Design and synthesis of phenylisoxazole derivatives as novel human acrosin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.04.118

Human acrosin is an attractive target for the discovery of novel male contraceptives. Isoxazole derivative ISO-1, a small-molecule weak human acrosin inhibitor, was used as the starting point for lead optimization. After two rounds of structure-based inhibitor design, a highly potent inhibitor B6 (IC₅₀ = 1.44 μM) was successfully identified, which showed good selectivity over trypsin and represents one of the most active human acrosin inhibitors up to date.

Full text of DOI:10.1016/j.bmcl.2014.04.118

Accepted Manuscript
Design and Synthesis of Phenylisoxazole Derivatives as Novel Human Acrosin
Inhibitors
Juntao Zhao, Wei Tian, Jingjing Qi, Diya Lv, Yang Liu, Yan Jiang, Guoqiang
Dong, Qianqian Chen, Youjun Zhou, Ju Zhu, Heling Wang, Chunquan Sheng,
Jiaguo Lv
PII:
S0960-894X(14)00485-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.118
BMCL 21611
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 February 2014
22 April 2014
29 April 2014
Please cite this article as: Zhao, J., Tian, W., Qi, J., Lv, D., Liu, Y., Jiang, Y., Dong, G., Chen, Q., Zhou, Y., Zhu,
J., Wang, H., Sheng, C., Lv, J., Design and Synthesis of Phenylisoxazole Derivatives as Novel Human Acrosin
Inhibitors, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.118
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Design and Synthesis of Phenylisoxazole Derivatives as Novel
Human Acrosin Inhibitors
Juntao Zhao §, Wei Tian§, Jingjing Qi§, Diya Lv, Yang Liu, Yan Jiang, Guoqiang Dong,
Qianqian Chen, Youjun Zhou, Ju Zhu, Heling Wang, Chunquan Sheng* and Jiaguo
Lv*
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai
200433, China
§These authors contributed equally to this work.
* To whom correspondence should be addressed. For C. Q. Sheng: Phone/Fax,
86-21-81871239, E-mail, shengcq@hotmail.com. For J. G. Lv: /Fax, 86-21-81870234,
E-mail, ljg19580808@163.com.
1

Abstract
Human acrosin is an attractive target for the discovery of novel male contraceptives.
Isoxazole derivative ISO-1, a small-molecule weak human acrosin inhibitor, was used
as the starting point for lead optimization. After two rounds of structure-based
inhibitor design, a highly potent inhibitor B6 (IC50 = 1.44 µM) was successfully
identified, which showed good selectivity over trypsin and represents one of the most
active human acrosin inhibitors up to date.
Keywords: Guanidinophenylpyrazole derivatives, Molecular hybridization, Male
contraceptive, Human acrosin inhibitors
2

It is estimated that human population in the world will reach about 9 billion in the
year 2050 [1]. Overpopulation is becoming one of the major global issues because of
limited resources and increased environmental burdens [1]. Contraception is one of
the best solutions to solve these problems. Currently, most of the contraceptives are
developed for women. However, choices for male contraception are rather limited (e.g.
condom and vasectomy). Discovery and development of effective male contraceptives
represents a challenging research area [2].
Acrosin is a typical serine endoprotease with trypsin-like activity, which is stored in
its inactive zymogen form (called proacrosin) and secreted as a serine protease after
sperm–zona pellucida (ZP) binding [3]. Acrosin plays multiple roles during
fertilization in several significant ways including activation of acrosome components,
secondary binding with ZP, and hydrolysis of ZP. Due to the importance of human
acrosin in fertilization, it is an attractive target for the development of human male
contraceptives. In 2000, crystal structures of ram and boar β-acrosins in complex with
p-aminobenzamidine were solved, which provided structural basis for rational design
of acrosin inhibitors [4].
Up to now, small molecule human acrosin inhibitors mainly included TLCK [5],
DV-1006, p-aminobenzamidine (pAB) [6], and isoxazolecarbaldehydes (ISO-1) [7]
(Fig. 1). However, most of these inhibitors showed relatively poor human acrosin
inhibitory activity (IC50 values in mM range). Structure-based drug design represents
an effetive stragegy to disocver potent human acorsin inhibitors. However, three
dimensional structure of human acrosin has not been solved up to date. Thus, we
3

