Analysis of structure-activity relationships with the N-(3-acyloxy-2- benzylpropyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea template for vanilloid receptor 1 antagonism

Article abstract of DOI:10.1016/j.bmc.2004.04.040

In a continuing effort to elucidate the structure-activity relationships of the lead antagonist N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N ^′-[4-(methylsulfonylamino)benzyl]thiourea (1), the distances between the proposed four pharmacophores in 1 have been varied by insertion or deletion of one carbon to optimize their fit to the receptor. In addition, the acyloxy group of the C region was replaced with amide and N-hydroxy amide to identify the pharmacophoric importance of the ester group in the C2 region. The results indicated that the pharmacophoric arrangement of 1 was optimal for receptor binding affinity and antagonism, and the ester of the C2 region was significant for receptor binding. Among the derivatives, compound 19 showed distinct behavior with a 2-fold improvement in antagonism but a 13-fold reduction in binding affinity compared to 1. The partial separation of pharmacophoric requirements of these two assays has been noted before and compound 19 is thus selective for the calcium entry-linked receptor population. The conformational analysis of 1 generated three distinct conformers having different types of hydrophobic interactions, which will be utilized for exploring the active conformation of the VR1 ligand.

Full text of DOI:10.1016/j.bmc.2004.04.040

Bioorganic & Medicinal Chemistry 12 (2004) 3411–3420
Analysis of structure–activity relationships with the N-(3-acyloxy-
2-benzylpropyl)-N0-[4-(methylsulfonylamino)benzyl]thiourea
template for vanilloid receptor 1 antagonism
Jeewoo Lee,a,* Su Yeon Kim,a Jiyoun Lee,a Myungsim Kang,a Min-Jung Kil,a
Hyun-Kyung Choi,a Mi-Kyung Jin,a Yun Wang,b Attila Toth,b Larry V. Pearce,b
Daniel J. Lundberg,b Richard Tranb and Peter M. Blumbergb
aResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,
Shinlim-Dong, Kwanak-Ku, Seoul 151-742, South Korea
bLaboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute,
NIH, Bethesda, MD 20892, USA
Received 23 March 2004; revised 30 April 2004; accepted 30 April 2004
Available online
Abstract—In a continuing effort to elucidate the structure–activity relationships of the lead antagonist N-[2-(3,4-dimethylbenzyl)-3-
pivaloyloxypropyl]-N0-[4-(methylsulfonylamino)benzyl]thiourea (1), the distances between the proposed four pharmacophores in 1
have been varied by insertion or deletion of one carbon to optimize their fit to the receptor. In addition, the acyloxy group of the C
region was replaced with amide and N-hydroxy amide to identify the pharmacophoric importance of the ester group in the C2
region. The results indicated that the pharmacophoric arrangement of 1 was optimal for receptor binding affinity and antagonism,
and the ester of the C2 region was significant for receptor binding. Among the derivatives, compound 19 showed distinct behavior
with a 2-fold improvement in antagonism but a 13-fold reduction in binding affinity compared to 1. The partial separation of
pharmacophoric requirements of these two assays has been noted before and compound 19 is thus selective for the calcium entry-
linked receptor population. The conformational analysis of 1 generated three distinct conformers having different types of
hydrophobic interactions, which will be utilized for exploring the active conformation of the VR1 ligand.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
cloned but are not believed to be sensitive to vanilloids.1
VR1, which is expressed predominantly on thin,
unmyelinated sensory nerve fibers (C-fibers) and small
Ad fibers in the dorsal root, trigeminal, and nodose
ganglia, is a molecular integrator of nociceptive stimuli.
VR1 is activated by protons,9 heat,10 natural exogenous
ligands such as capsaicin (CAP)11 or resiniferatoxin
(RTX),12 and endogenous substances such as anand-
amide13 and the lipoxygenase product 12-HPETE.14 Since
VR1 functions as a nonselective cation channel with
high Ca2þ permeability, its activation by these agents
leads to an increase in intracellular Ca2þ that results in
excitation of primary sensory neurons and ultimately the
central perception of pain. Chronic stimulation of VR1
leads to desensitization/defunctionalization of the neu-
rons, probably reflecting multiple mechanisms.
The vanilloid receptor (VR1) is a member of the tran-
sient receptor potential (TRP) superfamily. Members of
this family are nonvoltage activated cation channel
proteins that play critical roles in processes ranging from
sensory physiology to vasorelaxation and male fertility.
They share structural similarities such as six trans-
membrane segments and an oligmeric structure.1;2 The
vanilloid or capsaicin receptor (VR13 or TRPV1) has
been cloned from dorsal root ganglia (DRG) of the rat,4
the human,5 the chicken,6 the guinea pig,7 and the rab-
bit.8 Vanilloid receptor homologues have also been
Keywords: Vanilloid receptor 1; TRPV1; Antagonist; Capsaicin; Res-
iniferatoxin.
The involvement of VR1 in both pathological and
physiological conditions suggests that the blocking of
* Corresponding author. Tel.: +82-2-880-7846; fax: +82-2-888-0649;
e-mail: jeewoo@snu.ac.kr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.040

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J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
this receptor, by desensitization or by antagonism,
would have considerable therapeutic utility. Among its
therapeutic targets, pain is of particular interest. The
validation of VR1 as a molecular target for the treat-
ment of chronic pain was confirmed using transgenic
mice lacking functional VR1 receptors. These mice
exhibited impairment in the perception of thermal and
inflammatory pain.15
P3
P1
R
NHSO2CH3
Z
H
N
Y
H
N
O
X
O
P4
S
P2
X = 0, 1, 2
Y = 0, 1
Z = 1, 2
The therapeutical advantage of VR1 antagonism over
desensitization subsequent to agonism is that it avoids
the initial excitatory effect preceding the desensitization.
