Synthesis, biological evaluation and 3D-QSAR studies of novel 5-phenyl-1 H -pyrazol cinnamamide derivatives as novel antitubulin agents

Article abstract of DOI:10.1016/j.ejmech.2015.02.018

A series of novel 5-phenyl-1H-pyrazol derivatives (5a-5x) containing cinnamamide moiety were synthesized and their biological activities as potential tubulin polymerization inhibitors were evaluated. Among them, compound 5j exhibited the most potent inhibitory activity with an IC₅₀ value of 1.02 μ1/4M for tubulin, which was superior to that of Colchicine (IC50 Combining double low line 1.34 μ1/4M). Docking simulation was performed to insert compound 5j into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. 3D-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent tubulin inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2015.02.018

Accepted Manuscript
Synthesis, biological evaluation and 3D-QSAR studies of novel 5-phenyl-1H-pyrazol
cinnamamide derivatives as novel antitubulin agents
Shu-Fu Wang, Yong Yin, Ya-Liang Zhang, Shan-Wei Mi, Meng-Yue Zhao, Peng-
Cheng Lv, Bao-Zhong Wang, Hai-Liang Zhu
PII:
S0223-5234(15)00114-2
10.1016/j.ejmech.2015.02.018
EJMECH 7700
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 October 2014
Revised Date: 8 February 2015
Accepted Date: 12 February 2015
Please cite this article as: S.-F. Wang, Y. Yin, Y.-L. Zhang, S.-W. Mi, M.-Y. Zhao, P.-C. Lv, B.-Z.
Wang, H.-L. Zhu, Synthesis, biological evaluation and 3D-QSAR studies of novel 5-phenyl-1H-pyrazol
cinnamamide derivatives as novel antitubulin agents, European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.02.018.
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ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and 3D-QSAR studies of novel
5-phenyl-1H-pyrazol cinnamamide derivatives as novel antitubulin agents
Shu-Fu Wang +, Yong Yin +, Ya-Liang Zhang, Shan-Wei Mi, Meng-Yue Zhao,
Peng-Cheng Lv*, Bao-Zhong Wang*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
+ These authors contributed equally to this work.
* Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Compound 5j exhibited the most potent inhibitory activity with an IC50
value of 1.02 µM for tubulin, 0.35 µM for MCF-7, 0.62 µM for A549 and
0.57µM for B16-F10.

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and 3D-QSAR studies of novel
5-phenyl-1H-pyrazol cinnamamide derivatives as novel antitubulin agents
Shu-Fu Wang +, Yong Yin+, Ya-Liang Zhang, Shan-Wei Mi, Meng-Yue Zhao,
Peng-Cheng Lv*, Bao-Zhong Wang*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
+ These authors contributed equally to this work.
Abstract
A series of novel 5-phenyl-1H-pyrazol derivatives (5a-5x) containing
cinnamamide moiety were synthesized and their biological activities as potential
tubulin polymerization inhibitors were evaluated. Among them, compound 5j
exhibited the most potent inhibitory activity with an IC50 value of 1.02 µM for tubulin,
which was superior to that of Colchicine (IC50 = 1.34 µM). Docking simulation was
performed to insert compound 5j into the crystal structure of tubulin at colchicine
binding site to determine the probable binding model. 3D-QSAR model was also built
to provide more pharmacophore understanding that could be used to design new
agents with more potent tubulin inhibitory activity.
Keywords: Pyrazole; Cinnamamide; Antitubulin agent; Molecular docking;
3D-QSAR
Abbreviations: SAR, structure-activity relationship; IC50, half maximal inhibitory
concentration; MTT, 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
* Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn
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1. Introduction
Microtubules are major cytoskeletal protein polymers in all eukaryotic cells and
involved in numerous cellular functions including chromosome segregation,
intracellular transport, cell motility, and cell shape.[1] Microtubules assemble and
disassemble by a reversible process involving discrete α/β tubulin heterodimers. This
dynamic instability feature along with treadmilling gives microtubules significant role
in mitosis and cell division.[2] During cell mitosis, microtubules mediate the timely
and correct attachment of chromosomes at their kinetochores to the spindle, the
complex movements of the chromosomes that bring them to their properly aligned
positions and the synchronous separation of the chromosomes.[3] Given the
importance of microtubules in cell mitosis, microtubules or tubulins serve as effected
molecular targets for cancer chemotherapeutic agents.
