H), 6.21 (br s, 1H), 5.93 (br s, 1H), 4.32 (m, 2H), 4.06 (m, 2H),

3.49 (q, J = 7.3 Hz, 1H), 3.32 (m, 2H), 2.66 (m, 2H), 1.76 (m, 2H),

1.51 (d, J = 7.0 Hz, 3H), 1.46 (s, 9H). MS (FAB) m/z 632 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(phenyla-

mino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)propanamide

1

(17). Yield 58%, white solid, mp 189−192 °C. H NMR (300 MHz,

CD₃OD) δ 7.67 (m, 2H), 7.59 (d, J = 8.2 Hz, 1H), 7.34 (dd, J = 8.2,

8.2 Hz, 1H), 7.19 (dd, J = 10.9, 1.9 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H),

7.06 (d, J = 7.7 Hz, 1H), 7.02−6.95 (m, 3H), 4.45 (m, 2H), 3.67 (q, J

= 7.1 Hz, 1H), 2.89 (s, 3H), 1.47 (d, J = 7.1 Hz, 3H). MS (FAB) m/z

511 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((4-

ﬂuorophenyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

1

propanamide (18). Yield 58%, white solid, mp 158 °C. H NMR

(300 MHz, CD₃OD) δ 7.90 (m, 2H), 7.59 (d, J = 7.5 Hz, 1H), 7.33

(dd, J = 8.3, 8.3 Hz, 1H), 7.24 (m, 1H), 7.21 (dd, J = 11.4, 1.8 Hz,

1H), 7.11 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.59 (m, 1H),

4.46 (m, 2H), 3.68 (q, J = 7.1 Hz, 1H), 2.87 (s, 3H), 1.47 (d, J = 7.1

Hz, 3H). MS (FAB) m/z 529 (MH⁺).

Method C (for Compounds 54, 55, 82, 84, 87, 88, 91, 92). A

suspension of nitrile compounds (5.0 mmol), 10% Pd/C (500 mg),

and concentrated HCl (3 mL) in MeOH (30 mL) was hydrogenated

under a balloon of hydrogen for 6 h at room temperature and ﬁltered

through Celite. The ﬁltrate was concentrated in vacuo, and the residue

was puriﬁed by ﬂash column chromatography on silica gel using

EtOAc as eluant.

General Procedure for Coupling (12−99). A mixture of acid

(10.0 mmol), amine (12.0 mmol), and 1-(3-dimethylaminopropyl)-3-

ethylcarbodiimide hydrochloride (12.0 mmol) in DMF (20 mL) was

stirred for 12 h at room temperature. The aqueous portion was

extracted with EtOAc (50 mL). The aqueous phase was saturated with

NaCl and extracted again with EtOAc (25 mL). The combined organic

extracts were washed with 1 M HCl (25 mL) and brine (25 mL), dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The residue was

puriﬁed by ﬂash column chromatography on silica gel using EtOAc/

hexane (1:2) as eluant.

N-((2-((4-Chlorophenyl)amino)-6-(triﬂuoromethyl)pyridin-3-

yl)methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

1

propanamide (19). Yield 58%, white solid, mp 196 °C. H NMR

(300 MHz, CD₃OD) δ 7.10 (d, J = 9.0 Hz, 2H) 7.62 (d, J = 7.5 Hz,

1H), 7.34 (dd, J = 8.3, 8.3 Hz, 1H), 7.23 (d, J = 9.0 Hz, 2H), 7.19 (dd,

J = 11.7, 2.0 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 7.5 Hz,

1H), 4.45 (m, 2H), 3.67 (q, J = 7.0 Hz, 1H), 2.88 (s, 3H), 1.47 (d, J =

7.1 Hz, 3H). MS (FAB) m/z 545 (MH⁺).

N-((2-((3,4-Dimethylphenyl)amino)-6-(triﬂuoromethyl)-

pyridin-3-yl)methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)-

1

phenyl)propanamide (20). Yield 58%, white solid, mp 176 °C. H

NMR (300 MHz, CDCl₃) δ 7.56 (m, 2H), 7.43 (dd, J = 8.4, 8.4 Hz,

1H), 7.39 (d, J = 7.8 Hz, 1H), 7.14 (dd, J = 11.0, 2.2 Hz, 1H), 7.06 (d,

J = 8.7 Hz, 1H), 7.03 (d, J = 7.7 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H),

