N-4-Substituted-benzyl-N′-tert-butylbenzyl thioureas as vanilloid receptor ligands: Investigation on the role of methanesulfonamido group in antagonistic activity

Article abstract of DOI:10.1016/j.bmcl.2003.11.019

A series of N-4-substituted-benzyl-N′-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 not only the two oxyge

Full text of DOI:10.1016/j.bmcl.2003.11.019

Bioorganic & Medicinal Chemistry Letters 14 (2004) 787–791
N-4-Substituted-benzyl-N0-tert-butylbenzyl thioureas as vanilloid
receptor ligands: investigation on the role of methanesulfonamido
group in antagonistic activity
Hyeung-geun Park,a,* Ji-yeon Choi,a Sea-hoon Choi,a Mi-kyung Park,a Jihye Lee,a
Young-ger Suh,a Hawon Cho,a Uhtaek Oh,a Jiyoun Lee,a Sang-Uk Kang,a Jeewoo Lee,a
a,
Hee-Doo Kim,b Young-Ho Park,c 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
Received8 September 2003; accepted5 November 2003
Abstract—A series of N-4-substituted-benzyl-N0-tert-butylbenzyl thioureas were preparedfor the study of their agonistic/antag-
onistic activities to the vanilloidreceptor in rat DRG neurons. Their structure–activity relationship reveals that not only the two
oxygens and amide hydrogen of sulfonamido group, but also the optimal size of methyl in methanesulfonamido group play an
integral role for the antagonistic activity on vanilloidreceptor.
# 2003 Elsevier Ltd. All rights reserved.
Capsaicin (1), proton, andheat initiate the activation of
the vanilloidreceptor (VR1), which has been cloned
from rat dorsal root ganglia and human.1 It is a non-
selective cation channel placedin the plasma membrane
of peripheral sensory neurons.2,3 The activation of VR1
by agonists such as capsaicin andresiniferatoxin (RTX)
initially induce excitation of primary sensory neuron by
influx of cations, especially Ca2+, into neuronal cell.4
The subsequent desensitization leads the agonists to
have the effect of analgesia.5 However, the excitatory
side effects, such as initial irritancy, hypothermia,
bronchoconstriction, andhypertension, is derivedfrom
its inherent mechanism andbecome obstacles to develop
as systemic analgesics.6 A complementary approach to
overcome the unavoidable excitatory side effect by ago-
nists is the competitive antagonism of VR1. Whereas
several potent synthetic agonists were developed through
the extensive structure–activity relationship studies,7,8
only a few synthetic antagonists have been reportedso
far. Among the antagonists, while capsazepine9
(IC50=0.65 mM), N-alkyl glycine trimer10 (IC50=2 mM),
pyrrolidine-thiourea11 (IC50=3 mM), and N,N0,N00-tri-
substituted-thiourea12 (IC50=0.32 mM), showedonly
modest potency of antagonism in rat DRG, 5-iodo-
RTX,13 preparedby iodination of RTX, was reportedto
have potent antagonism with an IC50 of 3.9 nM for the
inhibition of rat VR1 so far.
As part of our program to develop novel VR antago-
nists as potent analgesics, we recently reporteda series
of N-4-(methylsulfonylamino)benzyl thiourea analo-
gues as highly potent VR1 antagonists with high affi-
nity.14,15 The previous results revealedthat the
agonistic activity of 4a was changedto antagonism by
the replacement of 3-methoxy-4-hydroxy group with 4-
methansulfonamido group (4b) (Fig. 1). In this
communication, we report the synthesis andfunctional
assay on receptor of N-4-substituted-benzyl- N0 -tert-
butylbenzyl thioureas, anddiscuss the role of sulfon-
amido group for receptor antagonism based on their
pharmacophoric analysis from systematic structure–
activity relationship study.
Basedon the previous results, we expectedthat the
agonistic/antagonistic properties of VR1 ligands could
be dramatically changed depending upon the functional
group substitutedon 4-position in vanilloidmoiety. The
importance of methansulfonyl group for antagonistic
* Corresponding authors. Fax: +82-2-872-9129; e-mail: hgpk@plaza.
snu.ac.kr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.019