There are two classes of these chemotherapeutic agents, microtubule stabilizers
(e.g., paclitaxel and docetaxel), and microtubule destabilizers (e.g., vinblastine and
vincristine). Both types interfere with the mitotic spindle assembly during cell
division and block cell cycle in G2/M phases, resulting in cell death.[4-6] Colchicine
(Figure 1) is the first identified antimitotic drug that inhibits microtubule
polymerization by binding to tubulin, and its binding site has been well
characterized.[7] Several other new compounds such as E7010, HMN-214, and CA-4
(Figure 1) have also attracted much interest as antitubulin agents. They bound
tubulins through colchicine binding site.[8]
Chalcones are the biogenetic precursors of all known flavonoids and exhibit a
variety of biological activities including anticancer,[9-11] anti-inflammatory,[12.13]
anti-tuberculosis,[12] anti-fungal and antiproliferative activities.[14-22] Antimitotic
agents
like
trans-1-(2,5-dimethoxy)-3-[4
(dimethylamino)
phenyl]-2-methyl-2-propen-1-one (MDL) possess a chalcone skeleton and display
rapid and reversible binding to the colchicine binding site of β-tubulin at the interface
with α-tubulin and cause inhibition of its assembly to microtubules.[23] Researches
showed that their broad biological properties are largely due to the α, β-unsaturated
ketone moiety.[24]
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As shown in
＞
Figure 2,＞chalcone and cinnamon amide share similar part of the
active structure. Cinnamic acids are abundant in various natural resources and its
natural analogues are unique as anticancer agents.[25] A lot of cinnamido compounds
were also synthesized and their anticancer abilities were evaluated by our research
groups.[26-28] Besides, Pyrazole-based heterocycles play a crucial role in the arena of
rigidified combretastatin analogues.[29] Lee and co-workers described a series of
3,5-diarylpyrazoles that display low cytotoxicity in tumor cell lines due to their planar
conformation.[30] These previous researches encouraged us to integrate cinnamon
amide with pyrazoles to screen compounds which have potent anticancer activities.
Herein we report the synthesis and bioactivities of
a
series of
5-phenyl-1H-pyrazol derivatives containing cinnamoyl moiety. Biological evaluation
indicated that some of the synthesized compounds were potent inhibitors of tubulin.
Docking simulation was performed using the X-ray crystallographic structure of
tubulin to explore the binding mode of the compound at the active site.
2. Results and discussion
2.1. Chemistry
The synthetic route of the pyrazol derivatives(5a-5x) was outlined in Scheme 1.
First of all, 2a-2c were synthesized in THF by treating p-substituted benzoate with
acetonitrile using NaH as catalyst. Secondly, compounds 2a-2c and hydrazine hydrate
were dissolved in ethanol and refluxed for 1 h to get compounds 3a-3c. Lastly, the
coupling reaction between the obtained compounds 3a-3c and nicotinic acids was
performed by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCl) and N-hydroxybenzotriazole (HOBt) in anhydrous methylene dichloride, and
refluxed to give the desired compounds 5a-5x. All of the synthesized compounds
were given satisfactory analytical and spectroscopic data, which were in full
accordance with their depicted structures.
2.2. Antiproliferative effects against cancer cells
To test the anticancer activities of the synthesized compounds, we evaluated
antiproliferative activities of compounds 5a-5x against MCF-7, A549 and B16-F10
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cancer cells. The results were summarized in Table 1. These 5-phenyl-1H-pyrazol
derivatives bearing cinnamoyl moiety displayed remarkable antiproliferative effects.
The results demonstrated that a p-substituted electron-withdrawing or halogen
group on phenyl ring A may have slightly improved antiproliferative activities in the
order 4-CF3ꢀ4-NO2ꢀ4-OMeꢀ4-Fꢀ4-Clꢀ4-BrꢀH>4-Me. In the case of constant
A ring substituents, change of substituents on B-ring could also affect the activities of
compounds and the inhibitory activities of compounds with different substituents on
ring B. The activities increased in the following order: 4-Me<H<4-Br, suggesting that
an electron-withdrawing could enhance the competence of the pyrazol derivatives.
2.3. Tubulin polymerization inhibition
To examine whether the compounds interact with tubulin and inhibit tubulin
polymerization in vitro, we performed the abilities of the synthesized compounds in
inhibiting the polymerization of tubulin. The IC50 of the compounds were summarized
in Table 2. It was observed that all the synthesized compounds showed inhibitory
activities of tubulin displaying IC50 values between 1.02 and 3.36 µM. Among them,
compound 5j displayed the most potent anti-tubulin polymerization activity with IC50
of 1.02 µM.
2.4. Effects on cell cycle against cancer cells
To gain further insight into the action mode of these compounds, the most potent
one was further assayed for its effects on cell cycle using flow cytometry (Figure 3).
As is shown in Figure 3, compound 5j strongly induced G2/M arrest in MCF-7 cells,
＞
and the effect was observed in a dose-independent manner after treatment with
increasing dose of the compound. About 45.45% of the cells were arrested in the
G2/M phase while 76.95% of the cells were found to be in the G2/M phase
＞
after
treatment with 5j of 1 and 2 ꢀg/mL for 24 h, respectively. These findings indicated a
＞
continuing impairment of cell division and confirmed compound 5j was a potent
antitubulin agent.
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2.5. Molecular docking study
To gain better understanding on the potency of the synthesized compounds and
guide further SAR studies, we proceeded to examine the interaction of the
5-phenyl-1H-pyrazol derivatives with tubulin (PDB code: 1SA0). The molecular
docking was performed by inserting synthetic compounds into the colchicine binding
site in tubulin. All docking runs were applied LigandFit Dock protocol of Discovery
Studio 3.5. The binding model of compound 5j and tubulin was depicted in Figure 4.
and the enzyme surface model was shown in Figure 5, which revealed that the
molecule is well filled in the active pocket.
In the binding mode, compound 5j is nicely bound to tubulin via two hydrogen
bonds. The hydrogen atom on the pyrazole ring with the oxygen atom on the main
chain of Ser178 (angle N-H…O = 160.6°, distance = 2.40 Å) and the fluorine atom of
CF3 with the hydrogen atom on the hydroxy of Tyr202 (angle F…H-O = 113.3°,
distance = 2.13 Å) contribute to the hydrogen bonding interaction together, being a
probable explanation for its nice activity. Meanwhile, the receptor surface model
showed in Figure 5 revealed that this candidate inhibitor was tightly embedded into
the active pocket of tubulin. This molecular docking result, along with the enzyme
assay data, suggested that compound 5j is a potential inhibitor of tubulin.
