N-(3-acyloxy-2-benzylpropyl)-N′-(4-hydroxy-3-methoxybenzyl)thiourea derivatives as potent vanilloid receptor agonists and analgesics

Article abstract of DOI:10.1016/S0968-0896(00)00216-9

A series of N-(3-acyloxy-2-benzylpropyl)-N′-(4-hydroxy-3-methoxybenzyl)thiourea derivatives were investigated as vanilloid receptor ligands in an effort to discover a novel class of analgesics. The proposed pharmacophore model of resiniferatoxin, which includes the C₂₀-homovanillic moiety, the C₃-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-inflammatory activity and pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among the new compounds, 23 and 28 were found to be the most potent receptor agonists of the series with K_i values of 19 nM and 11 nM, respectively. Their strong in vitro potencies were also reflected by an excellent analgesic profile in animal tests with ED₅₀ values of 0.5 μg/kg for 23 and 1.0 μg/kg for 28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both 23 and 28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show significant anti-inflammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and 35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in this investigation, which were designed as simplified resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor agonists endowed with potent analgesic activity and reduced pungency.

Full text of DOI:10.1016/S0968-0896(00)00216-9

Bioorganic & Medicinal Chemistry 9 (2001) 19±32
N-(3-Acyloxy-2-benzylpropyl)-N0-(4-hydroxy-3-methoxybenzyl)
thiourea Derivatives as Potent Vanilloid Receptor
Agonists and Analgesics
Jeewoo Lee,a,* Jiyoun Lee,a Jiyoung Kim,a Soo Yeon Kim,a
Moon Woo Chun,a Hawon Cho,b Sun Wook Hwang,b Uhtaek Oh,b
Young Ho Park,c Victor E. Marquez,d Maryam Beheshti,e
Tamas Szaboe and Peter M. Blumberge
aLaboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Shinlim-Dong,
Kwanak-Ku, Seoul 151-742, South Korea
bSensory Research Group, CRI, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku,
Seoul 151-742, South Korea
cPaci®c R&D Center, 314-1, Bora-Ri, Kiheung-Eup, Yougin-Si, Kyounggi-Do 449-900, South Korea
dLaboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute,
National Institute of Health, Bethesda, MA 20892, USA
eLaboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences,
National Cancer Institute, National Institute of Health, Bethesda, MA 20892, USA
Received 12 June 2000; accepted 17 July 2000
AbstractÐA series of N-(3-acyloxy-2-benzylpropyl)-N0-(4-hydroxy-3-methoxybenzyl)thiourea derivatives were investigated as
vanilloid receptor ligands in an e€ort to discover a novel class of analgesics. The proposed pharmacophore model of resiniferatoxin,
which includes the C20-homovanillic moiety, the C3-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized
as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor
binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-in¯ammatory activity and
pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among
the new compounds, 23 and 28 were found to be the most potent receptor agonists of the series with Ki values of 19 nM and 11 nM,
respectively. Their strong in vitro potencies were also re¯ected by an excellent analgesic pro®le in animal tests with ED50 values of
0.5 mg/kg for 23 and 1.0 mg/kg for 28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both
23 and 28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show sig-
ni®cant anti-in¯ammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and
35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in
this investigation, which were designed as simpli®ed resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor
agonists endowed with potent analgesic activity and reduced pungency. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
receptor functions as a cation-selective ion channel with
a preference for calcium, and its functional subtype VR1,
activated by both CAP and noxious heat, has recently
been cloned.3 Its desensitization caused by speci®c
ligands has been recognized as a promising therapeutic
approach to mitigate neuropathic pain and other patho-
logical conditions in which neuropeptides released from
primary sensory neurons play a crucial role.3 6
The vanilloid receptor (VR) (or capsaicin receptor) is a
speci®c neuronal membrane recognition site for capsaicin
(CAP) and related irritant compounds.1 It is expressed
almost exclusively by primary sensory neurons involved
in nociception and neurogenic in¯ammation.2 The
Most exogenous VR agonists which are being developed
or used as analgesics are structurally related to CAP
*Corresponding author. Tel.: +82-2-880-7846; fax: +82-2-888-06469;
e-mail: jeewoo@snu.ac.kr
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00216-9

20
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
(i.e., Zostrix, Olvanil, SDZ-249482, and DA-5018) and
resiniferatoxin.7 Their structures include a common
vanilloid ring which appears to be important for agonist
activity. However, in a recent report, it was demon-
strated that compounds lacking the vanilloid moiety,
such as sesquiterpenoid unsaturated dialdehydes or tri-
prenyl phenol, may also activate the receptor.8 Although
receptor antagonists are fewer, several compounds such
as capsazepine, which acts competitively at the CAP
binding site,9 the channel blocker ruthenium red10 and
capsazocaine11 have been reported.
Resiniferatoxin (RTX), a tricyclic diterpene isolated
from Euphorbia resinifera, has been regarded as an ultra-
potent CAP analogue (Chart 1). Indeed, the speci®c
binding of RTX to the CAP binding site in dorsal root
ganglia has been demonstrated with labelled [3H]RTX.12
RTX is being developed as an ultrapotent sensory neu-
ron desensitising agent for the treatment of urinary urge
incontinence and the pain associated with diabetic neu-
ropathy.13 Recently, a totally enantiocontrolled synthesis14
and a conformational analysis of RTX15 have been
reported. However, the pharmacophoric groups of RTX
have not yet been clearly de®ned, although structure±
activity studies suggest that the C20-homovanillic moi-
ety, the C3-keto group, and the orthoester phenyl group
on ring C are crucial structural elements responsible for
the extremely high potency of RTX.16 24 The lower
potency of CAP relative to RTX may be rationalized by
the lack of some of these critical pharmacophoric
groups, especially the C3-keto group. Although a num-
ber of vanilloid agonist based on the structures of CAP
and RTX have been reported as potential analgesics,7
CAP-like analogues are limited by their intrinsic lower
potency and narrow therapeutic index. RTX, on the
other hand, is of limited availability from natural sour-
ces and is dicult to obtain synthetically due to its
structural complexity (Fig. 1).
Chart 2.
the hydroxyl group of the vanilloid ring), the C3-car-
bonyl, and the orthoester phenyl moiety as essential
groups for recognition and binding.16
We have recently reported a series of 3-acyloxy-2-ben-
zylpropyl homovanillate analogues (Chart 3, templates I
and II) designed as simpli®ed vanilloid receptor ligands
of RTX that were based on our pharmacophore
model.25 The amide analogues proved to be quite potent
vanilloid agonists in terms of the receptor binding assay
and the CAP-activated single channels assay, and thus
represent good leads for the development of a novel class
of analgesics that function as VR agonists. Previous
structure±activity relationship studies on the amide
region of CAP, the so-called B-region, have shown that
the thiourea moiety confers high potency to most CAP
analogues in terms of CAP-like activities.26 This high
potency could be explained by the capacity of the
thiourea moiety to engage in multiple hydrogen bond-
ing interactions with the receptor and its increased con-
formational rigidity. However, unlike CAP analogues,
thiourea surrogates of RTX were reported to exhibit
much diminished potency in receptor binding anity
and other biological responses.19
As part of an ongoing program in our laboratory aimed
at the discovery of novel analgesic agents targeted to the
vanilloid receptor, we have directed our synthetic e€orts
toward the preparation of simpli®ed analogues of RTX
according to our proposed RTX-pharmacophore model
depicted in Chart 2. This pharmacophore model was
derived from previous structure±activity relationship
studies of RTX that included the C20-homovanillic
moiety (encompassing the carbonyl of the C20-ester and
In this paper, we wish to describe the results of a structure
activity relationship study of N-(3-acyloxy-2-benzylpro-
pyl)-N0-(4-hydroxy-3-methoxybenzyl)thioureas (Chart 3,
template III) as vanilloid receptor agonists and potential
analgesics which can be considered as thiourea surro-
gates of template I.
Chart 1.

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
21
Chemistry
late diethylmalonate under similar conditions to those
described above for Scheme 1 (Scheme 4).
The syntheses of 3-acyloxy-2-benzylpropyl thiourea
derivatives (18±29) are outlined in Schemes 1 and 2.
Monoalkylation of diethyl malonate with various benzyl
halides, followed by LiAlH4-reduction, produced the cor-
responding diols (2a±d). Monoacetylation of 2,27 conver-
sion of the remaining alcohol function to an azide, and
deprotection of the acetyl group a€orded the inter-
mediate 3-azido-2-benzyl-1-propanols (5a±d). Acylation
of 5a±d with various acyl halides produced the corre-
sponding esters 6±17, which following the reduction of the
azide group were condensed with 4-methoxymethyloxy-
3-methoxybenzylisothiocyanate and hydrolyzed to the
®nal targets 18±29, respectively. The alternative ether
analogues, represented by the 3-benzyloxy-2-benzylpro-
pyl thiourea derivatives, 32 and 33, were synthesized
from 5a,c as shown in Scheme 2. Two chiral analogues
of 18 (35 and 36) were synthesized from (R)-3-acetoxy-2-
benzyl-1-propanol ((R)-3a)-obtained from the enantio-
selective enzymatic acetylation of racemic 2-benzyl-1,3-
propanediol28 following the same procedure for the
synthesis of 18 (Scheme 3). For the synthesis of the
conformationally rigid analogues of 18 and 22 (38 and
39) bis-1,2-chloromethylbenzene was employed to alky-
Results and Discussion
Receptor binding anity and CAP-activated channel
assay
The CAP-like activity of the target compounds was
measured by an in vitro receptor binding assay20 and a
CAP-activated single channel activation assay.29 In the
receptor binding assay, the compounds were evaluated
for their ability to compete with [3H]RTX for binding to
the receptor. The results are expressed in terms of Ki
values (meanÆSEM, three experiments) which represent
the concentration of the non-radioactive ligand that
would give half-maximal receptor occupancy. In the
CAP-activated single channel activation assay, the
increase of inward current resulting from the non-
selective cation in¯ux was measured following the
extracellular application of the compounds to cultured
neonatal rat dorsal root ganglion (DRG) neurons. The
activities of the compounds are expressed in terms of the
relative di€erence in ion conductance compared to CAP
Chart 3.
Scheme 1.

