N-(3-acyloxy-2-benzylpropyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea analogues: Novel potent and high affinity antagonists and partial antagonists of the vanilloid receptor

Article abstract of DOI:10.1021/jm030089u

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N′-[3-fluoro-4- (methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K_i value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy2-benzylpropyl moieties in the C-region favored agonism.

Full text of DOI:10.1021/jm030089u

3116
J . Med. Chem. 2003, 46, 3116-3126
N-(3-Acyloxy-2-ben zylp r op yl)-N′-[4-(m eth ylsu lfon yla m in o)ben zyl]th iou r ea
An a logu es: Novel P oten t a n d High Affin ity An ta gon ists a n d P a r tia l An ta gon ists
of th e Va n illoid Recep tor
J eewoo Lee,*,† J iyoun Lee,† Myungshim Kang,† Myoungyoup Shin,† J i-Min Kim,† Sang-Uk Kang,† J u-Ok Lim,†
Hyun-Kyung Choi,† Young-Ger Suh,† Hyeung-Geun Park,† Uhtaek Oh,† Hee-Doo Kim,‡ Young-Ho Park,§
Hee-J in Ha,| Young-Ho Kim,| Attila Toth, Yun Wang, Richard Tran, Larry V. Pearce,
Daniel J . Lundberg, and Peter M. Blumberg
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku,
Seoul 151-742, Korea, College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Korea, AmorePacific R&D Center,
Yongin-Si, Kyounggi-Do 449-900, Korea, Digital Biotech, Ansan, Kyounggi-Do 425-839, Korea, and Laboratory of Cellular
Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892
Received February 24, 2003
Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists
with the alkylsulfonamido group provides a series of compounds which are effective antagonists
to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary
(CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N′-
[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin,
displayed a Ki value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX),
and showed excellent analgesic activity in mice. Structure-activity analysis of the influence
of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/
antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety
in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-
2-benzylpropyl moieties in the C-region favored agonism.
In tr od u ction
urologic problems such as detrusor hyperreflexia and
bladder hypersensitivity, and pruritus.1
The vanilloid receptor VR11 is a cation permeable ion
channel present on polymodal nociceptors that is acti-
vated by protons, heat,2 and ligands such as capsaicin
(CAP),3 the pungent ingredient in chilli peppers, and
resiniferatoxin (RTX),4 a natural diterpene isolated from
the cactuslike succulent Euphobia resinifera. VR1 has
been cloned from rat dorsal root ganglia (DRG)5 and
more recently from the human.6 It is a member of the
Trp family of channel proteins,7 oligomerizing as a
tetramer8 and functioning as a relatively nonselective
cation channel with some preference for calcium ions.9
The vanilloid receptor is expressed predominantly on
unmyelinated pain-sensing nerve fibers (C-fibers) and
small Αδ fibers in the dorsal root, trigeminal, and
nodose ganglia. The activation of the vanilloid receptor
by pungent agonists triggers cation influx resulting in
excitation of primary sensory neurons, action potentials,
and ultimately the central perception of pain. Following
excitation, the C-fiber sensory neurons may become
desensitized, and this desensitization forms a basis for
the therapeutic use of vanilloid receptor agonists.1,10
Potential therapeutic applications include chronic pain,
such as that associated with diabetic neuropathy, post-
herpetic neuralgia, arthritis and cluster headache,
A strategy complementary to desensitization of C-
fiber sensory neurons by VR1 agonists is the blocking
of pain-producing endogenous agonists by VR1 antago-
nists. Proof of principle for this approach has been pro-
vided with capsazepine and with some N-alkylglycine
trimers.11,12 Because of the long duration of desensitiza-
tion following treatment with VR1 agonists, antagonists
may be of particular utility where short-term blockade
of VR1 is desired. Whereas several agonists are cur-
rently marketed (e.g., capsaicin) or undergoing clinical
trial (e.g., resiniferatoxin,13 DA-5018,14 SDZ-24966515),
only a few VR1 antagonists have been described so far.
Capsazepine,16 the most extensively characterized, has
only modest potency and is somewhat nonspecific, also
antagonizing voltage-sensitive calcium channels and the
nicotinic cholinergic receptor.17,18 The N-alkylglycine
trimers appear to function as noncompetitive antago-
nists, presumably as channel blockers, with potencies
around 1 µM.12 Iodo-RTX, prepared semisynthetically
from RTX by iodination, was also reported to display
potent antagonism in rat and human VR1.19
The identification of RTX as a novel vanilloid with a
binding potency approximately 4 orders of magnitude
greater than that of capsaicin (RTX: Ki ) 0.13 nM,
CAP: Ki ) 1,700 nM in CHO/VR1)20 suggested a more
complex pharmacophore for vanilloids than that ap-
preciated previously. In the basis of part on previously
published SAR studies on RTX,21 we have proposed a
hypothetical pharmacophore model for interaction with
the capsaicin binding site of VR1 in which four func-
* To whom correspondence should be addressed. Phone: 82-2-
880-7846. Fax: 82-2-888-0649. E-mail: jeewoo@snu.ac.kr.
† Seoul National University.
‡ Sookmyung Women’s University.
§ AmorePacific R&D Center.
| Digital Biotech.
National Cancer Institute.
10.1021/jm030089u CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/30/2003

Antagonists of the Vanilloid Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3117
with the alkylsulfonamido group led to a series of potent
antagonists with high binding affinities. We have previ-
ously reported a different strategy for design of antago-
nists in which we incorporated our potent agonist
templates with bicycles such as tetrahydrobenzazepine
or tetrahydroisoquinoline as an A-region derived from
capsazepine.16 This approach afforded several novel
antagonists with potencies similar to or modestly better
than capsazepine; however, further modification of the
structures was problematic because their agonistic/
antagonistic character was very sensitive to small
modifications.26
In the present study, we have synthesized and
characterized in detail, using CHO cells heterologously
expressing rat VR1, a series of the potent and selective
VR1 antagonists having a N-4-alkylsulfonylamino-
benzyl thiourea template. We have also analyzed SAR
on the effect of A and C-regions in 4-alkylsulfonylamino
ligands and describe significant substitution aspects on
agonism/antagonism.
Ch em istr y
F igu r e 1.
The syntheses of target thioureas were achieved by
the general coupling methods between the correspond-
ing isothiocyanates and amines.22 The syntheses of
benzylamines with a different A-region were described
in Schemes1-3. 4-Alkylsulfonylamino and 4-acylamino-
benzylamines (13-20) were prepared from 4-amino-
benzylamine (3) in three steps in good yields as shown
in Scheme 1. 3-Methylsulfonylamino (24) and 4-(methyl-
sulfonylamino)methylbenzylamines (28) were also pre-
pared from the corresponding aminobenzyl alcohols (21,
25) in a conventional manner as shown in Scheme 2.
For the synthesis of 3-methoxy-4-(methylsulfonylamino)-
benzylamine as outlined in Scheme 3, 3-methoxy-4-
nitrobenzyl alcohol (29) was converted to the corre-
sponding N-Boc amine (31) in three steps. The nitro
group of 31 was reduced and then mesylated to furnish
33 whose protecting group was hydrolyzed to afford
amine 34. The synthesis of the 3-fluoro-4-(methyl-
sulfonylamino)benzylamine (35) was described in a
previous report.27 For the synthesis of sulfonamide
thiourea analogues, 2-(3,4-dimethylbenzyl)-3-pivaloyl-
oxypropyl isothiocyanate (39) was coupled with the
amines (13-20, 24, 28) to afford thioureas (40-49) as
shown in Scheme 5. For the synthesis of different A/C
region analogues as outlined in Scheme 6, 3-acyloxy-2-
benzylpropyl azides (50-53)26 were reduced under the
Lindler’s catalyzed hydrogenation and then coupled
with appropriate isothiocyanates (36-38), which were
prepared from the corresponding amines (13, 34, 35) by
the reaction of 1,1′-thiocarbonyldiimidazole as shown in
Scheme 4 to afford the desired thioureas (54-61).
tional groups, 4-hydroxy-3-methoxyphenyl, C20-ester,
C3-keto, and orthophenyl, represent principal pharma-
cophores. On the basis of this model, we have syn-
thesized and recently described the ultrapotent VR1
agonists 1 and 2, with Ki values of 19 nM and 11 nM,
respectively, in a [3H]RTX binding assay on DRG
neurons; the compounds are thus approximately 280-
and 480-fold more potent than capsaicin, respectively,
for receptor binding.22 The Novartis group3,24 has des-
ignated the structural regions of vanilloids as regions
A-C shown in Figure 1, and our current work involves
replacement of the isodecenoyl moiety of the C-region
with a 3-acyloxy-2-benzylpropyl moiety, which we de-
signed based on our RTX-derived pharmacophore model
and which represents a motif conferring significantly
enhanced binding affinity in RTX competition for VR1
compared to capsaicin.