6.41(br s, 1H), 5.85 (br t, 1H), 4.47 (d, J = 6.4 Hz, 2H), 3.52 (q, J =

7.1 Hz, 1H), 2.96 (s, 3H), 2.27 (s, 3H), 2.23 (s, 3H), 1.52 (d, J = 7.1

Hz, 3H). MS (FAB) m/z 539 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((6-

(triﬂuoromethyl)pyridin-3-yl)methyl)propanamide (12). Yield

12%, white solid, mp 70 °C. ¹H NMR (600 MHz, DMSO-d₆) δ

9.50 (s, 1H), 8.63 (br t, 1H), 8.56 (s, 1H), 7.82 (t, 2H), 7.32 (t, J =

8.28 Hz, 1H), 7.20 (d, J = 12.12 Hz, 1H), 7.12 (d, J = 8.34 Hz, 1H),

8400

dx.doi.org/10.1021/jm300780p | J. Med. Chem. 2012, 55, 8392−8408

Journal of Medicinal Chemistry

Article

N-((2-((5-Chloro-2-methylphenyl)amino)-6-(triﬂuoromethyl)-

pyridin-3-yl)methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)-

phenyl)propanamide (21). Yield 58%, pale yellow solid, mp 169 °C.

¹H NMR (300 MHz, CDCl₃) δ 7.68 (d, J = 2.2 Hz, 1H), 7.45 (d, J =

4H), 1.31−1.10 (m, 4H), 0.87 (t, J = 7.1 Hz, 6H). MS (FAB) m/z 547

(MH⁺).

N-((2-(Butyl(methyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

propanamide (31). Yield 60%, white solid, mp 66 °C. ¹H NMR (300

MHz, CDCl₃) δ 7.53 (dd, J = 8.3, 8.3 Hz, 1H), 7.45 (d, J = 7.9 Hz,

1H), 7.05−7.19 (m, 3H), 6.52 (br s, 1H), 6.13 (br t, 1H), 4.46 (d, J =

5.9 Hz, 2H), 3.56 (q, J = 7.1 Hz, 1H), 3.05−3.12 (m, 2H), 3.04 (s,

3H), 2.80 (s, 3H), 1.42−1.58 (m, 5H), 1.20−1.38 (m, 2H), 0.90 (t, J =

7.3 Hz, 3H). MS (FAB) m/z 505 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(methyl-

(phenyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (32). Yield 83%, white solid, mp 76−84 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.55 (d, J = 7.9 Hz, 1H), 7.53 (dd, J = 8.2, 8.2

Hz, 1H) 7.29−7.23 (m, 3H), 7.10−7.01 (m, 3H), 6.83 (m, 2H),

6.48(br s, 1H), 5.42 (br t, 1H), 3.88 (d, J = 6.0 Hz, 2H), 3.38 (s, 3H),

3.37 (q, J = 7.1 Hz, 1H), 3.04 (s, 3H), 1.43 (d, J = 7.1 Hz, 3H). MS

(FAB) m/z 525 (MH⁺).

7.5 Hz, 1H), 7.42 (dd, J = 8.3, 8.3 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H),

7.08 (dd, J = 9.0, 2.2 Hz, 1H), 7.03−7.00 (m, 3H), 6.43 (br s, 1H),

5.87 (br t, 1H), 4.49 (m, 2H), 3.51 (q, J = 7.1 Hz, 1H), 3.02 (s, 3H),

2.25 (s, 3H), 1.48 (d, J = 7.1 Hz, 3H). MS (FAB) m/z 559 (MH⁺).

