4,5-diaryl-3-aminopyrazole derivatives as analogs of combretastatin A-4: Synthesis and biological evaluation

Article abstract of DOI:10.1002/ardp.201000069

A series of cis-restricted 4,5-diaryl-3-aminopyrazole derivatives were synthesized and tested for their cytotoxic activity in vitro against five human cancer cell lines (K562, ECA-109, A549, SMMC-7721, and PC-3). Compounds 5a, 5b, 5d, and 6b showed potent

Full text of DOI:10.1002/ardp.201000069

Arch. Pharm. Chem. Life Sci. 2011, 11, 279–286
279
Full Paper
4,5-Diaryl-3-aminopyrazole Derivatives as Analogs of
Combretastatin A-4: Synthesis and Biological Evaluation
Tao Liu1, Rong Cui1,2, Jing Chen1, Jun Zhang3, Qiaojun He3, Bo Yang3, and Yongzhou Hu1
1 ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, P.R. China
2 JinHua College of Vocation And Technology, Jinhua, P.R. China
3 Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, P.R. China
A series of cis-restricted 4,5-diaryl-3-aminopyrazole derivatives were synthesized and tested for their
cytotoxic activity in vitro against five human cancer cell lines (K562, ECA-109, A549, SMMC-7721, and
PC-3). Compounds 5a, 5b, 5d, and 6b showed potent cytotoxicity against all tested cell lines. Primary
mechanism research on compound 5a indicated that it was a potent inhibitor of tubulin
polymerization, arresting cell cycle in G2/M phase. The docking research showed the
conformation of 5a overlaps well with CA-4 in the crystallized protein complex, suggesting the
4,5-diaryl-3-aminopyrazoles were good mimics of CA-4.
Keywords: Antitumor agents / Combretastatin A-4 analogs / Cytotoxicity / 4,5-Diaryl-3-aminopyrazole derivatives
Received: May 16, 2010; Accepted: May 21, 2010
DOI 10.1002/ardp.201000069
Introduction
heterocyclic bridges such as imidazole, pyrazoline, and triazole
[6, 7, 19, 20] et al. have been introduced into CA-4 to replace the
double bond. In our previous investigations of novel tubulin
polymerization inhibitors as potential antitumor agents, two
series of cis-restricted analogs of CA-4 with imidazole-2-one [21]
(2, Fig. 1) and 5-aminoisoxazole [22] (3, Fig. 1) rings in place of
the cis-double bond were reported in recent years. Some of
these compounds demonstrate significant antitumor and/or
antitubulin activities. For instance, most of N1-acetyl-imidazole-
2-one analogs show more effective cytotoxicity against HL-60
cancer cells than the corresponding N-free derivatives, and a
representative compound exhibits excellent antitumor activi-
ties in vivo [21]. Some 5-aminoisoxazoles and N1-acetyl-5-amino-
isoxazoles are potent inhibitors of tubulin polymerization [22].
Taking these results into account, we describe herein the
design and synthesis of 4,5-diaryl-3-aminopyrazoles (5a–g)
and N1-acetyl-4,5-diaryl-3-aminopyrazoles (6a–e). In addition,
we report their in-vitro cytotoxic activities, and the antitubulin
activity of the most cytotoxic compound 5a.
Microtubules are cytoskeletal components and play impor-
tant roles in a number of cellular processes [1–4]. Therefore,
compounds that target microtubules have long been inves-
tigated as anticancer drugs [5–10]. Typically, such com-
pounds arrest cells, at least transiently, in the mitotic
phase of the cell cycle. Combretastatin A-4 (CA-4, 1, Fig. 1),
a natural product isolated from the bark of the South African
bush willow tree Combretum caffrum, exhibits strong antitu-
bulin activity by binding to tubulin at the colchicine binding
site [11, 12]. It has attracted much attention owing to its
rather simple structure and potent cytotoxicity as well as
vascular disrupting activities [13–15].