22
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
as a control. The results are presented in Table 1 and
can be summarized as follows: (1) all the compounds
tested showed stronger agonist activity and receptor
binding anity than CAP; (2) the thiourea analogue 18
(Ki=96 nM) exhibited more potent agonist activity than
the CAP-related amide analogue (Ki=404nM) reported
in our earlier study;25 (3) the ester group in R1, which was
designed to mimic the C3-carbonyl of RTX, appears to be
important for potent agonist activity relative to the
benzyl ether analogues in R1 (compare 22 (Ki=31.2 nM)
with 32 (Ki=148 nM) and 27 (Ki=149 nM) with 33
(Ki=660 nM)) suggesting an important role for the car-
bonyl moiety in hydrogen-bonding to the receptor; (4)
the pivaloyl group at R1 and the 4-t-butylbenzyl group
at R2 proved to be e€ective hydrophobic groups result-
ing in optimal agonist activity for compounds 23 and 28
(Ki=19 and 10.8 nM, respectively) which are ca. 280
and 490 times more potent than CAP; (5) binding of
thiourea to the receptor appears to be quite stereospeci®c
as the two optically active enantiomers of 18 showed sub-
stantial di€erences in receptor binding anity relative to
the racemate (compare 18 (Ki=96 nM) with 35 (Ki=
18.4nM) and 36 (Ki=74 nM)); and (6) a conformationally
restricted benzyl moiety in R2 diminishes agonistic
potency (compare 18 (Ki=96 nM) ) with 38 (Ki=480nM)
and 22 (Ki=31.2 nM) with 39 (Ki=1820 nM)).
Analgesic and anti±in¯ammatory activity
The analgesic activities of some of the most potent CAP
agonists, selected from the receptor binding and single
channel assays, were evaluated in the PBQ-induced
writhing assay and the results are shown in Table 2. As
expected, two of the most potent compounds based on
Scheme 2. Method A: (1) H2, Lindler's cat; (2) SCNCH2PL(3-OMOM, 4-OCH3); (3) SF3COOH.
Scheme 3.

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
23
Scheme 4.
Table 1. Anities and channel activations
Compound
R1
R2
R3
Anity, Ki (nM)
5310 (Æ370)
0.023
Activationsa
CAP
RTX
Amideb
18
19
20
21
22
23
24
25
26
27
28
29
32
33
404 (Æ37)
++
+++
+++
++
+
++
++
++
+++
++
++
++
++
++
++
+
(CH3)3CCO
(CH3)2CHCO
CH3(CH2)4CO
CH3(CH2)16CO
PhCO
(CH3)3CCO
PhCO
(3,4-Me2)PhCO
(CH3)3CCO
PhCO
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
H
H
H
H
H
H
H
H
H
H
H
H
96 (Æ31)
133 (Æ75)
632 (Æ48)
1870 (Æ350)
31.2 (Æ9.0)
19 (Æ4.3)
410 (Æ180)
184 (Æ69)
54 (Æ16)
CH2Ph(3,4-Me2)
CH2Ph(3,4-Me2)
CH2Ph(3,4-Me2)
CH2Ph(4-Cl)
CH2Ph(4-Cl)
CH2Ph(4-t-Bu)
CH2Ph(4-t-Bu)
CH2Ph
CH2Ph(4-Cl)
CH2Ph
CH2Ph
CH2Ph
gem-(CH2)2Ph
gem-(CH2)2Ph
149 (Æ11)
10.8 (Æ4.0)
60 (Æ10)
(CH3)3CCO
PhCO
PhCH2
H
148 (Æ11)
660 (Æ290)
146 (Æ48)
18.4 (Æ5.1)
74 (Æ16)
PhCH2
H
CH2OCH3
34
(CH3)3CCO
(CH3)3CCO
(CH3)3CCO
(CH3)3CCO
PhCO
35,c
36,d
38
H
H
H
H
+++
++
++
++
480 (Æ170)
1820 (Æ790)
39
a+=CAP, ++=10 CAP, +++=100 CAP.
b
cR-isomer.
dS-isomer.
the in vitro assay, 23 and 28, exhibited excellent
analgesic activities with ED50 values of 0.5 and 1 mg/kg,
respectively. Such values make these compounds 300 to
600 times more potent than CAP. Both compounds
were likewise more potent than olvanil or DA-5018,
which are either currently available in the market or
undergoing clinical trial as topical analgesics. However,
in the TPA-induced ear edema assay,31 even the most
potent agonists of this series showed weak antiin-
¯ammatory activity compared to the strong anti-edema
activity of CAP (Table 2). We had originally speculated
that the thiourea analogues, with their higher intrinsic
potency, might dramatically reduce the TPA-induced
ear edema. In this respect, their activity resembles that
of olvanil or DA-5018, both of which display weak
topical activity in the TPA ear edema assay. Poor bioa-
vailability through transcutaneous penetration may
explain in part the weak topical antiin¯ammatory
properties observed with these compounds.
Analgesic and anti-in¯ammatory activity
The analgesic activities of some of the most potent CAP
agonists, selected from the receptor binding and single
channel assays, were evaluated in the PBQ-induced wri-
thing assay and the results are shown in Table 2. Though

24
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
they displayed distinctive potencies, tested compounds
could attain the same maximal antinociceptive activity
(100% inhibition) as did capsaicin. As expected, two of
the most potent compounds based on the in vitro assay,
23 and 28, exhibited excellent analgesic activities with
ED50 values of 0.5 and 1 mg/kg, respectively. Such
values make these compounds 300 to 600 times more
potent than CAP. It should be noted that the biological
data in Table 2 are not adjusted for their respective
molecular weights of tested compounds. Both compounds
were likewise more potent than olvanil or DA-5018,
which is a orally active capsaicin analogue with less
pungency or undergoing clinical trial as topical anal-
gesics. However, in the TPA-induced ear edema assay,31
even the most potent agonists of this series showed
weak antiin¯ammatory activity compared to the strong
anti-edema activity of CAP (Table 2). We had originally
speculated that the thiourea analogues, with their higher
intrinsic potency, might dramatically reduce the TPA-
induced ear edema. In this respect, their activity resem-
bles that of olvanil or DA-5018, both of which display
weak topical activity in the TPA ear edema assay. Poor
bioavailability through transcutaneous penetration may
explain in part the weak topical anti-in¯ammatory
properties observed with these compounds.
less potent in evoking acute pain. A possible explana-
tion for the reduced excitatory properties of these syn-
thetic compounds (23 and 28) could lie in their rate of
excitation of the sensory neuron.33 Separation of
intrinsic agonist activity from acute irritancy should
confer an important therapeutic advantage.
To check for the development of tachyphylaxis or cross-
tachyphylaxis, a group of rats treated with the selected
synthetic compounds were challenged 6 h postreatment
with 10 mg/mL CAP.8 CAP, RTX and the synthetic
analogues displayed an attenuated eye-wiping response
in the test challenge of 10 mg/mL CAP, a result that is
indicative of cross-tachyphylaxis between these classes
of compounds. However, it should be noted that for
CAP analogues a prior eye-wiping response is a pre-
requisite for the development of after-desensitization. In
other words, non-irritating doses of CAP analogues
were without e€ect to protect the eye-wiping movement
by test treatment with 0.001% CAP.
Molecular modelling
A search for a common pharmacophore pattern between
the thiourea analogues and RTX was performed by
molecular modelling using the program Sybyl 6.4 from
Tripos on a Silicon Graphics R4600 INDY work-
station. Energy-minimized structures of RTX and 35
were obtained from molecular dynamics simulation.
Four pharmacophoric groups in 35, namely the carbonyl
of the ester, the sulfur of the thiourea, the centre of
vanilloid ring and the centre of phenyl ring were matched
with the C3-keto carbonyl, the C20-ester carbonyl, the
centre of vanilloid ring and the centre of orthoester
phenyl in RTX, respectively. The ®tting demonstrated a
good correlation in the spatial disposition of the corre-
sponding key pharmacophores with 1.494 A rms values.
The result indicated that target thiourea compounds
serve as good surrogates of RTX by containing their
crucial pharmacophores.
Pungency and tachyphylaxis
The focus of medicinal chemistry in developing vanil-
loid-derived therapeutics has been the improvement of
the bioavailability and the reduction of the excitatory
properties. In line with these objectives, the rat eye-
wiping test was employed as an in vivo pungency test to
assess the pain-producing e€ects of the compounds.32
As shown in Table 3, pungency pro®les of selected
potent agonists, 22, 23 and 28 did not follow the trend
of the intrinsic agonistic activity measured by the two in
vitro assays. In this assay, RTX, an ultrapotent capsai-
cin analogue, was only 3-fold more potent than CAP
and the selected compounds from our series were much
Table 2. Writhing test and ear edema assay
d
Table 3. Eye-Wiping Testa
Compound
Writhing test Relative Ear edema assay Relative
ED50 (mg/kg) potency
ID50 (mg/ear)
potency
Compound
MPP (mg/mL)
RPP
100
Tachyphylaxis
CAP
RTX
Olvanil
DA-5018
Indomethacin
Aspirin
Morphine
18
19
20
21
22
23
24
25
26
27
300
0.01
30
3
400
3500
1000
2
15
5
20
20
0.5
7
2
5
12
1
1.5
1
30,000
10
3
0.002
30
1
1500
0.1
CAP
3
55 (1000)
35 (100)
85 (100)
20 (10)
RTX
1
300
100
200
0.015
0.75
0.086
0.3
150
20
60
15
15
600
43
150
60
25
Olvanil
DA-5018
WP
30
Ð
10
0 (1000)
90 (1000)
35 (100)
0 (1000)
90 (1000)
30 (100)
5 (10)
25
0.12
22
23
WP
20
Ð
15
20
18
50
50
22
17
5
0.15
0.17
0.06
0.06
0.14
0.18
0.6
28
30
10
NTe
aMPP, moderate pain-producing potency.
bRPP, relative pain-producing potency.
cWP, weakly pungent (less pungent than 3 mg/mL capsaicin at 100 mg/
mL).
dTachyphylaxis percent reduction of eye wiping number after test
challenge of 10 mg/mL capsaicin. The number in parentheses indicates
concd (mg/mL) of desensitising challenge.
eNT, not tested.
28
29
35
36
300
200
38
30
0.1
8
9
33

