2-(3-Fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists: Structure activity relationships of the 2-oxy pyridine C-region

Article abstract of DOI:10.1016/j.ejmech.2013.04.003

The structure activity relationships of 2-oxy pyridine derivatives in the C-region of N-(6-trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-4- methylsulfonylaminophenyl)propanamides as hTRPV1 antagonists were investigated. The analysis indicated that the lipophilicity of the 2-oxy substituents was critical for potent antagonism and 4 or 5 carbons appeared to be optimal for activity. Multiple compounds proved to have comparable activity to 1, which had been reported as the most potent antagonist for capsaicin activity among the previous series of compounds. Further analysis of compounds 22 (2-isobutyloxy) and 53 (2-benzyloxy) in the formalin test in mice demonstrated strong analgesic activity with full efficacy. Docking analysis of 53S using our hTRPV1 homology model indicated that the A- and B-region 2-(3-fluoro-4- methylsulfonylaminophenyl)propanamide made important hydrophobic and hydrogen bonding interactions with Tyr511 and that the C-region 6-trifluoromethyl and 2-benzyloxy groups of pyridine occupied the two hydrophobic binding pockets, respectively.

Full text of DOI:10.1016/j.ejmech.2013.04.003

Accepted Manuscript
2-(3-Fluoro-4-methylsulfonylaminophenyl) Propanamides as Potent TRPV1
Antagonists: Structure Activity Relationships of the 2-Oxy Pyridine C-region
Shivaji A. Thorat, Dong Wook Kang, HyungChul Ryu, Myeong Seop Kim, Ho Shin
Kim, Jihyae Ann, Tae-Hwan Ha, Sung Eun Kim, Karam Son, Sun Choi, Peter M.
Blumberg, Robert Frank, Gregor Bahrenberg, Klaus Schiene, Thomas Christoph,
Jeewoo Lee
PII:
S0223-5234(13)00235-3
10.1016/j.ejmech.2013.04.003
EJMECH 6113
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2013
Revised Date: 1 April 2013
Accepted Date: 2 April 2013
Please cite this article as: S.A. Thorat, D.W. Kang, H. Ryu, M.S. Kim, H.S. Kim, J. Ann, T.-H. Ha, S.E.
Kim, K. Son, S. Choi, P.M. Blumberg, R. Frank, G. Bahrenberg, K. Schiene, T. Christoph, J. Lee, 2-(3-
Fluoro-4-methylsulfonylaminophenyl) Propanamides as Potent TRPV1 Antagonists: Structure Activity
Relationships of the 2-Oxy Pyridine C-region, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.04.003.
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ACCEPTED MANUSCRIPT
Graphical Abstract
LP
Phe543
0.14
0.12
0.09
0.07
0.05
0.03
0.01
F3C
H
N
N
F
Leu547
Thr550
O
O
O
N S
R
Met514
H
O
-0.02
-0.04
-0.06
Leu515
Tyr511
-0.08
-0.11
-0.13
Tyr555
Ser512
-0.15
-0.17
-0.19
Ile564

ACCEPTED MANUSCRIPT
2-(3-Fluoro-4-methylsulfonylaminophenyl)
Propanamides as Potent TRPV1 Antagonists:
Structure Activity Relationships of the 2-Oxy Pyridine
C-region
Shivaji A. Thorat,a Dong Wook Kang,a,e HyungChul Ryu,a Myeong Seop Kim,a Ho Shin Kim,a
Jihyae Ann,a Tae-Hwan Ha,a Sung Eun Kim,a Karam Son,b Sun Choi,b Peter M. Blumberg,c
Robert Frank,d Gregor Bahrenberg,d Klaus Schiene,d Thomas Christoph,d Jeewoo Lee*,a
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 151-742, Korea
b
National Leading Research Lab (NLRL) of Molecular Modeling & Drug Design, College of
Pharmacy, Division of Life & Pharmaceutical Sciences, and National Core Research Center for
Cell Signaling & Drug Discovery Research, Ewha Womans University, Seoul 120-750, Korea
c
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer
Institute, NIH, Bethesda, MD 20892, USA
d Grunenthal Innovation, Grunenthal GmbH, D-52078 Aachen, Germany
e Department of Pharmaceutical Science and Technology, College of Health and Medical Science,
Catholic University of Deagu, Gyeongsan-si, Gyeongsangbuk-do 712-702, Korea
1

ACCEPTED MANUSCRIPT
Key Words: TRPV1 antagonists, analgesic, molecular modeling, capsaicin
Abstract
The structure activity relationships of 2-oxy pyridine derivatives in the C-region of N-(6-
trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamides as
hTRPV1 antagonists were investigated. The analysis indicated that the lipophilicity of the 2-oxy
substituents was critical for potent antagonism and 4 or 5 carbons appeared to be optimal for
activity. Multiple compounds proved to have comparable activity to 1, which had been reported
as the most potent antagonist for capsaicin activity among the previous series of compounds.
Further analysis of compounds 22 (2-isobutyloxy) and 53 (2-benzyloxy) in the formalin test in
mice demonstrated strong analgesic activity with full efficacy. Docking analysis of 53S using our
hTRPV1 homology model indicated that the A- and B-region 2-(3-fluoro-4-
methylsulfonylaminophenyl)propanamide made important hydrophobic and hydrogen bonding
interactions with Tyr511 and that the C-region 6-trifluoromethyl and 2-benzyloxy groups of
pyridine occupied the two hydrophobic binding pockets, respectively.
2

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1. Introduction
The transient receptor potential V1 (TRPV1) receptor is a non-selective cation ch
annel with high Ca2+ permeability which functions as a molecular integrator of nociceptiv
e stimuli.1 The receptor is activated by endogenous agonists including protons,2 noxious
heat,3 and inflammatory lipid mediators4,5 as well as by natural products such as capsaici
n (CAP)6 and resiniferatoxin (RTX)7. The increase in intracellular Ca2+ upon TRPV1 acti
vation causes excitation of the primary sensory neurons and the consequent central perce
ption of pain. TRPV1 antagonists, by inhibiting this transmission of nociceptive signaling
from the periphery to the CNS, have thus attracted much attention as potential analgesic
s. In recent years a number of TRPV1 antagonists have been developed as novel analges
ic and anti-inflammatory agents, particularly for the treatment of neuropathic pains.8 The
clinical development and therapeutic potential of TRPV1 antagonists have been extensivel
y reviewed.9-15
Recently, we investigated a series of N-(2-amino-6-trifluoromethyl-pyridin-3-ylmeth
yl) 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides, designed by combining previous
ly identified pharmacophoric elements, as hTRPV1 antagonists.16 Among them, compound
1 showed the most potent antagonism with Ki(CAP) = 0.3 nM and IC50(pH) = 8.4 nM, be
ing thus 150-fold and 230-fold better than precedent lead 217 for CAP and pH antagonis
m, respectively (Figure 1). In addition, the compound 1S, the S-form of 1, was found t
o be highly selective for TRPV1 with Ki(CAP) = 0.2 nM and displayed strong analgesic a
ctivity in a neuropathic pain model with almost no side effects. Consistent with its mech
anism of action in vivo being through TRPV1, compound 1S blocked capsaicin-induced h
ypothermia but caused modest TRPV1-related hyperthermia in mice.
3