contructed a structural model of human acrosin using homology modeling [8].
Moreover, three important sub-sites (P1, P2 and G) for inhibitor binding were
identified by computational analysis of the active site of human acrosin [9]. Based on
information obtained from computer modeling, we successfully designed a number of
novel
acrosin
inhibitors
including
[10],
4-guanidinobenzoates
benzenesulfonamides
[8],
diaminomethylene-benzenesulfonamides
[11],
phenylpyrazoles [12], imidazolesulfonamides [13],
phenylpyrazoles [14] and
quinazolinons [15] (Fig. 1).
Continuing our efforts in the discovery of novel acrosin inhibitors, herein the
isoxazolecarbaldehyde inhibitor ISO-1 was used as a starting point for lead
optimization. ISO-1 is a fragment-like lead compound with a low molecular weight
(MW = 240). Although its activity was low, structure-based methods can be used to
improve its potency. A series of phenylisoxazole derivatives were designed and
synthesized and most of them showed significantly increased human acrosin
inhibitory activity. Particularly, a highly potent inhibitor (compound B6) was
identified and its binding mode was clarified by molecular docking.
Chemical synthesis of phenylisoxazole derivatives A1-A5 is outlined in Scheme 1.
1-(4-Nitrophenyl)ethanone (1) was oxalylated by diethyl oxalate in the presence of
sodium ethanol to afford diketoester 2. The product was condensed with
hydroxylamine hydrochloride to yield the isoxazole scaffold 3. Then, the nitro group
of intermediate 3 was reduced by SnCl2 and followed by acylation with various acyl
chlorides in the presence of triethylamine to give the target compounds A1-A5. Target
4

compounds B1-B25 were synthesized by the method outlined in Scheme 2. The
guanidino intermediate 5 was prepared by reacting the amino group of intermediate 4
with NH2CN using EtOH as the solvent and HCl as the acid. Catalyzed by NaOH, the
ester group of compound 5 was hydrolyzed and finally condensed with various
amines in the presence of
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDC·HCl) and N-hydroxybenzotriazole (HOBt) to give the target
compounds B1-B25.
Lead optimization of ISO-1 was based on three subsites (P1, P2 and G) in the
active site of human acrosin [9]. Docking studies revealed that ISO-1 was located in
the P1 pocket (a hydrophobic hydrogen-bonding site) with its aldehyde oxygen atom
forming a hydrogen bond with Thr216 (Fig. 2A). On the basis of the binding model, a
series of phenylisoxazole derivatives (A1-A5) were designed. First, the
phenylisoxazole scaffold of ISO-1 was extended into the P2 pocket to form additional
interactions by introducing a substituted phenyl amide group. Second, the chemically
unstable aldehyde group was replaced by an ester group, which was expected to form
hydrogen bonding interaction with Gln218.
In vitro human acrosin inhibitory assay was performed using the protocols
according to our previous study [11]. The results revealed that most of the target
compounds were more active than the lead compound ISO-1 with IC50 values in the
range of 60 µM to 460 µM (Table 1). Particularly, the 2-chlorine derivative A2
showed the best activity (IC50 = 60 µM), which was 6 fold more potent than ISO-1.
Compound A2 was docked into the active site of human acrosin using LibDock
5

