A class of novel tubulin polymerization inhibitors exert effective anti-tumor activity via mitotic catastrophe

Article abstract of DOI:10.1016/j.ejmech.2018.12.030

In current work, a class of novel 4,5-dihydro-1H-pyrazole-1-carboxylate derivatives (E01-E28) were designed, synthesized and evaluated. Among them, the most potent compound E24 exhibited comparable activity against a panel of cancer cells (GI₅₀

Full text of DOI:10.1016/j.ejmech.2018.12.030

Accepted Manuscript
A class of novel tubulin polymerization inhibitors exerts effective anti-tumor activity via
mitotic catastrophe
Ya-Liang Zhang, Bo-Yan Li, Rong Yang, Lin-Ying Xia, A-Li Fan, Yi-Chun Chu, Lin-
Jian Wang, Zhong-Chang Wang, Ai-Qin Jiang, Hai-Liang Zhu
PII:
S0223-5234(18)31067-5
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.030
EJMECH 10963
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 November 2018
Revised Date: 10 December 2018
Accepted Date: 12 December 2018
Please cite this article as: Y.-L. Zhang, B.-Y. Li, R. Yang, L.-Y. Xia, A.-L. Fan, Y.-C. Chu, L.-J. Wang,
Z.-C. Wang, A.-Q. Jiang, H.-L. Zhu, A class of novel tubulin polymerization inhibitors exerts effective
anti-tumor activity via mitotic catastrophe, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2018.12.030.
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A class of novel tubulin polymerization inhibitors exerts effective anti-tumor activity via
mitotic catastrophe
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Ya-Liang Zhang , Bo-Yan Li , Rong Yang , Lin-Ying Xia , A-Li Fan , Yi-Chun Chu ,
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Lin-Jian Wang , Zhong-Chang Wang *, Ai-Qin Jiang *, Hai-Liang Zhu *
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State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210023, People's Republic of China.
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Medical School, Nanjing University, Nanjing 210093, People's Republic of China.
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Clinical Pharmacokinetics Laboratory, School of Basic Medicine and Clinical
Pharmacy, China Pharmaceutical University, Nanjing, 211198, People's Republic of
China.
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*Corresponding authors:
Hai-Liang Zhu, Tel. & fax: +86-25-89682572; E-mail: zhuhl@nju.edu.cn;
Zhong-Chang Wang, E-mail: wangzhongchang2006@163.com;
Ai-Qin Jiang, E-mail: jianaq@nju.edu.cn.

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Abstract
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In current work, a class of novel 4,5-dihydro-1H-pyrazole-1-carboxylate derivatives
(E01-E28) were designed, synthesized and evaluated. Among them, the most potent
compound E24 exhibited comparable activity against a panel of cancer cells (GI50
ranging 0.05~0.98 µM) and tubulin polymerization inhibition (IC50 = 1.49 µM) with
reference drug CA-4(P) (GI ranging 0.019~0.32 µM, IC = 2.18 µM). The following
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assays indicated that compound E24 disturbed the dynamics of tubulin catastrophe and
rescue, which triggered G2/M arrest, leading to ROS accumulation, cleavage of PARP
and apoptosis. Molecular dynamics simulation validated that compound E24 could
tightly bind into tubulin heterodimers with β Lys 254 and β Cys 241 of tubulin in the
docking pose. Metabolic stability and pharmacokinetics parameters were also determined.
The half time (t ) displayed species differences in three microsomes. The plasma
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elimination half-life (t ), peak plasma concentration (Cmax), mean retention time (MRT),
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the area under the curve (AUC0- ) and distribution volume (V ) of E24 after intravenous
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administration were 0.90 ± 0.22 h, 594.50 ± 97.23 ng/mL, 1.09 ± 0.22 h, 413.67 ± 105.64
ng/mL*h and 5.03 ± 1.82 L/kg, respectively. In HeLa-xenografts, compound E24
exhibited obvious antitumor efficacy via the suppression of tumor growth without weight
loss of body or organ. In brief, compound E24 might be a hopeful candidate with
excellent properties for oncotherapy as tubulin polymerization inhibitor.
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Keywords
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Mitotic
catastrophe;
Tubulin
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inhibitors;
Chemotherapy;
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Pharmacokinetics; Xenograft tumors

