Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives coupling with a thiourea moiety as novel CDKs inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.07.003

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 (5a-10d), 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 μM to 4.21 μM), In addition, flow cytometry indicated that compound 10b could induce cycle G ₀/G₁ phase arrest in A549 cells with a dose dependent. Taken together, compound 10b could be selected for further preclinical evaluation.

Full text of DOI:10.1016/j.ejmech.2013.07.003

Accepted Manuscript
Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives
coupling with a thiourea moiety as novel CDKs inhibitors
Jian Sun, Xian-Hai Lv, Han-Yue Qiu, Yan-Ting Wang, Qian-Ru Du, Dong-Dong Li,
Yong-Hua Yang, Hai-Liang Zhu
PII:
S0223-5234(13)00441-8
10.1016/j.ejmech.2013.07.003
EJMECH 6290
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 April 2013
Revised Date: 7 July 2013
Accepted Date: 8 July 2013
Please cite this article as: J. Sun, X.-H. Lv, H.-Y. Qiu, Y.-T. Wang, Q.-R. Du, D.-D. Li, Y.-H. Yang, H.-
L. Zhu, Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives coupling
with a thiourea moiety as novel CDKs inhibitors, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.07.003.
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ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and molecular docking studies of pyrazole
derivatives coupling with a thiourea moiety as novel CDKs inhibitors
Jian Sun a†, Xian-Hai Lv b†, Han-Yue Qiu a, Yan-Ting Wang a, Qian-Ru Du a, Dong-
Dong Li a, Yong-Hua Yang a*, Hai-Liang Zhu a*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,Nanjing
210093, People’s Republic of China
b College of Science, Anhui Agricultural University, Hefei 230036, People’s Republic
of China
[+] These authors contributed equally to this work.
* Corresponding author. Tel.: +86-25-8359 2572; Fax: 0086+25+8359 2672;
E-mail address: zhuhl@nju.edu.cn
Twenty new pyrazole derivatives have been designed and evaluated for their
anticancer and CDKs inhibitory activity. Compound 10b demonstrated the most
potent inhibitory activity.

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and molecular docking studies of pyrazole
derivatives coupling with a thiourea moiety as novel CDKs inhibitors
Jian Sun a†, Xian-Hai Lv b†, Han-Yue Qiu a, Yan-Ting Wang a, Qian-Ru Du a, Dong-
Dong Li a, Yong-Hua Yang a*, Hai-Liang Zhu a*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,Nanjing
210093, People’s Republic of China
b College of Science, Anhui Agricultural University, Hefei 230036, People’s Republic
of China
[†] These authors contributed equally to this work.
* Corresponding authors. Tel./fax: +86-25-83592672;
E-mail address: zhuhl@nju.edu.cn
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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 (5a-10d), 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 ꢀM to
4.21 ꢀM), 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.
Keywords:
Cyclin-dependent kinases;
CDK2;
Cell cycle profile;
Structure-activity relationship;
Molecular docking
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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 serine-
threonine 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.
CDK2, one significant member of CDKs family, have been proved to participate
in the majority of cancer cases mainly due to it could play a vital role during the G1/S
transition of the cell cycle when combined with cyclin E. Besides, plenty of reports
also illustrated that the inhibition of CDK2 could be an important way for the
treatment of cancers [4-5]. However, recent evidences indicated that the highly
selective inhibitors of individual CDK may not be therapeutically effective because of
the functional redundancy within the CDK family [6]. Additionally, several small
molecule candidates which have already undergone clinical evaluation are all broad-
range CDKs inhibitors which could meanwhile inhibit more than one member of the
CDK family [7-9]. Here are some representative compounds illustrated in Figure 1,
such as PNU-292137 (CDK1, 2, 4 inhibitor), 1H-indazole-3-carboxylic acid (4-
sulfamoylphenyl)amide (CDK1, 2 inhibitor), AT7519 (CDK1, 2, 4, 5 inhibitor),
olomoucine (CDK1, 2, 5 inhibitor), and roscovitine (CDK1, 2, 5, 7 inhibitor) [2].
(Fig 1)
In terms of the mechanism that the development of new anticancer therapeutic
agents is one of the fundamental goals in medicinal chemistry field, we tried to design
a series of novel broad-range CDKs inhibitors. As shown above, three inhibitors
(PNU-292137, 1H-indazole-3-carboxylic acid (4-sulfamoylphenyl)amide, AT7519)
shared the same pyrazole scaffold that facilitates us preliminarily in considering the
pyrazole core as the basic skeleton for designing novel pan-CDKs inhibitors as
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antitumor agents. Besides, given the remarkable pharmacological activities
(antibacterial, antifungal, tumor necrosis and antiangiogenic) of pyrazole derivatives,
[10-13] and those inhibitors coupling with pyrazole ring reported by Brasca et al. [6]
displayed highly activity against various CDKs and impressive selectivity against a
panel of serine-threonine and tyrosine kinases, we envisioned pyrazole ring actually is
a promising skeleton when involved in the inhibition of CDKs in cell cycle
progression. Thus, we attempted to design a series of pyrazole derivatives as broad-
range CDKs inhibitors.
With respect to the linkers between the core and the hydrophobic blocks of
designed compounds in the ATP-binding sites of CDKs proteins, we attempted to
employ the common thiourea linkers. It is noted that this linker as well as amido
linkage could bind tightly to the hinge part of the binding site, forming several various
interactions with certain important residues, such as hydrogen bond, or charge
interaction. In addition, thiourea derivatives have attracted continuing interest over the
years because they displayed a wide range of biological activity including anti-fungal,
antibacterial, insecticidal, antitubercular, herbicidal, and plant growth regulator
properties [14-16]. Especially recent years, thiourea derivatives with their antitumor
activity have become a new hot spot. Such as diarylsulfonylurea derivatives [17-18]
have been reported to possess a broad spectrum of anticancer activity in several tumor
models and showing good growth inhibition; hydroxyurea [19] has been described as
a clinically useful drug for the treatment of a wide range of solid tumors. Besides, our
previous research have illustrated that 1-(3-chloro-2-hydroxybenzyl)-1-(4-
hydroxybenzyl)-3-phenylthiourea possessed remarkable anti-proliferative activity
[20].
