Design and synthesis of novel benzoheterocyclic derivatives as human acrosin inhibitors by scaffold hopping

Article abstract of DOI:10.1016/j.ejmech.2012.11.005

Human acrosin is an attracting target for the development of novel male contraceptives. Scaffold hopping was used to optimize the isoxazolecarbaldehyde human acrosin inhibitors and extend their structure-activity relationships. Four kinds of scaffolds, namely benzimidazole, benzothiazole, 3H-indazole, and 5-phenyl-1H-pyrazole, were designed and synthesized. Most of the synthesized compounds showed potent human acrosin inhibitory activity and their binding modes were investigated by molecular docking. The scaffold of the compounds was found to be important for the inhibitory activity. Several compounds were more active than the positive control TLCK, suggesting that they can serve as good starting points for the discovery of novel male contraceptive agents.

Full text of DOI:10.1016/j.ejmech.2012.11.005

European Journal of Medicinal Chemistry 59 (2013) 176e182
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of novel benzoheterocyclic derivatives as human acrosin
inhibitors by scaffold hopping
Qianqian Chen a, Wei Tian a, Guangqian Han b, Jingjing Qi a, Canhui Zheng a, Youjun Zhou a, Lili Ding a,
,
a,
**
Juntao Zhao a, Ju Zhu a, Jiaguo Lv a , Chunquan Sheng
*
a Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
b Department of Pharmacognosy, School of Food Science, Fujian Agriculture and Forestry University, Fujian 350002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human acrosin is an attracting target for the development of novel male contraceptives. Scaffold hopping
was used to optimize the isoxazolecarbaldehyde human acrosin inhibitors and extend their structure
eactivity relationships. Four kinds of scaffolds, namely benzimidazole, benzothiazole, 3H-indazole, and
5-phenyl-1H-pyrazole, were designed and synthesized. Most of the synthesized compounds showed
potent human acrosin inhibitory activity and their binding modes were investigated by molecular
docking. The scaffold of the compounds was found to be important for the inhibitory activity. Several
compounds were more active than the positive control TLCK, suggesting that they can serve as good
starting points for the discovery of novel male contraceptive agents.
Received 28 June 2012
Received in revised form
18 October 2012
Accepted 7 November 2012
Available online 17 November 2012
Keywords:
3-Phenylisoxazole
Scaffold hopping
Synthesis
Ó 2012 Published by Elsevier Masson SAS.
Molecular docking
Human acrosin inhibitors
1. Introduction
hydrolyzes the zona pellucida, which helps sperm enter and fusion
to ovum [5]. Insufficient levels of acrosin are linked with infertility
Fertility control is an important and global issue because of
overpopulation and unintended pregnancy. The contraceptive pill
is one of the most important methods of contraception, protecting
millions of couples worldwide from unwanted pregnancy and
allowing them to take control over their reproduction. However,
the oral contraceptive pill is only available for women and most of
these pills have serious side effects, such as breakthrough
menstrual bleeding, nausea, vomiting, acne, breast tenderness,
cramping, weight gain, heavy bleeding and headaches [1e4].
Meanwhile, condoms do provide dual protection if used correctly
and consistently, but they are not women controlled. Therefore, it is
necessary to search for new contraceptive agents which are easier
to use with fewer side effects and available for both men and
women.
and inhibition of acrosin activity by protease inhibitors has been
shown to reduce the success of fertilization [6]. Therefore contra-
ception can be achieved through acrosome enzyme inhibition. In
recent years, acrosin has become an attractive target in contra-
ceptive drug discovery. Since the function of acrosin was reported,
there have been a large number of research interests focusing on
the discovery of novel acrosin inhibitors. A number of small
molecule acrosin inhibitors have been reported, such as TLCK
(C14H21ClN2O3S, CAS No. 2104-86-1), AGB (C16H16N4O3, CAS No.
79126-29-7), DV-1006 (C17H23NO4, CAS No. 34675-84-8), iso-
xazolecarbaldehydes, and guanidylbenzenesulfonamides (KF950,
C16H15N3O4, CAS No. 99450-778) (Fig. 1) [7e9]. Among them, iso-
xazolecarbaldehyde presents a promising lead structure for the
development of potent spermicidal agents with complementary
properties that add to their utility as safe, effective and prophylactic
topical contraceptives [9]. Thus, the extension of the structuree
activity relationships (SARs) of the isoxazolecarbaldehyde inhibi-
tors is of great importance.
Acrosin is a multifunctional protein located in acrosome, which
plays a critical role in the reproduction process. It not only affects
motility of sperm but also disperses acrosomal matrix and
In our previous studies, a homologous three-dimensional (3D)
model of human acrosin was built based on the crystal structures of
ram and boar acrosins [10]. The active site of human acrosin was
explored by multiple copy simultaneous search (MCSS) calculations
* Corresponding author. Tel./fax: þ86 21 81871234.