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Figure 1. Lead TRPV1 antagonists
F3C
H
H
N
N
F
N
F
O
O
N
O
O
N S
N S
H
O
H
O
1
2
CAP Ki(ant) = 0.3 nM
pH IC50 = 8.4 nM
CAP Ki(ant) = 44.5 nM
pH IC50 = 1990 nM
Our structural analysis indicated that the enhanced potency of 1 compared to 2
was attributable to a new hydrophobic interaction with the receptor, afforded by the addit
ional 4-methylpiperidine moiety in 1. Specifically, docking analysis using the hTRPV1 ho
mology model which we developed indicated that the 4-methylpiperidinyl group in the
C-region of 1 interacted with a hydrophobic region on the receptor composed of Met514
and Leu515.16
As a continuation of our SAR analysis of the 2-substitutent in the N-(6-trifluoro
methyl-pyridin-3-ylmethyl) C-region, we have investigated its 2-oxy derivatives (Figure 2)
as hTRPV1 antagonists. For a selected potent antagonist in the series, we further charac
terized analgesic activity in the formalin pain model and performed molecular modeling
with our hTRPV1 homology model.
Figure 2. General structure of the designed compounds
F3C
H
N
N
F
O
O
O
N S
R
H
O
4

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2. Results and Discussion
2.1. Chemistry
The syntheses of the final compounds are represented in Scheme 1. 2-Oxy substit
uted pyridines 5 were synthesized either by O-alkylation of pyridone 316 or by the nucle
ophilic substitution of various alcohols with 2-chloropyridine 416. Pyridines 5 containing c
yclopropyl substituents were prepared from the corresponding alkenes by the Simmons–S
mith reaction, as shown in Scheme 2. The nitrile groups of 5 were reduced to yield the
corresponding primary amines 6. The amines were coupled with 2-(3-fluoro-4-methylsulf
onylaminophenyl)propionic acid 717 to provide the final compounds 8-71.
F3C
F3C
F3C
a for 3
b for 4
c
N
NH2
N
N
CN
CN
OR
X
OR
6
3 X=OH
4 X=Cl
5
+
F3C
H
N
N
F
NHSO2CH3
HO
F
d
OR
O
O
NHSO2CH3
8-71
7
Scheme 1. Syntheses of 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamide analogues
Reagents and conditions: (a) [Method A] RBr, K2CO3, 18-crown-6, DMF, heat; (b) [Method B]
ROH, DBU, 1,4-dioxane or CH3CN, reflux: [Method C] ROH, NaH, THF, reflux: [Method D]
ROH, Cs2CO3, DMF, heat: [Method E] ROH, KF, DMF, heat; (c) [Method F] 2M BH3-SMe2 in
THF: [Method G] H2, 10% Pd-C, AcOH, MeOH: [Method H] LAH, diethylether: [Method I]
NaBH4, NiCl2-6H2O, MeOH; (d) EDC, HOBt, DMF
5

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F3C
F3C
a
N
N
CN
CN
O
O
R
R
R = H, CH3
Scheme 2. Syntheses of the 2-cyclopropylmethyloxy pyridine C-region
Reagents and conditions: (a) Diiodomethane, Diethylzinc, CH2Cl2
2.2. Structure-Activity Relationship (SAR) Analysis
The synthesized TRPV1 ligands were evaluated in vitro for antagonism as measured by
inhibition of activation by capsaicin (CAP) and pH as indicated. The assays were conducted
using a fluorometric imaging plate reader (FLIPR) with human TRPV1 heterologously expressed
in Chinese hamster ovary (CHO) cells.16 The results are summarized in Tables 1-5, together with
the potencies of the previously reported parent antagonist 1.
To investigate the SAR for 2-oxy derivatives of the pyridine C-region we began
with the straight 2-alkyloxy derivatives (Table 1). Starting from the 2-methoxy derivative
8, the antagonistic activity was enhanced sharply as the number of carbons in the chain
increased until reaching a maximum with the 2-butoxy derivative 11, which was as pote
nt as 1 with Ki(CAP) = 0.3 nM. The activity stayed constant upon further chain lengtheni
ng through the hexyloxy derivative 13, with all showing highly potent antagonism (11-13
). The two stereoisomers of 13 showed stereospecificity for their antagonistic activity. 13
S was the eutomer and 13R was the distomer, consistent with previous findings.16,18 Uns
aturation of the alkyl chain led to a decrease in activity. For example, the Z-alkene isom
ers 14 and 15 were ca. 5-fold and 1.5-fold less potent than the alkyl surrogates 11 and
12, respectively, for CAP antagonism. Introduction of oxygen to the alkyl chain caused a
dramatic loss of activity. For example, the oxygenated analogues 16 and 19 exhibited 1
60-fold and 20-fold less potent CAP antagonism than did the parents 11 and 13, respecti
vely. Similarly, 17 and 18, analogues of 12 oxygenated at different positions along the al
kyl chain, were 70-fold and 22-fold less potent than the parent, respectively. However, a
6

ACCEPTED MANUSCRIPT
ddition of a lipophilic phenyl group to 18 to yield compound 20 led to potent antagonis
m. The SAR analysis for this series of straight 2-alkyloxy group thus indicated that the l
ipophilicity of side chain was critical for antagonism, probably due to its hydrophobic int
eraction with the receptor, and the optimal number of carbons appeared to be four.
Table 1. In vitro hTRPV1 Antagonistic Activities for Straight 2-Alkyloxy Derivatives
F3C
H
N
N
F
O
O
O
N S
R
H
O
Ki [CAP]
(nM)
IC50 [pH]
(nM)
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
0.3
68.8
12.7
0.9
8.4
WE
WE
1
8
13R
14
15
16
17
18
19
20
WE
277
1.6
1
183
43
9
90.1
30.5
27.3
29.9
12.3
49
WE
WE
WE
462
161
10
11
12
13
13S
O
0.3
50
O
0.7
15.3
9.7
1.5
O
0.5
O
O
0.4
WE: weakly effective (1 µM<Ki or IC50<10 µM)
NE: not effective (Ki or IC50>10 µM)
Next, the SAR of branched 2-alkyloxy derivatives was investigated (Table 2). Si
7

ACCEPTED MANUSCRIPT
milar to the findings for the derivatives with straight alkyl chains, the branched 4 carbon
derivative 22 showed the most potent antagonism, but with high potency being retained
with longer substituents up to the 9-carbon derivative 28. The comparison of activity be
tween straight and branched alkyl derivatives indicated that the straight alkyl derivatives
generally showed slightly better antagonism than did the corresponding branched ones (fo
r example, 10 vs 21, 11 vs 22, 12 vs 23/25 and 13 vs 24). Interestingly, compounds 25-
28 were found to have a unique profile in which they highly antagonized the activation
by capsaicin but not by pH. This is an important distinction. Hyperthermia associated wit
h antagonism is a side effect of concern. SAR studies have shown that antagonists may
differ in their relative activities against agonism by capsaicin, by low pH, and by elevate
d temperature. Antagonism of the response to low pH is the predominant predictor of w
hether an antagonist may induce hyperthermia as a side effect, so antagonists blocking th
e response to capsaicin but not low pH may be of particular interest for further evaluati
on.19
Table 2. In vitro hTRPV1 Antagonistic Activities for Branched 2-Alkyloxy Derivatives
Ki [CAP]
(nM)
IC50 [pH]
(nM)
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
2.1
0.5
0.8
0.9
93.4
52.6
15.1
23.8
1.1
NE
21
22
23
24
25
26
27
28
0.7
0.5
0.7
WE
WE
WE
WE: weakly effective (1 µM<Ki or IC50<10 µM)
NE: not effective (Ki or IC50>10 µM)
The SAR of 2-cycloalkyloxy derivatives was investigated next (Table 3). The ana
8

