TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

Article abstract of DOI:10.1016/j.bmc.2013.08.015

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4- methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.

Full text of DOI:10.1016/j.bmc.2013.08.015

Accepted Manuscript
TRPV1 Antagonist with High Analgesic Efficacy: 2-Thio Pyridine C-region
Analogues of 2-(3-Fluoro-4-methylsulfonylaminophenyl)propanamides
Tae-Hwan Ha, HyungChul Ryu, Sung-Eun Kim, Ho Shin Kim, Jihyae Ann,
Phuong-Thao Tran, Van-Hai Hoang, Karam Son, Minghua Cui, Sun Choi, Peter
M. Blumberg, Robert Frank, Gregor Bahrenberg, Klaus Schiene, Thomas
Christoph, Sven Frormann, Jeewoo Lee
PII:
S0968-0896(13)00697-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.015
BMC 11031
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 July 2013
5 August 2013
5 August 2013
Please cite this article as: Ha, T-H., Ryu, H., Kim, S-E., Kim, H.S., Ann, J., Tran, P-T., Hoang, V-H., Son, K., Cui,
M., Choi, S., Blumberg, P.M., Frank, R., Bahrenberg, G., Schiene, K., Christoph, T., Frormann, S., Lee, J., TRPV1
Antagonist with High Analgesic Efficacy: 2-Thio Pyridine C-region Analogues of 2-(3-Fluoro-4-
methylsulfonylaminophenyl)propanamides, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/
10.1016/j.bmc.2013.08.015
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TRPV1 Antagonist with High Analgesic Efficacy:
2-Thio Pyridine C-region Analogues of 2-(3-Fluoro-4-
methylsulfonylaminophenyl)propanamides
Tae-Hwan Ha,a HyungChul Ryu,a Sung-Eun Kim,a Ho Shin Kim,a Jihyae Ann,a Phuong-Thao
Tran,a Van-Hai Hoang,a Karam Son,b Minghua Cui,b Sun Choi,b Peter M. Blumberg,c Robert
Frank,d Gregor Bahrenberg,d Klaus Schiene,d Thomas Christoph,d Sven Frormann,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 and Pharmaceutical Sciences, and Global Top5 Research Program,
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
* To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
1

Key Words: TRPV1 antagonists, analgesic, capsaicin, resiniferatoxin
Abstract
A
series
of
2-thio
pyridine
C-region
analogues
of
2-(3-fluoro-4-
methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among
them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-
induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model.
Consistent with its action in vitro being through TRPV1, compound 24S blocked capsacin-
induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was
performed to identify its binding mode.
2

1. Introduction
The transient receptor potential V1 (TRPV1) receptor is a molecular integrator of
nociceptive stimuli predominantly expressed on sensory neurons.1 It is activated by multi
ple endogenous agonists including protons,2 noxious heat,3 and inflammatory lipid mediat
ors4,5 as well as by natural products such as capsaicin (CAP)6 and resiniferatoxin (RTX)7.
The pharmacological blockade of TRPV1, by inhibiting the transmission of nociceptive s
ignaling from the periphery to the CNS, provides a promising strategy for the developme
nt of novel analgesics.8 Since the molecular cloning of TRPV1 in 1997,3 a massive effor
t to identify potent and selective TRPV1 antagonists has been made to develop clinical c
andidates as novel analgesic and antiinflammatory agents, particularly for neuropathic pain
. The clinical development and therapeutic potential of TRPV1 antagonists have been ext
ensively reviewed.9-15
Figure 1. Lead TRPV1 antagonists
Recently, we reported that compound 2 showed highly selective and potent antagonism
of TRPV1 with Ki(CAP) = 0.2 nM and IC50(pH) = 6.3 nM, being thus approximately 100-fold and
20-fold better than prototype 116 for CAP and pH antagonism, respectively (Figure 1).17 In
addition, compound 2 demonstrated strong anti-allodynic activity in a neuropathic pain model
with almost no off target effects and blocked capsacin-induced hypothermia in mice. The
structural analysis indicated that the enhanced potency of 2 was attributed to the additional 4-
methylpiperidine moiety in 2 compared to 1, which provided a new hydrophobic interaction with
3

the receptor. Indeed, the docking analysis using our hTRPV1 homology model indicated that the
4-methylpiperidinyl group in the C-region of 2 made additional hydrophobic interactions with
the hydrophobic region composed of Met514 and Leu515, an interaction not occurring with 1.18
Figure 2. General structure of the designed compounds
In order to further optimize the 2-substitutent in the N-(6-trifluoromethyl-pyridin-
3-ylmethyl) C-region, we have investigated the structure activity relationship of 2-thio der
ivatives (Figure 2) as hTRPV1 antagonists. With a selected potent antagonist in the serie
s, we further characterized target engagement by assessing its potential to inhibit capsaici
n-induced hypothermia and its anti-allodynic activity in animal models and performed mo
lecular modeling with our hTRPV1 homology model.
2. Result and discussion
2.1. Chemistry
The key intermediate of the C-region 3 was prepared as described in the previous
report.17 A library of thiols were reacted with 3 to afford 2-thio substituted pyridines 4
and then their nitrile groups were reduced to yield the corresponding primary amines 5.
The amines were coupled with racemic or chiral 2-(3-fluoro-4-methylsulfonylaminophenyl)
propanamide 6 to provide the final compounds 7-39, respectively.
4

3
3
3
2
HO
F
3
*
O
S
O
N
H
O
6
Scheme 1. Syntheses of 2-thio pyridine C-region analogues
Reagents and conditions: (a) [Method A] RSH, DBU, CH3CN, rt: [Method B] RSH, NaH, DMF, rt (b) 2M
BH3-SMe2 in THF (c) 6, EDC, HOBt, DMF
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.17 The results are summarized in Tables 1-3, together with
the potencies of the previously reported antagonists 1 and 2.16,17
To investigate the SAR for 2-thio derivatives of the pyridine C-region, we began
with the 2-alkylthio derivatives (Table 1). Previous SAR analysis with a series of 2-alk
yloxy derivatives indicated that the 2-butyloxy group was among the most optimal with l
ow lipophilicity and potent antagonism.18 The 2-butylthio derivative 7 likewise proved to
be a potent antagonist with Ki(CAP) = 0.7 nM and IC50(pH) = 40.4 nM but was nonetheless
ca. 2-fold and 5-fold less potent than the racemate of 2 (Ki(CAP) = 0.3 nM and IC50(pH)
= 8.4 nM), respectively. As the number of carbons in the chain further increased, antago
nistic activity decreased as seen with 8 and 9. The branched derivative 10 showed better
antagonism than the corresponding straight derivative 8 by 3-fold. With potent antagonis
t 7, we explored the SAR at its terminal position by introducing different types of group
5