The series of compounds described here arise out of
our ongoing program to find potent VR1 agonists and
antagonists. This series is based on the structural
modification on the A-region of the potent agonists, N-(3-
acyloxy-2-benzylpropyl)-N′-(4-hydroxy-3-methoxybenzyl)-
thioureas, which we had previously shown to have high
affinities for rat VR1.22 Extensive structure activity
studies on the capsaicinoids have indicated that the
4-phenolic hydroxyl group represents a crucial pharma-
cophore although it also contributes to the metabolic
lability in vivo.23,24 We reasoned that isosteric replace-
ment of the pungent phenolic OH group in our potent
agonists might maintain their strong affinities, change
their metabolic lability, and modify the biological char-
acteristic of the ligands. The alkylsulfonylamido group
has been utilized as a bioisostere of the phenolic OH
because the acid dissociation constant of the NH group
in alkylsulfonamides is close to that of the phenolic OH
group (both are approximately pKa ) 9). Its effectiveness
as a bioisostere implies that the NH portion of the
alkylsulfonamide hydrogen bonds with a receptor site
by aligning itself in a manner closely approximating the
phenolic OH group with respect to both bond distances
and bond angles.25 Consequently, the isosteric replace-
ment of the phenolic OH group in our potent agonists
Biologica l Resu lts
The agonistic and antagonistic activities of the syn-
thesized VR1 ligands were assessed in vitro by a 45Ca2+
uptake assay, which was carried out using rat VR1
heterologously expressed in Chinese hamster ovary cells
(CHO/VR1 cells) as previously described.20,27,28 The in
vitro antagonistic potencies of the compounds were
evaluated by measuring antagonism of the 45Ca2+
uptake induced by 50 nM capsaicin and expressed as
the Ki ( SEM, respectively, correcting for competition

3118 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Lee et al.
Sch em e 1
Sch em e 2
Sch em e 3
Sch em e 4
an agonist, compound 40 proved not to be a true, full
antagonist but rather retained weak efficacy as an
agonist, yielding about 7% of the 45Ca2+ uptake of a
maximally effective dose of capsaicin (Table 1). The
extent of the partial agonism depended on assay condi-
tions. The detailed characterization of this weak partial
agonism and its modulation by VR1 coregulators is
presented elsewhere.28 The receptor binding affinity of
40 yielded a Ki value of 29.3 nM, which was significantly
improved compared to capsazepine (Ki ) 1300 nM) and
was almost unchanged compared to that of the corre-
sponding agonist, 1. Compound 40 established that
replacement of the 4-hydroxyl with a 4-methylsulfon-
amido group in a vanilloid can change the biological
behavior of the ligand from agonist to antagonist while
preserving binding affinity.
by capsaicin. All compounds were also evaluated as
agonists. Potencies as agonists were expressed as EC50
( SEM, and absolute levels of 45Ca2+ 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 represent the
mean of at least three experiments. The results are
summarized in Table 1.
Replacement of the 4-phenolic OH group in our
previously reported high affinity and potent agonist 122
with a 4-methylsulfonamido group provided a potent
antagonist 40 which inhibited the 45Ca2+ uptake in-
duced by capsaicin with a Ki of 67 nM. The antagonistic
potency of 40 was thus approximately 10-fold better
than that of capsazepine (Ki ) 520 nM). Evaluated as
To determine the optimal size of the alkyl group of
the 4-alkylsulfonamide, derivatives with alkyl groups
of ethyl, propyl, isopropyl, and phenyl (41-44) were
prepared. Their binding affinities and antagonistic
potencies gradually decreased with increasing size
although they showed partial agonism as did 40. Indeed,
for the 4-phenylsulfonylamino analogue (44), the an-
tagonistic activity was completely abolished and the
binding affinity was found to be weak. These results

Antagonists of the Vanilloid Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3119
Sch em e 5
Sch em e 6
Ta ble 1. Potencies of Vanilloid Ligands for Binding to Rat
VR1 and for Inducing Calcium Influx in CHO/VR1 Cells
groups. The 4-trifluoromethylsulfonylamino analogue
(45) exhibited little binding affinity to the receptor. In
this case, the size of the alkyl group was almost the
same as that for the methylsulfonylamino group, and
the dramatic loss in receptor potency may be attributed
to the markedly reduced pKa value of the NH group in
NHSO2CF3 (calculated pKa ) 9.47 for NHSO2CH3; pKa
) 4.83 for NHSO2CF3 in a substituted benzene) due to
a strong withdrawing effect of CF3. As another isostere
of the phenolic OH, the amide analogues (46, 47) of the
potent agonist, 1, were also characterized. However,
they were devoid of any antagonism in blocking the
entry of calcium induced by capsaicin and proved to be
pure agonists with moderate binding affinities. To dis-
cern the positional importance of the 4-methylsulfonyl-
amino in 40 for antagonism, the corresponding 3-
methylsulfonylamino analogue (48) and one-carbon
elongated analogue (49) were examined; they proved to
be full agonists with binding affinities similar to cap-
saicin. These results indicate that the binding and
antagonistic potency of 40 is attenuated by the above
structural and positional modifications on the 4-
methylsulfonylamino group.
Ki (nM)
binding affinity
EC50 (nM)
agonism
Ki(nM)
antagonism
capsaicin
capsazepine
1800 ((270)
1300 ((150)
17.4 ((4.1)
6.35 ((0.48)
29.3 ((7.6)
108 ((11)
44.8 ((3.8)
NE
NE
520 ((12)
NEa
1
2
1.97 ((0.56)
2.83 ((0.55)
WEb
NE
40
41
42
43
44
45
46
47
48
49
54
55
56
57
58
59
60
61
67 ((25)
309 (( 91)
940 ((130)
WE
NE
NE
NE
NE
NE
NE
86 ((17)
NE
WEb
313 ((52)
WEb
227 ((40)
790 ((210)c
VWE
2500 ((660)
15.5% at 10 µM
1700 ((500)
2600 ((780)
355 ((61)
VWE
450 ((140)
1140 ((260)
990 ((79)
1700 ((150)
WEb
3000 ((980)
64 ((21)
410 ((110)
239 ((69)
1730 ((450)c
2790 ((860)b
22 ((10)c
40 ((11)
166 ((15)
252((13)
NE
NE
NE
NE
NE
49 ((15)
25.2 ((6.9)
148 ((19)
77.2 ((7.5)
54 ((28)
NE
7.8 ((3.0)
a
NE: not effective, VWE: very weakly effective, WE: weakly
b
effective at 30 µM. Only fractional calcium uptake compared to
Starting with the lead antagonist 40, we explored
structural modifications in the C region and elsewhere
in the A region. Substitution of the 3,4-dimethylphenyl
on 40 with the 4-tert-butylphenyl to provide 54 (Ki )
86 nM) resulted in a little decrease in the antagonistic
potencies. Likewise, the binding affinity of 54 with a Ki
value of 64 nM was reduced by 2-fold relative to 40. This
300 nM capsaicin (38, 7%; 39, 14%; 40, 15%; 52, 30%). c Only
fractional calcium uptake compared to 300 nM capsaicin (23-59%).
suggest that the capsaicin binding site of the receptor
probably possesses a small pocket for the phenolic OH
group and is subject to steric interference by bulky

3120 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Lee et al.
F igu r e 2. Comparison of the analgesic activities of compound
40 and 61 with ketorolac in the acetic acid-induced writhing
model in mice.
finding is somewhat different from our previous obser-
vation22 that a 4-tert-butylphenyl-substituted agonist,
2, exhibited a higher affinity than the corresponding 3,4-
dimethylphenyl-substituted derivative, 1, as shown in
Table 1. Like compound 40, compound 54 showed
partial agonism, 30% of the level of calcium uptake
induced by 300 nM capsaicin. This difference of partial
agonism is consistent with effects of C-region modifica-
tions on the pattern of antagonism for this template.
Incorporation of a 3-methoxy group on compounds 40
and 54 provided 57 and 58, which were found to be
agonists, without antagonistic activity, and with good
potency. The EC50 values were 22 nM and 40 nM,
respectively. The binding affinities of 57 and 58, with
Ki values of 49 nM and 25.2 nM, were 2-fold weaker
and 3-fold stronger than those of 40 and 54, respectively.
A structural comparison between 40 and 57 (or 54 and
58) reveals that the 3-methoxy group on the A-region
plays an important role in eliciting VR1 agonistic
activity. It is noteworthy that in the structure-activity
studies of a series of capsaicinoids, a 3-methoxy group
on the A-region proved to be a crucial pharmacophore
in terms of agonism in the calcium uptake assay. For
example, the 3-demethoxy analogue of N-octylhomo-
vanillic amide was ca. 20-fold less potent than that of
the parent compound.3 Replacement of 3-pivaloyl groups
of 40, 54, 57, and 58 with 3-benzoyl groups, compounds
55, 56, 59 and 60, respectively, led to a decrease in their
binding affinities and, for the first two, a shift toward
greater agonism. It should be noted that several of the
compounds in this series, namely 55, 56, and 57,
induced a lower level of calcium uptake than did 300
nM capsaicin. This did not represent partial agonism,
in that no corresponding activity was observed in assays
for antagonism. The mechanistic basis for this behavior
remains under investigation.
F igu r e 3. The SAR diagram of N-[4-(methylsulfonylamino)-
benzyl]thiourea analogues.
pound 40. Furthermore, unlike 40, it did not show
residual weak efficacy as an agonist. We conclude that
the 3-fluoro-4-methylsulfonylaminophenyl group is a
promising antagonistic pharmacophore in the A-region.
The analgesic activities of potent antagonists (40 and
61) were evaluated in the acetic acid (AA)-induced
writhing model in mice, following the previously re-
ported procedure,29 and were compared to that of the
clinically used analgesic ketorolac (Figure 2). Whereas
compound 40 showed a comparable potency (ED50
)
2,620 ( 2380 µg/kg) relative to ketorolac (ED50 ) 2800
( 1400 µg/kg), compound 61 exhibited dramatically
enhanced analgesic potency, with an ED50 of 7.43 ( 6.4
µg/kg that was approximately 350-fold more potent than
that of compound 40.
