Acrosin structure-based design, synthesis and biological activities of 7-azaindol derivatives as new acrosin inhibitors

Article abstract of DOI:10.1016/j.cclet.2010.09.033

A series of 7-azaindol derivatives were designed based on the homologous 3D model of human acrosin. These compounds were synthesized and evaluated for their human acrosin inhibitory activities in vitro. Compounds 7a, 7i, 7j, 7k and 7n showed highly inhibitory activity against human acrosin. The three-dimensional structure-activity relationship was investigated through a CoMFA model, which provided valuable information to further study of potential human acrosin inhibitors.

Full text of DOI:10.1016/j.cclet.2010.09.033

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 272–275
www.elsevier.com/locate/cclet
Acrosin structure-based design, synthesis and biological activities
of 7-azaindol derivatives as new acrosin inhibitors
Jun Hang Jiang 1, Xue Fei Liu 1, Can Hui Zhen, You Jun Zhou,
*
Ju Zhu, Jia Guo Lv , Chun Quan Sheng
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
Received 8 June 2010
Available online 22 December 2010
Abstract
A series of 7-azaindol derivatives were designed based on the homologous 3D model of human acrosin. These compounds were
synthesized and evaluated for their human acrosin inhibitory activities in vitro. Compounds 7a, 7i, 7j, 7k and 7n showed highly
inhibitory activity against human acrosin. The three-dimensional structure–activity relationship was investigated through a CoMFA
model, which provided valuable information to further study of potential human acrosin inhibitors.
# 2010 Published by Elsevier B.V. on behalf of Chinese Chemical Society.
Keywords: Rational design; Azaindole; Synthesis; Acrosin; CoMFA
Acrosin is a multifunctional protein located in acrosome. It not only affects motility of sperm but also disperses
acrosomal matrix and hydrolyzes the zona pellucida, which helps sperm enter and fusion to ovum. Therefore
contraception can be achieved through acrosome enzyme inhibition. In recent years, acrosin has become an attractive
target in contraceptive drug discovery [1].
In our previous studies, the homologous 3D model of human acrosin was built based on the crystal structures of ram
and boar acrosins [2], and the active site was searched by Insight II/binding site analysis. On the basis of these studies,
we further analyzed the properties of the active site by the multiple copy simultaneous search (MCSS) method [3]. It
was demonstrated that the active site of human acrosin is an irregular groove, which could be divided into three
subsites (P1, P2 and G) [4].
We have found that lonidamine, an antineoplastic agent with indazole skeleton, had anti-acrosin activity in vitro [5].
Based on the principle of bioisosterism, a series 7-azaindol compounds were designed in the present study. The
binding mode of azaindole was further investigated using the docking program Autodock (Fig. 1) [6]. The results
showed that azaindole could be well accommodated by the active site of human acrosin. The azaindole ring resided at
the entrance of the P1 subsite, which supported the whole structure through the hydrophobic interaction with the
residues Val245 and Gly246. The 3-ethylester group was located in the P1 subsite, forming hydrogen-bonds with the
key residue Gln218. Other 1-substituents were extended to the P2 subsite, which were surrounded by a hydrophobic
* Corresponding authors.
E-mail addresses: ljg20060508@yahoo.com.cn (J.G. Lv), shengcq@hotmail.com (C.Q. Sheng).
1
Co-first author.
1001-8417/$ – see front matter # 2010 Published by Elsevier B.V. on behalf of Chinese Chemical Society.
doi:10.1016/j.cclet.2010.09.033

[()TD$FIG]
J.H. Jiang et al. / Chinese Chemical Letters 22 (2011) 272–275
273
Fig. 1. Binding model of 7-azaindole with the active site residues of human acrosin.
center lined with the residues Val245, Gly244 and Trp243. Therefore, we synthesized a series of 7-azaindol derivatives
and tested their biological activities.
The chemical structures and synthetic route of the azaindole derivatives were depicted in Fig. 2 and Scheme 1.
Succinonitrile, ethyl formate and t-butylamine were condensed to afford compound 1, which was subsequently
converted to compound 2 by ring closure reaction. Cyclization reaction of 2 with 1,1,3,3-tetramethoxypropane formed
compound 3 [7]. De-tert-butylation of 3 provided compound 4, which was hydrolyzed to get compound 5.
Intermediate 5 was condensed to yield compound 6. Acylation of 6 provided the target compounds 7 [8].
Preliminary in vitro acrosin inhibitory activity of the novel compounds was shown in Table 1. The data demonstrated
that most of the compounds had potent in vitro acrosin inhibitory activity. So we further investigate their anti-acrosin
[()TD$FIG]
activity (Table 2). The result showed that compounds 7a, 7i, 7j, 7k and 7n showed highly stronger anti-acrosin activity
COOEt
COOEt
N
N
N
N
O
R2
O
R1
7a: R1 = H; 7b: R1 = 2-F; 7c: R1 = 3-F; 7d: R1 = 2-Cl; 7e: R1 = 2-CH3; 7f: R1 = 2-CH3O; 7g: R1 = 2-CH3O, 4-CH3O; 7h: R1 = 2-Cl, 6-Cl; 7i: R1 =
2-Cl, 4-Cl; 7j: R1 = 2-Cl, 4-F; 7k: R1 = 2-Br, 4-F; 7l: R1 = 3-Cl, 4-Cl; 7m: R2 = 2,4-dichloro-1-benzyl; 7n: R2 = 2-thienyl.
Fig. 2. Compounds with azaindole ring.
[()TD$FIG]
H
NC
CN
NC
NC
NC
NC
b
a
c
NH
+ HCOOEt
N
NH2
N
N
1
2
3
COOEt
O
CN
COOH
COOEt
g
d
e
f
N
N
N
N
H
N
N
N
N
H
H
R
7
5
4
Scheme 1. Synthesis of compounds 7a–7n. Reagents and conditions: (a) NaH, t-butylamine, reflux, 5hr; (b) KOH, EtOH; (c) 1,1,3,3-
tetramethoxypropane, toluene, p-TosOH, 95 8C; (d) AlCl3, xylene, reflux, 8hr; (e) HCl, 70 8C; (f) condensing agent EDC, EtOH, 50 8C; (g)
NaH, substituted acyl chloride.