Extensive studies have been conducted to examine the
structure-activity relationships (SAR) of variously modified
CA-4 analogs. The 3,4,5-trimethoxyphenyl (ring A) and the cis-
orientation of the bridge between the two aryl rings are
fundamental for efficient binding to tubulin [16–18]. To
retain the cis-olefin configuration of CA-4, five-membered
Results and discussion
Chemistry
Correspondence: Yongzhou Hu, ZJU-ENS Joint Laboratory of Medicinal
Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, P.R. China.
E-mail: huyz@zju.edu.cn
Fax: 086-571-88208460
The synthetic route of designed compounds 4,5-diaryl-3-ami-
nopyrazole derivatives (5a–g, 6a–f) is illustrated in Scheme 1.
a-Cyanoketones 4a–f, synthesized according to literature [23],
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Arch. Pharm. Chem. Life Sci. 2011, 11, 279–286
Figure 1. Structures of CA-4 (1), imidazole-2-one analog (2), and 5-aminoisoxazole derivative (3).
were converted to the 4,5-diaryl-3-aminopyrazoles (5a–g) by
the treatment with hydrate hydrazine in refluxing ethanol
[24]. Then, 4,5-diaryl-3-aminopyrazoles (5a–e, g) were acety-
lated by acetic anhydride to provide the corresponding N-
acetyl-4,5-diaryl-3-aminopyrazoles (6a–f).
The cytotoxic activities of synthesized compounds (5a–g
and 6a–f) and the control are summarized in Table 1. As
shown in Table 1, in general, 4,5-diaryl-3-aminopyrazoles
(5a, 5b, 5d, and 5e) showed more potent activities than that
of N-acetylated 4,5-diaryl-3-aminopyrazoles (6a, 6b, 6d, and
6e) against most tested cancer cell lines, except compound 5c.
And most tested compounds were more active against K562
cell line as compared with other cancer cell lines, with IC50
values in low micromolar range (IC50 0.08-15.98 mM). In
addition, it revealed that compounds 5a (IC50 0.08–12.07 mM),
5b (IC50 0.49–15.20 mM), 5d (IC50 0.31–13.79 mM), and 6b
(IC50 2.15–36.71 mM) are more potent than other tested
compounds.
Pharmacology
The synthesized 4,5-diaryl-3-aminopyrazole derivatives 5a–g
and 6a–f were tested for their cytotoxic activities in vitro
against five human cancer cell lines including human
myeloid leukemia cells K562, human esophageal carcinoma
cells ECA-109, human non-small lung cancer cells A549,
human hepatocellular carcinoma cells SMMC-7721, and
human prostate carcinoma cells PC-3 by MTT assay [25]. CA-
4 was employed as the positive control. Cell cycle progression
was detected using flow cytometry, which was performed on
FACScan (BD Biosciences, San Jose, CA). In-vitro microtubule
polymerization assay was conducted in a 96-well plate accord-
ing to the kit instruction manual (Cytoskeleton, Cat.#BK011),
using the Fluorence Microplate reader.
A scrutiny of the MTT assay results showed that some 4,5-
diaryl-3-aminopyrazoles possess potent cytotoxic activities.
Compared the cytotoxicities of compounds 5a with 5e, and
5b with 5f, the former compounds showed more potent
activities than the latter ones, which demonstrated that
the 3,4,5-trimethoxyphenyl (ring A) located on 5-position
of pyrazole ring (near N1-position) promote cytotoxicities.
Scheme 1. Reagents and conditions: (a) NaH, THF, reflux, 5 h; (b) 50% NH2NH2 ꢀ H2O, conc. HCl, EtOH, reflux, 9 h; (c) Ac2O, pyridine,
r.t., 12 h.
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4,5-Diaryl-3-aminopyrazole Derivatives
281
Table 1. In vitro cytotoxicities of the synthesized compounds against five human cancer cell lines
Compd.