But to our knowledge, few reports have been dedicated to design and synthesize
an antitumor compound which contains pyrazole and thiourea simultaneously. The
pyrazole ring along with the thiourea ring, the two combined substructures, might
exhibit synergistic anticancer effect. All of these encouraged us to integrate these two
structures and screen new pyrazole-thiourea derivatives as potential broad-range
CDKs inhibitory agents. Herein, we described the synthesis and the structure-activity
relationship (SAR) of 1, 3-diphenyl-N-(phenylcarbamothioyl)-1H-pyrazole-4-
carboxamide derivatives. Biological evaluation indicated that some of the targeted
compounds could be potential inhibitors of broad-range CDKs. Docking simulations
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were performed using the X-ray crystallographic structure of the CDK2 in complex
with an inhibitor to explore the binding modes of these compounds at the active site.
2. Results and discussion
2.1. Chemistry
The synthetic route of the cyanic 1, 3-diphenyl-1H-pyrazole-4-carboxylic
thioanhydride derivatives (6-10) was outlined in Scheme 1. [3, 21] Compounds 6-10
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 (6a-10d) was shown in Scheme 2. [21] As reports, the
synthesis of compounds (6a-10d) 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.
(Scheme 1)
(Scheme 2)
These compounds were all reported for the first time. All of the synthesized
compounds 6a-10d (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 Figure 2 gave a
perspective view of this compound together with the atomic labeling system.
(Table 1)
(Table 2)
(Fig. 2)
2.2. Bioassay
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To test the anticancer activities of the synthesized compounds, we evaluated anti-
proliferative activities of compounds 6a-10d against A549 (carcinomic human
alveolar basal epithelial cell), MCF-7 (breast cancer cell line) and H460 (lung
carcinoma cell line). The results were summarized in Table 3. With few exception,
the active analogs showed a remarkable potential antitumor activity, suggesting that 1,
3-diphenyl-N-(phenylcarbamothioyl)-1H-pyrazole-4-carboxamide derivatives could
significantly enhance anticancer potency. For the given compounds, it was observed
that compound 10b showed the most potent biological activity (IC50 = 2.57 ± 0.12 ꢀM
for H460 and IC50 = 0.75 ± 0.03 ꢀM for A549).
According to the data presented in Table 3, we could arrive at the conclusion that
the activity of the tested compounds may be correlated to the variation and
modifications of structure. For instance, different substitutes on the A-ring determine
the primary order of potency. Among the compounds, the compounds with OMe and
Me group as electron-donating substituents on ring A were of better antitumor activity
comparing to those with electron-withdrawing, the potency of para-substituents on the
A-ring was ordered as: OMe > Me > H > F> Cl. In addition, change of substituents on
the B-ring under constant A-ring substituents could also affect the activities of these
compounds. A comparison of the para-substituents on the B-ring demonstrated that
OMe group could dramatically improve anti-proliferative activity, with the
presumption that OMe group at the para-position of ring B might bind potently into
targeting protein. An overview of the compounds, compounds 10b meanwhile have
para-OMe group on the A-ring and para-OMe on the B-ring. Therefore, it displayed
the most potent anticancer activity.
On the basis of previous research, it was revealed that the class of N-((1, 3-
diphenyl-1H-pyrazol-4-yl)methyl)aniline inhibitors possessed exclusively CDK2
inhibitory activity. [3] To examine whether the synthesized compounds could also
inhibit CDK2, we screened compounds 6a-10d against the CDK2. An analysis of the
potency data we obtained by screening compounds 6a-10d against the CDK2 (Table
3), the analysis results manifested that compounds 7b, 8b and 10b showed strong
inhibitory effect (IC50 = 0.047 ꢀM, 0.037 ꢀM and 0.025 ꢀM, respectively) and the IC50
values of enzyme assay shared a similar tendency with that of anti-proliferative assay.
These results indicated the anti-proliferative effect might be produced partly by
interaction between CDK2 protein and the compounds. An analysis between the anti-
proliferative activity against A549 cell line and the CDK2 inhibitory activity of 8
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compounds (6b, 7b, 8b, 9b, 10a, 10b, 10c, and 10d) verified that there indeed was a
moderate correlation between CDK2 inhibition and inhibition of cancer cellular
proliferation, as evidenced in Figure 3, with a R square value 0.84.
(Table 3)
(Fig. 3)
To further investigate kinase selectivity of these compounds for CDKs,
compounds 6b, 7b, 8b, 9b, and 10b were evaluated as potential inhibitors of other
seven protein kinases relevant to cancers: CDK1, CDK4, CDK5, CDK6, CDK7,
EGFR, and VEGFR2. Complexes of CDK1 can regulate both the G2 to M phase
transition and mitosis; CDK2, CDK4 and CDK6 primarily regulate progression from
the G1 (Gap1) phase to the S phase (DNA synthesis) of the cell cycle; CDK5 plays a
role in neuronal and secretory cell function; CDK7 is a component of the CDK-
activating kinase (CAK), which acts upstream of cell-cycle CDKs; [22] EGFR is the
cell-surface receptor for members of the epidermal growth factor family of
extracellular protein ligands; [23] VEGFR2 is a receptor tyrosine kinase expressed on
the endothelial cells which can mediate endothelial cell proliferation, differentiation,
and micro-vascular permeability. [24] As expected, the results in Table 4 showed that
all the selected compounds (6b, 7b, 8b, 9b, and 10b) lowered at least one order of
magnitude in the inhibition for EGFR and VEGFR2, when compared to CDKs.
Besides, the data of Table 4 also demonstrated that those five compounds would
prefer to CDK2, CDK4, CDK6 (IC50 : 25-50 nM) than the other three members CDK1,
CDK5 and CDK7 (IC50 : 500-2000 nM). Therefore, we tended to conclude that the
synthesized molecule inhibitors can blockade the function of CDK2, CDK4, CDK6
and might have involved in the process of G1 to S phase transition of the cell cycle.
The detailed results were summarized in Table 4.
(Table 4)
To gain better understanding on the preliminary mechanism of the studied
compounds with potent inhibitory activity, the cell cycle profile experiment was
performed to assay the effect of compound 10b in Figure 4. The cell cycle profile of
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the treated cells showed obvious effect at micromolar range: a subsequent increase of
the G0/G1 population compared to the control cells. The presence of cells with G0/G1
DNA content (from 57.07% in control cells to 64.00% in cells treated with compound
10b at 2 ꢀM). This result indicated that compound 10b could induce cycle G0/G1
phase arrest and affect G1 to S phase transition.