The initial acute pain associated with capsaicin treat-
ment has proven to be the limiting toxicity. After the
Figure 2. The four principal pharmacophores of antagonist.
stead a full agonist with an EC50 ¼ 22 nM.26 The results
generalized that the A region makes a major contribu-
tion to the extent of agonism/antagonism in a series
of thiourea analogues; namely, substitution of the
4-phenolic hydroxyl of the A region in agonists with a
4-methylsulfonamido group shifted the ligands from
agonism toward antagonism, and a 3-fluoro substitution
further favored antagonism, whereas a 3-methoxy group
favored agonism.
discovery of capsazepine as the first VR1 antagonist,16
a
number of antagonists have been reported both with
structures related and unrelated to agonists.17 Among
them, 5-Iodo-RTX,18 SC0030,19 halogenated capsaicin
analogues,20 BCTC,21 SB-366791,22 7-hydroxynaphtha-
len-1-yl urea,23 and IBTU24 were characterized in detail
as potent VR1 competitive antagonists.
We have previously reported that isosteric replacement
of the phenolic hydroxyl group in potent vanilloid
receptor agonists25 with the alkylsulfonamido group
provided a series of compounds, which are effective
antagonists to the action of capsaicin on rat VR1 het-
erologously expressed in Chinese hamster ovary (CHO)
cells (Fig. 1).26 As a prototype, N-[2-(3,4-dimethylbenz-
yl)-3-pivaloyloxypropyl]-N0-[4-(methylsulfonylamino)-
benzyl]thiourea (1) showed a high binding affinity with a
Ki value of 29.3 nM for the inhibition of [3H]RTX
binding and potent antagonism with an IC50 value of
67 nM for the inhibition of 45Ca2þ uptake in response to
capsaicin, displaying partial agonism.27 The SAR of the
prototype has been examined extensively based on the
pharmacophoric regions represented in Figure 1.26;28
For the SAR of the B region, we modified the thiourea
group of 1 with diverse isosteres, including N-hydroxy
thiourea, thiocarbamate, amide, and ester groups.28
Structure–activity analysis indicated that the thiourea
group provided the best in vitro profile and the A region
was a primary factor in determining the agonistic/
antagonistic activities regardless of the B region. Inter-
estingly, the NC-hydroxy thiourea analogues showed
excellent analgesic activities in the acetic acid writhing
assay compared to the parent thiourea analogues despite
relatively less potency in agonism and in binding affin-
ity.28
In a continuation of these studies, we have now exam-
ined further aspects of the SAR. The four pharmaco-
phores in the lead antagonist 1, denoted as P1–P4 in
Figure 2, were derived from our RTX-derived phar-
macophore model, which incorporates the 4-hydroxy-
3-methoxyphenyl (A region), C20-ester (B region),
orthophenyl (C1 region) and C3-keto (C2 region) groups.
For the purpose of optimizing the spatial arrangement
of these pharmacophores, we have varied the distances
between the four pharmacophores by lengthening or
shortening them by one carbon, respectively. A similar
approach was examined previously in a series of agon-
ists and provided a plausible active conformation of
RTX.29 In addition, structural modification of the
acyloxy group of the C region, the so-called C2 region,
was examined by replacing it with amide and N-hydroxy
amide to define its pharmacophoric importance. In this
paper, we describe the synthesis and biological activity
profiles of these VR1 ligands, and analyze their SAR.
In the SAR of the A region, the 3-fluoro analogue of 1
was a full and potent antagonist with an IC50 ¼ 7.8 nM
and, conversely, the 3-methoxy analogue of 1 was in-
C
B
A
O
Resiniferatoxin
O
O
H
H
O
OCH3
OH
OH
O
O
R
NHSO2CH3
H
H
N
O
N
O
S
1 R =3,4-Me2
2 R =4-t-Bu
2. Chemistry
General syntheses of the final target thioureas were
achieved by the coupling between the corresponding
isothiocyanates and amines. The amines (4, 5, 7) and
Figure 1. The pharmacophoric comparison between RTX and N-(2-
benzyl-3-pivaloyloxypropyl)-N0-[4-(methylsulfonylamino)benzyl]thio-
urea.

J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
3413
isothiocyanates (6, 8) corresponding to the A region
R
were prepared starting, respectively, from 4-nitroaniline
(X ¼ 0), 4-aminobenzyl amine (X ¼ 1), and 4-nitro-
phenylacetonitrile (X ¼ 2) by conventional methods26
(Scheme 1). The isothiocyanates of the C region having
Y ¼ 1 (10) were synthesized from ethyl malonate as
reported previously25;29 and then coupled with amines
(4, 5, 7) to provide thioureas 11–14 (Scheme 2). The
amines of the C region having Y ¼ 0 (16) were prepared
from N-(diphenylmethylene)glycine ethyl ester by the
procedure reported previously29 and then reacted with
isothiocyanates 6 and 8 to afford thioureas 17–20,
respectively (Scheme 3). For the synthesis of the amide
congener of the pivaloyloxy group in 1, the hydroxyl
group of 2-(3,4-dimethylbenzyl)-3-hydroxypropyl azide
(21)25 was converted to the corresponding amine by the
Gabriel method, and was then acylated with pivaloyl
chloride to give 23. Azido reduction of 23 followed by
coupling with isothiocyanate 6 afforded the target amide
analogue 24 (Scheme 4). For the synthesis of the N-
hydroxyamide congener, the hydroxyl group of 21 was
transformed into the corresponding hydroxylamine 26
a
Z
EtO2C
CO2Et
O
NCS
O
Z
9
10
b
R
NHSO2CH3
H
N
H
N
O
X
O
S
11 X = 1, Z = 2, R = 3,4-Me2
12 X = 1, Z = 2, R = 4-t-Bu
13 X = 0, Z = 1, R = 3,4-Me2
14 X = 2, Z = 1, R = 3,4-Me2
Scheme 2. Synthesis of target compounds with Y ¼ 1. (a) Ref. 25; (b) 4
(or 5, 7), CH2Cl2.
R
EtO
a
CPh2
Z
N
O
S
O
NH2
O
H2N
15
O2N
a, b
16
b
NHSO2CH3
NH2
NH2
4
R
Z
O
H2N
c
R
N
H
N
H
X
O
NHSO2CH3
NHSO2CH3
5 R = NH2
6 R = NCS
17 X = 1, Z = 1, R = 3,4-Me2
18 X = 1, Z = 1, R = 4-t-Bu
19 X = 1, Z = 2, R = 3,4-Me2
20 X = 2, Z = 1, R = 3,4-Me2
b, a, d-e
R
NC
NO2
NHSO2CH3
Scheme 3. Synthesis of target compounds with Y ¼ 0. (a) Ref. 29; (b) 6
(or 8), CH2Cl2.