s. The lipophilic moieties such as trifluoromethyl (11), bromo (12) and phenoxy (13) cau
sed a reduction in activity compared to 7 but still provided good antagonism. The polar
moieties such as hydroxyl (14) and dimethylamine (15) led to a further reduction in acti
vity. However, the introduction of cyclic amines such as pyrrolidine (16), piperidine (17)
and 4-methylpiperidine (18) produced reasonable antagonism in the range of Ki(CAP) = 2-
3 nM. The morpholine derivative (19) showed weak antagonism due to its polar oxygen,
as was the case with 14. The carboxylate moieties (20-22) similarly reduced the potenc
y because of their polarity. The SAR analysis in this series of 2-alkylthio groups indicate
d that the number of 4 carbons (7) appeared to be optimal for antagonism, as had been
observed for the 2-alkyloxy groups, and the incorporation of any moiety at the terminus
of the 2-alkylthio group led to a reduction in potency.
Table 1. In vitro hTRPV1 Antagonistic Activities for 2-Alkylthio Derivatives
F3C
H
N
N
F
O
O
R
N S
H
O
Ki [CAP] IC50 [pH]
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
(nM)
(nM)
1
2
7
8
28
1281
14
15
16
17
WE
WE
0.2
0.7
2.5
6.3
27.4
3.3
1498
312
40.4
95.6
3.0
250
6

9
5.6
0.7
4.3
7
719
82
18
19
20
21
22
2.9
WE
WE
48.2
85
217
NE
10
11
12
13
WE
68
WE
1099
WE
2.3
118
Next, the SAR of 2-cycloalkylthio derivatives was investigated (Table 2). 2-Cycl
opentylthio (23) and 2-cyclhexylthio (24) analogues exhibited potent antagonism with Ki(C
= 1.2 and 0.9 nM, respectively, which were 2- and 4-fold better than the correspondi
AP)
ng acyclic 2-alkylthio derivatives 8 and 9. The result was contrary to that of the 2-alkyl
oxy and 2-cycloalkyloxy derivatives and the comparison will be discussed in detail in Ta
ble 4. The two stereoisomers of 24 showed stereospecific antagonism, as previously repor
ted,19 in which eutomer 24S proved to have excellent antagonism with Ki(CAP) = 0.4 nM.
To examine the effect of substituents on potent antagonist 24, its derivatives were furth
er investigated. The sulfur oxidation of 24, providing sulfoxide 25 and sulfone 26, led to
a dramatic loss of activity. Whereas the 4-methyl group in 27 enhanced antagonistic pot
ency by 2-fold, the 2-methyl group in 28 reduced potency by 2-fold compared to 24, res
pectively. The 4-ethyl derivative 29 showed similar activity to that of the parent 24, whil
e the 4-t-butyl group caused a slight decrease of activity. The 2-cyclohexylmethylthio der
ivative 31, the one carbon elongated product of 24, was 2.5-fold less potent than 24. Th
e SAR analysis in this series of 2-cycloalkylthio groups indicated that the 2-cyclohexylthi
7

o derivative 24 appeared to be optimal for antagonism and the 4-methyl and 4-ethyl gro
ups on the ring were tolerable for potency.
Table 2. In vitro hTRPV1 Antagonistic Activities for 2-Cycloalkylthio Derivatives
Ki [CAP]
(nM)
IC50 [pH]
(nM)
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
23
24
1.2
0.9
WE
WE
WE
WE
NE
27
28
29
30
31
0.5
180
1.9
1.0
2.6
2.3
NE
WE
89.6
254
24S
24R
25
0.4
43.5
WE
WE
26
NE
Next, we sought to evaluate the SAR of 2-benzylthio and 2-arylmethylthio derivat
ives since previous SAR analysis in a series of 2-alkyloxy derivatives indicated that a 2-
benzyloxy derivative showed potent antagonism (Table 3).18 Substituted 2-benzylthio deriv
atives (32-36) exhibited similar and reasonable antagonism with a range of Ki(CAP) = 2.3-
8.7 nM but were less potent than the corresponding 2-benzyloxy surrogates. 2-Arylmethyl
thio derivatives (37, 38) showed further reduced activities. The 2-phenylethylthio derivativ
e (39) displayed good antagonism.
Table 3. In vitro hTRPV1 Antagonistic Activities for 2-Arylmethylthio Derivatives
8

Ki [CAP] IC50 [pH]
Ki [CAP] IC50 [pH]
R
R
(nM)
(nM)
(nM)
(nM)
32
33
34
35
8.7
674
36
37
38
39
3.9
71
3.2
2.3
4.9
764
279
620
14.6
15
WE
WE
111
1.9
Detailed in vitro activity of 24S, the most promising antagonist in this study, was
investigated for multiple TRPV1 activators including capsaicin, N-arachidonoyl dopamine
(NADA), pH and heat (45oC), and compared to the activity of lead 2 (Table 4). Comp
ound 24S showed excellent antagonism of capsaicin and NADA comparable to 2, but sh
owed poor antagonism toward pH and heat, in contrast to 2. The differential activity of
24S compared to 2 is consistent with the different in vivo responses described below.
Table 4. In Vitro hTRPV1 Antagonistic Activities for 2 and 24S to Multiple Activators
Activators, parameter
CAP (f)Ki (nM)
2
24S
0.4
0.2
0.01
6.3
0.8
NADA (f)Ki (nM)
pH, IC50 (nM)
heat 45oC, IC50 (nM)
0.05
>1,000
22.3
2.3. In vivo Activity
The analgesic activity of compound 24S was evaluated orally in the rat Bennett
model20 as a neuropathic pain model and its activity was compared to those of reference
9

compounds 1, 2 and pregabalin (Figure 3). Previously, we reported that antagonist 2 sh
owed excellent anti-allodynic efficacy in that model with ED50 = 0.9 mg/Kg po (max 60
% at 3.16 mg/Kg) and was better than 1 (ED50 = 26.6 mg/Kg, max 59% at 46.4 mg/Kg
) and pregabalin (ED50 = 42.2 mg/Kg, max 69% at 100 mg/Kg).17 We now find that the
analgesic potency of 24S showed dose-dependent efficacy with ED50 = 0.19 mg/Kg po
(max 75% at 10 mg/Kg) and was superior to 2.
100
*
1
75
% MPE
50
*
*
*
2
*
24S
*
*
*
Pregabalin
*
*
*
*
*
25
0
*
0.1
1
0.01
10
100
1000
Dose (mg/kg)
Figure 3. Comparison of analgesic activities of compound 24S and reference compounds on
CCI-induced cold allodynia after oral administration in rats. Data, n=10, mean SEM, * p<0.05
vs vehicle. MPE, maximal possible effect.
Consistent with its in vitro mechanism of action, in vivo 24S also blocked response t
o capsaicin (Figure 4). The intraperitoneal injection of 3 mg/kg capsaicin resulted in a d
ecrease of body temperature as expected,21,22 with a reduction from 37.6
0.1 °C (p < 0.05) at 15 min post capsaicin injection and to 35.8 0.4 °C (p < 0.05)
at 30 min post capsaicin injection. By 60 min body temperature had returned to normal
(37.1 0.1 to 37.0 0.1 °C; p = 1.00). The oral administration of 0.3 mg/kg 24S (15
0.1 to 34.0
min before capsaicin injection) completely inhibited the effect of capsaicin on body temp
erature at 30 min, with partial inhibition of the effect at 15 min.
10