Discu ssion
Recently, we have demonstrated detailed biochemical
characterization of N-(4-tert-butylbenzyl)-N′-[4-(methyl-
sulfonylamino)benzyl]thiourea, its 3-fluoro and 3-meth-
oxy analogues as VR1 antagonists or partial ago-
nists.27,28 Structurally, they have the same A and
B-regions relative to the VR1 ligands described above;
however, their C-region contains a 4-tert-butylbenzyl
group different from the 3-acyloxy-2-benzylpropyl moi-
ety in above compounds. To investigate the effect on
VR1 agonism/antagonism of 3-substituents in the A re-
gion and their modulation by the C-regions in 4-methyl-
sulfonamide ligands, we compared the structure-activ-
ity relationships of two series of N-4-methylsulfonyl-
amino thioureas. Their structures and biochemical
properties as VR1 ligands are described in Figure 3.
Their pattern of activity was consistent with our emerg-
ing understanding of vanilloid structure-activity rela-
tionships. Our findings can be summarized as follows:
When, as a replacement of the 3-methoxy group, a
3-fluoro group was introduced to produce compound 61,
there was little change in its affinity as measured in
the binding assay (Ki of 54 nM compared to 49 nM for
57). On the other hand, consistent with the important
role of the 3-methoxy group for agonism, its replacement
with a 3-fluoro group generated a potent antagonist,
with a Ki of 7.8 nM. 61 thus represents a significant
improvement in antagonist potency compared to com-

Antagonists of the Vanilloid Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3121
mesh, Merck. Proton NMR spectra were recorded on a J EOL
J NM-LA 300 at 300 MHz. Chemical shifts are reported in ppm
units with Me4Si as a reference 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, and were within 0.4% of the calculated values
unless otherwise noted.
ter t-Bu tyl N-(4-Am in oben zyl)ca r ba m a te (4). A mixture
of 4-aminobenzylamine (3) (10 g, 82 mmol) and di-tert-butyl
dicarbonate (20 g, 90 mmol) in THF (100 mL) was stirred for
2 h at room temperature and concentrated in vacuo. The
residue was purified by flash column chromatography over
silica gel with hexane/ethyl acetate (3:2) as eluant to afford 4
as a yellow solid (17.5 g, 96%); mp ) 74-75 °C; 1H NMR
(CDCl3) δ 7.06 (d, 2 H, J ) 8.3 Hz, H-2,6), 6.63 (dt, 2 H, J )
2.7, 8.3 Hz, H-3,5), 4.73 (bs, 1 H, NHBoc), 4.18 (d, 2 H, J )
5.6 Hz, CH2NH), 3.60 (bs, 2 H, NH2), 1.45 (s, 9 H, C(CH3)3).
Gen er a l P r oced u r e for th e Syn th esis of 5-12. A cooled
solution of 4 (5 mmol) at 0 °C in pyridine (10 mL) was treated
with the corresponding acyl chloride (6 mmol) and stirred for
16 h at room temperature. The reacton mixture was cooled in
an ice-bath, carefully neutralized with 1 M hydrochloric acid
solution, diluted with water, and extracted with dichloro-
methane several times. The combined organic layers were
washed with water and brine, dried over magnesium sulfate,
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 5-12.
ter t-Bu tyl N-[(4-m eth ylsu lfon yla m in o)ben zyl]ca r ba m -
a te (5): 92% yield, 1H NMR (CDCl3) δ 7.28 (d, 2 H, J ) 8.3
Hz, H-2,6), 7.18 (dd, 2 H, J ) 2.2, 6.3 Hz, H-3,5), 6.76 (s, 1 H,
NHSO2), 4.88 (bs, 1 H, NHBoc), 4.28 (d, 2 H, J ) 5.6 Hz,
CH2NH), 2.99 (s, 3 H, SO2CH3), 1.46 (s, 9 H, C(CH3)3).
(1) we have demonstrated that substitution of the
4-phenolic hydroxyl of the A-region in agonists with an
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. (2) Although the A-region makes a
major contribution to the extent of agonism/antagonism,
it is clear that the C-region also contributes. Thus, the
N-(4-tert-butylbenzyl) moiety (top series in Figure 3)
tended to give more extensive antagonism than did the
N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl] moiety
(bottom series in Figure 3).
In the 4-tert-butylbenzyl series (top series), the re-
placement of 4-hydroxy-3-methoxyphenyl in a potent
and full agonist (65) previously reported by Wriggles-
worth et al.23 with 4-methylsulfonamide produced a full
antagonist (63). Interestingly, compound 63 was a full
antagonist of capsaicin stimulation but was only a
partial antagonist for stimulation by temperature or
pH.27 The addition of a 3-fluoro substituent to the
A-region in 63 further enhanced antagonism, as evi-
denced by compound 62 being a full antagonist for
stimulation not only by capsaicin but also by tempera-
ture (44 °C) or pH 6.0.27 It is noted that the 3-fluoro-4-
hydroxyphenylacetamide analogue, generated by the
replacement of 3-methoxy in 65 with 3-fluoro, was
shown to have 10-fold less agonistic potency in calcium
influx than the corresponding 3-methoxy analogue but
was a weak partial agonist with ca. 40% efficacy.3
Conversely, the incorporation of a 3-methoxy substitu-
ent to the A-region in 63 enhanced agonism, indicating
that compound 64 proved to be a partial agonist like
40.28 Recently, we have reported in detail the biochemi-
cal characterization of 40 and compound 64 as partial
agonists, including the demonstration that their frac-
tional efficacy reflects their functioning as partial ago-
nists with a corresponding degree of partial antago-
nism.28
In a similar manner, in the 2-(3,4-dimethylbenzyl)-
3-(pivaloyloxy)propyl series (bottom series), in which 40
showed a low level of partial agonism, the corresponding
derivative with a 4-hydroxy-3-methoxyphenyl group in
the A-region was a full and potent agonist (compound
1) as reported previously.22 Conversely, the addition of
a 3-fluoro group rendered it a full antagonist (61). The
incorporation of a 3-methoxy substituent to 40 shifted
its partial agonism to the full agonist 57.
In conclusion, we investigated the isosteric replace-
ment of the phenolic hydroxyl group in potent VR1
agonists with alkylsulfonamido groups and their struc-
ture-activity relationships as agonists and antagonists.
We obtained compounds which are antagonists of cap-
saicin in CHO cells heterologously expressing VR1.
Compound 61 with a 3-fluoro-4-methylsulfonylamino
pattern of substitution in the A-region represents a
potent and full VR1 antagonist. In vivo, it produced an
excellent analgesic effect in the acetic-acid induced
writhing test compared to ketorolac and 40 with partial
agonism.
ter t-Bu tyl N-[(4-eth ylsu lfon yla m in o)ben zyl]ca r ba m a te
(6): 73% yield, 1H NMR (CDCl3) δ 7.25 (d, 2 H, J ) 8.5 Hz,
H-2,6), 7.17 (d, 2 H, J ) 8.5 Hz, H-3,5), 6.85 (s, 1 H, NHSO2),
4.89 (bs, 1 H, NHBoc), 4.28 (d, 2 H, J ) 5.6 Hz, CH2NH), 3.09
(q, 2 H, J ) 7.2 Hz, SO2CH2CH3), 1.46 (s, 9 H, C(CH3)3), 1.34
(t, 3 H, J ) 7.2 Hz, SO2CH2CH3).
ter t-Bu tyl N-[(4-pr opylsu lfon ylam in o)ben zyl]car bam ate
(7): 99% yield, 1H NMR (CDCl3) δ 7.25 (d, 2 H, J ) 8.5 Hz,
H-2,6), 7.17 (d, 2 H, J ) 8.5 Hz, H-3,5), 6.85 (s, 1 H, NHSO2),
4.89 (bs, 1 H, NHBoc), 4.28 (d, 2 H, J ) 5.6 Hz, CH2NH), 3.05
(t, 2 H, J ) 7.8 Hz, SO2CH2CH2), 1.85 (m, 2 H, SO2CH2CH2),
1.46 (s, 9 H, C(CH3)3), 1.01 (t, 3 H, J ) 7.6 Hz, CH3).
ter t-Bu t yl N-[(4-isop r op ylsu lfon yla m in o)b en zyl]ca r -
1
ba m a te (8): 78% yield, H NMR (CDCl3) δ 7.25 (d, 2 H, J )
8.3 Hz, H-2,6), 7.17 (d, 2 H, J ) 8.3 Hz, H-3,5), 6.85 (s, 1 H,
NHSO2), 4.87 (bs, 1 H, NHBoc), 4.27 (d, 2 H, J ) 5.6 Hz,
CH2NH), 3.28 (m, 1 H, SO2CH), 1.46 (s, 9 H, C(CH3)3), 1.38
(d, 6 H, J ) 6.8 Hz, CH(CH3)3).
ter t-Bu tyl N-[(4-p h en ylsu lfon yla m in o)ben zyl]ca r ba m -
a te (9): 78% yield, 1H NMR (CDCl3) δ 7.75 (d, 2 H, J ) 8.5
Hz, Ph), 7.54 (t, 1 H, J ) 7.3 Hz, Ph), 7.44 (t, 2 H, J ) 7.8 Hz,
Ph), 7.15 (d, 2 H, J ) 8.5 Hz, H-2,6), 7.02 (d, 2 H, J ) 8.5 Hz,
H-3,5), 6.68 (s, 1 H, NHSO2), 4.80 (bt, 1 H, NHBoc), 4.23 (d, 2
H, J ) 5.8 Hz, CH2NH), 1.44 (s, 9 H, C(CH3)3).
ter t-Bu tylN-[(4-tr iflu or om eth ylsu lfon yla m in o)ben zyl]-
1
ca r ba m a te (10): 15% yield, H NMR (CDCl3) δ7.46 (d, 2 H,
J ) 8.3 Hz, H-2,6), 7.28 (d, 2 H, J ) 8.3 Hz, H-3,5), 4.98 (bs,
1 H, NHBoc), 4.30 (d, 2 H, J ) 5.8 Hz, CH2NH), 1.47 (s, 9 H,
C(CH3)3).
ter t-Bu t yl N-[(4-a cet yla m in o)b en zyl]ca r ba m a t e (11):
96% yield, 1H NMR (CDCl3) δ 7.45 (d, 2 H, J ) 8.3 Hz, H-2,6),
7.23 (d, 2 H, J ) 8.3 Hz, H-3,5), 4.81 (bs, 1 H, NHBoc), 4.27
(d, 2 H, J ) 5.1 Hz, CH2NH), 2.18 (s, 3 H, COCH3), 1.46 (s, 9
H, C(CH3)3).
ter t-Bu tyl N-[(4-isobu tyla m in o)ben zyl]ca r ba m a te (12):
99% yield, 1H NMR (CDCl3) δ 7.48 (d, 2 H, J ) 8.3 Hz, H-2,6),
7.23 (d, 2 H, J ) 8.3 Hz, H-3,5), 7.11 (s, 1 H, NHSO2), 4.79
(bs, 1 H, NHBoc), 4.26 (d, 2 H, J ) 5.1 Hz, CH2NH), 2.50 (m,
1 H, COCH), 1.46 (s, 9 H, C(CH3)3), 1.25 (d, 6 H, J ) 6.8 Hz,
CH(CH3)2).
