N-(3-acyloxy-2-benzylpropyl)-N′-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues as vanilloid receptor ligands

Article abstract of DOI:10.1016/S0968-0896(01)00068-2

The vanilloid receptor represents a promising target for drug development. Building on our previous strategies which have generated potent agonists for VR1, we now describe a series of novel N-(3-acyloxy-2-benzylpropyl)-N′-dihydroxytetrahydrobenzazepine a

Full text of DOI:10.1016/S0968-0896(01)00068-2

Bioorganic & Medicinal Chemistry 9 (2001) 1713–1720
N-(3-Acyloxy-2-Benzylpropyl)-N0-
Dihydroxytetrahydrobenzazepine and Tetrahydroisoquinoline
Thiourea Analogues as Vanilloid Receptor Ligands
Jeewoo Lee,a,* Jiyoun Lee,a Tamas Szabo,b Adamar F. Gonzalez,b
Jacqueline D. Welterb and Peter M. Blumbergb
aLaboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku,
Seoul 151–742, Republic of Korea
bLaboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute,
National Institute of Health, Bethesda, MD 20892, USA
Received 4 January 2001; accepted 26 February 2001
Abstract—The vanilloid receptor represents a promising target for drug development. Building on our previous strategies which
have generated potent agonists for VR1, we now describe a series of novel N-(3-acyloxy-2-benzylpropyl)-N0-dihydroxytetrahydro-
benzazepine and tetrahydroisoquinoline thiourea analogues, several of which are potent VR1 antagonists. We report here the
rationale for the design, the synthesis, and the in vitro characterization of activity in assays for [3H]resiniferatoxin binding and 45Ca
influx using heterologously expressed rat VR1. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Among a number of VR agonists studied, only a few
compounds are currently marketed (e.g., capsaicin) or
undergoing clinical trial (e.g., resiniferatoxin, DA-
5018).11 The primary mechanistic rationale of vanilloids
for treatment of pain is through C-fiber desensitization.
The initial response of VR1 to vanilloids is to permit a
large influx of Ca++ and C-fiber depolarization. Sub-
sequently, depending on dose of vanilloid, time of
exposure, and other factors, desensitization, potentially
reflecting multiple mechanisms, develops. For capsaicin,
the initial phase of acute stimulation, causing a severe
burning sensation, represents the limiting toxicity.
Apparently, pungency, reflecting the acute pain
response, and the subsequent desensitization are not
directly coupled. Thus, RTX is much more potent as a
ligand at VR1 but is only modestly more pungent.1 One
direction for drug development, therefore, is design of
analogues in which pungency and desensitization have
been dissociated. An inherent advantage/problem in this
approach is the potential long duration of desensitiza-
tion. A single treatment with RTX, for example, can
cause desensitization for multiple weeks.1
The vanilloid receptor (VR) is a specific neuronal
membrane recognition site for vanilloids like capsai-
cin and resiniferatoxin (RTX),1 and its expression is a
prominent feature of nociceptive primary afferents of
the C-fiber ‘pain’ pathway.2 Desensitization of the
receptor caused by specific ligands has long been recog-
nized as a promising therapeutic approach for the
treatment of neuropathic pain and other pathological
conditions involving C-fiber neurons.3ꢀ6
The functional vanilloid receptor subtype VR1, which is
activated not only by capsaicin but also by noxious heat
and low pH, has recently been cloned.7 The VR1 pro-
tein is structurally a distant homologue of the transient
release potential (TRP) family of store-operated calcium
channels, comprising six transmembrane domains and
three ankyrin repeats at the N-terminus.8 VR1 is a
nonselective cation channel with a preference for Ca++
and was shown, in studies with knockout mice, to be
essential for response to vanilloids as well as a major
mediator of thermal nociception.2,9,10
An alternative strategy, optimal when only a short
duration of inhibition is desirable, is to block VR1
function either through a channel blocker specific for
VR1 or else through a competitive receptor antago-
nist.3,12 Currently, capsazepine is the only commercially
*Corresponding author. Tel.:+82-2-880-7846; fax: +82-2-888-0649;
e-mail: jeewoo@snu.ac.kr
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00068-2

1714
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
available competitive antagonist for VR1, inhibiting
biological responses of capsaicin and RTX in a compe-
titive manner in a variety of bioassays.13,14 Capsazepine
has several drawbacks such as moderate potency, sus-
ceptibility to metabolic clearance, inhibitory activity on
several channels other than VR1, and poor solubility.
Nonetheless, the inhibition of nociceptive and hyper-
algesic responses by capsazepine proves in principle that
VR1 antagonists have an analgesic potential.15
These compounds are thus approximately 280 and 480
times more potent than capsaicin.21 The particular fea-
ture of these compounds is a 3-acyloxy-2-benzylpropyl
moiety as a C region which we designed to mimic key
putative pharmacophores of the diterpene moiety in
RTX. In the present study, we have sought to combine
features of the key pharmacophores of capsazepine and
our simplified RTX analogues as a possible route to
novel, potent antagonists (Fig. 1). We incorporated the
benzazepine ring of capsazepine as an A region and the
3-acyloxy-2-benzylpropyl group as a C region. We rea-
soned that the benzazepine (or tetrahydroisoquinoline)
template of the target compounds might induce antag-
onistic binding to the receptor conformationally and the
3-acyloxy-2-benzylpropyl group might confer tight
binding as shown previously.
