Structure-activity relationship of benzo[b]thiophene-2-sulfonamide derivatives as novel human chymase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.08.040

We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously discovered by in silico screening. TY-51076 (7) showed high potency (IC₅₀=56 nM) and excellent selectivity for chymase compared to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure-activity relationship of this class are described.

Full text of DOI:10.1016/j.bmcl.2003.08.040

Bioorganic & MedicinalChemistry Letters 13 (2003) 4085–4088
Structure–Activity Relationship of Benzo[b]thiophene-2-
sulfonamide Derivatives as Novel Human Chymase Inhibitors
Hidekazu Masaki,a,* Yusuke Mizuno,a Akira Tatui,b Akira Murakami,b
Yuuki Koide,a Shoji Satoha and Atsuo Takahashia
aDrug Research Department, Tokyo Research Laboratories, Toa Eiyo Ltd., 2-293-3 Amanuma-cho, Omiya-ku,
Saitama-shi, Saitama 330-0834, Japan
bDrug Research Department, Fukushima Research Laboratories, Toa Eiyo Ltd., 1 Tanaka, Yuno, Iizaka-cho,
Fukushima-shi, Fukushima 960-0280, Japan
Received 23 July 2003; accepted 15 August 2003
Abstract—We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously
discovered by in silico screening. TY-51076 (7) showed high potency (IC50=56 nM) and excellent selectivity for chymase compared
to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure–activity relationship of this class are described.
# 2003 Elsevier Ltd. All rights reserved.
Chemistry
Introduction
Chymase (EC 3.4.21.39) is a chymotrypsin-like serine
protease, and is a major non-ACE, angiotensin (Ang)
II-generating enzyme in human tissue.1,2 Chymase also
produces transforming growth factor-beta 1 (TGF-beta
1), collagen, endothelin-1 (1–31) and inflammatory
cytokines through the processing of precursors.3ꢀ6
These chymase-induced growth factors and cytokines
have been shown to play an important role in tissue
remodeling and inflammation. Thus, chymase inhibitors
are expected to be usefultherapeutic agents in disorders
such as congestive heart failure, allergy, vascular pro-
liferation and chronic inflammation following fibrosis.7,8
Compounds 7–19 were generally accessed by reacting
the corresponding benzo[b]thiophene-2-sulfonyl chlor-
ides with aniline intermediates. Benzo[b]thiophene-2-
sulfonyl chlorides were obtained commercially or
prepared from the corresponding benzothiolvia
alkylation,
cyclization
and
sulfonylchlorination
Our research group recently used a pharmacophore
hypothesis modelof chymase inhibitor to discover a ben-
zo[b]thiophene-2-sulfonamide compound, MWP00965.9
Although MWP00965 shows poor chymase inhibitory
activity, it is stable in human plasma. Interestingly,
MWP00965 also had a unique manner of binding that is
different from previously reported chymase inhibitors in
a docking study (Fig. 1).10ꢀ15 In this study, we examined
the structure–activity relationships (SARs) of a class of
benzo[b]thiophene-2-sulfonamide derivatives to identify
more potent and specific chymase inhibitors.
*Corresponding author. Tel.: +81-48-647-7971; fax: +81-48-648-
0078; e-mail: hidekazu.masaki@toaeiyo.co.jp
Figure 1. Binding mode between MWP00965 and chymase at the
active site.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.040