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
25
were performed with an EA 1110 Automatic Elemental
Analyzer, CE Instruments.
General procedure for the synthesis of 1a±d. A cooled
solution of diethylmalonate (6.4 g, 40 mmol) in DMF
(20 mL) at 0 ꢀC was treated with sodium hydride (60%,
1.92 g, 48 mmol) portionwise and stirred for 40 min at
room temperature. The reaction mixture was treated
with the corresponding substituted benzyl chlorides
(48 mmol) and stirred overnight at room temperature.
The 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 puri®ed by ¯ash
column chromatography on silica gel with EtOAc:hexane
(1:10) as eluant to give 1.
Diethyl 3,4-dimethylbenzylmalonate (1b). 70% yield,
colorless oil; 1H NMR (CDCl3) d 6.9±7.05 (m, 3H), 4.14
(m, 4H, 2ÂCO2CH2), 3.61 (t, 1H, CH), 3.25 (d, 1H,
CH2Ar), 3.14 (d, 1H, CH2Ar), 2.2±2.25 (m, 6H,
2ÂCH3), 1.21 (m, 6H, 2ÂCO2CH2CH3).
Diethyl 4-chlorobenzylmalonate (1c). 74% yield, color-
1
less oil; H NMR (CDCl3) d 7.25 (d, 2H), 7.16 (d, 2H),
4.14 (m, 4H, 2ÂCO2CH2), 3.60 (t, 1H, CH), 3.18 (d, 2H,
Figure 1. Superposition of 35 on resiniferatoxin.
CH2Ar), 1.21 (t, 6H, 2ÂCO2CH2CH3).
Diethyl 4-t-butylbenzylmalonate (1d). 74% yield, color-
1
Conclusion
less oil; H NMR (CDCl3) d 7.29 (d, 2H), 7.13 (d, 2H),
A series of N-(3-acyloxy-2-benzylpropyl)-N0-(4-hydroxy-
3-methoxybenzyl)thiourea analogues were constructed
as novel agonists of the vanilloid receptor with the
intent of mimicking the spatial disposition of the C20-
homovanillate, the C3-carbonyl, and the phenyl ortho-
ester side chain of RTX. Two compounds, 23 and 28,
showed very potent CAP-like activities in terms of the
binding assay and CAP-activated single channel assay.
Both compounds also showed excellent analgesic activ-
ity as shown by the PBQ-induced writhing test and were
less pungent than CAP. However, all the thioureas had
weak antiin¯ammatory activities. These compounds,
however, represent excellent leads for the development
of novel VR ligands as analgesics.
4.16 (q, 4H, 2ÂCO2CH2), 3.63 (t, 1H, CH), 3.18 (d, 2H,
CH2Ar), 1.30 (s, 9H, C(CH3)3), 1.20 (t, 6H, 2ÂCO2
CH2CH3).
General procedure for the synthesis of 2a±d. A cooled
solution of lithium aluminium hydride (3.64 g, 96 mmol)
in diethyl ether (80 mL) at 0 ꢀC was treated dropwise
with a solution of 1 (24 mmol) in diethyl ether (20 mL).
After stirring for 3 h at room temperature, the reac-
tion mixture was cooled over an ice-bath and treated
successively by the dropwise addition of 3.5 mL of
H2O, 7 mL of 15% NaOH solution, and 10.5 mL of
H2O. The mixture was ®ltered by washing with EtOAc
and the ®ltrate was concentrated in vacuo. The resi-
due was puri®ed by ¯ash column chromatography
over silica gel with EtOAc:hexane (3:1) as eluant to
give 2.
2-Benzyl-1,3-propanediol (2a). 98% yield, white solid,
mp=67 ꢀC; 1H NMR (CDCl3) d 7.15±7.30 (m, 5H,
phenyl), 3.74 (dd, 2H, CH2OH), 3.62 (dd, 2H, CH2OH),
2.9±3.0 (bs, 2H, OH), 2.58 (d, 2H, CH2Ph), 2.03 (m, 1H,
CH).
Experimental
General
All chemical reagents were commercially available.
Melting points were determined on a Melting Point
Buchi B-540 apparatus, and are uncorrected. Silica gel
column chromatography was performed on silica gel 60,
230±400 mesh, Merck. Proton and Carbon NMR spec-
tra were recorded on a JEOL JNM-LA 300 at 300 MHz
and JEOL JNM-GCX 400 at 100 MHz, respectively.
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
2-(3,4-Dimethylbenzyl)-1,3-propandiol (2b). 92% yield,
colorless oil; 1H NMR (CDCl3) d 6.88±7.06 (m, 3H,
phenyl), 3.6±3.8 (m, 4H, 2ÂCH2OH), 2.5±2.7 (bs, 2H,
OH), 2.60 (d, 1H, CH2Ph), 2.52 (d, 1H, CH2Ph), 2.2±
2.28 (m, 6H, 2ÂCH3), 2.02 (m, 1H, CH).
2-(4-Chlorobenzyl)-1,3-propandiol (2c). 75% yield, white
solid, mp=64 ꢀC; 1H NMR (CDCl3) d 7.24 (d, 2H),
7.10 (d, 2H), 3.75 (dd, 2H, CH2OH), 3.61 (dd, 2H,