Exp er im en ta l Section
Gen er a l Meth od . All chemical reagents were commercially
available. Melting points were determined on a Melting Point
Bu¨chi B-540 apparatus and are uncorrected. Silica gel column
chromatography was performed on silica gel 60, 230-400

3122 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Lee et al.
Gen er a l p r oced u r e for th e syn th esis of 13-20. A cooled
solution of 5-12 (4 mmol) in dichloromethane (20 mL) in an
ice-bath was slowly treated with trifluoroacetic acid (5 mL)
and stirred for 1.5 h at 0 °C. The mixture was carefully
concentrated in vacuo to give 13-20 in a quantitative yield.
The amine salts were washed with ethyl ether and used for
the next step without further purification.
N-[3-(Am in om eth yl)p h en yl]m eth a n esu lfon a m id e (24).
A suspension of 23 (100 mg, 0.442 mmol) and palladium on
carbon (20 mg) in MeOH (5 mL) was hydrogenated under a
balloon of hydrogen for 2 h. The reaction mixture was filtered,
and the filtrate was concentrated in vacuo to give the corre-
sponding amine (88 mg, 99%) as a white solid which was pure
enough to be used for the next step.
[4-(Am in om eth yl)p h en yl]m eth a n ol (25). To a suspen-
sion of lithium aluminum hydride (0.58 g, 15.25 mmol) in THF
(40 mL) was added dropwise a solution of 4-cyanobenzaldehyde
(0.5 g, 3.8 mmol) in THF (10 mL), and the mixture was
refluxed for 5 h. The reaction mixture was cooled in an ice
bath, quenched by successive addition of H2O (15 mL), 15%
aqueous NaOH (3 mL), and H2O (20 mL), and stirred for 30
min. The resulting suspension was filtered, and the residue
was washed with CH2Cl2. The combined filtrate and washings
were washed successively with H2O and brine, dried over
MgSO4, and concentrated in vacuo to give 25 as a solid (0.5 g,
96%), which was used for the next step without further
purification: 1H NMR (CDCl3) δ 7.33 (dd, 4 H, Ar), 4.69 (s, 2
H, CH2OH), 3.88 (s, 2 H, CH2NH2).
N-[4-(Ch lor om eth yl)ben zyl]m eth a n esu lfon a m id e (26).
The compound was prepared from 25 by following the proce-
dure described for the synthesis of 22 as a white solid in 52%
yield: mp ) 75 °C; 1H NMR (CDCl3) δ 7.35 (dd, 4 H, Ar), 4.58
(s, 2 H, CH2Cl), 4.34 (s, 2 H, CH2NH2SO2), 2.91 (s, 3 H,
NHSO2CH3); IR (KBr) 3235 (NH), 1302, 1134 (NHSO2)
N-[4-(Azid om eth yl)ben zyl]m eth a n esu lfon a m id e (27).
The compound was prepared from 26 by following the proce-
dure described for the synthesis of 23 as an oil in 78% yield:
1H NMR (CDCl3) δ 7.35 (dd, 4 H, Ar), 4.35 (m, 4 H, CH2N3
and CH2NH2SO2), 2.90 (s, 3 H, NHSO2CH3); IR (KBr) 3266
(NH), 2101 (N3), 1305, 1424 (NHSO2)
N-[4-(Am in om eth yl)p h en yl]m eth a n esu lfon a m id e tr i-
flu or oa ceta te (13): 1H NMR (DMSO-d6) δ 9.87 (s, 1 H,
NHSO2), 8.14 (bs, 3 H, NH3), 7.40 (d, 2 H, J ) 8.5 Hz, H-2,6),
7.22 (d, 2 H, J ) 8.5 Hz, H-3,5), 3.97 (s, 2 H, CH2), 2.99 (s, 3
H, SO2CH3).
N-[4-(Am in om et h yl)p h en yl]et h a n esu lfon a m id e t r i-
flu or oa ceta te (14): 1H NMR (DMSO-d6) δ 9.92 (s, 1 H,
NHSO2), 8.21 (bs, 3 H, NH3), 7.39 (d, 2 H, J ) 8.5 Hz, H-2,6),
7.22 (d, 2 H, J ) 8.5 Hz, H-3,5), 3.96 (s, 2 H, CH2), 3.08 (q, 2
H, J ) 7.3 Hz, SO2CH2CH3), 1.17 (t, 3 H, J ) 7.3 Hz,
SO2CH2CH3).
N-[4-(Am in om eth yl)ph en yl]-1-pr opan esu lfon am ide tr i-
flu or oa ceta te (15): 1H NMR (DMSO-d6) δ 9.92 (s, 1 H,
NHSO2), 8.21 (bs, 3 H, NH3), 7.39 (d, 2 H, J ) 8.5 Hz, H-2,6),
7.21 (d, 2 H, J ) 8.5 Hz, H-3,5), 3.96 (s, 2 H, CH2), 3.06 (t, 2
H, J ) 7.5 Hz, SO2CH2CH2), 1.66 (m, 2 H, SO2CH2CH2), 0.91
(t, 3 H, J ) 7.3 Hz, SO2CH2CH3).
N-[4-(Am in om eth yl)ph en yl]-2-pr opan esu lfon am ide tr i-
flu or oa ceta te (16): 1H NMR (DMSO-d6) δ 9.88 (s, 1 H,
NHSO2), 8.17 (bs, 3 H, NH3), 7.38 (d, 2 H, J ) 8.4 Hz, H-2,6),
7.24 (d, 2 H, J ) 8.4 Hz, H-3,5), 3.95 (s, 2 H, CH2), 3.20 (m, 1
H, SO2CH), 1.22 (d, 6 H, J ) 6.6 Hz, CH(CH3)2).
N-[4-(Am in om et h yl)p h en yl]b en zen esu lfon a m id e t r i-
flu or oa ceta te (17): 1H NMR (DMSO-d6) δ 8.11 (bs, 3 H, NH3),
7.78 (dd, 2 H, J ) 1.5, 6.8 Hz, Ph), 7.5-7.65 (m, 3 H, Ph), 7.28
(d, 2 H, J ) 8.4 Hz, H-2,6), 7.11 (d, 2 H, J ) 8.4 Hz, H-3,5),
3.89 (s, 2 H, CH2).
N-[4-(Am in om et h yl)p h en yl]t r iflu or om et h a n esu lfon -
a m id e tr iflu or oa ceta te (18): 1H NMR (DMSO-d6) δ 8.12 (bs,
3 H, NH3), 7.28 (d, 2 H, J ) 8.4 Hz, H-2,6), 7.11 (d, 2 H, J )
8.4 Hz, H-3,5), 3.98 (s, 2 H, CH2).
N-[4-(Am in om eth yl)ben zyl]m eth a n esu lfon a m id e (28).
The compound was prepared from 27 by following the proce-
dure described for the synthesis of 24 as a solid in a quantita-
tive yield and was directly used for the next step.
3-Meth oxy-4-n itr oben zyl Azid e (30). A cooled solution of
3-methoxy-4-nitrobenzyl alcohol (29, 1 g, 5.46 mmol) and
triphenylphosphine (2.146 g, 8.19 mmol) in DMF (30 mL) at 0
°C was treated with carbon tetrabromide (2.716 g, 8.19 mmol)
and stirred for 10 min at 0 °C. The reaction mixture was
treated with sodium azide (1.065 g, 16.38 mmol) and 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 magnesium sulfate, and filtered. The filtrate was
concentrated in vacuo, and the residue was purified by flash
column chromatography over silica gel with EtOAc/hexanes
(1:2) as eluant to afford 30 as a yellow solid (1.055 g, 93%):
N-[4-(Am in om et h yl)p h en yl]a cet a m id e t r iflu or oa ce-
1
ta te (19): H NMR (D2O) δ 7.50 (d, 2 H, J ) 8.5 Hz, H-2,6),
7.46 (d, 2 H, J ) 8.5 Hz, H-3,5), 4.18 (s, 2 H, CH2), 2.70 (m, 1
H, SO2CH), 1.21 (d, 6 H, J ) 7.1 Hz, CH(CH3)2).