N-((2-(Benzylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

1

propanamide (22). Yield 68%, white solid, mp 154−157 °C. H

NMR (300 MHz, CD₃OD) δ 7.42−7.14 (m, 8H), 6.82 (d, J = 7.2 Hz,

1H), 6.69 (br t, 1H), 4.68−4.44 (m, 2H), 4.25 (m, 2H), 3.62 (q, J =

7.1 Hz, 1H), 2.94 (s, 3H), 1.37 (d, J = 7.3 Hz, 3H). MS (FAB) m/z

525 (MH⁺).

N-((2-((4-Chlorobenzyl)amino)-6-(triﬂuoromethyl)pyridin-3-

yl)methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

1

propanamide (23). Yield 58%, white solid, mp 145 °C. H NMR

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(pyrroli-

(300 MHz, CDCl₃) δ 7.48 (dd, J = 8.3, 8.3 Hz, 1H), 7.33 (d, J = 8.6

Hz, 2H), 7.25 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 7.5 Hz, 1H), 7.07 (dd, J

= 11.2, 2.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H),

6.71 (br t, 1H), 6.47 (br s, 1H), 5.72 (br s, 1H), 4.58 (m, 2H), 4.32

(m, 2H), 3.44 (q, J = 7.1 Hz, 1H), 3.03 (s, 3H), 1.42 (d, J = 7.1 Hz,

3H). MS (FAB) m/z 559 (MH⁺).

din-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)propanamide

1

(33). Yield 80%, white solid, mp 130−135 °C. H NMR (300 MHz,

CDCl₃) δ 7.51 (dd, J = 8.1, 8.1 Hz, 1H), 7.38 (d, J = 7.5 Hz, 2H), 7.13

(dd, J = 11.1, 2.0 Hz, 1H), 7.07 (dd, J = 7.8, 1.8 Hz, 1H), 6.94 (d, J =

7.5 Hz, 1H), 5.72 (br t, 1H), 4.47 (d, J = 5.3 Hz, 2H), 3.52 (q, J = 6.9

Hz, 1H), 3.42−3.46 (m, 4H), 3.02 (s, 3H), 1.82−1.89 (m, 4H), 1.50

(d, J = 6.9 Hz, 3H). MS (FAB) m/z 489 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((pyridin-

2-ylmethyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

1

(2S)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(2-

methylpyrrolidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (34). Yield 75%, white solid, mp 80−85 °C. [α]_D²⁵

propanamide (24). Yield 62%, white solid, mp 155−165 °C. H

NMR (300 MHz, CD₃OD) δ 8.44 (m, 1H), 7.68 (td, J = 1.65, 7.68

Hz, 1H), 7.32−7.43 (m, 3H), 7.09−7.26 (m, 3H), 6.85 (d, J = 7.53

Hz, 1H), 4.72 (m, 2H), 4.33 (s, 2H), 3.63 (q, J = 7.14 Hz, 1H), 2.99

(s, 3H), 1.45 (d, J = 7.14 Hz, 3H). MS (FAB) m/z 526 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((pyridin-

3-ylmethyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (25). Yield 65%, white solid, mp 80−100 °C. ¹H NMR

(300 MHz, CDCl₃) δ 8.44−8.56 (m, 2H), 7.77 (m, 1H), 7.48 (m,

1H), 7.25 (m, 1H), 6.98−7.12 (m, 2H), 6.80−6.87 (m, 2H), 4.62 (m,

2H), 4.33 (m, 2H), 3.48 (m, 1H), 3.04 (s, 3H), 1.44 (d, J = 6.78 Hz,

3H). MS (FAB) m/z 526 (MH⁺).

1

−12.740 (c 1.0, CHCl₃). H NMR (300 MHz, CDCl₃) δ 7.53−7.35

(m, 2H), 7.17−6.98 (m, 3H), 6.52 (br s, 1H), 5.85 (br s, 1H), 4.57

(m, 1H), 4.28 (m, 2H), 3.53 (m, 3H), 3.14 (m, 1H), 3.02 (d, J = 3.66

Hz, 3H), 2.14 (m, 1H), 1.90 (m, 2H), 1.59 (m, 1H), 1.50 (d, J = 1.65

Hz, 3H), 1.10 (d, J = 6.03 Hz, 3H). MS (FAB) m/z 503 (MH⁺).

