Structure-activity relationships and molecular modeling of the N-(3-pivaloyloxy-2-benzylpropyl)-N′-[4-(methylsulfonylamino)benzyl] thiourea template for TRPV1 antagonism

Article abstract of DOI:10.1016/j.bmcl.2012.04.034

The structure-activity relationships of N-(3-acyloxy-2-benzylpropyl)- N′-4-[(methylsulfonylamino)benzyl] thioureas, which represent simplified RTX-based vanilloids, were investigated by varying the distances between the four principal pharmacophores and a

Full text of DOI:10.1016/j.bmcl.2012.04.034

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3656–3660
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships and molecular modeling of the
N-(3-pivaloyloxy-2-benzylpropyl)-N0-[4-(methylsulfonylamino)benzyl]
thiourea template for TRPV1 antagonism
Rahul S. Bhondwe a, Dong Wook Kang b, Myeong Seop Kim a, Ho Shin Kim a, Seul-gi Park c, Karam Son c,
Sun Choi c, Krystle A. Lang Kuhs d, Vladimir A. Pavlyukovets d, Larry V. Pearce d, Peter M. Blumberg d,
Jeewoo Lee a,
⇑
a Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
b Department of Pharmaceutical Science and Technology, College of Health and Medical Science, Catholic University of Deagu, Gyeongsan-si, Gyeongsangbuk-do 712-702,
Republic of Korea
c 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, Republic of Korea
d Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
The structure–activity relationships of N-(3-acyloxy-2-benzylpropyl)-N0-4-[(methylsulfonylamino)
benzyl] thioureas, which represent simplified RTX-based vanilloids, were investigated by varying the dis-
tances between the four principal pharmacophores and assessing binding and antagonistic activity on
rTRPV1. The analysis indicated that a 3-pivaloyloxy-2-benzylpropyl C-region conferred the best potency
in binding affinity and antagonism. The molecular modeling of this best template with the tetrameric
homology model of rTRPV1 was performed to identify its binding interactions with the receptor.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 20 March 2012
Revised 5 April 2012
Accepted 7 April 2012
Available online 13 April 2012
Keywords:
Vanilloid receptor 1
TRPV1 antagonist
Capsaicin
Resiniferatoxin
Molecular modeling
The transient receptor potential V1 (TRPV1) receptor1 is a
molecular integrator of nociceptive stimuli, including protons,2
heat,3 inflammatory mediators such as anandamide4 and lipoxyge-
nase products,5 and vanilloids such as capsaicin (CAP)6 and resinif-
eratoxin (RTX).7 The receptor functions as a non-selective cation
channel with high Ca2+ permeability and its activation leads to
an increase in intracellular Ca2+ that results in excitation of pri-
mary sensory neurons and ultimately the central perception of
pain.
TRPV1 antagonists are promising drug candidates. Therapeutic
applications include inhibiting the transmission of nociceptive sig-
naling from the periphery to the CNS as well as blocking other
pathological states associated with this receptor. TRPV1 antago-
nists have thus emerged as novel and promising analgesic and
antiinflammatory agents, particularly for chronic pain and inflam-
matory hyperalgesia.8 The number of antagonists reported contin-
ues to increase and their clinical development has been extensively
reviewed.9–13
Previously, we have reported that a series of N-(3-acyloxy-
2-benzylpropyl)-N0-4-[(methylsulfonylamino)benzyl]
thiourea
analogues (e.g. 1 and 2) were effective antagonists of the action of
C
B
A
O
O
O
H
H
O
OCH3
OH
Resiniferatoxin
20
3
OH
O
S
O
R2
R1=H R2=3,4-Me2
R1=H R2=4-t-Bu
R1=F R2=3,4-Me2
R1=F R2=4-t-Bu
1
2
3
4
NHSO2CH3
R1
H
N
H
N
O
O
⇑
Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
Figure 1. Pharmacophoric regions of RTX and sRTX.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.034

R. S. Bhondwe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3656–3660
3657
thiocarbamate) and amines. For the template having Y = 0 and
Z = 1, compounds 10 and 11 were synthesized by the coupling of
the C-region amines 7 and 8 previously reported15 with the phen-
ylthiocarbamate of A-region 9 (Scheme 1).