N-[4-(Am in om eth yl)p h en yl]-2-m eth ylp r op a n a m id e tr i-
flu or oa ceta te (20): 1H NMR (D2O) δ 7.52 (d, 2 H, J ) 8.5
Hz, H-2,6), 7.46 (d, 2 H, J ) 8.5 Hz, H-3,5), 4.18 (s, 2 H, CH2),
2.19 (s, 3 H, COCH3).
N-[3-(Ch lor om eth yl)p h en yl]m eth a n esu lfon a m id e (22).
A cooled suspension of 3-aminobenzyl alcohol (21) (0.2 g, 1.6
mmol), lithium chloride (0.136 g, 3.2 mmol), and 2,6-lutidine
(0.410 g, 3.52 mmol) in DMF (5 mL) at 0 °C was treated
dropwise with methanesulfonyl chloride (0.272 g, 3.52 mmol)
and stirred for 10 min. After being stirred for 5 h at room
temperature, the reaction mixture was diluted with H2O and
extracted with ether several times. The combined organic layer
was washed successively with H2O and brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified
by flash column chromatography over silica gel with EtOAc/
hexanes (3:1) as eluent to afford 22 (0.27 g, 76%) as a white
solid: mp ) 99.5 °C; 1H NMR (CDCl3) δ 7.15-7.4 (m, 4 H,
Ar), 6.38 (bs, 1 H, NH), 4.57 (s, 2 H, CH2Cl), 3.04 (s, 3 H,
SO2CH3); IR (KBr) 3258 (NH), 1326, 1149 (SO2N) cm-1
N-[3-(Azid om eth yl)p h en yl]m eth a n esu lfon a m id e (23).
A mixture of 22 (0.279 g, 1.27 mmol) and sodium azide (0.247
g, 3.81 mmol) in DMF (5 mL) was refluxed for 7 h. The reaction
mixture was cooled to room temperature, diluted with H2O
and extracted with ethyl acetate several times. The combined
organic layer was washed successively with H2O and brine,
dried over MgSO4, and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel with
EtOAc/hexanes (3:1) as eluent to afford 23 (0.273 g, 95%) as
an oil.;1H NMR (CDCl3) δ 7.15-7.4 (m, 4 H, Ar), 6.46 (bs, 1 H,
NH), 4.36 (s, 2 H, CH2N3), 3.04 (s, 3 H, SO2CH3); IR (neat)
3265 (NH), 2101 (N3), 1326, 1150 (SO2N) cm-1
1
mp ) 35-37 °C, H NMR (CDCl3) δ 7.87 (d, 1 H, J ) 8.3 Hz,
H-5), 7.04 (s, 1 H, H-2), 6.97 (dd, 1 H, J ) 1.7, 8.3 Hz, H-6),
4.45 (s, 2 H, CH2N3), 3.99 (s, 3 H, OCH3).
ter t-Bu tyl N-(3-Meth oxy-4-n itr oben zyl)ca r ba m a te (31).
A mixture of 30 (0.833 g, 4 mmol), triphenylphosphine (1.573
g, 6 mmol), and water (0.144 g, 8 mmol) in tetrahydrofuran
(30 mL) was stirred for 24 h at room temperature and
concentrated in vacuo. The residue was dissolved in ethanol
(20 mL) and treated with di-tert-butyl dicarbonate (1.746 g, 8
mmol). After being stirred for 2 h at room temperature, the
mixture was concentrated in vacuo, and the residue was
purified by flash column chromatography over silica gel with
EtOAc/hexanes (1:2) as eluant to afford 31 as a yellow oil
(0.971 g, 86%): 1H NMR (CDCl3) δ 7.84 (d, 1 H, J ) 8.3 Hz,
H-5), 7.02 (s, 1 H, H-2), 6.93 (dd, 1 H, J ) 8.3 Hz, H-6), 4.99
(bs, 1 H, NHBoc), 4.35 (d, 2 H, J ) 6.1 Hz, CH2NH), 3.96 (s,
3 H, OCH3), 1.47 (s, 9 H, C(CH3)3).
ter t-Bu tyl N-(4-Am in o-3-m eth oxyben zyl)car bam ate (32).
A suspension of 31 (0.847 g, 3 mmol) and 10% palladium on
carbon (0.085 g) in methanol (20 mL) was hydrogenated under
a balloon of hydrogen for 1 h at room temperature. The mixture
was filtered and concentrated in vacuo. The residue was
purified by flash column chromatography over silica gel with
EtOAc/hexanes (1:1) as eluant to afford 32 as a white solid

Antagonists of the Vanilloid Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3123
(0.684 g, 90%): mp ) 76-77.5 °C; 1H NMR (CDCl3) δ 6.6-
6.75 (m, 3 H, Ar), 4.20 (d, 2 H, J ) 5.6 Hz, CH2NH), 3.84 (s,
3 H, OCH3), 1.46 (s, 9 H, C(CH3)3).
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 magnesium sulfate,
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 in 85-95% yields.
ter t-Bu tyl N-[3-Meth oxy-4-(m eth ylsu lfon yla m in o)ben -
zyl]ca r ba m a te (33). A cooled solution of 32 (0.634 g, 2.5
mmol) in pyridine (5 mL) at 0 °C was treated with methane-
sulfonyl chloride (0.23 mL, 3 mmol) and stirred for 16 h at
room temperature. The reaction mixture was cooled in an ice-
bath, carefully neutralized with 1 M hydrochloric acid solution,
diluted with water, and extracted with dichloromethane
several times. The combined organic layers were washed with
water and brine, dried over magnesium sulfate, 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 33 as a white solid
(0.595 g, 72%): mp ) 125.5-127.5 °C; 1H NMR (CDCl3)) δ 7.46
(d, 1 H, J ) 8.5 Hz, H-5), 6.86 (m, 2 H, H-2,6), 6.74 (s, 1 H,
NHSO2), 4.88 (bs, 1 H, NHBoc), 4.28 (d, 2 H, J ) 5.8 Hz,
CH2NH), 3.88 (s, 3 H, OCH3), 2.94 (s, 3 H, SO2CH3), 1.47 (s, 9
H, C(CH3)3).
3-Meth oxy-4-(m eth ylsu lfon yla m in o)ben zyla m in e Tr i-
flu or oa ceta te (34). A cooled solution of 33 (1 mmol) in
dichloromethane (4 mL) in an ice-bath was slowly treated with
trifluoroacetic acid (1 mL) and stirred for 1.5 h at 0 °C. The
mixture was carefully concentrated in vacuo to give 34 as a
white solid in a quantitative yield. The amine salts were
washed with ethyl ether and used for the next step without
further purification, respectively: mp ) 161-163.5 °C; 1H
NMR (DMSO-d6) δ 8.16 (bs, 3 H, NH3), 7.28 (d, 1 H, J ) 8.0
Hz, H-5), 7.20 (s, 1 H, H-2), 6.99 (d, 1 H, J ) 8.0 Hz, H-6),
4.00 (s, 2 H, CH2NH3), 3.82 (s, 3 H, OCH3), 2.95 (s, 3 H,
SO2CH3).
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(m eth ylsu lfon ylam in o)ben zyl]th iou r ea (40): white solid,
mp ) 47 °C; 1H NMR (CDCl3) δ 7.29 (d, 2 H, J ) 8.3 Hz), 7.18
(d, 2 H, J ) 8.3 Hz), 6.85-7.05 (m, 3 H), 6.68 (s, 1 H, NHSO2),
6.34 (m, 1 H, NHCS), 6.09 (bs, 1 H, NHCS), 4.51 (bs, 2 H,
CSNHCH2Ar), 4.17 (m, 1 H, CH2OCO), 3.7-3.85 (m, 2 H,
CH2OCO and CHCH2NHCS), 3.22 (m, 1 H, CHCH2NHCS),
2.99 (s, 3 H, SO2CH3), 2.5-2.7 (m, 2 H, CH2Ar), 2.2-2.4 (m, 7
H, 2 × CH3 and CH), 1.23 (d, 9 H, J ) 3.9 Hz, C(CH3)3); MS
(FAB) m/z 520 (MH+); Anal. (C26H37N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(eth ylsu lfon yla m in o)ben zyl]th iou r ea (41): white solid,
mp ) 48 °C; 1H NMR(CDCl3) δ 7.25 (d, 2 H, J ) 8.6 Hz), 7.16
(d, 2 H, J ) 8.6 Hz), 6.85-7.1 (m, 3 H), 6.40 (m, 1 H, NH),
6.21 (bs, 1 H, NH), 4.50 (bs, 2 H, CSNHCH2Ar), 4.15 (m, 1 H,
CH2OCO), 3.65-3.85 (m, 2 H, CH2OCO and CHCH2NHCS),
3.24 (m, 1 H, CHCH2NHCS), 3.09 (q, 3 H, J ) 7.2 Hz,
SO2CH2CH3), 2.5-2.7 (m, 2 H, CH2Ar), 2.15-2.4 (m, 7 H, 2 ×
CH3 and CH), 1.34 (t, 3 H, J ) 7.2 Hz, SO2CH2CH3), 1.24 (d,
9 H, J ) 3.9 Hz, C(CH3)3); MS (FAB) m/z 534 (MH+); Anal.
(C27H39N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(pr opylsu lfon ylam in o)ben zyl]th iou r ea (42): white solid,
mp ) 54 °C; 1H NMR(CDCl3) δ 7.25 (d, 2 H, J ) 8.6 Hz), 7.15
(d, 2 H, J ) 8.6 Hz), 6.85-7.1 (m, 3 H), 6.45 (bs, 1 H, NH),
6.30 (bs, 1 H, NH), 4.51 (bs, 2 H, CSNHCH2Ar), 4.15 (m, 1 H,
CH2OCO), 3.7-3.85 (m, 2 H, CH2OCO and CHCH2NHCS),
3.24 (m, 1 H, CHCH2NHCS), 3.04 (t, 3 H, SO2CH2CH2CH3),
2.5-2.7 (m, 2 H, CH2Ar), 2.15-2.4 (m, 7 H, 2 × CH3 and CH),
1.82 (m, 2 H, SO2CH2CH2CH3), 1.24 (d, 9 H, J ) 3.7 Hz,
C(CH3)3), 1.00 (t, 3 H, SO2CH2CH2CH3); MS (FAB) m/z 548
(MH+); Anal. (C28H41N3O4S2) C, H, N, S.
