Discovery and structural optimization of pyrazole derivatives as novel inhibitors of Cdc25B

Article abstract of DOI:10.1016/j.bmcl.2010.03.040

Structural optimization and preliminary structure-activity relationship studies of a series of N-substituted maleimide fused-pyrazole analogues with Cdc25B inhibitory activity, starting from a high-throughput screening hit, are illustrated. A simplified 3,5-diacyl pyrazole analogue was obtained as the most potent compound (118, IC₅₀ = 0.12 μM) with a 270-fold increase in potency.

Full text of DOI:10.1016/j.bmcl.2010.03.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2876–2879
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and structural optimization of pyrazole derivatives as novel
inhibitors of Cdc25B
*
*
Hai-Jun Chen , Yong Liu , Li-Na Wang, Qiang Shen, Jia Li , Fa-Jun Nan
The National Center for Drugs Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural optimization and preliminary structure–activity relationship studies of a series of N-substi-
tuted maleimide fused-pyrazole analogues with Cdc25B inhibitory activity, starting from a high-through-
put screening hit, are illustrated. A simplified 3,5-diacyl pyrazole analogue was obtained as the most
Received 16 November 2009
Revised 30 January 2010
Accepted 6 March 2010
Available online 11 March 2010
potent compound (118, IC50 = 0.12
l
M) with a 270-fold increase in potency.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Structural optimization
Cdc25B inhibitor
Pyrazole derivatives
Cdc25 phosphatases are key regulators of the cell cycle.1 Three
Cdc25 homologues have been identified in humans—Cdc25A,
Cdc25B, and Cdc25C. The overexpression of Cdc25A and Cdc25B
has been reported in numerous human tumors,1–4 and the inhibi-
tion of Cdc25 phosphatases may represent a promising therapeutic
approach for treatment of cancer.5,6 So far, a number of small-mol-
ecule Cdc25 phosphatase inhibitors have been reported.7–22
Among these known series of Cdc25 inhibitors, the most potent
inhibitors reported to date are quinonoids, such as NSC6632847
and BN8268521 (Fig. 1), which have IC50 values in the high nano-
molar range. However, the redox properties of the quinones can
generate toxic oxygen species, which may be toxic to normal tis-
sues and thus limit their prospect for application.20 In addition,
many Cdc25 phosphatase inhibitors showed low selectivity against
other phosphatases. A novel structural scaffold and selective inhib-
itor activity are needed for antitumor application of these
inhibitors.
which contains a maleimide fused-pyrazole core, was identified
with moderate inhibitory activity of Cdc25B (IC50 = 32 M, Fig. 2).
l
To understand the structure–activity relationship and improve
the potency, we initially prepared a series of compounds with scaf-
fold I, as outlined in Figure 2, in which we changed the substituted
group R1 on the phenyl ring, R2 on the carbonyl group, and R3 on
the nitrogen of the pyrazole ring.
The general synthetic strategy23–26 of N-substituted maleimide
fused-pyrazole analogues is illustrated in Scheme 1. Starting from
O
O
O
H
N
Cl
N
S
O
N
N
N
N
H
O
Herein, we describe the discovery and structural optimization
of N-substituted maleimide fused-pyrazole analogues as a novel
series of Cdc25B inhibitors with submicromolar inhibitory potency
in vitro against Cdc25B. We also describe the discovery of more
simplified 3,5-diacylpyrazol analogues as more potent Cdc25B
inhibitors.
The original hit compound 1 was obtained by high-throughput
screening of an in-house compound library against a recombinant
Cdc25B expressed and purified in our laboratory. Compound 1,
NSC663284
BN82685
Figure 1. Known Cdc25B inhibitors.
O
H
O
R3
N
N
N
N
N
O2N
R1
N
R2
O
O
OEt
O
O
scaffold
1, IC50=32µM
* Corresponding authors. Tel./fax: +86 21 50800954.
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), fjnan@mail.shcnc.ac.cn (F.-J. Nan).
Authors contributed equally to this work.
Figure 2. The structure of the original hit compound 1 and new designed scaffold I.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.040

