Design and synthesis of 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles and pyrazolo[3,4-b]pyridines for Aurora-A kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.083

Two series of 3-aminopyrazole compounds including 24 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles and 16 pyrazolo[3,4-b]pyridines were synthesized and evaluated against HCT116, A549, and A2780 tumor cell lines. Among them, three compounds were found to have the ideal anti-proliferative activities in vitro. Docking experiments showed that the novel pyrazolo[3,4-b]pyridines share the similar interaction mode with Aurora-A kinase as PHA739358.

Full text of DOI:10.1016/j.bmcl.2010.04.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4273–4278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles and
pyrazolo[3,4-b]pyridines for Aurora-A kinase inhibitors
Jianyou Shi a,b, , Guobin Xu a, , Wei Zhu a, Haoyu Ye a, Shengyong Yang a, Youfu Luo a, Jing Han a,
a
a,
*
Jincheng Yang a,b, Rui Li a,c, , Yuquan Wei , Lijuan Chen
*
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu,
Sichuan 610041, PR China
b Pharmacy College of West China of Sichuan University, Chengdu, Sichuan 610065, PR China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, 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 December 2009
Revised 8 April 2010
Accepted 20 April 2010
Available online 10 May 2010
Two series of 3-aminopyrazole compounds including 24 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles and
16 pyrazolo[3,4-b]pyridines were synthesized and evaluated against HCT116, A549, and A2780 tumor
cell lines. Among them, three compounds were found to have the ideal anti-proliferative activities in
vitro. Docking experiments showed that the novel pyrazolo[3,4-b]pyridines share the similar interaction
mode with Aurora-A kinase as PHA739358.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazoles
Pyrazolo[3,4-b]pyridines
Antitumor
Aurora-A kinase
A large number of 3-aminopyrazole derivatives have been stud-
ied in the treatment of proliferative diseases,1–9 such as cancer,
inflammation, and arthritis. 3-Aminopyrazole is an important
structure presented in a number of pharmaceutically compounds
(Fig. 1). For example, 3-amino-1,4,5,6-tetrahydropyrrolo[3,4-c]pyr-
azoles (2) have been discovered as a new class of CDK2 inhibitors
and are able to efficiently inhibit CDK2-mediated tumor cell prolif-
eration.10 The skeleton of pyrazolo[3,4-b]pyridine (3) has been
identified as potent inhibitors for glycogen synthase kinase-3
(GSK-3).11 In addition, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles
(1), reported by Daniele Fancelli, can inhibit the activity of Aur-
ora-A kinase.1,12 40-(3-(Thiazol-5-ylamino)-1H-pyrazol-5-yl)biphe-
nyl-2,4-diol (4), the Checkpoint 1 (G1) inhibitor, was proved as an
attractive compound to fight cancer.13
The 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles and pyrazolo-
[3,4-b]pyridines share the common nucleus as 3-aminopyrazole
moiety, and the former have been discovered as effective Aurora
kinase inhibitors. Recently, PHA739358 (1) has advanced into
phase II clinical trials for the treatment of cancer. In order to find
out more effective derivatives containing 3-aminopyrazole nu-
cleus, a new series of derivatives were designed and synthesized.
It was demonstrated that 14p, 14s, and 20i have the ideal inhibi-
Scheme 1. Preparation of 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles 14a–x.
Reagents and conditions: (a) MeOH, H2SO4,7 h, reflux; (b) di-tert-butyl dicarbonate,
DCM/aq NaHCO3 (1:1), 24 h, 22 °C; (c) MeONa, toluene, 3 h, 80 °C, then 2 N HCl; (d)
hydrazine hydrochloride, EtOH, 3 h, 60 °C, then aq NaHCO3; (e) EtCOOCl, THF, 20 h,
0–5 °C; (f) RCOCl, DIEA, THF, 12 h, 22 °C; (g) TFA/DCM (1:1) 10 equiv, 1 h, 22 °C; (h)
RCOCl, DIEA, THF, 6 h, 22 °C; (i) Et3N 10% in MeOH, 3–6 h, 22 °C.
* Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060 (L.C.).
E-mail addresses: Lirui@scu.edu.cn (R. Li), lijuan17@hotmail.com (L. Chen).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.083