For the synthesis of a template having Y = 0 and Z = 2, the
amines of the C-region 12 and 13 were prepared starting from N-
(diphenylmethylene)glycine ethyl ester by the procedure reported
previously15 and then were converted to the corresponding iso-
thiocyanates, 14 and 15. The isothiocyanates reacted with the
amines of the A-region 16 and 1714 to afford thioureas 18–21,
respectively (Scheme 2).
P3
O
S
O
P1
H
N
Z
H
N
Y
H
N
O
X
O
P4
S
P2
Figure 2. The four principal pharmacophores of sRTX.
capsaicin on rat TRPV1 (Fig. 1).14–16 We further found that 3-fluoro
substitution on the 4-(methylsulfonylamino)phenyl group in the A-
region enhanced the extent of antagonism.14 Thus, the compounds 3
and 4 showed high binding affinities and potent antagonism in the
rat TRPV1/CHO system.
The principal pharmacophores in the template, denoted as P1–
P4 in Figure 2, were derived from our RTX-derived pharmacophore
model in which 4-hydroxy-3-methoxyphenyl (A-region), C20-ester
(B-region), orthophenyl (C1-region) and C3-keto (C2-region) groups
in RTX correspond to P1–P4, respectively.17
In order to find the optimal template and its active conforma-
tion for an sRTX (a simplified RTX having the four principal phar-
macophores of RTX), we have investigated the structure–activity
relationships of the template with regard to the spatial arrange-
ment of key pharmacophores by lengthening or shortening the dis-
tances between the pharmacophores. We have already established
from previous analysis that the distance between P1 and P2 was
optimal when the linker is one-carbon (X = 1).15
In a continuation of these efforts, we have now optimized the
distances between P2 and P4 in the template (corresponding to Y
and Z in Fig. 2) with a (3-fluoro-4-methylsulfonylamino)benzyl
A-region to obtain the preferred active spatial arrangement of four
pharmacophores. We describe here the syntheses and functional
TRPV1 activities of the designed ligands and our docking study of
the resulting active template with our homology model.
For the synthesis of a template having Y = 0 and Z = 0, the isothi-
ocyanate of the C-region 25 was prepared starting from vinylben-
zene 22 in four steps (Scheme 3). The dihydroxyation of 22
followed by selective pivaloylation provided 23 whose hydroxyl
group was converted to the corresponding azide 24. The azide 24
was then converted to the isothiocyanate 25 efficiently with car-
bon disulfide and triphenylphosphine.18 The isothiocyanate 25
was coupled with the amines of the A-region 16 and 17 to afford
the final compounds 26 and 27.
For the synthesis of a template having Y = 1 and Z = 0, the isothi-
ocyanate of the C-region 31 was prepared starting from 2-phenyl-
acetate 28 in five steps (Scheme 4). The C-acylation of 28, the
reduction of the diester followed by selective pivaloylation pro-
vided alcohol 29. By following the route represented in Scheme
3, compound 29 was converted to the final thioureas 32 and 33.
The binding affinities and potencies as agonists/antagonists of
the synthesized TRPV1 ligands were assessed in vitro by a binding
competition assay with [3H]RTX and by a functional 45Ca2+ uptake
assay using rat TRPV1 heterologously expressed in Chinese ham-
ster ovary (CHO) cells, as previously described.19 The results are
summarized in Table 1, together with the potencies of the previ-
ously reported thiourea antagonists 1–6.14–16
Previously, the analysis of the SAR for the spacing between P1
((4-methylsulfonylamino)phenyl) and P2 (thiourea) (the A and B-
regions, respectively) indicated that either one-carbon elongation
or one-carbon deletion between them caused a dramatic loss of
binding affinity regardless of the distances between P2 and P4.