within Discovery Studio 2.5 software package (DS 2.5) [16]. As shown in Fig. 2B, the
isoxazole scaffold was located into the P1 pocket with its nitrogen atom and oxygen
atom forming two hydrogen bonds with Ser221 and Gln218, respectively. As expected,
the carbonyl oxygen atom of the ester group also formed a hydrogen bond with the
backbone amide of Gln218. The 2-chlorophyenyl amide group was extended into the
P2 pocket and formed hydrophobic contacts.
The guanidyl group was an important pharmacophore observed in several human
acrosin inhibitors (e.g. KF950), because it could form hydrogen bonding interactions
with the P1 pocket [9]. Therefore, the guanidyl group was introduced onto the
phenylisoxazole inhibitors to increase the inhibitory potency. A series of
guanidinophenylisoxazole derivatives (B1-B25) were designed and synthesized. To
our delight, most of the target compounds showed significantly improved human
acrosin inhibitory activities (Table 2). Notably, nine highly active compounds (i.e. B5,
B6, B7, B8, B10, B15, B18, B19 and B22) were successfully identified with IC50 values
lower than 10 µM. In particular, compound B6 showed the best activity (IC50 = 1.4
µM), which was far more potent than the lead compound ISO-1. In order to
investigate the selectivity of compound B6, we determined its inhibitory activity
against pig trypsin (a member of the serine protease family). The IC50 value was 4200
µM, indicating that compound B6 exhibited good specificity toward human acrosin.
Structure-activity relationship (SAR) analysis indicated that the guanidine group
was essential for acrosin inhibitory activity. Moreover, the phenyl substitution
attached to the amine group was also important because the alkyl derivatives B1 and
6

B2 were inactive. The type and position of the substitutions on the terminal phenyl
group had various effects on the inhibitory activities. As compared with the
unsubstituted phenyl derivative B3 (IC50 = 24.6 µM), the introduction of various
substitutions generally led to the increase of the activity. In particular, compounds
with di-methoxyl substitutions, namely B5 and B6, showed excellent inhibitory
activities at low micromolar concentrations. The 2,5-dimethoxyl derivative B6 (IC50 =
1.4 µM) was the most active compound in this series. In contrast, mono-methoxyl
derivatives B18 (IC50 = 8.4 µM) and B9 (IC50 = 6.6 µM) showed decreased activity.
Moreover, di-methyl derivatives (e.g. B10, B12 and B24) were also less active than
the di-methoxyl derivatives. When the phenyl group was enlarged to naphthalene
(B21, IC50 = 29.9 µM), the activity was largely decreased. Moreover, ligand
efficiency (LE) values of the guanidinophenylisoxazole derivatives were calculated
(Table 2). Most of the compounds had similar LE values ranging from 0.22 to 0.28.
Compound B6 showed the best LE value (0.28) and o deserved to be further
optimized.
Molecular docking studies revealed that the general conformation of compound B6
was different from that of the phenylisoxazole inhibitor A2 (Fig. 2C). It mainly
interacted with the P1 and G pocket, whose conformation was directed by the
hydrogen bonding interaction between the guanidyl group and acrosin. As expected,
the guanidyl group was located deep into the P1 pocket and formed five hydrogen
bonds with Tyr256, Trp243, Thr216, and Val225, respectively. Moreover, cation-π
interaction was also observed between the guanidyl group and Tyr256 (Fig. 3). The
7

dimethoxylphenyl group formed hydrophobic interactions with Val245 and cation-π
interaction with Arg248.
In summary, structure-based drug design was successfully applied to optimize the
isoxazole human acrosin inhibitor ISO-1. Most of the target compounds showed
significantly improved human acrosin inhibitory activities. After two round of
inhibitor design, a highly potent guanidinophenylisoxazole inhibitor B6 (IC50 = 1.44
µM) with good selectivity toward trypsin was identified. Molecular docking studies
revealed that it formed hydrogen bonding, hydrophobic and cation-π interactions with
the active site of human acrosin. Moreover, compound B6 has suitable
physicochemical properties [17], showing its potential as an orally active agent.
Thus, compound B6 represented a promising lead to develop novel male
contraceptives. Further structural optimization studies are in progress.
Supplementary Material
Experimental Protocols and Structural Characterization of Target Compounds.
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).
References and Notes
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8

contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian
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526 (2013) 75-79.
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structure of mammalian sperm beta-acrosin, Structure, 8 (2000) 1179-1188.
[5] R. Pakzad, [The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on
the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro], Z
Mikrosk Anat Forsch, 103 (1989) 8-13.
[6] L.R. Fraser, p-Aminobenzamidine, an acrosin inhibitor, inhibits mouse sperm
penetration of the zona pellucida but not the acrosome reaction, J Reprod Fertil, 65
(1982) 185-194.
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Human Acrosin Active Site and Binding Modes with Its Inhibitors, Chem J Chinese U,
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9