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lysis indicated that the SAR pattern was similar to that of the above series, and compou
nd 30 with 5 carbons was optimal for antagonism. To examine the effect of substitution
on the cyclic ring, the cyclohexyl derivatives of 31 were further investigated. Whereas th
e 4-trifluoromethyl group in 32 enhanced antagonism compared to 31, the 4-methyl grou
p in 33 reduced activity, suggesting that lipophilicity is a contributor to the activity. The
4-ethyl and 4-t-butyl derivatives, 34 and 35, reduced the antagonism, probably due to st
eric repulsion with the receptor. However, the 4,4-dimethyl and 3,5-dimethyl analogues, 3
6 and 37, retained good antagonism. The results indicated that lipophilicity at the 4-posit
ion of the cyclohexyl ring was important for potent antagonism, but there were steric co
nstraints such that size bigger than methyl led to the reduction of activity. A similar SA
R pattern was found for the 4-position of piperidine ring in the C-region previously.16 In
troduction of nitrogen at the 4-position in 38 led to dramatic loss of activity. However, i
ts activity was recovered by adding the lipophilic Boc group to the nitrogen, as in 39.
Compound 40, the phenyl-fused analogue of 30, still displayed good CAP antagonism, in
dicating that the phenyl group was tolerated.
Table 3. In vitro hTRPV1 Antagonistic Activities for 2-Cycloalkyloxy Derivatives
Ki [CAP]
(nM)
IC50 [pH]
(nM)
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
1.4
0.9
1.3
0.3
2.9
69.8
28.7
43.4
14
7.7
242
29
30
31
32
33
35
36
37
38
39
0.9
3
140
174
NE
293
CF3
NH
WE
1.3
43.1
9

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4.8
125
2.7
WE
34
40
WE: weakly effective (1 µM<Ki or IC50<10 µM)
NE: not effective (Ki or IC50>10 µM)
Next, the SAR of 2-cycloalkylmethyloxy derivatives was investigated. This series was
designed by inserting a methyl group into the compounds of the 2-cycloalkyloxy series of Table
3. Generally the insertion led to a 2- to 5-fold enhancement in antagonism compared to that of
the corresponding parent compounds (for example, 29 vs 44, 30 vs 45, 31 vs 46, and 33 vs 47).
Of particular interest, compounds 42, 44-46 and 50 displayed high potency in the sub-nanomolar
range. Compounds 48-50 demonstrated a similar SAR pattern to that shown in table 3 in which
the introduction of a nitrogen at the 4-position abolished the activity. Conversely, compound 50
exhibited excellent antagonism, suggesting that the N-Boc moiety made an appropriate
hydrophobic interaction with the receptor.
Table 4. In vitro hTRPV1 Antagonistic Activities for 2-Cycloalkylmethyloxy Derivatives
Ki [CAP]
(nM)
IC50 [pH]
(nM)
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
1.1
0.3
1.1
0.3
0.4
115
5.1
0.7
19.7
41
42
43
44
45
46
47
48
49
50
1.0
WE
3.4
0.3
44.4
NE
161
40.5
NH
342
16.6
11.6
10

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WE: weakly effective (1 µM<Ki or IC50<10 µM)
NE: not effective (Ki or IC50>10 µM)
Finally, we sought to evaluate the SAR of 2-aryloxy and 2-arylmethyloxy derivatives. In
the 2-aryloxy series, the 2-phenyloxy derivative 51 exhibited reasonable antagonism and its 4-
methylation, providing 52, further increased activity. The 2-arylmethyloxy derivatives showed
better antagonism compared to the corresponding 2-aryloxy ones (51 vs 54, 52 vs 55), as
described above for the 2-cycloalkyloxy and 2-cycloalkylmethyloxy series. The 2-
phenylethyloxy derivative 54 was found to be as potent as 53. Further optimization was
conducted with the 2-benzyloxy derivative 53 to examine the effect of substitution. Introduction
of 4-substituents on the benzyl group, including alkyls (55-58), halogens (59-60), trifluoromethyl
(61) and methoxy (62), decrease the antagonism slightly. In the other hand, 3-substituted benzyl
derivatives showed better antagonism compared to the corresponding 4-substituted surrogates
(for example, 59 vs 63 for F, 60 vs 64 for Cl, 62 vs 65 for OCH3). 3,5-Disubstituted derivatives
displayed activity similar to that of the corresponding 3-substituted ones (for example, 63 vs 66
for F, 64 vs 67 for Cl, 65 vs 68 for OCH3). 2-Pyridinylmethyloxy derivatives (69-71) were much
less potent than the 2-benzyloxy derivative 53, confirming that incorporation of nitrogen into the
ring was detrimental to antagonism.
Table 5. In vitro hTRPV1 Antagonistic Activities for 2-Aryloxy and Arylmethyloxy Derivatives
Ki [CAP] IC50 [pH]
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
(nM)
(nM)
6.4
WE
2.6
653
51
52
62
63
OCH3
2.8
WE
0.8
69
F
11

ACCEPTED MANUSCRIPT
53
54
55
56
57
58
59
60
61
0.5
0.5
1.5
3.7
16
18.6
27.6
53
64
65
66
67
68
69
70
71
4.9
0.8
1.0
2.6
0.7
29.5
142
51
95
43.7
93
Cl
OCH3
F
F
Cl
165
337
182
25
114
76
Cl
OCH3
H3CO
N
2.1
2.5
2.5
3.2
WE
WE
WE
N
F
132
66.1
N
Cl
CF3
WE: weakly effective (1 µM<Ki or IC50<10 µM)
NE: not effective (Ki or IC50>10 µM)
Detailed in vitro activity of 22 and 53, the two selected antagonists in this study,
was investigated for multiple TRPV1 activators including capsaicin, pH, heat (45oC) and
N-arachidonoyl dopamine (NADA), and compared to the activity of lead 1 (Table 6). B
oth showed excellent antagonism of all four TRPV1 activators and comparable activities
12

ACCEPTED MANUSCRIPT
to 1. Particularly, compound 53 exhibited excellent potency toward NADA activation.
Table 6. In vitro hTRPV1 Antagonistic Activities for 1, 22 and 53 to multiple activators
Activators
CAP (f)Ki
1
22
(nM)
0.5
53
(nM)
(nM)
0.3
0.5
18.6
52.3
0.001
pH (IC50)
8.4
52.6
67.5
0.29
Heat 45oC (IC50)
NADA (f)Ki
4.5
0.23
2.3. Analgesic Activity
We evaluated the in vivo analgesic activities of two selected antagonists, 22 and
53, in the formalin test20 in mice upon oral administration. Compound 22 showed a signi
ficant antinociceptive effect, with 63.4 ± 11.5 % and 60.8 ± 9.9 % (mean ± SEM) inhibitio
n of response at the doses of 0.1 and 0.3 mg/kg, respectively (p < 0.05). Compound 53 inh
ibited the nociceptive response by 41.8 ± 17.3 % and 54.1 ± 26.3 % at the doses of 0.1 an
d 0.3 mg/kg, respectively (p < 0.05). Since we had observed that TRPV1 knock-out mice
showed approximately 50% of the magnitude of response in the formalin test as was se
en in wild-type mice (unpublished observations), the inhibition of the formalin response t
hat we found for the two antagonists would correspond to the expected result for full T
RPV1 blockade.
2.4. Molecular Modeling
Using our human TRPV1 (hTRPV1) model16, built based on our rat TRPV1 (rTRPV1)
model21, we performed a flexible docking study of compound 53S, the active isomer of 53. As
13

