Synthesis, biological evaluation, and molecular docking studies of N,1,3-triphenyl-1H-pyrazole-4-carboxamide derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2012.04.066

A series of N,1,3-triphenyl-1H-pyrazole-4-carboxamide derivatives have been designed, synthesized and evaluated for their potential antiproliferation activity and Aurora-A kinase inhibitory activity. Among all the compounds, compound 10e possessed the most potent biological activity against HCT116 and MCF-7 cell lines with IC₅₀ values of 0.39 ± 0.06 μM and 0.46 ± 0.04 μM, respectively, which were comparable to the positive control. Compound 10e also exhibited significant Aurora-A kinase inhibitory activity (IC₅₀ = 0.16 ± 0.03 μM). Docking simulation was performed to position compound 10e into the active site of Aurora-A kinase, in order to get the probable binding model for further study. The results of Western-blot assay demonstrated that compound 10e possessed good Aurora-A kinase inhibitory activity against HCT116. Based on the preliminary results, it is deduced that compound 10e with potent Aurora-A kinase inhibitory activity may be a potential anticancer agent.

Full text of DOI:10.1016/j.bmcl.2012.04.066

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3589–3593
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, and molecular docking studies of
N,1,3-triphenyl-1H-pyrazole-4-carboxamide derivatives as anticancer agents
Xi Li a, , Xiang Lu a, , Man Xing a, Xian-Hui Yang a, Ting-Ting Zhao a, Hai-Bin Gong b, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N,1,3-triphenyl-1H-pyrazole-4-carboxamide derivatives have been designed, synthesized and
evaluated for their potential antiproliferation activity and Aurora-A kinase inhibitory activity. Among all
the compounds, compound 10e possessed the most potent biological activity against HCT116 and MCF-7
Received 5 March 2012
Revised 30 March 2012
Accepted 13 April 2012
Available online 21 April 2012
cell lines with IC50 values of 0.39 0.06
the positive control. Compound 10e also exhibited significant Aurora-A kinase inhibitory activity
(IC50 = 0.16 0.03 M). Docking simulation was performed to position compound 10e into the active site
lM and 0.46 0.04 lM, respectively, which were comparable to
l
Keywords:
N,1,3-triphenyl-1H-pyrazole-4-
carboxamide
Aurora-A kinase
Structure–activity relationship
Molecular docking
of Aurora-A kinase, in order to get the probable binding model for further study. The results of Western-
blot assay demonstrated that compound 10e possessed good Aurora-A kinase inhibitory activity against
HCT116. Based on the preliminary results, it is deduced that compound 10e with potent Aurora-A kinase
inhibitory activity may be a potential anticancer agent.
Ó 2012 Elsevier Ltd. All rights reserved.
Cancer is such a class of diseases that a group of cells display
uncontrolled growth, invasion, and sometimes metastasis.1,2 Strat-
egies to block cell division by affecting the mitotic spindle have
been a successful area of research for the advancement of cancer
drugs for a long time.3,4
(B) contains a core of pyrrolo[2,3-b] pyrimidine; (C) contains a
core of quinoline; and (D) contains a core of 2-anilino-diamino-
pyrimidine.16 Given the above information we have designed a
series of new compounds by sharing a similar template with scaf-
fold B and C, with the presumption that the pyrazole group is a
common nucleus of Aurora-A kinase inhibitors which inhibits
Aurora-A kinase by binding to its ATP side.17On the other hand,
benzamide is an important pharmacophore of natural products
and the synthetic precursors to various drugs. A variety of phar-
macological activities of benzamide have been reported, including
anticancer, anti-inflammatory and anti-fungal activities.18,19 Gi-
ven the previous reports, the phenyl group and carboxide group
of benzamide template can also closely bind to the ATP side of
Aurora-A kinase through the synergistic effect of hydrophobic
The Aurora kinases are a family of highly conserved serine/thre-
onine protein kinases that play a key role in regulating many pivotal
processes of mitosis and completion of cell division.5–9 In mammals,
three Aurora kinases are found: Aurora-A, Aurora-B and Aurora-C.10
Aurora A and B, are well known for their distinct roles in regulating
mitosis, but the role of Aurora C is still unclear. Aurora A is involved
in centrosome maturation and separation, bipolar spindle assembly,
and mitotic entry, while Aurora B is essential for accurate chromo-
some segregation and cytokinesis.11 Overexpression of Aurora-A ki-
nase contributes to genetic instability and tumorigenesis by
disrupting the proper assembly of the mitotic checkpoint complex.
