Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2017.08.020

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.

Full text of DOI:10.1016/j.bmcl.2017.08.020

Accepted Manuscript
Pyrazole C-region Analogues of 2-(3-Fluoro-4-methylsulfonylaminophen-
yl)propanamides as Potent TRPV1 Antagonists
Sunho Lee, Changhoon Kim, Jihyae Ann, Shivaji A. Thorat, Eunhye Kim,
Jongmi Park, Sun Choi, Peter M. Blumberg, Robert Frank-Foltyn, Gregor
Bahrenberg, Hannelore Stockhausen, Thomas Christoph, Jeewoo Lee
PII:
S0960-894X(17)30819-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.020
BMCL 25214
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
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8 August 2017
10 August 2017
Please cite this article as: Lee, S., Kim, C., Ann, J., Thorat, S.A., Kim, E., Park, J., Choi, S., Blumberg, P.M., Frank-
Foltyn, R., Bahrenberg, G., Stockhausen, H., Christoph, T., Lee, J., Pyrazole C-region Analogues of 2-(3-Fluoro-4-
methylsulfonylaminophenyl)propanamides as Potent TRPV1 Antagonists, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.020
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Graphical Abstract
Pyrazole C-region Analogues of 2-(3-Fluoro-4-methyl Leave this area blank for abstract info.
sulfonylaminophenyl)propanamides as Potent TRPV1 Antagonists
Sunho Lee, Changhoon Kim, Jihyae Ann, Shivaji A. Thorat, Eunhye Kim, Jongmi Park, Sun Choi, Peter M.
Blumberg, Robert Frank-Foltyn, Gregor Bahrenberg, Hannelore Stockhausen, Thomas Christoph, Jeewoo Lee*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Pyrazole C-region Analogues of 2-(3-Fluoro-4-
methylsulfonylaminophenyl)propanamides as Potent TRPV1 Antagonists
Sunho Lee a, Changhoon Kim a, Jihyae Ann a, Shivaji A. Thorat a, Eunhye Kim b, Jongmi Park b, Sun Choi
b, Peter M. Blumberg c, Robert Frank-Foltyn d, Gregor Bahrenberg d, Hannelore Stockhausen d, Thomas
Christoph d, Jeewoo Lee a,*
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Korea
b National Leading Research Laboratory of Molecular Modeling & Drug Design, College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha
Womans University, Seoul 03760, Korea
c Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA
d Grünenthal Innovation, Grünenthal GmbH, D-52078 Aachen, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-
(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1
antagonism. The structure activity relationship indicated that the 3-chlorophenyl group
at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic
potency and the activity was stereospecific to the S-configuration, providing
exceptionally potent antagonists 13S and 16S with Ki(CAP) = 0.1 nM. Particularly
significant, 13S exhibited antagonism selective for capsaicin and NADA and not for
low pH or elevated temperature. Both compounds also proved to be very potent
antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin,
consistent with their in vitro mechanism. The docking study of compounds 13S and
16S in our hTRPV1 homology model indicated that the binding modes differed
somewhat, with that of 13S more closely resembling that of GRT12360.
Keywords:
Vanilloid Receptor 1
TRPV1 Antagonist
Molecular Modeling
2017 Elsevier Ltd. All rights reserved.
TRPV1, the target of capsaicin action, has emerged as a
promising therapeutic target for the treatment of neuropathic pain
and a range of other conditions, reflecting the central role of this
nociceptor in the function of C-fiber sensory neurons.1-3 Building
on the demonstration of a receptor for capsaicin,4 its cloning as
TRPV1,5 and insights arising from its structural analysis by cryo-
EM6,7 and homology modeling,8,9 dramatic strides have been
made in understanding the structure activity relations for
antagonist binding to TRPV1.10 These advances have been
coupled with an appreciation of the complexity of TRPV1
regulation, such as the the finding that different antagonists may
be differentially effective against different classes of TRPV1
activators such as capsaicin, low pH, or elevated temperature.11
Such differences, in term, may translate into different
activators (Figure 1).14-24 The pharmacophore of the antagonistic
template (Template I) can be divided into the A, B and C-regions,
analogous to the corresponding designation of regions for the
agonist capsaicin. The structure activity relationships (SAR) of
the template have been investigated in most detail for the
pyridine C-region, in which a variety of functional groups
including the amino, oxy, thio, alkyl, aryl and sulfonamido
groups were incorporated at the 2-position of pyridine (R2) along
with substitution at the 6-position (R1) with a trifluoromethyl14-
18,22 or tert-butyl group24 as well as with the pyridine core
modified by its isomers19 or by a phenyl group.21,24 In these
studies, a number of compounds demonstrated highly potent
antagonism toward TRPV1 activators including capsaicin (CAP),
N-arachidonoyl dopamine (NADA), low pH, and heat (45 oC)
and their antagonism were stereospecific to the S-configuration.