Cytotoxicity (IC50, mM)a
K562
ECA-109
A549
SMMC-7721
PC-3
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
0.08 ꢂ 0.04
0.49 ꢂ 0.12
3.97 ꢂ 0.67
0.31 ꢂ 0.16
ndb
12.01 ꢂ 2.45
1.56 ꢂ 0.37
>50
8.00 ꢂ 1.35
25.04 ꢂ 6.25
>50
1.38 ꢂ 0.94
8.38 ꢂ 1.38
22.39 ꢂ 4.25
6.03 ꢂ 2.51
40.38 ꢂ 3.21
10.04 ꢂ 2.84
>50
16.36 ꢂ 2.69
7.46 ꢂ 3.45
21.18 ꢂ 7.39
>50
12.07 ꢂ 2.66
15.20 ꢂ 2.10
>50
6.95 ꢂ 0.74
1.27 ꢂ 0.63
47.78 ꢂ 8.43
0.61 ꢂ 0.53
7.68 ꢂ 5.27
>50
13.79 ꢂ 5.24
ndb
>50
0.49 ꢂ 0.37
ndb
>50
ndb
>50
0.20 ꢂ 0.15
2.15 ꢂ 1.31
3.98 ꢂ 0.25
15.98 ꢂ 4.33
ndb
4.73 ꢂ 1.23
4.21 ꢂ 1.69
13.97 ꢂ 3.57
>50
>50
38.67 ꢂ 10.1
5.54 ꢂ 3.22
15.00 ꢂ 6.92
17.97 ꢂ 6.50
>50
36.71 ꢂ 3.64
24.68 ꢂ 8.65
>50
35.58 ꢂ 4.32
38.15 ꢂ 6.52
ndb
11.65 ꢂ 2.62
>50
4.50 ꢂ 1.63
>50
0.42 ꢂ 0.36
>50
3.22 ꢂ 0.36
50 ꢂ 0
CA-4
0.046 ꢂ 0.01
5.40 ꢂ 2.20
a
IC50 Values are means of three experiments.
nd: not determined.
b
The similar result was obtained when comparing the cyto-
toxic activity of 6a with 6e. Additionally, attempts were
conducted to explore the influences of several variations
on ring B while maintaining 3,4,5-trimethoxy phenyl group
(ring A) on 5-position of pyrazole moiety. We found that
compounds retaining 4-methoxy or 3,4-dimethoxy group
on ring B (i.e., 5a and 5b) show more potent cytotoxicities
against most cancer cell lines, compared to compound 5c
(3,4,5-trimethoxy group on ring B). The same reduction in
cytotoxicity was seen for N-acetyl-4,5-diaryl-3-aminopyrazoles
(6a, and 6b vs. 6c). Replacement of 3,4,5-trimethoxy phenyl
(ring B) of 5c with 4-chlorophenyl ring (5d) dramatically
increases cytotoxicity. But this result was not applicable to
N-acetyl-4,5-diaryl-3-aminopyrazoles (6c vs. 6d).
To investigate whether the cytotoxic activities of these
compounds were related to an interaction with the micro-
tubule system, compound 5a, the most promising compound
against A549 and SMMC-7721 cancer cell lines, was evaluated
for its inhibitory effects on tubulin polymerization. The
result showed that compound 5a (IC50 ¼ 2.4 mM) displays
similar tubulin polymerization inhibitory activity in com-
parison with CA-4 (IC50 ¼ 1.2 mM) (Table 2).
On the basis of our previous biological activity, we per-
formed the docking simulations on compound 5a in order to
investigate the binding pose of the pyrazoles to the colchi-
cines binding site of a,b-tubulin by using the reported high-
resolution crystal structure of tubulin–DAMAcolchicine com-
plex [26]. The results showed the docking conformation of 5a
overlaps well with CA-4 in the crystallized protein complex.