H.-J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2876–2879
2877
O
O
O
O
O
O
N2
O
O
H
N
H
N
R2
N
a,b
R1
N
N
d
R1
N
N
N
N
2a-2e
c
e
R1 NH2
R1
N
R2
R2
C17H35
O
O
O
O
30-69
3-29
70-109
110
Scheme 1. Reagents and conditions: (a) maleic anhydride, Et2O, rt; (b) NaOAc, Ac2O, 120 °C; (c) toluene, 110 °C; (d) MnO2, acetone, rt; (e) Cs2CO3, CH3I, acetone, rt.
primary amines, acylation with maleic anhydride yielded N-substi-
tuted maleimides 3–29, which were used as dipolarophile to carry
Table 2
Inhibitory activity for Cdc25B of compounds 86–109
out 1,3-dipolar cycloaddition reactions with a-diazo carbonyl com-
O
H
pounds 2a–e23,27 under refluxing conditions in toluene, and substi-
tuted dicyclo-pyrazolines compounds 30–69 were obtained.
Treatment of pyrazolines with MnO2 in acetone provided the de-
sired products 70–109 bearing structural variations in the R1 and
R2 positions.
N
R1
N
N
O
C17H35
O
86-109
For the initial 12 compounds prepared (compounds 70–81),
which have a different R1 group on the phenyl ring. As shown in
Table 1, these modifications led to the total loss of potency except
that the 4-Cl-phenyl-substituent compound (compound 76) dis-
played nearly the potency of a hit. Therefore, the 4-NO2-phenyl
substituent of compound 1 was retained. Compounds 82–85 were
synthesized by replacing R2. Interestingly, introducing a saturated
long carbon chain (compounds 83–85) improved the potency dra-
matically, and the 17-carbon chain substitution produced an 80-
Compds
R1
Cdc25B IC50 SD (lM)
86
87
88
89
90
91
92
93
94
95
96
97
98
3-NO2-Ph
2-F-Ph
3-F-Ph
1.11 0.20
0.45 0.04
1.47 0.29
0.54 0.20
0.24 0.04
0.17 0.01
0.25 0.07
1.01 0.02
0.96 0.03
0.52 0.12
0.54 0.22
0.23 0.01
2.13 0.37
2.60 0.95
4.33 1.09
0.41 0.16
0.49 0.20
0.32 0.10
1.15 0.25
0.73 0.32
2.08 0.44
0.34 0.08
2.92 1.50
0.31 0.06
4-F-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
3-MeO-Ph
4-MeO-Ph
Ph
fold increase in potency (compound 85, IC50 = 0.40 lM).
Encouraged by this result, we retained the saturated 17-carbon
chain on the right side of the molecule and diversified the R1 group
to synthesize compounds 86–109 using the same synthetic meth-
od as described in Scheme 1. The results of the biological activity
assay are presented in Table 2 and are summarized as follows.
(1) The compound that has no substituent on the phenyl ring
2-Me-Ph
3-Me-Ph
4-Me-Ph
99
100
101
102
103
104
105
106
107
108
109
2-MeOOC-Ph
4-MeOOC-Ph
2-CF3-Ph
3-Cl, 4-F-Ph
Bn
(compound 97, IC50 = 0.23 lM) was most potent (compounds 90,
91, and 92 showed similar potency). (2) Replacing the phenyl ring
with a benzyl or cyclohexyl substituent visibly decreased the po-
tency (compounds 105, 106, and 108). However, compounds 107
and 109, bearing substituted thiophene and naphthalene at R1,
maintained the inhibitory activity against Cdc25B.
The greatly reduced potency of compound 110 (Table 3), which
was synthesized by methylating compound 97 with iodomethane
in the presence of cesium carbonate (Scheme 1), suggests that
the N–H on the pyrazole ring is essential for the inhibitory activity
against Cdc25B.
4-F-Bn
2-MeOOC-3-thiophene
Cyclohexyl
1-Naphthalene
Hydrolysis of compounds 101 and 107 was next tried to intro-
duce carboxylic group on the aromatic ring, but these failed, prob-
ably because of the poor stability of the maleimide ring under base
conditions. To investigate the relationship between the maleimide
ring and Cdc25B inhibitory activity, the maleimide ring was
opened and substituted pyrazole compounds 115 and 116 were
synthesized using a similar method as described above (Scheme
2). As shown in Table 3, the potency of these two compounds de-
creased markedly.
Table 1
Inhibitory activity for Cdc25B of compounds 70–85
Compd
R1
R2
Cdc25B IC50 SD (lM)
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
3-NO2-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
3-Br-Ph
2-MeO-Ph
4-MeO-Ph
2-MeOOC-Ph
4-NO2-Ph
4-NO2-Ph
4-NO2-Ph
4-NO2-Ph
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
NA
NA
NA
NA
NA
NA
37.4 0.87
NA
NA
NA
NA
NA
NA
1.39 0.31
0.50 0.12
0.40 0.03
Interestingly, the corresponding acid 11728 and 11829 of com-
pounds 115 and 116 showed more potent inhibitory activity
(117, IC50 = 0.16 lM, 200-fold increase and 118, IC50 = 0.12 lM,
270-fold increase), indicating that introducing a carboxylic group
in the left side of the molecule improves the water solubility of
the two compounds and increases the potency against Cdc25B.
The most important compounds 117 and 118 present a novel
and simplified scaffold with more potent inhibitory activity than
the original maleimide fused-pyrazole scaffold. Further enzyme ki-
netic study shown that compound 118 binds reversely with
Cdc25B (Fig. 3) and inhibits in a mix-type mode (Fig. 4). These re-
sults should provide new ways to modify the structure further and
lead to new discoveries.
Ph
C9H19
C13H27
C17H35