Consistent with the in vitro mechanism of action of the
physiological responses of animals or humans upon treatment
with different TRPV1 antagonists.12,13
Over the past several years we have described an extensive
series of N-(pyridin-3-ylmethyl) 2-(3-fluoro-4-(methylsulfon
amido)phenyl)propanamides that showed potent and
compounds as hTRPV1 antagtonists, in vivo studies of selected
potent antagonists indicated that these compounds blocked
capsaicin-induced hypothermia and that they produced strong
antiallodynic effects in neuropathic pain models. Docking studies
stereospecific human TRPV1 (hTRPV1) antagonism for multiple

using our established hTRPV1 homology model14 indicated that
the 6-trifluoromethyl group and the 2-substituent in the pyridine
C-region made hydrophobic interactions with pockets composed
of Leu547/Thr550 and Met514/Leu515, respectively, that were
critical for their potent antagonism.14-18
Scheme 2. Synthesis of 3-t-butyl pyrazole C-region (Method B).
Reagents and conditions: (a) LiHMDS, diethyloxalate, THF, -78 oC to rt,
overnight, 97%; (b) NH2NH2·2HCl, TEA, EtOH, rt, 2 h, 77%; (c) NaOH,
MeOH:H2O (5:1), rt, 2 h, 70%; (d) SOCl2, MC, rt, overnight; then NH4OH,
MC, 0 oC, 1 h, 80%; (e) LiAlH4, THF, 0 oC to rt, 3 h; (f) Boc2O, MC, rt, 1 h,
50%; (g) Ar-B(OH)2, Cu(OAc)2, pyridine, MC, rt, 2 d; (h) TFA, MC, rt,
overnight
Figure 1. Design of pyrazole C-region TRPV1 antagonists
As part of our continuing effort to optimize TRPV1
antagonists as clinical candidates for neuropathic pain, we herein
have investigated a series of pyrazole C-region derivatives of 2-
(3-fluoro-4-methylsulfonylaminophenyl)propanamides
(Template II) as a new antagonistic template. Since the pyrazole
is a weak base and has a pyridine-like nitrogen (N2) with a lone
electron pair, it serves as a bioisostere of pyridine. Furthermore,
we anticipated that 1,3-substituents, R1 and R2, on pyrazole are
better positioned to interact with the two hydrophobic pockets
identified by the previous modeling than do the 2,6-substituents
of pyridine.
In this paper, we synthesized a series of 1-substituted 3-
(trifluoromethyl/t-butyl)pyrazole C-region derivatives and
evaluated their antagonism toward activation of hTRPV1 by
capsaicin. With selected potent antagonists in the series, we
characterized in detail their in vitro activities and mode of action
in vivo. Finally, we carried out a docking study using our
hTRPV1 homology model to identify their mode of binding to
the receptor.
Scheme 3. Synthesis of 3-trifluoromethyl pyrazole C-region (Method C).
Reagents and conditions: (a) LiAlH4, Et2O, -78 oC, 2 h; (b) R-NHNH2, EtOH,
reflux, 5 h, (15: 90%, 16: 67%); (c) NCS, DMF, rt, (15: 48%, 16: 26%); (d)
2-chloroacylonitrile, TEA, toluene, 80 oC, 20 h, (15: 44%, 16: 80%); (e)
LiAlH4, THF, 0 oC to rt, 3 h, (15: 64%, 16: 62%)
N1-Substituted 3-t-butyl-1H-pyrazole amines for the C-
region were synthesized by one of two methods. In Method A,
4,4-dimethyl-3-oxopentanenitrile was condensed with different
hydrazines to provide N1-substituted 5-amino-3-t-butylpyrazoles
whose amine was converted to the corresponding aminomethyl
group in 3 steps to give the t-butylpyrazole C-region amine
(Scheme 1). In Method B, N-Boc (3-t-butyl-pyrazol-5-
yl)methanamine as a key intermediate for coupling with the
corresponding boronic acids was prepared from 3,3-
dimethylbutan-2-one in 6 steps. A Chan-Lam coupling reaction
between pyrazole 1H-amine and aryl boronic acid followed by
deprotection provided the t-butylpyrazole C-region amine
(Scheme 2). N1-Substituted 3-trifluoromethyl-1H-pyrazole
amines for the C-region were synthesized from ethyl
Scheme 4. General synthesis of 2-(3-fluoro-4-(methylsulfonamido)phenyl)
propanamide analogues
Reagents and conditions: (a) EDC, HOBt, DMF, room temperature, 12 h.
