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.