Since its discovery, Aurora-A kinase has been identified as a colon-
cancer-associated kinase that is overexpressed in a wide range of
human tumors such as breast, colorectal, ovarian, as well as gli-
oma.12–14 Thus, targeted inhibition of Aurora-A kinase has become
an attractive therapeutic strategy in cancer therapy, and more than
10 Aurora inhibitors have entered early clinical assessment.15
The scaffolds of the known inhibitors of Aurora-A kinase can
be divided into four main groups labeled A–D, as shown in Figure
1: (A) contains a core of 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole;
interaction and p
-cation interaction.20 On that basis, a benzamide
moiety was introduced into the pyrazole moiety to construct a N-
phenyl-1H-pyrazole-4-carboxamide template with the reasoning
that this template could react potently with the residues in Aur-
ora-A kinase domain through the hydrophobic interactions. To
sum up, in continuation to extend our research on antitumor
compounds with Aurora-A kinase inhibitory activity, it is sup-
posed to be worthful to synthesize a series of N,1,3-triphenyl-
1H-pyrazole-4-carboxamide derivates as a novel class of Aurora-A
kinase inhibitors combined with pyrazole and benzamide consid-
ering that they might exhibit synergistic effect in anticancer
activities by the inhibition against Aurora-A kinase. Given the
suggestions, we directed our research to evaluate their anticancer
activities in HCT116 to further study the preliminary mechanism
of their role in mitosis.
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: ghbxzh@yahoo.com.cn (H.-B. Gong), zhuhl@nju.edu.cn (H.-L.
Zhu).
These two authors equally contributed to this paper.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.066

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X. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3589–3593
Figure 1. Four main types of scaffolds found in reported ATP competitive inhibitors of Aurora-A kinase and the potent Aurora-A kinase inhibitors under clinical study. (a)
Four main types of scaffold found in reported ATP competitive inhibitors of Aurora-A kinase. (A) 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold; (B) pyrrolo[2,3-b]
pyrimidine scaffold; (C) quinoline scaffold; (D) 2-anilino-diaminopyrimidine scaffold. Here, the groups R, R1 and R2 are variable. (b) Examples of potent Aurora-A kinase
inhibitors current under clinical study.
Scheme 1. General synthesis of N,1,3-triphenyl-1H-pyrazole-4-carboxamide derivatives (6a–10e). Reagents and conditions: (i) ethanol, 50–60 °C, 3 h; (ii) DMF, POCl3, 50–
60 °C, 5 h; (iii) NaClO2/NH2SO3, 40–50 °C, 5 h; (iv) DMAP, K2CO3, dichloromethane, 50–60 °C, 12 h.
The synthetic route of the N,1,3-triphenyl-1H-pyrazole-4-car-
boxamide derivatives 6a–10e is outlined in Scheme 1. As reported,
the synthesis of N,1,3-triphenyl-1H-pyrazole-4-carboxamide be-
gins with the interaction of substituted 1,3-diphenyl-1H-pyra-
zole-4-carboxylic and substituted anilines with the help of DMAP
and K2CO3 in anhydrous methylene dichloride to give the desired
compounds.21 Among these compounds, 7e, 8e, 9b, 9e and 10a–
10e are reported for the first time. All of the synthetic compounds
(Table 1) gave satisfactory elementary analytical and spectroscopic
data. 1H NMR and ESI-MS spectra were consistent with the as-
signed structures.