(Fig. 4)
2.3. Molecular docking
Paul group [25] given a detail analysis of the ATP binding site of CDK2 (PDB
code: 2VTO) and identified a number of key regions including a hydrophobic pocket
(defined by Ile10, Phe82, Asp86, and Leu134); the relatively small region between
the gatekeeper residue (Phe80) and the DFG motif (Asp145) and the solvent
accessible region toward Lys89.
A docking study was performed to fit compound 10b and reference compound
(LZ8) [25] into the active center of 2VTO. The obtained results were presented in
Figures 5. Figures 5A and 5B showed the binding mode of compound 10b interacting
with 2VTO protein and the docking results revealed that three amino acids Phe80,
Asp86, Lys89 located in the binding pocket of protein played a vital roles in the
combination with compound 10b, which were stabilized by one Pi-Pi bond, two
hydrogen bonds. One Pi-Pi bond, of which their lengths were 4.5 Å, was formed by
the C ring and Phe80; One hydrogen bond was provided by the nitryl group beside A
ring and Asp86 while another hydrogen bond was involved in thiourea linkers and
Lys89 which verified that thiourea linkers could bind tightly to active center and
employing thiourea structure was reasonable. Figure 5C and 5D displayed 2D and 3D
interactional maps between compound LZ8 and CDK2 protein crystal structure.
Insight into this picture, we can see that only one hydrogen bond which was consisted
by nitryl and Leu83 involved in the binding between the compound and the active site.
Therefore, we anticipated compound 10b could well embed in the active pocket and
have much better physicochemical properties than reference compound LZ8. In
addition, the enzyme surface model was showed in Figure 6, which revealed that the
molecule 10b occupies the ATP-binding pocket and binds to an active conformation
of CDK2.
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(Fig. 5)
(Fig. 6)
3. Conclusion
In this study, a series of 1, 3-diphenyl-N-(phenylcarbamothioyl)-1H-pyrazole-4-
carboxamide derivatives have been synthesized and evaluated for their antitumor
activities. These compounds exhibited potent anti-proliferative activities against A549
and CDK2, 4, 6 inhibitory activities. Among all of the synthesized compounds,
compound 10b demonstrated the most potent inhibitory activity (IC50 of 25 nM, 35
nM, 29 nM for CDK2, 4, 6). Anti-proliferative assay results also showed that
compound 10b (IC50 = 0.75 ꢀM for A549) had the potential to be developed as a anti-
proliferative agent against A549. Docking simulation was performed to position
compound 10b into the human CDK2 active site to determine the probable binding
model. Analysis of the compound 10b binding conformation in active site showed
that the compound 10b was stabilized by one Pi-Pi bond with PHE80 and two
hydrogen bonds, respectively, with ASP86 and LYS89. Cell cycle profile experiment
also showed the compound 10b was a potential antitumor agent. The information of
this work might be helpful for the design and synthesis of a leading compound 10b
towards the development of new therapeutic agent to fight against cancer.
4. Experimental Section
4.1. Materials and Methods.
All of the synthesized compounds were chemically characterized by thin layer
chromatography (TLC), proton nuclear magnetic resonance (1H NMR) and elemental
1
microanalyses (CHN). H NMR spectra were measured on a Bruker AV-300 or AV-
500 spectrometer at 25 and referenced to Me4Si. Chemical shifts were reported in
ppm (δ) using the residual solvent line as internal standard. Splitting patterns were
designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS spectra were
recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument and were within ± 0.4 % of the theoretical
values. Melting points were determined on a XT4 MP apparatus (Taike Corp., Beijing,
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China) and were as read. Analytic thin-layer chromatography (TLC) was performed
on the glass-backed silica gel sheets (silica gel 60 Å GF254). All compounds were
detected using UV light (254 nm or 365 nm).
4.2. General method for the preparation of target compounds 6-10
The starting material substituted 1, 3-diphenyl-1H-pyrazole-4-carboxylic
thioanhydride derivatives (6-10) was synthesized as following: para-substituted
acetophenone (1-5) (20 mmol) interact with phenylhydrazine hydrochloride (25 mmol)
couple with sodium acetate (40 mmol) in anhydrous ethanol to form 1-phenyl-2-(1-
phenylethyli-dene) hydrazine, which was then dissolved in a cold mixed solution of
DMF (20 mL) and POCl3 (16 mL), stirred at 50-60 °C for 5 h. The resulting mixture
was poured into ice-cold water, a saturated solution of sodium hydroxide was added
to neutralize the mixture, then the obtained solid precipitate were oxidized to the
corresponding carboxylic acids by treatment with potassium permanganate (10 mmol),
stirred at 70-80 °C for 3 h. while the transformation of acids into the appropriate acid
chlorides was accomplished with thionyl chloride in refluxing toluene for 3 h. A
solution of substituted acid chlorides (4 mmol) in anhydrous acetone (10 ml) and 3%
TBAB in acetone was added drop wise to a suspension of ammonium thiocyanate in
acetone (10 ml) and the reaction mixture was refluxed for 1 h to give the desired
compounds 6-10.
4.3. General procedure for 1,3-diphenyl-N-(phenylcarbamothioyl)-1H-pyrazole-
4-carboxamide derivatives (6a-10d)
Compounds 6a-10d were synthesized from a stirring mixture of the starting
material substituted 1, 3-diphenyl-1H-pyrazole-4-carboxylic thioanhydride derivatives
(6-10) (2 mmol) and substituted anilines (2 mmol) with the help of K2CO3 in
anhydrous methylene dichloride (15ml) at the room temperate for 1 h. The reaction
mixture was poured into five times its volume of cold water when the thiourea
precipitated as a solid. The solid product was washed with water and purified by
recrystallization from an ethanol-dichloromethane mixture (1:2).