7 R = NH2
8 R = NCS
via the Mitsunobu reaction with di-(t-butoxycar-
bonyl)hydroxylamine followed by acid hydrolysis. The
pivaloylation and coupling with 6 as described in
Scheme 1. Synthesis of A region. (a) MsCl, pyridine; (b) H2, Pd–C,
MeOH; (c) Ref. 26; (d) H2, c-HCl, MeOH; (e) 1,10-thiocarbonyldiim-
idazole, CH2Cl2.
O
a
N
N3
HO
N3
O
21
22
b, c
NHSO2CH3
d, e
H
N
H
H
N
H
N
N
N3
O
S
O
24
23
Scheme 4. Synthesis of amide congener. (a) Phthalimide, PPh3, DEAD, THF; (b) NH2NH2, EtOH; (c) Me3CCOCl, Et3N, CH2Cl2; (d) H2, Lindler
cat, EtOH; (e) 6, CH2Cl2.

3414
J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
a, b
OR
N
N3
HO
N3
R
21
25 R=Boc
26 R=H
c
NHSO2CH3
d, e
OH
N
OH
N
H
N
H
N
N3
O
S
O
28
27
Scheme 5. Synthesis of N-hydroxyamide congener. (a) NH(Boc)OBoc, PPh3, DEAD, THF; (b) CF3CO2H; (c) Me3CCOCl, Et3N, CH2Cl2; (d) H2,
Lindler cat, EtOH; (e) 6, CH2Cl2.
Scheme 3 provided the target N-hydroxyamide analogue
28 (Scheme 5).
levels of 45Ca2þ uptake were compared with that induced
by a maximally effective concentration of capsaicin in
this system, namely 300 nM. Receptor binding affinities
were assessed in terms of the ability of the compounds to
compete for specific binding of [3H]RTX in the CHO/
VR1 system and were expressed as the Ki SEM. All
values SEM represent the mean of at least three
experiments. The results are summarized in Tables 1 and
2. In the CHO/VR1 system, the parent antagonists, 1
and 2 (X ¼ Y ¼ Z ¼ 1), showed high binding affinities
with Ki values of 29.3 and 64 nM, respectively, and
potent antagonism with Ki values of 67 and 86 nM, dis-
playing partial agonism,26 respectively. Their potencies
represented 44- and 20-fold enhancements in binding
affinity and 8- and 6-fold enhancements in antagonism
compared to capsazepine (Ki ¼ 1300 nM in binding
affinity, Ki ¼ 520 nM in antagonism).
3. Results and discussion
3.1. Biological activity
The potencies and activities as agonists and antagonists
of the synthesized VR1 ligands were assessed in vitro by
assay of 45Ca2þ uptake, which was carried out using rat
VR1 heterologously expressed in Chinese hamster ovary
cells (CHO/VR1 cells) as previously described.19;26 The
in vitro antagonistic potencies of the compounds were
evaluated by measuring antagonism of the 45Ca2þ up-
take induced by 50 nM capsaicin and expressed as the
Ki SEM, correcting for competition by capsaicin. All
compounds were also evaluated as agonists. Potencies as
agonists were expressed as EC50 SEM, and absolute
In the variation of the Z part of the template, one-car-
bon lengthening of the benzyl group in the parent
Table 1. Potencies of vanilloid ligands for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
R
NHSO2CH3
Z
H
N
H
N
O
X
Y
O
S
X
Y
Z
R
Ki (nM) binding affinity
EC50 (nM) agonism
Ki (nM) antagonism
Capsazepine
1
1300 ( 150)
29.3 ( 7.6)
64 ( 21)
NE
WEa
520 ( 12)
67 ( 25)
86 ( 17)
121 ( 39)
NE
1
1
1
1
0
2
1
1
1
2
1
1
1
1
1
1
0
0
0
0
1
1
2
2
1
1
1
1
2
1
3,4-Me2
4-t-Bu
2
WEa
11
12
13
14
17
18
19
20
3,4-Me2
4-t-Bu
1430 ( 390)
690 ( 250)
4670 ( 800)
1850 ( 360)
580 ( 130)
416 ( 44)
WEa
1675 ( 106)
WEa
3,4-Me2
3,4-Me2
3,4-Me2
4-t-Bu
WEb
WEa
790 ( 260)
WEb
WEb
WEa
WEa
3,4-Me2
3,4-Me2
390 ( 100)
3170 ( 590)
WEa
NE
33.7 ( 2)
1750 ( 580)
NE: not effective, WE: weakly effective at 30 lM.
a Only fractional calcium uptake compared to 30 lM capsaicin 1 (at 300 nM), 15%; 2, 40%; 11, 23%; 13, 21%; 14, 6%; 17, 19%; 18, 8%; 19, 5%.
b Only fractional inhibition 13, 72%; 17, 48%; 18, 31%.

J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
3415
Table 2. Potencies of vanilloid ligands for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
NHSO2CH3
H
N
H
N
X
O
S
X
Ki (nM) binding affinity
EC50 (nM) agonism
Ki (nM) antagonism
1
O
29.3 ( 7.6)
7700 ( 2900)
2270 ( 120)
WE
NE
NE
67 ( 25)
NE
1870 ( 180)
24
28
NH
NOH
NE: not effective, WE: weakly effective at 30 lM.