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J.H. Jiang et al. / Chinese Chemical Letters 22 (2011) 272–275
Table 1
Preliminary anti-acrosin activity data of the target compounds at concentration of 60 mmol/mL.
Compounds
Acrosin activitya (IU/106 sperm)
Inhibition ratiob
7a
7b
7c
7d
7e
7f
À12.768
18.424
7.567
1.190
0.727
0.888
0.535
0.002
0.214
0.748
0.077
0.961
0.988
1.124
1.068
0.946
1.173
31.334
67.254
52.969
16.978
62.181
2.619
7g
7h
7i
7j
0.780
7k
7l
À8.337
À4.606
3.619
7m
7n
À11.676
a
Standard activity is 67.376 IU/106 Sperm.
b
Inhibition ratio = (standard activity À experimental activity)/standard activity.
Table 2
In vitro inhibitory activities of human acrosin.
Compounds
IC50 (mmol/mL)
7a
7i
2.21
0.29
7j
7k
3.47
1.74
7n
TLCK
3.15
142.6
than the standard control compound TLCK (N-tosyl-L-lysine chloromethyl ketone). Then we performed a CoMFA
research (except compound 7m) to establish the 3D-QSAR model based on the data of activity (Fig. 3). The CoMFA
result (q2 = 0.516, r2 = 0.776, SEE = 0.300, F = 19.044, n = 2, steric and electronic field contribution were 0.279 and
0.721, respectively) indicated that electron-withdrawing group was favorable in the region of position C-2 at the benzene
ring (blue part), and large group was unfavorable in the region of position C-4 at the benzene ring (yellow part). The
docking model also revealed that small groups were beneficial to be stretched into the P2 subsite, which was in
accordancewith the CoMFA results. The anti-acrosin activity data demonstrated that halogens at position C-2 and C-4 of
the benzene ring were more favorable for the inhibitory activity than the methoxyl group, which was in consistence with
the CoMFA results. The bioassay results also hinted that the parent scaffold of these compounds could play an important
role in the inhibition of human acrosin.
[()TD$FIG]
Fig. 3. (a) Superposition of the compounds 7a–7l, and 7n. (b) Steric (yellow and green part) and electronic (blue part) field of CoMFA model. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

J.H. Jiang et al. / Chinese Chemical Letters 22 (2011) 272–275
275
In summary, the novel 7-azaindol derivatives were designed and synthesized and their anti-acrosin activity in vitro
were tested. The result showed that compound 7i had the highest activity. Subsequently, we built a CoMFA model to
help us to discuss the three-dimensional structure–activity relationship to guide further structure modification.
Acknowledgments
This work was supported by the Shanghai Family Planning Commission Research Fund (No. 2007JG02) and
Shanghai Leading Academic Discipline Project (No. B906).
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[8] Yield, physical and spectral data for some synthesized compounds: 7a Full yield 95.0%, mp: 137–138 8C. 1H NMR (300 MHz, CDCl3): d 1.41–
1.46(t, 3H, J = 7.2 Hz), 4.39–4.46(q, 2H, J = 7.2 Hz), 7.26–7.85(m, 9H). ESI-MS (m/z): 295.47 [M+1]. 7i Full yield 85.8%, mp: 116–118 8C. 1H
NMR (300 MHz, CDCl3): d 1.42–1.46(t, 3H, J = 7.2 Hz), 4.39–4.47(q, 2H, J = 7.2 Hz), 7.26–8.43(m, 7H). ESI-MS (m/z): 364.60 [M+1]. 7j Full
yield 75.3%. mp: 126–127 8C. 1H NMR (300 MHz, CDCl3): d 1.42–1.46(t, 3H, J = 7.2 Hz), 4.39–4.46(q, 2H, J = 7.2 Hz), 7.13–8.22(m, 7H).
ESI-MS (m/z): 347.89 [M+1]. 7k Full yield 74.1%. mp: 122–124 8C. 1H NMR (300 MHz, CDCl3): d 1.42–1.47(t, 3H, J = 7.2 Hz), 4.40–4.47(q,
2H, J = 7.2 Hz), 7.18–8.23(m, 7H). ESI-MS (m/z): 393.65 [M+1]. 7n Full yield 91.0%. mp: 144–146 8C. 1H NMR (300 MHz, CDCl3): d 1.42–
1.47(t, 3H, J = 7.2 Hz), 4.40–4.48(q, 2H, J = 7.2 Hz), 8.47(s, 1H), 7.23–8.60(m, 5H). ESI-MS (m/z): 301.37 [M+1].