4.3.1
1,3-Diphenyl-N-(p-tolylcarbamothioyl)-1H-pyrazole-4-carboxamide (6a)
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1
Mp: 175-177 ; H NMR (300 MHz, CDCl3, δ ppm): 2.28 (s, 3H), 7.12 (d, J = 8.40
Hz, 2H), 7.34 (t, J = 7.22 Hz, 1H), 7.43-7.55 (m, 7H), 7.66 (dd, J1 = 2.10 Hz, J2 = 1.20
Hz, 2H), 7.72 (d, J = 8.10 Hz, 2H), 8.55 (s, 1H), 8.67 (s, 1H), 12.28 (s, 1H). 13C NMR
(100 MHz, DMSO-d6, δ ppm): 179.55, 163.92, 153.63, 139.19, 136.11, 135.84,
133.29, 132.30, 130.29, 129.55, 129.34, 129.16, 128.59, 128.00, 124.81, 119.39,
114.62, 21.09. MS (ESI): 412.14 (C24H20N4OS, [M+H]+). Anal. Calcd for
C24H19N4OS: C, 69.88; H, 4.89; N, 13.58 Found: C, 69.80; H, 4.85; N, 13.68.
4.3.2
N-((4-methoxyphenyl)carbamothioyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide
(6b)
Mp: 177-179 ; 1H NMR (300 MHz, CDCl3, δ ppm): 3.30 (s, 3H), 7.27-7.35 (m, 6H),
7.45 (t, J = 8.22 Hz, 2H), 7.51-7.58 (m, 4H), 7.70 (d, J = 8.07 Hz, 2H), 8.53 (s, 1H),
13
8.71 (s, 1H), 12.43 (s, 1H). C NMR (100 MHz, DMSO-d6, δ ppm): 179.56, 163.95,
158.86, 153.63, 139.82, 132.81, 132.31, 130.54, 130.30, 129.75, 129.37, 129.21,
128.57, 127.74, 124.87, 120.34, 114.62, 55.34. MS (ESI): 428.13 (C24H20N4O2S,
[M+H]+). Anal. Calcd for C24H19N4O2S: C, 67.27; H, 4.70; N, 13.07 Found: C, 67.13;
H, 4.53; N, 13.37.
4.3.3
N-((4-fluorophenyl)carbamothioyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (6c)
Mp: 137-139 ; 1H NMR (300 MHz, CDCl3, δ ppm): 6.96 (t, J = 8.43 Hz, 2H), 7.07 (t,
13
J = 8.43 Hz, 2H), 7.34-7.79 (m, 10H), 8.62 (s, 1H), 8.78 (s, 1H), 12.39 (s, 1H). C
NMR (100 MHz, DMSO-d6, δ ppm): 179.55, 163.96, 162.87, 153.62, 139.84, 134.06,
132.83, 131.23, 130.79, 129.36, 129.23, 127.75, 127.59, 124.86, 120.26, 115.67,
114.61. MS (ESI): 416.11 (C23H17FN4OS, [M+H]+). Anal. Calcd for C23H16FN4OS: C,
66.33; H, 4.11; N, 13.45 Found: C, 66.31; H, 4.01; N, 13.65.
4.3.4
N-((4-chlorophenyl)carbamothioyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide
(6d)
Mp: 184-186 ; 1H NMR (300 MHz, CDCl3, δ ppm): 7.33 (t, J = 8.22 Hz, 2H), 7.43 (t,
J = 7.50 Hz, 2H), 7.50-7.80 (m, 10H), 8.63 (s, 1H), 8.74 (s, 1H), 12.49 (s, 1H). 13C
NMR (100 MHz, DMSO-d6, δ ppm): 179.55, 163.91, 153.63, 139.85, 136.62, 133.65,
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132.84, 131.26, 130.78, 129.35, 129.22, 129.07, 127.74, 124.85, 120.25, 115.58,
114.62. MS (ESI): 432.08 (C23H17ClN4OS, [M+H]+). Anal. Calcd for C23H16ClN4OS:
C, 63.81; H, 3.96; N, 12.94 Found: C, 63.51; H, 3.91; N, 12.99.
4.3.5
1-Phenyl-3-(p-tolyl)-N-(p-tolylcarbamothioyl)-1H-pyrazole-4-carboxamide (7a)
Mp: 182-184 ; 1H NMR (300 MHz, CDCl3, δ ppm): 2.35 (s, 3H), 2.45 (s, 3H), 7.19
(d, J = 8.25 Hz, 2H), 7.37-7.53 (m, 7H), 7.61 (d, J = 8.04 Hz, 2H), 7.78 (d, J = 8.22
Hz, 2H), 8.61 (s, 1H), 8.80 (s, 1H), 12.37 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 179.55, 164.03, 153.62, 139.22, 138.59, 136.10, 135.84, 133.24, 130.27,
129.56, 129.43, 129.21, 129.16, 127.93, 124.78, 119.36, 114.51, 21.39, 21.09. MS
(ESI): 426.15 (C25H22N4OS, [M+H]+). Anal. Calcd for C25H21N4OS: C, 70.40; H, 5.20;
N, 13.14 Found: C, 70.06; H, 5.34; N, 13.18.
4.3.6
N-((4-methoxyphenyl)carbamothioyl)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (7b)
Mp: 181-183 ; 1H NMR (300 MHz, CDCl3, δ ppm): 2.45 (s, 3H), 3.81 (s, 3H), 6.91
(q, J = 6.96 Hz, 2H), 7.37-7.59 (m, 7H), 7.61 (d, J = 8.04 Hz, 2H), 7.80 (d, J = 8.07
Hz, 2H), 8.61 (s, 1H), 8.80 (s, 1H), 12.29 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 179.55, 164.03, 159.34, 153.62, 139.76, 136.21, 135.83, 133.24, 130.84,
129.65, 129.57, 128.46, 127.59, 124.77, 121.21, 119.93, 114.52, 55.75, 21.08. MS
(ESI): 442.15 (C25H22N4O2S, [M+H]+). Anal. Calcd for C25H21N4O2S: C, 67.85; H,
5.01; N, 12.66 Found: C, 67.63; H, 5.08; N, 12.56.
4.3.7
N-((4-fluorophenyl)carbamothioyl)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (7c)
Mp: 172-174 ; 1H NMR (300 MHz, CDCl3, δ ppm): 2.47 (s, 3H), 7.09 (t, J = 8.58 Hz,
2H), 7.39-7.65 (m, 9H), 7.80 (d, J = 8.07 Hz, 2H), 8.62 (s, 1H), 8.80 (s, 1H), 12.42 (s,
1H). 13C NMR (100 MHz, DMSO-d6, δ ppm): 179.54, 164.05, 163.23, 153.63, 139.78,
136.23, 135.83, 133.25, 130.21, 129.66, 129.52, 128.45, 124.78, 121.23, 119.94,
115.74, 114.51, 21.13. MS (ESI): 430.13 (C24H19FN4OS, [M+H]+). Anal. Calcd for
C24H18FN4OS: C, 66.96; H, 4.45; N, 13.01 Found: C, 66.61; H, 4.60; N, 13.06.