compounds 1 and 2 (Z ¼ 1), providing N-(3-pivaloyl-2-
phenethylpropyl) analogues 11 and 12 (Z ¼ 2), led to
reduced potencies in binding affinity with values of
Ki ¼ 1430 and 690 nM (ca. 50-fold in 11, ca. 10-fold in
12) as compared to 1 and 2, respectively. Interestingly,
this modification showed a complicated result. Whereas
the 3,4-dimethyl analogue 11 showed antagonism with
weak partial agonism like the parent compound, the 4-
tert-butyl analogue 12 exhibited full agonism although
the potency was low. This finding is yet another example
that functional receptor activity is sensitive to even small
structural modification, as we reported previously.30
to VR1 by less than 10%. These distinct potencies reflect
different fractions of VR1 in the two assay systems:
namely, a plasma membrane fraction controlling 45Ca2þ
uptake, and an intracellular fraction that dominates the
[3H]RTX binding measurements. Therefore, as with
IBTU, the divergence of 19 in binding affinity and
antagonism may be explained by marked selectivity
between subpopulations of VR1, and a prediction is that
while it blocks the increase in intracellular calcium from
the medium by occupying the VR1 at the plasma
membrane level, it will be less effective in blocking that
released from internal stores. Unlike compound 19,
compound 20, derived from the variation of X and Y of
1, exhibited much reduced potencies in binding affinity
and antagonism by 108-fold and 26-fold, respectively.
In the variation of the X part of the template, one-
carbon shortening of 1 (X ¼ 0), producing the N-[4-
(methylsulfonylamino)phenyl] derivative 13, resulted
insubstantial loss of potencies in binding affinity and
antagonism. Likewise, one-carbon lengthening of 1
The acyloxy group of the C region, defined as the C2
region in the lead 1, has been proposed as a pharma-
cophore derived from the C3-keto group in RTX. In
order to identify the pharmacophoric importance of its
ester group, it was replaced with amide and N-hydroxy-
amide to provide 24 and 28, respectively. The amide
analogue 24 showed almost complete loss of activity in
binding affinity and in the functional assay, and the N-
hydroxyamide analogue 28, whose hydroxyl was de-
signed to mimic the C4-hydroxyl in RTX, also exhibited
weak activities. These results indicated that a hydrogen
bonding acceptor may be required to interact with the
receptor.
(X ¼ 2),
producing
the
N-[4-(methylsulfonyl-
amino)phenethyl] derivative 14, also led to reduced
potencies in binding affinity (63-fold, Ki ¼ 1850 nM) and
antagonism (12-fold, Ki ¼ 790 nM), but their activities
were a little better than those of the shortened derivative
13.
In the variation of the Y part of the template, one-car-
bon shortening of 1 and 2 (Y ¼ 0) provided the N-(2-
pivaloyl-1-benzylethyl) analogues 17 and 18, for which
the binding affinities were reduced 20-fold and 7-fold
(Ki ¼ 580 nM in 17, Ki ¼ 416 nM in 18), respectively, and
the potencies as agonists and antagonists were sub-
stantially lost.
3.2. Conformational analysis
The simultaneous variation of Y and Z of 1 to provide
19 (Y ¼ 0 and Z ¼ 2) showed a complicated but very
interesting result. Whereas the binding affinity of 19 was
reduced by 13-fold (Ki ¼ 390 nM) compared to that of 1,
its potency as an antagonist increased by 2-fold
(Ki ¼ 33.7 nM). This finding is yet another example that
receptor binding affinity is not fully correlated with
antagonist activity. Such divergence between binding
affinity and antagonism was recently described in detail
with biochemical characterization of an antagonist,
N-(4-chlorobenzyl)-N0-(4-hydroxy-3-iodo-5-methoxy-
benzyl)thiourea (IBTU).24 Whereas IBTU showed
potent antagonism of vanilloid-induced calcium uptake
(Ki ¼ 99 and 93 nM for capsaicin and RTX, respec-
tively), at 30 lM it inhibited [3H]resiniferatoxin binding
Since compound 1 showed the highest binding affinity
among the synthesized derivatives with diverse disposi-
tions of the pharmacophoric groups, conformational
analysis was performed to identify its active conforma-
tion for binding to VR1. The structure of 1 was mini-
mized in the
Sybyl program and 48 unique
conformations of 1 were found after eliminating the
other conformations based on energy cutoff (3 kcal/mol),
on chirality, or steric interference (bump check). The
conformation with the lowest energy (conformer A) has
an energy value of )14.61 kcal/mol. All 48 conformers
were matched with the lowest energy conformer A, and
the RMS overlap values were calculated. The RMS
overlap versus energy values for all conformers were
plotted in Figure 3. The energy values ranged from

3416
J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
RMS value
-4
-6
0
1
2
3
4
5
-8
-10
-12
-14
-16
Figure 3. RMS versus energy values for the conformers of 1.
)6.27 to )14.61 kcal/mol. The maximum and minimum
RMS values were 0.00 and 4.21 A, respectively. The
ꢀ
lowest energy (conformer A) and highest energy (con-
former C) conformers and the conformer with the most
dissimilar topology (conformer B), that is with the
highest RMS value, were selected as representative
conformations of 1, and are indicated with the red
symbols in Figure 3.
The pharmacophore model of 1 consists of two hydro-
phobic ring systems and two hydrogen bond acceptors
as indicated in Table 3. The interatomic distances of the
center of the (4-methylsulfonylamino)phenyl ring (A
region), the sulfur atom of the thiourea (B region), the
center of the 3,4-dimethylphenyl ring (C1 region), and
the carbonyl of the ester (C2 region) in the three con-
formers were measured for the structural analysis (Table
3). Their three-dimensional conformations were dis-
played in Figure 4. The structures of the three con-
formers feature different types of hydrophobic
interactions between the three hydrophobic groups: the
A ring, the C1 ring, and the pivaloyl group of the C2
region. While the two phenyls of the A region and the
Figure 4. Proposed representative conformations of 1. (a) Conformer
A; (b) conformer B; (c) conformer C.
Table 3. Interatomic distances of four pharmacophoric features
C1-region
ꢀ
C1 region are close (distance of 5.95 A) in conformer A
A-region
and the phenyl of the A region and the pivaloyl group of
the C2 region are close in conformer B, the hydrophobic
groups of the C1 and C2 regions are as far away as
possible from the phenyl of the A region in conformer
C. This finding indicates that hydrophobic interactions
in 1 play a crucial role in energy stabilization of the
conformations. The conformational analysis of 1 pro-
vided three representative conformations with unique
structural features characteristic of different hydropho-
bic interactions between the three hydrophobic groups;
these will be utilized for providing a plausible model of
the active conformation.