The trimethoxyphenyl moiety (ring A) and 4-methoxyphenyl
moiety (ring B) of pyrazole 5a is positioned in the hydrophobic
pocket between Alab250ꢁAlab316 and Vala181ꢁMetb259,
respectively. In particular, the trimethoxyphenyl moiety is
situated in close proximity to Cysb241, the oxygen atom of 4-
methoxy group of ring A forms a hydrogen bond with the
thiol group of Cysb241, which was also found in CA-4.
Besides, the hydrogen atom of NH in pyrazole ring forms
one hydrogen bond with the NH of Alab250 and the hydro-
gen atom of 3-NH2 forms another hydrogen bond with the NH
of Asna101 (Fig. 3). These results demonstrated that com-
pound 5a would lead to strong biological activity, and this
was confirmed by the experimental results.
Besides, compounds 5g and 6f, bearing a thiophene as ring
B, were devoid of cytotoxic activity, suggesting that substi-
tuted phenyl is required for the cytotoxic activity.
Owing to tubulin inhibitors could cause alteration of cell
cycle parameters, with preferential G2/M blockade, flow
cytometry analysis was performed to determine the effect
of compound 5a on cell cycle of A549 cells and SMMC-7721
cells (Fig. 2). The percentages of G2/M population were 98.6%
in 0.1 mM 5a-exposed A549 cells for 48h. While only
about 12.8% cells were in G2/M phase in control group.
Similarly, in 1.0 mM 5a treatment SMMC-7721 cells for
48 h, 80.5% cells were arrested in G2/M phase, 10.9%
cells were arrested in G0/G1 phase, and 8.6% cells were
arrested in S phase, while in control group, 6.3%, 69.1%,
and 24.6% cells were observed in G2/M, G0/G1, and S phase,
respectively. Taken together, these results demonstrated
that compound 5a could strongly accumulate cells in
G2/M phase.
In addition, docking studies of compound 6a had also been
performed. Compound 6a has a changeable conformation in
the colchicine binding site compared with 5a because of the
introduction of N1-acetyl substitution (Fig. 4). The RMSD value
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Arch. Pharm. Chem. Life Sci. 2011, 11, 279–286
Figure 2. Effect of 5a on the cell cycle of A549 cells and SMMC-7721 cells as determined by flow cytometry. (A), A549 cells were treated with
5a at the concentrations of 0 mM (control) and 0.1 mM for 48 h, (B), SMMC-7721 cells were treated with 5a at the concentrations of 0 mM
(control) and 1.0 mM for 48 h.
(heavy atom) between the conformation of 5a and 6a is
˚
1.06 A. Obviously, compound 6a only forms two hydrogen
bonds with tubulin, less than that of 5a (three hydrogen
bonds as shown in Fig. 3). So we deduced that the inhibitory
effects on tubulin polymerization of compound 6a may be
less potent than that of 5a.
Table 2. Inhibition of tublin polymerization by compound 5a and
CA-4
Compd.
IC50 (mM)a
5a
CA-4
2.4 ꢂ 0.4
1.2 ꢂ 0.2
Figure 3. Docking pose of compound 5a and CA-4 in the colchicine
a
IC50 values are means of three experiments.
site of tubulin.
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4,5-Diaryl-3-aminopyrazole Derivatives
283
General procedure for synthesis of 4,5-diaryl-3-
aminopyrazoles (5a–g)
To a solution of a-cyanoketone 4 (1.4 mmol) in EtOH (10 mL),
conc. HCl (1 mL) was added. Then the mixture was heated
to reflux, and 50% hydrazine hydrate solution (0.4 mL,
4.1 mmol) was added dropwise into the reaction mixture.
The mixture was refluxed for 9 h. Then the solvent was
removed under reduced pressure. Water (10 mL) was added,
and the mixture was basified with aq. NaHCO3, and extracted
with EtOAc (3 ꢃ 20 mL). The organic phase was washed with
brine (2 ꢃ 20 mL), dried over anhydrous Na2SO4, and con-
centrated in vacuo. The residue was purified over silica col-
umn chromatography using petroleum ether (PE):EtOAc (1:2
to 1:3, v/v) as eluent to afford 5.