26
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
CH2OH), 2.85 (bs, 2H, OH), 2.58 (d, 2H, CH2Ph), 1.97
(m, 1H, CH).
over MgSO4 and concentrated in vacuo. The residue
was puri®ed by ¯ash column chromatography on silica
gel with EtOAc:hexane (1:15) as eluant to give 4.
2-(4-t-Butylbenzyl)-1,3-propandiol (2d). 80% yield, white
solid, mp=67 ꢀC; 1H NMR (CDCl3) d 7.31 (d, 2H),
7.11 (d, 2H), 3.81 (dd, 2H, CH2OH), 3.68 (dd, 2H,
CH2OH), 2.58 (d, 2H, CH2Ph), 2.23 (bs, 2H, OH), 2.05
(m, 1H, CH), 1.30 (s, 9H, C(CH3)3).
3-Azido-2-benzylpropyl acetate (4a). 92% yield, color-
1
less oil; H NMR (CDCl3) d 7.15±7.35 (m, 5H, phenyl),
4.05 (ddd of AB, 2H, CH2OAc), 3.34 (m, 2H, CH2N3),
2.68 (d, 2H, CH2Ph), 2.22 (m, 1H, CH), 2.08 (s, 3H,
COCH3)
General procedure of the synthesis of 3a±d. A mixture of
2 (20 mmol), trimethylorthoacetate (3.6 g, 30 mmol) and
a catalytic amount of p-toluenesulfonic acid in CH2Cl2
(40 mL) was stirred for 2 h at room temperature and then
treated with H2O (0.54 g, 30 mmol). After stirring over-
night at room temperature, the reaction mixture was
concentrated in vacuo. The residue was puri®ed by ¯ash
column chromatography over silica gel with EtOAc:
hexane (1:2) as eluant to give 3.
3-Azido-2-(3,4-dimethylbenzyl)propyl acetate (4b). 85%
1
yield, colorless oil; H NMR (CDCl3) d 6.87±7.07 (m,
3H), 4.04 (m, 2H, CH2OAc), 3.33 (m, 2H, CH2N3), 2.69
(d, 1H, CH2Ph), 2.60 (d, 1H, CH2Ph), 2.1±2.3 (m, 7H,
2ÂCH3 and CH), 2.07 (s, 3H, COCH3).
3-Azido-2-(4-chlorobenzyl)propyl acetate (4c). 88%
1
yield, colorless oil; H NMR (CDCl3) d 7.26 (d, 2H),
7.12 (d, 2H), 4.03 (ddd of AB, 2H, CH2OAc), 3.34 (m,
2H, CH2N3), 2.65 (d, 2H, CH2Ph), 2.17 (m, 1H, CH),
2.07 (s, 3H, COCH3).
2-Benzyl-3-hydroxypropyl acetate (3a). 97% yield, col-
orless oil; 1H NMR (CDCl3) d 7.1±7.25 (m, 5H, phenyl),
4.06 (ddd of AB, 2H, CH2OAc), 3.48 (m, 2H, CH2OH),
2.58 (ddd of AB, 2H, CH2Ph), 2.10 (m, 1H, CH), 2.01
(s, 3H, COCH3).
3-Azido-2-(4-t-butylbenzyl)propyl acetate (4d). 92%
1
yield, colorless oil; H NMR (CDCl3) d 7.32 (d, 2H),
7.08 (d, 2H), 4.05 (ddd of AB, 2H, CH2OAc), 3.35 (ddd
of AB, 2H, CH2N3), 2.64 (d, 2H, CH2Ph), 2.20 (m, 1H,
CH), 2.07 (s, 3H, COCH3), 1.30 (s, 9H, C(CH3)3).
(R)-2-Benzyl-1,3-propanediol ((R)-3a). This compound
was obtained from 2a by a published literature pro-
cedure.28
General procedure for the synthesis of 5a±d. A solution
of 4 (8 mmol) and a catalytic amount of K2CO3 in
MeOH (10 mL) was treated with a couple of drops of
H2O. After stirring for 2 h at room temperature, the
reaction mixture was quenched with several drops of
acetic acid and concentrated in vacuo. The residue was
puri®ed by ¯ash column chromatography on silica gel
with EtOAc:hexane (1:3) as eluant to give 5.
2-(3,4-Dimethylbenzyl)-3-hydroxypropyl acetate (3b). 90%
1
yield, colorless oil; H NMR (CDCl3) d 6.88±7.06 (m,
5H, phenyl), 4.13 (m, 2H, CH2OAc), 3.54 (m, 2H,
CH2OH), 2.62 (m, 2H, CH2Ph), 2.2±2.3 (m, 6H, 2Â
CH3), 2.10 (m, 1H, CH), 2.07 (s, 3H, COCH3)
2-(4-Chlorobenzyl)-3-hydroxypropyl acetate (3c). 86%
1
yield, colorless oil; H NMR (CDCl3) d 7.25 (d, 2H),
7.12 (d, 2H), 4.12 (ddd of AB, 2H, CH2OAc), 3.53 (ddd
of AB, 2H, CH2OH), 2.63 (ddd of AB, 2H, CH2Ph),
2.10 (m, 1H, CH), 2.06 (s, 3H, COCH3).
3-Azido-2-benzyl-1-propanol (5a). 98% yield, colorless
oil; 1H NMR (CDCl3) d 7.15±7.35 (m, 5H, phenyl), 3.65
(m, 2H, CH2OH), 3.40 (ddd of AB, 2H, CH2N3), 2.67
(ddd of AB, 2H, CH2Ph), 2.04 (m, 1H, CH).
2-(4-t-Butylbenzyl)-3-hydroxypropyl acetate (3d). 84%
1
yield, colorless oil; H NMR (CDCl3) d 7.30 (d, 2H),
3-Azido-2-(3,4-dimethylbenzyl)-1-propanol (5b). 92%
1
7.11 (d, 2H), 4.13 (ddd of AB, 2H, CH2OAc), 3.56 (ddd
of AB, 2H, CH2OH), 2.62 (ddd of AB, 2H, CH2Ph),
2.28 (s, 1H, OH), 2.10 (m, 1H, CH), 2.01 (s, 3H,
COCH3), 1.30 (s, 9H, C(CH3)3).
yield, colorless oil; H NMR (CDCl3) d 6.88±7.08 (m,
5H, phenyl), 3.65 (m, 2H, CH2OH), 3.42 (m, 2H,
CH2N3), 2.68 (dd, 1H, CH2Ph), 2.60 (dd, 1H, CH2Ph),
2.2±2.3 (m, 6H, 2ÂCH3), 2.03 (m, 1H, CH).
General procedure of the synthesis of 4a±d. A cooled
solution of 3 (12 mmol) and triethylamine (3.64 g,
36 mmol) in CH2Cl2 (20 mL) at 0 ꢀC was treated drop-
wise with methanesulfonylchloride (2.06 g, 18 mmol).
After stirring for 6 h at room temperature, the mixture
was diluted with CH2Cl2. The organic layer was washed
with 1N HCl, H2O and brine, dried over MgSO4, and
concentrated in vacuo. The residue was puri®ed by ¯ash
column chromatography on silica gel with EtOAc:hex-
ane (1:3) as eluant to give the corresponding mesylate as
an oil. The mesylate was dissolved in DMF (10 mL) and
treated with sodium azide (2.2 g, 34 mmol). After 8 h of
stirring at 80 ꢀC, the reaction mixture was diluted with
H2O and extracted with EtOAc several times. The
combined organic layer was washed with H2O, dried
3-Azido-2-(4-chlorobenzyl)-1-propanol (5c). 88% yield,
1
colorless oil; H NMR (CDCl3) d 7.25 (d, 2H), 7.10 (d,
2H), 3.58 (m, 2H, CH2OH), 3.36 (m, 2H, CH2N3), 2.63
(dd, 2H, CH2Ph), 2.1 (bs, 1H, OH), 1.98 (m, 1H, CH).
3-Azido-2-(4-t-butylbenzyl)-1-propanol (5d). 98% yield,
1
colorless oil; H NMR (CDCl3) d 7.32 (d, 2H), 7.10 (d,
2H), 3.63 (ddd of AB, 2H, CH2OH), 3.39 (ddd of AB,
2H, CH2N3), 2.62 (m, 2H, CH2Ph), 2.02 (m, 1H, CH),
1.78 (bs, 1H, OH), 1.30 (s, 9H, C(CH3)3).
General procedure for the synthesis of 6±17. A cooled
solution of 5 (1 mmol), triethylamine (4 mmol) and a
catalytic amount of 4-dimethylaminopyridine in CH2Cl2
(5 mL) at 0 ꢀC was treated with the corresponding acyl

