Synthesis of 2-substituted-pyrrolidinethiourea derivatives and their antagonist effect on vanilloid receptor.

Article abstract of DOI:10.1016/S0960-894X(02)00887-9

Four pyrrolidine derivatives were prepared by the formation of a 5-membered ring based on capsazepine. Among them, the two carbon extended derivatives, 4a (IC(50)=55 microM) and 4b (IC(50)=3 microM), both showed different levels of antagonist activity aga

Full text of DOI:10.1016/S0960-894X(02)00887-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 197–200
Synthesis of 2-Substituted-Pyrrolidinethiourea Derivatives and
Their Antagonist Effect on Vanilloid Receptor
Hyeung-geun Park,a,* Mi-kyung Park,a Ji-yeon Choi,a Sea-hoon Choi,a Jihye Lee,a
Young-ger Suh,a Uhtaek Oh,a Jeewoo Lee,a Hee-Doo Kim,b Young-Ho Park,c
a,
Yeon Su Jeong,c Jin Kyu Choic andSang-sup Jew *
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
Received29 August 2002; accepted15 October 2002
Abstract—Four pyrrolidine derivatives were prepared by the formation of a 5-membered ring based on capsazepine. Among them,
the two carbon extended derivatives, 4a (IC50=55 mM) and 4b (IC50=3 mM), both showeddifferent levels of antagonist activity
against the vanilloidreceptor in a 45Ca2+-influx assay.
# 2002 Elsevier Science Ltd. All rights reserved.
Capsaicin (1), the pungent component of chili pepper,
activates the vanilloidreceptor (VR1) which is a
polymodal nociceptor.1 The activation opens a novel
cation selective ion channel in the plasma membrane
of peripheral sensory neurons.2 Capsaicin induces
pain upon topical application in the early stage,
regions are virtually orthogonal, in contrast to the ago-
nist structure, in which they are approximately
coplanar.7 This conformational difference was sup-
portedby an X-ray crystallographic analysis as well
as a molecular modeling technique. Recently, tetra-
hydrobenzazepine
and
tetrahydroisoquinoline
3
which is followedby a periodof edsensitization.
thiourea derivatives have been prepared as antago-
nists by the replacement of the p-chlorophenethyl
group with 3-acetoxy-2-benzylpropyl groups.5d As
part of our program to finda new scaffoldfor a
competitive antagonist against the capsaicin receptor,
we modified capsazepine by introducing a 5-mem-
beredpyrroliidne ring ( 3, 4), which has a similar
conformation basedon molecular moedling. In this
communication, we report the synthesis of 2-sub-
stituted-pyrrolidinethiourea derivatives and their bio-
logical activities. The molecular modeling studies
proposed the pyrrolidine derivatives (3) as target
molecules with an orthogonal conformation between
the A andB regions. The energy-minimizedcon-
formation of 3a was very similar to that of capsaze-
pine, as shown in Figure 1. Both structures showed
the E,E-thiourea-p–p-stacking form (3a: 5.91 kcal/mol;
2: 4.24 kcal/mol) rather than the E,E-thiourea-p–p-
nonstacking form (3a: 8.86 kcal/mol; 2: 6.68 kcal/mol)
because of the stabilizing effect induced by the p–p
stacking interaction of the two corresponding benzene
moieties.8
Since this agent became a target for the design of a
novel class of analgesics, its structure andactivity
relationships have been studied by modifications of
capsaicin.4,5 However, the potent synthetic agonists
(SDZ-249-482,4e KR-250185a) couldnot be developedas
orally active analgesics, because of their initial irritancy.6
Since it has been impossible to remove the initial excita-
tory side effect in the agonist, a competitive antagonist
has been pursuedas a novel pharmacological agent for
analgesics, rather than an agonist.
In 1994, the first competitive antagonist, capsazepine
(2), was reportedby introducing a saturated7-mem-
7
beredrigidring system.
The rationale for the major
difference between the agonist and the antagonist is in
the structural relationship between the catechol or
vaniloidaromatic ring (A region) andthe amide bond
(B region). In the antagonist structure, the A andB
*Corresponding author. E-mail: hgpk@plaza.snu.ac.kr
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00887-9