Table 2. As illustrated in Table 2, the active analogs showed a
remarkable potential antitumor activity. Particularly, it should be
noticed that compound 10e showed the most potent biological
activity (IC50 = 0.39 0.06
for MCF-7), comparable to the positive control VX-680 (IC50
0.30 0.03 M for HCT116 and IC50 = 0.38 0.02 M for MCF-7
cells respectively).
lM for HCT116 and IC50 = 0.46 0.04 lM
=
l
l
From the results listed in Table 2 we can conclude that the
activity of the tested compounds may be correlated to structure
variation and modifications. By investigating the variation in selec-
tivity of the tested compounds over two cell lines, it was obviously
revealed that different substitutes on the B-ring led to different
antitumor activities, a comparison of the para substituents on
the B-ring demonstrated that an electron-donating group have
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 6a–10e
against HCT116 and MCF-7 cells. The results were summarized in

X. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3589–3593
3591
Table 1
Structure of compounds 6a–10e
R1
O
A
R2
B
N
H
Figure 2. Compound 10e was examined by Western blotting. Data are represen-
tative of three independent experiments.
N
N
could also affect the activities of these compounds. Among the
compounds, the compounds with NO2 and Cl group as electron-
withdrawing substituents on ring A are of better antitumor activity
comparing to those with electron-donating substituents, quite dif-
ferent from ring B, the potency of para-substituents on the A-ring is
ordered as: NO2 > Cl > H > Me > MeO, followed that 8c showed the
lowest activity. Among all the compounds, 10e with para-NO2
group on the A-ring and para-OEt on the B-ring respectively, led
to a noteworthy best activity.
To examine whether the compounds inhibit Aurora-A kinase,
we screened compounds 6a–10e against the Aurora-A kinase.
The results were summarized in Table 2. Most of the tested com-
pounds displayed potent Aurora-A kinase inhibitory. Among them,
compounds 10e showed the most potent inhibitory with IC50 of
Compound
R1
R2
Compound
R1
R2
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
H
H
H
H
H
Cl
Br
OMe
OEt
H
Cl
Br
OMe
OEt
H
Cl
Br
8d
8e
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
OMe
OMe
Cl
Cl
Cl
OMe
OEt
H
Cl
Br
OMe
OEt
H
Cl
Br
H
Me
Me
Me
Me
Me
OMe
OMe
OMe
Cl
Cl
NO2
NO2
NO2
NO2
NO2
0.16 0.03
0.13 0.01
l
l
M (the positive control VX-680 with an IC50 of
M for Aurora-A kinase). The results of Aurora-A kinase
OMe
OEt
inhibitory activity of the tested compounds corresponded to the
structure relationships (SAR) of their antitumor activities. This
demonstrated that the potent antitumor activities of the synthetic
compounds were probably correlated to their Aurora-A kinase
inhibitory activities.
Table 2
In an effort to study the preliminary mechanism of the com-
pound with potent inhibitory activity, Western-blot experiment
was performed to assay the effect of compound 10e. The
Western-blot results are summarized in Figure 2. The results
indicated that compound 10e showed excellent Aurora-A kinase
inhibitory activity against HCT116.
To gain better understanding of the potency of the studied com-
pounds and guide further SAR studies, we proceeded to examine
the interaction of compound 10e with Aurora A crystal structure
(2BMC.pdb). The molecular docking was performed by inserting
compound 10e into ATP binding site of Aurora-A kinase. All dock-
ing runs were applied in LigandFit Dock protocol of Discovery Stu-
dio 3.1.