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
27
chloride (2 mmol). After 2±12 h of stirring at room tem-
perature, the mixture was diluted with CH2Cl2. The
organic layer was washed with 1N HCl, H2O and brine,
dried over MgSO4, and concentrated in vacuo. The
residue was puri®ed by ¯ash column chromatography
over silica gel with EtOAc:hexane (1:10±20) as eluant to
give 6±17, respectively.
CH2N3), 2.66 (d, 2H, CH2Ph), 2.18 (m, 1H, CH), 1.23
(s, 9H, C(CH3)3).
3-Azido-2-(4-chlorobenzyl)propyl benzoate (15). 92%
1
yield, colorless oil; H NMR (CDCl3) d 8.01 (m, 2H),
7.58 (m, 1H), 7.46 (m, 2H), 7.28 (d, 2H), 7.13 (d, 2H),
4.29 (ddd of AB, 2H, CH2OCO), 3.43 (m, 2H, CH2N3),
2.75 (d, 2H, CH2Ph), 2.33 (m, 1H, CH).
3-Azido-2-benzylpropyl pivalate (6). 92% yield, colorless
oil; 1H NMR (CDCl3) d 7.15±7.35 (m, 5H, phenyl), 4.04
(ddd of AB, 2H, CH2OCO), 3.34 (ddd of AB, 2H,
CH2N3), 2.68 (d, 2H, CH2Ph), 2.21 (m, 1H, CH), 1.23
(s, 9H, C(CH3)3)
3-Azido-2-(4-t-butylbenzyl)propyl pivalate (16). 99% yield,
1
colorless oil; H NMR (CDCl3) d 7.31 (d, 2H), 7.08 (d,
2H), 4.04 (ddd of AB, 2H, CH2OCO), 3.35 (m, 2H,
CH2N3), 2.65 (d, 2H, CH2Ph), 2.20 (m, 1H, CH), 1.30
(s, 9H, C(CH3)3), 1.23 (s, 9H, C(CH3)3).
3-Azido-2-benzylpropyl 2-methylpropanoate (7). 93%
1
yield, colorless oil; H NMR (CDCl3) d 7.15±7.35 (m,
3-Azido-2-(4-t-butylbenzyl)propyl benzoate (17). 88%
1
5H, phenyl), 4.05 (ddd of AB, 2H, CH2OCO), 3.34 (m,
2H, CH2N3), 2.68 (d, 2H, CH2Ph), 2.58 (m, 1H, CHMe2),
2.21 (m, 1H, CH), 1.18 (dd, 6H, 2ÂCH3).
yield, colorless oil; H NMR (CDCl3) d 8.04 (m, 2H),
7.57 (m, 1H), 7.45 (m, 2H), 7.32 (d, 2H), 7.12 (d, 2H),
4.31 (ddd of AB, 2H, CH2OCO), 3.44 (m, 2H, CH2N3),
2.75 (d, 2H, CH2Ph), 2.35 (m, 1H, CH), 1.30 (s, 9H,
C(CH3)3).
3-Azido-2-benzylpropyl hexanoate (8). 90% yield, color-
1
less oil; H NMR (CDCl3) d 7.14±7.35 (m, 5H, phenyl),
4.05 (ddd of AB, 2H, CH2OCO), 3.34 (m, 2H, CH2N3),
2.68 (d, 2H, CH2Ph), 2.32 (t, 2H, OOCCH2), 2.20 (m,
1H, CH), 1.64 (m, 2H), 1.2±1.4 (m, 4H), 0.88 (distorted
t, 3H).
General procedure for the synthesis of 18±29. A solution
of 6±17 (0.5 mmol) and Lindler's catalyst (50 mg) in
EtOH (5 mL) was hydrogenated under a hydrogen bal-
loon for 2 h. The reaction mixture was ®ltered and the
®ltrate was concentrated in vacuo. The residue was dis-
solved in CH2Cl2 (5 mL) and treated with 4-[(methoxyl-
methyl)oxy)]-3-methoxybenzyl isothiocyanate (0.5 mmol).
After stirring overnight at room temperature, the reac-
tion mixture was concentrated and the residue was
puri®ed by ¯ash column chromatography on silica gel
with EtOAc:hexane (1:1) as eluant to give the corre-
sponding thiourea. The thiourea was dissolved in
CH2Cl2 (2 mL) and treated with tri¯uoroacetic acid
(1 mL). After 1 h of stirring at room temperature, the
mixture was quenched with solid NaHCO3, ®ltered, and
the ®ltrate was concentrated. The residue was diluted
with EtOAc, washed with NaHCO3, H2O and brine,
and concentrated in vacuo. The residue was puri®ed by
¯ash column chromatography on silica gel with EtOAc:
hexane (1:1) as eluant to give 18±29.
3-Azido-2-benzylpropyl stearate (9). 78% yield, colorless
oil; 1H NMR (CDCl3) d 7.14±7.35 (m, 5H, phenyl), 4.05
(ddd of AB, 2H, CH2OCO), 3.33 (m, 2H, CH2N3), 2.68
(d, 2H, CH2Ph), 2.32 (t, 2H, OOCCH2), 2.20 (m, 1H,
CH), 1.2±1.7 (m, 30H), 0.88 (distorted t, 3H).
3-Azido-2-benzylpropyl benzoate (10). 90% yield, color-
less oil; 1H NMR (CDCl3) d 8.04 (m, 2H), 7.15±7.60 (m,
8H), 4.35 (dd, 1H, J=5.1 and 11.2 Hz, BzOCH2), 4.26
(dd, 1H, BzOCH2), 3.43 (ddd of AB, 2H, J=5.61, 5.85
and 12.42 Hz, CH2N3), 2.78 (m, 2H, CH2Ph), 2.35 (m,
1H, CH).
3-Azido-2-(3,4-dimethylbenzyl)propyl pivalate (11). 94%
1
yield, colorless oil; H NMR (CDCl3) d 6.86±7.07 (m,
3H), 4.04 (m, 2H, CH2OCO), 3.36 (m, 2H, CH2N3),
2.70 (d, 1H, CH2Ph), 2.61 (d, 1H, CH2Ph), 2.1±2.3 (m,
7H, 2ÂCH3 and CH), 1.23 (s, 9H, C(CH3)3).
2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)amino]carbo-
1
thioyl}propyl pivalate (18). 40% yield, colorless oil; H
NMR (CDCl3) d 7.1±7.32 (m, 5H, phenyl), 6.75±6.9 (m,
3H, Ar), 6.27 (bt, 1H, NH), 6.05 (bs, 1H, NH), 5.63 (s,
1H, OH), 4.40 (bd, 2H, J=4.38 Hz, NHCH2Ar), 4.17
(dd, 1H, J=3.9 and 11.46 Hz, CH2OCO), 3.87 (s, 3H,
OCH3), 3.7±3.85 (m, 2H, 1H of CH2OCO and
CHCH2NHC=S), 3.24 (ddd, 1H, J=5.37, 8.07 and
13.89 Hz, CHCH2NHC=S), 2.61 (ddd of AB, 2H,
CH2Ph), 2.33 (m, 1H, CH), 1.23 (s, 9H, C(CH3)3); IR
(neat): 3362, 1715, 1278, 1157; MS m/e 445 (M++1).
Anal. calcd for C24H32N2O4S: C, 64.84; H, 7.25; N,
6.30; S, 7.21. Found: C, 65.12; H, 7.28; N, 6.32; S, 7.19.
3-Azido-2-(3,4-dimethylbenzyl)propyl benzoate (12). 95%
1
yield, colorless oil; H NMR (CDCl3) d 8.02 (m, 2H),
7.57 (m, 1H), 7.46 (m, 2H), 6.8±7.07 (m, 3H), 4.32 (m,
2H, CH2OCO), 3.45 (m, 2H, CH2N3), 2.80 (d, 1H,
CH2Ph), 2.71 (d, 1H, CH2Ph), 2.2±2.4 (m, 7H, 2ÂCH3
and CH).
3-Azido-2-(3,4-dimethylbenzyl)propyl 3,4-dimethylbenzo-
1
ate (13). 84% yield, colorless oil; H NMR (CDCl3) d
7.77 (m, 2H), 7.21 (m, 1H), 6.9±7.07 (m, 3H), 4.30 (m,
2H, CH2OCO), 3.44 (m, 2H, CH2N3), 2.79 (d, 1H,
CH2Ph), 2.70 (d, 1H, CH2Ph), 2.2±2.4 (m, 13H, 4ÂCH3
and CH).
2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)amino]carbo-
thioyl}propyl 2-methylpropanoate (19). 35% yield, col-
orless oil; 1H NMR (CDCl3) d 7.13±7.30 (m, 5H, phenyl),
6.75±6.87 (m, 3H, Ar), 6.34 (bt, 1H, NH), 6.30 (bs, 1H,
NH), 5.75 (s, 1H, OH), 4.40 (bs, 2H, NHCH2Ar), 4.17
(dd, 1H, J=3.9 and 10.95 Hz, CH2OCO), 3.84 (s, 3H,
3-Azido-2-(4-chlorobenzyl)propyl pivalate (14). 86% yield,
1
colorless oil; H NMR (CDCl3) d 7.27 (d, 2H), 7.09 (d,
2H), 4.02 (ddd of AB, 2H, CH2OCO), 3.33 (m, 2H,