3-F lu or o-4-(m et h ylsu lfon yla m in o)b en zyla m in e (35).
This compound was prepared from 2-fluoro-4-iodoaniline in
three steps by our previously reported procedure.27
Gen er a l P r oced u r e for th e Syn th esis of Isoth iocya n -
a te. Meth od A. A solution of amine salt (10 mmol) in DMF
(5 mL) was treated with triethylamine (1.4 mL, 10 mmol),
stirred for 1 h at room temperature, and added with 1,1′-
thiocarbonyldi-2(1H)-pyridone (2.326 g, 10 mmol). After being
stirred for 24 h at room temperature, the mixture was diluted
with water and extracted with ethyl acetate. The combined
organic layers were washed with water and brine, dried over
magnesium sulfate, and filtered. The filtrate was concentrated
in vacuo, and the residue was purified by flash column
chromatography on silica gel with EtOAc/hexanes (1:1) as
eluant to afford isothiocyanate.
Meth od B. To a solution of 1,1′-thiocarbonyl diimidazole
(12 mmol) dissolved completely in DMF (20 mL) at 50 °C was
added dropwise a preprepared solution of amine salt (10 mmol)
and triethylamine (10 mmol) in DMF (3 mL) for 5 min. After
the mixture was stirred for 20 min at room temperature, the
usual workup and purification described above afforded isothio-
cyanate.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(isop r op ylsu lfon yla m in o)ben zyl]th iou r ea (43): white
1
solid, mp ) 48 °C; H NMR(CDCl3) δ 7.34 (s, 1 H, NHSO2),
7.22 (d, 2 H, J ) 8.5 Hz), 7.16 (d, 2 H, J ) 8.5 Hz), 6.8-7.05
(m, 3 H), 6.48 (bs, 1 H, NH), 6.40 (bs, 1 H, NH), 4.50 (bs, 2 H,
CSNHCH2Ar), 4.13 (m, 1 H, CH2OCO), 3.65-3.85 (m, 2 H,
CH2OCO and CHCH2NHCS), 3.2-3.35 (m, 2 H, CHCH2NHCS
and SO2CH(CH3)2), 2.5-2.7 (m, 2 H, CH2Ar), 2.15-2.4 (m, 7
H, 2 × CH3 and CH), 1.36 (s, 3 H, SO2CH(CH3)2), 1.34 (s, 3 H,
SO2CH(CH3)2), 1.23 (d, 9 H, J ) 3.6 Hz, C(CH3)3); MS (FAB)
m/z 548 (MH+); Anal. (C28H41N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(ph en ylsu lfon ylam in o)ben zyl]th iou r ea (44): white solid,
mp ) 66 °C; 1H NMR(CDCl3) δ 7.76 (d, 2 H, J ) 7.3 Hz), 7.51
(t, 1 H), 7.41 (t, 2 H), 7.14 (d, 2 H, J ) 8.3 Hz), 6.8-7.1 (m, 5
H), 6.40 (bs, 1 H, NH), 6.19 (bs, 1 H, NH), 4.43 (bs, 2 H,
CSNHCH2Ar), 4.13 (m, 1 H, CH2OCO), 3.65-3.85 (m, 2 H,
CH2OCO and CHCH2NHCS), 3.20 (m, 1 H, CHCH2NHCS),
2.5-2.7 (m, 2 H, CH2Ar), 2.1-2.4 (m, 7 H, 2 × CH3 and CH),
1.23 (d, 9 H, J ) 3.8 Hz, C(CH3)3); MS (FAB) m/z 582 (MH+);
Anal. (C31H39N3O4S2) C, H, N, S.
4-(Meth ylsu lfon yla m in o)ben zyl isoth iocya n a te (36):
1
63% yield, white solid, mp )122-124 °C; H NMR(CDCl3) δ
7.32 (d, 2 H, J ) 8.5 Hz) 7.24 (d, 2 H, J ) 8.5 Hz), 6.62 (s, 1
H, NHSO2), 4.70 (s, 2 H, CH2) 3.04 (s, 3 H, SO2CH3).
3-Met h oxy-4-(m et h ylsu lfon yla m in o)b en zyl isot h io-
1
cya n a te (37): 59% yield, white solid, mp ) 100-103 °C; H
NMR (CDCl3) δ 7.46 (d, 1 H, J ) 8.5 Hz, H-5), 6.86 (m, 2 H,
H-2,6), 6.74 (s, 1 H, NHSO2), 4.68 (s, 2 H, CH2), 3.88 (s, 3 H,
OCH3), 2.98 (s, 3 H, SO2CH3).
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(tr iflu or om eth ylsu lfon yla m in o)ben zyl]th iou r ea (45):
1
white solid, mp ) 51-52 °C; H NMR(CDCl3) δ 7.28 (d, 2 H,
3-F lu or o-4-(m eth ylsu lfon yla m in o)ben zyl isoth iocya n -
a te (38): 62% yield, white solid, mp ) 93-96 °C; 1H NMR
(CDCl3) δ 7.61 (t, 1 H, J ) 8.3 Hz, H-5), 7.1-7.2 (m, 2 H,
H-2,6), 6.58 (bs, 1 H, NHSO2), 4.71 (s, 2 H, CH2), 3.05 (s, 3 H,
SO2CH3).
Gen er a l P r oced u r e for th e Syn th esis of Th iou r ea (40-
49). A solution of amine salt (13-20, 0.5 mmol) in dimethyl-
formamide (1 mL) was treated with triethylamine (0.5 mmol)
(not needed for free amine 24 and 28) and stirred for 30 min
at room temperature. To the mixture was added {[2-(3,4-
dimethylbenzyl)-3-pivaloyloxy]propyl} isothiocyanate (39) (0.5
J ) 8.7 Hz), 7.22 (d, 2 H, J ) 8.7 Hz), 6.85-7.05 (m, 3 H),
6.36 (bs, 1 H, NH), 6.16 (bs, 1 H, NH), 4.51 (bs, 2 H,
CSNHCH2Ar), 4.08 (dd, 1 H, CH2OCO), 3.65-3.85 (m, 2 H,
CH2OCO and CHCH2NHCS), 3.20 (m, 1 H, CHCH2NHCS),
2.5-2.7 (m, 2 H, CH2Ar), 2.15-2.3 (m, 7 H, 2 × CH3 and CH),
1.22 (d, 9 H, J ) 3.8 Hz, C(CH3)3); MS (FAB) m/z 574 (MH+);
Anal. (C26H34F3N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(a cetyla m in o)ben zyl]th iou r ea (46): white solid, mp )
74 °C; 1H NMR (CDCl3) δ 7.40 (d, 2 H, J ) 8.3 Hz), 7.23 (d, 2

3124 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Lee et al.
H, J ) 8.3 Hz), 6.8-7.05 (m, 3 H), 6.28 (m, 1 H, NH), 6.08 (bs,
1 H, NH), 4.44 (bs, 2 H, CSNHCH2Ar), 4.17 (m, 1 H, CH2OCO),
3.7-3.85 (m, 2 H, CH2OCO and CHCH2NHCS), 3.27 (m, 1 H,
CHCH2NHCS), 2.5-2.7 (m, 2 H, CH2Ar), 2.1-2.4 (m, 10 H, 2
× CH3, COCH3 and CH), 1.23 (d, 9 H, J ) 3.9 Hz, C(CH3)3)
MS (FAB) m/z 484 (MH+); Anal. (C27H37N3O3S) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[4-(isobu tylca r bon yla m in o)ben zyl]th iou r ea (47): white
H, NHSO2), 6.39 (m, 1 H, NH), 6.12 (bs, 1 H, NH), 4.53 (bs, 2
H, CSNHCH2Ar), 4.40 (m, 1 H, CH2OCO), 4.08 (m, 1 H,
CH2OCO), 3.80 (m, 1 H, CHCH2NHCS), 3.38 (m, 1 H, CHCH2-
NHCS), 2.97 (s, 3 H, SO2CH3), 2.6-2.75 (m, 2 H, CH2Ar), 2.48
(m, 1 H, CH), 1.29 (s, 9 H, C(CH3)3); MS (FAB) m/z 568 (MH+);
Anal. (C30H37N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[3-m eth oxy-4-(m eth ylsu lfon ylam in o)ben zyl]th iou r ea (57):
white solid, mp ) 49 °C; 1H NMR (CDCl3) δ 7.48 (d, 1 H, J )
8.0 Hz), 6.85-7.1 (m, 5 H), 6.77 (s, 1 H, NHSO2), 6.31 (m, 1
H, NH), 5.98 (bs, 1 H, NH), 4.49 (bs, 2 H, CSNHCH2Ar), 4.18
(m, 1 H, CH2OCO), 3.7-3.85 (m, 2 H, CH2OCO and CHCH2-
NHCS), 3.88 (s, 3 H, OCH3), 3.22 (m, 1 H, CHCH2NHCS), 2.93
(s, 3 H, SO2CH3), 2.5-2.7 (m, 2 H, CH2Ar), 2.2-2.4 (m, 7 H, 2
× CH3 and CH), 1.23 (d, 9 H, J ) 4.1 Hz, C(CH3)3); MS (FAB)
m/z 550 (MH+); Anal. (C27H39N3O5S2) C, H, N, S.
