Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.09.060

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-l-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.

Full text of DOI:10.1016/j.bmcl.2011.09.060

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6674–6677
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and acrosin inhibitory activity of substituted
4-amino-N-(diaminomethylene) benzenesulfonamide derivatives
Lili Ding a, Ju Zhu a, Canhui Zheng a, Chunquan Sheng a, Jingjing Qi a, Xuefei Liu a, Guangqian Han b,
a,
Juntao Zhao a, Jiaguo Lv a, , Youjun Zhou
⇑
⇑
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:
Received 14 June 2011
Revised 8 September 2011
Accepted 16 September 2011
Available online 21 September 2011
A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and
their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin
inhibitory activities with compounds 4o and 4p being significantly more potent than the control com-
pound N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for
the development of acrosin inhibitory agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Acrosin inhibitory agents
Synthesis
4-Amino-N-(diaminomethylene)
benzenesulfonamide
Modern contraceptive methods have protected millions of cou-
ples worldwide from unwanted pregnancy and allowed them to
control reproduction. One of the most important methods of con-
traception is the contraceptive pill. However, the oral contracep-
tive pill is only available for women and most of these pills can
have serious side effects, including breakthrough menstrual bleed-
ing, nausea, mood swings, headaches, and an increased risk of rec-
tal cancer.1–4 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.
(TLCK), suramin, DV-1006, isoxazolecarbaldehydes, and 4-guani-
dinobenzoates (Fig. 1)8–11 have been reported.
In recent years, our group has been searching for novel small
acrosin inhibitors. Since no crystal structures of human acrosin
have been reported, in a previous study, we constructed a homolo-
gous three-dimensional model of human acrosin based on the crys-
tal structures of the ram and boar forms.12 Using this model, we
searched for novel small acrosin inhibitors. In these studies, one
of the potent acrosin inhibitory agents, such as KF950 (Fig. 2, Table
1), was designed and synthesized.13
Sperm acrosin is the major trypsin-like protease present in the
acrosome of all mammalian spermatozoa.5 Acrosin is also a sperm-
specific enzyme. In fertilization, acrosin hydrolyzes the zona pellu-
cida and the vitelline membrane, and facilitates the penetration of
the sperm through the innermost glycoprotein layers of the
oocytes.6 Consequently, acrosin is a key enzyme in fertilization.
Inhibition of acrosin activity should block fertilization and reduce
the success of fertilization.7 Thus; acrosin represents a potential
target for the design and development of novel male contraceptive
agents. Recently, research on acrosin inhibitors as potential contra-
ceptives has been carried out. Several classes of small acrosin
The previous molecular docking study showed that most of
known acrosin inhibitors were located in the P1 pocket, and inter-
acted with the hydrophobic residues of active site of human acro-
sin.14 However, KF950, with the highest inhibitory activity, was
located in P1 and G pockets and its guanidine group formed strong
hydrogen-bonding interactions with the residues Cys217, Trp243,
Ser221, and Thr216 of P1 pocket, respectively. It suggested that
the guanidine group was an important pharmacophore for the high
acrosin inhibitory activity. As KF950 is unstable, a series of novel
amide acrosin inhibitors containing guanidine group were
designed.
inhibitors such as N-alpha-tosyl-L-lysyl-chloromethyl-ketone
Here, we report the synthesis and in vitro acrosin inhibitory
activities of 4-amino-N-(diaminomethylene) benzenesulfonamide
derivatives (Scheme 1, Table 1).
The chemical synthesis of the target compounds is outlined in
Scheme 1. The key intermediate 4-amino-N-(diaminomethylene)
⇑
Corresponding authors. Tel.: +86 021 81871234.
E-mail addresses: ljg20060508@yahoo.com.cn (J. Lv), zhouyoujun2005@yahoo.
com.cn (Y. Zhou).
benzenesulfonamide (3) was obtained by
a
reported
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.060

