Novel non-vanilloid VR1 antagonist of high analgesic effects and its structural requirement for VR1 antagonistic effects

Article abstract of DOI:10.1016/j.bmcl.2003.09.024

A novel non-vanilloid VR1 antagonist consisting of a new vanilloid equivalent exhibits excellent analgesic effects as well as highly potent antagonistic activities in both capsaicin single channel and calcium uptake assays. In addition, the structural requirement for the vanilloid equivalent of the potent VR1 antagonist has also been elucidated.

Full text of DOI:10.1016/j.bmcl.2003.09.024

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4389–4393
Novel Non-vanilloid VR1 Antagonist of High Analgesic Effects
and Its Structural Requirement for VR1 Antagonistic Effects
Young-Ger Suh,a,* Yong-Sil Lee,a Kyung-Hoon Min,a Ok-Hui Park,a Ho-Sun Seung,a
Hee-Doo Kim,c Hyoung-Geun Park,a Ji-Yeon Choi,a Jeewoo Lee,a Sang-Wook Kang,a
Uh-taek Oh,b Jae-yeon Koo,b Yung-Hyup Joo,d Sun-Young Kim,d Jin Kwan Kimd
and Young-Ho Parkd
aCollege of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea
bSensory Research Center, CRI, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea
cCollege of Pharmacy, Sookmyung Women’s University, 53-12, Chungpa-Dong, Yongsan-Gu, Seoul 140-742, South Korea
dR&D Center of AmorePacific Corp., 314-1, Bora-Ri, Giheung-Eup, Yongin-Si, Gyounggi-Do 449-900, South Korea
Received 13 August 2003; revised 6 September 2003; accepted 12 September 2003
Abstract—A novel non-vanilloid VR1 antagonist consisting of a new vanilloid equivalent exhibits excellent analgesic effects as well
as highly potent antagonistic activities in both capsaicin single channel and calcium uptake assays. In addition, the structural
requirement for the vanilloid equivalent of the potent VR1 antagonist has also been elucidated.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
thermia, bronchoconstriction, increased GI mobility,
and hypertension, precluded the development of cap-
saicin as a systemic agent. Thus, recent studies on VR1
agonists or antagonists have focused on separating the
excitatory effects of capsaicin analogues from the anti-
nociceptive properties of these molecules.
Over the past few years, the amount of information
from studies on pain transmission by capsaicin has
dramatically increased, revealing novel targets for the
advent of new pain therapies. Recently, a giant step
forward came with the identification of a protein called
the vanilloid receptor 1 (VR1). VR1 can be activated
not only by vanilloid ligands including capsaicin and
endocannabinoid, but also by noxious heat (>43 ꢀC)
and protons (extracelluar pH<6). Particularly, vanil-
loids and low pH are known to reduce the temperature
threshold for VR1 activation.1À3 Our recent studies have
also revealed several 12-lipoxygenase metabolites of
arachidonic acid, including 12-(S)-HPETE, as endogen-
ous activators of the neuronal vanilloid receptor.4
In particular, the idea that VR1 functions as an inte-
grator of multiple pain-producing stimuli implies that
VR1 antagonists or channel blockers should have pro-
found antinociceptive effects,5 especially in inflamma-
tory pain models.6 Many research groups, including
ours,7 are engaged in developing potent and novel VR1
antagonists, although the therapeutically useful antago-
nists are not currently available.2,8,9
On the basis of the previous studies on vanilloid recep-
tor agonists and antagonists, as well as our recent
exciting findings,4 we have looked for the non-vanilloid
VR1 antagonists by developing the ideal vanilloid equiva-
lents, which might provide the perfect analgesic effects
without the side effects caused by vanilloid receptor
agonists. Consequently, we have developed an excel-
lent VR1 antagonist (SC0030) and revealed a part of the
mechanistic aspect of SC0030.10 We herein report the
results of our recent studies on SC0030 from the view-
Since capsaicin was found as an excellent vanilloid
receptor agonist, considerable efforts toward the devel-
opment of a novel analgesic have been continued.
However, the small therapeutic window between these
effects and the excitatory side effects, such as hyper-
*Corresponding author. Tel.: +82-2880-7875; fax: +82-2888-0649;
e-mail: ygsuh@snu.ac.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.024