Figure 4. Docking pose of compound 5a and 6a in the colchicine
4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-amine (5a)
site of tubulin.
Yellow solid (58%), mp: 169–1718C. 1H-NMR (400 MHz, CDCl3):
d 3.61 (s, 6H, 2 OCH3), 3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
4.40 (br s, 2H, NH2), 6.58 (s, 2H, Ar-H), 6.90 (d, 2H, J ¼ 8.4 Hz,
Ar-H), 7.21 (d, 2H, J ¼ 8.4 Hz, Ar-H). 13C-NMR (100 MHz,
CDCl3): d 55.4, 56.0, 56.8, 104.2, 110.8, 115.2, 126.3, 128.5,
128.6, 140.1, 142.0, 150.4, 152.6, 162.3. HRMS calcd.
for C19H22N3O4 [M þ H] 356.1610. Found: 356.1608.
Conclusions
In summary, the synthesis and biological evaluation of a new
class of 4,5-diaryl-3-aminopyrazole derivatives as analogs
of CA-4 were described. The SAR analysis demonstrated that
the 3,4,5-trimethoxyphenyl (ring A) located on 5-position of
pyrazole ring (near N1-position) promotes cytotoxic activity
and incorporating an acetyl group at N1 of the pyrazole
ring is detrimental to cytotoxicity. The flow cytometry
analysis showed the most promising compound 5a causes
accumulation of cells in G2/M phase. Primary mechanism
research on 5a indicated its potential inhibitory effect
against microtubule, with IC50 value of 2.4 mM, comparable
with that of CA-4 (1.2 mM). Molecular modeling studies
revealed that compound 5a exhibits similar binding pose
as that of CA-4 in the crystallized protein complex,
suggesting the 4,5-diaryl-3-aminopyrazoles are good mimics
of CA-4.
4-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-amine (5b)
Yellow solid (65%), mp: 178–1808C. 1H-NMR (400 MHz, CDCl3):
d 3.62 (s, 6H, 2 OCH3), 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 4.68 (brs, 2H, NH2), 6.74 (s, 2H, Ar-H), 6.76 (s,
1H, Ar-H), 6.84 (d, 1H, J ¼ 8.0 Hz, Ar-H), 6.92 (d, 1H,
J ¼ 8.0 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3): d 56.0, 56.4,
104.9, 111.2, 112.6, 116.1, 121.3, 127.3, 130.1, 140.2, 141.3,
149.8, 150.0, 150.8, 152.7. HRMS calcd. for C20H24N3O5
[M þ H] 386.1716 Found: 386.1705.
4,5-Bis(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine (5c)
Yellow solid (71%), mp: 178–1808C. 1H-NMR (400 MHz, CDCl3):
d 3.83 (s, 6H, 2 OCH3), 3.84 (s, 6H, 2 OCH3), 3.85 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 4.98 (br s, 2H, NH2), 6.50 (s, 2H, Ar-H), 6.61
(s, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3): d 55.9, 56.4, 105.1,
111.7, 126.5, 130.6, 141.2, 143.3, 151.9, 153.4. HRMS calcd.
for C21H26N3O6 [M þ H] 416.1822. Found: 416.1820.
Experiments
Chemistry
Solvents were freshly distilled from Na/benzophenone (THF)
or KOH (pyridine). Reactions were carried out under N2,
unless otherwise stated. Melting points were obtained on a
B-540 Buchi melting-point apparatus and are uncorrected.
NMR spectra were recorded on a Bruker AM 400 instrument
(400 MHz and 100 MHz, for 1H and 13C, respectively).
ESI (positive) was recorded on an Esquire-LC-00075 spec-
trometer. HRMS were performed on a MAT-95 instrument.
The molecular model studies were performed using the
Discovery Studio 2.1.