General syntheses of the final target thioureas were achieved by
the coupling between the corresponding isothiocyanates (or
R2
R2
R1
R1
S
S
F
a
+
PhO
N
H
O
F
O
N
H
N
H
NH2
NHMs
O
O
NHMs
9
7 R1 = Me
8 R2 = H
R
R
2 = Me
2 = t-Bu
10 R1 = Me R2 = Me
2 = t-Bu
R
11 R2 = H
Scheme 1. Reactions and conditions: (a) NEt3, CH2Cl2, 90–95%.
R
R2
R2
R2
H2N
R1
R1
R1
NHMs
16 R = H
17 R = F
a
b
S
O
R
O
O
O
N
H
N
NH2
N
C S
H
O
O
NHMs
R2 = Me
12 R1 = Me
13 R1 = H
R
2 = Me
R2 = Me
14 R1 = Me
15 R1 = H
R1 = Me
18 R = H
19 R = H
20 R = F
21 R = F
R
2 = t-Bu
R2 = t-Bu
R
2 = t-Bu
2 = Me
R
1 = H
R
R1 = Me
R2 = t-Bu
R1 = H
Scheme 2. Reactions and conditions: (a) 1,10-thiocarbonyl diimidazole, NEt3, DMF, 80–85%; (b) NEt3, CH2Cl2, 90–95%.

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R. S. Bhondwe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3656–3660
a, b
c
O
O
N3
OH
O
O
23
22
24
d
e
S
O
R
O
N
H
N
H
N
C S
O
O
NHMs
25
26 R = H
27 R = F
Scheme 3. Reactions and conditions: (a) OsO4, NMO, acetone–H2O (1:1), 84%; (b) pivaloyl chloride, NEt3, DMAP, CH2Cl2, 95%; (c) DPPA, DBU, toluene, 65%; (d) CS2, PPh3, THF,
74%; (e) 16 or 17, NEt3, CH2Cl2, 90–95%.
a-c
d, e
EtO
O
OH
O
N3
O
O
O
28
30
29
f
g
NHMs
R
H
N
H
N
O
O
N C S
O
S
O
31
32 R = H
33 R = F
Scheme 4. Reactions and conditions: (a) NaOEt, diethyl carbonate, toluene, 58%; (b) LiAlH4, ether, 85%; (c) pivaloyl chloride, NEt3, DMAP, CH2Cl2, 95%; (d) MsCl, NEt3, CH2Cl2,
92%; (e) NaN3, DMF, 82%; (f) CS2, PPh3, THF, 78%; (e) 16 or 17, NEt3, CH2Cl2, 90–95%.
Thereby, the SAR in the present study was conducted based on the
template of the N-[(4-methylsulfonylamino)benzyl]thiourea tem-
plate corresponding to X = 1 in Figure 2.
extremes were a 2-fold enhancement in antagonistic potency for
18 compared to 1 and a 14-fold reduction in potency for 20 com-
pared to 3. On the other hand, one-carbon shortening of both the
Y and Z parts from 2 and 4 (Y = 1, Z = 1) afforded N-(2-pivaloyl-1-
phenylethyl) analogues 26 and 27 (Y = 0, Z = 0), which also showed
weaker binding affinities but became partial agonists/antagonists
in contrast to the above template.
Overall, the SAR analysis of Y and Z parts in the C-region pro-
vided the order of potencies in their binding affinities as follows:
(Y, Z) = (1, 1) > (1, 0) > (0, 0 ꢀ 0, 1 ꢀ 0, 2). We conclude that the
C-region having Y = 1, Z = 1 together with X = 1 for the A-region
provided the optimal sRTX for interacting with the rTRPV1.