** Corresponding author. Tel./fax: þ86 21 65516994.
E-mail addresses: ljg20060508@yahoo.com.cn (J. Lv), shengcq@hotmail.com
(C. Sheng).
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.11.005

Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
177
H
COOH
H3C
N
CH3
O
O
O
O
O
S
NH
O
NH
O
CH2Cl
NH2
O
H2N
N
NH2
H
DV-1006
TLCK
AGB
O
CH3
N
O
O
O
O
O
H2N
N
CHO
S
O
NH
O
NH2
Cl
Cl
N
R
H2N
N
H
H
KF950
isoxazolecarbaldehyde
guanidylbenzenesulfonamides
Fig. 1. Chemical structures of representative acrosin inhibitors.
[11]. Guided by the results from molecular modeling, a highly
potent acrosin inhibitor C16H15N3O4 (CAS.99450-778, KF950) was
designed and synthesized, which is under pre-clinical trial [10].
Continuing our efforts in rational design of novel acrosin inhibitors
[12e14], the isoxazolecarbaldehyde acrosin inhibitor was used as
a lead for further structural optimization. Herein, a series of ben-
zoheterocyclic derivatives were designed and synthesized by
scaffold hopping (Fig. 2). In vitro assay indicated that several
compounds showed potent human acrosin inhibitory activity.
(Et3N) gave compounds 4, which were subsequently treated with
various alkanolamines in pyridine to afford the target compounds
5aei. 2-Amino-5-bromobenzothiazole 7 [16] was synthesized
according to the method of Gourley [17]. Then, it was treated with
2-chloroacetyl chloride in the presence of Et3N and THF to afford
intermediate 8. Target compounds 9aee were obtained by reacting
compound 8 with various amines in the presence of Et3N and DMF.
Claisen condensation of 1-(4-methoxyphenyl)ethanone (10) with
diethyl oxalate in the presence of Na and ethanol afforded
compound 11, which was subsequently reacted with NH2NH2 to
give phenylpyrazole 12. Then, compounds 13aed were obtained by
substitution of the acetate group of compound 12 by various
alkanolamines. 1H-indazole-3-carboxylic acid (14) was reacted
with ethanol and methylamine in the presence of EDC,HCl, oxalyl
dichloride and Et3N to give compounds 15. Free amine of
compounds 15 was converted to sodium salt, and then treated with
2, 4-dichlorobenzoyl chloride in THF to afford target compounds
17aeb.
2. Chemistry
The synthetic route of the target compounds is outlined in
Schemes 1e4. In general, the target compounds were obtained via
a two-step strategy. First, the heterocyclic scaffolds were con-
structed by various ring closure reactions. Then, various side chains
were substituted on the scaffold to afford the target compounds
[15]. Benzoimidazol-2-amine 1 reacted with dimethyl carbonate in
the presence of tetrabutylammonium bromide under reflux to
afford the carbamate intermediate 2. Chlorosulfonyl benzoimida-
zole 3 was readily prepared by the reaction of chlorosulfonic acid
and compound 2. The reaction of intermediate 3 with different
benzyl amines or aliphatic amines in the presence of triethylamine
3. Pharmacology
The in vitro acrosin inhibitory activities of the target compounds
were evaluated by a modification of the method of Kennedy et al.
[18], and the detailed protocols can be found in our previous studies
[12e14].
H
N
S
N
4. Results and discussion
R
NH
R
NH
N
4.1. Design rationale
Previous molecular modeling studies indicated that the active
site of human acrosin can be divided into three subsites (P1, P2 and
G) [10]. Docking model of the isoxazolecarbaldehyde inhibitor with
human acrosin revealed that the ligand was located in the P1
pocket, which was a hydrophobic hydrogen-bonding site for
inhibitor binding [10]. Its aldehyde oxygen atom formed a hydrogen
bond with Thr216. Based on the binding mode, a series of benzo-
heterocyclic derivatives were rationally designed. First, scaffold
hopping was used to replace the isoxazole core by benzoimidazole,
benzothiazole, pyrazole, and indazole in order to form stronger
hydrophobic and hydrogen bonding interactions with the P1
pocket. Second, the chemically unstable aldehyde group was
removed. Its hydrogen bonding interaction with Thr216 could be
compensated by the hydrogen bonds between the scaffold or side
chain and the P1 pocket. Third, various side chains were introduced
lead
O
N
CHO
R
NH
N
R
N
R
N
Fig. 2. Scaffold hopping of the isoxazolecarbaldehyde human acrosin inhibitors.