The synthesized compounds were evaluated in vitro for
TRPV1 antagonism as measured by inhibition of activation by
capsaicin (100 nM). The assays were conducted using a
fluorometric imaging plate reader (FLIPR) with hTRPV1
heterologously expressed in Chinese hamster ovary (CHO)
cells.14 The results are summarized in Tables 1-3.
First, we investigated the SAR of the N1-substituent in the
3-t-butylpyrazole C-region (Table 1). The unsubstituted
derivative (1) was found to be inactive, suggesting that the two
hydrophobic groups were indispensable for activity by providing
needed interactions with the receptor pockets as previously
characterized for the pyridine C-region.14-24 The incorporation of
an alkyl group (2-4) enhanced the antagonism progressively as
the size increased. The rigidity of alkyl groups further increased
the potency (3 vs 5, 4 vs 6). Most phenyl derivatives (7-14)
showed potent antagonism. Among them, the 3-chlorophenyl
trifluoroacetic acid in 5 steps using different hydrazines (Scheme
3). A library of synthesized pyrazole C-region amines were
coupled with 2-(3-fluoro-4-(methylsulfonamido)phenyl)
propionic acids14 to provide the final compounds.
derivative (13) displayed excellent antagonism with a Ki(CAP)
=
0.3 nM and its S-stereoisomer (13S) exhibited a stereospecific
activity with a Ki(CAP) = 0.1 nM. The 4-fluoro-3-chlorophenyl
derivatives (14, 14S) also showed high potency like those of the
3-chlorophenyl derivatives. The analysis of 4-substituted phenyl
derivatives indicated that whereas the chloro group provided
promising antagonism, electron-donating (9) and bulky (11,12)
groups led to the dramatic reduction in activity.
Scheme 1. Synthesis of 3-t-butyl pyrazole C-region (Method A).
Reagents and conditions: (a) R-NHNH2·HCl, EtOH:H2O (1:1), reflux,
overnight, 44-94%; (b) t-BuONO, CuI, CH3CN, 0 oC to 65 oC; (c) Zn(CN)2,
Pd(PPh3)4, DMF, reflux, overnight; (d) LiAlH4, THF, 0 oC to rt, 1 h, 12-26%
(3 steps yield)

Table 1. In vitro hTRPV1 antagonistic activities for 3-t-butylpyrazole
derivatives
Ki(CAP) = 0.3 and 0.1 nM, versus isomers 15R, 16R with Ki(CAP)
31.7 and 26.5 nM.
=
Finally, we investigated α-substituted acetamide B-region
derivatives of the potent antagonist 13 (Table 3) because some of
these B-region derivatives previously had provided high
potency20. Unfortunately, they all displayed weak antagonism.
Ki(CAP)
(nM)
Ki(CAP)
(nM)
R
R
Table 3. In vitro hTRPV1 antagonistic activities for α-substituted acetamide
derivatives
H
1
2
3
4
5
6
7
8
NE
9
WE
8.0
75.2
819
0.3
0.1
0.5
0.1
WE
50.2
27.7
5.1
10
11
12
13
13S
14
14S
Ki(CAP)
(nM)
Ki(CAP)
(nM)
R
R
17
18
WE
WE
19
WE
WE: weakly active (17: 46%, 18: 12%, 19: 49% inhibition % at 5 µM)
21.6
8.0
Detailed in vitro activities of 13S and 16S, the most potent
antagonists in this series, were investigated for four different
TRPV1 activators, viz. capsaicin, N-arachidonoyl dopamine
(NADA), pH and heat (45oC), and compared to the activity of the
previous lead GRT12360 (Table 4). Both 13S and 16S showed
excellent antagonism of hTRPV1 activation by capsaicin and
NADA comparable to that of GRT12360. However, 13S
exhibited poor antagonism toward pH and heat, in contrast to 16S
and GRT12360, indicating that the size of the hydrophobic group
at the 3-position in the pyrazole C-region affected the selectivity
of antagonism for different activators.
3.7
WE: weakly active, NE: not effective
Table 2. In vitro hTRPV1 antagonistic activities for 3-
trifluoromethylpyrazole derivatives
Both antagonists proved to be highly potent antagonists of
capsaicin action in vivo against rat TRPV1 (rTRPV1). Consistent
with its in vitro mechanism of action (Ki(CAP) = 0.1 nM in
r/hTRPV1), they were able to block the acute hypothermic
response to capsaicin (3 mg/kg, ip). The oral administration of 3
mg/kg 13S and 16S almost completely antagonized the effect of
capsaicin on body temperature, with 95% and 85% inhibition,
respectively, of the decrease in body temperature induced by
capsaicin.