4-(4-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-amine (5d)
White solid (68%), mp: 199–2008C. 1H-NMR (400 MHz, CDCl3):
d 3.70 (br s, 2H, NH2), 3.69 (s, 6H, 2 OCH3), 3.91 (s, 3H, OCH3),
7.24 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.32 (d, 2H, J ¼ 8.0 Hz, Ar-H),
7.35 (s, 2H, Ar-H), 7.36 (br s, 1H, pyrazole-NH). 13C-NMR
(100 MHz, CDCl3): d 56.0, 56.5, 104.6, 111.2, 127.0, 128.5,
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129.9, 131.5, 134.4, 140.0, 142.3, 151.6, 152.9. HRMS calcd.
(s, 3H, OCH3), 3.83 (s, 3H, OCH3), 5.54 (br s, 2H, NH2),
6.74 (s, 2H, Ar-H), 6.94 (d, 2H, J ¼ 8.8 Hz, Ar-H), 7.17
(d, 2H, J ¼ 8.8 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3):
for C18H19ClN3O3 [M þ H] 360.1115. Found: 360.1111.
5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-amine (5e)
d 24.7, 55.5, 56.0, 56.6, 103.9, 114.9, 116.2, 126.8,
128.7, 129.0, 138.7, 142.5, 150.0, 151.4, 158.9, 177.6.
HRMS calcd. for C21H24N3O5 [M þ H] 398.1716. Found:
398.1710.
White solid (58%), mp: 174–1768C. 1H-NMR (400 MHz, CDCl3):
d 3.63 (s, 6H, 2 OCH3), 3.68 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
6.02 (br s, 2H, NH2), 6.41 (s, 2H, Ar-H), 6.71 (d, 2H, J ¼ 8.4 Hz,
Ar-H), 7.26 (d, 2H, J ¼ 8.4 Hz, Ar-H). 13C-NMR (100 MHz,
CDCl3): d 55.9, 56.3, 103.1, 109.8, 114.8, 124.5, 128.0, 130.6,
139.0, 142.8, 150.4, 151.9, 163.6. HRMS calcd. for C19H22N3O4
[M þ H] 356.1610. Found: 356.1608.
1-(3-Amino-4-(3,4-dimethoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-pyrazol-1-yl) ethanone (6b)
Yellow solid (39%), mp: 162–1638C. 1H-NMR (400 MHz,
–
CDCl ): d 2.29 (s, 3H, O C-CH ), 3.63 (s, 6H, 2 OCH ),
3
–
3
3
3.72 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
5.57 (br s, 2H, NH2), 6.68 (s, 2H, Ar-H), 6.73 (d, 1H,
J ¼ 2.0 Hz, Ar-H), 6.80 (dd, 1H, J ¼ 2.0, 8.0 Hz, Ar-H),
6.86 (d, 1H, J ¼ 8.0 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3):
5-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-amine (5f)
White solid (63%), mp: 191–1938C. 1H-NMR (400 MHz, CDCl3):
d 3.68 (s, 3H, OCH3), 3.75 (s, 6H, 2 OCH3), 3.87 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 6.54 (s, 2H, Ar-H), 6.83 (d, 1H, J ¼ 8.0 Hz,
Ar-H), 6.88 (s, 1H, Ar-H), 7.56 (d, 1H, J ¼ 8.0 Hz, Ar-H). 13C-NMR
(100 MHz, CDCl3): d 56.0, 56.6, 104.1, 110.9, 113.4, 116.0,
121.1, 127.3, 130.6, 139.9, 141.3, 150.5, 151.0, 151.3, 154.0.
HRMS calcd. for C20H24N3O5 [M þ H] 386.1716. Found:
386.1710.
d
23.2, 56.0, 56.3, 105.1, 100.9, 112.3, 115.9, 120.9,
126.4, 128.9, 138.3, 140.8, 145.2, 148.7, 150.0, 152.1, 179.5.
HRMS calcd. for C22H26N3O6 [M þ H] 428.1822. Found:
428.1817.