178
Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
H
N
H
N
O
S
O
H
N
a
H
N
Cl
H
N
O
b
NH2
O
N
N
O
N
O
O
1
2
3
O
S
H
N
O
H
N
O
S
H
N
O
H
N
H
N
R
c
d
H
N
H
N
O
R
(CH2)2
OH
N
O
N
5
4
Scheme 1. Reagents and conditions: (a) dimethyl carbonate, tetrabutylammonium bromide, calcium chloride, reflux, 90e100 ꢀC, 6 h, yield 72.3%; (b) chlorosulfonic acid, 0 ꢀC, ethyl
acetate, yield 90.2%; (c) tetrahydrofuran, triethylamine, amine derivatives, rt, overnight, yield 50.2e70.3%; (d) ethanolamine, pyridine, reflux, 90e100 ꢀC, 3 h, yield 70.3e85.1%.
on the benzoheterocyclic scaffold to interact with the residues in
the P2 or G pocket, which was important to improve the binding
affinity of the ligands. As a result, compounds 5aei, 9aed, 13ae
d and 17aeb were synthesized and assayed.
decreased by about 5 fold and 2 fold, respectively. As compared
with the alkyl derivatives 9aec, compound 9d, bearing a methyl
piperazinyl substitution, showed obviously decreased inhibitory
activity. Phenylpyrazole derivatives 13aed generally showed
moderate acrosin inhibitory activity (IC50 range: 1.43e8.92 mM). 3-
Hydroxypropyl group was found to be more favorable for the side
chain amine substitution than the 2-hydroxypropyl (compound
13d) or 3-hydroxyethyl (compounds 13a and 13b) group. When the
phenylpyrazole scaffold was replaced by indazole (compounds 17a
and 17b), similar inhibitory activity was observed.
4.2. Human acrosin inhibitory activities and SARs
As shown in Table 1, imidazole derivatives 5aei generally
showed moderate to good human acrosin inhibitory activities with
IC50 values ranging from 0.08 mM to 7.32 mM. Notably, most of
them were more potent than the standard reference drug TLCK
(IC50
¼
142.6 mM) and the isoxazolecarbaldehyde lead
4.3. The binding modes of the new human acrosin inhibitors
(IC50 ¼ 39 mM). It was observed that the type and position of the
substitutions played an important role for the inhibitory activities.
As compared with the unsubstituted benzyl derivative 5g
(IC50 ¼ 0.28 mM), the alkyl derivative 5i showed decreased activity
(IC50 ¼ 1.62 mM). On the other hand, the introduction of a fluorine
atom on the ortho-position of the benzyl group (compound 5a) led
to the substantial increase of the inhibitory activities. The IC50 value
of compound 5a was 0.08 mM, which was far more potent than
TLCK and the isoxazolecarbaldehyde lead. The replacement of the
fluorine atom of compound 5a by a chlorine atom (compound 5h,
In order to obtain the binding modes of the benzoheterocyclic
inhibitors, representative compounds 5a, 9c, 13a and 17a were
docked into the active site of human acrosin. As shown in Fig. 3A,
compound 5a bound with the active site of human acrosin mainly
through hydrophobic and hydrogen bonding interactions. Its ben-
zoimidazole core and C2-side chain was located in the P1 pocket.
The urea carbonyl group, amine group and terminal hydroxyl group
formed three hydrogen bonds with Gln218, Trp243 and Val245,
respectively. Moreover, the benzyl sulfonamide group of compound
5a was extended to the G pocket and formed hydrophobic inter-
actions. The substitution on the terminal phenyl could interact with
Arg199. For example, the difluoro derivative 5d showed better
activity than the dichloro derivative 5c because it formed stronger
electrostatic interaction with Arg199. Compound 13a showed
a similar binding mode (Fig. 3C) to that of compound 5a. The pyr-
azole core and amide side chain interacted with the P1 pocket. The
pyrazole nitrogen atom and terminal hydroxyl group formed
hydrogen bonding interactions with Ser221 and Thr216, respec-
tively. The methoxyl phenyl group of compound 13a mimicked the
benzyl group of compound 5a and formed similar hydrophobic
interaction with the G pocket. In contrast, two pentyl groups of
compound 9c could form hydrophobic interactions with the P2 and
IC50
¼
1.06 mM) or methoxyl group (Compound 5e,
IC50 ¼ 6.04 mM) resulted in the decreased activity. Two meta-
substituted derivatives 5b and 5f were moderately active. In
contrast, 3, 5-disubstituted derivatives 5c and 5d showed increased
inhibitory activity. Moreover, the 3, 5-difluoro derivative 5d
(IC50 ¼ 0.34 mM) was more potent than the 3, 5-dichloro derivative
5c (IC50 ¼ 2.45 mM), but it was less active than the unsubstituted
compound 5g.