(S)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((S)-2-

(hydroxymethyl)pyrrolidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-

yl)methyl)propanamide (35). Yield 42%, white solid, mp 123−129

°C. [α]²_D⁵−45.620 (c 0.1, CHCl₃). H NMR (300 MHz, CDCl₃) δ

1

7.44 (m, 2H), 7.13−7.00 (m, 3H), 6.68 (br s, 1H), 6.17 (br s, 1H),

4.59 (m, 2H), 4.35 (m, 1H), 3.78 (m, 1H), 3.66−3.45 (m, 4H), 3.19

(m, 2H), 3.10 (s, 3H), 2.04−1.72 (m, 4H), 1.50 (d, J = 7.14 Hz, 3H).

MS (FAB) m/z 519 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((pyridin-

4-ylmethyl)amino)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (26). Yield 68%, white solid, mp 80−100 °C. ¹H NMR

(300 MHz, CDCl₃) δ 8.46−8.49 (m, 2H), 7.47 (t, J = 8.24 Hz, 1H),

7.24−7.31 (m, 3H), 7.02−7.09 (m, 2H), 6.84 (m, 1H), 5.92 (m, 1H),

4.64 (m, 2H), 4.38 (m, 2H), 3.46 (m, 1H), 3.03 (s, 3H), 1.45 (d, J =

7.14 Hz, 3H). MS (FAB) m/z 526 (MH⁺).

N-((2-(Dimethylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

propanamide (27). Yield 75%, white solid, mp 65−67 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.46−7.40 (m, 2H), 7.20−7.00 (m, 3H), 6.60

(br t, 1H), 4.50 (br d, 2H), 3.60 (m, 1H), 3.00 (s, 3H), 2.80 (s, 6H),

1.49 (d, J = 7.0 Hz, 3H). MS (FAB) m/z 463 (MH⁺).

(S)-1-(3-(((S)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-

propanamido)methyl)-6-(triﬂuoromethyl)pyridin-2-yl)-N,N-di-

methylpyrrolidine-2-carboxamide (36). Yield 55%, white solid,

mp 90−100 °C. [α]²_D⁵−170.019 (c 1.0, CHCl₃). ¹H NMR (300 MHz,

CDCl₃) δ 8.06 (m, 1H), 7.46 (d, J = 7.32 Hz, 1H), 7.40 (t, J = 8.25

Hz, 1H), 7.26 (m, 1H), 7.18 (m, 1H), 6.99 (d, J = 7.50 Hz, 1H), 5.40

(t, J = 7.35 Hz, 1H), 4.52 (dd, J = 15.03, 6.42 Hz, 1H), 4.35 (d, J =

14.82 Hz, 1H), 3.75 (m, 2H), 3.23 (s, 3H), 2.98 (s, 3H), 2.95 (s, 3H),

2.35 (m, 1H), 2.15 (m, 1H), 2.04 (m, 1H), 1.89 (m, 1H), 1.50 (d, J =

7.14 Hz, 3H). MS (FAB) m/z 560 (MH⁺).

N-((2-(Diethylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

propanamide (28). Yield 74%, white solid, mp 58−60 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.50 (dd, J = 8.0, 8.0 Hz, 1H), 7.50 (d, J = 8.0

Hz, 1H), 7.20−7.00 (m, 3H), 6.50 (br s, 1H), 6.30 (br s, 1H), 4.50 (m,

2H), 3.50 (q, J = 7.0 Hz, 1H), 3.20 (m, 2H), 3.00 (s, 3H), 1.50 (d, J =

7.0 Hz, 3H), 0.99 (t, J = 7.2 Hz, 6H). MS (FAB) m/z 491 (MH⁺).

N-((2-(Dipropylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

propanamide (29). Yield 58%, white solid, mp 96−98 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.54−7.44 (m, 2H), 7.18−7.07 (m, 3H), 6.63

(br s, 1H), 6.20 (br t, 1H), 4.44 (m, 2H), 3.55 (q, J = 7.3 Hz, 1H),

3.12−3.07 (m, 4H), 3.02 (s, 3H), 1.54−1.40 (m, 4H), 0.83 (t, J = 7.3

Hz, 6H). MS (FAB) m/z 519 (MH⁺).