2.6. 3D-QSAR
In order to acquire a systematic SAR profile on the synthesized compounds and
to explore the more powerful and selective tubulin inhibitors, 3D-QSAR model was
built to choose activity conformation of the designed molecular and reasonably
evaluated the designed molecules by using the corresponding pIC50 values which
were converted from the obtained IC50 (µM) values of tubulin inhibition and
performed by built-in QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co.
Ltd). By convention, the pIC50 scale (-logIC50), in which higher values indicate
exponentially greater potency, is used as a method to measure inhibitory activity. The
training and test set was chosen by the Diverse Molecules method in Discovery
Studio. Considering a good alignment is very important for the analysis of molecular
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fields, we applied CDOCKER protocol to explore each molecule with lowest energy
before alignment conformation. 5-phenyl-1H-pyrazol was selected as substructure to
build alignment conformation before building the QSAR model.
The correlation coefficient r2 between observed and predicted activity of training
set was found to be 0.991, while that of test set was found to be 0.796, which proved
that the QSAR model built by us was acceptable. The q2 of 5-fold cross validation
result is 0.603 while the q2 of validation result using external test set is 0.796. It
means that the model performs well with compounds aligned by docking and the
predictive ability of our QSAR model is acceptable. Predicted pIC50 values and
residual errors of 24 compounds by this QSAR model had been given in Table 3. The
well agreement between predicted pIC50 value and experimental pIC50 value for both
test sets and training sets are shown in Figure 6.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR model
coefficients on electrostatic potential grids (Figure 7a) and van der Waals grids
(Figure 7b) are listed. Electrostatic map indicates red contours around regions where
high electron density (negative charge) is expected to increase activity, and blue
contours represent areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates areas where steric bulk is
predicted to increase (green) or decrease (yellow) activity.
According to the maps, it was suggested that the compound with high negative
charged and bulky R2 group would show higher activity, validating that CF3
substituted group being a better choice than mono fluorine substituted group and F
substituent better than Cl as well as Cl substituent better than Br. Meanwhile, we can
get from the maps that compound with high negative charged R1 group would show
better activity than compound with low negative charged R1 group, validating that Br
substituent better than H and CH3. As a result, data summarized above demonstrated
that compounds 5j, the most potent tubulin inhibitor (IC50 = 1.02 µM), which contains
suitable substituents, had an outstanding activity.
3. Conclusion
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In this paper, a series of novel tubulin inhibitors beared pyrazole core and
cinnamamide moiety have been synthesized and their biological activities were
evaluated. Among them, compound 5j exhibited the most potent tubulin inhibition
activities (IC50 = 1.02 µM for tubulin) and antiproliferative activities (IC50 = 0.35 µM
for MCF-7, IC50 = 0.62 µM for A549 and IC50 = 0.57 µM for B16-F10). Docking
simulation was performed to put compound 5j into the colchicine binding site of
tubulin to determine the potential binding model and found that several interactions
with the protein residues in the colchicine binding site might play an important role in
its antitubulin polymerization and antiproliferative activities. QSAR model was built
to provide a reliable tool for reasonable design of novel tubulin inhibitors in future.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were analytical grade. The
reactions were monitored by glass-backed silica gel sheets (Silica Gel 60 GF254).
Column chromatography was performed using silica gel (200–300 mesh) eluting with
ethyl acetate and petroleum ether. Melting points (uncorrected) were determined on a
XT4MP apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a
1
Mariner System 5304 mass spectrometer, and H NMR spectra were collected on a
Bruker DPX400 or DPX300 model spectrometer at room temperature with TMS and
solvent signals allotted as internal standards. The solvent signals allotted as internal
stands. Elemental analyses were performed on a CHN-O-Rapid instrument. All the
compounds gave satisfactory chemical analyses (±0.4%).
4.2. General procedure for the synthesis of 2a-2c
Mixed p-substituted methyl benzoate (7 mmol) with NaH (14 mmol, 0.35 g) in
boiling tetrahydrofuran (5 mL) and then followed by dropwise addition of solution of
acetonitrile (7 mmol, 0.29g, 0.4 mL) in tetrahydrofuran (1 mL). The resulting mixture
was refluxed for 4 h and then cooling down to room temperature, after which the
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solution was diluted with diethylether (15 mL) and left to stand at room temperature
for 48 h. The precipitated sodium salt was filtered and washed with diethylether. The
dry compound was dissolved in water (5 mL) and acidified with HCl (1mol/L) to pH
2. The collected extracts were crystallized from toluene, filtered and dried with
Na2SO4.
4.3. General procedure for the synthesis of 3a-3c
To a stirred mixture of compound 2a-2c (5 mmol) in EtOH (25 mL) were added
NH2NH2·H2O (0.3 mL, 6 mmol) and CH3SO3H (0.1 mL, 1 mmol) at room
temperature and the mixture was stirred under reflux for 1h. The precipitate was
purified by column chromatograghy over silica gel to give the compound 3a-3c.