Ki(CAP)
(nM)
Ki(CAP)
(nM)
R
R
15
0.4
16
0.3
Table 4. Antagonistic activities of 13S and16S for multiple activators in
hTRPV1 and rTRPV1.
Activators, parameter
15S
15R
0.3
16S
16R
0.1
13S
16S
GRT12360b
hTRPV1
31.7
26.5
CAP (f)Ki (nM)
NADA (f)Ki (nM)
pH, IC50 (nM)
heat 45oC, IC50 (nM)
rTRPV1
0.1
0.1
0.1
8.0
8.1
0.2
0.01
6.3
0.8
0.04
WE
WE
Next, we explored the SAR of N1-substituents in the 3-
trifluoromethylpyrazole C-region (Table 2). Since 4- or 3-
chlorophenyl derivatives in the 3-t-butylphenyl series provided
the most promising activity, their surrogates were examined. 4-
Chlorophenyl (15) and 3-chlorophenyl (16) derivatives displayed
highly potent antagonism with Ki(CAP) = 0.4 and 0.3 nM,
respectively, in line with those of the 3-t-butylpyrazole series. In
addition, the S-configuration was confirmed as the active
configuration, as dramatically shown by isomers 15S, 16S with
CAP (f)Ki (nM)
Anti-hypothermia
0.1
95%a
0.1
85%a
at 3 mg/kg, po
at 3 mg/kg, po
a Inhibition percent to hypothermic response by 3 mg/kg ip capsaicin
b compound 49S in ref 9

Figure 2. Binding modes of GRT12360 and 16S in the hTRPV1 model.25
(Top) 2-D representation of the binding interactions between GRT12360 (A) and 16S (B) with hTRPV1. Hydrogen bonding interactions are drawn in blue
dashed line arrows, and hydrophobic interactions are displayed with curved patches. (Bottom) The Fast Connolly surface of hTRPV1 and the van der Waals
surface of GRT12360 and 16S. Using MOLCAD, the hTRPV1 molecular surface was created and the surface with the lipophilic potential property is presented.
For clarity, the surface of hTRPV1 is Z-clipped and that of the ligands are colored individually by magenta and purple
In order to investigate the binding interactions of 13S and
16S, flexible docking studies were carried out using our hTRPV1
model14 constructed on the basis of our rTRPV1 model8. 13S and
16S share with the previously reported GRT12360 (Figure
2A)14 the identical A,B-region structure of 2-(3-fluoro-4-
methylsulfonamidophenyl)propanamide but are distinguished by
different C-regions. Compound 16S has a 3-
(trifluoromethyl)pyrazole ring and a chlorobenzene ring in the C-
region and showed a different binding mode in the C-region
compared to GRT12360. As shown in Figure 2B, the
sulfonaminobenzyl group in the A-region occupied the deep
bottom hole and participated in the hydrophobic interactions
with Val508, Tyr511, Leu515, Ile564, Tyr565, and Ile569. In
addition, the fluorine atom in the A-region engaged in hydrogen
bonding with Lys571 while the NH of the sulfonamide group
formed a hydrogen bond with Ile564. The amide group in B-
region was able to form a hydrogen bond with Tyr511 and
contributed to the proper positioning of the C-region for the
hydrophobic interactions. In the C-region, the 3-
(trifluoromethyl)pyrazole ring formed hydrophobic interactions
with Tyr554 and with Phe587 and Leu588 of the adjacent
monomer. Instead of the 3-(trifluoromethyl)pyrazole ring, the
chlorobenzene ring oriented towards the upper hydrophobic
region composed of Leu547, Thr550 and was involved in
hydrophobic interactions with Phe587 and Phe591 in the
adjacent monomer, which might have caused the flipped
positioning of the two rings in the C-region.
well to the deep bottom area of the binding site, which consisted
of Val508, Tyr511, Leu515, Tyr555, Ile564, Tyr565, and Ile569,
forming hydrophobic interactions. Additionally, the NH of the
sulfonamide group engaged in hydrogen bonding with Ser512.
The amide group in the B-region made a hydrogen bond with the
OH of Tyr511 and assisted the appropriate positioning of the C-
region. The t-butyl pyrazole ring in the C-region extended
toward the hydrophobic pocket composed of Leu515, Leu518,
and Leu547, along with Phe587 from the adjacent monomer.
