N-4-Methansulfonamidobenzyl-N′-2-substituted-4-tert-butyl-benzyl thioureas as potent vanilloid receptor antagonistic ligands

Article abstract of DOI:10.1016/j.bmcl.2004.01.041

A series of N-4-methansulfonamidobenzyl-N′-2-substituted-4-tert- butylbenzyl thioureas were prepared for the study of their agonistic/ antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that

Full text of DOI:10.1016/j.bmcl.2004.01.041

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1693–1696
N-4-Methansulfonamidobenzyl-N0-2-substituted-4-tert-butyl-benzyl
thioureas as potent vanilloid receptor antagonistic ligands
Hyeung-geun Park,a,* Ji-yeon Choi,a Sea-hoon Choi,a Mi-kyung Park,a Jihye Lee,a
c
Young-Ger Suh,a Hawon Cho,a Uhtaek Oh,a Hee-Doo Kim,b Yung HyupJoo,
Sun-Young Kim,c Young-Ho Park,c Yeon Su Jeong,c Jin Kyu Choi,c Jin Kwan Kimc
and Sang-supJew *
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
a,
cAmorePacific R & D Center, Youngin-Si, Kyounggi-do 449-900, South Korea
Received 25 November 2003; accepted 20 January 2004
Abstract—A series of N-4-methansulfonamidobenzyl-N0-2-substituted-4-tert-butylbenzyl thioureas were prepared for the study of
their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure–activity relationship reveals that
there is a space for another hydrophobic binding interaction around 2-position in 4-tert-butylbenzyl region. Among the prepared
derivatives, 6n show the highest antagonistic activity against the vanilloid receptor (IC50=15 nM).
# 2004 Elsevier Ltd. All rights reserved.
Vanilloid receptor (VR1), a nonselective cation channel
placed in the plasma membrane of peripheral sensory
neurons,1,2 has been regarded as a new target for
the treatment of pain.3 The agonists initially induce the
excitation of primary sensory neuron by influx of
cations, especially Ca2+, into neuronal cell,4 but finally
desensitize the peripheral sensory neurons, which leads
analgesic effect.3 However, the initial excitatory side
effects of agonists, such as initial irritancy, hypothermia,
bronchoconstriction, and hypertension, derived from its
inherent mechanism, become obstacles to developas
systemic analgesics.5 Recently, several research groups
have focused on the investigation of antagonist to
overcome the unavoidable excitatory side effect. Several
synthetic and semi-synthetic antagonists have been
reported so far, such as capsazepine6 (IC50=0.65 mM),
N-alkyl glycine trimer7 (IC50=2 mM), pyrrolidine-
thiourea8 (IC50=3 mM), and N,N0,N00-trisubstituted-
thiourea9 (IC50=0.32 mM), 4-(2-pyridyl)piperazine-1-
carboxamides (IC50=0.03 mM),10 and 5-iodo-RTX
(IC50=4 nM),11 prepared by iodination of resinifer-
atoxin (RTX).
We recently reported a series of N-4-methylsulfonami-
dobenzyl thiourea analogues (1,12 IC50,=70 nM; 2,13,14
IC50=110 nM; 3,13,15,16 IC50=37 nM) as highly potent
VR1 antagonists with high affinity. These results
revealed that the agonism could be dramatically chan-
ged to antagonism by the replacement of 3-methoxy-4-
hydroxy groupwith 4-methansulfonamido group( Fig.
1).12ꢀ14 In addition, the further introduction of fluoro
15
groupin 3-position enhanced the antagonistic activity.
As a subsequent research, we attempted to optimize the
other part of the thioureas, N0-4-tert-butylbenzyl group,
by the combination of the structure of 1 and 2. In this
communication, we report the synthesis and functional
assay on receptor of N-4-methansulfonamidobenzyl-N0-
2-substituted-4-tert-butylbenzyl thioureas, and discuss
on the another-binding for receptor antagonism based
on their pharmacophoric analysis from structure–activ-
ity relationshipstudy.
Based on the structure of 1 and 2, the 3,4-dimethylben-
zyl groupof N0-3-acetoxy-2-benzylpropyl group of 1
could be equivalent to the N0-4-tert-butylbenzyl group
of 2 in hydrophobic binding with VR1. So we expected
that the introduction of another potential binding moi-
ety into 2, such as the pivaloyl group in the case of 1,
might increase the antagonistic potency (Fig. 1). For the
Keywords: Vanilloid receptor; Antagonist; Structure–activity relationship.
* Corresponding authors. Tel.: +82-2880-7871; fax: +82-2872-9129;
e-mail: hgpk@plaza.snu.ac.kr
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.041