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J. Shi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4273–4278
Figure 1. Aurora kinase inhibitor 1 (PHA739358) in clinical trials as anticancer treatment, CDK2 inhibitor 2, GSK-3 inhibitor 3 and G1 inhibitor 4.
Scheme 2. Preparation of pyrazolo[3,4-b]pyridines 20a–p. Reagents and conditions: (a) AcOH, 125 °C (100%); (b) Br2, AcOH (60%); (c) POCl3, PCl5, reflux (75%); (d) N2H4ꢀH2O,
EtOH, reflux (85%); (e) RCOCl, pyridine, rt (80%).
Table 1
Inhibition of cell proliferation by compounds 14a–x and 1
Compound
R1
R2
Cell line (IC50, lM)
HCT116
135
A549
A2780
>200
O
O
OH
O
O
O
O
14a
14b
84
O
O
O
>200
>200
>200
>200
>200
O
O
O
O
O
N
14c
14d
118
O
O
O
O
>200
>200
>200
>200
>200
O
O
O
O
O
O
O
O
O
14e
>200
O
O
O
N
14f
104
207
>200
72
>200
>200
O
O
O
O
14g
O

J. Shi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4273–4278
4275
Table 1 (continued)
Compound
R1
R2
Cell line (IC50
A549
, lM)
HCT116
>200
A2780
O
O
N
14h
14i
>200
99
>200
>200
O
O
O
O
O
O
O
O
103
236
143
107
O
O
O
O
O
N
O
O
O
14j
14k
14l
>200
>200
>200
>200
OH
O
76
N
O
161
O
14m
14n
168
26
175
>200
>200
N
O
O
O
O
O
>200
O
N
14o
14p
14q
>200
13
>200
42
>200
82
O
O
OH
O
O
O
O
>200
>200
>200
O
O
O
O
O
14r
14s
>200
4
>200
25
>200
35
OH
O
O
O
O
O
14t
>200
38
>200
16
>200
23
N
O
O
O
14u
(continued on next page)

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Table 1 (continued)
Compound
R1
R2
Cell line (IC50, lM)
HCT116
45
A549
A2780
54
O
O
O
O
O
14v
14w
14x
34
O
O
>200
>200
>200
>200
13
>200
43
>200
11
O
O
O
N
N
1 (Cont)
Cont, positive control.
tion activity on HCT116, A549, and A2780 cell lines. Meanwhile,
the interaction mode and SAR (structural and activity relationship)
of this kind of compounds were also concluded.
The target compounds 14a–x were synthesized according to the
literatures with minor revision (Scheme 1).1,2 Treatment of
2-(2-cyanoethylamino)acetic acid 5 with sulfuric acid and methanol
afforded methyl ester 6, which was protected with di-tert-butyl
dicarbonate to give 7. Reaction of 7 with sodium methoxide fur-
nished 4-oxo-pyrrolidine-3-carbonitriles 8, while cyclization of 8
to provide tetrahydropyrrolopyrazole 9 was accomplished by
treatment with hydrazine in ethanol. To obtain 10, ethyl chlorocar-
bonate was added to 9 in dry THF and DIEA under stirring at 0–5 °C.
The reaction was kept at the same temperature for 2 h, allowed to
reach roomtemperature, and stirredovernight. Acylationoftheami-
no group of 10 yield 11a. Then, the dihydropyrrole nitrogen of 11a
was unmasked with TFA to give the intermediate 12a. Next, acyl
chloride was added to 12a to acquire 13a. A solution of 13a in MeOH
and Et3N was stirred at 30 °C for 3 h to obtain 14a. Compounds 14b–
x were obtained by using the same method. The compounds 20a–p
were synthesized using a modified literature method (Scheme
2).11,12,14 2-Chloronicotinonitrile 15 was transformed into 2-
hydroxynicotinonitrile 16 by glacial acetic acid. Bromination of 16
by bromine in acetic acid afforded bromide 17. Then, treatment of
17withphosphorusoxychloride andphosphoruspentoxideat reflux
afforded the corresponding chloropyridine 18. Furthermore, 18 was
treated with hydrazine hydrate in ethanol at reflux to yield the pyr-
azolopyridine 19. Selective acylation of the C-3 amino group in pyr-
idine would afford the corresponding amides 20a–p.
With regard to 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles (Table
1), the structures of R2 of 14a and 14b are similar with PHA739358,
but their activities are lower than that of PHA739358 by one mag-
nitude, illustrating that the piperonylic acid moiety is not a good
substituent for R1 as Aurora inhibitor. The activity of 14c is also
low. Moreover 14d–f, with the benzyloxy group attached on the
3-position of piperonylic acid moiety, did not reach the ideal activ-
ity when varying different substituents of R2. It is demonstrated
that the R1 group should not be too large, otherwise they cannot
reach to the active site of Aurora-A successfully. When one or
two methoxy groups attached to the phenyl group of R1, the activ-
ities of compounds 14g–l improved but still lower than
PHA739358. When R1 substituent was tolyl or benzyl, the activity
of 14n and 14p improved greatly. However, 14m and 14o showed
Figure 2. The binding mode of 14s with Aurora-A kinase.
inactive, indicating that the methine of R2 on the benzene ring
played a key role in the conjugation of benzene ring with
P–P
HIS280. As for R1, apart from benzyl derivatives, furan and naph-
thalene substituents were also tested, and it was found that 14s
and 14u had the ideal activity. As for 14s, the formation of extra
hydrogen bonding between furan O atom and TYR212 (2.41 Å, ob-
tained by docking experiment) (Fig. 2) may contribute to the bind-
ing energy. Moreover, 14v–x bearing the same naphthalene group
were synthesized and found that 14v in which R2 substituent is
furan ring also showed good activity.
From the docking experiments by Autodock 4,15 it was found that
theinteraction ofpyrazolo[3,4-b]pyridinederivativeswithAurora-A
kinase is similar to that of PHA739358 (Fig. 3). The 3-aminopyrazole
group would form three hydrogen bonds with GlU211 and AlA213
residues. So the pyrazolo[3,4-b]pyridine derivatives should bear