Based on structure–activity relationships for stimulation
of Ca2+ uptake by capsaicin derivatives, a model for
ligand interaction at the receptor has been proposed in
which three pharmacophoric elements, the so-called A,
B and C regions, are crucial for activity.16ꢀ18 Molecular
modeling of capsazepine suggests that the twisted orien-
tation of the A and C regions imposed by the benzaze-
pine ring is responsible for its activity as an antagonist.14
In this paper, we describe the syntheses of 3-acyloxy-2-
benzyl-propyl
thiourea
analogues
of
tetra-
The identification of RTX as a novel vanilloid with
potency 3–4 orders of magnitude greater than that of
capsaicin suggested exciting opportunities for exploita-
tion through medicinal chemistry.19 Structurally, RTX
represents a typical vanilloid in the A and B regions, but
with a tetracyclic diterpene in place of the typical C
region substituents in capsaicin and its congeners. Over
the past few years, we have explored simplified RTX
analogues as VR1 ligands based on our hypothetical
pharmacophore model.20 Using this approach, we have
recently reported ultrapotent VR agonists, 27 and 28,
with Ki values of 19 and 11 nM, respectively, in an
[3H]RTX binding assay on dorsal root ganglion neurons.
hydrobenzazepine and tetrahydroisoquinoline and
characterize their in vitro receptor activities by calcium
influx and binding affinity assays. The analysis was car-
ried out using rat VR1 heterologously expressed in
CHO cells in place of the more problematic dorsal root
ganglion cultures used previously.
Chemistry
The thiourea VR1 ligands were conventionally synthe-
sized from the condensation between amines of the A
region and isothiocyanates of the C region. Iso-
thiocyanates 1–8 were prepared from the corresponding
Figure 1.

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
1715
azides, whose syntheses were reported in a previous
paper,21 by catalytic hydrogenation with Lindler’s catalyst
followed by condensation with 1,10-thiocarbonyldi-2(1H)-
pyridone22 (Scheme 1). Two tetrahydro-2-benzazepines, 9
and 10, were efficiently prepared from isovanillin in
seven steps by our procedure.23 Catechols 11 and 12
were obtained from commercially available 6,7-
dimethoxytetrahydroisoquinoline and 4-hydroxy-3-
methoxybenzylamine, respectively, under refluxing 48%
hydrobromic acid.14 The condensation between iso-
thiocyanates (1–8) and amines (9–12) proceeded
smoothly in THF or DMF to afford thioureas; the
reaction was carried out in the presence of base if amine
salts were used.
According to the calcium uptake assay, 13, 14 and 18
among the tested VR ligands displayed antagonistic
activity, with Ki values of 0.884, 0.966, and 0.194 mM,
respectively. The antagonist 18 was ca. 3-fold more
potent than capsazepine under these assay conditions.
The binding assays of 13 (Ki=0.884 mM), 14
(Ki=0.223 mM) and 18 (Ki=0.81 mM) showed enhanced
affinities compared to capsazepine (Ki=3.22 mM ) of
between 15- and 4-fold. It is interesting that while the
6,7-dihydroxytetrahydroisoquinoline surrogate of cap-
sazepine was an agonist rather than an antagonist,14 the
6,7-dihydroxytetrahydroisoquinoline
analogue
18,
which is generated from the replacement of the chlo-
rophenylethyl moiety in capsazepine by a 3-benzoyloxy-
2-benzylpropyl group, is an antagonist. This suggests
that the 3-benzoyloxy-2-benzylpropyl moiety in the C
region might be responsible for its antagonistic activity.
Results and Discussion
The activities of synthesized VR ligands as agonists and
antagonists were assessed from assay of 45Ca influx and
their affinities for receptor binding were measured by
competition of [3H]RTX binding. Both assays were
carried out using rat VR1 expressed in Chinese hamster
ovary cells (CHO/VR1 cells) as previously described.24
Compounds with measurable affinity for [3H]RTX
binding were first tested as possible agonists for 45Ca
uptake. Compounds without agonist activity were eval-
uated as possible antagonists. Antagonists were assayed
in the presence of 50 nMcapsaicin ( KD for 45Ca uptake
is 81 nM). Ki values generated from these experiments
can be compared with that for capsazepine
(Ki=0.56 mM), the one competitive antagonist of VR1.
The results are presented in Table 1.
The antagonistic activities of 14 and 18 were lost upon
quite small structural modifications in the A and C
regions. Within the A region, the corresponding mono-
methoxy analogues, 15 and 17, were full agonists. In
contrast, the 8-methoxy analogue of capsazepine 13,
retained its activity as an antagonist. This result indi-
cated that a free catechol moiety may be important for
antagonist activity in this series, but its importance
depends on the specific structural context. In the C
region, any modifications at the R1 and R2 groups
(R1=pivaloyl, 3,4-dimethylphenyl or 4-t-butyl; R2=3,4-
dimethylphenyl or 4-t-butylphenyl) also switched both
14 and 18 into agonists (16–17 and 19–23), most of
whose potencies were comparable to capsaicin in cal-
cium influx and receptor binding assays. Removal of the
Scheme 1.

1716
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
Scheme 2.
carbonyl group in antagonist 18 to afford 24 also con-
verted its activity from an antagonist to an agonist.
Apparently, the interaction of the 3-benzoyloxy-2-ben-
zylpropyl group shared commonly in 14 and 18 with the
receptor seems to be very specific for conferring their
antagonistic activity. The acyclic surrogate 25 of the
two antagonists 14 and 18, which represents a con-
formationally flexible analogue, was also prepared and
found to be a full agonist with an EC50=0.165 mMin
the calcium influx assay and a Ki=0.253 mMin binding
affinity. This agonist was of equal or greater potency
than the other tetrahydrobenzazepine and tetra-
hydroisoquinoline surrogates in both assays, but was
8-fold less potent in the binding assay than the corre-
sponding 7-methoxy analogue 26, for which the
21
Ki=0.031 mMwas reported previously.