Inhibition (IC50) of HCT116 and MCF-7 cells proliferation and inhibition of Aurora-A
by compounds 6a–10e
Compound
IC50 SD (
MCF-7a
lM)
HCT116a
Aurora-A
b
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
VX-680
2.31 0.14
3.52 0.33
4.47 0.45
1.35 0.07
0.62 0.03
2.36 0.14
3.88 0.16
5.35 0.27
1.45 0.18
0.78 0.04
2.54 0.20
4.06 0.38
5.87 0.42
1.52 0.09
0.97 0.10
2.18 0.15
2.99 0.32
4.36 0.42
1.33 0.09
0.42 0.08
2.01 0.11
2.92 0.26
4.08 0.31
1.02 0.12
0.39 0.06
0.30 0.03
2.34 0.17
3.86 0.21
5.75 0.32
1.71 0.08
0.88 0.04
2.45 0.09
4.01 0.10
6.08 0.24
1.62 0.04
1.03 0.11
2.53 0.19
4.35 0.47
8.21 0.35
1.77 0.15
1.10 0.08
2.34 0.27
3.75 0.38
5.31 0.50
1.55 0.27
0.56 0.06
2.26 0.26
3.24 0.13
5.30 0.62
1.21 0.05
0.46 0.04
0.38 0.02
2.34 0.36
3.57 0.35
7.25 0.81
1.33 0.17
0.42 0.12
2.65 0.29
4.79 0.83
7.32 0.69
1.45 0.05
0.65 0.09
2.78 0.14
5.59 0.37
12.37 0.85
1.62 0.11
0.78 0.13
2.10 0.13
3.18 0.17
5.71 0.52
0.84 0.10
0.22 0.08
2.10 0.15
3.04 0.48
5.52 0.55
0.79 0.07
0.16 0.03
0.13 0.01
The binding modes of compound 10e and Aurora-A kinase are
depicted in Figure 3. All the amino acid residues which had inter-
actions with Aurora-A kinase are exhibited in Figure 4. In the bind-
ing mode, compound 10e is potently bound to the ATP binding site
of Aurora-A kinase via hydrophobic interactions and binding is sta-
bilized by two hydrogen bonds and one
p-cation interaction. The
oxygen atom of the OEt group formed one hydrogen bond with
the amino hydrogen of Arg F:137 (bond length: 1.79 Å; bond angle:
153.9°) and the hydrogen atom of the amide bond formed another
with the carboxylic oxygen of Leu F:139 (bond length: 2.28 Å; bond
angle: 129.6°). The enzyme surface model was showed in Figure
3b, which revealed that the molecule was well embedded in the ac-
tive pocket. This molecular docking result, along with the biologi-
cal assay data, suggested that compound 10e was a potential
inhibitor of Aurora-A kinase.
a
Inhibition of the growth of tumor cell lines.
Inhibition of Aurora-A kinase.
b
In conclusion, a series of N,1,3-triphenyl-1H-pyrazole-4-car-
boxamide derivatives have been synthesized and evaluated for
their antitumor activities. These compounds exhibited potent
antiproliferative activities against HTC116, MCF-7 cells and
Aurora-A kinase inhibitory activities. Among all of the com-
pounds, 10e showed the most potent inhibition activity which
inhibited the growth of HTC116 and MCF-7 cell lines with IC50
improved antiproliferative activity, and the potency order is OE-
t>OMe, whereas Cl group substituent and Br group substituent
had minor effects, which showed even lower activity compared
with those without substituents on the B-ring. In the case of con-
stant B ring substituents, change of substituents on the A-ring

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X. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3589–3593
Figure 3. (a) Compound 10e (colored by atom: carbons: gray; nitrogen: blue; oxygen: red) is bond into Aurora-A (entry 2BMC in the Protein Data Bank). The dotted lines
show the hydrogen bond and the yellow line show the
p-cation interaction interaction. Arg F:137 is the mean of Arg 137 in F chain. (b) The surface model structure of
compound 10e binding model with Aurora-A complex.
