Synthesis of N,N′,N″-trisubstituted thiourea derivatives and their antagonist effect on the vanilloid receptor

Article abstract of DOI:10.1016/S0960-894X(02)01040-5

Twenty-seven N,N′,N″-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(4′-hydroxy-3′-methoxy-phenyl)-propyl]-1,3-diphenethyl- thiourea (8l, IC₅₀=0.32 μM), showed 2-fold higher antagonistic activity than that of capsazepine (3

Full text of DOI:10.1016/S0960-894X(02)01040-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 601–604
Synthesis of N,N0,N00-Trisubstituted Thiourea Derivatives
and Their Antagonist Effect on the Vanilloid Receptor
Hyeung-geun Park,a,* Mi-kyung Park,a Ji-yeon Choi,a Sea-hoon Choi,a Jihye Lee,a
Boon-saeng Park,a Myoung Goo Kim,a Young-ger Suh,a Hawon Cho,a Uhtaek Oh,a
Jeewoo Lee,a Hee-Doo Kim,b Young-Ho Park,c Hyun-Ju Koh,c Kyung Min Limc
Joo-Hyun Mohc and Sang-sup Jewa,*
aResearch Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University,
Seoul 151-742, South Korea
bCollege of Pharmacy, Sookmyung Women’s University, Seoul 140-742, South Korea
cAmorePacific R & D Center, Youngin-Si, Kyounggi-do 449-900, South Korea
Received 29 October 2002; accepted 2 December 2002
Abstract—Twenty-seven N,N0,N00-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(40-hydroxy-30-methoxy-
phenyl)-propyl]-1,3-diphenethyl-thiourea (8l, IC50=0.32 mM), showed 2-fold higher antagonistic activity than that of capsazepine
(3, IC50=0.65 mM) against the vanilloid receptor in a 45Ca2+-influx assay.
# 2003 Elsevier Science Ltd. All rights reserved.
Capsaicin (1), the pungent component of chili pepper,
opens a novel cation selective ion channel in the plasma
membrane of peripheral sensory neurons via binding to
the vanilloid receptor (VR1), which is a polymodal
nociceptor.1,2 Resiniferatoxin (RTX, 2), isolated from
the cactus-like succulent Euphobia resinifera, shows
thousands-fold greater agonistic activity than that of
capsaicin.3 These agonists induce pain upon topical
application in the early stage, which is followed by a
period of desensitization.4 The desensitization led to the
consideration of the agonists as potential analgesic
agents. Through extensive structure–activity relation-
ship studies,5,6 several potent synthetic agonists were
introduced, such as SDZ-249-482,5e KR-25018.6a How-
ever, their initial irritation7 makes them less appealing
as orally active analgesics. Due to the unavoidable
initial excitatory side effect of the agonists, competitive
antagonists have been pursued as novel analgesic
agents. Only a few antagonists have been reported so
far. The first competitive antagonist, capsazepine (3,
IC50=0.65 mM), was prepared by introducing a satu-
rated seven-membered rigid ring system, which main-
tains a virtually orthogonal conformation between
each N-substituent in the thiourea structure.8 5-Iodo-
RTX,9 prepared from RTX by semi-synthesis, showed
the most potent antagonistic activity (IC50, 3.9 nM),
but there is a limitation in the RTX supply from the
natural source. Tetetrahydrobenzazepine and tetra-
hydroisoquinoline thiourea derivatives have been pre-
pared as antagonists by the replacement of the p-
chlorophenethyl group with 3-acyloxy-2-benzylpropyl
groups.6d We also recently reported the five-membered
pyrrolidine derivative (4, IC50=3.0 mM) as a moderate
antagonist by the modification of capsazepine.10 As part
of our program to develop new efficient analgesic
agents, we continued the structure–activity relationship
studies on 4 by introducing further modifications. In
this communication, we report the synthesis of N,N0,N00-
trisubstituted thioureas and their potent antagonistic
effects against VR1.
Our previous studies revealed that the orthogonal con-
formation of capsazepine (3) is not as critical as the
lengths of the N(2)-substituents, which could play more
important roles in the antagonistic activity.10 Based on
these results, we designed the ring-opened thiourea 5,
with variation in the lengths of the two N(2)-sub-
stituents. Our rationale was that the increased flexibility
*Corresponding author. Fax:+82-2-872-9129; e-mail: hgpk@plaza.
snu.ac.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01040-5