L. Ding et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6674–6677
6675
COOH
O
O
ClH2C
O
O
S
H2N
N
O
H
NH2
CH3
TLCK
DV-1006
H
H2N
N
N
O
N
Cl
O
NH
HCl
O
CHO
OHC
O
O2N
NHCCH3
O
Cl
ISO-2
ISO-1
AGB
Figure 1. Chemical structures of representative human acrosin inhibitors.
H
as the control. The results showed that all title compounds had bet-
ter inhibitory activities than TLCK.
H2N
N
NH
CH3SO3H
O
In general, compounds with aromatic rings (4a–4p) were more
potent than compounds with aliphatic chains (4q–4r). The com-
pound 4s, with a longer chain, showed better inhibitory activity
than 4q and 4r. For the aromatic series, the compounds bearing
an electron donating substituted phenyl ring (–CH3, –OCH3,
–C2H5, –OC2H5) were more active than compounds bearing an
electron withdrawing substituted phenyl ring (–CF3, –NO2). The
O
O
O
KF950
Figure 2. Chemical structure of KF950.
compounds bearing
a phenyl ring with bi-halogen groups
(4e–4g) were more active than the single-halogenated substituted
compounds (4c, 4d). The para or ortho positions in the phenyl ring
(4c) were favorable substitutions for bioactivity, and compounds
with a chloro group on the phenyl ring (4c–4d) showed better
inhibitory activity than their fluoro (4i) and bromo (4h) analogs.
Interestingly, of all the compounds, 4o and 4p, with para substitu-
tion and an electron-donating group on the phenyl ring,
demonstrated much higher inhibitory activity than the other syn-
Table 1
In vitro acrosin inhibitory activities of the target compounds (IC50 in
l
mol/ml)
IC50
Compound
IC50
Compound
IC50
Compound
4a
4b
4c
4d
4e
4f
4g
4h
4i
2.21
2.06
6.30
7.00
5.63
5.96
3.80
7.63
7.11
7.18
3.84
2.45
0.027
4m
4n
4o
4p
4q
4r
7.05
2.50
0.21
0.13
9.00
9.58
1.58
7.41
9.90
4.98
9.30
2.99
7c
3.31
7d
7e
7f
8a
8b
8c
8d
8e
8f
8g
TLCK
2.35
9.50
2.51
2.30
9.30
8.92
8.98
9.35
9.84
4.20
thesized compounds, with IC50 values of 0.21 and 0.13 lmol/ml,
4s
respectively. Of compounds 6a–6c, 6c bearing the para substitu-
tion had better bioactivity than the non-substituted compound
6a and the ortho substituted compound 6b. For compounds
7a–7f, compounds with a branch chain (7d, 7f) were more potent
than compounds with a linear chain (7a–7c, 7e). For compounds
8a–8g, unsubstituted compound 8a showed better acrosin inhibi-
tory activity than the substituted analogs (8b–8g).
6a
6b
6c
7a
7b
4j
4k
4l
142.6
KF950
A molecular docking study was carried out using the Discovery
Studio 2.5 software package.19 The binding mode of compounds
TLCK, 4o and 4p with the active site of human acrosin are pre-
sented in Figures 3 and 4, respectively.
procedure.15,16 Acetylation of aniline (1) was used to obtain acet-
anilide (2) with VB1 as the catalyst by simply mixing aniline and
acetic acid. The compound 3 was synthesized by the one-pot reac-
tion of acetanilide (2) with ClSO3H, NH2CN, (NH4)2SO4, and NaOH.
The reaction of compound 3 with different substitute acyl chlo-
rides in the presence of 1.2 M equiv K2CO3 with DMF as the solvent
yielded target compounds 4a–4s. The target compounds 6a–6c
were prepared by the reaction of piperidine with compound 5,
which was obtained by the Friedel–Crafts reaction of 3 with chlo-
roacetylchloride in DMF. The target compounds 7a–7f were pre-
pared using a similar procedure. The target compounds 8a–8g
were synthesized by the reaction of compound 5 with phenol in
the presence of 1.05–1.2 M equiv K2CO3. The structures of all target
compounds were determined by 1H NMR, 13C NMR, and HRMS
analysis.17
Analysis of the binding mode finds that the control compound
TLCK, 4o and 4p are all located in P1 and G pockets. Figure 3 shows
that TLCK only forms hydrophobic interaction with the P1 pocket,
while Figure 4 reveals that the target compounds 4o and 4p could
form hydrogen-bond interactions with the key residues Ser221,
Trp243, and Thr216 of the P1 pocket through the guanidine group,
which shows that the hydrogen-bond interactions with the P1
pocket increase the acrosin inhibitory activity of the target com-
pounds and the guanidine group is an essential pharmacophore
for the high acrosin inhibitory activity. Moreover, the terminal
substituted benzene group of compound 4o extends into the G
pocket, which is surrounded by hydrophobic residues lined with
Arg199, Ala118, and Ser117. The terminal substituted benzene
group of compound 4p extends into both the P2 and G pockets,
and interacts with the hydrophobic residues Arg199, Ala118,
Ser117, and Tyr196. The added interaction with the residue
The acrosin inhibitory activities of the title compounds, KF950
and TLCK were assessed according to the method of Kennedy
et al.18 and the results are listed in Table 1, in which TLCK was used