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1. Introduction
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Mitotic catastrophe, a mechanism of delayed mitosis-linked cell death, was identified as a
regulated onco-suppressive mechanism that impedes the proliferation and/or survival of
cells that unable to complete mitosis [1]. It has proven to be a highly attractive
therapeutic endpoint [2]. A sizeable fraction of cancer cells is more sensitive to mitotic
catastrophe, resulting from their common polyploidy or aneuploidy renders more prone to
mitotic aberrations. Mitotic catastrophe is triggered by much lower doses of cytotoxic
agents and distinct from cell death manners activated by multiple agents at relatively high
doses [2,3]. Furthermore, it is an essential mechanism to overcome chemo-resistance in
various cancers [4]. Mitotic catastrophe can be initiated by ionizing radiation and
chemical agents, especially tubulin polymerization inhibitors, leading to the drastic
change of microtubule dynamics [3,5–7].
In eukaryotic cells, the polymerization-depolymerization dynamics of microtubules
(MTs) is deeply relied on by various fundamental cellular processes, including cell shape
maintenance, cell division, cell signaling and even the transport of vesicles throughout
cells [8,9]. In dividing mammalian cells, MTs forming the mitotic spindle are highly
dynamic and incisively sensitive to destabilizing agents [10], such as colchicine
(compound 1, Fig. 1) and vinblastine. Interference with microtubule dynamics turns out
to be an efficient strategy in the clinic [11,12].
A large number of agents could bind to tubulin and interfere with microtubule assembly.
Colchicine, one of the earliest tubulin-targeting drugs, could effectively obstruct
microtubule polymerization. Its binding site on tubulin has been characterized already

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[13]. However, the clinical development of colchicine for cancer therapy did not succeed
due to its highly side effects [12].
Another well-known anti-tubulin agent is CA-4 (compound 2, Fig. 1), which was isolated
from the bark of the South African tree, Combretum caffrum. It can potently bind to the
colchicine binding site, leading to severe cytotoxicity against multiple human cancer cell
lines [14]. To improve the water solubility of CA-4, its soluble phosphate prodrug, CA-4P
(compound 3, Fig. 1) was investigated [15] and has been approved for the treatment of
various cancers, such as non-small-cell lung cancer, prostate adenocarcinoma and
platinum-resistant ovarian cancer [16,17]. On the another hand, CA-4 easily undergoes
cis-trans isomerization under natural light, causing a sharp reduction in the anti-tubulin
activity, which limits its further applications [18]. Currently, it has been an effective
strategy to overcome the configuration shift that to replace double-bond with a
heterocyclic motif (compound 4-6, Fig. 1), leading to fixed cis-configuration [19–22].
In previous work, a series of tubulin polymerization inhibitors were obtained by
employing pyrazoline as the linker [23,24]. And pharmacokinetics properties of partial
compounds were also investigated further [25,26]. To double-check the previous
compounds (7 & 8; Fig. 1), two changes have been made to correspond to the features of
Colchicine-Domain Inhibitors (CDIs) [27]: 1) move 3,4,5-trimethoxyphenyl from 3-
position of pyrazoline to 5-position; 2) introduce oxygen atom to mimic the
pharmacophore of colchicine and CA-4 [28]. Thus, novel series of tubulin polymerization
inhibitors E01-E28 were designed. Here, we report current work is commented to
evaluate the bioactivities of these compounds. Compound E24 was found more potential
and induced mitotic catastrophe to cause HeLa cell death. Furthermore, its