198
H.-g. Park et al. / Bioorg. Med. Chem. Lett. 13 (2003) 197–200
Compounds 3a and 3b were preparedin 5 and6 steps,
respectively, from vanillin, as shown in Scheme 1.
Reductive amination of vanillin with 3-chloro-
propylamine, using 10% Pd/C under atmospheric H2,
followedby sequential N-butyloxycarbonyl (Boc) pro-
tection and O-methoxymethyl (MOM) protection, gave
7. The pyrrolidine ring system (8) was constructedby
intramolecular cyclization of 7 under basic conditions.9
The deprotection of the Boc and MOM groups with
trifluoroacetic acidgave 9, which was coupledwith
phenethylisothiocyanate to provide 3b. Compound 3a
also couldbe preparedby demethylation of 9, using c-
HBr andthe same coupling reaction as above.
The biological activities of 3a and 3b were evaluatedas
both agonists andantagonists in the 45Ca2+-influx assay
by using the neonantal rat culturedspinal sensory neu-
rons.10 Unexpectedly, none of derivatives showed both
agonist andantagonist activity, in spite of their similar
conformations to capsazepine basedon the molecular
modeling (Table 1). A possible explanation may come
from the unfavorable rotation of C(10)–C(2) of 3, which
may affect the binding interaction of the catechol or
Figure 1. Stereopair modeling representation of the preferred three-
dimensional conformation. (a) Energy-minimized conformation of 2;
(b) energy-minimizedconformation of 3a.
Scheme 1. Reaction andconditions: (a) 3-chloropropylamine, 10% Pd/C, H 2, MeOH, rt; Boc2O, CH2Cl2, TEA, rt, 95%, (b) MOMCl, K2CO3, DMF,
50 ꢀC, 93%, (c) n-BuLi, THF, ꢁ78–0 ꢀC, 84%, (d) TFA, CH2Cl2, rt, 95%, (e) phenethylisothiocyanate, CH2Cl2, rt, 85%, (f) c-HBr, 100ꢀC, 57%.