NHSO2CH3
H
N
H
N
O
O
S
C2-region
B-region
Conformer A Conformer B Conformer C
Energy (kcal/mol) )14.61
)11.18
4.21
3.79
9.52
3.38
6.90
4.13
7.69
)6.27
4.11
3.90
10.95
8.14
7.47
4.49
5.31
ꢀ
RMS (A)
0.00
5.91
5.95
8.47
5.27
7.20
5.43
Distance (A–B)
Distance (A–C1)
Distance (A–C2)
Distance (B–C1)
Distance (B–C2)
Distance (C1–C2)
In summary, we have modified the prototype antagonist
(1) to expand the analysis of SAR in two ways. (1) The
distances between the four pharmacophores were varied

J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
3417
by lengthening or shortening of one carbon between
4.2. General procedure for the synthesis of thiourea
them for the purpose of optimizing the spatial
arrangement. (2) The acyloxy group of the C region was
replaced with amide and with N-hydroxy amide for
identifying the pharmacophoric importance of the ester
group of the C2 region. We conclude that the pharma-
cophoric distances of 1 are optimal for binding affinity
and antagonism and that the ester of the C2 region is
significant for receptor binding. Compound 19 showed
partial separation of potencies, with enhanced potency
as an antagonist but with weaker potency in the binding
assay compared to 1. Based on our current under-
standing, this suggests that 19 should be more selective
for the calcium entry-linked VR1 receptor subpopula-
tion. The conformational analysis of 1 generated three
distinct conformers with different hydrophobic interac-
tions, which should be of use for modeling of the active
conformation.
A solution of amine salt (0.5 mmol) in dimethylforma-
mide (1 mL) was treated with triethylamine (0.5 mmol)
and stirred for 30 min at room temperature. To the
mixture was added isothiocyanate (0.5 mmol). After
being stirred for 24 h at room temperature, the mixture
was diluted with water and extracted with ethyl acetate
several times. The combined organic layers were washed
with water and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo and the residue
was purified by flash column chromatography over silica
gel with EtOAc/hexanes (1:1) as eluant to afford the
thiourea.
4.2.1. N-[4-(3,4-Dimethylphenyl)-2-(pivaloyloxymethyl)-
butyl]-N0-[4-(methylsulfonylamino)benzyl]thiourea (11,
1
KJM-481). 73% yield, white solid, mp ¼ 50–51 ꢁC; H
NMR (CDCl3) d 7.33 (d, 2H, J ¼ 8:5 Hz), 7.18 (d, 2H,
J ¼ 8:5 Hz), 6.9–7.1 (m, 3H), 6.42 (s, 1H, NHSO2), 6.36
(t, 1H, NH), 6.06 (br s, 1H, NH), 4.59 (d, 2H,
J ¼ 5:3 Hz, CSNHCH2Ar), 4.18 (dd, 1H, J ¼ 3:7,
11.5 Hz, CH2OCO), 3.95 (dd, 1H, J ¼ 5:1, 11.5 Hz,
CH2OCO), 3.75 (m, 1H, CHCH2NHCS), 3.22 (m, 1H,
CHCH2NHCS), 3.00 (s, 3H, SO2CH3), 2.65 (m, 2H,
CH2CH2Ar), 2.2–2.3 (m, 7H, 2 · CH3 and CH), 1.5–1.65
(m, 2H, CH2CH2Ar), 1.20 (s, 9H, C(CH3)3); MS (FAB)
534 (MHþ); Anal. Calcd for C27H39N3O4S2: C, 60.76; H,
7.36; N, 7.87; S, 12.02. Found: C, 60.96; H, 7.38; N,
7.83; S, 11.98.
4. Experimental section
4.1. General method
All chemical reagents were commercially available.
melting points were determined on a melting point
€
Buchi B-540 apparatus and are uncorrected. Silica gel
column chromatography was performed on silica gel 60,
230–400 mesh, Merck. Proton NMR spectra were re-
corded on a JEOL JNM-LA 300 at 300 MHz. Chemical
shifts are reported in ppm units with Me4Si as a refer-
ence standard. Mass spectra were recorded on a VG
Trio-2 GC–MS. Combustion analyses were performed
on an EA 1110 Automatic Elemental Analyzer, CE
Instruments.
4.2.2. N-[4-(4-tert-Butylphenyl)-2-(pivaloyloxymethyl)-
butyl]-N0-[4-(methylsulfonylamino)benzyl]thiourea (12,
KJM-459). 77% yield, white solid, mp ¼ 34 ꢁC; 1H
NMR (CDCl3) d 7.33 (d, 2H, J ¼ 8:3 Hz), 7.31 (d, 2H,
J ¼ 8:5 Hz), 7.18 (d, 2H, J ¼ 8:5 Hz), 7.13 (d, 2H,
J ¼ 8:3 Hz), 6.54 (br s, 1H, NHSO2), 6.41 (t, 1H, NH),
6.14 (br s, 1H, NH), 4.60 (d, 2H, J ¼ 4:9 Hz,
CSNHCH2Ar), 4.19 (dd, 1H, J ¼ 3:7, 11.5 Hz,
CH2OCO), 3.96 (dd, 1H, J ¼ 4:9, 11.5 Hz, CH2OCO),
3.76 (m, 1H, CHCH2NHCS), 3.24 (m, 1H,
CHCH2NHCS), 2.99 (s, 3H, SO2CH3), 2.70 (m, 2H,
CH2CH2Ar), 2.04 (m, 1H, CH), 1.5–1.7 (m, 2H,
CH2CH2Ar), 1.30 (s, 9H, C(CH3)3), 1.20 (s, 9H,
C(CH3)3); MS (FAB) 562 (MHþ); Anal. Calcd for
C29H43N3O4S2: C, 62.00; H, 7.71; N, 7.48; S, 11.42.
Found: C, 62.20; H, 7.74; N, 7.44; S, 11.36.
4.1.1. 4-(Methylsulfonylamino)aniline (4, KMJ-308).