Enhanced
agonistic activity associated with conformational flexi-
bility was also evident in the conversion of the benza-
zepine 17 (Ki=1.42 mM) into the acyclic analogue 27
(Ki=0.0033 mM), resulting in a dramatic increase in
binding affinity of 430-fold. The SAR in this series
revealed that, whereas the antagonistic activity at VR1
requires appropriate conformational restriction of the
3,4-dihydrohydroxybenzyl amine, agonistic activity was
enhanced when the flexibility was increased and when
the 3-hydroxy group of catechol was methylated. Of

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
Table 1. In vitro assays of synthesized compounds toward VR1 receptor
1717
Compounds
45Ca uptake
[3H]RTX binding
) Ki, mM
Relative potency
Agonist (ED50, mM)
Antagonist (Ki, mM
(
)
++ uptake veCrsaus Binding
0.02a
Capsaicin
0.081 (ꢁ0.011)
1.7a
0.038a
0.14a
6.6a
Capsazepine
0.56 (ꢁ0.12)
0.884 (ꢁ0.152)
0.966 (ꢁ0.118)
3.22 (ꢁ0.48)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
NEc
NE
1.15 (ꢁ0.42)
5.01 (ꢁ0.74)
0.57 (ꢁ0.29)
NE
0.36 (ꢁ0.11)
0.80 (ꢁ0.32)
0.187 (ꢁ0.033)
0.21 (ꢁ0.076)
0.36 (ꢁ0.24)
0.12 (ꢁ0.040)
0.165 (ꢁ0.032)
NTd
0.865 (ꢁ0.051)
0.223 (ꢁ0.052)
0.59 (ꢁ0.18)
1.9
4.3
0.4
1.156 (ꢁ0.047)
1.42 (ꢁ0.049)
0.81 (ꢁ0.065)
0.40 (ꢁ0.068)
0.52 (ꢁ0.15)
0.196 (ꢁ0.05)
0.9
1.5
0.3
0.9
0.05
0.45
0.65
0.627 (ꢁ0.074)
0.233 (ꢁ0.003)
7.1 (ꢁ2.4)
0.267 (ꢁ0.059)
0.253 (ꢁ0.063)
0.031 (ꢁ0.009)b
0.0067 (ꢁ0.0009)
0.0032 (ꢁ0.0001)
0.0033 (ꢁ0.0015)
0.49
0.97
0.0031 (ꢁ0.0002)
aLiterature value in ref 24.
bLiterature value in ref 21.
cNot effective.
dNot tested.
course, constraint in the conformation optimized for
agonist activity might yield yet greater potency, as sug-
gested by the Ki of 0.00001 mMfor RTX, an analogue
with a rigid C region but still with flexibility between the
A and B regions (Scheme 2).24
indicated that both conformational constraint at the A
region and the 3-benzoyloxy-2-benzylpropyl group at
the C region were crucial for the antagonistic activities
of 14 and 18 and their binding affinities to the receptor;
their acyclic analogues and any structurally modified
analogues in the C region were full agonists with an
activity comparable to capsaicin. Our results suggest
novel opportunities for the design of VR1 antagonists, a
promising therapeutic objective.
It has been noted that whereas capsaicinoids seem to be
consistently less potent for inhibiting RTX binding than
for inducing 45Ca2+-uptake, RTX analogues are recip-
rocally more potent for inhibiting RTX binding (usually
calcium uptake/binding affinity=EC50/Ki<1 for cap-
saicinoids and >1 for resiniferanoids).1,25 The relative
potencies for calcium uptake and binding affinity of the
compounds in the series described are shown in Table 1.
Most are between 0.5 and 1. These values reveal that
these ligands bind to VR1 in CHO cells and induce calcium
uptake by them with similar affinities as do the RTX
amide and thiourea analogues, with ratios of 1 and 1.2,
respectively, for assay in cultured DRG neurons.26
Experimental
All chemical reagents were commercially available. Melt-
ing 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 recorded on a
JEOL JNM-LA 300 at 300 MHz. Chemical shifts are
reported in ppm units with Me4Si as a reference standard.
Infrared spectra were recorded on a Perkin-Elmer 1710
Series FTIR. Mass spectra were recorded on a VG Trio-2
GC–MS. Elemental analyses were performed with an EA
1110 Automatic Elemental Analyzer, CE Instruments.
Conclusion
Thirteen thiourea compounds having a dihydroxy-
tetrahydrobenzazepine or dihydroxy tetrahydroiso-
quinoline template for the A region and a 3-acyloxy-2-
benzyl-propyl moiety for the C region were synthesized
as VR1 ligands and their in vitro receptor activities were
measured by 45Ca influx and [3H]RTX binding assays.
Among them, 14 and 18 were found to be potent VR
antagonists. Compared to capsazepine, the only com-
petitive VR1 antagonist described so far, 14 and 18 were
0.6- and 3-fold as potent when assayed as antagonists
for calcium influx, and they were 15- and 4-fold as
potent in binding affinity. The SAR study of this series
General procedure for the synthesis of isothiocyanates
(1–8)
A suspension of Lindler’s catalyst (0.05 g) in a solu-
tion of azide (0.5 mmol) in ethanol (5 mL) was
hydrogenated under a balloon of hydrogen for 2 h.