4.4.General procedure for the synthesis of 5-phenyl-1H-pyrazol derivatives
(5a-5x)
Compounds 5a-5x were synthesized by coupling substituted 3a-3c with cinnamic
acids, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCl)
and N-hydroxybenzotriazole (HOBt) as condensing agent. The mixture was refluxed
in anhydrous CH2Cl2 for 1-3 h. The products were extracted with ethyl acetate. The
extract was washed successively with 5% HCl, then evaporated and purified by
column chromatograghy over silica gel to give the compound.
4.4.1
(E)-3-(4-nitrophenyl)-N-(5-phenyl-1H-pyrazol-3-yl)acrylamide(5a)
Yellow powder, yield: 74%. m.p. 229-230 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.90 (s, 1H); 6.88 (s, 2H); 7.46 (m, 3H); 7.97 (m, 3H); 8.14 (m, 3H); 8.32 (d, J
= 8.32 Hz, 2H). MS (ESI): 335.43 ([M+H]+). Anal. Calcd for C18H14N4O3: C, 64.66;
H, 4.22; N, 16.76%; Found: C, 66.49; H, 4.37; N, 16.51%.
4.4.2
de(5b)
Yellow powder, yield: 69%. m.p. 183-184 °C. 1H NMR (300 MHz, DMSO, δ
(Z)-N-(5-phenyl-1H-pyrazol-3-yl)-3-(4-(trifluoromethyl)phenyl)acrylami
ppm): 5.89 (s, 1H); 6.87 (s, 2H); 7.43-7.50 (m, 3H); 7.85 (d, J = 5.97 Hz, 2H);
7.92-8.00 (m, 3H); 8.07-8.12 (m, 3H). MS (ESI): 358.13 ([M+H]+). Anal. Calcd for
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C19H14F3N3O: C, 63.86; H, 3.95; N, 11.76%; Found: C, 63.63; H, 3.82; N, 11.54%.
4.4.3
N-(5-phenyl-1H-pyrazol-3-yl)cinnamamide(5c)
Yellow powder, yield: 59%. m.p. 198-199 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.88 (s, 1H); 6.85 (s, 2H); 7.47 (m, 6H); 7.84-8.02 (m, 6H). MS (ESI): 290.24
([M+H]+). Anal. Calcd for C18H15N3O: C, 74.72; H, 5.23; N, 14.52%; Found: C, 74.48;
H, 5.46; N, 14.37%.
4.4.4
(E)-3-(4-methoxyphenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5d)
Yellow powder, yield: 69%. m.p. 166-167 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.88 (s, 1H); 6.84 (s, 2H); 7.34 (t, J =13.2, 2H); 7.45 (m, 3H); 7.94 (m, 6H).
MS (ESI): 308.53 ([M+H]+). Anal. Calcd for C18H14FN3O: C, 70.35; H, 4.59; N,
13.67%; Found: C, 70.23; H, 4.54; N, 13.34%.
4.4.5
(E)-3-(4-chlorophenyl)-N-(5-phenyl-1H-pyrazol-3-yl)acrylamide(5e)
Yellow powder, yield: 75%. m.p. 185-186 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.88 (s, 1H); 6.85 (s, 2H); 7.46 (m, 3H); 7.57 (d, J = 6.42 Hz, 2H); 7.95 (m,
6H). MS (ESI): 324.78 ([M+H]+). Anal. Calcd for C18H14ClN3O: C, 66.77; H, 4.36; N,
12.98%; Found: C, 66.49; H, 4.55; N, 13.14%.
4.4.6
(E)-N-(5-phenyl-1H-pyrazol-3-yl)-3-(p-tolyl)acrylamide(5f)
Yellow powder, yield: 55%. m.p. 189-190 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.38 (s, 3H); 5.88 (s, 1H); 6.84 (s, 2H); 7.32 (d, J = 5.97 Hz, 2H); 7.45 (m, 3H);
7.34 (d, J = 6.06 Hz, 2H); 7.92 (m, 4H); MS (ESI): 304.34 ([M+H]+). Anal. Calcd for
C19H17N3O: C, 75.23; H, 5.65; N, 13.85%; Found: C, 75.43; H, 5.44; N, 13.74%.
4.4.7
(Z)-3-(4-methoxyphenyl)-N-(5-phenyl-1H-pyrazol-3-yl)acrylamide(5g)
Yellow powder, yield: 69%. m.p. 166-167 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 3.84 (s, 3H); 5.87 (s, 1H); 6.83 (s, 2H); 7.06 (d, J = 6.18 Hz, 2H); 7.42-7.49 (m,
3H); 7.81-7.87 (m, 4H); 7.92 (d, J = 5.85 Hz, 2H). MS (ESI): 320.41 ([M+H]+). Anal.
Calcd for C19H17N3O2: C, 71.46; H, 5.37; N, 13.16%; Found: C, 71.63; H, 5.44; N,
13.34%.
4.4.8
(E)-3-(4-bromophenyl)-N-(5-phenyl-1H-pyrazol-3-yl)acrylamide(5h)
Yellow powder, yield: 58%. m.p. 209-210 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.88 (s, 1H); 6.85 (s, 2H); 7.46 (m, 3H); 7.70 (d, J = 6.36 Hz, 2H); 7.81-7.93
(m, 5H); 7.99 (d, J = 12.09 Hz, 1H). MS (ESI): 369.32 ([M+H]+). Anal. Calcd for
C18H14BrN3O: C, 58.71; H, 3.83; N, 11.41%; Found: C, 58.61; H, 3.76; N, 11.67%.