For the thiazole derivatives 9aed, their IC50 values were in the
range of 0.54e5.08 mM. Analysis of the SAR of the N-substitutions
revealed that the pentyl group was the most favorable. When the
pentyl group of compound 9c was replaced by butyl (compound 9a)
or isopentyl (compound 9b), the acrosin inhibitory activity was
O
Cl
NH2
Br
S
N
Br
a
b
S
N
NH2
NH
Br
6
7
8
R
N
O
Br
S
N
c
R
NH
9
Scheme 2. Reagents and conditions: (a) 1.1 equiv of NH4NCS and 1.1 equiv of benzoyl chloride in acetone, reflux 1.5 h, then 1 equiv of Br2 in dichloroethane <32 ꢀC for 18 h, yield
65.2%; (b) 2-chloroacetyl chloride, triethylamine, THF, rt, 1 h, yield 90%; (c) various amines, DMF, triethylamine, reflux, 85 ꢀC, 3e5 h, yield 45.2e67.3%.

Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
179
N
HN
O
O
O
COOC2H5
b
a
COOC2H5
O
O
O
10
11
12
N
HN
O
c
R
O
13
Scheme 3. (a) Diethyl oxalate, ethanol, Na, rt, 8 h, yield 70%; (b) NH2NH2, ethanol, reflux, 80e90 ꢀC, 4e8 h, yield 78.2%; (c) various alkanolamines, pyridine, reflux, 90e100 ꢀC, 3 h,
yield 70.2e89.1%.
G pocket (Fig. 3B), respectively. Thus, compound 9c showed better
inhibitory activity than the butyl derivative 9a because it formed
stronger hydrophobic interaction with acrosin. Moreover, its amide
NH formed a hydrogen bond with the backbone of Val245. The
indazole derivative 17a formed similar interactions with the P1 and
G pocket (Fig. 3D) with its N-carbonyl group and ester group
forming hydrogen bonding interaction with Gln218 and His69,
respectively. The binding mode of the reference drug TLCK with
human acrosin has been investigated in our previous studies [11].
TLCK could also interact with the P1, P2 and G pocket, but it formed
relatively weak hydrophobic interactions. Thus, the designed
compounds showed higher inhibitory activities.
performed with Silica gel 60G (Qindao Haiyang Chemical, China).
Commercial solvents were used without any pretreatment.
6.1.1. Chemical synthesis of N-(2-fluorobenzyl)-2-(3-(2-hydroxy-
ethyl)ureido)-1H-benzo[d]imidazole-5-sulfonamide (5a, Scheme 1)
A
solution of compound 4 (0.5 g, 1.38 mmol) and 2-
aminoethanol (3.04 g, 50 mmol) in pyridine (15 mL) was stirred
under reflux for 3 h. Then, the mixture was stirred at room
temperature for 10 h. The solid was filtrated and was purified by
column chromatography (CH2Cl2:MeOH ¼ 60:2, v/v) to give 5a as
white solid (0.48 g, yield 90%). M.p. 198.3e198.9 ꢀC 1H NMR
(300 MHz, DMSO) d 10.72 (s, 2H, NH), 7.80 (s, 1H, Ar-H), 7.47 (s, 2H,
Ar-H), 7.33 (s, 1H, Ar-H), 7.23 (d, 1H, J ¼ 7.1 Hz, Ar-H), 7.15e7.02 (m,
2H, Ar-H), 4.84 (s, 1H, OH), 3.94 (s, 2H, PhCH2), 3.49e3.47 (m, 2H,
urea-CH2), 3.28e3.24 (m, 2H, CH2-OH). MS (ESI) m/z: 408 (M þ 1).
5. Conclusion
13C NMR (75 MHz, DMSO)
d 161.48, 158.23, 154.03, 150.65, 131.89,
In summary, scaffold hopping was used to optimize the iso-
xazolecarbaldehyde human acrosin inhibitors. A series of benzoi-
midazole, benzothiazole, pyrazole, and indazole derivatives were
rationally designed and synthesized. Most of them showed
moderate to good human acrosin inhibitory activities. Molecular
docking studies revealed that the heterocyclic inhibitors formed
hydrophobic and hydrogen bonding interactions with the active
site of human acrosin. The benzoimidazole derivative 5a was far
more potent than the standard reference drug TLCK, which repre-
sents a good lead structure to develop male contraceptives.
130.14, 129.28, 129.17, 124.78, 124.59, 124.19, 119.29, 115.12, 114.83,
þ
59.98, 41.93, 41.93. HRMS: m/z (%): 408.1132 [M þ H]
The synthetic procedure for the target compounds 5bei and
13aed was similar to the synthesis of compound 5a.
6.1.2. Chemical synthesis of N-(6-bromobenzo[d]thiazol-2-yl)-2-
(dibutylamino) acetamide (9a, Scheme 2)
Et3N (3 drops) was added to a solution of compound 8 (1.0 g,
3.3 mmol) and dibutylamine (0.51 g, 3.96 mmol) in DMF (15 mL).