The reaction mixture was filtered and the filtrate was
concentrated in vacuo. The residue was dissolved in
CH2Cl2 (5 mL) and treated with 1,10-thiocarbonyldi-
2(1H)-pyridone (0.5 mmol). After stirring for 16 h at room
temperature, the reaction mixture was concentrated in

1718
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
vacuo. The residue was purified by flash column chro-
matography over silica gel with EtOAc/hexane (10:1) as
eluant to give the corresponding isothiocyanate.
column chromatography over silica gel with EtOAc/
hexane (1:1) as eluant to give the thiourea.
N-(4-Chlorophenethyl)-7-hydroxy-8-methoxy-1,3,4,5-tet-
rahydro-2H-2-benzazepine-2-carbothioamide (13). 90%
2-Benzyl-3-isothiocyanatopropyl benzenecarboxylate (1).
1
ꢃ
1
90% yield; a colorless oil; H NMR (CDCl3) d 8.04 (d,
yield; a white solid; mp 58 C; H NMR (CDCl3) d 7.23
(d, 2H, Ph), 6.96 (d, 2 H, Ph), 6.75 (s, 1H, ArH), 6.53 (s,
1H, ArH), 5.59 (s, 1H, ArOH), 5.37 (t, 1H, NH), 4.60 (s,
2H, ArCH2N), 4.08 (m, 2H, PhCH2CH2NH), 3.88 (dd,
2H, ArCH2CH2CH2N), 3.73 (s, 3H, ArOCH3), 2.8–2.9
(m, 4H, ArCH2CH2CH2N and CH2Ph), 1.81 (m, 2H,
ArCH2CH2CH2N); IR (KBr) 3396, 1591, 1489, 1288,
759 cmꢀ1; Anal. calcd for C20H23ClN2O2S: C, 61.45; H,
5.93; N, 7.17; S, 8.20. Found: C, 61.65; H, 5.95; N, 7.15;
S, 8.17.
2H), 7.60 (t, 1H), 7.47 (t, 2H) 7.2–7.35 (m, 5H, phenyl),
4.43 (dd, 1H, J=4.8 and 11.4 Hz, BzOCH2), 4.28 (dd,
1H, BzOCH2), 3.63 (ddd of AB, 2H, CH2NCS), 2.82
(dd of AB, 2H, CH2Ph), 2.47 (m, 1H, CH).
2-Benzyl-3-isothiocyanatopropyl 3,4-dimethylbenzenecar-
boxylate (2). 82% yield; a colorless oil; 1H NM R
(CDCl3) d 7.78 (m, 2H), 7.2–7.35 (m, 6H), 4.40 (dd, 1H,
OCOCH2), 4.34 (dd, 1H, OCOCH2), 3.67 (ddd of AB,
2H, CH2NCS), 2.7–2.9 (m, 2H, CH2Ph), 2.46 (m, 1H,
CH), 1.35 (s, 9H, C(CH3)3).
2-Benzyl-3-{[(7-hydroxy-8-methoxy-1,3,4,5-tetrahydro-
2H-2-benzazepin-2-yl)carbothioyl]amino}propyl benzoate
(15). 87% yield; a white solid; mp 58 ꢃC; 1H NM R
(CDCl3) d 8.06 (d, 2H, Bz), 7.64 (t, 1H, Bz), 7.49 (t, 2H,
Bz), 7.2–7.34 (m, 5H, Ph), 7.12 (s, 1H, Ar), 6.73 (s, 1H,
Ar), 6.43 (t, 1H, NH), 5.50 (s, 1H, OH), 4.73 (s, 2 H,
ArCH2NH), 4.60 (dd, 1H, CH2OBz), 3.97 (dd, 2 H,
CH2OBz and CH2NHCSN), 3.81 (s, 3H, ArOCH3),
3.23 (m, 1H, CH2CH2N), 2.88 (m, 4 H, ArCH2CH2 and
CH2Ph), 2.50 (m, 1H, CH),1.88 (m, 2H, ArCH2CH2);
IR (KBr) 3408, 1514, 1276, 1100 cmꢀ1; Anal. calcd for
C29H32N2O4S: C, 69.02; H, 6.39; N, 5.55; S, 6.35.
Found: C, 69.27; H, 6.41; N, 5.53; S, 6.33.
2-Benzyl-3-isothiocyanatopropyl 4-tert-butylbenzenecarb-
oxylate (3). 73% yield; a colorless oil; 1H NM R
(CDCl3) d 7.99 (d, 2 H), 7.50 (d, 2H) 7.2–7.35 (m, 5 H,
phenyl), 4.43 (dd,1 H, BzOCH2), 4.24 (dd, 1H,
BzOCH2), 3.62 (ddd of AB, 2H, CH2NCS), 2.7–2.9
(m, 2H, CH2Ph), 2.46 (m, 1H, CH), 1.35 (s, 9H,
C(CH3)3).
2-(3,4-Dimethylbenzyl)-3-isothiocyanatopropyl pivalate
1
(4). 94% yield; a colorless oil; H NMR (CDCl3) d 6.8–
7.3 (m, 3H), 4.04 (m, 2H, OCOCH2), 3.36 (m, 2H,
CH2NCS), 2.63 (m, 2H, CH2Ph), 2.2–2.3 (m, 7H, 2ꢂ
CH3 and CH), 1.23 (s, 9H, C(CH3)3).