We previously constructed the tetramer homology model of rat
TRPV1 (rTRPV1) and reported the docking results of representative
TRPV1 ligands, including capsaicin, resiniferatoxin (RTX) and
sRTX.20 Using this rTRPV1 model, we performed a flexible docking
study with compound 4, which possesses the highest binding affin-
ity in this series.21 As shown in Figure 3, this molecule fitted well
into the binding site located at the interface of the two monomers.
The 4-(methylsulfonylamino) phenyl group (A-region) occupied
the deep bottom hole at the binding site, and the two oxygen
atoms and NH of the sulfonamide group participated in hydrogen
In the variation of the Y part of the template, one-carbon short-
ening of 1 and 2 (Y = 1, Z = 1) provided N-(2-pivaloyl-1-benzyleth-
yl) analogues 5 and 6 (Y = 0, Z = 1), which were found to be partial
antagonists with weaker binding affinities by 20- and 6.5-fold,
respectively. Modification of the A-region through incorporation
of a fluoro group into 5 and 6 at the 3-position in the A-region
led not only to stronger binding affinities (2.5-fold for 10, 3.6-fold
for 11) but also to enhanced antagonism, as we would predict from
previous studies.14
In the variation of the Z part of the template, one-carbon short-
ening of 2 and 4 (Y = 1, Z = 1) provided N-(3-pivaloyloxy-2-phenyl-
propyl) analogues 32 and 33 (Y = 1, Z = 0), which were found to be
full antagonists despite a modest loss in binding affinities of 2.2-
and 3-fold, respectively.
The combination of one-carbon shortening of the Y part and
one-carbon elongation of the Z part of analogues 1–4 (Y = 1,
Z = 1) provided N-[2-pivaloyl-1-(phenylethyl)ethyl] analogues
18–21 (Y = 0, Z = 2), which exhibited reduced potencies in binding
affinity and variable effects on antagonistic potencies. The

R. S. Bhondwe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3656–3660
3659
Table 1
Potencies of vanilloid ligands for binding to rat TRPV1 and for inducing calcium influx in CHO/TRPV1 cells
O
S
O
H
N
R2
Z
H
N
Y
H
N
O
R1
O
S
Y
Z
R1
R2
Binding affinity Kia (nM)
Agonismb (%)
Antagonism Kic (nM) (%)
1
2
3
4
5
1
1
1
1
0
0
0
0
1
1
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
0
2
2
2
2
0
0
H
H
F
3,4-Me2
4-t-Bu
3,4-Me2
4-t-Bu
3,4-Me2
4-t-Bu
3,4-Me2
4-t-Bu
4-t-Bu
4-t-Bu
3,4-Me2
4-t-Bu
3,4-Me2
4-t-Bu
4-t-Bu
4-t-Bu
29.3
64
54
22.6
580
416
233
115
143
69
390
366
120
190
197
244
( 7.6)
(15)
(40)
NE
(5)
(19)
(8)
NE
NE
NE
NE
(5)
NE
NE
NE
(46)
(35)
67
86
7.8
( 25)
( 17)
( 3.0)
( 17)
(48)
(31)
( 210)
( 20)
( 25)
( 40)
( 2)
( 48)
( 43)
( 46)
( 420) (46)
( 18) (72)
( 21)
( 28)
( 2.7)
( 130)
( 44)
( 18)
( 2.7)
( 43)
( 18)
( 100)
( 8.1)
( 4.6)
( 38)
( 56)
( 46)
F
52
H
H
F
F
H
F
H
H
F
F
H
F
6
10
11
32
33
18
19
20
21
26
27
650
179
91
116
33.7
286
111
152
940
213
NE: not effective, WE: weakly effective at 30 lM.
a
Mean SEM of at least three experiments.
If any agonism was detected, value in parentheses indicates fractional calcium uptake compared to that by 30
Mean SEM of at least three experiments for measuring Ki. Value in parenthesis indicates measured extent of partial antagonism if observed.
b
c
lM capsaicin.