J. Shi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4273–4278
4277
Table 2
Inhibition of cell proliferation by compounds 20a–p
Compound
R
Cell line (IC50
,
lM)
HCT116
A549
A2780
O
O
20a
20b
39.82
51.5
>200
>200
>200
>200
O
20c
20d
25.49
34.69
>200
51.4
49.90
51.56
O
O
Cl
20e
20f
20g
32.27
49.19
40.29
>200
>200
75.1
46.69
>200
>200
Cl
Cl
Cl
O
O
F3C
O
20h
20i
20j
21.88
44.1
>200
>200
>200
>200
50.03
>200
CF3
F
O
O
O
24.85
O
20k
20l
15.63
19.31
>200
53.7
27.01
37.86
O
O
O
O
O
O
20m
20n
43.48
31.7
61.9
42.00
22.82
Br
43.10
O
O
20o
20p
23.02
>200
>200
N
Figure 3. The binding mode of 20p with ATP pocket of Aurora-A kinase obtained by
molecular docking experiments (PDB code 2J4Z) (Fig. 3-1 and Fig. 3-2) and the
molecular overlay of 20p with PHA739358 (Fig. 3-3).
O
14.29
13
45.25
43
46.45
11
N
1 (Cont)
the similar anti-proliferative activity to PHA739358. Hence, a series
of its derivatives were synthesized (Table 2). As for tolyl group, the
activity of methyl in meta position (20c) is higher than para (20a)
and ortho (20b) in HCT116 cell line. In terms of chlorine atom, the
location and number have little effect on the activities (20d–f). As
for the electron-withdrawing groups CF3 and F, para position
showed good activities. With regard to the electron-donating groups
Cont, positive control.
such as methoxy, ortho position 20k is superior to para position 20j,
however 20k was invalid on A549 cell line. Furthermore, compound
20l having two methoxy groups substituted on the meta position

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J. Shi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4273–4278
2. Fancelli, D.; Berta, D.; Bindi, S.; Cameron, A.; Cappella, P.; Carpinelli, P.; Catana,
C.; Forte, B.; Giordano, P.; Giorgini, M. L.; Mantegani, S.; Marsiglio, A.; Meroni,
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activities in three kinds of cell lines. In addition, furan, pyridine,
and heterocyclic derivatives were also tested, and their activities
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had the best activity, which might be ascribed to the formation of
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nol hydroxyl moiety of TYR212 (2.675 Å) (Fig. 3) besides the com-
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reported by Fancelli et al.1 and Talele and McLaughlin.16
In conclusion, a series of new compounds bearing pyrazol-
o[3,4-b]pyridine scaffold, represented as a novel class of com-
pounds to inhibit the Aurora-A’s activity, were synthesized and
evaluated. They should interact with the Aurora-A kinases in
the similar mode to PHA739358. The ongoing work aimed to ex-
plore the efficacy of compounds 14p, 14s, and 20p in a range of
in vivo models, will be the subjects of future reports.
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The authors are grateful to the support of National Program of
China (2009ZX09501-015), the National Science Foundation of
China (30901743), and the State Key Laboratory of Drug Research.
10. Pevarello, P.; Fancelli, D.; Vulpetti, A.; Amici, R.; Villa, M.; Pittalàs, V.; _Vianello,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.083.
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