28
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
OCH3), 3.65±3.85 (m, 2H, CH2OCO and CHCH2
NHC=S), 3.28 (m, 1H, CHCH2NHC=S), 2.60 (m, 3H,
CH2Ph and CHMe2), 2.30 (m, 1H, CHCH2Ph), 1.18
(dd, 6H, 2ÂCH3); IR (neat): 3361, 1715, 1273, 1157; MS
m/e 430 (M+). Anal. calcd for C23H30N2O4S: C, 64.16;
H, 7.02; N, 6.51; S, 7.45. Found: C, 64.42; H, 7.05; N,
6.49; S, 7.42.
2-(3,4-Dimethylbenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)
amino]carbothioyl}propyl benzoate (24). 45% yield,
ꢀ
1
white solid, mp=44 C; H NMR (CDCl3) d 8.02 (m,
2H), 7.59 (m, 1H), 7.46 (m, 2H), 6.9±7.06 (m, 3H), 6.72±
6.85 (m, 3H, Ar), 6.33 (m, 1H, NH), 6.13 (bs, 1H, NH),
5.65 (s, 1H, OH), 4.35±4.44 (m, 3H, NHCH2Ar and
CH2OCO), 4.05 (m, 1H, CH2OCO), 3.7±3.85 (m, 4H,
OCH3 and CHCH2NHC=S), 3.40 (m, 1H, CHCH2
NHC=S), 2.6±2.78 (m, 2H, CH2Ph), 2.44 (m, 1H, CH),
2.2-2.3 (m, 6H, 2ÂCH3); IR (neat): 3360, 1713, 1274,
1121; MS m/e 492 (M+). Anal. calcd for C28H32N2O4S:
C, 68.27; H, 6.55; N, 5.69; S, 6.51. Found: C, 68.53; H,
6.58; N, 5.67; S, 6.49.
2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)amino]carbo-
thioyl}propyl hexanoate (20). 34% yield, colorless oil;
1H NMR (CDCl3) d 7.1±7.3 (m, 5H, phenyl), 6.75±6.9
(m, 3H, Ar), 6.23 (bt, 1H, NH), 6.11 (bs, 1H, NH), 5.66
(s, 1H, OH), 4.40 (bs, 2H, NHCH2Ar), 4.15 (dd, 1H,
J=3.9 and 11.43 Hz, CH2OCO), 3.86 (s, 3H, OCH3),
3.65±3.85 (m, 2H, CH2OCO and CHCH2NHC=S),
3.29 (m, 1H, CHCH2NHC=S), 2.61 (m, 2H, CH2Ph),
2.30 (m, 3H, CH2COO and CH), 1.65 (m, 2H), 1.2±1.4
(m, 4H), 0.88 (distorted t, 3H); IR (neat): 3360, 1715,
1274, 1122; MS m/e 458 (M+). Anal. calcd for C25H34
N2O4S: C, 65.47; H, 7.47; N, 6.11; S, 6.99. Found: C,
65.72; H, 7.50; N, 6.09; S, 6.96.
2-(3,4-Dimethylbenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)
amino]carbothioyl}propyl 3,4-dimethylbenzoate (25). 55%
yield, white solid, mp=58 ꢀC; H NMR (CDCl3) d 7.75
1
(m, 2H), 7.20 (m, 1H), 6.9±7.06 (m, 3H), 6.74±6.85 (m,
3H, Ar), 6.39 (m, 1H, NH), 6.07 (bs, 1H, NH), 5.63 (s,
1H, OH), 4.35±4.43 (m, 3H, NHCH2Ar and CH2OCO),
4.05 (m, 1H, CH2OCO), 3.7±3.85 (m, 4H, OCH3 and
CHCH2NHC=S), 3.37 (m, 1H, CHCH2 NHC=S),
2.6±2.74 (m, 2H, CH2Ph), 2.41 (m, 1H, CH), 2.2±2.35
(m, 12H, 4ÂCH3); IR (neat): 3373, 1711, 1266, 1124;
MS m/e 520 (M+). Anal. calcd for C30H36N2O4S: C,
69.20; H, 6.97; N, 5.38; S, 6.16. Found: C, 69.47; H,
7.00; N, 5.36; S, 6.14.
2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)amino]carbo-
1
thioyl}propyl stearate (21). 30% yield, colorless oil; H
NMR (CDCl3) d 7.1±7.3 (m, 5H, phenyl), 6.75±6.9 (m,
3H, Ar), 6.28 (bt, 1H, NH), 6.17 (bs, 1H, NH), 5.69 (s,
1H, OH), 4.39 (bs, 2H, NHCH2Ar), 4.14 (dd, 1H,
J=3.9 and 11.67 Hz, CH2OCO), 3.87 (s, 3H, OCH3),
3.65±3.85 (m, 2H, CH2OCO and CHCH2NHC=S),
3.30 (m, 1H, CHCH2NHC=S), 2.60 (m, 2H, CH2Ph),
2.30 (m, 3H, CH2COO and CH), 1.2±1.7 (m, 30H), 0.88
(distorted t, 3H); IR (neat): 3363, 1731, 1274, 1031; MS
m/e 626 (M+). Anal. calcd for C37H58N2O4S: C, 70.89;
H, 9.32; N, 4.47; S, 5.11. Found: C, 71.15; H, 9.36; N,
4.45; S, 5.10.
2-(4-Chlorobenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)ami-
no]carbothioyl}propyl pivalate (26). 40% yield, white
solid, mp=62 ꢀC; 1H NMR (CDCl3) d 7.25 (d, 2H),
7.10 (d, 2H), 6.77±6.90 (m, 3H, Ar), 6.38 (t, 1H, NH),
6.25 (bs, 1H, NH), 5.72 (s, 1H, OH), 4.42 (bs, 2H, NH
CH2Ar), 4.16 (dd of AB, 1H, CH2OCO), 3.86 (s, 3H,
OCH3), 3.7±3.85 (m, 2H, CH2OCO and CHCH2 NHC=
S), 3.19 (m, 1H, CHCH2NHC=S), 2.59 (ddd of AB,
2H, CH2Ph), 2.32 (m, 1H, CH), 1.22 (s, 9H, C(CH3)3);
IR (neat): 3360, 1714, 1279, 1159; MS m/e 479 (M+).
Anal. calcd for C24H31ClN2O4S: C, 60.17; H, 6.52; N,
5.85; S, 6.69. Found: C, 60.40; H, 6.55; N, 5.83; S, 6.66.
2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)amino]carbo-
thioyl}propyl benzoate (22). 50% yield, white solid,
mp=90 ꢀC; 1H NMR (CDCl3) d 7.98±8.02 (m, 2H), 7.58
(m, 1H), 7.4±7.5 (m, 2H), 7.1±7.32 (m, 5H, phenyl),
6.72±6.85 (m, 3H, Ar), 6.42 (bt, 1H, NH), 6.31 (bs, 1H,
NH), 5.73 (s, 1H, OH), 4.34±4.42 (m, 3H, NHCH2Ar
and CH2OCO), 4.05 (dd, 1H, CH2OCO), 3.7±3.85 (m,
4H, OCH3 and CHCH2NHC=S), 3.38 (m, 1H, CH
CH2NHC=S), 2.6±2.78 (m, 2H, CH2Ph), 2.46 (m, 1H,
CH); IR (neat): 3360, 1714, 1274, 1121; MS m/e 464
(M+). Anal. calcd for C26H28N2O4S: C, 67.22; H, 6.07;
N, 6.03; S, 6.90. Found: C, 67.50; H, 6.10; N, 6.01; S,
6.88.
2-(4-Chlorobenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)ami-
no]carbothioyl}propyl benzoate (27). 55% yield, white
solid, mp=57 ꢀC; 1H NMR (CDCl3) d 8.00 (m, 2H), 7.60
(m, 1H), 7.47 (m, 2H), 7.27 (d, 2H), 7.15 (d, 2H), 6.76±6.90
(m, 3H, Ar), 6.36 (t, 1H, NH), 6.15 (bs, 1H, NH), 5.61 (s,
1H, OH), 4.40 (m, 3H, NHCH2Ar and CH2OCO), 4.00
(dd of AB, 1H, CH2OCO), 3.86 (s, 3H, OCH3), 3.85 (m,
1H, CHCH2NHC=S), 3.31 (m, 1H, CHCH2NHC=S),
2.67 (ddd of AB, 2H, CH2Ph), 2.46 (m, 1H, CH); IR
(neat): 3373, 1711, 1266, 1124; MS m/e 499 (M+). Anal.
calcd for C26H27ClN2O4S: C, 62.58; H, 5.45; N, 5.61; S,
6.43. Found: C, 62.83; H, 5.47; N, 5.59; S, 6.40.
2-(3,4-dimethylbenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)
amino]carbothioyl}propyl pivalate (23). 40% yield, white
solid, mp=47 ꢀC; H NMR (CDCl3) d 6.93±7.05 (m,
1
3H), 6.77±6.9 (m, 3H, Ar), 6.22 (m, 1H, NH), 5.98 (bs,
1H, NH), 5.61 (s, 1H, OH), 4.37 (bs, 2H, NHCH2Ar),
4.17 (ddd of AB, 1H, CH2OCO), 3.87 (s, 3H, OCH3),
3.7±3.85 (m, 2H, CH2OCO and CHCH2NHC=S), 3.27
(m, 1H, CHCH2NHC=S), 2.60 (m, 2H, CH2Ph), 2.2±
2.32 (m, 7H, 2ÂCH3 and CH), 1.22 (s, 9H, C(CH3)3);
IR (neat): 3360, 1714, 1279, 1159; MS m/e 474
(M++2). Anal. calcd for C26H36N2O4S: C, 66.07; H,
7.68; N, 5.93; S, 6.78. Found: C, 66.30; H, 7.71; N, 5.91;
S, 6.76.
2-(4-t-Butylbenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)ami-
no]carbothioyl}propyl pivalate (28). 40% yield, yellow
solid, mp=52 ꢀC; 1H NMR (CDCl3) d 7.30 (d, 2H),
7.09 (d, 2H), 6.75±6.90 (m, 3H, Ar), 6.27 (t, 1H, NH),
6.10 (bs, 1H, NH), 5.66 (s, 1H, OH), 4.40 (bs, 2H,
NHCH2Ar), 4.15 (dd of AB, 1H, CH2OCO), 3.86 (s,
3H, OCH3), 3.7±3.85 (m, 2H, CH2OCO and CHCH2
NHC=S), 3.27 (m, 1H, CHCH2NHC=S), 2.58 (ddd of
AB, 2H, CH2Ph), 2.30 (m, 1H, CH), 1.29 (s, 9H,

