28-05-2014, 11:41 AM
Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives coupling with a thiourea moiety as novel CDKs inhibitors
Synthesis, biological evaluation.pdf (Size: 1.41 MB / Downloads: 24)
ABSTRACT
It was discovered that a number of cyclin dependent kinase inhibitors containing the pyrazole core
structure exhibited high inhibitory potency against broad-range CDKs and corresponding anti-
proliferative activities. This information guided us to design and synthesize a series of 1,3-diphenyl-N-
(phenylcarbamothioyl)-1H-pyrazole-4-carboxamide derivatives (5ae10d), and evaluate their biological
activities as CDKs inhibitors. Among all the synthesized compounds, compound 10b inhibited CDK2 with
an IC50 value of 25 nM, counteracting tumor cell proliferation of three cancer cell lines (H460, MCF-7,
A549) in the micromolar range (from 0.75 mM to 4.21 mM), In addition, flow cytometry indicated that
compound 10b could induce cycle G0/G1 phase arrest in A549 cells with a dose dependent. Taken
together, compound 10b could be selected for further preclinical evaluation.
Introduction
Along with the living habits and environment changes, cancer
has become the major cause of death in both developing and
developed countries [1]. Until now one significant way to induce
the occurrence of cancers is still by mutation or mis-regulation of
cell cycle regulatory genes and proteins to guide an abnormal
control of cell proliferation [2]. The cyclin-dependent kinases
(CDKs) are a family of serineethreonine protein kinases, which are
key regulatory elements in cell cycle progression. Inhibition of
CDKs activity has turned out to be the most effective strategy for
the discovery of novel anticancer agents specifically targeting the
cell cycle proteins [3]. To this end, we attempted to design and
synthesize new molecule inhibitors targeting these kinases and
related kinase-overexpression cancers.
Results and discussion
Chemistry
The synthetic route of the cyanic 1,3-diphenyl-1H-pyrazole-4-
carboxylic thioanhydride derivatives (6e10) was outlined in
Scheme 1 [3,21]. Compounds 6e10 were prepared by the simple
condensation and cyclization of phenylhydrazine and various
substituted acetophenones.
The synthetic route of 1,3-diphenyl-N-(phenylcarbamothioyl)-
1H-pyrazole-4-carboxamide derivatives (6ae10d) was shown in
Scheme 2 [21]. As reports, the synthesis of compounds (6ae10d)
began with the interaction of substituted cyanic 1,3-diphenyl-1H-
pyrazole-4-carboxylic thioanhydride and substituted anilines with
the help of K2CO3 in anhydrous methylene dichloride.
These compounds were all reported for the first time. All of the
synthesized compounds 6ae10d (Table 1) gave satisfactory
elementary analytical and spectroscopic data. 1H NMR, 13C NMR
and ESI-MS spectra were consistent with the assigned structures.
Additionally, the structure of compound 7a was further confirmed
by X-ray diffraction. Its crystal data were presented in Table 2 and
Fig. 2 gave a perspective view of this compound together with the
atomic labeling system.
Flow cytometry
Approximately 105 cells/well were plated in a 24 well plate and
allowed to adhere. After 12 h, the medium was replaced with fresh
culture medium containing compounds 10b at final concentrations
of 0.5, 1, 2 mM. Nontreated wells received an equivalent volume of
ethanol (<0.1%). After 36 h, Cells in the supernatant and adherent
cells were collected using 0.25% Trypsin, 0.02% EDTA. Cells were
washed with PBS and were fixed in 70% ethanol, centrifuged for
1 min at 3000 g at 4 C, washed once with PBS, treated with 1 mg/
mL ribonuclease (Sigma Chemical Co.) for 15 min at 37 C and
stained with 50 mg/mL propidium iodide (Sigma Chemical Co.) for
30 min at room temperature. Flow cytometry analyses were per-
formed on a Becton Dickinson FaCS-Calibur using the Becton
Dickinson Cell Quest program.