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H. Masaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4085–4088
ular, the introduction of a methanesulfonyl group (15)
exhibited in 8-fold higher potency than 11. On the other
hand, the introduction of substituents at the meta-posi-
tion only reduced inhibitory activity (16 and 17). Next,
we examined the effect of substituents at the 5-position
of the benzo[b]thiophene ring. Replacement of the
chlorine atom with a more electronegative fluorine atom
(7) resulted in a 4-fold greater inhibitory activity than
15, while replacement of the chlorine atom with methyl
group or hydrogen atom slightly reduced the activity
(18 and 19).
Scheme 1. Synthesis of 5-fluorobenzo[b]thiophene-2-sulfonylchloride
3. Reagents and conditions: (a) chloroacetone, K2CO3, acetone, 94%;
(b) P2O5, H3PO4, 69%; (c) ClSO3H, CHCl3, 74%.
To elucidate the effect of a methoxycarbonyl group at
the para-position of the N-phenylring as an H-bond
acceptor subunit, severalcarbonylcompounds,
hydroxymethyland methoxymethylgroups were exam-
ined. As a result, replacement of a methoxycarbonyl
group with a acetylgroup did not atler the potency ( 20).
Replacement of a methoxycarbonyl group with a
hydroxymethylgroup ( 21), methoxymethylgroup ( 22),
or carboxamide as amino acid derivatives (23, 24)
Scheme 2. Synthesis of benzo[b]thiophene-2-sulfonamide derivative 7.
Reagents and conditions: (a) ClSO3H, 130 ꢁC; (b) 40% NaOH, 4 M
Na2SO3; (c) MeI, 40% NaOH/MeOH/H2O, three steps 42%; (d)
BnNH2, 140 ꢁC, 55%; (e) H2, 5% Pd/C, dioxane/MeOH, quant; (f)
cH2SO4, MeOH, reflux, 90%; (g) 3, 60% NaH, THF–DMF, 68%.
Table 1. Effect of substitution on the benzo[b]thiophene ring and
N-phenylring
Compd
X
R1
R2
R3
IC50 (nM)a
Human chymase
(Scheme 1). Compound 7 was synthesized by coupling
of 5-fluorobenzo[b]thiophenesulfonylchloride 3 with 4-
aminobenzoic acid 6. 4-Aminobenzoic acid 6 was prepared
in 6 steps as described in the literature (Scheme 2).16 Other
aniline compounds were obtained commercially or pre-
pared by standard methods. Compounds 20–27 were syn-
thesized by starting from compound 7 in a conventional
sequence.
1
8
9
ClH
ClH
ClH
ClH
ClH
ClNO
ClOMe
ClCO 2Me
ClSO 2Me
ClH
SO
S(CH
CN
NO
CO
2(CH2)2CH3
H
H
10,600
NIb
NI
2)2CH3
H
10
11
12
13
14
15
16
17
18
19
7
H
H
H
H
H
NI
2
2Me
CO2Me
2Me
CO2Me
CO2Me
2Me
CO2Me
CO2Me
CO2Me
CO2Me
1647
880
663
NI
203
4208
594
325
305
56
2
CO
H
CO
CO2Me
Me
H
ClSO 2Me
Me SO2Me
H
F
Results and Discussion
SO2Me
SO2Me
H
H
The compounds were tested for their in vitro inhibitory
activity using purified recombinant human chymase.17
The results are given as IC50 values, as shown in Tables
1 and 2. Based on our reported Catalyst/HypoGen
model and molecular docking model,9 we focused on
investigating the effect of substituents on the N-phenyl
ring and the benzo[b]thiophene ring in MWP00965. In
particular, we initially changed the propylsulfonyl
group at the para-position of the N-phenylring to
identify an unfavorable substituent in the S10 region.
Changing the propylsulfonyl group to a propylthio-,
cyano-, or nitro-group resulted in loss of activity (8–10),
whereas changing it to a simple methoxycarbonyl group
(11) dramatically enhanced the inhibitory activity to
about 6-fold greater than that of MWP00965 (1). This
improvement in the potency of a methoxycarbonyl
group may be the result of increased hydrogen-bond
(H-bond) accepting ability. We then examined the
ortho- and meta-positions of the N-phenylring. Intro-
duction of an electron-donating group or electron-
withdrawing group at the ortho-position resulted in
increased inhibitory activity, except for the ortho-meth-
oxycarbonyl-substituted derivatives (12–15). In partic-
aValues are means of three experiments.
bNo inhibition at 10 mM.
Table 2. Modifications at the para-position in the N-phenylring
Compd
R2
IC50 (nM)a
Human chymase
7
CO2Me
COMe
56
82
337
192
154
159
20
21
22
23
24
25
26
27
CH2OH
CH2OMe
CONHGly
CONHSer
CONH2
CO2H
>1000
>1000
>1000
CHO
aValues are means of three experiments.

H. Masaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4085–4088
Table 3. Enzyme selectivity and stability of compounds 7, 15 and 20
4087
Compd
Stabilityb (%)
IC50 (nM)a
Bovine chymotrypsin
Human chymase
Human cathepsin G
Human elastase
7
15
20
74.3
93.9
96.2
56
203
82
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
aValues are means of three experiments.
bStability was defined as the percent remaining in rat plasma after incubation for 60 min at 37 ꢁC.
crystalstructure anayl sis.
These SAR studies and the docking modeling suggest
that H-bond acceptors at the ortho- and para-positions
of the N-phenylring are necessary to increase the inhi-
bitory activity against chymase. In particular, the ability
to interact with the Lys40 region is sensitively depen-
dent on the property of the H-bond acceptor at the
para-position. Furthermore, we found that substitution
at the meta-position of the N-phenylring coudl steri-
cally hinder the S0 subsite. We also observed that the
introduction of electron-withdrawing groups at the 5-
position of the benzo[b]thiophene ring and the ortho-
position of the N-phenylring enhanced interaction with
the enzyme. This effect may be due to an increase in the
H-bond-accepting ability of the sulfonamide–NH and
sulfonyl group.
Figure 2. Proposed binding of 7 with chymase at the active site. Red,
blue, yellow and green represent oxygen, nitrogen, sulfur, and fluorine
atoms, respectively.
In conclusion, based on our hypothesis modeling, we
improved the potency of lead compound 1. Compounds
7, 15 and 20 showed increased potency and greater
selectivity than chymotrypsin and cathepsin G, and
were stable in plasma. Thus, these compounds should
enable more precise studies of the biological roles of
chymase, and may lead to new drugs against cardiovas-
cular diseases and inflammation. Additional studies are
currently underway, and further modifications and
examinations of potency in vivo will be reported in the
near future.
decreased the inhibitory activity to 3- to 7-fold less than
that of 7. In addition, replacement of a methoxy-
carbonylgroup with a carbamoy,l carboxy,l or formyl
group dramatically decreased the potency (25–27).
The enzyme selectivity and stability of selected com-
pounds in rat plasma are shown in Table 3. All of the
compounds tested (7, 15, 20) showed excellent selectivity
versus chymotrypsin, cathepsin G and elastase (>400-
fold), and were sufficiently stable in rat plasma.
Acknowledgements
To predict the binding mode of this class of inhibitor,
an originalhypothesis docking modelfor 7 with chy-
mase was constructed (Fig. 2).9 As shown in Figure 2, a
benzo[b]thiophene ring occupies the S1 specificity
pocket, and a sulfonamide moiety is located in the oxy-
anion hole and forms strong H-bond interactions with
Ser195 and Gly193 in a manner similar to MWP00965.
In addition, two H-bond interactions are observed
between basic amino acid residues and substituents of
the N-phenylring. (1) The methoxycarbonylgroup at
the 4-position was found close to the S10–S20 region and
formed a H-bond interaction with Lys40. (2) The
methanesulfonyl group at the 2-position formed an H-
bond interaction with His57. These interactions are
important for maintaining potency, respectively, and H-
bond interactions in oxy-anion hole especially would be
related to enzyme specificity. To determine the actual
shape of the complex, we will need to obtain further
experimental evidence including the result of an X-ray
We would like to thank Katakura Industries Co. for
supplying recombinant chymase.
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