H.-g. Park et al. / Bioorg. Med. Chem. Lett. 13 (2003) 197–200
199
Scheme 2. Reaction andconditions: (a) 4 0-benzyoxy-30-methoxybenzyltriphenylphosphoniumbromide, THF, n-BuLi, 0 ꢀC, 85%, (b) 10% Pd/C, H2,
CH3OH, rt, 95%, (c) TFA, CH2Cl2, rt, 95%, (d) phenethylisothiocynate, CH2Cl2, rt, 82%, (e) c-HBr, 100 ꢀC, 67%.
Table 1. 45Ca2+-influx activity of the pyrrolidine derivatives
difference in potency between the vanilloid derivative (4b)
andthe catechol derivative ( 4a) is congruent with the
reportedgeneral tendency in agonists, but opposite to
11
that of capsazepine andits vanilloidderivative.
These
cumulative findings may suggest that the binding site of
the vanilloidor catechol moiety in 4 may same as that of
the general agonists, such as capsaicin.
In conclusion, four pyrrolidine derivatives12 were pre-
paredby the formation of a 5-memberedring basedon
capsazepine. Even though the energy minimizedmole-
cular conformations of 3 are very similar to that of
capsazepine, they show no biological activities. How-
ever, the two carbons extended derivatives, 4a and 4b,
both show antagonist effects at different levels. This
result suggests that the orthogonal conformation may
not so critical for the antagonist activity but the varia-
tion in the length of the ligandcouldplay more impor-
tant role for the design of new scaffold as potent
antagonist. The studies on the vanilloid type acyclic
antagonist are currently being investigated.
No.
R
n
45Ca2+ influx activity (mM)a
Agonist (EC50 Antagonist (IC50
)
)
3a
3b
4a
4b
2
H
CH3
H
CH3
—
0
0
2
2
—
>100
>100
>100
>100
>100
>100
>100
55
3
0.6
aEC50 (the concentration of derivatives necessary to produce 50% of
the maximal response) andIC values (the concentration of deriva-
50
tives necessary to reduce the response to 0.5 mM capsaicin by 50%)
were estimatedwith at least three replicates at each concentration.
Each compoundwas testedin two independent experiments. Antago-
nist data were fitted with a sigmodial function.
Acknowledgements
This research was supportedby a grant from Amore
Pacific Co. Ltd., Korea, and a grant of the Korea
Health 21 R+D Project, Ministry of Health andWel-
fare, Republic of Korea: O2-PJ2-PG4-PT01-0014.
vanilloidmoiety of 3 with the receptor. We extended
two carbons between C(10)–C(2) of 3, which could
give sufficient flexibility. The preparation of 4a and
4b is shown in Scheme 2. Wittig coupling with N-
Boc-2-formypyrrolidine 10 and3 0-methoxy-40-benzy-
loxybenzyltriphenylphosphoniumbromide, followed by
hydrogenation, gave 12. The deprotection of the Boc
group with trifluoroacetic acid, followed by coupling
with phenethylisothiocyanate, provided 4b. Demethy-
lation of 13 with c-HBr followedby treatment with
phenethylisothiocyanate gave 4a. As shown in Table
1, the 45Ca2+-influx assay revealedthat both 4a and
4b show antagonist activity andthe vanilloidderiva-
tive, 4b (IC50=3 mM), is more active than the cate-
chol derivative, 4a (IC50=55 mM). Interestingly, the
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10. The uptake andaccumulation of 45Ca2+ by the pyrroli-
dine derivatives was studied in neonatal rat cultured spinal
sensory neurons by the method described in detailed by the
reference, 3b.
11. See the reference 7 and5d N-(3,4-Dihydroxybenzyl)-N0-
[(4-chlorophenyl)ethyl]thiourea (EC50, 0.10 mM), N-(4-
Hydroxy-3-methoxybenzyl)-N0-[(4-chlorophenyl)ethyl]thiourea
(EC50, 0.06 mM); Capsazepine (IC50=0.56 mM), the vanilloid
derivative of Capsazepine (IC50=0.97 mM).
12. All compounds gave satisfactory spectroscopic data con-
sistent with the proposedstructures. Data for 3a: mp 197 ꢀC. 1H
NMR (CDCl3, 300 MHz), d 6.98 (m, 8H), 4.60 (m, 1H), 3,99 (m,
2H), 3.85 (m, 2H), 2.72 (m, 2H), 2.40 (m, 1H), 1.87 (m, 3H). IR
(KBr) 3384, 2924, 1644, 1618 cmꢁ1. MS (EI) m/e, 342 [M+].
Anal. calcdfor C 19H22N2O2S: C, 66.64; H, 6.48; N, 8.18. Found:
C, 66.59; H, 6.42; N, 8.09. 3b: mp 148 ꢀC. 1H NMR(CDCl3,
300 MHz), d 7.06 (m, 5H), 6.65 (m, 3H), 4.61 (s, 1H), 4.01 (d, 2H,
J=7.14 Hz), 3.81 (m, 2H), 3.82 (s, 3H), 2.72 (m, 2H), 2.38 (m,
1H), 1.90 (m, 3H). IR (KBr) 3390, 2938, 1602, 1516 cmꢁ1. MS
(EI) m/e, 356 [M+]. Anal. calcdfor C 20H24N2O2S: C, 67.38; H,
6.79; N, 7.86. Found: C, 66.75; H, 6.70; N, 7.65. 4a: mp 158ꢀC.
1H NMR (CDCl3, 300 MHz) d 7.12 (m, 5H), 6.56 (m, 2H), 6.39
(d, 1H, J=8.04 Hz), 3.67 (m, 3H), 3.58 (m, 1H), 3.21 (t, 1H,
J=1.58 Hz), 2.76 (m, 2H), 2.36 (t, 2H, J=7.32 Hz), 1.87 (m, 6H),
1.50 (m, 1H). IR (KBr) 3399, 2920, 1604, 1531, 1455 cmꢁ1. MS
(EI) m/e, 370 [M+]. Anal. calcdfor C 21H26N2O2S: C, 68.08; H,
7.07; N, 7.56. Found: C, 67.89; H, 7.17; N, 7.05. 4b: mp 121ꢀC.
1H NMR (CDCl3, 300 MHz) 7.18 (m, 5H), 6.75 (d , 1H,J=8.07
Hz), 6.64 (s, 1H), 6.50 (d, 1H, J=7.86 Hz), 3.97 (m, 7H), 3.52, (m,
1H), 2.81 (d, 2H, J=3.84 Hz), 2.48 (m, 2H), 1.93 (m, 6H). IR
(KBr) 3397, 2920, 1516, 1456, 1384 cmꢁ1. MS (EI) m/e, 384.
Anal. calcdfor C 22H28N2O2S: C, 68.72; H, 7.34; N, 7.29. Found:
C, 68.48; H, 7.40; N, 7.05.