2-Benzyl-3-{[(7-hydroxy-8-methoxy-1,3,4,5-tetrahydro-
2H-2-benzazepin-2-yl)carbothioyl]amino}propyl pivalate
(17). 81% yield; a white solid; mp 60 ꢃC; 1H NM R
(CDCl3) d 6.9–7.2 (m, 4H, Ph and ArH), 6.73 (s, 1H,
Ar), 6.43 (m, 1H, NH), 5.50 (s, 1H, OH), 4.68 (s, 2H,
ArCH2NH), 4.60 (ddd, 2H, CH2OCO), 3.99 (m,
1H, CH2NHCSN), 3.84 (s, 3H, ArOCH3), 3.72 (m, 1H,
CH2CH2N), 2.83 (m, 2 H, ArCH2CH2 ), 2.23 (m, 7H,
2ꢂCH3 and CH), 1.86 (m, 2 H, ArCH2CH2), 1.30 (s, 9H,
2-(3,4-Dimethylbenzyl)-3-isothiocyanatopropyl benzene-
carboxylate (5). 58% yield; a colorless oil; 1H NM R
(CDCl3) d 8.05 (d, 2H), 7.61 (t, 1H), 7.57 (t, 2H) 6.9–7.1
(m, 3 H), 4.42 (dd,1 H, BzOCH2), 4.28 (dd, 1H,
BzOCH2), 3.64 (ddd of AB, 2H, CH2NCS), 2.85 (m,
2H, CH2Ph), 2.4–2.5 (m, 7H, 2ꢂ CH3 and CH).
2-(4-tert-Butylbenzyl)-3-isothiocyanatopropyl benzene-
carboxylate (6). 94% yield; a colorless oil; 1H NM R
(CDCl3) d 8.05 (d, 2H), 7.61 (t, 1H), 7.57 (t, 2H), 7.33 (d,
2H) 7.15 (d, 2H), 4.44 (dd, 1H, BzOCH2), 4.27 (dd, 1H,
BzOCH2), 3.64 (ddd of AB, 2H, CH2NCS), 2.7–2.9 (m,
2H, CH2Ph), 2.45 (m, 1H, CH), 1.31 (s, 9H, C(CH3)3).
C(CH3)3); IR (KBr) 3408, 1713, 1515, 1287, 1164 cmꢀ1
;
Anal. calcd for C29H40N2O4S: C, 67.94; H, 7.86; N, 5.46; S,
6.25. Found: C, 68.15; H, 7.88; N, 5.48; S, 6.27.
Method B. A solution of amine salt (1.0 mmol) and
triethylamine (1.5 mmol) in DMF (1 mL) was stirred for
1 h and treated with isocyanate (1.2 mmol). After stir-
ring for 20 h at room temperature, the reaction mixture
was diluted with H2O and extracted with EtOAc several
times. The combined organic layer was washed with
H2O and brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by flash column chro-
matography over silica gel with EtOAc/hexane (2:1) as
eluant to give the thiourea.
1-[(2-Benzyl-3-isothiocyanatopropoxy)methyl]benzene (7).
1
51% yield; a colorless oil; H NMR (CDCl3) d 7.1–7.4
(m, 10H, phenyl), 4.50 (s, 2H, OCH2Ph), 3.3–3.6 (m,
4H, CH2N3 and CH2O), 2.6–2.7 (m, 2H, CH2Ph), 2.24
(m, 1H, CH).
1-Chloro-4-(2-isothiocyanatoethyl)-benzene (8). 95% yield;
a colorless oil; 1H NMR (CDCl3) d 7.33 (d, 2H, Ph), 7.16
(d, 2H, Ph), 3.71 (t, 2H, CH2Ph), 2.95 (t, 2H, CH2NCS).
2-Benzyl-3-{[(7,8-dihydroxy-1,3,4,5-tetrahydro-2H-2-ben-
zazepin-2-yl)carbothioyl]amino}propyl benzoate (14).
41% yield; a white solid; mp 123 ꢃC; H NMR (CDCl3)
1
General procedure for the synthesis of thioureas
d 8.10 (d, 2H, Bz), 7.66 (t, 1H, Bz), 7.52 (t, 2H, Bz), 7.2–
7.3 (m, 5H, Ph), 6.89 (s, 1H, Ar), 6.73 (s, 1H, Ar), 6.01
(bs, 1H, NH), 5.50 (bs, 1H, OH), 4.50 (m, 3H,
ArCH2NH and CH2OBz), 4.03 (dd, 2H, CH2OBz and
CH2NHCSN), 3.32 (m, 1H, CH2CH2N), 2.85 (m, 4H,
ArCH2CH2 and CH2Ph), 2.49 (m, 1H, CH), 1.85 (m,
Method A. A mixture of isocyanate (0.5 mmol) and
7-hydroxy-8-methoxy-2,3,4,5-tetrahydro-1H-2-benzaze-
pine (0.5 mmol) in THF (5 mL) was stirred for 18 h at
room temperature. The reaction mixture was con-
centrated in vacuo and the residue was purified by flash

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
1719
2H, ArCH2CH2). IR (KBr) 3407, 1539, 1277 cmꢀ1
Anal. calcd for C28H30N2O4S: C, 68.55; H, 6.16; N,
5.71; S, 6.54. Found: C, 68.72; H, 6.18; N, 5.69; S, 6.52.