1-(3-Amino-4,5-bis(3,4,5-trimethoxyphenyl)-1H-
pyrazol-1-yl)ethanone (6c)
Yellow solid (47%), mp: 160–1628C. 1H-NMR (400 MHz, CDCl3):
–
d 2.31 (s, 3H, O C–CH ), 3.69 (s, 6H, 2 OCH ), 3.70 (s, 6H, 2
–
3 3
4-(Thiophen-3-yl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-
3-amine (5g)
OCH3), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.37 (s, 2H, Ar-H),
6.58 (s, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3): d 23.7, 56.1, 56.4,
104.5, 104.8, 100.6, 128.1, 130.6, 139.4, 141.8, 148.3, 153.4,
177.9. HRMS calcd. for C23H28N3O7 [M þ H] 458.1927. Found:
458.1924.
Red solid (78%), mp: 130–1328C. 1H-NMR (400 MHz, CDCl3): d
3.68 (s, 6H, 2 OCH3), 3.84 (s, 3H, OCH3), 4.06 (br s, 2H, NH2),
6.60 (s, 2H, Ar-H), 7.00 (s, 1H, thiophene-H), 7.05 (s, 1H,
pyrazole-NH), 7.20–7.22 (m, 1H, thiophene-H), 7.23–7.24 (m,
1H, thiophene-H). 13C-NMR (100 MHz, CDCl3): d 56.0, 56.3,
100.7, 105.1, 122.3, 127.0, 128.9, 138.6, 139.9, 144.4, 152.5,
155.7. HRMS calcd. for C16H18N3O3S [M þ H] 332.1069.
Found: 332.1066.
1-(3-Amino-4-(4-chlorophenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-pyrazol-1-yl) ethanone (6d)
Yellow solid (38%), mp: 157–1588C. 1H-NMR (400 MHz, CDCl3):
–
d 2.84 (s, 3H, O C–CH ), 3.67 (s, 6H, 2 OCH ), 3.82 (s, 3H,
3
–
3
General procedure for synthesis of N-acetylated 4,5-diaryl-
3-aminopyrazoles (6a–e)
OCH3), 5.63 (br s, 2H, NH2), 6.66 (s, 2H, Ar–H), 7.19 (d, 2H,
J ¼ 8.4 Hz, Ar-H), 7.35 (d, 2H, J ¼ 8.4 Hz, Ar–H). 13C-NMR
(100 MHz, CDCl3): d 22.9, 56.0, 56.4, 100.1, 109.8, 127.0,
127.4, 130.1, 134.9, 135.2, 140.0, 142.1, 149.8, 153.6, 176.9.
HRMS calcd. for C20H21ClN3O4 [M þ H] 402.1221. Found:
402.1216.
4,5-Diaryl-3-aminopyrazoles 5 (2 mmol) was treated with ace-
tic anhydride (3 mmol) and pyridine (0.3 mL). The mixture
was stirred for 24 h at room temperature. Then, water
(10 mL) was added and the mixture was extracted with
EtOAc (3 ꢃ 15 mL). The organic phase was combined, washed
with aq. NaHCO3 and then brine (2 ꢃ 15 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue
was purified over silica column chromatography using PE/
EtOAc (2:1 to 1:1, v/v) as eluent to provide corresponding
acetylated compound 6.
1-(3-Amino-5-(4-methoxyphenyl)-4-(3,4,5-
trimethoxyphenyl)-1H-pyrazol-1-yl) ethanone (6e)
Yellow solid (42%), mp: 162–1648C. 1H-NMR (400 MHz,
–
CDCl ): d 2.28 (s, 3H, O C-CH ), 3.66 (s, 3H, OCH ), 3.75
3
–
3
3
(s, 6H, 2 OCH3), 3.80 (s, 3H, OCH3), 6.44 (s, 2H, Ar-H),
6.81 (d, 2H, J ¼ 8.8 Hz, Ar-H), 7.37 (d, 2H, J ¼ 8.8 Hz, Ar-H).