ACCEPTED MANUSCRIPT
shown in Figure 3, a binding mode generally similar to that of 1S16 was obtained with the S form
of compound 53. The sulfonylaminobenzyl group (A-region) occupied the deep bottom hole and
was involved in a hydrophobic interaction with Tyr511. A fluorine atom of the A-region
participated in hydrogen bonding with Ser512 and Tyr555 and NH of the sulfonamide group
made hydrogen bonds with Ser512. 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. In addition, the 3-trifluoromethyl group (C-region) extended toward the upper
hydrophobic areas composed of Leu547 and Thr550, forming hydrophobic interactions.
Furthermore, the 2-benzyloxy group in the C-region made an additional hydrophobic interaction
with the hydrophobic region composed of Met514 and Leu515.
Figure 3. Flexible docking result of 53S in the hTRPV1 model.
(A)
(B)
(C)
LP
Phe543
0.14
0.12
0.09
0.07
0.05
0.03
0.01
Leu547
Thr550
Met514
-0.02
-0.04
-0.06
Leu515
-0.08
-0.11
-0.13
Tyr511
-0.15
-0.17
-0.19
Ser512
Ile564
(A) Binding mode of 53S, the S isomer of 53. The key interacting residues are marked and
displayed as capped-stick with carbon atoms in white. The helices are colored by gray and the
helices of the adjacent monomer are displayed in line ribbon. Compound 53S 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 non-
polor 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
14

ACCEPTED MANUSCRIPT
lipophilic potential property. The surface of hTRPV1 is Z-clipped and that of the ligand is in its
carbon color for clarity. (C) Van dar Waals surface of the ligand colored by its lipophilic potential
property.
3. Conclusion
The structure activity relationship of 2-oxy pyridine derivatives in the C-region of N-(6-
trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-4-methylsulfonylaminophenyl) propanamides as
hTRPV1 antagonists was investigated. The analysis indicated that the lipophilicity of the 2-oxy
substituents was a key determinant of antagonism. Generally, as the lipophilicity of the 2-oxy
substituents increased, the antagonism was enhanced until it reached a maximal value, after
which it remained constant. The number of 4 or 5 carbons appeared to be optimal for activity.
Numerous compounds (11, 13, 22, 27, 32, 42, 44, 45, 50, 53 and 54) were found to have
comparable potency to 1, which had recently been reported as the most potent antagonist of a
previous series, with a range of Ki(CAP) = 0.3-0.5 nM. Among the compounds, 22 (2-isobutyloxy)
and 53 (2-benzyloxy) demonstrated strong analgesic activity in the formalin test in mice with full
efficacy. Docking analysis of 53S with our hTRPV1 homology model indicated that the 2-(3-
fluoro-4-methylsulfonylaminophenyl)propanamide (A and B-regions) made critical hydrophobic
and hydrogen bonding interactions with Tyr511, and the 6-trifluoromethyl and 2-benzyloxy
groups of pyridine (C-region) interacted the two hydrophobic regions composed of
Leu547/Thr550 and Met514/Leu515, respectively.
4. Experimental
4.1. Chemistry
All chemical reagents were commercially available. Melting points were determined on a Büchi
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)
spectra were recorded on a JEOL JNM-LA 300 and Bruker Avance 400 MHz FT-NMR
15

ACCEPTED MANUSCRIPT
spectrometer. Chemical shifts are reported in ppm units with Me4Si as a reference standard. Mass
spectra were recorded on a VG Trio-2 GC-MS and 6460 Triple Quad LC/MS. Elemental
analyses were performed with an EA 1110 Automatic Elemental Analyzer, CE Instruments.
4.1.1. General Procedure for the Synthesis of 5
4.1.1.1. Method A (for 29-40)
°
To a stirred solution of 3 (1.0 mmol) in N,N-dimethylformamide (6 mL) at 0 C was added 18-
crown-6 ether (cat.), potassium carbonate (2 mmol) followed by bromide compound (1.2
mmol). The reaction mixture was heated at 60-90 °C for overnight and 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 EtOAc:hexane (1:2) as eluant.
4.1.1.2. Method B (for 8-13, 21-28 and 51-58)
To a stirred solution of 4 (1.0 mmol) in 1,4-dioxane or acetonitrile (5 mL) at 0 °C was added 1,8-
diazabicyclo[5.4.0]undec-7-ene (2 mmol) followed by alcohol compound (1.2 mmol). The
°
reaction mixture was stirred at 50 C for overnight, and extracted with EtOAc for 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 EtOAc:hexane (1:2) as eluant.
4.1.1.3. Method C (for 16-20 and 41-43)
°
To a stirred solution of 4 (1.0 mmol) in anhydrous THF (5 mL) at 0 C was added sodium
hydride (2.5 mmol) followed by alcohol compound (1.2 mmol). The reacton mixture was
°
stirred at 50 C for 5 h, and extracted with EtOAc several times. The combined organic
layers were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue
16

ACCEPTED MANUSCRIPT
was purified by flash column chromatography on silica gel using EtOAc:hexane (1:2) as eluant.
4.1.1.4. Method D (for 14-15, 48-50 and 59-71)
°
To a stirred solution of 4 (1.0 mmol) in N,N-dimethylformamide (5 mL) at 0 C was added
cesium carbonate (3.0 mmol) followed by alcohol compound (1.2 mmol). The reaction mixture
°
was stirred at 60-90 C for overnight, and 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
EtOAc:hexane (1:2) as eluant.
4.1.1.5. Method E (for 44-47)
To a stirred solution of 4 (1.0 mmol) in N,N-dimethylformamide (5 ml) was added potassium
fluoride (2.0 mmol) followed by alcohol compound (1.2 mmol). The reaction mixture was
°
stirred at 60-90 C for overnight and 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 EtOAc:hexane
(1:2) as eluant.
4.1.2. General Procedure for Nitrile Reduction
4.1.2.1. Method F (for 16-20, 29-37 and 59-68)
To a stirred solution of nitrile (2.0 mmol) in anhydrous THF (1 mL) was added 2 M BH3·SMe2
in THF (3 mL, 3 equiv) at room temperature. After being refluxed for 8 h, the reaction mixture
was cooled to room temperature and 2 M HCl solution was added. Then the mixture was
refluxed for 30 min, cooled to to room temperature, neutralized by 2 M NaOH solution and
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
17

ACCEPTED MANUSCRIPT
chromatography on silica gel using CH2Cl2:MeOH (10:1) as eluant.
4.1.2.2. Method G (for 8-13, 21-28 and 51-58)
A suspension of nitrile (5.0 mmol) and 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 eluant.
4.1.2.3. Method H (for 14-15, 41-47 and 69-71)
To a cooled solution of nitrile (1.0 mmol) in anhydrous THF at 0 °C (5 mL) was added slowly
lithium aluminium hydride (3.0 mmol) in portion wise. The mixture was refluxed for
overnight, cooled to room temperature and quenched by dropwise addition of water. 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.
.
4.1.2.4. Method I (for 38-40 and 48-50)
To a stirred solution of nitrile (1.0 mmol) and NiCl2·6H2O (2.0 mmol) in MeOH (8 mL) was
added sodium borohydride (4.0 mmol) slowly. 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.
4.1.3. General Procedure for Amide coupling
A mixture of acid (5.0 mmol), amine (5.5 mmol),
N-(3-dimethylaminopropyl)-N’-ethyl-
carbodiimide hydrochloride (6.0 mmol), and 1-hydrxybenzotriazole hydrate in
N,N-
18