;
(CDCl3) d 8.08 (m, 2H, Bz), 7.4–7.6 (m, 3H, Bz), 6.9–7.1
(m, 5H, Ph), 6.67 (d, 2H, Ar), 6.32 (t, 1H, NH), 4.69 (s,
2H, ArCH2N), 4.57 (dd of AB, 1H, CH2OBz), 4.0–4.15
(m, 2H, CH2OBz and CH2NHCSN), 3.74 (t, 2H,
CH2CH2N), 3.53 (m, 1H, CH2NHCSN), 2.7–2.8 (m,
4H, CH2CH2NCSNH and CH2Ph), 2.57 (m, 1H, CH),
2.21 (m, 6H, 2ꢂ CH3); IR (KBr) 3391, 1523, 1276,
1101 cmꢀ1; Anal. calcd for C29H32N2O4S: C, 69.02; H,
6.39; N, 5.55; S, 6.35. Found: C, 69.25; H, 6.42; N, 5.53;
S, 6.33.
2-(3,4-Dimethylbenzyl)-3-{[(7,8-dihydroxy-1,3,4,5-tetra-
hydro-2H-2-benzazepin-2-yl)carbothioyl]amino}propyl piva-
late (16). 35% yield; a yellow solid; mp 76 ꢃC; 1H
NMR (CDCl3) d 6.9–7.2 (m, 3H, Ph), 6.86 (s, 1H, Ar),
6.73 (s, 1H, Ar), 5.94 (bs, 1H, NH), 5.88 (bs, 1H, NH),
4.45 (m, 2H, ArCH2NH), 4.12 (dd, 1H, CH2OCO), 3.8–
4.0 (m, 2H, CH2OCO and CH2NHCSN), 3.22 (m, 1H,
CH2NHCSN), 2.83 (d, 2H, ArCH2CH2), 2.6 (m, 2H,
CHCH2Ph), 2.23 (m, 7H, 2ꢂ CH3 and CH), 1.81 (m,
2H, ArCH2CH2), 1.30 (s, 9H, C(CH3)3); IR (KBr) 3401,
1537, 1293 cmꢀ1; Anal. calcd for C28H38N2O4S: C,
67.44; H, 7.68; N, 5.62; S, 6.43. Found: C, 67.66; H,
7.70; N, 5.60; S, 6.42.
2-(4-tert-butylbenzyl-3-{[(6,7-dihydroxy-3,4-dihydro-2(1H)-
isoquinolinyl)carbothioyl]amino}propyl benzoate (22).
94% yield; a yellow solid; mp 59 ꢃC; H NMR (CDCl3)
1
d 8.05 (d, 2H, Bz), 7.60 (t, 1H, Bz), 7.48 (t, 2H, Bz), 7.2–
7.35 (m, 5H, Ph), 6.88 (bs, 1H, ArOH), 6.67 (s, 2H, Ar),
6.39 (t, 1H, NH), 4.75 (d, 2H, ArCH2N), 4.57 (dd of
AB, 1H, CH2OBz), 4.11 (ddd, 2H, CH2OBz and
CH2NHCSN), 3.74 (t, 2H, CH2CH2N), 3.48 (m, 1H,
CH2NHCSN), 2.7–2.79 (m, 4H, CH2CH2NCSNH
and CH2Ph), 2.57 (m, 1H, CH), 1.29 (s, 9H,
C(CH3)3); IR (KBr) 3390, 1523, 1275, 1100 cmꢀ1
;
Anal. calcd for C31H36N2O4S: C, 69.90; H, 6.81; N,
5.26; S, 6.02. Found: C, 70.12; H, 6.83; N, 5.24; S,
6.00.
2-Benzyl-3-{[(6,7-dihydroxy-3,4-dihydro-2(1H)-isoquino-
linyl)carbothioyl]amino}propyl benzoate (18). 62% yield;
a yellow solid; mp 53 ꢃC; H NMR (CDCl3) d 8.06 (d,
1
2H, Bz), 7.60 (t, 1H, Bz), 7.47 (t, 2H, Bz), 7.2–7.35 (m,
5H, Ph), 6.88 (bs, 1H, ArOH), 6.67 (s, 1H, Ar), 6.66 (s,
1H, ArH), 6.53 (bs, 1H, ArOH), 6.36 (t, 1H, NH), 4.69
(ArCH2N), 4.57 (dd of AB, 1H, CH2OBz), 4.15 (m, 2H,
CH2OBz and CH2NHCSN), 3.78 (t, 2H, CH2CH2N),
3.50 (m, 1H, CH2NHCSN), 2.7-2.83 (m, 4H,
CH2CH2NCSNH and CH2Ph), 2.60 (m, 1 H, CH); IR
(KBr) 3375, 1712, 1524, 1275, 1101 cmꢀ1; Anal. calcd
for C27H28N2O4S: C, 68.04; H, 5.92; N, 5.88; S, 6.73.
Found: C, 68.29; H, 5.94; N, 5.86; S, 6.71.
2-(3,4-Dimethylbenzyl)-3-{[(6,7-dihydroxy-3,4-dihydro-
2(1H)-isoquinolinyl)carbothioyl]amino} propyl pivalate
(23). 61% yield; a yellow solid; mp 78 ꢃC; 1H NM R
(CDCl3) d 6.9–7.5 (m, 3H, Ph), 6.63 (d, 2H, ArH), 6.29
(t, 1H, NH), 4.67 (s, 2H, ArCH2N), 4.32 (dd of AB,
1H, CH2OCO), 3.93 (m, 2H, CH2OCO and
CH2NHCSN), 3.67 (m, 2H, CH2CH2N), 3.41 (m, 1H,
CH2NHCSN), 2.55–2.7 (m, 4H, CH2CH2NCSNH and
CH2Ph), 2.41 (m, 1H, CH), 2.20 (m, 6H, 2ꢂ CH3), 1.26
(s, 9H, C(CH3)3). IR (KBr) 3390, 1697, 1523,
1279 cmꢀ1; Anal. calcd for C27H36N2O4S: C, 66.91; H,
7.49; N, 5.78; S, 6.62. Found: C, 67.14; H, 7.46; N, 5.76;
S, 6.60.