(A)
40
39
38
37
36
35
34
33
C
°
m
ea
n
(+/
-
s.
e.
m.
)
bo
dy
te
m
24S 0.3 mg/kg p.o
+ capsaicin 3 mg/kg i.p. n = 5
vehicle p.o.
+ vehicle i.p. n = 5
vehicle p.o.
+ capsaicin 3 mg/kg i.p. n = 5
min
pv
15
30
60
(B)
*
100
75
% MPE
50
*
25
0
15
30
time after capsaicin (min)
Figure 4. Effect of 24S on capsaicin-induced hypothermia in mice. Data, n=5, mean SEM, * p
< 0.05 vs vehicle. MPE, maximal possible effect of compound.
2.4. Molecular Modeling
In order to investigate the binding interactions of 24S, a flexible docking study was
performed using our human TRPV1 (hTRPV1) model17 built based on our rat TRPV1 (rTRPV1)
model23. The docking result demonstrated that 24S binds very well at the capsaicin binding site,
as we had previously described for 2.17 The 4-(methylsulfonylamino)phenyl group (A-region)
11

occupied the deep bottom hole and was involved in hydrophobic interactions with Tyr511 and
Ile564 (Figure 5). The fluorine atom of the A-region participated in hydrogen bonding with
Lys571. The amide group (B-region) made a hydrogen bond with Tyr511 and also contributed to
the appropriate positioning of the C-region for hydrophobic interactions.
In the C-region, the cyclohexylthio group initially was expected to orient toward the
hydrophobic region composed of Met514 and Leu515 as did the 4-methylpiperidinyl group of
2.17 However, the modeling indicated that the cyclohexyl ring was extended toward the upper
hydrophobic pocket composed of Leu547 and Thr550 to generate the hydrophobic interaction,
and the sulfur atom made the hydrophobic interaction with Leu515. It seems that the bent
conformation of the thioether linker allowed the cyclohexylthio group to make hydrophobic
interactions with both hydrophobic regions. In addition, the pyridine ring nitrogen in the C-
region made a hydrogen bond with Tyr511.
(A)
(B)
(C)
LP
Phe543
0.14
0.12
0.09
0.07
0.05
0.03
0.01
Leu547
Thr550
-0.02
-0.04
-0.06
Leu515
-0.08
-0.11
-0.13
Tyr511
Lys571
Ile564
-0.15
-0.17
-0.19
Figure 5. Flexible docking result of 24S in the hTRPV1 model.
(A) Binding mode of 24S. The key interacting 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 depicted in line ribbon. 24S is displayed 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
12

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 dar Waals surface of the ligand colored by its lipophilic potential
property.
3. Conclusion
The structure activity relationships of 2-thio pyridine C-region derivatives of 2-(3-
fluoro-4-methylsulfonylaminophenyl)propanamides as hTRPV1 antagonists were investigate
d. The analysis indicated that 2-cycloalkylthio derivatives showed better antagonism than
the corresponding 2-alkylthio ones, in contrast to the findings for a series of 2-alkyloxy
derivatives. Among the 2-thio pyridine derivatives, compound 24S showed stereospecific
and excellent TRPV1 antagonism with Ki(CAP) = 0.4 nM. It demonstrated dose-dependent
and strong anti-allodynic activity with ED50 = 0.19 mg/Kg po (max 75% at 10 mg/Kg) i
n a neuropathic pain model in the rat and was superior to previous lead 2. Consistent w
ith its action in vitro being through TRPV1, compound 24S blocked capsacin-induced hy
pothermia. Docking analysis of 24S with our hTRPV1 homology model indicated that the
2-(3-fluoro-4-methylsulfonylaminophenyl)propanamide (A and B-regions) made critical hy
drophobic and hydrogen bonding interactions with Tyr511, and the cyclohexyl group and
the sulfur atom in the C-region made two hydrophobic interactions with the pocket com
posed of Leu547/Thr550 and Leu515, respectively.
4. Experimental
4.1. Chemistry
4.1.1. General
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 spectra were
13

recorded on a JEOL JNM-LA 300 and Bruker Avance 400 MHz FT-NMR 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. 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 a Agilent
Eclipse Plus C18 column (4.6 x 250 mm, 5 µm) and Daicel Chiralcel OD-H column (4.6 x 250
mm, 5 µm). Optical rotations were measured in a JASCO DIP-2000 digital polarimeter.
4.1.2. General Procedure for the Synthesis of 4
4.1.2.1. Method A
A mixture of compound 3 (2 mmol), thiol (4 mmol) and DBU (4 mmol) were dissolved in
acetonitrile (15 mL) and stirred for 12 h at room temperature. The reaction mixture was diluted
with water and extracted with EtOAc several times. 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: hexanes =1:15 as eluant.
4.1.2.2. Method B
A solution of thiol (2.4 mmol) in DMF (5 mL) was treated with sodium hydride (2.6 mmol). The
o
resulting suspension was cooled to 0 C and compound 3 (2 mmol) was added. After stirring
overnight at room temperature, the reaction mixture was diluted with water and extracted with
EtOAc several times. 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: hexanes =1:15 as eluant.
4.1.3. General Procedure for the Synthesis of 5
14