1
White solid, H NMR (CDCl3) d 7.06 (bd, 2H), 6.66
(bd, 2H), 6.06 (br s, 1H, NHSO2), 3.73 (br s, 2H, NH2),
2.94 (s, 3H, SO2CH3).
4.1.2. 4-(Methylsulfonylamino)benzyl amine (5) and 4-
(methylsulfonylamino)benzyl isothiocyanate (6). The
compounds were prepared according to the previous
procedure.26
4.2.3. N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-
N0-[4-(methylsulfonylamino)phenyl]thiourea (13, KMJ-
246). 64% yield, white solid, mp ¼ 43 ꢁC; 1H NMR
(CDCl3) d 7.86 (br s, 1H), 7.27 (d, 2H, J ¼ 8:5 Hz), 7.16
(d, 2H, J ¼ 8:5 Hz), 6.85–7.05 (m, 3H), 6.49 (br s, 1H),
4.10 (m, 1H, CH2OCO), 3.80 (m, 2H, CH2OCO and
CH2NHCS), 3.34 (m, 1H, CH2NHCS), 3.01 (s, 3H,
SO2CH3), 2.5–2.7 (m, 2H, CH2Ar), 2.34 (m, 1H, CH),
2.15–2.25 (m, 6H, 2 · CH3), 1.17 (d, 9H, C(CH3)3); MS
(FAB) 506 (MHþ); Anal. Calcd for C25H35N3O4S2: C,
59.38; H, 6.98; N, 8.31; S, 12.68. Found: C, 59.62; H,
7.01; N, 8.27; S, 12.61.
4.1.3. 2-[4-(Methylsulfonylamino)phenyl]ethyl amine
hydrochloride (7, KMJ-276). White solid, 1H NMR
(DMSO-d6) d 7.20 (dd, 4H), 3.15 (m, 2H, CH2NH3þ),
2.7–3.0 (m, 5H, SO2CH3 and CH2Ar).
4.1.4. 2-[4-(Methylsulfonylamino)phenyl]ethyl isothiocya-
1
nate (8, KMJ-338). White solid, H NMR (CDCl3) d
7.22 (s, 4H), 6.51 (br s, 1H, NHSO2), 3.73 (t, 2H,
J ¼ 6:7 Hz, CH2NCS), 3.02 (s, 3H, SO2CH3), 2.98 (t,
2H, J ¼ 6:7 Hz, CH2Ar).

3418
J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
4.2.4. N-[2-(3,4-Dimethylbenzyl)-3-(pivaloyloxy)propyl]-
N0-{2-[4-(methylsulfonylamino)phenyl]ethyl}thiourea (14,
KMJ-282). 72% yield, white solid, mp ¼ 46 ꢁC; 1H NMR
(CDCl3) d 7.18 (s, 4H, Ar), 7.15 (br s, 1H, NHSO2), 6.9–
7.1 (m, 3H, Ar), 6.45 (br s, 1H, NHCS), 6.00 (br s, 1H,
NHCS), 4.24 (m, 1H, CH2OCO), 3.82 (m, 2H,
CH2OCO, and CH2NHCS), 3.53 (m, 2H, CSNHCH2),
3.20 (m, 1H, CH2NHCS), 2.98 (s, 3H, SO2CH3), 2.85
(m, 2H, CH2CH2Ar), 2.5–2.7 (m, 2H, CHCH2Ar), 2.2–
2.35 (m, 7H, 2 · CH3, and CH), 1.25 (d, 9H, C(CH3)3);
MS (FAB) 534 (MHþ); Anal. Calcd for C27H39N3O4S2:
C, 60.76; H, 7.36; N, 7.87; S, 12.02. Found: C, 61.00; H,
7.39; N, 7.83; S, 11.97.
NMR (CDCl3) d 7.16 (m, 4H, Ar), 6.9–7.1 (m, 3H, Ar),
6.82 (br s, 1H, NHSO2), 6.20 (br s, 1H, NHCS), 6.03 (br
t, 1H, NHCS), 4.50 (br s, 1H, CHNH), 4.16 (m, 1H,
CH2OCO), 3.94 (m, 1H, CH2OCO), 3.61 (m, 2H,
CSNHCH2), 2.98 (s, 3H, SO2CH3), 2.86 (t, 2H,
CH2CH2Ar), 2.7–2.8 (m, 2H, CHCH2Ar), 2.2–2.3 (m,
6H, 2 · CH3), 1.20 (d, 9H, C(CH3)3); MS (FAB) 520
(MHþ); Anal. Calcd for C26H37N3O4S2: C, 60.09; H,
7.18; N, 8.09; S, 12.34. Found: C, 60.29; H, 7.21; N,
8.04; S, 12.29.
4.2.9. N-[3-Azido-2-(3,4-dimethylbenzyl)propyl]phthal-
imide (22, JYL-1575). A cooled mixture of 21 (0.55 g,
2.3 mmol), phthalimide (0.508 g, 3.45 mmol), triphenyl-
phospine (0.905 g, 3.45 mmol) in THF (10 mL) at 0 ꢁC
was treated with diethyl azodicarboxylate (1.5 mmol,
0.543 mL) and stirred at room temperature for 18 h. The
solvent was removed in vacuo and the residue was
purified by flash column chromatography over silica gel
with EtOAc/hexanes (1:10) as eluant to afford 22 as a
4.2.5. N-[3-(3,4-Dimethylphenyl)-1-pivaloyloxy-2-prop-
yl]-N0-[4-(methylsulfonylamino)benzyl]thiourea
(17,
1
MSK-483). 66% yield, white solid, mp ¼ 63–65 ꢁC; H
NMR (CDCl3) d 7.27 (d, 2H, J ¼ 8:5 Hz), 7.18 (d, 2H,
J ¼ 8:5 Hz), 6.9–7.1 (m, 3H), 6.25 (br s, 1H, NH), 6.00
(d, 1H, J ¼ 7:8 Hz, NH), 4.56 (br s, 3H, NHCH2Ar, and
CHNHCS), 4.18 (dd, 1H, J ¼ 4:9, 11.5 Hz, CH2OCO),
3.98 (dd, 1H, J ¼ 5:1, 11.5 Hz, CH2OCO), 3.00 (m, 4H,
SO2CH3, and CH2Ar), 2.75 (m, 1H, CH2Ar), 2.27 (d,
3H, J ¼ 5:4 Hz, CH3), 2.23 (s, 3H, CH3), 1.19 (s, 9H,
C(CH3)3); MS (FAB) 506 (MHþ); Anal. Calcd for
C25H35N3O4S2: C, 59.38; H, 6.98; N, 8.31; S, 12.68.