2-Benzyl-3-{[(6,7-dihydroxy-3,4-dihydro-2(1H)-isoquino-
linyl)carbothioyl]amino}propyl 3,4-dimethylbenzoate (19).
92% yield; a yellow solid; mp 45 ꢃC; H NMR (CDCl3)
1
d 7.80 (m, 2H, Bz), 7.2–7.35 (m, 6H, Ph), 6.88 (m, 1H,
ArOH), 6.65 (s, 1H, Ar), 6.63 (s, 1H, ArH), 6.43 (t, 1H,
NH), 4.75 (d, 2H, ArCH2N), 4.57 (dd of AB, 1H,
CH2OCO), 4.0–4.16 (m, 2H, CH2OCO and
CH2NHCSN), 3.78 (t, 2H, CH2CH2N), 3.45 (m, 1H,
CH2NHCSN), 2.7–2.81 (m, 4H, CH2CH2NCSNH and
CH2Ph), 2.57 (m, 1 H, CH), 2.32 (m, 6H, 2ꢂ CH3); IR
(KBr) 3389, 1696, 1524, 1267 cmꢀ1; Anal. calcd for
C29H32N2O4S: C, 69.02; H, 6.39; N, 5.55; S, 6.35.
Found: C, 69.27; H, 6.41; N, 5.54; S, 6.42.
N-(2-Benzyl-3-benzyloxypropyl)-6,7-dihydroxy-3,4-dihy-
dro-2(1H)-isoquinoline carbothioamide (24). 80% yield;
a yellow solid; mp 45 ꢃC; 1H NMR (CDCl3) d 7.17–7.33
(m, 10H, 2ꢂ Ph), 6.88 (m, 2H, ArOH), 6.56 (s, 1H, Ar),
6.42 (s, 1H, Ar), 4.54 (s, 2H, PhCH2O), 4.41 (s, 2H,
OCH2CH), 3.97 (m, 1H, CH2NCS), 3.78 (m, 2H,
ArCH2CH2), 3.65 (t, 2H, CH2CH2N), 2.67 (t, 3H,
CH2Ph), 2.37 (m, 3 H, CH2CH2NCS and CH); IR
(KBr) 3349, 1521, 1372, 1278 cmꢀ1; Anal. calcd for
C27H30N2O3S: C, 70.10; H, 6.54; N, 6.06; S, 6.93.
Found: C, 70.31; H, 6.56; N, 6.04; S, 6.91.
2-Benzyl-3-{[(6,7-dihydroxy-3,4-dihydro-2(1H)-isoquino-
linyl)carbothioyl]amino}propyl 4-tꢃert-butylbenzoate (20).
1
85% yield; a yellow solid; mp 66 C; H NMR (CDCl3)
d 8.06 (d, 2H, Bz), 7.60 (t, 1H, Bz), 7.47 (t, 2H, Bz), 7.2–
7.35 (m, 5H, Ph), 6.65 (s, 2H, Ar), 6.38 (t, 1H, NH),
4.80 (d, 2H, ArCH2N), 4.58 (dd of AB, 1H, CH2OBz),
4.11 (m, 2H, CH2OBz and CH2NHCSN), 3.74 (t, 2H,
CH2CH2N), 3.50 (m, 1H, CH2NHCSN), 2.6–2.8 (m,
4H, CH2CH2NCSNH and CH2Ph), 2.57 (m, 1H,
CH),1.29 (s, 9H, C(CH3)3); IR (KBr) 3375, 1699, 1522,
1277 cmꢀ1; Anal. calcd for C31H36N2O4S: C, 69.90; H,
6.81; N, 5.26; S, 6.02. Found: C, 70.09; H, 6.83; N, 5.24;
S, 6.00.
2-Benzyl-3-({[(3,4-dihydroxybenzyl)amino]carbothionyl}-
amino)propyl benzoate (25). 81% yield; a yellow solid;
mp 30 ꢃC; 1H NMR (CDCl3) d 8.02 (d, 2H, Ar), 7.60 (t,
1H, Ar), 7.47 (t, 2H, Ar), 7.1–7.3 (m, 5H, Ph), 6.84 (m,
2H, Ar), 6.67 (m, 1H, Ar), 6.36 (m, 2H, NH), 4.36 (m,
2H, ArCH2N and CH2OCO), 4.15 (m, 2H, CH2OCO
and CH2NHCSN), 3.48 (m, 1H, CH2NHCSN), 2.69 (m,
2H, CH2Ph), 2.43 (m, 1H, CH); IR (KBr) 3363, 1602,
1556, 1280, 1113 cmꢀ1; Anal. calcd for C25H26N2O4S:
C, 66.64; H, 5.82; N, 6.22; S, 7.12. Found: C, 66.83; H,
5.84; N, 6.20; S, 7.10.
2-(3,4-Dimethylbenzyl)-3-{[(6,7-dihydroxy-3,4-dihydro-
2(1H)-isoquinolinyl)carbothioyl]amino}propyl benzoate
(21). 66% yield; a yellow solid; mp 62 ꢃC; 1H NM R

1720
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 1713–1720
Protocol of 45Ca uptake by Tet-off VR1 expressing
CHO cells
4. Williams, M.; Kowaluk, E. A.; Arneric, S. P. J. Med.
Chem. 1999, 42, 1481.