ACCEPTED MANUSCRIPT
dimethylformamide (20 mL) was stirred for 12 h at room temperature. The reaction
mixture was extracted with EtOAc several times. 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.
4.1.3.1.
N-((2-Methoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-Fluoro-4-
(methylsulfonamido)phenyl)propanamide (8)
Yield 81%, white solid, mp 122-124 °C; 1H NMR (CDCl3)
δ 7.60 (d, J = 7.3 Hz, 1H), 7.52 (dd, J
= 8.3, 8.3 Hz, 1H), 7.21 (d,
J
= 7.3 Hz, 1H), 7.12-7.06 (m, 2H), 6.50 (bs, NH), 6.00 (bt, NH),
= 7.1 Hz, 1H), 3.04 (s, 3H), 1.49 (d, = 7.0 Hz,
4.37 (d, = 6.1 Hz, 2H), 3.95 (s, 3H), 3.52 (q,
J
J
J
3H); MS (FAB) m/z 450 (M+H). Anal. Calcd for C18H19F4N3O4S: C, 48.10; H, 4.26; N, 9.35.
Found: C, 48.27; H, 4.24; N, 9.33.
4.1.3.2
N-((2-Ethoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (9)
Yield 68%, white solid, mp 138-140 °C; 1H NMR (CDCl3)
δ 7.58 (d, J = 7.3 Hz, 1H), 7.51 (dd, J
= 8.2, 8.2 Hz, 1H), 7.19 (d,
4.44-4.36 (m, 4H), 3.53 (q,
J
= 7.3 Hz, 1H), 7.12-7.05 (m, 2H), 6.58 (bs, NH), 6.02 (bt, NH),
J
= 7.0 Hz, 1H), 3.03 (s, 3H), 1.49 (d, = 7.1 Hz, 3H), 1.34 (t, 3H, J
J
= 7.1 Hz); MS (FAB) m/z 464 (M+H). Anal. Calcd for C19H21F4N3O4S: C, 49.24; H, 4.57; N,
9.07. Found: C, 49.40; H, 4.56; N, 9.04.
4.1.3.3.
N-((2-Propoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (10)
Yield 78%, white solid, mp 88-90 °C; 1H NMR (CDCl3)
δ 7.58 (d, J = 7.5 Hz, 1H), 7.52 (dd, J =
8.2, 8.2 Hz, 1H), 7.19 (d,
J
= 7.3 Hz, 1H), 7.12-7.05 (m, 2H), 6.50 (bs, NH), 5.97 (bt, NH), 4.39-
19

ACCEPTED MANUSCRIPT
4.23 (m, 4H), 3.52 (q,
J
= 7.1 Hz, 1H), 3.03 (s, 3H), 1.74 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); MS
(FAB) m/z 478 (M+H). Anal. Calcd for C20H23F4N3O4S: C, 50.31; H, 4.86; N, 8.80. Found: C,
50.14; H, 4.88; N, 8.83.
4.1.3.4.
N-((2-Butoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (11)
°
1
Yield 77%, white solid, mp 77-79 C; H NMR (300 MHz, CDCl3)
7.52 (dd, = 8.1, 8.1 Hz, 1H), 7.19 (d, = 7.5 Hz, 1H), 7.12-7.05 (m, 2H), 6.46 (bs, NH), 5.96
(bt, NH), 4.39-4.28 (m, 4H), 3.51 (q, = 7.1 Hz, 1H), 3.03 (s, 3 H), 1.75-1.66 (m, 2H), 1.49 (d,
= 7.1 Hz, 3H), 1.49-1.37 (m, 2H), 0.98 (t, = 9.5 Hz, 3H); MS (FAB) m/z 492 (M+H). Anal.
Calcd for C21H25F4N3O4S: C, 51.32; H, 5.13; N, 8.55. Found: C, 51.49; H, 5.11; N, 8.52.
δ 7.57 (d, J = 7.3 Hz, 1H),
J
J
J
J
J
4.1.3.5.
N-((2-Pentyloxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (12)
Yield 71%, white solid, mp 58-60 °C; 1H NMR (CDCl3)
δ 7.57 (d, J = 7.5 Hz, 1H), 7.52 (dd, J =
8.2, 8.2 Hz, 1H), 7.19 (d,
4.29 (m, 4H), 3.51 (q, = 7.0 Hz, 1H), 3.03 (s, 3H), 1.77-1.67 (m, 2H), 1.49 (d,
1.43-1.35 (m, 4H), 0.93 (t, = 7.1 Hz, 3H); MS (FAB) m/z 506 (M+H). Anal. Calcd for
C22H27F4N3O4S: C, 52.27; H, 5.38; N, 8.31. Found: C, 52.41; H, 5.36; N, 8.28.
J
= 7.4 Hz, 1H), 7.12-7.05 (m, 2H), 6.45 (bs, NH), 5.98 (bt, NH), 4.38-
= 7.1 Hz, 3H),
J
J
J
4.1.3.6.
N-((2-Hexyloxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (13)
Yield 90%, white solid, mp 84-86 °C; 1H NMR (CDCl3)
δ 7.60 (d, J = 7.3 Hz, 1H), 7.52 (dd, J =
8.3, 8.3 Hz, 1H), 7.21 (d,
J
= 7.3 Hz, 1H), 7.12-7.06 (m, 2H), 6.50 (bs, NH), 6.00 (bt, NH), 4.37
= 7.1 Hz, 1H), 3.04 (s, 3H), 1.49 (d, = 7.0 Hz, 3H);
(d, = 6.1 Hz, 2H), 3.95 (s, 3H), 3.52 (q,
J
J
J
MS (FAB) m/z 520 (M+H). Anal. Calcd for C23H29F4N3O4S: C, 53.17; H, 5.63; N, 8.09. Found:
20

ACCEPTED MANUSCRIPT
C, 53.00; H, 5.64; N, 8.13.
4.1.3.7. (Z)-N-((2-(But-2-en-1-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (14)
°
1
Yield 72%, white solid, mp 110 C; H NMR (CDCl3)
8.3 Hz, 1H), 7.20 (d, = 7.5 Hz, 1H), 7.09 (dd, = 11.5, 1.8 Hz, 1H), 7.06 (d,
6.48 (bs, 1H), 6.01 (bt, 1H), 5.76 (m, 1H), 5.58 (m, 1H), 4.94 (d, = 6.8 Hz, 2H), 4.37 (d,
Hz, 2H), 3.50 (q, = 7.0 Hz, 1H), 3.02 (s, 3H), 1.77 (d, = 7.0 Hz, 3H), 1.48 (d, = 7 .1 Hz,
δ
7.59 (d,
J
= 8.2 Hz, 1H), 7.52 (dd,
= 9.1 Hz, 1H),
= 6.0
J =
J
J
J
J
J
J
J
J
3H); MS (FAB) m/z 490 (M+H). Anal. Calcd for C21H23F4N3O4S: C, 51.53; H, 4.74; N, 8.58.
Found: C, 51.36; H, 4.75; N, 8.61.
4.1.3.8. (Z)-N-((2-(Pent-2-en-1-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (15)
°
1
Yield 66%, white solid, mp 107 C; H NMR (CDCl3)
8.4 Hz, 1H), 7.20 (d, = 8.4 Hz, 1H), 7.09 (dd, = 11.5, 1.8 Hz, 1H), 7.06 (d,
6.49 (s, 1H), 6.01 (bt, 1 H), 5.67 (m, 1H), 5.52 (m, 1H), 4.92 (d, = 4.7 Hz, 2H), 4.37 (d,
Hz, 2H), 3.50 (q, = 6.8 Hz, 1H), 3.03 (s, 3H), 2.20 (m, 2H), 1.48 (d, = 7.1 Hz, 3H), 1.02 (t, J
δ
7.59 (d,
J
= 7.4 Hz, 1H), 7.51 (dd,
= 9.2 Hz, 1H),
= 6.4
J =
J
J
J
J
J
J
J
= 7.5 Hz, 3H); MS (FAB) m/z 504 (M+H). Anal. Calcd for C22H25F4N3O4S: C, 52.48; H, 5.00; N,
8.35. Found: C, 52.69; H, 4.99; N, 8.33.
4.1.3.9.
N-((2-(2-Methoxyethoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (16)
Yield 68%, white solid, mp 109 °C; 1H NMR (CDCl3)
δ 7.65 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 8.4
Hz, 1H), 7.23 (d,
J
= 7.2 Hz, 1H), 7.12 (m, 2H), 6.54 (bt, 1H), 6.45 (bs, 1H), 4.56 (m, 2H), 4.41(t,
J
= 3.6 Hz, 2H), 3.74 (t, = 4.5 Hz, 2H), 3.48 (q, = 7.2 Hz, 1H), 3.43 (s, 3H), 3.01 (s, 3H), 1.47
J
J
(d,
J
= 6.9 Hz, 3H); MS (FAB) m/z 494 (M+H). Anal. Calcd for C20H23F4N3O5S: C, 48.68; H,
21