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H. Park et al. / Bioorg. Med. Chem. Lett. 13 (2003) 601–604
of the ring-opened thiourea 5 might enhance the affinity
for VR1 receptor. As shown in Scheme 1, a series of
N,N0,N00-trisubstituted thiourea derivatives (8a–a0)11
were prepared from 6 via reductive amination with the
corresponding amines followed by reaction with the
appropriate isothiocynates in 75–95% yield (two
steps).12 The biological activities of the prepared com-
pounds were evaluated as both agonists and antagonists
in the 45Ca2+-influx assay, by using neonatal rat cul-
tured spinal sensory neurons (Tables 1–3).13 As shown
in Table 1, most of the ring-opened thiourea derivatives
showed comparable or higher antagonistic activities
than 4. The optimal length of the carbon chain between
N(2) and the vanilloid (40-methoxy-30-hydroxyphenyl)
ring was C3 (C1, 8a, NE at 30 mM; C2, 8b, NE at 30 mM;
C3, 8l, IC50=0.32 mM; C4, 8o, IC50=6.1 mM). Depend-
ing on the R group, quite wide variations in the activity
were observed in the series of N(2)-(40-hydroxy-30-
methoxyphenyl) propyl derivatives (8c–n). When the
bulkiness of R was increased from H (8c, NE at 30 mM)
to n-butyl (8g, IC50=10 mM), the activities changed
from low-level agonists to modest antagonists, but the
longer hydrocarbon chain (8h, IC50>30 mM) dramati-
cally decreased the antagonistic activity. The a-bran-
ched alkyl or benzyl derivatives (8i, 8j, 8n) showed
relatively higher antagonistic activities than those of the
non-branched derivatives (8e, 8g, 8k), respectively.
These findings suggest that the bulky R groups could
assume more preferable molecular conformations for
the antagonistic binding, but the size is limited for
efficient binding. Interestingly, most R groups invol-
ving a phenyl ring provided quite high activities,
implying that there might be an extra binding affi-
nity, such as p–p stacking or a hydrophobic interac-
tion, between the phenyl ring in R and the aromatic or
hydrophobic amino acid residues in the receptor (8k–n).
The best result was obtained with the phenethyl deriva-
tive, 8l (IC50=0.32 mM), which had 10-fold higher
potency as compared with the compound, 4 (IC50=3.0
mM). Table 2 clearly shows that the vanilloid group is
very important for the antagonistic activity, in accor-
dance with the previous results.10 The electronic effects
on the phenyl ring in each of the N(1) and N(2) phe-
nethyl groups against the antagonistic activities are
shown in Table 3 (8u–a0). There was no significant dif-
ference in the N(2)-phenethyl groups (8x–8a0) except
for the p-fluoro derivative (8w); however introduction
of the p-Cl (8u) and p-F (8v) groups on the phenyl ring
of the N(1)-phenethyl group reduced their antagonistic
activities.
Scheme 1. Reactions and conditions: (i) RNH2, 10% Pd/C, H2, MeOH, rt; (ii) 4-X-PhCH2CH2NCS, CH2Cl2, rt.