5. Szallasi, A. Drug News Perspectives 1997, 10, 522.
6. Szallasi, A.; Blumberg, P. M. Pain 1996, 68, 195.
7. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.;
Rosen, T. A.; Levine, J. D.; Julius, D. Nature 1997, 389, 816.
8. Clapham, D. E. Neuron 1996, 16, 1069.
9. Oh, U.; Kwang, S. W.; Kim, D. J. Neurosci. 1996, 16, 1659.
10. Davies, J. B.; Gray, J.; Gunthorpe, M. J.; Hatcher, J. P.;
Davey, P. T.; Overend, P.; Harries, M. H.; Latcham, J.;
Clapham, C.; Atkinson, K.; Hughes, S. A.; Rance, K.; Gran,
E.; Harper, A. J.; Pugh, P. L.; Rogers, D. C.; Bingham, S.;
Randall, A.; Sheardown, S. A. Nature 2000, 405, 183.
11. Wrigglesworth, R.; Walpole, C. S. J. Drug of the Future
1998, 23, 531.
12. Kress, M.; Zeilhofer, H. U. TIPS 1999, 20, 112.
13. Walpole, C. S. J.; Wrigglesworth, R. Capsaicin in the
Study of Pain; Academic: San Diego, CA, 1993: pp 63–82.
14. Walpole, C. S.; Bevan, S.; Bovermann, G.; Boelsterli, J. J.;
Breckkenridge, R.; Davies, J. W.; Hughes, G. A.; James, I.;
Oberer, L.; Winter, J.; Wrigglesworth, R. J. Med. Chem. 1994,
37, 1942.
15. Kwak, J. Y.; Jung, J. Y.; Hwang, S. W.; Lee, W. T.; Oh,
U. Neuroscience 1998, 86, 619.
16. Szolcsanyi, A.; Jancso-Gabor, A. Arzneimittel-Forsch
1975, 25, 1877.
17. Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S. J.; Cam-
pell, E. A.; Dray, A.; James, I. F.; Perkins, M. N.; Masdin,
K. J.; Winter, J. J. Med. Chem. 1993, 36, 2373.
18. Klopman, G.; Li, J-Y. J. Comp-Aided Mol. Design 1995,
9, 283.
Cells (CHO-VR1 Tet-off cells)22 were plated in 24-well
plates to yield a cell density 20–40% of confluent. The
next day, the medium was changed to remove the tetra-
cycline and induce VR1 expression. Experiments were
done approximately 36–40 h after induction. For assay
of 45Ca uptake, cells were incubated for 10 min at 37 ꢃC
in a total volume of 500 :L of serum free DMEM (con-
taining 1.8 mMCaCl ) in the presence of 0.25 mg/mL
2
BSA (Sigma), 1 :Ci/mL 45Ca (5–30 Ci/g from ICN, CA),
and increasing concentrations of the compound to be
tested. Immediately after the incubation, extracellular
45Ca was removed by washing the cells three times with
cold DPBS (containing 1.8 mMCaCl 2). Then 400 :L
RIPA buffer (50 mMTris pH 7.4; 150 mMNaCl; 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 :L 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 was
analyzed by computer fit to the Hill equation. At least
three separate experiments were carried out for each
compound.
19. Szallasi, A.; Blumberg, P. M. Neuroscience 1989, 30, 515.
20. Lee, J.; Park, S-U.; Kim, J-Y.; Kim, J-K.; Lee, J.; Oh, U.;
Marquez, V. E.; Beheshti, M.; Wang, Q. J.; Modarres, S.;
Blumberg, P. M. Bioorg. Med. Chem. Lett. 1999, 9, 2909.
21. (a) Lee, J.; Lee, J.; Kim, J.; Kim, S. Y.; Chun, M. W.;
Cho, H.; Hwang, S. W.; Oh, U.; Park, Y. H.; Marquez, V. E.;
Beheshti, M.; Szabo, T.; Blumberg, P. M. Bioorg. Med. Chem.
2001, 9, 19. (b) PCT/KR00/00137.
Acknowledgements
This research was supported by a Fund 2000 grant from
the Korea Research Foundation and BK21 project for
Medicine, Dentistry and Pharmacy.
22. Kim, S.; Yi, K. Y. J. Org. Chem. 1986, 51, 2615.
23. Lee, J.; Lee, J. Syn. Commun. 1999, 29, 4127.
24. Szallasi, A.; Blumberg, P. M.; Annicelli, L. L.; Krause,
J. E.; Cortright, D. N. Mol. Pharm. 1999, 56, 581.
25. Acs, G.; Lee, J.; Marquez, V. E.; Blumberg, P. M. Mol.
Brain Res. 1996, 35, 173.
26. Acs, G.; Lee, J.; Marquez, V. E.; Wang, S.; Milne,
G. W. A.; Lewin, N. E.; Blumberg, P. M. J. Neurochem. 1995,
65, 301.
References and Notes
1. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51,
159.
2. Caterina, M. J.; Leffler, A.; Malmberg, A. B.; Martin, W. J.;
Trafton, J.; Petersen-Zeitz, K. R.; Koltzenburg, M.; Basbaum,
A. I.; Julius, D. Science 2000, 288, 241.
3. Chizh, B. A.; Dickenson, A. H.; Wnendt, S. TIPS 1999, 20,
354.