ACCEPTED MANUSCRIPT
4.70; N, 8.52. Found: C, 48.49; H, 4.71; N, 8.55.
4.1.3.10.
N-((2-(2-Ethoxyethoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (17)
Yield 70% white solid, mp 118 °C; 1H NMR (CDCl3)
δ 7.63 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 8.3
Hz, 1H), 7.24 (d,
= 3.6 Hz, 2H), 3.73 (t,
(s, 3H), 3.01 (s, 3H), 1.42 (d,
J
= 7.4 Hz, 1H), 7.11 (m, 2H), 6.55 (bt, 1H), 6.46 (bs, 1H), 4.55 (m, 2H), 4.40(t,
= 4.5 Hz, 2H), 3.63 (q, = 7.2 Hz, 2H), 3.47 (q, = 7.2 Hz, 1H), 3.44
= 6.8 Hz, 3H); MS (FAB) m/z 508 (M+H). Anal. Calcd for
J
J
J
J
J
C21H25F4N3O5S: C, 49.70; H, 4.97; N, 8.28. Found: C, 49.51; H, 4.99; N, 8.31.
4.1.3.11. N-((2-(3-Methoxypropoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (18)
°
1
Yield 77%, white solid, mp 112 C; H NMR (CDCl3)
8.1 Hz, 1H), 7.20 (d, = 7.3 Hz, 1H), 7.02-7.11 (m, 2H), 6.44 (bt, 1H), 4.47-4.50 (m, 2H), 4.34
(d, = 6.0 Hz, 2H), 3.42-3.61 (m, 3H), 3.36 (s, 3H), 3.03 (s, 3H), 1.89-2.01 (m, 2H), 1.47 (d, J =
δ 7.61 (d, J = 7.3 Hz, 1H), 7.49 (dd, J =
J
J
7.1 Hz, 3H); MS (FAB) m/z 508 (M+H). Anal. Calcd for C21H25F4N3O5S: C, 49.70; H, 4.97; N,
8.28. Found: C, 49.88; H, 4.96; N, 8.24.
4.1.3.12.
N-((2-(3-Ethoxypropoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (19)
°
1
Yield 60%, white solid, mp 113 C; H NMR (CDCl3)
8.3 Hz, 1H), 7.20 (d, = 7.5 Hz, 1H), 7.01-7.12 (m, 2H), 6.35 (bt, H), 4.37-4.50 (m, 2H), 4.35 (d,
= 6.0 Hz, 2H), 3.47-3.60 (m, 5H), 3.03 (s, 3H), 1.90-2.01 (m, 2H), 1.47 (d, = 7.0 Hz, 3H),
= 7.1 Hz, 3H); MS (FAB) m/z 522 (M+H). Anal. Calcd for C22H27F4N3O5S: C, 50.67;
δ 7.61 (d, J = 7.5 Hz, 1H), 7.50 (dd, J =
J
J
J
1.20 (t,
J
H, 5.22; N, 8.06. Found: C, 50.49; H, 5.24; N, 8.09.
22

ACCEPTED MANUSCRIPT
4.1.3.13.
N-((2-(2-Phenoxyethoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (20)
Yield 68%, white solid, mp 70 °C; 1H NMR (CDCl3)
δ 7.66 (d, J = 7.3 Hz, 1H), 7.41 (dd, J = 8.4,
8.4 Hz, 1H), 7.22-7.35 (m, 3H), 6.88-7.05 (m, 5H), 6.42 (bs, 1H), 6.21 (bt, 1H), 4.63-4.82 (m,
2H), 4.27-4.42 (m, 4H), 3.34 (q, = 7.1 Hz, 1H), 2.99 (s, 3H), 1.38 (d, = 7.0 Hz, 3H); MS
J
J
(FAB) m/z 556 (M+H). Anal. Calcd for C25H25F4N3O5S: C, 54.05; H, 4.54; N, 7.56. Found: C,
54.23; H, 4.52; N, 7.53.
4.1.3.14.
N-((2-Isopropoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (21)
Yield 81%, white solid, mp 66-68 °C; 1H NMR (CDCl3)
δ 7.56 (d, J = 7.5 Hz, 1H), 7.50 (dd, J =
8.1 Hz, 1H), 7.16 (d,
J
= 7.5 Hz, 1H), 7.12-7.05 (m, 2H), 6.70 (bs, NH), 6.05 (bt, NH), 5.36 (m,
= 7.1 Hz, 3H), 1.30 (t, 6H); MS (FAB) m/z
1H), 3.53 (q, = 7.1 Hz, 1H), 3.03 (s, 3H), 1.49 (d,
J
J
478 (M+H). Anal. Calcd for C20H23F4N3O4S: C, 50.31; H, 4.86; N, 8.80. Found: C, 50.49; H,
4.84; N, 8.77.
4.1.3.15.
N-((2-Isobutoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (22)
Yield 84%, white solid, mp 62-64 °C; 1H NMR (CDCl3)
δ 7.60 (d, J = 7.3 Hz, 1H), 7.52 (dd, J =
8.3, 8.3 Hz, 1H), 7.21 (d,
J
= 7.3 Hz, 1H), 7.12-7.06 (m, 2H), 6.50 (bs, NH), 6.00 (bt, NH), 4.37
= 7.1 Hz, 1H), 3.04 (s, 3H), 1.49 (d, = 7.0 Hz, 3H);
(d, = 6.1 Hz, 2H), 3.95 (s, 3H), 3.52 (q,
J
J
J
MS (FAB) m/z 492 (M+H). Anal. Calcd for C21H25F4N3O4S: C, 51.32; H, 5.13; N, 8.55. Found:
C, 51.15; H, 5.15; N, 8.58.
4.1.3.16.
N-((2-Isopentyloxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (23)
23