1694
H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1693–1696
Figure 1.
convenient preparation of target compounds (Fig. 2)
with various functional groups at 2-position, we
employed 2 as a basic template, which has been proven
as a potent antagonist.14
dent upon the length of carbon chain. The optimal
length of alkyl carbon chain was 4 (5d, IC50=60 nM) or
5 (5e, IC50=70 nM). The shorter or the longer carbon
chain decreases the antagonistic activity. Similar ten-
dency was observed in the methoxy ether derivatives, 5j
(IC50=500 nM) and 5k (IC50=60 nM). Among the
branched alkyl groups, the neopentyloxy derivative (5h,
IC50=60 nM) showed the higher antagonistic activity
than that of isopropyloxy derivative (5g, IC50=350
nM). The benzyloxy derivative (5i, IC50=770 nM)
showed far less antagonistic activity than that of the
similar sized aliphatic ones (5d,e). O-Pivaloyl ester deri-
vative 5l (IC50=220 nM) exhibited relatively low
antagonistic activity, although the pivaloyl group is
We prepared fourteen 2-substituted derivatives (5a–l)
(Scheme 1). Compounds 5a–k were prepared in 4 steps
from 7. The introduction of cyano groupinto 7 by using
trichloroacetonitrile,15 followed by O-alkylation in basic
condition gave 9. The reduction of cyano groupof
9
with LiAlH4 and the subsequent coupling with 4-tert-
butylbenzylisothiocyanate provided 5a–k. Compound 5l
was prepared in 3 steps from 8. The reduction of 8, fol-
lowed by the coupling with 4-tert-butylbenzylisothio-
cyanate gave 12, which was treated with pivaloyl
anhydride to afford 5l. Compounds 5m and 5n were
obtained in 5 steps from 8 (Scheme 2). The O-triflate
formation of 8, followed by the insertion of CO in the
presence of methanol and the catalytic amount of
Pd(OAc)2 gave the methyl ester 14. The reduction of 14
with LiAlH4 and the followed coupling with 4-tert-
butylbenzylisothiocyanate provided 16. Finally, the
acylation with acetic anhydride and pivaloyl anhydride
afforded 5m and 5n, respectively.
Table 1. In vitro biological activity of the derivatives by 45Ca2+
influx assay in rat DRG neurons
-
No
R1
45Ca2+-influx activity (mM)a
Agonist (IC50
)
Antagonist (IC50)
NEb
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
0.11
2.40
1.10
0.25
0.06
0.07
0.15
0.35
0.06
0.77
0.50
0.06
0.22
0.58
0.07
The agonistic or antagonistic activities of the prepared
2
H
OCH3
OCH2CH3
derivatives17 on receptor were evaluated by the 45Ca2+
-
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
influx assay previously reported18 by using the neonatal
rat cultured spinal sensory neurons (Table 1). As shown
in Table 1, the antagonistic activities were quite depen-
OCH2CH2CH3
OCH2(CH2)2CH3
OCH2(CH2)3CH3
OCH2(CH2)4CH3
OCH(CH3)2
OCH2C(CH3)3
OCH2C6H5
OCH2OCH3
OCH2CH2OCH3
OC(O)C(CH3)3
CH2OC(O)CH3
CH2OC(O)C(CH3)3
a EC50 (the concentration of derivative necessary to produce 50% of
the maximal response) and IC50 values (the concentration of deriva-
tive necessary to reduce the response to 0.5 mM capsaicin by 50%)
were estimated with at least 3 replicates at each concentration. Each
compound was tested in two independent experiments. Antagonist
data were fitted with a sigmoidal function.
Figure 2.
b NE: not effective at 30 mM.