Figure 3. Predicted binding mode of compound 4 in the rTRPV1 model with surface representations. (A) Binding mode of compound 4. The key residues are marked and
displayed as capped-stick with carbon atoms in white. The helices are colored in gray and the helices of the adjacent monomer are displayed in line ribbon. Compound 4 is
depicted as ball-and-stick with carbon atoms in magenta. The van der Waals surface of the ligand is presented with the lipophilic potential property. Hydrogen bonds are
shown in black dashed lines, and non-polar hydrogens are undisplayed for clarity. (B) Surface representations of the docked compound 4 and rTRPV1. The Fast Connolly
surface of rTRPV1 was generated by MOLCAD and colored by the lipophilic potential property. The surface of rTRPV1 is Z-clipped, and that of the ligand is in its carbon color
for clarity. (C) Van der Waals surface of compound 4 is colored by the lipophilic potential property.
bonding with Ser512 and Gly558, respectively. The sulfur of the
thiourea group (B-region) made a hydrogen bond with Tyr511
and also contributed to the appropriate positioning of the C-region
for the hydrophobic interactions. The bulky and hydrophobic
4-t-butylbenzyl and pivaloyloxymethyl groups in the C-region ex-
tended toward Met547 in the upper hydrophobic area and the
adjacent monomer’s hydrophobic region, composed of Phe587
and Phe591, and formed hydrophobic interactions.
in binding affinity and antagonism for rTRPV1, reflecting that its
pharmacophores provided the optimal spacing for rTRPV1 interac-
tion. The docking study of compound 4 with the tetrameric homol-
ogy model established by us provided useful information on the
binding interactions with the receptor, including the hydrophilic
interactions with Tyr511 and Ser512 and the hydrophobic ones
with the two separate pockets composed of Met547 and Phe587/
591, respectively.
In summary, we have investigated the structure–activity rela-
tionship of a series of N-(3-acyloxy-2-benzylpropyl)-N0-4-[(meth-
ylsulfonylamino)benzyl] thioureas as TRPV1 antagonists in which
we varied the distances between the four principal pharmaco-
phores (P1–P4). The analysis indicated that the C-region template
having X = 1, Y = 1, and Z = 1 in Figure 2 displayed the best potency
Acknowledgments
This research was supported by Grants from the National Re-
search Foundation of Korea (NRF) (R11-2007-107-02001-0), Grants
from the NLRL program (2011-0028885), Brain Research Center of

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R. S. Bhondwe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3656–3660
15. Lee, J.; Kim, S. Y.; Lee, Ji; Kang, M.; Kil, M-J.; Choi, H-K.; Jin, M-K.; Wang, Y.;
Toth, A.; Pearce, L. V.; Lundberg, D. J.; Tran, R.; Blumberg, P. M. Bioorg. Med.
Chem. 2004, 12, 3411.
16. Ryu, H.; Jin, M.-K.; Kang, S.-U.; Kim, S. Y.; Kang, D. W.; Lee, J.; Pearce, L. V.;
Pavlyukovets, V. A.; Morgan, M. A.; Tran, R.; Toth, A.; Lundberg, D. J.; Blumberg,
P. M. J. Med. Chem. 2008, 51, 57.
17. Lee, J.; Lee, J.; Kim, J.; Kim, S. Y.; Chun, M. W.; Cho, H.; Hwang, S. W.; Oh, U.;
Park, Y. H.; Marquez, V. E.; Beheshti, M.; Szabo, T.; Blumberg, P. M. Bioorg. Med.
Chem. 2001, 9, 19.
the 21st Century Frontier Research Program (2011-K000289)
funded by MEST and NRF, the Ewha Global Top 5 Grant 2011 of
Ewha Womans University, and was supported in part by the Intra-
mural Research Program of the National Institutes of Health, Cen-
ter for Cancer Research, National Cancer Institute (Project Z1A BC
005270).
18. Isoda, T.; Hayashi, K.; Tamai, S.; Kumagai, T.; Nagao, Y. Chem. Pharm. Bull. 2006,
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