(S)-tert-Butyl 1-(3-(((S)-2-(3-Fluoro-4-(methylsulfonamido)-

phenyl)propanamido)methyl)-6-(triﬂuoromethyl)pyridin-2-yl)-

pyrrolidine-2-carboxylate (37). Yield 62%, white solid, mp 80−95

°C. [α]²_D⁵−33.2 (c 0.15, MeOH). H NMR (300 MHz, CDCl₃) δ

1

6.83−7.56 (m, 5H), 6.58 (s, 1H), 4.69 (s, 1H), 4.43 (m, 2H), 3.91 (m,

1H), 3.58 (m, 4H), 3.00 (s, 3H), 1.85 (m, 2H), 1.50 (d, J = 7.1 Hz,

3H), 1.44 (s, 9H). MS (FAB) m/z 589 (MH⁺).

(S)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-((R)-3-

hydroxypyrrolidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)propanamide (38). Yield 62%, white solid, mp 88−90 °C.

[α]²_D⁵−1.6 (c 0.05, CHCl₃). H NMR (300 MHz, CDCl₃) δ 7.49 (m,

1

2H), 7.13−7.05 (m, 2H), 6.99 (d, J = 7.5 Hz, 1H), 5.97 (br s, 1H),

4.56−4.37 (m, 2H), 3.75−3.36 (m, 6H), 3.03 (s, 3H), 2.04−1.97 (m,

1H), 1.83−1.74 (m, 2H), 1.50 (d, J = 7.0 Hz, 3H). MS (FAB) m/z

505 (MH⁺).

N-((2-(Dibutylamino)-6-(triﬂuoromethyl)pyridin-3-yl)-

methyl)-2-(3-ﬂuoro-4-(methylsulfonamido)phenyl)-

(S)-N-((2-((R)-3-(Benzyloxy)pyrrolidin-1-yl)-6-

(triﬂuoromethyl)pyridin-3-yl)methyl)-2-(3-ﬂuoro-4-

(methylsulfonamido)phenyl)propanamide (39). Yield 62%,

white solid, mp 109−112 °C. [α]²_D⁵−31.108 (c 1.0, MeOH). ¹H

NMR (300 MHz, CDCl₃) δ 7.40 (dd, J = 8.1 Hz, 2H), 7.33−7,28 (m,

1

propanamide (30). Yield 70%, white solid, mp 102−104 °C. H

NMR (300 MHz, CDCl₃) δ 7.51−7.43 (m, 2H), 7.19−7.07 (m, 3H),

6.96 (br s, 1H), 6.40 (br t, 1H), 4.50 (m, 2H), 3.56 (q, J = 7.1 Hz,

1H), 3.13 (m, 4H), 3.02 (s, 3H), 1.52 (d, J = 7.1 Hz, 3H) 1.50 (m,

8401

dx.doi.org/10.1021/jm300780p | J. Med. Chem. 2012, 55, 8392−8408

Journal of Medicinal Chemistry

Article

5H), 7.10 (dd, J = 11 Hz, 1H), 6.97 (dd, J = 7.8 Hz, 2H), 6.72 (s, 1H),

5.98 (br s, 1H), 4.54 (d, 2H), 4.20 (br s, 1H), 3.75−3.39 (m, 5H),

2.97 (s, 1H), 2.16−2.11 (m, 1H), 2.05−1.94 (m, 1H), 1.76 (s, 1H),

1.45 (d, J = 7.0 Hz, 3H). MS (FAB) m/z 595 (MH⁺).