Moreover, the chlorobenzene ring in the C-region participated in
the hydrophobic interactions with Tyr511, Met514, and Leu515.
Additionally, 13S presented another comparable binding mode
by flipping the two rings in the C-region as shown in Figure 3B.
The 3-fluoro-4-methylsulfonamidophenyl group in the A-region
fitted in the deep bottom region and showed hydrophobic
interactions with Val508, Tyr511, Ile564, Tyr565, and Ile569.
The NH of the amide group in the B-region formed a hydrogen
bond with Asn551. Moreover, the t-butyl pyrazole ring in the C-
region participated in hydrophobic interactions with Tyr511,
Met514, and Leu515 as did the 4-methylpiperidine ring in
GRT12360. The chlorobenzene ring approached the upper
hydrophobic area, which included Leu518, Leu547, Thr550 and
Phe587 from the adjacent monomer as did the 6-
trifluoromethylpyridine ring in GRT12360.
On the other hand, 13S, which has the t-butyl pyrazole and
chlorobenzene rings in the C-region, exhibited interactions
similar to those of GRT1236014. As shown in Figure 3A, the 3-
fluoro-4-methylsulfonamidophenyl group in the A-region bound

Figure 3. Binding modes of 13S in the hTRPV1 model.25
(Top) 2-D illustrations of the two binding modes of 13S with hTRPV1. Hydrogen bonding interactions are shown in blue dashed line arrows and hydrophobic
interactions are presented with curved patches. (Bottom) The Fast Connolly surface representation of hTRPV1 and the van der Waals surface representation of
the ligand 13S. MOLCAD was used to generate the molecular surface of hTRPV1 and the surface is displayed with the lipophilic potential property. For clarity,
the surface of hTRPV1 is Z-clipped and that of 13S is in magenta color.
In summary, the structure activity relationship of 3-(t-
butyl)pyrazole and 3-(trifluoromethyl)pyrazole C-region
derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl)
propanamides as antagonists for hTRPV1 was investigated. The
3-chlorophenyl group at the 1-position of pyrazole was the
optimal hydrophobic group for antagonism and the activity was
stereospecific for the S-configuration, providing the
exceptionally potent antagonists 13S and 16S with Ki(CAP) = 0.1
nM. Whereas 16S showed full antagonism to all activators as did
the previous lead, 13S exhibited antagonism selective for
capsaicin and NADA but not low pH or elevated temperature.
Both compounds also proved to be very potent antagonists for
rTRPV1 and blocked the hypothermic effect of capsaicin in vivo,
consistent with their in vitro mechanism. The docking study of
compounds 13S and 16S in our hTRPV1 homology model
indicated that they displayed different binding interactions and
that the mode of 13S was more like that of GRT12360.
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Acknowledgments
This research was supported by research grants from Grünenthal
in Germany, a grant from the National Research Foundation
(NRF) of Korea (NRF-2016M3A9B5939892), a grant from the
Mid-career Researcher Program (NRF-2017R1A2B4010084)
funded by the Ministry of Science, ICT and Future Planning
(MSIP) and the NRF, and in part by the Intramural Research
Program of the National Institutes of Health, Center for Cancer
Research, National Cancer Institute (Project Z1A BC 005270) in
the USA.

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25. The 3D structures of the 13S and 16S were generated using
Concord and energy minimized with MMFF94s force field and
MMFF94 charge until the rms of Powell gradient was 0.05 kcal
mol-1A-1 in SYBYL-X 2.0 (Tripos Int., St. Louis, MO, USA).
The flexible docking study on our hTRPV1 model was carried
out using GOLD v.5.2 (Cambridge Crystallographic Data
Centre, Cambridge, UK), which employes 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 important side
chains of the nine residues (i.e., Tyr511, Ser512, Met514,
Leu515, Leu518, Phe543, Leu547, Thr550, and Asn551) were
set to be flexible with ‘crystal mode’ in GOLD. 13S and 16S was
docked with the GoldScore scoring function with 30 GA runs,
and the other parameters were remainded as default. All the
computation calculations were undertaken on an Intel® XeonTM
Quad-core 2.5 GHz workstation with Linux Cent OS release 5.5.
20. Tran, P-T. Kim, H. S.; Ann, J.; Kim, S-E.; Kim, C.; Hong,
M.; Hoang, V-H.; Ngo, V. T. H.; Hong, S.; Cui, M.; Choi, S.;
Blumberg, P. M.; Frank-Foltyn, R.; Bahrenberg, G.; Stockhausen,