1
solid, mp ) 65 °C; H NMR (CDCl3) δ 7.50 (d, 2 H, J ) 8.3
Hz), 7.24 (d, 2 H, J ) 8.3 Hz), 6.8-7.05 (m, 3 H), 6.25 (m, 1 H,
NH), 6.00 (bs, 1 H, NH), 4.42 (bs, 2 H, CSNHCH2Ar), 4.12 (m,
1 H, CH2OCO), 3.7-3.85 (m, 2 H, CH2OCO and CHCH2-
NHCS), 3.25 (m, 1 H, CHCH2NHCS), 2.5-2.7 (m, 3 H, CH2Ar
and CHMe2), 2.15-2.3 (m, 7 H, 2 × CH3 and CH), 1.25 (m, 15
H, C(CH3)3 and CH(CH3)2); MS (FAB) m/z 512 (MH+); Anal.
(C29H41N3O3S) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[3-(m eth ylsu lfon ylam in o)ben zyl]th iou r ea (48): white solid,
mp ) 51-54 °C; H NMR (CDCl3) δ 6.85-7.35 (m, 7 H, Ar),
N-[2-(4-ter t-Bu t ylb en zyl)-3-p iva loyloxyp r op yl]-N′-[3-
m eth oxy-4-(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (58):
white solid, mp ) 62 °C; 1H NMR (CDCl3) δ 7.43 (d, 1 H, J )
8.1 Hz), 7.30 (d, 2 H, J ) 8.3 Hz), 7.10 (d, 2 H, J ) 8.3 Hz),
6.93 (s, 1 H), 6.88 (d, 1 H, J ) 8.1 Hz), 6.81 (s, 1 H, NHSO2),
6.39 (t, 1 H, NH), 6.24 (bs, 1 H, NH), 4.52 (bs, 2 H,
CSNHCH2Ar), 4.15 (dd, 1 H, J ) 3.6, 11.2 Hz, CH2OCO), 3.7-
3.9 (m, 2 H, CH2OCO and CHCH2NHCS), 3.87 (s, 3 H, OCH3),
3.25 (m, 1 H, CHCH2NHCS), 2.92 (s, 3 H, SO2CH3), 2.5-2.7
(m, 2 H, CH2Ar), 2.30 (m, 1 H, CH), 1.29 (s, 9 H, C(CH3)3),
1.23 (s, 9 H, C(CH3)3); MS (FAB) m/z 578 (MH+); Anal.
(C29H43N3O5S2) C, H, N, S.
1
6.3-6.4 (bs, 3 H, NH), 4.57 (bs, 2 H, NHCH2Ar), 4.12 (m, 1 H,
OdCOCH2), 3.85 (m, 1 H, OdCOCH2), 3.71 (m, 1 H, CHCH2-
NHCdS), 3.29 (m, 1 H, CHCH2NHCdS), 2.99 (s, 3 H, SO2CH3),
2.55-2.64 (m, 2 H, CH2Ph), 2.2-2.3 (m, 7 H, 2 × CH3 and
CHCH2NHCdS), 1.24 (s, 9 H, (CH3)3CO2); IR (KBr) 3364 (NH),
3250 (NH), 1715 (CdO), 1326 (SO2N), 1286 (N-CS-N), 1150
(SO2N) cm-1; MS (FAB) m/z 520 (MH+); Anal. (C26H37N3O4S2)
C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
{[4-(m eth ylsu lfon yla m in o)m eth yl]ben zyl}th iou r ea (49):
N-[3-Ben zoyloxy-2-(3,4-d im eth ylben zyl)p r op yl]-N′-[3-
m eth oxy-4-(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (59):
white solid, mp ) 62 °C; 1H NMR (CDCl3) δ 8.02 (m, 2 H),
7.60 (m, 1 H), 7.4-7.5 (m, 3 H), 6.8-7.1 (m, 5 H), 6.77 (s, 1 H,
NHSO2), 6.38 (m, 1 H, NH), 6.10 (bs, 1 H, NH), 4.50 (bs, 2 H,
CSNHCH2Ar), 4.42 (m, 1 H, CH2OCO), 4.05 (m, 1 H, CH2OCO),
3.82 (m, 1 H, CHCH2NHCS), 3.85 (s, 3 H, OCH3), 3.37 (m, 1
H, CHCH2NHCS), 2.92 (s, 3 H, SO2CH3), 2.6-2.8 (m, 2 H,
CH2Ar), 2.45 (m, 1 H, CH), 2.2-2.3 (m, 6 H, 2 × CH3); MS
(FAB) m/z 570 (MH+); Anal. (C29H35N3O5S2) C, H, N, S.
1
white solid, mp ) 39 °C; H NMR (CDCl3) δ 7.25-7.35 (m, 4
H), 6.85-7.05 (m, 3 H, aromatic), 6.31 (bs, 1 H, NH), 6.22 (bs,
1 H, NH), 5.04 (bs, 1 H, NH), 4.49 (bs, 2 H, NHCH2Ar), 4.35
(d, 2 H, J ) 5.7 Hz, CH2NHSO2CH3), 3.92 (m, 1 H, OdCOCH2),
3.72 (m, 1 H, OdCOCH2), 3.48 (m, 1 H, CHCH2NHCdS), 3.22
(m, 1 H, CHCH2NHCdS), 2.89 (s, 3 H, SO2CH3), 2.5-2.64 (m,
2 H, CH2Ph), 2.2-2.3 (m, 7 H, 2 × CH3 and CHCH2NHCdS),
1.22 (s, 9 H, (CH3)3CO2); IR (KBr) 3355 (NH), 1716 (CdO),
1318 (SO2N), 1286 (N-CS-N), 1150 (SO2N) cm-1; MS (EI) m/z
532 (M+ - 1); Anal. (C27H39N3O4S2) C, H, N, S.
N-[3-Ben zoyloxy-2-(4-ter t-b u t ylb en zyl)p r op yl]-N′-[3-
m eth oxy-4-(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (60):
white solid, mp ) 57 °C; 1H NMR (CDCl3) δ 8.02 (dd, 2 H, J )
7.1, 1.5 Hz), 7.60 (m, 1 H), 7.47 (m, 3 H), 7.32 (d, 2 H, J ) 8.3
Hz), 7.15 (d, 2 H, J ) 8.3 Hz), 6.85-6.95 (m, 2 H), 6.77 (s, 1
H, NHSO2), 6.38 (t, 1 H, NH), 6.08 (bs, 1 H, NH), 4.51 (bs, 2
H, CSNHCH2Ar), 4.42 (dd, 1 H, J ) 3.9, 11.7 Hz, CH2OCO),
4.05 (m, 1 H, CH2OCO), 3.82 (m, 1 H, CHCH2NHCS), 3.86 (s,
3 H, OCH3), 3.37 (m, 1 H, CHCH2NHCS), 2.92 (s, 3 H,
SO2CH3), 2.5-2.7 (m, 2 H, CH2Ar), 2.45 (m, 1 H, CH), 1.29 (s,
9 H, C(CH3)3); MS (FAB) m/z 598 (MH+); Anal. (C31H39N3O5S2)
C, H, N, S.
Gen er a l P r oced u r e for t h e Syn t h esis of Th iou r ea s
(54-61). A suspension of azide (50-53) (0.5 mmol) and Lindlar
catalyst (50 mg) in ethanol (5 mL) was hydrogenated under a
balloon of hydrogen for 2 h. The mixture was filtered, and the
filtrate was concentrated in vacuo. The residue was dissolved
in dichloromethane (5 mL) and added with isocyanate (36-
38) (0.5 mmol). After being stirred for 24 h at room temper-
ature, the 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 thiourea.
N-[2-(4-ter t-Bu t ylb en zyl)-3-p iva loyloxyp r op yl]-N′-[4-
(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (54): white solid,
mp ) 61 °C; 1H NMR (CDCl3) δ 7.30 (d, 4 H, J ) 8.5 Hz), 7.18
(d, 2 H, J ) 8.6 Hz), 7.11 (d, 2 H, J ) 8.6 Hz), 6.51 (s, 1 H,
NHSO2), 6.33 (m, 1 H, NH), 6.02 (bs, 1 H, NH), 4.53 (bs, 2 H,
CSNHCH2Ar), 4.16 (dd, 1 H, CH2OCO), 3.7-3.85 (m, 2 H,
CH2OCO and CHCH2NHCS), 3.22 (m, 1 H, CHCH2NHCS),
2.99 (s, 3 H, SO2CH3), 2.6-2.7 (m, 2 H, CH2Ar), 2.32 (m, 1 H,
CH), 1.29 (s, 9 H, C(CH3)3), 1.23 (s, 9 H, C(CH3)3); MS (FAB)
m/z 548 (MH+); Anal. (C28H41N3O4S2) C, H, N, S.
N-[2-(3,4-Dim et h ylb en zyl)-3-(p iva loyloxy)p r op yl]-N′-
[3-flu or o-4-(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (61):
white solid, mp ) 56 °C; 1H NMR (CDCl3) δ 7.45 (d, 2 H, J )
8.3 Hz), 6.85-7.15 (m, 5 H), 6.53 (bs, 2 H, NHCS), 4.58 (bs, 2
H, CSNHCH2Ar), 4.14 (dd, 1 H, J ) 3.9, 11.7 Hz, CH2OCO),
3.6-3.9 (m, 2 H, CH2OCO and CHCH2NHCS), 3.25 (m, 1 H,
CHCH2NHCS), 2.99 (s, 3 H, SO2CH3), 2.5-2.7 (m, 2 H, CH2Ar),
2.2-2.4 (m, 7 H, 2 × CH3 and CH), 1.23 (d, 9 H, J ) 3.9 Hz,
C(CH3)3); MS (FAB) m/z 538 (MH+); Anal. (C26H36FN3O4S2) C,
H, N, S.