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4.4.9
(5i)
Yellow powder, yield: 55%. m.p. 230-231 °C. 1H NMR (300 MHz, DMSO, δ
(E)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(4-nitrophenyl)acrylamide
ppm): 5.91 (s, 1H); 6.91 (s. 2H); 7.67 (d, J = 6.36 Hz, 2H); 7.90 (d, J = 6.39 Hz, 2H);
7.99 (d, J = 12.12 Hz, 1H); 8.13 (m, 3H); 8.32 (d, J = 6.57 Hz, 2H). MS (ESI): 414.22
([M+H]+). Anal. Calcd for C18H13BrN4O3: C, 52.32; H, 3.17; N, 13.56%; Found: C,
52.49; H, 3.22; N, 13.79%.
4.4.10 (E)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(4-(trifluoromethyl)phenyl
)acrylamide(5j)
Yellow powder, yield: 82%. m.p. 180-181 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.90 (s, 1H); 6.91 (s, 2H); 7.66 (d, J = 6.33 Hz, 2H); 7.87 (m, 4H); 7.95-8.09
(m, 4H). MS (ESI): 437.62 ([M+H]+). Anal. Calcd for C19H13BrF3N3O: C, 52.31; H,
3.00; N, 9.63%; Found: C, 52.49; H, 2.77; N, 9.58%.
4.4.11 N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)cinnamamide(5k)
Yellow powder, yield: 65%. m.p. 215-216 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.90 (s, 1H); 6.89 (s, 2H); 7.51 (t, J =4.74, 3H); 7.66 (d, J = 6.39 Hz, 2H);
7.84-8.00 (m, 6H). MS (ESI): 369.11 ([M+H]+). Anal. Calcd for C18H14BrN3O: C,
58.71; H, 3.83; N, 11.41%; Found: C, 58.49; H, 3.65; N, 11.14%.
4.4.12 (Z)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(4-fluorophenyl)acrylamid
e(5l)
Yellow powder, yield: 75%. m.p. 199-200 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.89 (s, 1H); 6.89 (s, 2H); 7.32-7.36 (m, 2H); 7.65 (d, J = 6.33 Hz, 2H);
7.88-7.96 (m, 6H). MS (ESI): 387.34 ([M+H]+). Anal. Calcd for C18H13BrFN3O: C,
55.98; H, 3.39; N, 10.88%; Found: C, 55.79; H, 3.22; N, 10.77%.
4.4.13 (E)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(4-chlorophenyl)acrylami
de(5m)
Yellow powder, yield: 66%. m.p. 179-180 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.89 (s, 1H); 6.88 (s, 2H); 7.49 (d, J = 5.55 Hz, 1H); 7.57 (d, J = 6.36 Hz, 2H);
7.66 (d, J = 6.39 Hz, 2H); 7.90 (m, 5H). MS (ESI): 403.57 ([M+H]+). Anal. Calcd for
C18H13BrClN3O: C, 53.69; H, 3.25; N, 10.44%; Found: C, 53.47; H, 3.43; N, 10.57%.
4.4.14 (E)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(p-tolyl)acrylamide(5n)
Yellow powder, yield: 36%. m.p. 213-214 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.38 (s, 3H); 5.89 (s, 1H); 6.87 (s, 2H); 7.33 (d, J = 5.91 Hz, 2H); 7.66 (d, J =
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6.39 Hz, 2H); 7.75 (d, J = 6.09 Hz, 2H); 7.89 (m, 4H). MS (ESI): 383.19 ([M+H]+).
Anal. Calcd for C19H16BrN3O2: C, 57.30; H, 4.05; N, 10.55%; Found: C, 57.21; H,
4.09; N, 10.32%.
4.4.15 (E)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)-3-(4-methoxyphenyl)acryla
mide(5o)
Yellow powder, yield: 65%. m.p. 185-186 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 3.85 (s, 3H); 5.88 (s, 1H); 6.86 (s, 2H); 7.06 (d, J = 6.57 Hz, 2H); 7.65 (d, J =
6.36 Hz, 2H); 7.84 (m, 6H). MS (ESI): 399.23 ([M+H]+). Anal. Calcd for
C19H16BrN3O2: C, 57.30; H, 4.05; N, 10.55%; Found: C, 57.49; H, 4.22; N, 10.77%.
4.4.16 (E)-3-(4-bromophenyl)-N-(5-(4-bromophenyl)-1H-pyrazol-3-yl)acrylami
de (5p)
Yellow powder, yield: 65%. m.p. 186-187 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 5.90 (s, 1H); 6.88 (s, 2H); 7.65 (m, 4H); 7.71 (d, J = 6.36 Hz, 3H); 7.82 (d, J =
6.39 Hz, 2H); 7.98 (d, J = 6.09 Hz, 1H). MS (ESI): 448.41 ([M+H]+). Anal. Calcd for
C18H13Br2N3O: C, 48.35; H, 2.93; N, 9.40%; Found: C, 48.54; H, 2.65; N, 9.14%.
4.4.17 (E)-3-(4-nitrophenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5q)
Yellow powder, yield: 54%. m.p. 209-210 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.86 (s, 1H); 6.86 (s, 2H); 7.28 (d, J = 5.97 Hz, 2H); 7.82 (d, J =
6.09 Hz, 2H); 7.98-8.15 (m, 4H); 8.32 (d, J = 6.57 Hz, 2H). MS (ESI): 349.35
([M+H]+). Anal. Calcd for C19H16N4O3: C, 65.51; H, 4.63; N, 16.08%; Found: C,
66.49; H, 4.47; N, 16.32%.