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pharmacokinetics characters and antitumor activity on HeLa xenograft tumors also were
evaluated.
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2. Results
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2.1. Chemistry
The target compounds were synthesized with method in Scheme 1. The substituted
benzaldehyde A and acetophenone B went on aldol condensation in alkaline
environment, forming condensation products chalcones C01-C16. Then the intermediates
pyrazolines D01-D16 were synthesized from cyclization between chalcones and
hydrazine hydrate. Under inert environment, the pyrazolines were put into next steps
immediately without any purification and reacted with substituted chloroformate to afford
the target materials E01-E28.
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2.2. Crystal structure of compound E04
All of synthesized compounds were cultivated to obtain single crystals. However, only
crystal of compound E04 was acquired and determined by X-ray diffraction analysis. The
data for structural analysis has been checked and deposited with the Cambridge
Crystallographic Data Centre No. 1558498 for E04. The crystallographic data is
presented in Table 1 and Fig. 2 giving view of E04 with an atomic labeling system.

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2.3. Anti-proliferative activities in vitro
To evaluate the activities of compounds E01-E28, a panel of six human cancer cell lines
were utilized, including HepG2, A549, HT29, HeLa, MDA-MB-231 and A2780, which
originated from different tissues of human. All of the cells were given a continuous dose-
exposure of 48 h and GI values were shown as mean ± SD of at least three independent
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Firstly, systematic changes to the R and R groups were applied to investigate the SAR,
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while 3,4,5-trimethoxyphenyl was set as R group (compounds E01-E16, Table 2).
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Obviously, introduction of a bigger or atomic group at R position showed a fierce loss of
cytotoxic activity. Carbomethoxy contributed to most potency of activity when
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compounds contain same R group, such as E01-E04, E05-E08, E09-E12 and E13-E16.
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hydroxyl gave a little improvement in these four groups.
As reported, 3,4,5-trimethoxyphenyl is essential to maintain cytotoxic potency for
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colchicine binding site (CBS) inhibitors [28]. To examine the hypothesis, R was changed
from 3,4,5-trimethoxyphenyl to alternative groups, such as no substituents and less or
different positions of methoxyls. Compared with E01, loss of all methoxyls (E17) led to
total loss of activity, as same as E19. Also, missing or position shift of 3- or 5-methoxyl
weakened the ability to inhibit cancer cells.
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substituents was screened in a bigger extension, including E01, E05, E09 and E13. It was
found that mono-substituent gave higher potency than multi-substituent. And bigger
ethoxy group (E05 and E23) decreased activity of anti-proliferation. E27 showed more

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potency comparing with E05, E09 and E13, and it suggested that hydroxyl in para-
position was suitable for the activity pocket. Interestingly, E24 and E25 displayed more
potency than E27. It indicated that hydrophobic group on this site was more helpful to
improve antiproliferative activity.
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2.4. Compound E24 inhibits colony formation in HeLa cell and tubulin polymerization
The cellular long-term effects of E24 and E25 were tested in HeLa cell by colony
formation assay, which displays cancer cells ability to produce a viable colony, to predict
efficacy in vivo. It was shown in Fig. 3A that HeLa cells treated with E24 and E25 were
inhibited in the dose dependent manner. At high concentration, colony formation treated
with E24 was inhibited up to 80%, while low concentration displayed less effect. And it
also implied the greater potency of E24 compared to E25.
The ability of compounds E24 to inhibit tubulin polymerization was evaluated in vitro to
investigate whether the antiproliferative activity was resulted from an interaction with
tubulin. The tubulin assembly time-course plots in OD340 of reaction mixtures in the
presence of compound E24 were shown in Fig. 3B. In the presence of E24, tubulin
polymerization was inhibited in the concentration-dependent fashion. The compound
concentration required to inhibit tubulin polymerization by 50% of E24 was 1.49
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µM, while that of CA-4 as reference compound was 2.18 ± 0.12 µM.
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2.5. Virtual simulations
Colchicine targets the β-tubulin and hinders the curve-to-straight transition of tubulin by
changing the conformations of β-subunit T7 loop [29]. To better understand the molecular