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4.3.8
N-((4-chlorophenyl)carbamothioyl)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (7d)
1
Mp: 181-183 ; H NMR (300 MHz, CDCl3, δ ppm): 2.39 (s, 3H), 6.91 (d, J = 8.94
Hz, 2H), 7.38-7.80 (m, 11H), 8.62 (s, 1H), 8.77 (s, 1H), 12.28 (s, 1H). 13C NMR (100
MHz, DMSO-d6, δ ppm): 179.86, 164.01, 163.23, 153.64, 139.76, 136.54, 135.84,
133.65, 130.23, 129.65, 129.18, 128.46, 124.77, 121.24, 119.93, 115.75, 114.53,
21.09. MS (ESI): 446.10 (C24H19ClN4OS, [M+H]+). Anal. Calcd for C24H18ClN4OS:
C, 64.49; H, 4.28; N, 12.54 Found: C, 64.35; H, 4.29; N, 12.44.
4.3.9
3-(4-Chlorophenyl)-1-phenyl-N-(p-tolylcarbamothioyl)-1H-pyrazole-4-
carboxamide (8a)
1
Mp: 182-184 ; H NMR (300 MHz, CDCl3, δ ppm): 2.35 (s, 3H), 7.20 (d, J = 8.22
Hz, 2H), 7.39-7.72 (m, 9H), 7.76 (t, J = 8.04 Hz, 2H), 8.59 (s, 1H), 8.78 (s, 1H),
13
12.30 (s, 1H). C NMR (100 MHz, DMSO-d6, δ ppm): 179.78, 164.21, 153.63,
139.77, 137.25, 135.56, 134.43, 133.25, 131.22, 129.68, 129.36, 129.28, 128.99,
126.57, 124.74, 119.91, 114.52, 21.08. MS (ESI): 446.10 (C24H19ClN4OS, [M+H]+).
Anal. Calcd for C24H18ClN4OS: C, 64.49; H, 4.28; N, 12.54 Found: C, 64.35; H, 4.29;
N, 12.44.
4.3.10
3-(4-Chlorophenyl)-N-((4-methoxyphenyl)carbamothioyl)-1-phenyl-1H-pyrazole-
4-carboxamide (8b)
1
Mp: 203-204 ; H NMR (300 MHz, CDCl3, δ ppm): 3.80 (s, 3H), 6.87 (d, J = 8.97
Hz, 2H), 7.27-7.79 (m, 11H), 8.55 (s, 1H), 8.77 (s, 1H), 12.33 (s, 1H). 13C NMR (100
MHz, DMSO-d6, δ ppm): 179.86, 164.23, 159.32, 153.65, 139.75, 134.45, 133.26,
131.22, 129.69, 129.36, 129.27, 128.97, 127.56, 124.76, 119.94, 115.11, 114.53,
55.76. MS (ESI): 462.09 (C24H19ClN4O2S, [M+H]+). Anal. Calcd for C24H18ClN4O2S:
C, 62.27; H, 4.14; N, 12.10 Found: C, 62.15; H, 4.20; N, 12.22.
4.3.11
3-(4-Chlorophenyl)-N-((4-fluorophenyl)carbamothioyl)-1-phenyl-1H-pyrazole-4-
carboxamide (8c)
13

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Mp: 169-171 ; 1H NMR (300 MHz, CDCl3, δ ppm): 7.10 (t, J = 8.94 Hz, 2H), 7.44-
7.80 (m, 11H), 8.60 (s, 1H), 8.76 (s, 1H), 12.35 (s, 1H). 13C NMR (100 MHz, DMSO-
d6, δ ppm): 179.83, 164.28, 163.32, 153.66, 139.74, 134.47, 134.15, 133.25, 131.27,
129.68, 129.37, 128.65, 124.79, 129.28, 119.96, 115.89, 114.54. MS (ESI): 450.07
(C23H16ClFN4OS, [M+H]+). Anal. Calcd for C23H15ClFN4OS: C, 61.26; H, 3.58; N,
12.43 Found: C, 61.11; H, 3.43; N, 12.48.
4.3.12
3-(4-Chlorophenyl)-N-((4-chlorophenyl)carbamothioyl)-1-phenyl-1H-pyrazole-4-
carboxamide (8d)
Mp: 201-203 ; 1H NMR (300 MHz, CDCl3, δ ppm): 7.36-7.73 (m, 11H), 7.79 (d, J =
8.04 Hz, 2H), 8.59 (s, 1H), 8.74 (s, 1H), 12.44 (s, 1H). 13C NMR (100 MHz, DMSO-
d6, δ ppm): 179.89, 164.20, 153.62, 139.75, 136.61, 134.48, 133.27, 131.28, 131.25,
129.78, 129.62, 129.39, 129.23, 128.66, 124.79, 119.97, 114.59. MS (ESI): 466.04
(C23H16Cl2N4OS, [M+H]+). Anal. Calcd for C23H15Cl2N4OS: C, 59.11; H, 3.45; N,
11.99 Found: C, 59.07; H, 3.41; N, 11.89.
4.3.13
3-(4-Fluorophenyl)-1-phenyl-N-(p-tolylcarbamothioyl)-1H-pyrazole-4-
carboxamide (9a)
1
Mp: 200-202 ; H NMR (300 MHz, CDCl3, δ ppm): 2.37 (s, 3H), 7.22 (d, J = 8.22
Hz, 2H), 7.30-7.55 (m, 7H), 7.73-7.80 (m, 4H), 8.60 (s, 1H), 8.71 (s, 1H), 12.32 (s,
1H). 13C NMR (100 MHz, DMSO-d6, δ ppm): 179.85, 164.23, 162.88, 153.69, 139.75,
137.24, 135.69, 133.27, 131.56, 130.31, 129.65, 128.14, 126.57, 124.77, 119.41,
115.62, 114.22, 21.09. MS (ESI): 430.13 (C24H19FN4OS, [M+H]+). Anal. Calcd for
C24H18FN4OS: C, 66.96; H, 4.45; N, 13.01 Found: C, 66.65; H, 4.51; N, 13.14.