To a stirred solution of nitrile (2 mmol) in anhydrous THF (10 mL) was added BH3-SMe2 (2.0 M
in THF, 3 mL, 6 mmol) at room temperature. After being refluxed for 8 hrs, the mixture was
cooled to room temperature, 2 N HCl was added, and the solution then refluxed for 30 min. After
cooling to room temperature, the mixture was neutralized with 2 N NaOH 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 CH2Cl2:MeOH=10:1 as eluant.
4.1.4. Generel Procedure for the Syntheses of 7-39
A mixture of amine 5 (1.2 mmol), acid 6 (1.0 mmol) and 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (1.2 mmol) in DMF (5 mL) was stirred for 12 h at room temperature.
The reaction mixture was diluted with water and extracted with EtOAc several times. The
aqueous phase was saturated with NaCl and extracted with EtOAc again. The combined organic
extracts were washed with 1N HCl and brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography on silica gel using
EtOAc:hexanes=1:2 as eluant.
4.1.4.1.
2-(3-Fluoro-4-methanesulfonylamino-phenyl)-(N-(2-(butylthio)-6-
(trifluoromethyl)pyridin-3-ylmethyl)propionamide (7)
o
1
71% yield, white solid, mp = 105-107 C, H NMR (300 MHz, CDCl3) δ 7.50-7.42 (m, 2 H),
7.27 (s, 1 H), 7.13 (dd, 1 H, J = 8.1 Hz), 7.07 (d, 1 H, J = 8.1 Hz), 6.98 (bs 1 H), 6.33 (bt, 1 H),
4.36 (m, 2 H), 3.56 (q, 1 H, J = 6.9 Hz), 3.22 (t, 2 H, J = 7.5 Hz), 3.01 (s, 3 H), 1.66 (m, 2 H),
1.50-1.38 (m, 5 H), 0.93 (t, 3 H, J = 7.2 Hz); MS (FAB) m/z 508 (M+H).
4.1.4.2.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(pentylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (8)
15

o
1
65% yield, white solid, mp = 126-129 C, H NMR (300 MHz, CDCl3) δ 7.52-7.42 (m, 2 H),
7.27 (s, 1 H), 7.13 (dd, 1 H, J = 8.0 Hz), 7.07 (d, 1 H, J = 8.2 Hz), 6.98 (bs 1 H), 6.33 (bt, 1 H),
4.36 (m, 2 H), 3.58 (q, 1 H, J = 6.9 Hz), 3.25 (t, 2 H, J = 7.5 Hz), 3.05 (s, 3 H), 1.66 (m, 2 H),
1.58-1.48 (m, 5 H), 1.23-1.09 (m, 2 H), 0.90 (t, 3 H, J = 7.1 Hz); MS (FAB) m/z 522 (M+H).
4.1.4.3.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(hexylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (9)
o
1
72% yield, white solid, mp = 102-105 C, H NMR (300 MHz, CDCl3) δ 7.51-7.40 (m, 2 H),
7.24 (s, 1 H), 7.11 (dd, 1 H, J = 8.1 Hz), 7.05 (d, 1 H, J = 8.0 Hz), 6.94 (bs 1 H), 6.31 (bt, 1 H),
4.31 (m, 2 H), 3.58 (q, 1 H, J = 6.7 Hz), 3.24 (t, 2 H, J = 7.3 Hz), 3.03 (s, 3 H), 1.66 (m, 2 H),
1.58-1.48 (m, 5 H), 1.24-1.02 (m, 4 H), 0.91 (t, 3 H, J = 7.2 Hz); MS (FAB) m/z 536 (M+H).
4.1.4.4.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(isopentylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (10)
o
1
81% yield, white solid, mp = 123-126 C, H NMR (300 MHz, CDCl3) δ 7.50-7.41 (m, 2 H),
7.28 (s, 1 H), 7.12 (dd, 1 H, J = 8.1 Hz), 7.07 (d, 1 H, J = 8.1 Hz), 6.98 (bs 1 H), 6.33 (bt, 1 H),
4.36 (m, 2 H), 3.56 (q, 1 H, J = 6.9 Hz), 3.22 (t, 2 H, J = 7.5 Hz), 3.01 (s, 3 H), 1.93-1.83 (m, 1
H), 1.66 (m, 2 H), 1.18 (d, 3 H, J = 7.2 Hz), 0.96 (d, 6 H, J = 7.1 Hz); MS (FAB) m/z 522 (M+H).
4.1.4.5.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(2,2,2-trifluoroethylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (11)
o
1
75% yield, white solid, mp = 164-166 C, H NMR (300 MHz, CDCl3) δ 7.55-7.42 (m, 2 H),
7.32 (s, 1 H), 7.16 (dd, 1 H, J = 8.0 Hz), 7.07 (d, 1 H, J = 8.1 Hz), 6.96 (bs 1 H), 6.32 (bt, 1 H),
4.36 (m, 2 H), 3.59 (q, 1 H, J = 6.9 Hz), 3.45 (s, 2 H), 3.00 (s, 3 H); MS (FAB) m/z 534 (M+H).
4.1.4.6. N-((2-(3-Bromopropylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
16

(methylsulfonamido)phenyl)propanamide (12)
o
1
40% yield, white solid, mp = 100-103 C, H NMR (300 MHz, CDCl3) δ 7.48-7.56 (m, 2 H),
7.32 (d, 1 H, J = 7.8 Hz), 7.15-7.07 (m, 2 H), 6.62 (bs, 1 H), 6.03 (bt, 1 H), 4.38 (d, 2 H, J = 5.7
Hz), 3.60-3.49 (m, 3 H), 3.37 (t, 2 H, J = 6.9 Hz), 3.03 (s, 3 H), 2.26 (m, 2 H), 1.50 (d, 3 H, J =
6.9 Hz); MS (FAB) m/z 573 (M+H).
4.1.4.7.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-hydroxypropylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (13)
65% yield, white solid, mp = 93-95 oC, 1H NMR (300 MHz, CDCl3) δ 7.57 (dd, 1 H, J = 7.8 and
7.8 Hz), 7.49 (d, 1 H, J = 7.8 Hz), 7.33 (d, 1 H, J = 7.8 Hz), 7.06-7.12 (m, 2 H), 6.61 (bs, 1 H),
5.97 (bt, 1 H), 4.39 (d, 2 H, J = 5.7 Hz), 3,70 (m, 2 H), 3.54 (q, 1 H, J = 6.9 Hz), 3.36 (m, 2 H),
3.03 (s, 3 H), 1.96 (m, 2 H), 1.50 (d, 3 H, J = 6.9 Hz); MS (FAB) m/z 510 (M+H).
4.1.4.8. N-((2-(3-(Dimethylamino)propylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (14)
o
1
33% yield, white solid, mp = 130-133 C, H NMR (300 MHz, CDCl3) δ 7.58-7.49 (m, 2 H),
7.33 (d, 1 H, J = 6 Hz), 7.15-7.08 (m, 2H), 5.99 (m, 1 H), 4.40-4.37 (m, 2 H), 3.56 (q, 1 H, J = 6
Hz), 3.21-3.17 (m, 2 H), 3.03 (s, 3 H), 2.90-2.84 (m, 2 H), 2.57 (s, 6 H), 2.22-2.14 (m, 2 H), 1.50
(d, 3 H, J = 6.0 Hz); MS (FAB) m/z 537 (M+H).
4.1.4.9. 2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-(pyrrolidin-1-yl)propylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (15)
o
1
78% yield, white solid, mp = 139-141 C, H NMR (300 MHz, CDCl3) δ 7.49-7.35 (m, 3 H),
7.23-7.12 (m, 2 H), 4.39-4.25 (m, 2 H), 3.74 ( q, 1 H, J = 6.0 Hz), 3.30 (m, 1 H), 3.21 (t, 1 H, J =
6.0 Hz), 3.10-3.03 (m, 1 H), 2.98 (s, 3 H), 2.89-2.84 (m, 1 H), 2.80 (s, 3 H), 2.25-2.14 (m, 1 H),
2.09-2.00 (m, 1 H), 1.94-1.85 (m, 1 H), 1.46 (d, 3 H, J = 6.0 Hz); MS (FAB) m/z 563 (M+H).
17