2878
H.-J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2876–2879
Table 3
100.0
Inhibitory activity for Cdc25B for compounds 110, 115–118
90.0
80.0
70.0
60.0
50.0
40.0
Compd
Structure
Cdc25B IC50 SD (lM)
O
N
O
N
N
110
30.0 4.41
O
C17H35
O
H
N
N
H
N
0
1
2
3
4
5
115
116
117
118
5.08 0.38
3.36 0.62
0.16 0.01
0.12 0.02
Dialysis Time (h)
COOMe
C
17H35
Figure 3. Reversible binding of compound 118 with Cdc25B.30
O
COOMe
O
H
S
N
N
H
N
0.012
0.01
0.008
0.006
0.004
0.002
0
C17H35
O
O
H
N
N
N
H
COOH
C
17H35
O
COOH
O
H
S
N
N
H
N
C
17H35
-0.2
0
0.2
0.4
O
1/[S]
-0.002
-0.004
To study the selectivity of these novel Cdc25B inhibitors for
other phosphatases, we also tested the inhibitory activity of six
compounds against PTP1B, TCPTP, SHP1, SHP2, and PTP-LAR. The
results (Table 4) indicated higher selectivity for Cdc25B than for
SHP1, SHP2, and PTP-LAR; minor selectivity for TCPTP; and almost
no selectivity for PTP1B. The exception was N-substituted malei-
mide fused-pyrazole compounds 1 and 97, which showed moder-
ate selectivity for Cdc25B compared with PTP1B.
Figure 4. Characterization of compound 118 (0, 0.1, 0.2, 0.3, and 0.4 lM) inhibition.
mization of the series provided compounds 117 (IC50 = 0.16
and 118 (IC50 = 0.12 M), which show 200-fold and 270-fold po-
tency of the original hit compound 1 (IC50 = 32 M), respectively,
and improved physical properties. These compounds provide a
new starting point for further structural modification, which may
lead to new discoveries of inhibitory agents against the Cdc25
phosphatases.
lM)
l
l
In conclusion, novel pyrazole derivatives including N-substi-
tuted maleimide fused-pyrazole and 3,5-diacylpyrazole derivatives
were identified as Cdc25B inhibitors. Preliminary structural opti-
O
O
O
H
N
H
N
N2
O
C17H35
R1
N
H
N
R1
N
H
N
2e
b
c
a
R1 NH2
R1
N
H
C17H35
C17H35
O
O
111, 112
113, 114
115, 116
COOR
117:
115: R1=
R= Me
R= H
d
d
S
116: R1=
COOR R= Me
118:
R= H
Scheme 2. Reagents and conditions: (a) acryloyl chloride, Et3N, DCM; (b) benzene, 80 °C; (c) MnO2, acetone, rt; (d) LiOH, THF/H2O, 0 °C to rt.

H.-J. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2876–2879
2879
M)
Table 4
Inhibitory activity for other phosphatases compared with Cdc25B31
Compd
PTP1B IC50 SD (
lM)
TCPTP IC50 SD (
lM)
SHP1 IC50 SD (lM)
SHP2 IC50 SD (
lM)
PTP-LAR IC50 SD (
lM)
Cdc25B IC50 SD (l
1
97
115
116
117
118
NA
NA
NA
NA
NA
NA
10.6 1.24
9.79 0.89
NA
9.59 0.75
NA
NA
15.2 1.97
12.2 0.76
NA
NA
NA
NA
NA
NA
31.9 3.08
0.23 0.01
5.08 0.38
3.36 0.62
0.16 0.01
0.12 0.02
1.56 0.16
7.49 1.79
7.16 0.24
0.35 0.03
0.41 0.02
3.31 0.19
18.7 2.63
18.6 3.17
1.30 0.16
1.16 0.10
18. Huang, W.; Li, J.; Zhang, W.; Zhou, Y.; Xie, C.; Luo, Y.; Li, Y.; Wang, J.; Li, J.; Lu,
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This work was supported by The National Natural Science Foun-
dation of China (Grants 30725049, 30801406 and 30721005) and
National Science & Technology Major Project ‘Key New Drug Crea-
tion and Manufacturing Program’, China (No. 2009ZX09301-001).
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28. Characterization data of compound 117: 1H NMR (DMSO-d6, 300 MHz): d 14.40
(s, 1H), 12.40 (s, 1H), 8.76 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.64 (t,
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116.4, 109.2, 39.8, 31.1, 28.9, 28.7 (8C), 28.6, 28.5, 28.4, 23.5, 21.9, 13.7; MS
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1H), 8.13 (d, J = 5.1 Hz, 1H), 7.89 (d, J = 5.4 Hz, 1H), 7.52 (s, 1H), 2.94 (t,
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