The mixture was refluxed for 6 h, then diluted with H2O (20 mL)
and stirred at room temperature for 12 h. The solid was filtrated
and was purified by column chromatography (CH2Cl2:MeOH
¼ 100:1, v/v) to give 9a as white solid (1.1 g, yield 85%). M.p. 69.2e
6. Experimental protocols
6.1. General procedure for the synthesis of compounds
69.4 ꢀC 1H NMR (300 MHz, DMSO)
d 8.25e8.24 (m, 1H, Ar-H), 7.67
(d, 1H, J ¼ 8.6 Hz, Ar-H), 7.58e7.57 (m, 1H, Ar-H), 3.42 (s, 2H,
NHCOCH2), 2.58e2.51 (m, 4H, 2 ꢁ NCH2CH2CH2), 1.50e1.32 (m, 4H,
2 ꢁ NCH2CH2CH2), 1.32e1.20 (m, 4H, 2 ꢁ NCH2CH2CH2), 0.85 (t,
J ¼ 7.3 Hz, 6H, 2 ꢁ CH3). MS (ESI) m/z: 398 (M þ 1). 13C NMR
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker 300 spectrometer with TMS as an internal standard and
CDCl3/DMSO as solvent. Chemical shifts (
d values) and coupling
constants (J values) are given in ppm and Hz, respectively. ESI mass
spectra were performed on an API-3000 LCeMS spectrometer.
TLC analysis was carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). Silica gel column chromatography was
(75 MHz, DMSO)
d 171.19, 158.38, 147.57, 134.01, 128.99, 124.06,
121.97, 115.35, 56.42, 53.58, 53.58, 28.82, 28.82, 20.13, 20.13, 14.04,
14.04. HRMS: m/z (%): 398.0893 [M þ H]þ.
COR
COOH
N
COR
N
COR
a
N
b
Cl
c
N
N
N
N
N
H
H
Na
O
Cl
14
15
16
17
Scheme 4. (a) Ethanol or methylamine, EDC,HCl/oxalyl dichloride, Et3N, reflux, 90e100 ꢀC, 1e10 h, yield 85e95%; (b) THF, 60% NaH, 0e5 ꢀC, 1e3 h, yield 81e88%; (c) 2, 4-
dichlorobenzoyl chloride, THF, rt, 0e5 ꢀC, 1e3 h, yield 71e80%.

180
Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
Table 1
(s, 1H), 7.17 (d, J ¼ 8.2 Hz, 1H), 6.88e6.83 (m, 2H), 4.84 (s, 1H), 3.85
(d, J ¼ 5.6 Hz, 2H), 3.67 (s, 3H), 3.51e3.46 (m, 2H), 3.27e3.22 (m,
2H). MS (ESI) m/z: 420 (M þ 1).
Structures and human acrosin inhibitory activities of the target compounds.
Compound
R
Formula
IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
9a
9b
9c
R ¼ (2-F)PhCH2e
R ¼ (3-OCH3)PhCH2e
R ¼ (3,5-Cl)PhCH2e
R ¼ (3,5-F)PhCH2e
R ¼ (2-OCH3)PhCH2e
R ¼ (3-CH3)PhCH2e
R ¼ PhCH2e
C17H18FN5O4S
C18H21N5O5S
C17H17Cl2N5O4S
C17H17F2N5O4S
C18H21N5O5S
C18H21N5O4S
C17H19N5O4S
C17H18ClN5O4S
C15H23N5O4S
C17H24BrN3OS
C19H28BrN3OS
C19H28BrN3OS
0.08
7.32
2.45
0.34
6.04
5.45
0.28
1.06
1.62
2.78
1.09
0.54
5.08
6.81
5.73
1.43
8.92
3.24
3.78
142.6
6.1.8. 2-(3-(2-Hydroxyethyl)ureido)-N-(2-methylbenzyl)-1H-benzo
[d]imidazole-5-sulfonamide (5f)
1H NMR (300 MHz, DMSO)
d 11.91 (s, 1H), 10.15 (s, 1H), 7.87
(t, J ¼ 5.8 Hz, 1H), 7.81 (s, 1H), 7.48 (s, 2H), 7.15e7.10 (m, 2H), 6.99
(d, J ¼ 8.3 Hz, 3H), 4.84 (s, 1H), 3.86 (d, J ¼ 6.1 Hz, 2H), 3.51e3.46
(m, 2H), 3.27e3.22 (m, 2H), 2.19 (s, 3H). MS (ESI) m/z: 404
R ¼ (2-Cl)PhCH2e
R ¼ CH3(CH2)5e
(M þ 1). 13C NMR (75 MHz, DMSO)
d 154.03, 150.47, 137.85, 137.74,
R ¼ CH3(CH2)3e
137.21, 128.18, 128.18, 128.06, 128.06, 127.62, 127.62, 124.67,
124.67, 119.47, 59.99, 46.20, 41.97, 20.89. HRMS: m/z (%): 404.1385
[M þ H]þ.