ACCEPTED MANUSCRIPT
°
1
Yield 68%, white solid, mp 136-139 C; H NMR (CD3OD)
δ
7.40-7.49 (m, 2H), 7.14-7.23 (m,
= 7.5 Hz, 2H), 3.71 (q, = 6.9 Hz, 1H), 2.97 (s, 3H),
= 6.9 Hz, 3H), 0.95 (d, = 6.6 Hz, 6H); MS
3H), 4.38 (t,
J
= 7.2 Hz, 2H), 4.31 (d,
J
J
1.76 (m, 1H), 1.64 (q,
J
= 6.6 Hz, 2H), 1.45 (d,
J
J
(FAB) m/z 506 (M+H). Anal. Calcd for C22H27F4N3O4S: C, 52.27; H, 5.38; N, 8.31. Found: C,
52.06; H, 5.40; N, 8.35.
4.1.3.17. N-((2-(3,3-Dimethylbutoxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (24)
Yield 70%, white solid, mp 64-66 °C; 1H NMR (CD3OD)
4.42 (t, = 7.5 Hz, 2H), 4.31 (d, = 7.2 Hz, 2H), 3.72 (q,
= 7.2 Hz, 2H), 1.45 (d,
C23H29F4N3O4S: C, 53.17; H, 5.63; N, 8.09. Found: C, 53.30; H, 5.62; N, 8.06.
δ
7.40-7.48 (m, 2H), 7.13-7.22 (m, 3H),
J
J
J
= 6.9 Hz, 1H), 2.97 (s, 3H), 1.68 (t,
J
J
= 6.9 Hz, 3H), 0.97 (s, 9H); MS (FAB) m/z 520 (M+H). Anal. Calcd for
4.1.3.18.
N-((2-(Pentan-3-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (25)
Yield 65%, colorless oil; 1H NMR (CDCl3)
δ
7.55 (d,
J
= 7.5 Hz, 1H), 7.49 (t,
= 6.9 Hz, 1H), 3.01
= 6.9 Hz, 3H), 0.90 (m, 6H); MS (FAB) m/z 506 (M+H). Anal.
Calcd for C22H27F4N3O4S: C, 52.27; H, 5.38; N, 8.31. Found: C, 52.09; H, 5.40; N, 8.34.
J = 8.4 Hz, 1H),
7.12 (m, 3H), 6.25 (t,
J = 5.7 Hz, 1H), 5.20 (m, 1H), 4.42 (m, 2H), 3.58 (q, J
(s, 3H), 1.70 (m, 4H), 1.49 (d,
J
4.1.3.19.
N-((2-(Heptan-4-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (26)
Yield 68%, colorless oil; 1H NMR (CDCl3)
δ
7.56 (d,
J
= 7.4 Hz, 1H), 7.48 (t,
= 6.8 Hz, 1H), 3.02
= 6.9 Hz, 3H), 1.25 (m, 4H), 0.92 (m, 6H); MS (FAB) m/z 534
J = 8.5 Hz, 1H),
7.11 (m, 3H), 6.24 (t,
J = 5.8 Hz, 1H), 5.21 (m, 1H), 4.43 (m, 2H), 3.59 (q, J
(s, 3H), 1.71 (m, 4H), 1.48 (d,
J
(M+H). Anal. Calcd for C24H31F4N3O4S: C, 54.02; H, 5.86; N, 7.88. Found: C, 54.22; H, 5.85; N,
24

ACCEPTED MANUSCRIPT
7.85.
4.1.3.20.
N-((2-(Nonan-5-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (27)
Yield 57%, pale yellow oil; 1H NMR (CDCl3)
δ
7.49-7.56 (m, 2H), 7.15 (d,
J
= 7.7 Hz, 1H), 7.08
= 7.0 Hz,
= 7.0 Hz, 3H), 1.24-1.31 (m, 8H), 0.88-0.90 (m, 6H);
(t,
J = 5.9 Hz, 1H), 6.47 (bs, 1H), 5.98 (bt, 1H), 5.29 (m, 1H), 4.37 (m, 2H), 3.49 (q, J
1H), 3.03 (s, 3H), 1.57 (m, 2H), 1.49 (d,
J
MS (FAB) m/z 562 (M+H). Anal. Calcd for C26H35F4N3O4S: C, 55.60; H, 6.28; N, 7.48. Found:
C, 55.78; H, 6.27; N, 7.45.
4.1.3.21. N-((2-(2,6-Dimethylheptan-4-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (28)
Yield 67%, colorless oil; 1H NMR (CDCl3)
7.12 (m, 3H), 6.25 (t, = 5.8 Hz, 1H), 5.22 (m, 1H), 4.44 (m, 2H), 3.57 (q,
(s, 3H), 1.71 (m, 4H), 1.49 (d, = 6.8 Hz, 3H), 1.24 (m, 2H), 0.92 (m, 12H); MS (FAB) m/z 562
δ
7.57 (d,
J
= 7.5 Hz, 1H), 7.49 (t,
J = 8.6 Hz, 1H),
J
J
= 6.9 Hz, 1H), 3.03
J
(M+H). Anal. Calcd for C26H35F4N3O4S: C, 55.60; H, 6.28; N, 7.48. Found: C, 55.78; H, 6.26; N,
7.44.
4.1.3.22.
N-((2-Cyclobutoxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (29)
Yield 80%, white solid, mp 76-78 °C; 1H NMR (CDCl3)
δ
7.58-7.51 (m, 2H), 7.18 (d,
= 6.2 Hz, 2H),
= 7.0 Hz, 1H), 3.03 (s, 3H), 2.50-2.40 (m, 2H), 2.05-1.65 (m, 4H), 1.50 (d, = 7.1 Hz,
J = 7.3 Hz,
1H), 7.13-7.07 (m, 2H), 6.50 (bs, NH), 6.00 (bt, NH), 5.20 (m, 1H), 4.37 (d,
3.56 (q,
J
J
J
3H); MS (FAB) m/z 490 (M+H). Anal. Calcd for C21H23F4N3O4S: C, 51.53; H, 4.74; N, 8.58.
Found: C, 51.35; H, 4.76; N, 8.61.
25

ACCEPTED MANUSCRIPT
4.1.3.23.
N-((2-Cyclopentyloxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (30)
Yield 79%, white solid, mp 67-69 °C; 1H NMR (CDCl3)
δ 7.56 (d, J = 7.5 Hz, 1H), 7.50 (dd, J =
8.1, 8.1 Hz, 1H), 7.16 (d,
J
= 7.5 Hz, 1H). 7.12-7.05 (m, 2H), 6.70 (bs, NH), 6.05 (bt, NH), 5.36
= 7.1 Hz, 3H), 1.30 (t, 6H); MS (FAB)
(m, 1H), 3.53 (q, = 7.1 Hz, 1H), 3.03 (s, 3H), 1.49 (d,
J
J
m/z 504 (M+H). Anal. Calcd for C22H25F4N3O4S: C, 52.48; H, 5.00; N, 8.35. Found: C, 52.67; H,
4.98; N, 8.33.
4.1.3.24.
N-((2-Cyclohexyloxy-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (31)
Yield 80%, white solid, mp 81-83 °C; 1H NMR (CDCl3)
δ 7.56 (d, J = 7.5 Hz, 1H), 7.50 (dd, J =
8.1 Hz, 1H), 7.16 (d,
J
= 7.5 Hz, 1H), 7.12-7.05 (m, 2H), 6.70 (bs, NH), 6.05 (bt, NH), 5.36 (m,
= 7.1 Hz, 3H), 1.30 (t, 6H); MS (FAB) m/z
1H), 3.53 (q, = 7.1 Hz, 1H), 3.03 (s, 3H), 1.49 (d,
J
J
518 (M+H). Anal. Calcd for C23H27F4N3O4S: C, 53.38; H, 5.26; N, 8.12. Found: C, 53.59; H,
5.23; N, 8.07.
4.1.3.25. N-((6-Trifluoromethyl-2-(4-(trifluoromethyl)cyclohexyloxy)pyridin-3-yl)methyl)-2-
(3-fluoro-4-(methylsulfonamido)phenyl)propanamide (32)
Yield 30%, white solid, mp 66-68 °C; 1H NMR (CDCl3)
8.2 Hz, 1H), 7.20 (d, = 7.5 Hz, 1H), 7.13-7.05 (m, 2H), 6.50 (bs, NH), 5.91 (bt, NH), 5.43 (m,
1H), 4.39 (m, 2H), 3.51 (q, = 6.6 Hz, 1H), 3.03 (s, 3H), 2.20-2.08 (m, 3H), 1.85-1.77 (m, 2H),
1.63-1.50 (m, 4H), 1.49 (d, = 7.1 Hz, 3H); MS (FAB) m/z 586 (M+H). Anal. Calcd for
C24H26F7N3O4S: C, 49.23; H, 4.48; N, 7.18. Found: C, 49.50; H, 4.51; N, 7.14.
δ 7.59 (d, J = 7.0 Hz, 1H), 7.51(dd, J =
J
J
J
4.1.3.26.
N-((2-(4-Methylcyclohexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (33)
26