H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1693–1696
1695
Scheme 1. Reaction and conditions: (a) (i) Cl3CCN, ClCH2CH2Cl, reflux, 17 h; (ii) KOH, tert-BuOH, reflux, 20 min, 65%; (b) RX, K2CO3, DMF,
reflux, 2 h, 95–99%; (c) LiAlH4, ether, reflux, 1 h; (d) 4-MsNHPhCH2NCS or 3-F-4-MsNHPhCH2NCS, Et3N, CH2Cl2, rt, 2 h, 60–70% from 9;
(e) 4-MsNHPhCH2NCS, Et3N, CH2Cl2, rt, 2 h, 55% from 8; (f) Et3N, CH2Cl2, Piv2O, rt, 12 h, 52%.
Scheme 2. Reaction and conditions: (a) Tf2O, Et3N, CH2Cl2, 0 ꢁC, 1 h, 93%; (b) Pd(OAc)2, dppf, Et3N, MeOH, CO, DMSO, 50 ꢁC, 3 h, 83%;
(c) LiAlH4, ether, reflux, 1 h; (d) 4-MsNHPhCH2NCS or 3-F-4-MsNHPhCH2NCS, Et3N, CH2Cl2, rt, 2 h, 50%; (e) Et3N, CH2Cl2, Ac2O or Piv2O,
rt, 12 h, 50–60%.
spatially located in similar position with that of 1. The
Table 2. In vitro biological activity of the derivatives by 45Ca2+
influx assay in rat DRG neurons
-
low activity might be come from the low stability of the
phenolic ester in 5l. In case of 2-acyloxymethyl deriva-
tives (5m, IC50=580 nM; 5n, IC50=70 nM), the piva-
loyl ester derivative 5n, showed
8 times higher
antagonistic activity than that of the acetyl ester deri-
vative 5m, which is in accord with the case in alkyl
derivatives (5c,h). Especially, 5n, one carbon extended
derivative of 5l, showed 3 times higher antagonist
activity than 5l itself. These accumulated findings may
imply that there is a space for another hydrophobic
binding, but it is not bigger than the size of C4– C5 alkyl
chain. To increase the potency by the introduction of
fluoro groupat 3-position, 3-fluoro derivatives of 5d,
5h, and 5n were prepared by using 3-fluoro-4-methan-
sulfonamidobenzylisothiocynate, instead of 4-methan-
sulfonamidobenzylisothiocynate in Schemes 1 and 2. As
shown in Table 2, 6d and 6n showed 3 times higher
No
R
45Ca2+-influx activity (nM)a
Agonist (IC50
)
Antagonist (IC50)
3
H
NEb
NE
NE
NE
37
20
66
15
6d
6h
6n
OCH2(CH2)2CH3
OCH2C(CH3)3
CH2OC(O)C(CH3)3
a EC50 and IC50 values were estimated by the same method described
in Table 1.
b NE: not effective at 30 mM; Only partial activity was observed at
30 mM.