4.4.18 (E)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)-3-(4-(trifluoromethyl)phenyl)acrylam
ide(5r)
Yellow powder, yield: 78%. m.p. 171-172 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.85 (s, 1H); 6.85 (s, 2H); 7.28 (d, J = 6.06 Hz, 2H); 7.83 (m, 4H);
8.03 (m, 4H). MS (ESI): 372.44 ([M+H]+). Anal. Calcd for C20H16F3N3O: C, 64.69; H,
4.34; N, 11.32%; Found: C, 64.79; H, 4.47; N, 11.48%.
4.4.19 N-(5-(p-tolyl)-1H-pyrazol-3-yl)cinnamamide(5s)
Yellow powder, yield: 69%. m.p. 196-197 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.85 (s, 1H); 6.83 (s, 2H); 7.27 (d, J = 5.94 Hz, 2H); 7.50 (t, J =
4.71 Hz, 3H); 7.8-8.0 (m, 6H). MS (ESI): 304.52 ([M+H]+). Anal. Calcd for
C19H17N3O: C, 75.23; H, 5.65; N, 13.85%; Found: C, 75.47; H, 5.77; N, 13.56%.
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4.4.20 (E)-3-(4-fluorophenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5t)
Yellow powder, yield: 65%. m.p. 186-187 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.84 (s, 1H); 6.82 (s, 2H); 7.31 (m, 4H); 7.80-7.96 (m, 6H). MS
(ESI): 322.63 ([M+H]+). Anal. Calcd for C19H16FN3O: C, 71.01; H, 5.02; N, 13.08%;
Found: C, 69.94; H, 5.65; N, 11.14%.
4.4.21 (E)-3-(4-chlorophenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5u)
Yellow powder, yield: 65%. m.p. 180-181 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.84 (s, 1H); 6.83 (s, 2H); 7.27 (d, J = 6.00 Hz, 2H); 7.56 (d, J =
6.36 Hz, 2H); 7.81 (d, J = 6.06 Hz, 2H); 7.94 (m, 4H). MS (ESI): 338.86 ([M+H]+).
Anal. Calcd for C19H16ClN3O: C, 67.56; H, 4.77; N, 12.44%; Found: C, 66.79; H,
4.52; N, 12.78%.
4.4.22 (E)-3-(p-tolyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5v)
Yellow powder, yield: 66%. m.p. 143-144 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.37 (d, J = 6.06 Hz, 2H); 5.84 (s, 1H); 6.82 (s, 2H); 7.27 (d, J = 6.00 Hz, 2H);
7.32 (d, J = 5.91 Hz, 2H); 7.74 (d, J = 6.00 Hz, 2H); 7.81 (d, J = 6.03 Hz, 2H); 7.90 (d,
J = 6.15 Hz, 2H). MS (ESI): 318.12 ([M+H]+). Anal. Calcd for C19H16BrN3O2: C,
75.69; H, 6.03; N, 13.24%; Found: C, 74.21; H, 6.09; N, 13.32%.
4.4.23 (E)-3-(4-methoxyphenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5w)
Yellow powder, yield: 69%. m.p. 166-167 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 3.84 (s, 3H); 5.83 (s, 1H); 6.81 (s, 2H); 7.06 (d, J = 6.60 Hz, 2H);
7.27 (d, J = 5.94 Hz, 2H); 7.84 (m, 6H). MS (ESI): 334.23 ([M+H]+). Anal. Calcd for
C20H19N3O2: C, 72.05; H, 5.74; N, 12.60%; Found: C, 72.23; H, 5.54; N, 12.34%.
4.4.24 (E)-3-(4-bromophenyl)-N-(5-(p-tolyl)-1H-pyrazol-3-yl)acrylamide(5x)
Yellow powder, yield: 78%. m.p. 187-188 °C. 1H NMR (300 MHz, DMSO, δ
ppm): 2.36 (s, 3H); 5.84 (s, 1H); 6.83 (s, 2H); 7.27 (d, J = 5.94 Hz, 2H); 7.70 (d, J =
6.36 Hz, 2H); 7.85 (m, 5H); 7.99 (d, J = 12.06 Hz, 1H). MS (ESI): 383.38 ([M+H]+).
Anal. Calcd for C19H16BrN3O: C, 59.70; H, 4.22; N, 10.99%; Found: C, 59.56; H,
4.35; N, 10.78%.
4.5.Antiproliferation activity
The antiproliferative activities of the prepared compounds against MCF-7, A549
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and B16-F10 cells was evaluated as described elsewhere with some modifications.[17]
Target tumor cell line was grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 × 104 cells/mL with the complete
medium, 100 µL of the obtained cell suspension was added to each well of 96-well
culture plates. The subsequent incubation was permitted at 37 oC, 5% CO2 atmosphere
for 24 h before the cytotoxicity assessments. Tested samples at pre-set concentrations
were added to 6 wells with colchicine and CSA-4 coassayed as positive reference.