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
29
C(CH3)3), 1.22 (s, 9H, C(CH3)3); IR (neat): 3360, 1714,
1277, 1158; MS m/e 500 (M+). Anal. calcd for C28H40
N2O4S: C, 67.17; H, 8.05; N, 5.59; S, 6.40. Found: C,
67.42; H, 8.08; N, 5.57; S, 6.38.
5.68 (s, 1H, OH), 4.32 (m, 4H, NHCH2Ar and
PhCH2O), 3.82 (s, 3H, OCH3), 3.48 (m, 2H, BnOCH2),
3.34 (m, 2H, CHCH2NHC=S), 2.58 (m, 2H, CH2Ph),
2.16 (m, 1H, CH); IR (neat): 3340, 1273, 1153; MS m/e
484 (M+). Anal. calcd for C26H29ClN2O3S: C, 64.38; H,
6.03; N, 5.78; S, 6.61. Found: C, 64.62; H, 6.02; N, 5.75;
S, 6.58.
2-(4-t-Butylbenzyl)-3-{[(4-hydroxy-3-methoxybenzyl)ami-
no]carbothioyl}propyl benzoate (29). 50% yield, white
solid, mp=54 ꢀC; 1H NMR (CDCl3) d 8.02 (m, 2H), 7.60
(m, 1H), 7.46 (m, 2H), 7.32 (d, 2H), 7.14 (d, 2H), 6.75±
6.90 (m, 3H, Ar), 6.28 (t, 1H, NH), 6.06 (bs, 1H, NH),
5.61 (s, 1H, OH), 4.40 (m, 3H, NHCH2Ar and CH2
OCO), 4.08 (dd of AB, 1H, CH2OCO), 3.86 (s, 3H,
OCH3), 3.80 (m, 1H, CHCH2NHC=S), 3.40 (m, 1H,
CHCH2NHC=S), 2.68 (m, 2H, CH2Ph), 2.46 (m, 1H,
CH), 1.29 (s, 9H, C(CH3)3); IR (neat): 3360, 1714, 1272,
1124; MS m/e 520 (M+). Anal. calcd for C30H36N2O4S:
C, 69.20; H, 6.97; N, 5.38; S, 6.16. Found: C, 69.48; H,
6.99; N, 5.36; S, 6.14.
(R or S)-2-Benzyl-3-{[(4-hydroxy-3-methoxybenzyl)ami-
no]carbothioyl}propyl pivalate (35, 36). 35, (R)-enantio-
mer of 18 and 36, (S)-enantiomer of 18 were obtained
from (R)-3a,28 following the same general procedure for
18; 35: [a]D= 714 (c, 0.22, CHCl3), 36: [a]D=+695 (c,
0.08, CHCl3).
Diethyl 1,3-dihydro-2H-indene-2,2-dicarboxylate (37). This
compound was prepared from bis-1,2-chloromethyl-
benzene by following general procedures for the syn-
thesis of 1 in 58% yield as a white solid. mp=68 ꢀC; 1H
NMR (CDCl3) d 7.12±7.26 (m, 4H, phenyl), 4.20 (q,
4H), 3.56 (s, 4H), 1.26 (t, 6H).
General procedure for the synthesis of 30±31. A solution
of 5a (or 5c) (0.1 g, 0.52 mmol) in THF (10 mL) was
treated with sodium hydride (60% dispersion, 0.032 g,
0.78 mmol) followed by benzyl bromide (0.92 mL, 0.78
mmol) and tetrabutylammonium iodide (0.096, 0.26
mmol), and stirred for 3 h at room temperature. The
mixture was diluted with ether, ®ltered through a short
pad of silica gel and the ®ltrate was concentrated in
vacuo. The residue was puri®ed by ¯ash column chro-
matography on silica gel with EtOAc:hexane (1:20) as
eluant to give 30 (or 31).
{2-[({[(4-Hydroxy-3-methoxybenzyl)amino]carbothioyl}
amino)methyl]-2,3-dihydro-1H-inden-2-yl}methyl pivalate
(38). This compound was prepared from 37 by follow-
ing general procedures of 18 in 28% yield as a white
solid. mp=60.5 ꢀC; H NMR (CDCl3) d 7.14 (s, 4H,
1
aromatic), 6.79±6.89 (m, 3H, Ar), 6.45 (bt, 1H, NH),
6.12 (bs, 1H, NH), 5.62 (s, 1H, OH), 4.38 (bs, 2H,
NHCH2Ar), 3.97 (s, 2H, O=COCH2), 3.88 (s, 3H,
OCH3), 3.69 (bs, 2H, CCH2NHC=S), 2.92 (d, 2H,
J=12 Hz, CH2C), 2.74 (d, 2H, J=12 Hz, CH2C), 1.21
(s, 9H, C(CH3)3); IR (neat): 3356, 1715, 1553, 1514,
1276, 1155; MS m/e 456 (M+). Anal. calcd for C25H32
N2O4S: C, 65.76 ; H, 7.06; N, 6.14; S, 7.02. Found: C,
66.00; H, 7.09; N, 6.11; S, 7.00.
3-Azido-2-benzylpropyl benzyl ether (30). 82% yield, col-
1
orless oil; H NMR (CDCl3) d 7.1±7.4 (m, 10H, phenyl),
4.49 (dd of AB, 2H, PhCH2O), 3.33±3.50 (m, 4H, BnO
CH2 and CH2N3), 2.68 (m, 2H, CH2Ph), 2.14 (m, 1H,
CH).
3-Azido-2-(4-chlorobenzyl)propyl benzyl ether (31). 84%
1
{2-[({[(4-Hydroxy-3-methoxybenzyl)amino]carbothioyl}
amino)methyl]-2,3-dihydro-1H-inden-2-yl}methyl benzene-
carboxylate (39). This compound was prepared from 37
by following general procedures of 22 in 30% yield as a
yield, colorless oil; H NMR (CDCl3) d 7.23±7.4 (m,
5H, phenyl), 7.21 (d, 2H), 7.05 (d, 2H), 4.47 (dd of AB,
2H, PhCH2O), 3.30±3.45 (m, 4H, BnOCH2 and CH2
N3), 2.64 (m, 2H, CH2Ph), 2.08 (dd, 1H, CH).
ꢀ
1
white solid. mp=62.6 C; H NMR (CDCl3) d 8.01 (d,
2H, J=5.5 Hz, Bz), 7.60 (t, 1H, J=5.6Hz, Bz), 7.46 (t,
2H, J=5.8 Hz, Bz), 7.16 (s, 4H, aromatic), 6.78±6.87 (m,
3H, Ar), 6.52 (bt, 1H, NH), 6.15 (bs, 1H, NH), 5.61 (s,
1H, OH), 4.40 (bs, 2H, NHCH2Ar), 4.22 (s, 2H, O=
COCH2), 3.86 (s, 3H, OCH3), 3.79 (bs, 2H, CCH2
NHC=S), 3.00 (d, 2H, J=12 Hz, CH2C), 2.85 (d, 2H,
J=12 Hz, CH2C); IR (neat): 3354, 1715, 1553, 1514,
1273, 1113; MS m/e 476 (M+). Anal. calcd for C27H28
N2O4S: C, 68.04; H, 5.92; N, 5.88; S, 6.73. Found: C,
68.30; H, 5.94; N, 5.86; S, 6.71.
N-[2-Benzyl-3-(benzyloxy)propyl]-N0-(4-hydroxy-3-meth-
oxybenzyl)thiourea (32). This compound was prepared
from 30 by following the general procedure for the
1
synthesis of 18±29 in 42% yield as a colorless oil. H
NMR (CDCl3) d 7.10±7.30 (m, 10H, 2ÂPh), 6.81 (d,
1H, Ar), 6.60 (bs, 1H, Ar), 6.56 (d, 1H, Ar), 6.54 (bs,
1H, NH), 5.64 (s, 1H, OH), 4.35 (m, 4H, NHCH2Ar
and PhCH2O), 3.82 (s, 3H, OCH3), 3.50 (m, 2H,
BnOCH2), 3.37 (bt, 2H, CHCH2NHC=S), 2.64 (m, 2H,
CH2Ph), 2.20 (m, 1H, CH); IR (neat): 3288, 1274, 1123;
MS m/e 450 (M+). Anal. calcd for C26H30N2O3S: C,
69.30; H, 6.71; N, 6.22; S, 7.12. Found: C, 69.59; H,
6.74; N, 6.20; S, 7.10.
Molecular modelling
Modelling studies on RTX and 35 were performed on a
Silicon Graphics R4600 INDY workstation. The mole-
cules were constructed using SYBYL 6.4, TRIPOS. The
conformational space available was explored using
molecular dynamic (MD) simulations. The MD simula-
tions were accomplished using the SYBYL Dynamic
program. Thus, each compound was submitted to the
following protocol: A starting structure was energy-
N-[3-Benzyloxy-2-(4-chlorobenzyl)propyl]-N0-(4-hydroxy-
3-methoxybenzyl)thiourea (33). This compound was
prepared from 30 by following the general procedure for
the synthesis of 18±29 in 46% yield as a white solid.
mp=37.5 ꢀC; 1H NMR (CDCl3) d 7.0±7.3 (m, 9H), 6.5±
6.84 (m, 3H, Ar), 6.54 (bs, 1H, NH), 6.30 (bs, 1H, NH),

30
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
minimized using the Tripos force ®eld and the conjugate
gradient method until the rms derivative of the energy was
below 0.005 kcal/mol/A. The system was then brought to
equilibrium, over 1 ps at 300 K, before a molecular simu-
lation study spanning a further 500 ps (at 300 K) was
undertaken. Snap shots taken at 1 ps intervals were
minimized using the optimization criteria outlined
above. For each compound, the 501 resulting minimized
structures were analyzed by superposition of the four
pharmacophore points (RTX: centre of the vanilloid
ring, centre of the orthoester phenyl, carbonyl oxygen of
the C20-ester, and carbonyl oxygen of the C3-carbonyl;
35: centre of the vanilloid ring, centre of the phenyl ring,
sulfur of the thiourea, and carbonyl oxygen of ester).
Binding was expressed as fmol/mg protein. Protein
concentration was measured using the Bio-Rad protein
assay according to the manufacturer's protocol (Bio-
Rad Laboratories, CA). Samples were equilibrated in
scintillation ¯uid for 10 h before scintillation counting
began and each sample was counted for 5 min. Binding
data were analyzed by ®tting to the following equation:
ꢀn 1†
B ˆ ꢀꢀLH ‡ LC à KdKI†
ꢀ1†
n
ꢀKnd ‡ ꢀLH ‡ LC à KdKI† ††ꢀLnH ꢀKnd ‡ LHn ††
1
where LC is the concentration of the non-radioactive
ligand and KI is the concentration of the free non-
radioactive ligand at which it occupies half of the bind-
ing sites. B represents speci®cally bound [3H]RTX, Bmax
is the receptor density, LH is the concentration of free
[3H]RTX, Kd is the concentration of [3H]RTX at which
half of the receptors are occupied and n is the coopera-
tivity index referred to as the Hill coecient.
[3H]Resiniferatoxin binding assay
[3H]RTX (37 Ci/mmol) was synthesized by the Chemical
Synthesis and Analysis Laboratory, NCI-FCRDC.
Nonradioactive RTX and capsazepine were purchased
from LC Laboratories (Woburn, MA).
Inhibition of [3H]RTX binding in the presence of com-
peting ligands was determined on membrane prepara-
tions from rat spinal cord. Membrane preparations were
prepared as described.34 Brie¯y, animals were euthani-
zed under general anesthesia and the entire spinal cord
was removed aseptically. Samples were disrupted with
the aid of an Omni 2000 tissue homogenizer in ice-cold
10 mM HEPES, pH 7.4, containing 5 mM KCl, 5.8 mM
NaCl, 2 mM MgCl2, 0.75 mM CaCl2, 12 mM D-glucose,
and 137 mM sucrose (bu€er A). Homogenates were
centrifuged at 1000Âg for 10 min at 4 ꢀC; pellets were
resuspended in Bu€er A and recentrifuged at 35,000Âg
for 40 min at 4 ꢀC. The pellets from the second cen-
trifugation were resuspended in the same bu€er at an
approximate protein concentration of 2 mg/mL, quick
frozen on dry ice as small aliquots, and stored at 70 ꢀC
until assayed.
CAP-activated single channel assay
Cell preparation. Cultured DRG neurons were prepared
as described previously.29 Brie¯y, DRGs were dissected
from all levels of lower cervical, thoracic and lumbar spinal
cord of 1- or 2-day-old neonatal rats. DRGs were collected
in cold culture medium (4 ꢀC) containing DMEM/F-12
mixture (Gibco, Grand Island, NY), fetal bovine serum
(10%, Gibco), 1 mM sodium pyruvate, 25 ng/mL nerve
growth factor (Sigma, St. Louis, MO), and 100 units/mL
of penicillin/streptomycin (Sigma). Ganglia were washed
three times with DMEM/F-12 medium and incubated
for 30 min in the DMEM/F-12 medium containing
1 mg/mL collagenase (Type II, Worthington Biomedi-
cal, Freehold, NJ). The ganglia were then washed three
times with Mg2+- and Ca2+-free Hank's solution and
incubated with gentle shaking in the warm (37 ꢀC)
Hank's solution containing 2.5 mg/mL trypsin (Gibco).
The solution was centrifuged at 1000 rpm for 10 min,
and the pellet was washed two or three times with the
culture medium to inhibit the enzyme. The pellet was
suspended in the culture medium and gently triturated
with a Pasteur pipette. The suspension was plated on
round glass coverslips (Fisher, Pittsburgh, PA) placed in
small Petri dishes. The glass coverslips were treated
overnight with poly-L-lysine (Sigma) and dried before
use. Cells were incubated at 37 ꢀC in 95% air/5% CO2
gas mixture. Cells were used 2±4 days after plating.
Experiments were designed to assess inhibition of spe-
ci®c [3H]RTX binding to membranes by non-radio-
active compounds. [3H]RTX (100 pM) was incubated in
the presence of competing ligand in a total volume of
300 mL with 100 mg membrane protein for 60 min at 37 ꢀC
in Bu€er A supplemented with 0.25 mg/mL bovine serum
albumin (type V, Sigma) . The bovine serum albumin
was included to reduce nonspeci®c adsorption of RTX
to surfaces. At the end of the incubation, tubes were
chilled on ice and 100 mg of `a1-acid glycoprotein'
(AGP, Sigma) in a 50 mL volume was added to each
tube to reduced nonspeci®c binding. Bound and free
[3H]RTX were then separated by pelleting the mem-
branes by centrifugation at 10,000Âg for 15 min at 4 ꢀC.
The tips of the tubes containing the pellets were cut o€,
and the bound radioactivity was determined by scintil-
lation counting. Nonspeci®c binding was determined in
the presence of 1 mM non-radioactive RTX. Measure-
ments of binding were determined in triplicate in each
experiment, and each experiment was performed at least
three times. In each experiment, competition curves
were determined typically using 5±6 concentrations of
competing ligand.
Current recording. Borosilicate glass pipettes (Narishige
Scienti®c Instrument Lab., Tokyo) were pulled and
coated with Sylgard (Dow Corning Co., Midland, MI).
Tip resistances were about 2 and 5 Mohms for whole-
cell and single-channel current recordings, respectively.
After gigaseals were formed with the glass pipettes, cell-
attached and inside-out patch con®gurations were used
to study single-channel currents as described by Hamill et
al.30 A salt bridge (1% agar in 300 mM KCl) immersed
in the bath and an Ag/AgCl reference electrode in the