R ¼ (CH3)2CH(CH2)2e
R ¼ CH3(CH2)4e
9d
R ¼ e(CH2)2N[(CH2)4CH3](CH2)2e C17H23BrN4OS
13a
13b
13c
13d
17a
17b
TLCK
R ¼ eN(CH3)CH2CH2OH
R ¼ eN(CH2CH2OH)2
R ¼ eNHCH2CH2CH2OH
R ¼ eNHCH2CH(OH)CH3
R ¼ eOCH2CH3
C14H17N3O3
C15H19N3O4
C14H17N3O3
C14H17N3O3
C17H12Cl2N2O3
C16H11Cl2N3O2
6.1.9. N-Benzyl-2-(3-(2-hydroxyethyl)ureido)-1H-benzo[d]
imidazole-5-sulfonamide (5g)
1H NMR (300 MHz, DMSO)
d 11.99 (s,1H), 10.13 (s,1H), 7.94e7.80
R ¼ eNHCH3
(m, 2H), 7.49 (s, 2H), 7.25e7.21 (m, 5H), 7.14 (s, 1H), 4.85 (s, 1H), 3.90
(d, J ¼ 6.2 Hz, 2H), 3.51e3.46 (m, 2H), 3.27e3.22 (m, 2H). MS (ESI)
m/z: 390 (M þ 1).
The synthetic procedure for the target compounds 9bed was
similar to the synthesis of compound 9a.
6.1.10. N-(2-Chlorobenzyl)-2-(3-(2-hydroxyethyl)ureido)-1H-benzo
[d]imidazole-5-sulfonamide (5h)
6.1.3. Chemical synthesis of ethyl 1-(2, 4-dichlorobenzoyl)-1H-
indazole-3-carboxylate (17a, Scheme 4)
1H NMR (300 MHz, DMSO)
d 10.60 (s, 2H), 7.81 (s, 1H), 7.51e7.48
(m, 2H), 7.44e7.40 (m, 1H), 7.38e7.35 (m, 1H), 7.28e7.21 (m, 3H),
4.84 (s, 1H), 3.98 (s, 2H), 3.51e3.45 (m, 2H), 3.27e3.22 (m, 2H). MS
(ESI) m/z: 424 (M þ 1).
2, 4-Dichlorobenzoyl chloride (2.07 g, 0.01 mol) in THF (5 mL)
was added dropwise to a solution of compound 16 (2.12 g, 0.01 mol)
in THF (10 mL). The mixture was stirred at 0e5 ꢀC for 2e6 h. After
removing THF, ice water (10 mL) was added to the residue, and
stirred at room temperature. The solid was filtrated and was puri-
fied by recrystallization to give 17a as white solid (2.72 g, 75%). 1H
6.1.11. N-Hexyl-2-(3-(2-hydroxyethyl)ureido)-1H-benzo[d]
imidazole-5-sulfonamide (5i)
1H NMR (300 MHz, DMSO)
d 11.94 (s, 1H), 10.13 (s, 1H), 7.77 (s,
NMR (300 MHz, CDCl3)
d
8.58 (d, J ¼ 9.0 Hz, 1H, Ar-H), 8.26 (d,
1H), 7.46 (s, 2H), 7.32 (t, J ¼ 5.9 Hz, 1H), 7.19e7.13 (m, 1H), 4.84 (s,
1H), 3.48 (q, J ¼ 9.7, 4.8 Hz, 2H), 3.24 (q, J ¼ 11.0, 5.5 Hz, 2H), 2.65 (q,
J ¼ 13.0, 6.7 Hz, 2H), 1.34e1.25 (m, 2H), 1.19e1.12 (m, 6H), 0.78 (t,
J ¼ 6.8 Hz, 3H). MS (ESI) m/z: 384 (M þ 1).
J ¼ 9.0 Hz, 1H, Ar-H), 7.73e7.67 (m, 1H, Ar-H), 7.57e7.53 (m, 3H, Ar-
H), 7.41 (dd, J ¼ 9.0, 3.0 Hz, 1H, Ar-H), 4.50 (q, J ¼ 7.2 Hz, 2H, CH2),
1.46 (t, J ¼ 6.0 Hz, 3H, CH3). MS (ESI) m/z: 363 (M þ 1). 13C NMR
(75 MHz, DMSO)
d 166.12, 160.67, 141.83, 139.90, 136.37, 132.45,
131.65, 131.49, 130.74, 129.25, 127.55, 126.75, 124.30, 122.40, 115.06,
6.1.12. N-(5-Bromobenzo[d]thiazol-2-yl)-2-(diisopentylamino)
acetamide (9b)
61.74, 14.05. HRMS: m/z (%): 363.0292 [M þ H]þ.