After 48 h exposure period,40 µL of PBS containing 2.5 mg/mL of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to each
well. Four hours later, 100 µL extraction solution (10% SDS-5% isobutyl
＞
＞
o
alcohol-0.01M HCl) was added. After an overnight incubation at 37 C, the optical
density was measured at a wavelength of 570 nm on an ELISA microplate reader. In
all experiments three replicate wells were used for each drug concentration. Each
assay was carried out for at least three times. The results were summarized in Table 1.
4.6.Effects on tubulin polymerization
Bovine brain tubulin was purified as described previously.[31] To evaluate the
effect of the compounds on tubulin assembly in vitro,[32] varying concentrations were
preincubated with 10 ꢀM tubulin in glutamate buffer at 30 ℃ and then cooled to
0 ℃. After addition of GTP, the mixtures were transferred to 0 ℃ cuvettes in a
recording spectrophotometer and warmed up to 30 ℃ and the assembly of tubulin
was observed turbidimetrically. The IC50 was defined as the compound concentration
that inhibited the extent of assembly by 50% after 20 min incubation. In all
experiments three replicate wells were used for each drug concentration. The results
were summarized in Table 2.
4.7.Docking simulations
The crystal structures of tubulin (PDB code: 1SA0) was obtained from the
Protein Data Bank (http://www.rcsb.org). Molecular docking of compound 5j into the
three-dimensional X-ray structure of tubulin was carried out using CDOCKER Dock
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protocol of Discovery Studio 3.5. All bound water and ligands were eliminated from
the protein and the polar hydrogen was added. The whole tubulin complex was
defined as a receptor and the site sphere was selected based on the ligand binding
location of colchicine, then the colchicine molecule was removed and 5j was placed
during the molecular docking procedure. Types of interactions of the docked protein
with ligand were analyzed after the end of molecular docking.
4.8.QSAR model
79% (that is 19) of the compound were utilized as a training set for QSAR
modeling and the remaining 21% (that is 5) were chosen as an external test subset for
validating the reliability of the QSAR model by the Diverse Molecules protocol in
Discovery Studio 3.5. The selected test compounds were: 5e, 5h, 5j, 5m, 5o.
The inhibitory activities of the compounds in literatures [IC50 (mol/L)] was
initially changed into the minus logarithmic scale [IC50 (mol/L)] and then used for
subsequent QSAR analysis as the response variable.
In Discovery Studio, the CHARMm force field is used and the electrostatic
potential and the van der Waals potential are treated as separate terms. A +1e point
charge is used as the electrostatic potential probe and distance-dependent dielectric
constant is used to mimic the solvation effect. For the van der Waals potential a
carbon atom with a 1.73 Å radius is used as a probe.
A Partial Least-Squares (PLS) model is built using energy grids as descriptors.
QSAR models were built by using the Create 3D QSAR Model protocol in Discovery
Studio 3.5.
Acknowledgements
This work was supported by Natural Science Foundation of Jiangsu Province
(No. BK20130554) and supported by Major Projects on Control and Rectification of
Water Body Pollution (No. 2011ZX07204-001-2214 004).
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Figure Captions
Table 1. Structural features, inhibition (IC50) of MCF-7, A549 and B16-F10 cells
proliferation by compounds 5a-5x
Table 2. Tubulin inhibitory activities of synthetic compounds.
Table 3. Experimental, predicted inhibitory activities of compounds 5a-5x by
3D-QSAR models based upon active conformation achieved by molecular docking.
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
Figure 2. Similar part of chalcone and cinnamon amide.
Figure 3. Effects of compound 5j on cell cycle progression of MCF-7 cells were
determined by flow cytometry analysis. MCF-7 cells were treated with different
concentrations of 5j for 24 h. The percentage of cells in each cycle phase was
indicated.
Figure 4. Binding mode of compound 5j with tubulin. For clarity, only interacting
residues were labeled. Hydrogen bonding interactions are shown in dotted lines. This
figure was made using Discovery Studio 3.5.
Figure 5. Binding mode of compound 5j with tubulin. The enzyme is shown as
surface; while 5j docked structures are shown as sticks. This figure was made using
Discovery Studio 3.5.
Figure 6. Plot of experimental versus predicted tubulin inhibitory activities of training
set and test set.
Figure 7. (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue
represents positive coefficients; red represents negative coefficients. (b) 3D-QSAR
model coefficients on van der Waals grids. Green represents positive coefficients;
yellow represents negative coefficients.
Scheme 1. Synthesis of compounds 5a-5x. Reagents and conditions: (a) CH3CN, NaH,
THF, reflux, 4 h; (b) NH2NH2·H2O, CH3SO3H, EtOH, reflux, 1 h; (c) EDCl, HOBt,
dichloromethane, reflux, 1 h.