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H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 787–791
activity couldbe confirmedby 4-methansulfonate deri-
vative (5b, IC50=0.30 mM in rat DRG) of SDZ249482
(5a) (Fig. 1). For the convenient preparation of target
compounds with various functional groups on 4-position
(Fig. 2), we employed4- tert-butylbenzyl group of
SDZ2494827e as a basic template, which has been
proven as a potent agonist (Norvatis’s group).
We prepared10 4-substitutederdivatives
(
6a–j)
(Scheme 1). Compounds 6a–d were easily preparedby
the coupling of 4-tert-butylbenzyl isothiocyanate with
the corresponding benzyl amines (8a–d), respectively.
The O-mesylation of 6b andthe hydrogenation of 6d
gave 6j and 6e, respectively. Compounds 6f–h were
preparedfrom 6e by the addition of methansulfonyl
Figure 1.
Figure 2.

H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 787–791
789
tively modest antagonistic activity. The result implies that
N–H of sulfonamide group, hydrogen bonding donor, is
more preferable to O of sulfonate group, hydrogen
bonding acceptor, for antagonism. However, the other
N–H types, such as amide 6g, carbamate 6h, andthiourea
6i, showedlittle activity as agonist or antagonist, sug-
gesting that the sulfonyl amide moiety is quite critical
andsensitive for its antagonistic activity.
To investigate further pharmacophoric analysis of sul-
fonamido group for antagonistic activity, we prepared
10 sulfonamido derivatives (7b–j, 7n) andthree sulfamide
derivatives (7k–m) (Scheme 2). Compound 7b–j were
preparedfrom 9 in three steps. The coupling of 9 with
various sulfonyl chlorides or sulfamoyl chlorides under
basic condition gave the corresponding sulfonamides or
sulfamides (10b–m), respectively. The deprotection of the
N-Boc group of 10b–m with trifluoroacetic acid, followed
by the coupling with 4-tert-butylbenzyl isothiocyanate
provided 7b–m. The N-methylation of 10a andthe fol-
lowing same procedure as above gave 7n.
Scheme 1. Reagents andconditions: (i) 4- t-Bu-BnNCS, CH2Cl2, rt,
98%; (ii) MsCl, pyridine, CH2Cl2, rt, 66%; (iii) Pd/C, H2, CH3OH, rt,
90%; (iv) Ac2O, Et3N, CH2Cl2, rt, 86%; (v) ClCO2Et, Et3N, CH2Cl2,
rt, 80%; (vi) DPT, CH2Cl2, rt, then c-NH4OH, 65%.
chloride, acetyl chloride, and ethyl chloroformate under
basic condition, respectively. The isothiocyanate for-
mation from 6e with dipyridylthiocarbonate (DPT),
followedby the addition of c-NH4OH provided 6i.
16
The 45Ca2+-influx activity of the preparedderivatives
was shown in Table 2. Generally, the bulkier R1 showed
the less antagonistic activity (7b–j). This finding is in
accordwith the previous results obtainedfrom a series
of 3-acyloxy-2-benzylpropyl analogues,15 andsuggests
that there is very limitedbinding space between sulfon-
amido group and VR1 receptor. Also the fluoro deriva-
tives 7f and 7g of 6f and 7b, respectively, exhibited
dramatic decrement in antagonistic activity, suggesting
that polar substituents are not favorable for antag-
onistic binding. The N-methylatedderivative of sulfon-
amido group (7n) ledto 20 times less antagonistic
activity than that of 6f, implying that the N–H, acting
as hydrogen bonding donor, may be important for the
antagonistic binding with the receptor. In a series of
sulfamides (7k–m), we expectedthat the potential
hydrogen bonding via the additional N–H in sulfamides
might enhance the antagonistic activity. However their