1H NMR (300 MHz, DMSO)
d
10.46 (s, 1H), 7.94 (d, J ¼ 1.9 Hz, 1H),
6.1.4. 2-(3-(3-Hydroxyethyl)ureido)-N-(2-methoxybenzyl)-1H-
7.65 (d, J ¼ 8.6 Hz, 1H), 7.53 (dd, J ¼ 8.6, 1.6 Hz, 1H), 3.23 (s, 2H),
2.61e2.56 (m, 4H), 1.65e1.51 (m, 2H), 1.42e1.25 (m, 4H), 0.90 (d,
J ¼ 6.6 Hz, 12H). MS (ESI) m/z: 426 (M þ 1). 13C NMR (75 MHz,
benzo[d]imidazole-5-sulfonamide (5b)
1H NMR (300 MHz, DMSO)
d 11.94 (s, 1H), 10.13 (s, 1H), 7.91 (t,
J ¼ 4.3 Hz, 1H), 7.81 (s, 1H), 7.50e7.48 (m, 2H), 7.19e7.13 (m, 2H),
6.80e6.74 (m, 3H), 4.84 (s, 1H), 3.88 (d, J ¼ 5.6 Hz, 2H), 3.65 (s, 3H),
3.51e3.46 (m, 2H), 3.27e3.22 (m, 2H). MS (ESI) m/z: 420 (M þ 1).
DMSO) d 170.81, 158.08, 147.43, 133.56, 128.90, 124.42, 121.96,
115.35, 56.74, 51.96, 51.96, 35.38, 35.38, 25.72, 25.72, 22.27, 22.27,
22.27, 22.27. HRMS: m/z (%): 426.121[M þ H]þ.
6.1.5. N-(3,5-Dichlorobenzyl)-2-(3-(2-hydroxyethyl)ureido)-1H-
6.1.13. N-(5-Bromobenzo[d]thiazol-2-yl)-2-(dipentylamino)
benzo[d]imidazole-5-sulfonamide (5c)
acetamide (9c)
1H NMR (300 MHz, DMSO)
d
11.07 (s, 1H), 10.25 (s, 1H), 8.02 (s,
1H NMR (300 MHz, DMSO)
d
10.22 (s, 1H), 7.94 (d, J ¼ 1.9 Hz, 1H),
1H), 7.79 (s, 1H), 7.52 (d, J ¼ 2.1 Hz, 1H), 7.48 (s, 2H), 7.46e7.40 (m,
1H), 7.37e7.33 (m, 1H), 7.20e7.15 (m, 1H), 4.85 (s, 1H), 3.97 (s, 2H),
3.48 (s, 2H), 3.28e3.22 (m, 2H). MS (ESI) m/z: 458 (M þ 1).
7.65 (d, J ¼ 8.6 Hz,1H), 7.53 (dd, J ¼ 8.6, 2.0 Hz,1H), 3.31 (s, 2H), 2.57
(t, J ¼ 15.4 Hz, 4H), 1.55e1.47 (m, 4H), 1.33e1.25 (m, 8H), 0.89 (t,
J ¼ 6.7 Hz, 6H). MS (ESI) m/z: 426 (M þ 1).
6.1.6. N-(3, 5-Difluorobenzyl)-2-(3-(2-hydroxyethyl)ureido)-1H-
benzo[d]imidazole-5-sulfonamide (5d)
6.1.14. N-(5-Bromobenzo[d]thiazol-2-yl)-2-(4-pentylpiperazin-1-
yl)acetamide (9d)
1H NMR (300 MHz, DMSO)
d
12.00 (s,1H),10.13 (s,1H), 7.96 (s,1H),
1H NMR (300 MHz, DMSO)
d
12.29 (s,1H), 8.26 (d, J ¼ 2.1 Hz,1H),
7.84e7.71 (m,1H), 7.46 (s, 2H), 7.35 (dd, J ¼ 15.6, 8.7 Hz,1H), 7.15e7.07
(m, 2H), 6.98 (t, J ¼ 8.2 Hz, 1H), 4.85 (s, 1H), 3.92 (d, J ¼ 5.8 Hz, 2H),
3.51e3.45 (m, 2H), 3.28e3.23 (m, 2H). MS (ESI) m/z: 426 (M þ 1).
7.67 (d, J ¼ 8.6 Hz, 1H), 7.57 (dd, J ¼ 8.6, 2.1 Hz, 1H), 3.42 (s, 2H),
3.38e3.30 (m, 8H), 2.71 (m, 2H), 1.54 (s, 2H),1.32e1.21 (m, 4H), 0.86
(t, J ¼ 6.9 Hz, 3H). MS (ESI) m/z: 426 (M þ 1).