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Table 1. Structural features, inhibition (IC50) of MCF-7, A549 and B16-F10 cells
proliferation by compounds 5a-5x
compound
R1
R2
IC50±SD (µM)a
MCF-7b
A549c
B16-F10d
5a
5b
H
H
4-NO2
4-CF3
H
0.63±0.08
0.54±0.23
1.36±0.34
0.84±0.16
0.97±0.24
1.78±0.18
0.72±0.33
1.24±0.14
0.56±0.22
0.35±0.08
1.12±0.16
0.64±0.25
0.77±0.17
1.17±0.06
0.59±0.08
0.92±0.15
0.84±0.09
0.72±0.21
1.63±0.03
0.95±0.17
1.19±0.14
1.72±0.25
0.86±0.07
1.37±0.34
0.39±0.05
0.21±0.06
0.97±0.11 0.72±0.04
0.88±0.09 0.64±0.06
1.58±0.14 1.56±0.17
1.22±0.16 1.25±0.15
1.39±0.24 1.27±0.34
1.77±0.21 1.68±0.36
1.06±0.18 0.95±0.07
1.46±0.31 1.38±0.14
0.74±0.24 0.63±0.09
0.62±0.18 0.57±0.17
1.44±0.06 1.27±0.22
0.93±0.17 0.85±0.16
1.21±0.08 1.08±0.08
1.62±0.05 1.48±0.09
0.82±0.22 0.77±0.16
1.36±0.24 1.28±0.18
1.13±0.16 1.32±0.27
1.02±0.11 1.21±0.32
1.67±0.13 1.68±0.21
1.25±0.09 1.43±0.44
1.49±0.35 1.52±0.38
1.88±0.33 1.75±0.34
1.19±0.41 1.37±0.25
1.59±0.21 1.57±0.36
0.58±0.08 0.48±0.07
0.46±0.04 0.42±0.06
5c
H
5d
H
4-F
5e
H
4-Cl
5f
H
4-Me
4-OMe
4-Br
4-NO2
4-CF3
H
5g
H
5h
H
5i
Br
5j
Br
5k
Br
5l
Br
4-F
5m
5n
Br
4-Cl
Br
4-Me
4-OMe
4-Br
4-NO2
4-CF3
H
5o
Br
5p
Br
5q
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
5r
5s
5t
4-F
5u
4-Cl
5v
4-Me
4-OMe
4-Br
5w
5x
Colchicine
CA-4
a The averaged values determined by at least three separate determinations.
b Inhibition of the growth of MCF-7 cell lines.
c Inhibition of the growth of A549 cell lines.
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d Inhibition of the growth of B16-F10 cell lines.
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Table 2. Tubulin inhibitory activities of synthetic compounds.
compound
R1
R2
IC50±SD(µM)a
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4-NO2
4-CF3
H
4-F
4-Cl
4-Me
4-OMe
4-Br
4-NO2
4-CF3
H
1.56±0.21
1.34±0.34
2.84±0.27
1.85±0.22
2.06±0.19
3.02±0.27
1.64±0.15
2.36±0.24
1.24±0.11
1.02±0.09
2.55±0.26
1.78±0.18
1.95±0.22
2.76±0.39
1.35±0.17
2.14±0.33
1.62±0.16
1.53±0.18
3.12±0.29
1.92±0.28
2.15±0.44
3.36±0.36
1.76±0.16
2.55±0.35
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
4-F
4-Cl
4-Me
4-OMe
4-Br
4-NO2
4-CF3
H
4-F
4-Cl
4-Me
4-OMe
4-Br
5s
5t
5u
5v
5w
5x
Colchicine
1.34±0.23
0.68±0.16
CA-4
a The averaged values determined by three separate determinations.
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Table 3. Experimental, predicted inhibitory activities of compounds 5a-5x by
3D-QSAR models based upon active conformation achieved by molecular docking
compound a
tubulin
Actual pIC50
Residual error
Predicted pIC50
5.845
5.888
5.633
5.757
5.690
5.523
5.763
5.648
5.781
5.962
5.539
5.712
5.739
5.519
5.817
5.696
5.790
5.816
5.555
5.687
5.717
5.467
5.760
5.623
5a
5b
5c
5d
5e
5f
5.807
5.873
5.547
5.733
5.686
5.52
-0.038
-0.015
-0.086
-0.024
-0.004
-0.003
0.022
-0.021
0.126
0.029
0.054
0.038
0.029
0.041
0.053
-0.026
0.000
-0.001
-0.049
0.030
-0.049
0.007
-0.006
-0.030
5g
5h
5i
5.785
5.627
5.907
5.991
5.593
5.75
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5.71
5.56
5.87
5.67
5.79
5.815
5.506
5.717
5.668
5.474
5.754
5.593
5t
5u
5v
5w
5x
a Underlined compounds were selected as the test sets while the rest ones were in the
training sets.
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MeO
MeO
OMe O
OMe
MeO
MeO
NHAc
O
OMe
OMe
OH
N
OMe
OMe
1 Colchicine
2 Combretastain A-4 (CA-4)
3 MDL-27048
H
O
S
N
O
O
O
N
O
HN
S
N
N O
O
O
OH
3 E7010
4 HMN-214
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
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Figure 2. Similar part of chalcone and cinnamon amide.
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Figure 3. Effects of compound 5j on cell cycle progression of MCF-7 cells were
determined by flow cytometry analysis. MCF-7 cells were treated with different
concentrations of 5j for 24 h. The percentage of cells in each cycle phase was
indicated.
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Figure 4. Binding mode of compound 5j with tubulin. For clarity, only interacting
residues were labeled. Hydrogen bonding interactions are shown in dotted lines. This
figure was made using Discovery Studio 3.5.
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Figure 5. Binding mode of compound 5j with tubulin. The enzyme is shown as
surface; while 5j docked structures are shown as sticks. This figure was made using
Discovery Studio 3.5.
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Figure 6. Plot of experimental versus predicted tubulin inhibitory activities of training
set and test set.
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Figure 7. (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue
represents positive coefficients; red represents negative coefficients. (b) 3D-QSAR
model coefficients on van der Waals grids. Green represents positive coefficients;
yellow represents negative coefficients.
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