4.1.4.10. 2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-(piperidin-1-yl)propylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (16)
o
1
76% yield, white solid, mp = 137-139 C, H NMR (300 MHz, CDCl3) δ 7.57 (d, 1 H, J = 7.7
Hz), 7.45 (t, 1 H, J = 8.0 Hz), 7.28-7.26 (m, 2 H), 7.07 (d, 1 H, J = 9.18 Hz), 6.87 (br s, 1 H),
4.43-4.25 (m, 2 H), 3.69 (q, 1 H), 3.15 (t, 2 H, J = 7.14 Hz), 2.99 (s, 3 H), 2.83-2.77 (m, 4 H),
2.05-1.95 (m, 2 H), 1.85 (t, 3 H, J = 5.31 Hz), 1.57 (br s, 1 H), 1.48 (d, 2 H, J = 7.14 Hz), 1.26 (s,
3 H), 0.87 (m, 1 H); MS (FAB) m/z 577 (M+H).
4.1.4.11.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-(4-methylpiperidin-1-
yl)propylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)propanamide (17)
o
82% yield, white solid, mp = 75-78 C, 1H NMR (300 MHz, CDCl3) δ 7.64-7.45 (m, 2 H), 7.32
(d, 1 H, J = 9.0 Hz), 7.16-7.09 (m, 2 H), 4.44-4.34 (m, 2 H), 3.56 (q, 1 H, J = 9.0 Hz), 3.25-3.17
(m, 2 H), 3.05-2.84 (m, 9 H), 2.17-2.07 (m, 2 H), 0.97 (d, 3 H, J = 6.0 Hz); MS (FAB) m/z 591
(M+H).
4.1.4.12.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-morpholinopropylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (18)
o
81% yield, white solid, mp = 80-85 C, 1H NMR (300 MHz, CDCl3) δ 7.59-7.48 (m, 2 H), 7.30
(d, 1 H, J = 7.68 Hz), 7.07-7.00 (m, 2 H), 5.97 (t, 1 H), 4.44-4.28 (m, 2 H), 3.75 (t, 6 H, J = 4.38
Hz), 3.55 (q, 1 H), 3.27-3.13 (m, 2H), 3.01 (s, 3 H), 2.47-2.42 (m, 6 H), 1.82-1.74 (m, 2 H),
1.51-1.48 (d, 4 H, J = 6.96 Hz); MS (FAB) m/z 579 (M+H).
4.1.4.13.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(2-phenoxyethylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (19)
69% yield, white solid, mp = 88-90 oC, 1H NMR (300 MHz, CDCl3) δ 7.58-7.45 (m, 2 H), 7.38-
18

7.26 (m, 4 H), 7.14-7.04 (m, 2 H), 6.98-6.92 (m, 3 H), 6.10-6.08 (m, 1 H), 4.46-4.30 (m, 1 H),
4.26 (t, 2 H, J = 6.39 Hz), 3.63 (t, 2 H, J = 6.39 Hz), 3.53 (q, 1 H, J = 6.0 Hz), 3.00 (s, 3 H), 1.48
(d, 3 H, J = 6.0 Hz); MS (FAB) m/z 572 (M+H).
4.1.4.14.
3-(3-((2-(3-Fluoro-4-(methylsulfonamido)phenyl)propanamido)methyl)-6-
(trifluoromethyl)pyridin-2-ylthio)propanoic acid (20)
o
1
60% yield, yellow solid, mp = 135-137 C, H NMR (300 MHz, CDCl3) δ 11.9 (s, 1 H), 7.56-
7.55 (m, 3 H), 7.30 (d, 1 H, J = 7.8 Hz), 7.34 (s, 1 H), 7.13-7.09 (m, 2 H), 6.72 (bs, 1 H), 6.11 (bt,
1 H), 4.32 (m, 2 H), 3.53 (q, 1 H, J = 6.9 Hz), 3.46 (t, 2 H, J = 6.6 Hz), 3.02 (s, 3 H), 2.81 (t, 2 H,
J = 6.7 Hz), 1.53 (d, 3 H, J = 6.9 Hz); MS (FAB) m/z 524 (M+H).
4.1.4.15. Methyl 3-(3-((2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamido)methyl)-6-(trifluoromethyl)pyridin-2-
ylthio)propanoate (21)
o
1
68% yield, white solid, mp = 117-120 C, H NMR (300 MHz, CDCl3) δ 7.57-7.46 (m, 3 H),
7.31 (d, 1 H, J = 7.8 Hz), 7.34 (s, 1 H), 7.14-7.01 (m, 2 H), 6.74 (bs, 1 H), 6.15 (bt, 1 H), 4.35 (m,
2 H), 3,70 (s, 3 H), 3.55 (q, 1 H, J = 6.9 Hz), 3.47 (t, 2 H, J = 6.6 Hz), 3.02 (s, 3 H), 2.78 (t, 2 H,
J = 6.6 Hz), 1.50 (d, 3 H, J = 6.9 Hz); MS (FAB) m/z 538 (M+H).
4.1.4.16.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(3-oxo-3-(pyrrolidin-1-
yl)propylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)propanamide (22)
42% yield, pale yellow solid, mp = 94-96 oC, 1H NMR (300 MHz, CDCl3) δ 7.56-7.55 (m, 3 H),
7.30 (d, 1 H, J = 7.8 Hz), 7.34 (s, 1 H), 7.13-7.09 (m, 2 H), 6.72 (bs, 1 H), 6.11 (bt, 1 H), 4.32 (m,
2 H), 3.53 (q, 1 H, J = 6.9 Hz), 3.49 (t, 4 H, J = 7.9 Hz), 3.36 (t, 2 H, J = 6.7 Hz), 3.04 (s, 3 H),
2.80 (t, 2 H, J = 6.7 Hz), 1.87-1.82 (m, 4 H), 1.54 (d, 3 H, J = 6.9 Hz); MS (FAB) m/z 573
(M+H).
19