4.3.14
3-(4-Fluorophenyl)-N-((4-methoxyphenyl)carbamothioyl)-1-phenyl-1H-pyrazole-
4-carboxamide (9b)
1
Mp: 196-198 ; H NMR (300 MHz, CDCl3, δ ppm): 3.83 (s, 3H), 6.93 (d, J = 8.97
Hz, 2H), 7.30-7.80 (m, 11H), 8.61 (s, 1H), 8.79 (s, 1H), 12.26 (s, 1H). 13C NMR (100
MHz, DMSO-d6, δ ppm): 179.82, 164.23, 162.85, 153.74, 139.68, 136.24, 135.24,
133.28, 130.91, 130.33, 129.66, 128.62, 127.56, 124.75, 119.95, 115.63, 114.66,
14

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55.79. MS (ESI): 446.12 (C24H19FN4O2S [M+H]+). Anal. Calcd for C24H18FN4O2S: C,
64.56; H, 4.29; N, 12.55 Found: C, 64.71; H, 4.34; N, 12.61.
4.3.15
3-(4-Fluorophenyl)-N-((4-fluorophenyl)carbamothioyl)-1-phenyl-1H-pyrazole-4-
carboxamide (9c)
Mp: 185-187 , 1H NMR (300 MHz, CDCl3, δ ppm): 7.10 (t, J = 8.65 Hz, 2H), 7.28-
7.81 (m, 11H), 8.61 (s, 1H), 8.74 (s, 1H), 12.36 (s, 1H). 13C NMR (100 MHz, DMSO-
d6, δ ppm): 179.82, 164.22, 163.35, 153.75, 139.74, 136.24, 135.14, 133.27, 131.28,
130.69, 129.67, 128.63, 127.57, 124.63, 119.46, 115.86, 114.29. MS (ESI): 434.10
(C23H16F2N4OS [M+H]+). Anal. Calcd for C23H15F2N4OS: C, 63.58; H, 3.71; N, 12.90
Found: C, 63.80; H, 3.69; N, 12.92.
4.3.16
N-((4-chlorophenyl)carbamothioyl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (9d)
1
Mp: 184-185 , H NMR (300 MHz, CDCl3, δ ppm): 7.28-7.57 (m, 6H), 7.66 (d, J =
13
8.79 Hz, 2H), 7.73-7.80 (m, 5H), 8.61 (s, 1H), 8.70 (s, 1H), 12.46 (s, 1H). C NMR
(100 MHz, DMSO-d6, δ ppm): 179.87, 169.24, 162.94, 153.76, 139.75, 137.69,
135.27, 133.75, 131.27, 130.69, 129.68, 128.64, 124.65, 119.96, 116.59, 115.96,
114.31. MS (ESI): 450.07 (C23H16ClFN4OS [M+H]+). Anal. Calcd for
C23H15ClFN4OS: C, 61.26; H, 3.58; N, 12.43 7.11 Found: C, 61.41; H, 3.53; N, 12.53.
4.3.17
3-(4-Methoxyphenyl)-1-phenyl-N-(p-tolylcarbamothioyl)-1H-pyrazole-4-
carboxamide (10a)
Mp: 178-180 , 1H NMR (300 MHz, CDCl3, δ ppm): 2.36 (s, 3H), 3.99 (s, 3H), 7.11
(d, J = 8.61 Hz, 2H), 7.21 (d, J = 8.40 Hz, 2H), 7.38-7.69 (m, 7H), 7.80 (d, J = 7.50
Hz, 2H), 8.61 (s, 1H), 8.84 (s, 1H), 12.39 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 179.86, 164.26, 160.74, 153.74, 139.75, 137.29, 135.61, 133.29, 130.32,
129.64, 128.59, 126.58, 125.36, 124.76, 119.98, 115.63, 114.86, 55.75, 21.09. MS
(ESI): 442.15 (C25H22N4O2S [M+H]+). Anal. Calcd for C25H21N4O2S: C, 67.85; H,
5.01; N, 12.66 Found: C, 67.60; H, 5.16; N, 12.68.
15

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4.3.18
3-(4-Methoxyphenyl)-N-((4-methoxyphenyl)carbamothioyl)-1-phenyl-1H-
pyrazole-4-carboxamide (10b)
Mp: 170-172 , 1H NMR (300 MHz, CDCl3, δ ppm): 3.75 (s, 3H), 3.91 (s, 3H), 6.87
(q, J = 9.00 Hz 2H), 7.09 (t, J = 7.68 Hz, 2H), 7.38-7.71 (m, 7H), 7.79 (d, J = 8.04 Hz,
2H), 8.62 (s, 1H), 8.90 (s, 1H), 12.31 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ ppm):
179.84, 164.23, 160.69, 153.75, 139.68, 135.64, 133.32, 130.91, 130.84, 129.65,
128.96, 127.56, 125.39, 124.77, 119.96, 115.23, 114.96, 55.74, 55.73. MS (ESI):
458.14 (C25H22N4O3S [M+H]+). Anal. Calcd for C25H21N4O3S: C, 65.48; H, 4.84; N,
12.22 Found: C, 65.25; H, 4.87; N, 12.27.
4.3.19
N-((4-fluorophenyl)carbamothioyl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-
4-carboxamide (10c)
1
Mp: 162-163 ; H NMR (300 MHz, CDCl3, δ ppm): 3.90 (s, 3H), 7.00 (t, J = 8.84
Hz, 2H), 7.11 (d, J = 8.92 Hz, 2H), 7.30-7.70 (m, 7H), 7.78 (d, J = 7.89 Hz, 2H), 8.61
(s, 1H), 8.83 (s, 1H), 12.41 (s, 1H). 13C NMR (100 MHz, DMSO-d6, δ ppm): 179.83,
164.26, 160.68, 153.76, 139.67, 134.23, 133.35, 131.24, 130.92, 129.67, 128.97,
127.64, 125.38, 124.77, 119.97, 115.86, 114.97, 55.75. MS (ESI): 446.12
(C24H19FN4O2S [M+H]+). Anal. Calcd for C24H18FN4O2S: C, 64.56; H, 4.29; N, 12.55
Found: C, 64.32; H, 4.33; N, 12.61.