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Y.-G. Suh et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4389–4393
point of the minimal structural requirement for its
potent VR1 antagonistic effects as well as its therapeutic
application.
The analogue 10 was synthesized from the commercially
available 4-fluoro-3-nitrobenzaldehyde 6. Reaction of
benzaldehyde 6 with hydroxylamine and reduction of the
resulting oxime followed by Boc-protection provided
the Boc-protected benzylamine 7. Bismesylation of 7
and Boc-deprotection followed by coupling of the ben-
zylamine salt with 4-t-butylbenzyl isothiocyanate affor-
ded the thiourea 9 in 67% yield. Finally, selective
removal of one mesyl group of 9 by NaOH treatment
afforded the desired thiourea 10 in 54% yield
(Scheme 2). The analogue 14 (MK056) was synthesized
from p-nitrobenzylamine 11 by a five steps sequence.
The sulfonamide intermediate 13 was prepared by Boc-
protection of nitrobenzylamine 11, catalytic reduction
of nitro group, followed by mesylation of the resulting
benzylamine. The sulfonamide 13 was converted to the
analogue 14 by the procedure previously described10
(Scheme 3). Analogues 17 and 18 were conveniently
prepared by coupling of the corresponding benzylamine
with 4-t-butylbenzyl isothiocyanate (Scheme 4).
Since our initial work was focused on the development
of novel vanilloid equivalents, which function as both
hydrogen bonding donors and/or acceptors, like the
vanilloid moiety of capsaicin, a large number of vanil-
loid equivalents have been designed and synthesized in
our laboratory. The representative analogues, shown in
Table 1, were selected for the comparison of their VR1
antagonistic effects because they have the characteristic
functions of the vanilloid equivalent.
Chemistry
The advanced synthesis of SC003010 is outlined in
Scheme 1. Cyanation of the methansulfonamide 2, pre-
pared by mesylation of the commercially available 2-
fluoro-4-iodoaniline, with CuCN at 130 ꢀC provided the
cyano intermediate 3 in 75% of two steps yield. Con-
version of the nitrile 3 to the benzylamine 4 was carried
out by BH3 reduction. Finally, the coupling of the
amine salt 4 with 4-t-butylbenzyl isothiocyanate affor-
ded the thiourea 5 (SC0030) in 75% of two steps yield.
Biological Assays
Dorsal root ganglion (DRG) neurons were prepared
from neonatal Sprague–Dawley rats by a modification
of the previously described method, and 45Ca2+ uptake
experiments were carried out by the reported proce-
dure.11 The IC50 is expressed as the concentration of the
tested compound required to reduce the response to 0.5
mM capsaicin by 50%.
Table 1. Ca2+ uptake inhibition of SC0030 and its representative
structural analogues in DRG neurons
In order to determine whether the tested compounds
activate or antagonize the vanilloid receptor, we recor-
ded the single-channel current of the vanilloid receptor
in inside-out membrane patches isolated from cultured
sensory neurons.4
Analogues
R1
R2
IC50 (mM)
5 (SC0030)
10
14 (MK056)
17
18
F
NHSO2CH3
NHSO2CH3
F
NHSO2CH3
0.037
24.30
0.110
The PBQ-induced writhing assay carried out with male
mice, which received an intraperitoneal injection of the
chemical irritant phenyl-p-quinone.12 A reduction in the
number of writhes responding to phenyl-p-quinone
relative to the number responding in the saline control
H
F
H
H
H
>30
Not active
0.59
Capsazepine

Y.-G. Suh et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4389–4393
4391
Scheme 1. Synthesis of analogue 5 (SC0030).
Scheme 2. Synthesis of analogue 10.
Scheme 3. Synthesis of analogue 14 (MK056).