4.1.4.17.
N-((2-(Cyclopentylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (23)
o
1
59% yield, yellow solid, mp = 126-129 C, H NMR (300 MHz, CDCl3) δ 7.50 (t, 2 H, J = 9.0
Hz), 7.27 (d, 1 H, J = 9.0 Hz), 7.14-7.06 (m, 2 H), 6.59 (s, 1 H), 6.03 (m, 1 H), 4.42-4.29 (m, 2
H), 4.16-4.07 (m, 1 H), 3.55 (q, 1 H, J = 9.0 Hz), 3.02 (s, 2 H), 2.27-2.17 (m, 4 H), 1.75-1.48 (m,
7 H); MS (FAB) m/z 520 (M+H).
4.1.4.18.
N-((2-(Cyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (24)
65% yield, white solid, mp =70-73 oC, 1H NMR (300 MHz, CD3OD) δ 7.48-7.35 (m, 3 H), 7.26
(d, 1 H, J = 7.8 Hz), 7.16 (dd, 1 H, J = 1.8 and 11.1 Hz), 7.10 (d, 1 H, J = 8.4 Hz), 6.13 (bs, 1 H),
4.35 (d, 2 H, J = 5.7 Hz), 3.82 (m, 1 H), 3.56 (q, 1 H, J = 7.2 Hz), 3.02 (s, 3 H), 2.06 (m, 2 H),
1.75 (m, 2 H), 1.49 (d, 3 H, J = 7.2 Hz), 1.33-1.26 (m, 6 H); MS (FAB) m/z 534 (M+H).
4.1.4.19. (S)-N-((2-(Cyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (24S).
[α]D25 = +18.2 (c 0.1, MeOH), 99% ee, The specta are identical to those of compound 24.
4.1.4.20. (R)-N-((2-(Cyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (24R).
[α]D25 = -18.5 (c 0.1, MeOH), 98.5% ee, The specta are identical to those of compound 24.
4.1.4.21. N-((2-(Cyclohexylsulfinyl)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (25)
1
78% yield, white solid, mp = 68-70 oC, H NMR (300 MHz, DMSO) δ 9.51 (s, 1 H), 8.61 (d, 1
20

H), 8.05 (d, 1 H), 8.00 (m, 1 H), 7.31 (m, 1 H), 7.22 (m, 1 H), 7.15 (m, 1 H), 4.74 (d, 2 H), 3.78
(m, 2 H), 3.02 (s, 3 H), 1.93 (brs, 2 H), 1.81 (d, 2 H), 1.65 (m, 2 H), 1.47 (m, 2 H), 1.31-1.21 (m,
5 H); MS (FAB) m/z 520 (M+H).
4.1.4.22. N-((2-(Cyclohexylsulfonyl)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (26)
1
67% yield, white solid, mp = 68-71 oC, H NMR (300 MHz, DMSO) δ 9.52 (s, 1 H), 8.63 (d, 1
H), 8.15 (d, 1 H), 8.00 (m, 1 H), 7.34 (m, 1 H), 7.24 (m, 1 H), 7.15 (m, 1 H), 4.63 (d, 2 H), 3.71
(m, 2 H), 3.01 (s, 3 H), 1.81-1.69 (m, 4 H), 1.59-1.50 (m, 4 H), 1.37 (d, 3 H), 1.25-1.15 (m, 4 H);
MS (FAB) m/z 566 (M+H)
4.1.4.23.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-methylcyclohexylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (27)
1
66% yield, white solid, mp = 70-75 oC, H NMR (300 MHz, DMSO) δ 9.52 (s, 1 H), 8.59 (d, 1
H), 7.51 (d, 1 H), 7.35 (m, 1 H), 7.25 (m, 1 H), 7.15 (m, 1 H), 4.25 (d, 2 H), 4.00 (m, 2 H), 3.75
(m, 2 H), 3.01 (s, 3 H), 1.85 (s, 2 H), 1.60-1.48 (m, 4 H), 1.28-1.18 (m, 3 H), 0.91 (d, 3 H); MS
(FAB) m/z 548 (M+H).
4.1.4.24.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(2-methylcyclohexylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (28)
1
65% yield, white solid, mp = 75-78 oC, H NMR (300 MHz, DMSO) δ 9.52 (s, 1 H), 8.59 (d, 1
H), 7.51 (d, 1 H), 7.35 (m, 1 H), 7.25 (m, 1 H), 7.15 (m, 1 H), 4.31 (d, 2 H), 4.25 (m, 2 H), 3.75
(m, 2 H), 3.01 (s, 3 H), 1.80 (s, 2 H), 1.60-1.48 (m, 4 H), 1.40-1.37 (m, 3 H), 0.91 (d, 3 H); MS
(FAB) m/z 548 (M+H).
4.1.4.25. N-((2-(4-Ethylcyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-
21

4-(methylsulfonamido)phenyl)propanamide (29)
o
1
77% yield, white solid, mp = 73-76 C, H NMR (300 MHz, DMSO) δ 9.52 (s, 1 H), 8.52 (t, 1
H), 7.51 (d, 1 H), 7.35 (t, 1 H), 7.25 (d, 1 H), 7.15 (d, 1 H), 4.18 (d, 2 H), 3.75 (m, 2 H), 3.01 (s,
3 H), 2.04 (m, 2 H), 1.75 (m, 3 H), 1.25-1.00 (m, 4 H), 0.91 (d, 5 H); MS (FAB) m/z 562 (M+H).
4.1.4.26.
N-((2-(4-tert-Butylcyclohexylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (30)
o
1
74% yield, white solid, mp = 86-90 C, H NMR (300 MHz, DMSO) δ 9.52 (s, 1 H), 8.52 (t, 1
H), 7.51 (d, 1 H), 7.35 (t, 1 H), 7.25 (d, 1 H), 7.15 (d, 1 H), 4.17 (d, 2 H), 3.75 (m, 2 H), 3.01 (s,
3 H), 2.14 (m, 2 H), 1.78 (m, 3 H), 1.41 (s, 2 H), 1.25-1.00 (m, 4 H), 0.75 (d, 9 H); MS (FAB)
m/z 590 (M+H).
4.1.4.27. N-((2-(Cyclohexylmethylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-
4-(methylsulfonamido)phenyl)propanamide (31)
o
1
75% yield, white solid, mp = 130-132 C, H NMR (300 MHz, CDCl3) δ 7.54-7.49 (m, 2 H),
7.28 (d, 1 H, J = 7.8 Hz), 7.15-7.07 (m, 2 H), 6.50 (bs, 1 H), 6.00 (bt, 1 H), 4.40 (m, 2 H), 3.55
(q, 1 H, J = 6.9 Hz), 3.14 (d, 2 H, J = 6.6 Hz), 3.03 (s, 3 H), 1.85 (m, 2 H), 1.70-1.53 (m, 4 H),
1.49 (d, 3 H, J = 6.9 Hz), 0.95-1.28 (m, 5 H); MS (FAB) m/z 548 (M+H).
4.1.4.28. N-((2-(4-Chlorobenzylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (32)
72% yield, white solid, mp = 96-98 oC, 1H NMR (300 MHz, CDCl3) δ 7.55-7.48 (m, 2 H), 7.36-
7.31 (m, 3 H), 7.24-7.23 (m, 1 H), 7.13-7.04 (m, 2 H), 6.47 (s, 1 H), 5.88 (m, 1 H), 4.42 (s, 2 H),
4.34 (t, 2 H, J=6.0 Hz), 3.49 (q, 1 H, J = 7.2 Hz), 3.02 (s, 3 H), 1.47 (d, 3 H, J = 7.2 Hz); MS
(FAB) m/z 576 (M+H).
22