N-[3-Ben zoyloxy-2-(3,4-d im eth ylben zyl)p r op yl]-N′-[4-
(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (55): white solid,
1
mp ) 46 °C; H NMR (CDCl3) δ 8.0 (m, 2 H), 7.59 (m, 1 H),
VR1 Bin d in g Assa ys. Cell Cu ltu r e. The pUHG102 VR1
plasmid was transfected into CHO cells containing the pTet
Off regulatory plasmid (Clontech). In these cells, expression
of the VR1 is repressed in the presence of tetracycline but is
induced upon removal of the antibiotic. Stable clones were
isolated in culture medium containing puromycin (10 µg/mL)
and maintained in HAM F12 medium supplemented with
tetracycline (1 µg/mL), 5 µg/mL geniticin, 25 mM HEPES, 10%
FBS. Cells utilized for assays were grown in culture medium
without antibiotic for 48 h before use. Cells were seeded in
T75 cell culture flasks in media without antibiotics and grown
to approximately 90% confluence. The flasks were then washed
with PBS and harvested in 0.25% trypsin, 1 mM EDTA. The
7.45 (m, 2 H), 7.29 (bs, 1 H, NHSO2), 7.24 (d, 2 H, J ) 8.3 Hz),
7.15 (d, 2 H, J ) 8.3 Hz), 6.85-7.05 (m, 3 H), 6.40 (m, 1 H,
NH), 6.09 (bs, 1 H, NH), 4.52 (bs, 2 H, CSNHCH2Ar), 4.37 (m,
1 H, CH2OCO), 4.10 (m, 1 H, CH2OCO), 3.80 (m, 1 H, CHCH2-
NHCS), 3.41 (m, 1 H, CHCH2NHCS), 2.94 (s, 3 H, SO2CH3),
2.6-2.8 (m, 2 H, CH2Ar), 2.44 (m, 1 H, CH), 2.2-2.4 (m, 6 H,
2 × CH3); MS (FAB) m/z 540 (MH+); Anal. (C28H33N3O4S2) C,
H, N, S.
N-[3-Ben zoyloxy-2-(4-ter t-b u t ylb en zyl)p r op yl]-N′-[4-
(m eth ylsu lfon yla m in o)ben zyl]th iou r ea (56): white solid,
mp ) 68 °C; 1H NMR (CDCl3) δ 8.02 (d, 2 H), 7.60 (t, 1 H)
7.47 (t, 2 H), 7.25-7.35 (m, 4 H), 7.1-7.2 (m, 4 H), 6.54 (s, 1

Antagonists of the Vanilloid Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3125
cells were pelleted by gentle centrifugation and stored at -20
compounds or vehicle (0.2 mL, i.p.) 30 min before the injection
of acetic acid. Test compounds were dissolved in either ethanol/
Tween-80/saline (10/10/80) mixture or Cremophor EL/DMSO/
distilled water (10/10/80) mixture. The effect of each compound
was tested at 4-7 different doses. A reduction in the number
of writhing movements compared to the vehicle-treatment
group (the mean number of writhing movements in this group
was 35) was considered to be indicative of an antinociceptive
effect of a compound. The percentage antinociceptive efficiency
(eff) was calculated as follows: % eff ) 100-[(# of writhing
movements/# of writhing movement control) × 100].
°C until assay.
Com p et it ion Bin d in g Assa y. Binding studies with
[3H]resiniferatoxin (RTX) were carried out as described previ-
ously with minor modifications (Szallasi et al., 1992). Binding
assay mixtures were set up on ice and contained 50-100 pM
[3H]RTX, various concentrations of competing ligands, 0.25 mg/
mL BSA (Cohn fraction V), and about 5 × 105 VR1-transfected
cells. The final volume was adjusted to 350 µL with DPBS with
Ca2+ and Mg2+ and 0.25 mg/mL bovine serum albumin.
Nonspecific binding was determined in the presence of 100 nM
nonradioactive RTX. The binding reaction was initiated by
transferring the assay mixtures to a 37 °C water bath and was
terminated after a 60 min incubation period by cooling the
tubes on ice. To reduce nonspecific binding, 200 µg/mL R-glyco-
protein was added. Membrane-bound RTX was then separated
from the free by pelleting the membranes in a Beckman 12
benchtop centrifuge (15 min, maximal velocity), the tips of the
tubes containing the pellets were cut off, and the radioactivity
was determined by scintillation counting. Equilibrium binding
parameters (Ki and cooperativity) were determined by fitting
the Hill equation to the measured values with the aid of the
program MicroCal Origin 6.0.
Data are expressed as ED50 values that indicate the
concentration at which a given compound reduces the number
of writhing by 50% compared to that of a vehicle-treatment
group. ED50 values were obtained based on dose-response
curves using mean data and fitted to by nonlinear regression
analysis (Winnonlin version 3.1, Pharsight Corp., Mountain-
view, CA) on a PC.
Ack n ow led gm en t. This work was supported by a
Fund 2002 grant (ES0024) from the Korea Research
Foundation.
Com p ou n d P r ep a r a tion . Initial stocks were dissolved in
DMSO. For the binding assays, compounds were diluted in
with DPBS with Ca2+ and Mg2+ and 0.25 mg/mL bovine serum
albumin. For the calcium uptake assays, compounds were
diluted in DMEM with 0.25 mg/mL bovine serum albumin.
Refer en ces
(1) Szallasi, A.; Blumberg, P. M. Vanilloid (Capsaicin) Receptors and
Mechanisms. Pharmacol. Rev. 1999, 51, 159-211.
F u n ction a l Ch a r a cter iza tion for Agon ist/An ta gon ist
Activity. 45Ca 2+ Up ta k e. Molecules were characterized to
determine whether they were full agonists, partial agonists,
or antagonists. For studies of 45Ca2+ uptake by CHO/VR1 cells
(Tet-off cells), the cells were plated in 24-well plates to yield a
cell density 20-40% of that required to produce confluence.
The next day the medium was changed to remove the
tetracycline and induce VR1 expression. Experiments were
performed approximately 36-40 h after induction. For 45Ca2+
uptake assay, cells were incubated for 5 min at 37 °C in a total
volume of 400 µL of serum free DMEM (containing 1.8 mM
CaCl2) in the presence of 0.25 mg/mL BSA (Sigma), 1 µCi/mL
45Ca2+ (5-30 Ci/g from ICN, CA), and increasing concentra-
tions of the compound to be tested. Immediately after the
incubation, extracellular 45Ca2+ was removed by washing the
cells three times with cold DPBS (containing 1.8 mM CaCl2).
Then 400 µL of RIPA buffer (50 mM Tris pH 7.4; 150 mM
NaCl; 1% Triton X-100; 0.1% SDS; 1% sodium deoxycholate)
was added to each well in order to lyse the cells. Plates were
shaken slowly for 20 min; then 300 µL of cell lysate was
transferred from each well into a scintillation vial and
radioactivity was determined by scintillation counting. For
each data point in each experiment, four wells were assayed.
Data from these experiments were analyzed by computer fit
to the Hill equation. At least three separate experiments were
carried out for each compound. To determine antagonist
activity, studies were performed in exactly the same fashion
with the exception that 50 nM capsaicin was added to the
assay mixture to stimulate 45Ca2+ uptake.
Wr ith in g Test. Experimental protocols involving animals
in this study were reviewed by the Animal Care and Use
Committee of the College of Pharmacy, Seoul National Uni-
versity, according to the NIH guidelines (NIH publication
number 85-23, revised 1985) of “Principles of Laboratory
Animal Care”. Male ICR mice (Bio Genomics, Korea), weighing
∼25 g, were maintained on a 12 h light-dark cycle (light on
between 6:00 p.m. and 6:00 a.m.) and allowed free access to
food and water. The temperature and humidity of the animal
room were maintained at 22 ( 2 °C and 50 ( 5%, respectively.
Mice were allowed to habituate for ∼30 min in the testing room
on the day of experimentation. Animals then received an
intraperitoneal injection of 0.3 mL of an acetic acid solution
(1.2%, diluted in 0.9% saline) and were placed in a transparent
acrylic cage. Five minutes later the number of writhing
movements (abnormal stretching) was counted for a 20 min
period. Animals (10 animals/dose) were pretreated with test
(2) Kress, M.; Zeihofer, H. U. Capsaicin, Protons and Heat: New
Excitement about Nociceptors. TIPS 1999, 20, 112-118.
(3) Walpole C. S. J .; Wrigglesworth, R. Capsaicin in the Study of
Pain; Academic Press: San Diego, CA, 1993; pp 63-82.
(4) Appendino, G.; Szallasi, A. Euphorbium: Modern Research on
Its Active Principle, Resiniferatoxin, Revives an Ancient Medi-
cine. Life Sci. 1997, 60, 681-696.
(5) Caterina, M. J .; Schumacher, M. A.; Tominaga, M.; Rosen, T.
A.; Levine, J . D.; J ulius, D. The Capsaicin Receptor: a Heat-
activated Ion Channel in the Pain Pathway. Nature 1997, 389,
816-824.
(6) Hayes, P.; Meadows, H. J .; Gunthorpe, M. J .; Harries, M. H.;
Duckworth, D. M.; Cairns, W.; Harrision, D. C.; Clarke, C. E.;
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