H. Park et al. / Bioorg. Med. Chem. Lett. 13 (2003) 601–604
603
Table 1. 45Ca2+-influx activity of the N,N0,N00-trisubstituted thiourea
derivatives
Table 3. 45Ca2+-influx activity of the N,N0,N00-trisubstituted thiourea
derivatives
No.
n
R
45Ca2+-influx activity (mM)a
Agonist (EC50
)
Antagonist (IC50)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
0
1
2
2
2
2
2
2
2
2
2
2
2
2
3
PhCH2CH2
PhCH2CH2
H
CH3
CH3CH2
CH3CH2CH2
CH3(CH2)2CH2
CH3(CH2)6CH2
(CH3)2CH
C6H11
PhCH2
PhCH2CH2
PhCH2CH2CH2
(Ph)2CH
NEb
NE
30<c
30<
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
30<
12
No.
X
Y
45Ca2+-influx activity (mM)a
Agonist (EC50 Antagonist (IC50
)
)
8l
H
F
H
H
H
F
NEb
NE
NE
NE
NE
NE
NE
NE
0.32
5.1
2.4
1.2
0.55
0.95
0.72
0.50
10
8u
8v
8w
8x
8y
8z
8a0
30<
2.5
3.0
Cl
H
H
H
H
H
Cl
0.95
0.32
3.2
0.46
6.1
CH3
OCH3
NO2
aEC50 and IC50 values were estimated by the same method described in
Table 1.
bNE, not effective at 30 mM.
PhCH2CH2
aEC50 (the concentration of test compound necessary to produce 50%
of the maximal response) and IC50 values (the concentration of test
compound necessary to reduce the response to 0.5 mM capsaicin by
50%) were estimated with at least three replicates at each concen-
tration. Each compound was tested in two independent experiments.
Antagonist data were fitted with a sigmoidal function.
activity relationship are now under way, including
replacement of the phenyl ring with other aromatic or
heterocyclic ring systems.
bNE, not effective at 30 mM.
cOnly partial activity was observed at 30 mM.
Acknowledgements
Table 2. 45Ca2+-influx activity of the N,N0,N00-trisubstituted thiourea
derivatives
This research was supported by a grant from Amore
Pacific Co. Ltd., Korea, and a grant of the Korea
Health 21 R&DProject, Ministry of Health & Welfare,
Republic of Korea: 02-PJ2-PG4-PT01-0014.
No.
R1
R2
45Ca2+-influx activity (mM)a
References and Notes
Agonist (EC50
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Antagonist (IC50)
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In conclusion, we prepared 27 N,N0,N00-trisubstituted
thiourea derivatives by the modification of 4. The
45Ca2+-influx assay provided that the 1-[3-(40-hydroxy-
30-methoxy-phenyl)-propyl]-1,3-diphenethyl-thiourea, 8l
showed the highest activity among the prepared com-
pounds, which is 2-fold higher than that of capsazepine.
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substituted thiourea would be a useful scaffold for
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11. All compounds gave satisfactory spectroscopic data con-
sistent with the proposed structures.
12. Representative procedure for the synthesis of 8l: A
methanolic mixture (3 mL) of 3-(30-methoxy-40-hydroxy-
phenyl) propionaldehyde (100 mg, 0.55 mmol), phenethyl-
amine (67 mg, 0.55 mmol), and a catalytic amount of 10%-Pd/C
was stirred under an atmosphere of H2 at room temperature (3
h). The methanol solvent was removed in vacuo. Without
further purification, the residue was adapted to the following
coupling reaction. To a methylenechloride solution (3 mL) of
the residue was added phenethylisothiocynate (98 mg, 0.60
mmol) at 0 ꢀC, and the reaction solution was stirred at room
temperature (3 h). The solvent was removed in vacuo and the
residue was purified by column chromatography (SiO2,
EtOAc/n-hexane=1:3) to afford 8l as white solid (216 mg,
87%); mp 107.7 ꢀC. H NMR (CDCl3, 300 MHz), d 7.17 (m,
1
10H), 6.79 (m, 1H), 6.55 (m, 2H), 5.50 (s, 1H), 5.05 (s, 1H),
3.81 (m, 5H), 3.68 (t, 2H, J=7.43 Hz), 3.30 (t, 2H, J=7.55
Hz), 2.80 (m, 4H), 2.43 (t, 2H, J=7.43 Hz), 1.76 (m, 2H). 13C
NMR (CDCl3, 75 MHz), d 180.82, 146.48, 143.94, 138.85,
138.54, 132.58, 128.76, 128.71, 128.69, 128.62, 126.59, 126.53,
120.66, 114.34, 110.88, 55.89, 53.20, 50.10, 46.64, 34.96, 33.73,
32.41, 28.50. IR (KBr) 3397, 2934, 1604, 1618, 1518, 1452
cmꢁ1. MS (EI) m/e, 448 [M+]. Anal. calcd for C27H32N2O2S:
C, 72.29; H, 7.19; N, 6.24. Found: C, 72.05; H, 7.32; N, 6.15.
13. The uptake and the accumulation of 45Ca2+ by the
N,N0,N00-trisubstituted thiourea derivatives were studied in
neonatal rat cultured spinal sensory neurons according to the
described method in detail in ref 4b.