Found: C, 59.60; H, 7.02; N, 8.26; S, 12.62.
1
colorless oil (0.753 g, 94%); H NMR (CDCl3) d 7.65–
7.9 (m, 4H), 6.85–7.0 (m, 3H, Ar), 3.65–3.8 (m, 2H,
CH2N), 3.2–3.4 (m, 2H, CH2N3), 2.72 (d, 1H,
J ¼ 7:8 Hz, CH2Ph), 2.64 (d, 1H, J ¼ 7:1 Hz, CH2Ph),
2.45 (m, 1H, CH), 2.1–2.3 (m, 6H, 2 · CH3).
4.2.10.
N-[3-Azido-2-(3,4-dimethylbenzyl)propyl]-2,2-
dimethylpropanamide (23, JYL-1581). A mixture of 22
(0.753 g, 2.16 mmol) and hydrazine monohydrate
(0.126 mL, 2.59 mmol) in ethanol (8 mL) was stirred at
room temperature for 16 h and concentrated in vacuo.
The residue was dissolved in CH2Cl2 (10 mL) and trea-
ted with pivaloyl chloride (0.317 mL, 2.59 mmol). After
3 h of stirring at room temperature, the mixture was
diluted with H2O and extracted with EtOAc several
times. The combined organic layers were dried over
MgSO4 and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel
with EtOAc/hexanes (1:3) as eluant to afford 23 as a
4.2.6. N-[3-(4-tert-Butylphenyl)-1-pivaloyloxy-2-propyl]-
N0-[4-(methylsulfonylamino)benzyl]thiourea (18, MSK-
1
423). 68% yield, white solid, mp ¼ 90–92 ꢁC; H NMR
(CDCl3) d 7.25–7.35 (m, 4H), 7.12–7.2 (m, 4H), 6.60 (s,
1H, NHSO2), 6.50 (t, 1H, NH), 6.08 (d, 1H, J ¼ 7:8 Hz,
NH), 4.59 (br s, 3H, NHCH2Ar, and CHNHCS), 4.20
(dd, 1H, J ¼ 4:9, 11.5 Hz, CH2OCO), 3.97 (dd, 1H,
J ¼ 5:1, 11.5 Hz, CH2OCO), 3.00 (m, 4H, SO2CH3, and
CH2Ar), 2.75 (dd, 1H, J ¼ 8:3, 13.6 Hz, CH2Ar), 1.30 (s,
9H, C(CH3)3), 1.18 (s, 9H, C(CH3)3); MS (FAB) 534
(MHþ); Anal. Calcd for C27H39N3O4S2: C, 60.76; H,
7.36; N, 7.87; S, 12.02. Found: C, 60.98; H, 7.39; N,
7.84; S, 11.98.
1
yellow oil (0.392 g, 60%); H NMR (CDCl3) d 6.85–7.1
(m, 3H, Ar), 5.75 (br s, 1H, NH), 3.2–3.4 (m, 4H,
CH2N, and CH2N3), 2.5–2.7 (m, 2H, CH2Ph), 2.2–2.3
(m, 6H, 2 · CH3), 2.09 (m, 1H, CH), 1.13 (s, 9H,
COC(CH3)3).
4.2.7. N-[4-(3,4-Dimethylphenyl)-1-pivaloyloxy-2-butyl]-
N0-[4-(methylsulfonylamino)benzyl]thiourea (19, JYL-
1413). 73% yield, white solid, mp ¼ 52 ꢁC; 1H NMR
(CDCl3) d 7.50 (br s, 1H, NHSO2), 7.32 (d, 2H,
J ¼ 8:5 Hz), 7.22 (d, 2H, J ¼ 8:5 Hz), 6.9–7.1 (m, 3H),
6.78 (br s, 1H, NH), 6.19 (br s, 1H, NHCS), 4.68 (br s,
2H, CSNHCH2Ar), 4.3–4.4 (m, 2H, CH2OCO, and
CH), 3.97 (br s, 1H, CH2OCO), 3.02 (s, 3H, SO2CH3),
2.6–2.8 (m, 2H, CH2Ar), 2.25 (m, 6H, 2 · CH3), 1.8–2.0
(m, 2H, CH2CH2Ar), 1.18 (s, 9H, C(CH3)3); MS (FAB)
520 (MHþ); Anal. Calcd for C26H37N3O4S2: C, 60.09; H,
7.18; N, 8.09; S, 12.34. Found: C, 60.28; H, 7.23; N, 8.03;
S, 12.27.