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activities
of
substituted
ethyl
5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives, Bioorg Med Chem Lett,
21 (2011) 5822-5825.
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Lv, C. Sheng, Design and synthesis of novel benzoheterocyclic derivatives as human
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acid amide derivatives, Bioorg Med Chem Lett, 23 (2013) 4177-4184.
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[16] S.D. Disocovery studio 2.5 software package AI, CA (USA):
http://www.accelrys.com.
10

[17] Calculated physicochemical properties of compound B6: molecular weight = 381,
number of hydrogen bond donors = 4, number of hydrogen bond donors = 9, LogP =
2.03, tPSA = 131.05.
11

Figure Legends
Figure 1. Chemical structures of known human acrosin inhibitors.
Figure 2. The binding modes of compounds ISO-1 (A), A2 (B), and B6 (C) with the
active site of human acrosin.
Figure 3. Schematic representation of the proposed interactions between inhibitor B6
and the active site of human acrosin.
12

Figure 1. Chemical structures of known human acrosin inhibitors.
13

(A)
(B)
14

(C)
Figure 2. The binding modes of compounds ISO-1 (A), A2 (B), and B6 (C) with the
active site of human acrosin.
15

Figure 3. Schematic representation of the proposed interactions between inhibitor B6
and the active site of human acrosin.
16

Table 1. Chemical structures and human acrosin inhibitory activities of the
phenylisoxazole derivatives
Compds
A1
R
IC50 (µM) Compds
R
IC50 (µM)
460.0
240.0
A4
60.0
380.0
390.0
A2
A3
A5
140.0
ISO-1
17

Table 2. Chemical structures, human acrosin inhibitory activities (IC50, µM) and
ligand efficiency of the guanidinophenylisoxazole derivatives
LEa
LEa
Compds
B1
R
IC50
Compds
B14
R
IC50
>100
14.4
0.26
0.26
>100
24.6
0.25
0.26
6.4
0.26
0.23
B2
B3
B15
B16
20.3
B4
B5
21.4
2.3
0.25
0.27
B17
B18
26.8
8.4
0.25
0.27
1.4
9.3
6.6
B6
B7
0.28
0.24
B19
B20
0.27
0.25
27.9
7.8
10.1
5.7
0.25
0.27
0.27
29.9
9.4
0.22
0.27
0.24
B8
B9
B21
B22
B23
CH3
23.9
B10
18

25.4
12.1
14.4
11.5
20.5
B11
B12
B13
0.25
0.26
0.26
B24
B25
0.26
0.26
390.0
ISO-1
a LE = (-∆G / HAC ≈ -RT ln(IC50) / HAC), HAC = heavy atom count.
19

Scheme 1. Reagents and conditions: (a) (COOC2H5)2, C2H5ONa, rt, 24 h, yield 90.6%;
(b) H2NOH⋅HCl, EtOH, reflux, 4 h yield 81.9%; (c) SnCl2⋅2H2O, EtOAc, reflux, 6 h,
yield 81.9%; (d) Acyl chlorides, Et3N, CH3CO2C2H5, yields 42.0%~81.0%.
20

Scheme 2. Reagents and conditions: (a) H2NCN, HCl, EtOH, reflux, 8h, yield 83.9%;
(b) NaOH, CH3OH/H2O (1:1), reflux, 2 h, yield 92.2%; (c) ROH or RNH2, EDC⋅HCl,
HOBt, rt, 48h, yields 43.0%~90.0%
21

Graphical Abstract
Discovery of Novel Phenylisoxazole Human Acrosin Inhibitors by
Structure-based Drug Design
Juntao Zhao §, Wei Tian§, Jingjing Qi §, Diya Lv, Yang Liu, Yan Jiang, Guoqiang Dong,
Qianqian Chen, Youjun Zhou, Ju Zhu, Heling Wang, Chunquan Sheng* and Jiaguo Lv*
A potent human acrosin inhibitor B6 (IC50 = 1.44 M) was identified by two-round of
structure-based drug design.