6676
L. Ding et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6674–6677
O
a
b
N
S
NH2
NHCOCH3
NH2
H2N
O
NH2
1
2
3
4a R1=4-ArOCH3
4b R1=4-ArCH3
4c R1=2-ArCl
4d R1=3-ArCl
4e R1=2,4-Ar2Cl
4f R1=2,6-Ar2Cl
4g R1=3,4-Ar2Cl
4h R1=2-ArBr
4i R1=4-ArF
c
O
N
H2N
S
3
O
O
NH2
4j R1=2,6-Ar2F
N
R1
4k R1=4-ArCH2Cl
4l R1=2,4,6-Ar3CH3
4mR1=3-ArCF3
4n R1=3,5-Ar2NO2
4o R1=4-ArCH2CH3
4p R1=4-ArOCH2CH3
4q R1=CH2CH3
H
4a-4s
4r R1=CH2CH2CH3
4s R1=CH2CH2CH2CH3
O
S
e
H2N
N
d
O
S
R2
H2N
N
O
N
O
3
O
NH2
O
NH2
Cl
N
H
N
H
5
6a-6c
6a R2=H
6b R2=2-CH3
6c R2=4-CH3
f
g
O
O
S
H2N
N
5
R3
O
O
O
NH2
N
N
H
7a-7f
7a R3=CH3
7b R3=CH2CH3
7c R3=CH2CH2CH3
7d R3=CH(CH3)2
7e R3=CH2CH2CH2CH3
7f R3=CH2CH2CH(CH3)2
O
O
R4
O
S
h
N
NH
8a R4=H
5
H2N
O
NH2
8b R4=2-CH3
8c R4=3-CH3
8d R4=4-CH3
8e R4=4-Cl
8a-8g
8f R4=4-OCH3
8g R4=4-NO2
Scheme 1. Reagents and conditions: (a) CH3COOH, VB1, 4 h, 100 °C; (b) ClSO3H, NH2CN, (NH4)2SO4, NaOH; (c) acylchloride, DMF, K2CO3, 0 °C, 5 h; (d) chloroacetylchloride,
DMF, K2CO3, 0 °C, 2 h; (e) piperidine, acetone, K2CO3, NaI, reflux, 1 h; (f) 4-piperidine formic acid, alcohol, concd H2SO4, 100 °C, 4 h; (h) 4-piperidine formic acid ester, acetone,
K2CO3, NaI, reflux, 1 h; (i) phenol, acetone, K2CO3, KI, rt, overnight.
Tyr196 in P2 may explain why compound 4p has slightly better
acrosin inhibitory activity when compared with compound 4o
(Table 1).
In summary, a series of novel 4-amino-N-(diaminomethylene)
benzenesulfonamide derivatives were designed and synthe-
sized. In vitro biological assays indicated that all target com-
pounds show more potent acrosin inhibitory activity than the
positive control compound TLCK. In particular, compounds 4o
and 4p had the most potent activities, and are worthy of further
investigation.
Acknowledgment
Financial support was provided from the Science and Technol-
ogy Support Program, Jiang Su 2010, BE2010682.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.060.

L. Ding et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6674–6677
6677
Figure 3. The docking result of TLCK in the active site of acrosin.
Figure 4. Docking results of compounds 4o and 4p in the active site of acrosin. Important residues involved in inhibitor binding are shown and hydrogen-bonds are displayed
by red dotted lines.
14. Zhang, J.; Zheng, C. H.; Sheng, C. Q.; Zhou, Y. J.; Zhu, J.; Lv, J. G. Chem. J. Chinese.
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