4.2.11. N-[2-(3,4-Dimethylbenzyl)-3-(N-pivaloylamino)-
propyl]-N0-[4-(methylsulfonylamino)phenyl]thiourea (24,
JYL-1585). A suspension of 23 (0.302 g, 1 mmol) and
Lindlar’s catalyst (0.5 g) in ethanol (50 mL) was hydro-
genated under a balloon of hydrogen for 1 h. The
reaction mixture was filtered and the filtrate was con-
centrated in vacuo. The residue was dissolved in CH2Cl2
(50 mL) and treated with 4-(methylsulfonylamino)benz-
yl isothiocyanate (0.242 g, 1 mmol). After being stirred
for 3 h at room temperature, the reaction mixture was
concentrated in vacuo and the residue was purified by
flash column chromatography over silica gel with
EtOAc/hexanes (1:1) as eluant to afford 24 as a white
4.2.8. N-[3-(3,4-Dimethylphenyl)-1-pivaloyloxy-2-prop-
yl]-N0-{2-[4-(methylsulfonylamino)phenyl]ethyl}thiourea (20,
KMJ-342). 76% yield, white solid, mp ¼ 54 ꢁC; 1H
1
solid (0.394 g, 76%); mp ¼ 86 ꢁC; H NMR (CDCl3) d
7.29 (d, 2H, J ¼ 8:3 Hz), 7.15 (d, 2H, J ¼ 8:3 Hz), 6.8–

J. Lee et al. / Bioorg. Med. Chem. 12 (2004) 3411–3420
3419
7.1 (m, 3H), 6.58 (m, 1H, NH), 6.08 (m, 1H, NH), 4.59
(br s, 2H, NHCH2Ar), 3.65–3.85 (m, 2H, CHCH2NH),
3.1–3.5 (m, 2H, CONHCH2), 2.96 (s, 3H, SO2CH3), 2.70
(ddd, 1H, CH2Ar), 2.1–2.5 (m, 8H, 2 · CH3, CH2Ar,
and CH), 1.12 (d, 9H, J ¼ 4:9 Hz, C(CH3)3); MS (FAB)
519 (MHþ); Anal. Calcd for C26H38N4O3S2: C, 60.20; H,
7.38; N, 10.80; S, 12.36. Found: C, 60.39; H, 7.42; N,
10.74; S, 12.29.
from 27 to provide 28 as a white solid in 54% yield;
mp ¼ 84 ꢁC; 1H NMR (CDCl3)
d 7.32 (d, 2H,
J ¼ 8:3 Hz), 7.19 (d, 2H, J ¼ 8:3 Hz), 6.9–7.1 (m, 3H),
5.58 (t, 1H, NH), 5.51 (t, 1H, NH), 4.7–4.9 (m, 2H,
CSNHCH2Ar), 3.5–3.8 (m, 2H, HONCH2), 2.75–3.0
(m, 2H, CHCH2NHCS), 2.99 (s, 3H, SO2CH3), 2.3–2.5
(m, 3H, CH2Ar, and CH), 2.24 (dd, 6H, 2 · CH3), 0.93
(d, 9H, J ¼ 6:6 Hz, C(CH3)3); MS (FAB) 535 (MHþ);
Anal. Calcd for C26H38N4O4S2: C, 57.78; H, 6.79; N,
10.78; S, 12.34. Found: C, 58.00; H, 6.82; N, 10.74; S,
12.29.
4.2.12. tert-Butyl N-(3-azido-2-(3,4-dimethylbenzyl)prop-
yl)-N-[(tert-butoxycarbonyl)oxy] carbamate (25, JYL-
1459). A cooled mixture of 21 (0.5 g, 2.3 mmol), tert-
butyl-N-(tert-butoxycarbonyloxy)carbamate
(0.536 g,
4.3. Molecular modeling
2.3 mmol) and triphenylphosphine (0.905 g, 3.45 mmol)
in THF (10 mL) at 0 ꢁC was treated with diethyl azodi-
carboxylate (0.543 mL, 1.5 mmol) and stirred at room
temperature for 18 h. The reaction mixture was con-
centrated in vacuo, and the residue was purified by flash
column chromatography over silica gel with EtOAc/
hexanes (1:10) as eluant to afford 25 as a colorless oil
(0.97 g, 97%); 1H NMR (CDCl3) d 6.85–7.1 (m, 3H, Ar),
3.5–3.75 (m, 2H, CH2NO), 3.3–3.5 (m, 2H, CH2N3),
2.5–2.8 (m, 2H, CH2Ph), 2.0–2.3 (m, 7H, CH, and
2 · CH3), 1.52 (s, 9H, C(CH3)3), 1.48 (s, 9H, C(CH3)3).
The conformational analysis of the lead antagonist 1
was performed to identify its active conformation on
binding to VR1. The structure of 1 was built and min-
imized using the Tripos force field (method: conjugate
gradient, termination: gradient 0.01 kcal/mol A, and
max iterations: 10,000) implemented in the Sybyl 6.5
ꢀ
program. The partial atomic charges were calculated by
€
the Gasteiger–Huckel method. Then, the three-dimen-
sional structure was subjected to conformational anal-
ysis using the Random Search method with the
following non-default options (Maximum Cycles: 3,000
and Chirality: R configuration). In the analysis, 48 un-
ique conformations of 1 were found, and the other
conformations were eliminated based on energy cutoff
(3 kcal/mol), chirality, or bump check.
4.2.13. N-[3-Azido-2-(3,4-dimethylbenzyl)propyl]hydroxyl-
amine (26, JYL-1527). To cooled trifluoroacetic acid
(3 mL) at 0 ꢁC was added 25 (0.97 g, 2.23 mmol). The
reaction mixture was stirred at room temperature for
20 min. The solvent was removed in vacuo to afford 26
as a brown oil (0.7 g, 90%), which was used in next step
1
without any further purification; H NMR (CDCl3) d
Acknowledgements
6.85–7.25 (m, 3H, Ar), 3.5–3.7 (m, 2H, CH2 NOH), 3.3–
3.5 (m, 2H, CH2N3), 2.45–2.8 (m, 3H, CH2Ph, and CH),
2.1–2.3 (m, 6H, 2 · CH3).
This work was supported by a Fund 2002 grant
(ES0024) from the Korea Research Foundation.
4.2.14. N-[3-Azido-2-(3,4-dimethylbenzyl)propyl]-N-hy-
droxy-2,2-dimethylpropanamide (27, JYL-1533).
A
References and notes
solution of 26 (0.7 g, 2 mmol) in H2O (2 mL) was treated
with sodium bicarbonate (0.2 g, 2.4 mmol) and stirred at
room temperature for 30 min. After addition of CH2Cl2
(5 mL), the reaction mixture was treated with pivaloyl
chloride (0.2 mL, 0.7 mmol) dropwise and stirred at
room temperature for 2 h. After 2 h of stirring, the
mixture was extracted with EtOAc several times. The
combined organic layers were dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel
with EtOAc/hexanes (1:4) as eluant to afford 27 as a
yellow oil (0.333 g, 52%); 1H NMR (CDCl3) d 7.40 (br s,
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1.25 (s, 9H, COC(CH3)3).
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