activities were dropped by 3–5 times lower than that of
the corresponding alkyl derivatives (6f, 7b, 7e), respec-
tively. There is another potential factor for the dramatic
variation in the antagonistic activity of 6f, 7f, and 7k,
The agonistic or antagonistic activities of the prepared
derivatives16 on receptor were evaluatedby the 45Ca2+
-
influx assay previously reported17 by using the neonatal
rat culturedspinal sensory neurons ( Table 1). As shown
in Table 1, 4-methanesulfonamido derivative, 6f
(IC50=0.11 mM), showedthe highest antagonistic
activity among the preparedderivatives, but 4-metha-
nesulfonate derivatives 6j (IC50=9.3 mM) showedrela-
Table 1. In vitro biological activity of the derivatives by 45Ca2+
influx assay in rat DRG neurons
-
No.
R
45Ca2+-influx activity (mM)a
Agonist (EC50 Antagonist (IC50
NEb
which have similar sizedsubstituents (CH , CF3, NH2,
3
)
)
respectively). The optimal pKa of the N–H in sulfon-
amido groups, depending on the electronic effect of the
substituents, might be important for the binding with
receptor. These cumulative findings might suggest that
the two oxygen (as hydrogen bonding acceptors) and
N–H (as a hydrogen bonding donor) of sufonamido
group play an integral role in the antagonistic binding
with VR1 as drawn in Figure 3 andthe spatial environ-
ment between sulfonamido group and binding site is
very limited.
1
2
Capsaicin
Capsazepine
H
0.03
NE
0.65
6a
6b
6c
6d
6e
6f
6g
6h
6i
NE
>30c
NE
NE
7.0
NE
NE
OH
CF3
NO2
NH2
12.4
>30c
NE
CH3SO2NH
CH3CONH
C2H5OCONH
NH2CSNH
CH3SO3
NE
NE
0.11
NE
NE
>30c
NE
>30c
NE
6j
9.3
In conclusion, 28 N-4-substituted-benzyl-N-tert-butyl-
benzyl thiourea derivatives were prepared for the sys-
tematic studies on the pharmacophoric information
requiredfor the antagonistic activity on VR1. Among
them, the best antagonistic activity was observedwith
the methanesulfonamide derivative (6f). Any modifi-
cation, such as N-methylation andthe replacement of
a EC50 (the concentration of derivative necessary to produce 50% of
the maximal response) andIC values (the concentration of deriva-
50
tive necessary to reduce the response to 0.5 mM capsaicin by 50%)
were estimatedwith at least three replicates at each concentration.
Each compound was tested in two independent experiments.
Antagonist data were fitted with a sigmoidal function.
b NE, not effective at 30 mM.
c Only partial activity was observedat 30 mM.

790
H. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 787–791
Scheme 2. Reagents andconditions: (i) RSO 2Cl, pyridine, CH2Cl2, rt, 70–95%; (ii) NaH, CH3I, THF, 0 ꢀC, 85%; (iii) TFA, CH2Cl2, rt; (iv) 4-t-Bu-
BnNCS, CH2Cl2, rt, 85–95% from 10.
Table 2. In vitro biological activity of the derivatives by 45Ca2+
influx assay in rat DRG neurons
-
wouldbe very useful to design more potent antagonistic
scaffolds for the development of potential analgesics.
Further investigation for new scaffolds based on these
results is now under way.
Acknowledgements
No.
R1
R2
45Ca2+-influx activity (mM)a
This research was supportedby a grant of the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea: 02-PJ2-PG4-PT01-0014.
Agonist (EC50
)
Antagonist (IC50)
1
2
Capsaicin
0.03
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NEb
0.65
0.11
1.3
4.5
4.1
Capsazepine
CH3
Et
n-Pr
n-Bu
6f
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
References and notes
i-Pr
CF3
CF3CH2
2-Thiophene
4-CH3Ph
Bn
3.8
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