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Y.-G. Suh et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4389–4393
Scheme 4. Synthesis of analogues 17 and 18.
group was considered to be indicative of an
antinociceptive effect.
the potency is quite low. However, the analogue 18,
which possesses neither fluoro nor methansulfonamido
substituent, completely loses the antagonistic activity.
In conclusion, the fluoro and methansulfonamido
groups for R1 and R2, respectively, are obviously
essential for the high Ca2+ uptake inhibition activity of
the vanilloid equivalent. The fluoro substituent itself
provides weak antagonistic activity. However, it is cru-
cial for the high antagonistic activity of the vanilloid
equivalents.
Results and Discussion
We initially examined the in vitro activities of more than
hundred synthetic compounds, which were designed
based on the structures of the reported natural and
unnatural agonists and antagonists. The analogue
SC0030 was finally selected as the best antagonist based
on the in vitro assays employing calcium uptake of the
vanilloid receptor. On the structural basis of the syn-
thesized ligands,13 the structural requirements of the
vanilloid equivalents for the potent VR1 antagonistic
effects were analyzed from Table 1.
Consistent with observation of calcium uptake results,
the VR1 antagonism of SC0030 was confirmed by inhi-
bition of the capsaicin-induced action on patch-clamped
rat DRG neurons. As shown in Figure 1, the applic-
ation of 1 mM of capsaicin greatly activates capsaicin
receptors in inside-out membrane patches. However,
SC0030 inhibited the channel activity (85.7Æ2.4%
reduction, n=4) evoked by capsaicin when 0.25 mM of
SC0030 was applied to the patch together with 1 mM
of capsaicin. After the SC0030 was removed, the appli-
cation of 1 mM of capsaicin again activated the channel
activity. Thus, these results suggest that 0.25 mM
SC0030 clearly antagonizes the action of capsaicin at
the capsaicin receptor in a reversible manner. Figure 2
shows the magnitude of inhibition by 0.25 mM of
SC0030 was comparable to the magnitude obtained
after the application of 10 mM of capsazepine.
Fluoride for R1, which displaces methoxy of the vanil-
loid moiety of capsaicin turned out to be the best sub-
stituent
as
a
H-bonding
acceptor
while
methansufonamide for R2, which displaces hydroxy of
the vanilloid moiety was the best substituent as a H-
bonding donor. In particular, SC0030, which possesses
both fluoro and methansulfonamido substituents was
16-fold more potent than capsazepine in Ca2+ uptake
inhibition, as a known VR1 antagonist. This novel
ligand deserves great attention as a potent VR antago-
nist with an IC50 of 0.037 mM in Ca2+ uptake inhibition
test, which makes it one of the most potent non-vanil-
loid VR antagonistic ligands described to date. The
analogues possessing other substituents for R1 and/or
R2 provided lower antagonistic effects or agonistic
effects. Switch of fluoro (R1) and methansulfonamido
(R2) groups significantly drops Ca2+ uptake inhibition
activity. Elimination of fluoro group for R1 (14)
decreases the antagonistic activity. However, the analo-
gue 14 still exhibits the higher potency than capsaze-
pine. The analogue 17, which possesses only fluoro
substituent for R1 retains antagonistic activity although
The analgesic effect of SC0030 was evaluated from the
viewpoint of the therapeutic applications and it was
confirmed by the PBQ-induced writhing antinociceptive
assay. The results presented in Figure 3 show that
Figure 2. Comparison of the antagonistic effects of SC0030 and cap-
sazepine on capsaicin receptor activity in sensory neurons. Numbers
represent concentrations in mM.
Figure 1. Reversible antagonistic effects of SC0030 on the capsaicin
receptor.

Y.-G. Suh et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4389–4393
4393
candidate for non-vanilloid analgesic development but
also as a tool to investigate the VR1-mediated pain
response.
Acknowledgements
This work was supported by grant 02-PJ2-PG4-PT01-
0014 from the Korean ministry of Health & Welfare.
References and Notes
Figure 3. Potent and dose-dependent analgesic effect of SC0030 on
PBQ-induced writhing in mice (n=10/group). Values (meanÆSEM)
represent % inhibition of writhing responses.
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