(m, 2H), 6.71 (br t, 1H), 6.58 (br s, 1H), 4.67−4.57 (m, 1H), 4.35 (m,

1H), 3.56−3.46 (m, 2H), 3.02 and 3.01 (s, 3H), 3.01−2.95 (m, 1H),

2.79 (m, 1H), 1.80−1.50 (m, 9H), 0.90 and 0.85 (d, 3H). MS (FAB)

m/z 517 (MH⁺).

tert-Butyl ((R)-1-(3-(((S)-2-(3-Fluoro-4-(methylsulfonamido)-

phenyl)propanamido)methyl)-6-(triﬂuoromethyl)pyridin-2-yl)-

pyrrolidin-3-yl)carbamate (40). Yield 65%, white solid, mp 78−79

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-methyl-

piperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (48). Yield 82%, white solid, mp 67−69 °C. ¹H NMR

(300 MHz, CDCl₃) δ 7.54−7.47 (m, 2H), 7.21 (d, J = 7.7 Hz, 1H),

7.15−7.07 (m, 2H), 6.64 (br s, 1H), 6.34 (br t, 1H), 4.48 (d, J = 5.9

Hz, 2H), 3.56 (q, J = 7.0 Hz, 1H), 3.32−3.17 (m, 2H), 3.03 (s, 3H),

2.74 (m, 1H), 2.46 (m, 1H), 1.82−1.61 (m, 4H), 1.53 (d, J = 7.1 Hz,

3H), 1.13−1.01 (m, 1H), 0.91 (m, 3H). MS (FAB) m/z 517 (MH⁺).

2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-methyl-

piperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (49). Yield 75%, white solid, mp 85 °C. ¹H NMR (300

MHz, CDCl₃) δ 7.47−7.55 (m, 2H), 7.07−7.22 (m, 3H), 6.29 (br t,

1H), 4.47 (d, J = 5.7 Hz, 2H), 3.54 (q, J = 6.9 Hz, 1H), 3.30 (m, 2H),

3.03 (s, 3H), 2.82 (m, 2H), 1.71 (m, 2H), 1.52 (d, J = 6.9 Hz, 3H),

1.24 (m, 3H), 0.97 (d, J = 6.6 Hz, 3H). MS (FAB) m/z 517 (MH⁺).

(S)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-

methylpiperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (49S). Yield 86%, white solid, mp 75−77 °C. [α]_D²⁰

°C. [α]²_D⁵10.8925 (c 0.8, MeOH). H NMR (300 MHz, CD₃OD) δ

1

7.48−7.41 (dd, J = 8.1 Hz, 2H), 7.22−7.14 (dd, J = 8.1 Hz, 2H), 6.97

(d, J = 7.68 Hz, 1H), 4.42 (q, 2H), 4.07 (q, 1H), 3.70 (m, 2H), 3.52

(m, 2H), 3.02 (s, 3H), 2.06 (m, 1H), 1.82 (m, 1H), 1.44 (m, 12H).

MS (FAB) m/z 604 (MH⁺).

tert-Butyl ((S)-1-(3-(((S)-2-(3-Fluoro-4-(methylsulfonamido)-

phenyl)propanamido)methyl)-6-(triﬂuoromethyl)pyridin-2-yl)-

pyrrolidin-3-yl)carbamate (41). Yield 58%, white solid, mp 78−80

°C. [α]²_D⁵−5.62 (c 0.1, MeOH). ¹H NMR (300 MHz, CD₃OD) δ 7.43

(d, J = 8.22 Hz, 2H), 7.41 (s, 1H), 7.22−7.13 (dd, J = 8.1 Hz, 2H),

6.97 (d, J = 7.5 Hz, 1H), 4.60 (q, 2H), 4.50−4.32 (m, 2H), 3.72 (m,

2H), 3.57−3.48 (m, 1H), 3.02 (s, 3H), 2.15−2.06 (m, 1H), 1.89−1.82

(m, 1H), 1.44 (m, 12H). MS (FAB) m/z 604 (MH⁺).