ACCEPTED MANUSCRIPT
Yield 78%, white solid, mp 107-109 °C; 1H NMR (CDCl3)
δ
7.57-7.49 (m, 2H), 7.16 (d,
J
= 7.3
Hz, 1H), 7.12-7.05 (m, 2H), 6.48 (bs, NH), 5.99 (bt, NH), 5.00 (m, 1H), 4.34 (d,
J = 5.8 Hz, 2H),
3.51 (q,
1.48 (d,
J
J
= 6.8 Hz, 1H), 3.03 (s, 3H), 2.12-2.00 (m, 2H), 1.80-1.72 (m, 2H), 1.50-1.10 (m, 5H),
= 7.1 Hz, 3H), 0.94 (d, = 6.6 Hz, 3H); MS (FAB) m/z 532 (M+H). Anal. Calcd for
J
C24H29F4N3O4S: C, 54.23; H, 5.50; N, 7.91. Found: C, 54.40; H, 5.51; N, 7.95.
4.1.3.27. N-((2-(4-Ethylcyclohexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-
4-(methylsulfonamido)phenyl)propanamide (34)
Yield 65%, white solid, mp 123-125 °C; 1H NMR (CDCl3)
Hz, 1H), 7.12-7.05 (m, 2H), 6.47 (bs, NH), 5.99 (bt, NH), 5.00 (m, 1H), 4.34 (m, 2H), 3.52 (q,
= 7.5 Hz, 1H), 3.03 (s, 3H), 2.13-2.03 (m, 2H), 1.87-1.80 (m, 2H), 1.49 (d, = 7.1 Hz, 3H), 1.32-
1.04 (m, 7H), 0.91 (t, = 7.1 Hz, 3H); MS (FAB) m/z 546 (M+H). Anal. Calcd for
C25H31F4N3O4S: C, 55.04; H, 5.73; N, 7.70. Found: C, 55.20; H, 5.72; N, 7.68.
δ 7.57-7.50 (m, 2H), 7.16 (d, J = 7.3
J
J
J
4.1.3.28.
N-((2-(4-tert-Butylcyclohexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (35)
Yield 69%, white solid, mp 83-85 °C; 1H NMR (CDCl3)
δ
7.57-7.50 (m, 2H), 7.16 (d, J = 7.3 Hz,
1H), 7.12-7.05 (m, 2H), 6.46 (bs, NH), 5.98 (bt, NH), 4.96 (m, 1H), 4.34 (m, 2H), 3.51 (q,
7.1 Hz, 1H), 3.03 (s, 3H), 2.20-2.10 (m, 2H), 1.88-1.80 (m, 2H), 1.49 (d, = 7.1 Hz, 3H), 1.30-
J =
J
1.00 (m, 5H), 0.89 (s, 9H); MS (FAB) m/z 574 (M+H). Anal. Calcd for C27H35F4N3O4S: C,
56.53; H, 6.15; N, 7.33. Found: C, 56.38; H, 6.17; N, 7.35.
4.1.3.29. N-((2-(4,4-Dimethylcyclohexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (36)
°
1
Yield 63%, white solid, mp 86 C; H NMR (CDCl3)
δ 7.56-7.50 (m, 2H), 7.17 (d, J = 7.2 Hz,
1H), 7.11-7.05 (m, 2H), 6.44 (bs, NH), 5.97 (bt, NH), 4.96 (m, 1H), 4.33 (m, 2H), 3.52 (q,
J =
27

ACCEPTED MANUSCRIPT
7.2 Hz, 1H), 3.03 (s, 3H), 2.20-2.10 (m, 2H), 1.88-1.80 (m, 2H), 1.49 (d,
J = 7.1 Hz, 3H), 1.30-
1.00 (m, 4H), 0.96 (d,
J = 6.3 Hz, 6H); MS (FAB) m/z 546 (M+H). Anal. Calcd for
C25H31F4N3O4S: C, 55.04; H, 5.73; N, 7.70. Found: C, 54.90; H, 5.74; N, 7.73.
4.1.3.30. N-((2-(3,5-Dimethylcyclohexyloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (37)
°
1
Yield 66%, white solid, mp 84 C; H NMR (CDCl3)
1H), 7.10-7.06 (m, 2H), 6.42 (bs, NH), 5.98 (bt, NH), 4.95 (m, 1H), 4.34 (m, 2H), 3.53 (q,
7.2 Hz, 1H), 3.03 (s, 3H), 2.22-2.10 (m, 2H), 1.88-1.80 (m, 2H), 1.48 (d, = 7.1 Hz, 3H), 1.29
(m, 1H), 1.28-1.00 (m, 3H), 0.96 (d, = 6.3 Hz, 6H); MS (FAB) m/z 546 (M+H). Anal. Calcd for
C25H31F4N3O4S: C, 55.04; H, 5.73; N, 7.70. Found: C, 54.89; H, 5.74; N, 7.73.
δ 7.55-7.51 (m, 2H), 7.18 (d, J = 7.4 Hz,
J
=
J
J
4.1.3.31.
N-((2-(Piperidin-4-yloxy)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (38)
Yield 69%, white solid, mp 80-82 °C; 1H NMR (CDCl3)
δ
7.58 (d,
J
= 7.5 Hz, 1H), 7.41 (dd,
J
J
=
=
8.3 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.19-7.11 (m, 2H), 5.29 (m, 1H), 4.36 (m, 2H), 3.71 (q,
7.0 Hz, 1H), 3.20 (m, 2H), 3.01-2.90 (m, 2H), 2.97 (s, 3H), 2.06 (m, 2H), 1.81 (m, 2H), 1.45 (d,
= 7.1 Hz, 3H); MS (FAB) m/z 519 (M+H). Anal. Calcd for C22H26F4N4O4S: C, 50.96; H, 5.05;
J
N, 10.81. Found: C, 50.78; H, 5.07; N, 10.84.
4.1.3.32. tert-Butyl 4-(3-((2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamido)methyl)-
6-(trifluoromethyl)pyridin-2-yloxy)piperidine-1-carboxylate (39)
Yield 75%, white solid, mp 82-84 °C; 1H NMR (CDCl3)
8.2 Hz, 1H), 7.20 (d, = 7.3 Hz, 1H), 7.13-7.04 (m, 2H), 5.87 (bt, NH), 5.24 (m, 1H), 4.36 (d,
2H), 3.70-3.62 (m, 2H), 3.54 (q, = 7.7 Hz, 1H), 3.28-3.17 (m, 2H), 3.04 (s, 3H), 1.98-1.88 (m,
δ 7.60 (d, J = 7.3 Hz, 1H), 7.50 (dd, J =
J
J
2H), 1.54-1.40 (m, 2H), 1.51 (d, 3H), 1.50 (s, 9H); MS (FAB) m/z 619 (M+H). Anal. Calcd for
28