4.1.4.29.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-methylbenzylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (33)
o
81% yield, white solid, mp = 145-147 C, 1H NMR (300 MHz, CD3OD) δ 7.47-7.36 (m, 3 H),
7.28-7.26 (m, 2 H), 7.22-7.12 (m, 2 H), 7.04 (d, 2 H, J = 7.68 Hz), 4.41 (s, 2 H), 4.28 (d, 2 H, J =
5.31 Hz), 3.71 (q, 1 H), 2.97 (s, 3 H), 2.27 (s, 3 H), 1.44 (d, 3 H, J = 6.96 Hz); MS (FAB) m/z
556 (M+H).
4.1.4.30.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(4-methoxybenzylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (34)
o
1
84% yield, white solid, mp = 158-161 C, H NMR (300 MHz, CDCl3) δ 7.55-7.45 (m, 2 H),
7.34-7.30 (m, 3 H), 7.17-7.03 (m, 2 H), 6.84-6.80 (m, 2 H), 6.60 (s, 1 H), 5.97 (m, 1 H), 4.42 (s,
2 H), 4.38-4.27 (m, 2 H), 3.78 (s, 3 H), 3.50 (q, 1 H, J = 6.0 Hz), 3.01 (s, 3 H), 1.46 (d, 3 H, J =
6.0 Hz); MS (FAB) m/z 572 (M+H).
4.1.4.31. N-((2-(2-Chlorobenzylthio)-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-
(methylsulfonamido)phenyl)propanamide (35)
o
1
85% yield, white solid, mp = 161-163 C, H NMR (300 MHz, CDCl3) δ 7.61-7.55 (m, 2 H),
7.51-7.46 (m, 1 H), 7.39-7.32 (m, 2 H), 7.23-7.14 (m, 1 H), 7.12-7.07 (m, 1 H), 7.05-7.03 (m, 1
H), 6.45 (s, 1 H), 5.95 (m, 1 H), 4.60 (s, 2 H), 4.32 (m, 2 H), 3.49 (q, 1 H, J = 7.5 Hz), 3.02 (s, 3
H), 1.45 (d, 3 H, J = 7.5 Hz); MS (FAB) m/z 576 (M+H).
4.1.4.32.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((6-(trifluoromethyl)-2-(3-
(trifluoromethyl)benzylthio)pyridin-3-yl)methyl)propanamide (36)
70% yield, white solid, mp = 165-167 oC, 1H NMR (300 MHz, CDCl3) δ 7.72 (s, 1 H), 7.63-7.48
(m, 4 H), 7.43-7.32 (m, 2 H), 7.14-7.05 (m, 2 H), 6.45 (s, 1 H), 5.89 (m, 1 H), 4.49 (s, 2 H), 4.34
(m, 2 H), 3.52 (q, 1 H, J = 6.0 Hz), 3.02 (s, 3 H), 1.48 (d, 3 H, J = 9.0 Hz); MS (FAB) m/z 610
23

(M+H).
4.1.4.33.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(furan-2-ylmethylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (37)
o
1
72% yield, white solid, mp =143-145 C, H NMR (300 MHz, CDCl3) δ 7.59-7.48 (m, 2 H),
7.35-7.33 (m, 2 H), 7.13-7.04 (m, 2 H), 6.49 (s, 1 H), 6.29-6.28 (m, 2 H), 5.95-5.93 (m, 1 H),
4.53 (s, 2 H), 4.35 (Septet, 2 H), 3.52 (q, 1 H), 3.03 (s, 2 H), 1.58 (s, 2 H), 1.48 (d, 3 H), 1.25 (m,
4 H), 0.95-0.8 (m, 3 H); MS (FAB) m/z 532 (M+H).
4.1.4.34.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(thiophen-2-ylmethylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (38)
o
1
84% yield, white solid, mp = 126-129 C, H NMR (300 MHz, CDCl3) δ 7.57 (d, 1 H, J = 6.0
Hz), 7.47 (t, 1 H, J = 6.0 Hz), 7.34 (d, 1 H, J = 6.0 Hz), 7.17-7.03 (m, 4 H), 6.90-6.87 (m, 1 H),
6.61 (s, 1 H), 6.02 (t, 1 H, J = 6.0 Hz). 4.71 (s, 2 H), 4.41-4.27 (septet, 2 H), 3.51 (q, 1 H, J = 6.0
Hz), 3.0 (s, 3 H), 1.46 (d, 3 H, J = 6.0 Hz); MS (FAB) m/z 548 (M+H).
4.1.4.35.
2-(3-Fluoro-4-(methylsulfonamido)phenyl)-N-((2-(phenethylthio)-6-
(trifluoromethyl)pyridin-3-yl)methyl)propanamide (39)
68% yield, white solid, mp = 135-139 oC, 1H NMR (300 MHz, CD3OD) δ 7.44 (d, 2 H, J = 8.25
Hz), 7.35-7.15 (m, 10 H), 4.33 (d, 2 H, J = 3.12 Hz), 3.71 (q, 1 H), 3.43 (t, 2 H), 3.34 (t, 2 H),
2.98 (s, 3 H), 1.49 (d, 3 H, J = 7.14 Hz); MS (FAB) m/z 556 (M+H).
4.2. Molecular modeling
The 3D structure of the ligand was generated with Concord and energy minimized using
MMFF94s force field and MMFF94 charge until the rms of Powell gradient was 0.05 kcal mol-
1A-1 in SYBYL-X 1.2 (Tripos Int., St. Louis, MO, USA). The flexible docking study on our
24