1696
H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1693–1696
antagonistic activity than that of 5d and 5n, respec-
tively, but the comparable potency was observed in case
of the neopentyloxy derivative (6h).
8. Park, H. g.; Park, M.-k.; Choi, J.-y.; Choi, S.-h.; Lee, J.;
Suh, Y.-g.; Oh, U.; Lee, J.; Kim, H.-D.; Park, Y.-H.;
Jeong, Y. S.; Choi, J. K.; Jew, S.-s. Bioorg. Med. Chem.
Lett. 2003, 13, 197.
9. Park, H. g.; Park, M.-k.; Choi, J.-y.; Choi, S.-h.; Lee, J.;
Suh, Y.-g.; Cho, H.; Oh, U.; Lee, J.; Kim, H.-D.; Park,
Y.-H.; Koh, H.-J.; Lim, K. M.; Moh, J.-H.; Jew, S.-s.
Bioorg. Med. Chem. Lett. 2003, 13, 601.
10. Sun, Q.; Tafesse, L.; Islam, K.; Zhou, X.; Victory, S. F.;
Zhang, C.; Hachicha, M.; Schmid, L. A.; Patel, A.;
Rotshteyn, Y.; Valenzano, K. J.; Kyle, D. J. Bioorg. Med.
Chem. Lett. 2003, 13, 3611.
In conclusion, seventeen N0-2-substituted-4-tert-butyl-
benzyl thioureas were prepared for the optimization of
N0-4-tert-butylbenzyl groupof 2. Among them, the best
antagonistic activity was observed with the N-3-fluoro-
4-methansulfonamidobenzyl-N0-2-pivaloyloxymethyl-4-
tert-butylbenzyl thiourea (6n). We believe this pharma-
cophoric information would be very useful to design
more potent antagonistic scaffolds for the development
of potential analgesics.
11. Wahl, P.; Foged, C.; Tullin, S.; Thomsen, C. Mol. Phar-
macol. 2001, 59, 9.
12. Lee, J.; Lee, J.; Kang, M.; Shin, M.-Y.; Kim, J.-M.; Kang,
S.-U.; Lim, J.-O.; Choi, H.-K.; Suh, Y.-G.; Park, H.-g.;
Oh, U.; Kim, H.-D.; Park, Y.-H.; Ha, H.-J.; Kim, Y.-H.;
Toth, A.; Wang, Y.; Tran, R.; Pearce, L. V.; Lundberg,
D. J.; Blumberg, P. M. J. Med. Chem. 2003, 46, 3116.
13. Wang, Y.; Szabo, T.; Welter, J. D.; Toth, A.; Tran, R.;
Lee, J.; Kang, S. U.; Lee, Y.-S.; Min, K. H.; Suh, Y-g.;
Park, M.-k.; Park, H.-g.; Park, Y.-H.; Kim, H.-D.; Oh,
U.; Blumberg, P. M.; Lee, J. Mol. Pharmacol. 2002, 62,
947 (Published erratum appears in Mol. Pharmacol. 2003,
63, 958).
Acknowledgements
This research was supported by a grant of the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea: 02-PJ2-PG4-PT01-0014
References and notes
14. Park, H. g.; Choi, J.-y.; Choi, S.-h.; Park, M.-k.; Lee, J.;
Suh, Y.-g.; Cho, H.; Oh, U.; Lee, J.; Kang, S.-U.; Lee, J.;
Kim, H.-D.; Park, Y.-H.; Jeong, Y. S.; Chou, J. K.; Jew,
S.-s. Bioorg. Med. Chem. Lett. 2004, 14, 787.
1. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51, 159.
2. Fitzgerald, M. Pain 1983, 15, 109.
3. (a) Virus, R. M.; Gebhart, G. F. Life Sci. 1979, 25, 1275.
(b) Wood, J. N.; Winter, J.; James, I. F.; Rang, H. P.;
Yeats, J.; Bevan, S. J. Neurosci. 1988, 8, 3208. (c) Wood,
J. N., Ed.; Capsaicin In the Study of Pain; Academic Press:
San Diego, CA, 1993.
15. Suh, Y.-G.; Lee, Y.-S.; Min, K.-H.; Park, O.-H.; Seung,
H.-S.; Kim, H.-D.; Park, H. g.; Choi, J.-y.; Lee, J.; Kang,
S.-W.; Oh, U.; Koo, J.-y.; Joo, Y.-H.; Kim, S.-Y.; Kim,
J.-K.; Park, Y.-H. Bioorg. Med. Chem. Lett. 2003, 13,
4389.
4. Appendino, G.; Szallasi, A. Life Sci. 1997, 60, 681.
5. (a) Wrigglesworth, R.; Walpole, C. S. J. Drugs of the
Future 1998, 23, 531. (b) Jancso, N.; Jancso-Gaber, A.;
Szolcsanyi, J. J. Br. Pharmacol. 1967, 31, 138. (c) Petsche,
U.; Fleischer, E.; Lembeck, F.; Handwerker, H. O. Brain
Res. 1983, 265, 233. (d) Dray, A.; Bettany, J.; Forster, P.
J. Pharmacol. 1990, 101, 727. (e) Dray, A.; Bettany, J.;
Reuff, A.; Walpole, C. S. J.; Wrigglesworth, R. Eur. J.
Pharmacol. 1990, 181, 289.
6. Walpole, C. S. J.; Bevan, S.; Bovermann, G.; Boelsterli,
J. J.; Breckenridge, R.; Davies, J. W.; Hughes, G. A.;
James, I.; Oberer, L.; Winter, J.; Wrigglesworth, R. J.
Med. Chem. 1994, 37, 1942.
16. Bigi, F.; Maggi, R.; Sartori, G.; Gasnati, G. Gazz. Chim.
Ital. 1992, 122, 283.
17. All compounds gave satisfactory spectroscopic data con-
sistent with the proposed structures. Selected spectral data
for 6n; mp 65 ꢁC. 1H NMR (CDCl3, 300 MHz), d 7.37 (m,
4H), 6.97 (t, J=7.8 Hz, 2H), 6.56 (br, 1H), 5.10 (s, 2H),
4.71 (s, 4H), 2.99 (s, 3H), 1.28 (s, 9H), 1.11 (s, 9H). 13C
NMR (CDCl3, 100 MHz), d 181.71, 178.98, 155.51,
153.07, 151.23, 137.45, 133.83, 131.71, 128.15, 125.76,
125.63, 124.08, 123.66, 123.37, 123.24, 114.78, 114.58,
63.37, 47.16, 44.98, 39.69, 38.67, 34.46, 31.13, 26.94. IR
(KBr) 3353, 2964, 1717, 1549, 1512 cmꢀ1. MS (FAB) m/e,
538 [M+H+]. Anal. calcd for C26H36FN3O4S2: C, 58.08;
H, 6.75; N, 7.31. Found: C, 57.37; H, 6.99; N, 7.02.
18. The uptake and accumulation of 45Ca2+ by the 4-sub-
stituted-benzyl-tert-benzyl thiourea derivatives was stud-
ied in neonatal rat cultured spinal sensory neurons by the
method described in detailed in the ref 3b.
7. Garcia-Martinez, C.; Humet, M.; Planells-Cases, R.;
Gomis, A.; Caprini, M.; Viana, F.; De le Pena, E.; San-
chez-Baeza, F.; Carbonell, T.; De Felipe, C.; Perez-Paya,
E.; Belmonte, C.; Messeguer, A.; Ferrer-Montiel, A. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 2374.