4.3.20
N-((4-chlorophenyl)carbamothioyl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-
4-carboxamide (10d)
Mp: 193-194 ; 1H NMR (300 MHz, CDCl3, δ ppm): 3.90 (s, 3H), 7.11 (d, J = 6.75
Hz, 2H), 7.35-7.44 (m, 3H), 7.54 (t, J = 7.32 Hz, 2H), 7.66 (d, J = 8.76 Hz, 4H), 7.81
13
(d, J = 7.50 Hz, 2H), 8.61 (s, 1H), 8.81 (s, 1H), 12.52 (s, 1H). C NMR (100 MHz,
DMSO-d6, δ ppm): 179.86, 164.25, 162.09, 153.76, 139.76, 136.68, 133.75, 131.24,
129.68, 129.14, 128.98, 127.75, 125.69, 124.76, 119.99, 114.99, 114.38, 55.74. MS
(ESI): 462.09 (C24H19ClN4O2S [M+H]+). Anal. Calcd for C24H18ClN4O2S: C, 62.27;
H, 4.14; N, 12.10 Found: C, 62.41; H, 4.03; N, 12.25.
4.4. Kinase Assays
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The inhibition studies of cell cycle dependent kinase 1, 2, 4, 5, 6, 7 were
performed for the synthesized compounds along with olomoucine and roscovitine as
reference compounds. CDK2 enzyme was purified from infected sf21 insect cells. For
baculoviral overexpression of proteins, human CDK2 c-DNA tagged by hexa-
histidine was subcloned on its N-terminal. CDK2 enzyme was purified using Ni 2+
affinity resin from sf21 insect cell culture. Enzyme assays were done in 20 mL of 50
mM Tris-HCl containing 10 ꢀM ATP, 0.2 ꢀCi of gamma-P32 ATP, 10 mM MgCl2, 5
mM DTT and 4 ꢀg of histone H1 was used as a substrate. The reaction was continued
for 10 min in the presence of inhibitors and stopped by adding 10 mL of 30%
phosphoric acid. The stopped mixtures were spotted onto P81 paper and were washed
with 10 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl for five times. The
radioactivity of each spot was quantified with BAS imager. The inhibition studies of
human EGFR tyrosine kinase activities were done using C-terminal human EGFR
tyrosine kinase domain and Erlotinib was used as a reference compound [26].The
inhibition studies of human VEGFR tyrosine kinase activities were done using a DNA
sequence encoding the human KDR tyrosine kinase domain (Asp807-Val1356) and
ZD-4190 was used as a reference compound [27]. The concentration of inhibitor that
gives 50% inhibition was designated as IC50 value.
4.5. Cell proliferation assay
CCK8 is much more convenient and helpful than MTT for analyzing cell
proliferation, because it can be reduced to soluble formazan by dehydrogenase in
mitochondria and has little toxicity to cells. Cell proliferation was determined using
CCK8 dye (Beyotime Inst Biotech, China) according to manufacture’s instructions.
Briefly, 1-5 × 103 cells per well were seeded in a 96-well plate, grown at 37 for 12
h, Subsequently, cells were treated with compounds (Table 2) at increasing
concentrations in the presence of 10% FBS for 24 or 48h. After 10 µL CCK8 dye was
added to each well, cells were incubated at 37 for 1-2 h and Plates were read in a
Victor-V multilabel counter (Perkin-Elmer) using the default europium detection
protocol. Percent inhibition or IC50 values of compounds were calculated by
comparison with DMSO-treated control wells. The results are shown in Table 3.
4.6. Flow cytometry
Approximately 105 cells/well were plated in a 24 well plate and allowed to
17

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adhere. After 12 h, the medium was replaced with fresh culture medium containing
compounds 10b at final concentrations of 0.5, 1, 2 ꢀM. 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
,
washed once with PBS, treated with 1 mg/mL ribonuclease (Sigma Chemical Co.) for
15 min at 37 and stained with 50 mg/mL propidium iodide (Sigma Chemi-cal Co.)
for 30 min at room temperature. Flow cytometry analyses were performed on a
Becton Dickinson FaCS-Calibur using the Becton Dickinson Cell Quest program.
4.7. Docking simulations
The three-dimensional X-ray structure of CDK2 crystal structure (PDB code:
2VTO) was chosen as the template for the modeling study of compound 10b bound to
CDK2. The crystal structure was obtained from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). The molecular docking procedure was
performed by using CDOCKER protocol within Discovery Studio 3.1. For ligand
preparation, the 3D structure of 10b was generated and minimized using Discovery
Studio 3.1. For protein preparation, the hydrogen atoms were added, and the water
and impurities were removed. The molecular docking was performed by inserting
compound 10b into the binding pocket of CDK2 based on the binding mode. Types of
interactions of the docked protein with ligand-based pharmacophore model were
analyzed after the end of molecular docking.
Acknowledgment
The work was financed by Natural Science Foundation of China (No. J1103512);
Universities Natural Science research projects of Anhui Province (KJ2013B088) and
the opening foundation of the Key Laboratory of Green Pesticide and Agricultural
Bioengineering, Ministry of Education, Guizhou University (No.2011GDGP0105).
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Figure caption:
Table 1. Structure of compounds 6a–10d
Table 2. Crystallographic data and structure refinements for compound 7a
Table 3. Inhibition (IC50) of A549, MCF-7 and H460 cells proliferation and inhibition
of CDK2 by compounds 6a-10d
Table 4. Selected kinase inhibition activities IC50 a(µM)
Figure 1. ATP-competitive inhibitors of CDKs
Figure 2. Crystal structure of compound 7a
Figure 3. Correlation between the anti-proliferative activity against A549 cell line
and the CDK2 inhibitory activity, R2 = 0.84, which indicated that there was a
moderate correlation between CDK2 inhibition and inhibition of cancer cellular
proliferation
Figure 4. Effects of 10b on the cell cycle distribution in A549 cells. A549 cells were
treated with various concentrations of 10b. A) control; B) concentrations of 10b was
0.5 ꢀM; C) concentrations of 10b was 1.0 ꢀM; D) concentrations of 10b was 2.0 ꢀM.