(S)-N-((2-((R)-3-(Dimethylamino)pyrrolidin-1-yl)-6-

(triﬂuoromethyl)pyridin-3-yl)methyl)-2-(3-ﬂuoro-4-

(methylsulfonamido)phenyl)propanamide (42). Yield 67%,

1

−2.73 (c 1.00, CHCl₃). H NMR (300 MHz, CDCl₃) δ 7.54 (d, J =

1

white solid, mp 75−77 °C. [α]²_D⁵16.294 (c 1.0, MeOH). H NMR

8.25 Hz, 1H), 7.48 (d, J = 8.43 Hz, 1H), 7.20 (d, J = 7.89 Hz, 1H),

7.15−7.07 (m, 2H), 6.44 (br s, 1H), 6.27 (br t, 1H), 4.47 (d, J = 5.49

Hz, 2H), 3.55 (q, J = 6.93 Hz, 1H), 3.30 (t, 2H), 3.03 (s, 3H), 2.82 (tt,

2H), 1.73 (m, 2H), 1.53 (d, J = 7.14 Hz, 3H), 1.23 (m, 3H), 0.97 (d, J

= 6.39 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 173.48, 161.14, 155.55−

153.10 (d, J = 244.6), 145.47−144.44 (q, J = 34.1), 140.25−140.18 (d,

J = 6.6), 137.64, 128.03, 124.16, 124.11−124.08 (d, J = 3.4), 123.68−

123.56 (d, J = 12.7), 125.51−117.34 (q, J = 272.1), 114.91−114.71 (d,

J = 20), 114.05−114.02 (d, J = 2.8), 50.49−50.42 (d, J = 6.3), 46.39,

39.84, 39.82, 34.34−34.30 (d, J = 3.8), 30.60, 21.81, 18.53. MS (FAB)

m/z 517 (MH⁺); HRMS calcd for C₂₃H₂₉F₄N₄O₃S (M + H),

517.1897, found 517.1904.

(300 MHz, CDCl₃) δ 7.47 (dd, J = 8.1 Hz, 1H), 7.42 (d, J = 8.1 Hz,

1H), 7.16 (dd, J = 10.5 Hz, 1H), 7.06 (d, J = 9.5 Hz, 1H), 6.97 (d, J =

6.9 Hz, 1H), 5.98 (br s, 1H), 4.79 (s, 3H), 4.55 (dd, J = 6.9 Hz, 1H),

4.40 (dd, J = 6.9 Hz, 1H), 3.64−3.48 (m, 4H), 3.36 (t, 1H), 3.02 (s,

3H), 2.71−2.66 (m, 4H), 2.29 (s, 6H), 2.11−2.05 (m, 1H), 1.83−1.76

(m, 2H), 1.49 (d, J = 7.0 Hz, 3H). MS (FAB) m/z 532 (MH⁺).

(S)-N-((2-((S)-3-(Dimethylamino)pyrrolidin-1-yl)-6-

(triﬂuoromethyl)pyridin-3-yl)methyl)-2-(3-ﬂuoro-4-

(methylsulfonamido)phenyl)propanamide (43). Yield 65%,

white solid, mp 78−79 °C. [α]²_D⁵−28.355 (c 0.6, MeOH). H NMR

1

(300 MHz, CDCl₃) δ 7.50 (dd, J = 8.1 Hz, 1H), 7.42 (d, J = 8.1 Hz,

1H), 7.13 (dd, J = 10.5 Hz, 1H), 7.08 (d, J = 9.5 Hz, 1H), 6.98 (d, J =

6.9 Hz, 1H), 5.98 (br s, 1H), 4.55 (dd, J = 6.9 Hz, 1H), 4.42 (dd, J =

6.9 Hz, 1H), 3.66−3.45 (m, 4H), 3.36 (t, 1H), 3.02, (s, 3H), 2.69 (m,

1H), 2.28 (s, 6H), 2.11−2.05 (m, 1H), 1.83−1.74(m, 2H), 1.49 (d, J =

7.0 Hz, 3H). MS (FAB) m/z 532 (MH⁺).

(R)-2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-

methylpiperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)methyl)-

propanamide (49R). Yield 50%, white solid, mp 78−80 °C. [α]_D²⁰

1

−2.30 (c 1.00, CHCl₃). H NMR (300 MHz, CDCl₃) δ 7.54 (d, J =