Figure 4. 2D Ligand interaction diagram of compound 10e with Aurora-A using Discovery Studio program with the essential amino acid residues at the binding site are
tagged in circles. The purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions and the green circles show the amino
acids which participate in the Van der Waals interactions.
values of 0.39 0.06
inhibited the Aurora-A kinase with IC50 of 0.16 0.03
was comparable to the positive control VX680(IC50 of 0.13
0.01 M). Molecular docking was further performed to study
the inhibitor- Aurora-A kinase interactions. After analysis of the
binding model of compound 10e with Aurora-A kinase, it was
found that compound 10e was potently bound to the Aurora-A
l
M and 0.46 0.04
l
M respectively and
Acknowledgments
lM, which
This work was supported by The Public Benefits Project
sponsored by Ministry of Environmental Protection of PRC
(201009023), Jiangsu Provincial Fund for Science and technology
support projects (BE2011355), and Natural Science Foundation of
Jiangsu Province (BK2009234).
l
kinase with two hydrogen bonds and a
p-cation interaction.
Western-blot results also showed compound 10e was a potential
antitumor agent. The information of this work might be helpful
for the design and synthesis of a leading compound 10e toward
the development of new therapeutic agent to fight against
cancer.
References and notes
1. Workman, P.; Kaye, S. Trends Mol. Med. 2002, 8, 1.
2. Chabner, B. A.; Roberts, T. G. Nat. Rev. Cancer 2005, 5, 65.
3. Honore, S.; Pasquier, E.; Braguer, D. Cell. Mol. Life Sci. 2005, 62, 3039.

X. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3589–3593
3593
4. Pellegrini, F.; Budman, D. R. Cancer Invest. 2005, 23, 264.
12. Coumar, M. S.; Leou, J.-S.; Wu, J.-S. J. Med. Chem. 2009, 52, 1050.
13. Zhong, M.; Bui, M.; Shen, W. Med. Chem. Lett. 2009, 17, 5158.
5. Ando, R.; Ikegami, H.; Sakiyama, M.; Ooike, S.; Hayashi, M.; Fujino, Y.; Abe, D.;
Nakamura, H.; Mishina, T.; Kato, H.; Iwase, Y. Bioorg. Med. Chem. Lett. 2010, 20,
4709.
6. Cee, V. J.; Schenkel, L. B.; Hodous, B. L. J. Med. Chem. 2010, 53, 6368.
7. Kishore, A. H.; Vedamurthy, B. M.; Mantelingu, K. J. Med. Chem. 2008, 51, 792.
8. Rawson, T. E.; Rüth, M.; Blackwood, El.; Burdick, D. J. Med. Chem. 2008, 51, 4465.
9. Coumar, M. S.; Chu, C. Y.; Lin, C. W. J. Med. Chem. 2010, 53, 4980.
10. Bischloff, J. R.; Plowman, G. D. Trends in Cell BioloEy. 1999, 9, 454.
11. Coumar, M. S.; Shukla, P. J. Med. Chem. 2010, 53, 4980.
14. Moriarty, K. J.; Koblish, H. K.; Garrabrant, T. J. Med. Chem. Lett. 2006, 16, 5778.
15. Howard, S.; Berdini, V.; Boulstridge, J. A. J. Med. Chem. 2009, 52, 379.
16. Aixia, Y.; Liyu, W.; Shuyu, X. Drug Discovery Today. 2011, 16, 5.
17. Daniele, F.; Daniela, B.; Simona, B. J. Med. Chem. 2005, 48, 3080.
18. Mitra, S. K.; Hanson, D. A.; Schlaepfer, D. Mol. Cell. Biol. 2005, 6, 56.
19. Valenti, P.; Lodi, L. Acta Oncol. 1987, 05, 149.
20. Fancelli, D. J. Med. Chem. 2005, 49, 4247.
21. Prakash, O.; Pannu, K.; Kumar, A. Molecules 2006, 11, 43.