13C-NMR (100 MHz, CDCl3): d 23.4, 55.5, 56.0, 56.3, 104.0,
110.6, 115.1, 124.3, 128.8, 131.0, 138.4, 142.7, 149.0, 152.3,
161.2, 175.9. HRMS calcd. for C21H24N3O5 [M þ H] 398.1716.
Found: 398.1711.
1-(3-Amino-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-pyrazol-1-yl) ethanone (6a)
Yellow solid (45%), mp: 158–1608C. 1H-NMR (400 MHz,
–
CDCl ): d 2.75 (s, 3H, O C-CH ), 3.65 (s, 6H, 2 OCH ), 3.82
3
–
3
3
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Arch. Pharm. Chem. Life Sci. 2011, 11, 279–286
4,5-Diaryl-3-aminopyrazole Derivatives
285
1-(3-Amino-4-(thiophen-3-yl)-5-(3,4,5-trimethoxyphenyl)-
1H-pyrazol-1-yl) ethanone (6f)
Red solid (42%), mp: 148–1508C. 1H-NMR (400 MHz, CDCl3): d
on a FACS-Calibur flow cytometer (BD Biosciences, San Jose,
CA).
–
2.74 (s, 3H, O C-CH ), 3.70 (s, 6H, 2 OCH ), 3.83 (s, 3H, OCH ),
3
Molecular modeling [26]
–
3
3
5.62 (br s, 2H, NH2), 6.75 (s, 2H, Ar-H), 6.95 (s, 1H, thiophene-
H), 7.19–7.20 (m, 1H, thiophene-H), 7.37–7.39 (m, 1H, thio-
phene-H). 13C-NMR (100 MHz, CDCl3): d 23.5, 56.0, 56.4, 100.4,
104.6, 120.9, 127.4, 128.1, 128.5, 138.6, 139.8, 144.4, 150.7,
152.3, 175.9. HRMS calcd. for C18H20N3O4S [M þ H] 374.1175.
Found: 374.1171.
Tubulin–DAMAcolchicine crystal structure (PDB ID: 1SA0)
was chosen as a template. The functional a,b-chains of tubu-
lin were kept, polar hydrogens were added, and CHARMm
force field was employed. Binding sphere (45.4407, 41.1246,
34.6219, 9) was selected from the active site using the binding
site tools. For all tested compounds, hydrogens were added
and CHARMm force fields were employed. Each of the com-
pounds was minimized by Dreiding Minimize tool. CDOCKER
(Discovery Studio 2.1) was used for the docking simulation.
For each compound, the docking parameters were as follows:
Top Hits: 25; Random Conformations: 10; Random
Conformations Dynamics Steps: 1000; Grid Extension: 8.0;
Random Dynamics Time Step: 0.002. Final docked confor-
mations were scored by Calculated Cdocker-Energy and
Cdocker interaction Energy.
Pharmacology
Cytotoxicity assay [25]
The cancer cell lines (K562, ECA-109, A549, SMMC-7721, and
PC-3) were obtained from Cell Bank of China Science
Academy (Shanghai, China). The cytotoxic activity in vitro
was measured using the MTT assay. MTT solution (5.0 mg/mL
in RPMI, 1640 medium; Sigma-Aldrich) was added (10.0 mL/well)
after cells were treated with drug for 48 h, and cells were
incubated for a further 4 h at 378C. The purple formazan
crystals were dissolved in 100.0 mL DMSO. After 5 min, the
plates were read on an automated microplate spectropho-
tometer (Bio-Tek Instruments, Winooski, VT) at 570 nm.
Assays were performed in triplicate in three independent
experiments. The concentration required for 50% inhibition
of cell viability (IC50) was calculated using the software of
‘Dose-Effect Analysis with Microcomputers’. In all of these
experiments, three replicate wells were used to determine
each point.
The authors have declared no conflict of interest.
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