J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
31
pipette solution was used to minimize changes in junc-
tional potentials. Junctional potentials were cancelled
before gigaseals were formed. For whole-cell recording,
the cell membrane under a glass pipette was ruptured by
a gentle suction. After forming a whole-cell patch, the
capacitative transient was cancelled. Single-channel cur-
rents were recorded using a patch-clamp ampli®er (Axo-
patch 200A, Axon Instruments, Foster City, CA) and
®ltered at 5 KHz with an 8-pole, low-pass Bessel ®lter.
Data were digitized at 37KHz with a digital data recorder
(VR-10B, Instrutech, Great Neck, NY) and stored on
videotapes for later analysis. For chart recording, output
of the ampli®er was ®ltered at 500 Hz (Frequency Device,
Havenhill, MA) and fed into a thermal array chart
recorder (TA-240, Gould Instrument System, Valley
View, OH). The digitized data stored on videotapes were
imported to a personal computer (IBM Pentium-com-
patible) for computer analysis of single-channel currents.
are expressed as an ED50 value representing a 50%
reduction in the number of writhes. ED50 values were
determined by linear regression analysis with at least
three data points in the log dose±response curve.
TPA-induced mouse ear edema assay.31 Male ICR mice
(25±30g) were treated topically on the right ear with 25mL
acetone or synthesized compound in acetone. Groups of
10 animals were used for each dose and for each treat-
ment. Initial treatment of vanilloid agonist caused ery-
thema in the treated mouse ear possibly through release
of neuropeptides. Approximately 17 h later, an identical
treatment was applied to release residual neuropeptides
and desensitize nociceptive sensory neurons which are
known to play an important role in neurogenic in¯am-
mation. One hour later, 25mL of 0.5 nmol TPA in acetone
was applied to the same ear. Five hours following the
application of TPA, the animals were sacri®ced and ear
punches (6 mm diameter) were weighed to the nearest
0.1 mg on a electrobalance. The increased weight of the ear
punch is a measure of in¯ammation (ear edema). Anti-
in¯ammatory e€ects were calculated as percent inhibition
of swelling in the compound-treated versus the control
group. The percent inhibition is de®ned by the following
equation.% inhibition=(C T)/CÂ100, where C and T
refer to increases in ear weight in TPA-treated and TPA+
drug-treated groups, respectively. From this data the IC50
value was determined as the dose required to produce a
50% reduction in the TPA-induced in¯ammation.
Channel open probability (Po), amplitude and mean
open time of single-channel currents were obtained
using the pCLAMP software (Version 6.02, Axon
Instruments). Po of single channels was obtained from
the ratio of the areas under the curves representing open
events divided by the sum of the areas under the curves
representing open and closed events. The half-amplitude
algorithm in FETCHAN (Axon Instruments) was used
for the threshold amplitude for detecting open events.
Channel activity (NPo) was calculated as a product of
the number of channel (N) in the patch and Po. NPo or
Po was collected only from patches that contained
power than ®ve functional CAP-activated channels.
Pungency and tachyphylaxis in the rat eye-wiping test
The pain-inducing potency of the compounds was deter-
mined in the eye-wiping assay as previously described,32
and expressed quantitatively as follows. Solutions (20 mL/
eye) in 10-fold increasing concentrations in physiological
saline, containing at most 5% ethanol, were dropped
into the eye of rats weighing 150±180 g, and the number
of protective movements (eye-wiping with the foreleg)
was counted in the period of 5 min. Each concentration
was applied to six rats and the dose±response curves
were obtained from the mean values. From the dose±
response curves the concentrations having a moderate
pain-producing potency (MPP), i.e., inducing an equal
response of 10 wipings, were calculated for each com-
pound. On the basis of these concentrations the relative
pain-producing potency (RPP) was determined as com-
pared to that of capsaicin, which was taken as 100.
Solutions. Solutions in bath and pipette for single-channel
recordings contained (in mM) 140 Na+, 2 Mg2+, 144 Cl ,
5 EGTA, and 10HEPES at pH 7.2. For whole cell
recording, pipette solution contained (in mM) 140 K+,
2 Mg2+, 144 Cl , 5 EGTA, 10HEPES, and 4 ATP at pH
7.2. The control perfusion solution for whole-cell record-
ing contained (in mM) 140 Na+, 5 K+, 2 Mg2+, 1 Ca2+
,
151 Cl , and 10 HEPES. The synthesized compounds
were dissolved and stored in 100% ethanol to make
10 mM stock solutions. All other reagents used in cell-
culture or in electrophysiological experiments were pur-
chased from Sigma.
Mouse writhing antinociceptive assay
Male ICR mice (weight 25 g) were maintained in a con-
trolled lighting environment (12 h on/12 h o€). Groups
of 10 animals were used for each dose and for each
treatment. Animals received an intraperitoneal injection
of 0.3 mL of the chemical irritant phenyl-p-quinone
(4.5 mg/kg dissolved in saline containing 5% ethanol),
and 6 min later the number of abdominal constrictions
was counted in the subsequent period of 6 min. Animals
received the synthesized compounds in 0.2 mL vehicle of
ethanol:Tween-80:saline (10:10:80) intraperitoneally
30 min before the injection of phenyl-p-quinone. A
reduction in the number of writhes responding in the
phenyl-p-quinone treated group relative to the number
responding in the saline control group was considered
to be indicative of an antinociceptive e€ect. The data
To check for the development of tachyphylaxis or cross-
tachyphylaxis, 6 h following the application of the test
compound (20 mL/eye), test challenge of 10 mg/mL
(20 mL/eye) capsaicin was applied to the same eye. The
percent reduction of response to 10 mg/mL CAP in rats
treated with the test compounds, compared to the vehi-
cle-treated rats, was estimated to be the index of desen-
sitization.
Acknowledgements
This research was supported in part by a Grant No.
971-0711-095-2 awarded by the Korea Science and

32
J. Lee et al. / Bioorg. Med. Chem. 9 (2001) 19±32
Engineering Foundation (KOSEF), by a KOSEF Grant
through the Research Centre for New Drug Develop-
ment at Seoul National University, and by the 1999
BK21 project for Medicine, Dentistry and Pharmacy.
16. Walpole, C. S. J.; Bevan, S.; Bloom®eld, G.; Breckenridge,
R.; James, I. F.; Ritchie, T.; Szallasi, A.; Winter, J.; Wriggles-
worth, R. J. Med. Chem. 1996, 39, 2939.
17. Appendino, G.; Cravotto, G.; Palmisano, G.; Annunziata,
R.; Szallasi, A. J. Med. Chem. 1996, 39, 3123.
18. Acs, G.; Lee, J.; Marquez, V. E.; Blumberg, P. M. Mol.
Brain Res. 1996, 35, 173.
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