hTRPV1 model17,23 was performed by GOLD v.5.0.1 (Cambridge Crystallographic Data Centre,
Cambridge, UK), which uses a genetic algorithm (GA) and allows for full ligand flexibility and
partial protein flexibility. The binding site was defined as 8 Å around the capsaicin docked in the
hTRPV1 model. The side chains of the nine residues, which are important for ligand binding,
(i.e., Tyr511, Ser512, Met514, Leu515, Leu518, Phe543, Leu547, Thr550, and Asn551) were set
to be flexible with ‘crystal mode’ in GOLD. The ligand was docked using the GoldScore scoring
function with 30 GA runs. Other parameters were set as default. All computation calculations
were undertaken on Intel® XeonTM Quad-core 2.5 GHz workstation with Linux Cent OS release
5.5.
4.3. Biolgical Assay
The methods for in vitro and in vivo assays were reported in the previous literature.17
Acknowledgements
This research was supported by Research Grants from Grunenthal, Grants from the National
Research Foundation of Korea (NRF) (R11-2007-107-02001-0), Grants from the National
Leading Research Lab (NLRL) program (2011-0028885), and in part by the Intramural Research
Program of NIH, Center for Cancer Research, NCI (Project Z1A BC 005270).
References and Notes
1. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51, 159.
2. Tominaga, M.; Caterina, M. J.; Malmberg, A. B.; Rosen, T. A.; Gilbert, H.; Skinner, K.;
Raumann, B. E.; Basbaum, A. I.; Julius, D. Neuron 1998, 21, 531.
3. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen, T. A.; Levine, J. D.; Julius, D.
Nature 1997, 389, 816.
4. Zygmunt, P. M.; Petersson, J.; Andersson, D. A.; Chuang, H-H.; Sorgard, M.; Di Marzo, V.;
25

Julius, D.; Hogestatt, E. D. Nature 1999, 400, 452.
5. Hwang, S. W.; Cho, H.; Kwak, J.; Lee, S. Y.; Kang, C. J.; Jung, J.; Cho, S.; Min, K. H.; Suh, Y.
G.; Kim, D.; Oh, U. Proc. Natl. Acad. Sci. USA 2000, 97, 6155.
6. Walpole, C. S. J.; Wrigglesworth, R. Capsaicin in the Study of Pain; Academic Press: San
Diego, CA, 1993; pp 63.
7. Appendino, G.; Szallasi, A. Life Sci. 1997, 60, 681.
8. Szallasi, A.; Cruz, F.; Geppetti, P. Trends in Mol. Med. 2006, 12, 545.
9. Kym, P. R.; Kort, M. E.; Hutchins, C. W. Biochem. Pharmacol. 2009, 78, 211.
10. Wong, G. Y.; Gavva, N. R. Brain Res. Rev. 2009, 60, 267.
11. Gunthorpe, M. J.; Chizh, B. A. Drug Disc. Today 2009, 14, 56.
12. Lazar, J.; Gharat, L.; Khairathkar-Joshi, N.; Blumberg, P. M.; Szallasi, A. Exp. Opin. Drug
Discov. 2009, 4, 159.
13. Voight, E. A.; Kort, M. E. Exp. Opin. Ther. Pat. 2010, 20, 1.
14. Szolcsányi, J.; Sándor, Z. Trend Pharmacol. Sci. 2012, 33, 646.
15. Szallasi, A.; Sheta, M. Exp. Opin. Investig. Drug 2012, 21, 1351.
16. Kim, Y. S.; Kil, M-J.; Kang, S-U.; Ryu, H.; Kim, M. S.; Cho, Y.; Bhondwe, R. S.;
Thorat, S. A.; Sun, W.; Liu, K.; Lee, J. H.; Choi, S.; Pearce, L. V.; Pavlyukovets, V. A.
; Morgan, M. A.; Tran, R.; Lazar, J.; Blumberg, P. M.; Lee, J. Bioorg. Med. Chem. 2012
, 20, 215.
17. Kim, M. S.; Ryu, H.; Kang, D. W.; Cho, S-H.; Seo, S.; Park, Y. S.; Kim, M-Y.; Kwak, E. J.;
Kim, Y. S.; Bhondwe, R. S.; Kim, H. S.; Park, S-g. Son, K.; Choi, S.; DeAndrea-Lazarus, I.;
Pearce, L. V.; Blumberg, P. M.; Frank, R.; Bahrenberg, G.; Stockhausen, H.; Kögel, B. Y.;
Schiene, K.; Christoph, T.; Lee, J. J. Med. Chem. 2012, 55, 8392.
18. Thorat, S. A.; Kang, D. W.; Ryu, H.; Kim, M. S.; Kim, H. S.; Ann, J.; Ha, T-H.; K
im, S. E.; Son, K.; Choi, S.; Blumberg, P. M.; Frank, R.; Bahrenberg, G.; Schiene, K.; C
hristoph, T.; Lee, J. Euro. J. Med. Chem. 2013, 64, 589.
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19. Ryu, H.; Jin, M-K.; Kang, S-U.; Kim, S. Y.; Kang, D. W.; Lee, J.; Pearce, L. V.; P
avlyukovets, V. A.; Morgan, M. A.; Tran, R.; Toth, A.; Lundberg, D. J.; Blumberg, P. M
. J. Med. Chem. 2008, 51, 57.
20. Bennett, G. J.; Xie, Y-K. Pain 1988, 33, 87.
21. Szikszay, M.; Obál F. Jr.; Obál, F. Naunyn Schmiedebergs Arch Pharmacol. 1982, 320, 97.
22. De Vries, D. J., Blumberg, P. M. Life Sci. 1989, 44, 711.
23. Lee, J. H.; Lee, Y.; Ryu, H.; Kang, D. W.; Lee, J.; Lazar, J.; Pearce, L. V; Pavlyukovets, V.
A.; Blumberg, P. M.; Choi, S. J. Comput. Aided Mol. Des. 2011, 25, 317.
27

TRPV1 Antagonist with High Analgesic Efficacy: 2-Thio Pyridine C-region Analogues of 2-(3-
Fluoro-4-methylsulfonylaminophenyl)propanamides
Tae-Hwan Ha, HyungChul Ryu, Sung-Eun Kim, Ho Shin Kim, Jihyae Ann, Phuong-Thao Tran,
Van-Hai Hoang, Karam Son, Minghua Cui, Sun Choi, Peter M. Blumberg, Robert Frank, Gregor
*
Bahrenberg, Klaus Schiene, Thomas Christoph, Sven Frormann, Jeewoo Lee
3
28