A549 cells were treated with alcohol for 36 h as a control group, Values represent the
mean ± S.D, n =3. P <0.05 versus control
Figure 5. A) 2D molecular docking model of compound 10b with 2VTO; B) Model
of compound 10b bound to CDK2; C) The 2D diagram of docking structure of LZ8
with 2VTO; D) Model of compound LZ8 bound to CDK2
Figure 6. The surface model of compound 10b with 2VTO
Scheme 1. Synthesis of compound 6-10. Reagents and conditions: (i) ethanol, 50-60
°C, 3h; (ii) DMF, POCl3, 50-60 °C, 5 h; (iii) KMnO4, 70-80 °C, 3 h; (iv) SOCl2,
toluene, 70-80 °C, 3h; (v) NH4SCN, 3% TBAB, acetone, rt, 1 h
Scheme 2. Synthesis of compounds 6a-10d. Reagents and conditions: (i) CH2Cl2,
K2CO3, rt,1 h
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Table 1. Structures of compounds 6a-10d
Compounds
R1
H
H
H
H
Me
Me
Me
Me
Cl
Cl
R2
Me
OMe
F
Compounds
R1
Cl
Cl
F
F
F
F
OMe
OMe
OMe
OMe
R2
F
Cl
Me
OMe
F
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
Cl
Me
OMe
F
Cl
Me
OMe
Cl
9d
Me
OMe
F
10a
10b
10c
10d
Cl
Table 2. Crystallographic data and structure refinements for compound 7a
Crystal parameters
Formula
Compound 7a
C25H22N4OS
0.30 x 0.25 x 0.21
426.53
Crystal size (mm)
Formula weight
Crystal system
Triclinic
ꢀ(°)
ꢁ(°)
ꢂ(°)
89.645(2)
80.356(2)
72.588(2)
a(Å)
b(Å)
c (Å)
V (Å3)
Z
9.7827(8)
10.2080(8)
11.7098(9)
1098.81(15)
2
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ꢃ limits(°)
2.55䏨h䏨26.00
-12䏨h䏨12, -12䏨k䏨12䟍-14䏨l䏨14
hkl limits
F(000)
448
Data/restraints/parameters
Absorption coefficient
Reflections collected
Independent reflections
R1/ wR2 [I > 2&Gs(I)]
R1/wR2 (all data)
GF
4221/0/290
0.172
10748
4221 [Rint= 0.0149]
0.0397/0.1065
0.0444/0.1117
1.022
Table 3. Inhibition (IC50) of A549, MCF-7 and H460 cells proliferation and inhibition
of CDK2 by compounds 6a-10d
ꢀ
M
IC50
䟉
䟊
H460 a
Compounds
A549 a
MCF-7 a
CDK2
2.80±0.16
2.50±0.14
3.50±0.23
3.20±0.21
2.83±0.19
2.39±0.16
3.42±0.33
3.17±0.27
2.69±0.18
2.27±0.22
3.98±0.43
3.15±0.29
2.08±0.17
1.98±0.14
3.54±0.26
5.90±0.35
5.46±0.36
6.82±0.43
6.23±0.41
5.66±0.26
5.12±0.31
6.42±0.53
6.21±0.49
5.98±0.41
5.24±0.46
5.99±0.43
5.36±0.39
5.44±0.45
4.89±0.47
5.61±0.58
3.96±0.22
3.54±0.16
4.21±0.25
4.02±0.23
3.76±0.21
3.24±0.19
3.98±0.24
3.65±0.33
3.12±0.27
3.01±0.21
3.64±0.29
3.26±0.22
2.98±0.20
2.86±0.19
3.24±0.25
0.069±0.0050
0.056±0.0030
0.075±0.0040
0.071±0.0060
0.052±0.0030
0.047±0.0090
0.041±0.0080
0.063±0.0060
0.042±0.0030
0.041±0.0040
0.059±0.0060
0.058±0.0070
0.043±0.0030
0.036±0.0030
0.049±0.0090
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
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2.99±0.18
5.47±0.52
4.63±0.43
4.21±0.41
4.99±0.39
4.76±0.35
142.00±2.12
-
3.09±0.24
0.044±0.0030
0.029±0.0030
0.025±0.0020
0.032±0.0020
0.030±0.0050
7.00±0.27
9d
10a
1.25±0.11
0.75±0.03
1.43±0.09
1.31±0.14
130.00±1.99
-
2.88±0.24
2.57±0.12
3.05±0.26
3.01±0.25
132.00±2.03
-
10b
10c
10d
Olomoucine
Roscovotine
0.50±0.021
a Cancer cells kindly supplied by KeyGen Biotech; A549 (carcinomic human alveolar
basal epithelial cell), MCF-7 (breast cancer cell line) and H460 (lung carcinoma cell
line).
b Olomoucine, Roscovotine were prepared according to the literatures [3, 28].
Table 4. Inhibition of selected kinases IC50 a(nM)
Compounds
CDK1 CDK4 CDK5 CDK6 CDK7 EGFR VEGFR2
6b
7b
610
584
571
569
522
42
40
40
39
35
1502
1420
1356
1178
1093
50
43
42
33
29
1876
1788
1645
1642
1423
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
8b
9b
10b
a Values are the averages from at least four independent dose response curves;
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Cl
Cl
NH
NH
N
NH
O
O
HO
N
N
N
N
H3C
Roscovitine
NH
N
N
N
HO
HN N
H
N
N
N
H
H
CH3
CH3
Olomoucine
AT7159
O
O
H2N
H
S
N
N
O
N
NH
NH
N
H
O
PNU-292137
1H-Indazole-3-carboxylic Acid (4-Sulfamoylphenyl)amide
Figure 1. ATP-competitive inhibitors of CDKs
Figure 2. Crystal structure of compound 7a
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Figure 3. Correlation between the anti-proliferative activity against A549 cell line
and the CDK2 inhibitory activity, R2 = 0.84, which indicated that there was a
moderate correlation between CDK2 inhibition and inhibition of cancer cellular
proliferation.
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Figure 4. Effects of 10b on the cell cycle distribution in A549 cells. A549 cells were
treated with various concentrations of 10b. A) control; B) concentrations of 10b was
0.5 ꢀM; C) concentrations of 10b was 1.0 ꢀM; D) concentrations of 10b was 2.0 ꢀM.
A549 cells were treated with alcohol for 36 h as a control group, Values represent the
mean ± S.D, n =3. P <0.05 versus control.
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Figure 5. A) 2D molecular docking model of compound 10b with 2VTO; B) Model
of compound 10b bound to CDK2; C) The 2D diagram of docking structure of LZ8
with 2VTO; D) Model of compound LZ8 bound to CDK2.
28