6.1.7. 2-(3-(2-Hydroxyethyl)ureido)-N-(2-methoxybenzyl)-1H-
6.1.15. N-(2-Hydroxyethyl)-5-(4-methoxyphenyl)-N-methyl-1H-
benzo[d]imidazole-5-sulfonamide (5e)
pyrazole-3-carboxamide (13a)
1H NMR (300 MHz, DMSO)
d
11.98 (s, 1H), 10.22 (s, 1H), 7.81
1H NMR (600 MHz, DMSO)
d
13.43 (s, 1H, NH), 7.71 (d, J ¼ 8.7 Hz,
(s, 1H), 7.77e7.73 (m, 1H), 7.48 (s, 2H), 7.25 (d, J ¼ 7.6 Hz, 1H), 7.21
2H, Ar-H), 7.03 (d, J ¼ 8.7 Hz, 2H, Ar-H), 6.98 (d, J ¼ 8.7 Hz, 1H, CH),

Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
181
Fig. 3. The binding modes of compounds 5a (A), 9c (B), 13a (C) and 17a (D) with the active site of human acrosin.
4.75 (s, 1H, OH), 3.77 (d, J ¼ 6.0 Hz, 2H, CH2OH), 3.61 (q, J ¼ 8.4 Hz,
2H, NCH2), 3.02 (s, 3H, CH3). MS (ESI) m/z: 276 (M þ 1).
3.47 (q, J ¼ 6.0 Hz, 2H), 3.34e3.26 (m, 2H),1.67 (s, 2H). MS (ESI) m/z:
276 (M þ 1). 13C NMR (75 MHz, DMSO)
d 161.85, 158.96, 148.10,
143.45, 126.38, 126.38, 121.77, 114.81, 114.81, 101.25, 57.33, 54.53,
6.1.16. N,N-bis(2-Hydroxyethyl)-5-(4-methoxyphenyl)-1H-
35.70, 31.62. HRMS: m/z (%): 276.1348 [M þ H]þ.
pyrazole-3-carboxamide (13b)
1H NMR (300 MHz, DMSO)
7.01 (d, J ¼ 8.2 Hz, 2H), 6.87 (s,1H), 4.79 (s, 2H), 3.80 (s, 2H), 3.78 (s, 3H),
3.60 (d, J¼ 5.6 Hz, 4H), 3.54 (d, J¼ 4.8 Hz, 2H). MS (ESI) m/z:306(Mþ 1).
d
13.46 (s, 1H), 7.71 (d, J ¼ 8.3 Hz, 2H),
6.1.18. N-(2-Hydroxypropyl)-5-(4-methoxyphenyl)-1H-pyrazole-3-
carboxamide (13d)
1H NMR (300 MHz, DMSO)
d 13.47 (s, 1H), 7.90 (s, 1H), 7.72 (d,
J ¼ 8.5 Hz, 2H), 7.02 (d, J ¼ 8.5 Hz, 2H), 6.95 (s, 1H), 4.81 (s, 1H), 3.78
6.1.17. N-(3-Hydroxypropyl)-5-(4-methoxyphenyl)-1H-pyrazole-3-
(s, 3H), 3.34 (s, 1H), 3.31e3.09 (m, 2H), 1.05 (d, J ¼ 6.0 Hz, 3H). MS
carboxamide (13c)
(ESI) m/z: 276 (M þ1). 13C NMR (75 MHz, DMSO)
d 161.84, 159.42,
1H NMR (600 MHz, DMSO)
d
13.43 (s, 1H), 8.11 (s, 1H), 7.72 (d,
147.75, 143.53, 126.78, 126.35, 121.47, 114.42, 114.42, 101.68, 65.28,
J ¼ 7.0 Hz, 2H), 6.98 (d, J ¼ 54.1 Hz, 3H), 4.50 (s, 1H), 3.80 (s, 3H),
55.17, 46.18, 21.14. HRMS: m/z (%): 236.1343 [M þ H]þ.

182
Q. Chen et al. / European Journal of Medicinal Chemistry 59 (2013) 176e182
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1H NMR (300 MHz, CDCl3)
d
10.30 (s, 1H), 8.43 (d, J ¼ 8.2 Hz, 1H),
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In our previous studies, Libdock within the Discovery Studio 2.5
[19] software package was found to be a good method to dock
human acrosin inhibitors [13]. The protein structure of human
acrosin was obtained from our previous report [19]. In the process
of Libdock docking, the docking preferences parameter was set to
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Other parameters were used as default.
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Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(dia-
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Acknowledgments
This work was supported in part by the National Natural Science
Foundation of China (Grant No. 81222044) and the Science and
Technology Support Program of Jiang Su Province of China (Grant
No. BE2010682).
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