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binding mode of E24 with tubulin, molecular docking simulations were proceeded by
inserting E24 into the colchicine binding site (CBS) of tubulin (PDB ID:1SA0). As
shown in Fig. 4A & 4B, the molecule E24 was located between α- and β- subunits and
well filled in the active pocket. 3,4,5-Trimethoxyphenyl of E24 was buried into the cave
of β-subunit, forming a 2.10 Å hydrogen bond with Cys 241. Similarly, two H-bonds
were found in the docking model of E25 with 1SA0 (Fig. 4C). According to literature
reports [30], Cys 241 in Helix 7 of β-subunit is a key amino acid that anchors most of all
CBS inhibitors. Same interaction of DAMA-colchicine (N-deacetyl-N-(2-
mercaptoacetyl)-colchicine) with tubulin was observed in the original co-crystal data of
1SA0, while the distance of H-bond was 1.80 Å (Fig. 4D). Differing from DAMA-
colchicine, M-tolyl of E24, extending to α-subunit nearby, formed π-cation interaction
with β Lys 254 instead of the H-bond with α Ser 178. The orientation and space of
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carbomethoxy was appropriate to interact with active pocket. Thus, if R group of E24
were longer, bigger or aromatic, the inhibitory potency would greatly diminish. And it
also proved to match the experimental data.
However, docking analysis showed a single glimpse of binding model rather than a
procedure. A 20 ns MD simulation was employed for E24-tubulin complex starting from
the docking pose (brilliant blue molecule in gray tubulin, Fig. 4E) above mentioned.
After simulation, the opening and ending poses were aligned. E24 hold original
orientation and conformation in the interaction with tubulin, despite of only a short shift.
The evolution in time of RMSD calculated on the backbone atoms of the protein to the
starting structure is represented in Fig. 4F. The E24-tubulin complex settled into
equilibrium soon with fluctuations wobbling in narrow range. Thus, it suggested the

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virtual simulations were convincible to demonstrate the binding model of E24 with
tubulin.
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2.6. Compound E24 & E25 arrested HeLa cells in G2/M phase
Generally, tubulin-target inhibitors could disrupt the mitosis to arrest normal cell cycle,
which is one typical characteristic of these agents. To confirm the hypothesis, compound
E24 and E25 also could arrest cell cycle to cause death, concerning assay was performed
by the method of PI staining.
Herein, HeLa cells also treated with different doses of E24 for 6, 12 and 24 h. As shown
in Fig. 5, E24 displayed forceful function to disorder cell cycle. Cells in G2/M phase
notably increased with the increase of doses or time of treatment, while percent of cells in
the other phases of the cell cycle declined. The effect reached plateau when treated with
the concentration above 0.10 µM at longer term. Meanwhile, the appearance of sub-G1
peaks implied the death or apoptosis might be caused by the arrest of cell cycle. E25
exhibited similar function of arresting mitosis, despite of higher effective dose.
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2.7. Compound E24 induced HeLa apoptosis and ROS accumulation
It is generally agreed that sub-G1 peaks are the marked indication that cells undergo
apoptosis. Because of obvious typical sub-G1 peaks in previous cell cycle analysis, there
was a hypothesis that synthesized compounds might induce cell apoptosis. To verify the
pro-apoptosis potency, double staining method with Annexin V-FITC and PI was carried
by flow cytometry. HeLa cells were treated with E24 (0.050, 0.10 µM), E25 (0.20, 0.40
µM) and CA-4P (0.050 µM) for 24h. Compared with untreated group (Fig. 6), the total

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percentage of apoptotic cells, FITC positive, raised up to about 2-5 folds after treated
with E24, which was a little weaker to induce apoptosis than CA-4P. To further prove this
hypothesis, the cleavage of poly ADP-ribose polymerase (PARP) was detected after 24 h
treatment with E24. It suggested that E24 could induce caspase 3 to cleave PARP,
causing cell apoptosis. However, there was no obvious evidence to suggest that E25 could
induce cell apoptosis. ROS plays an eventful role in the apoptosis and its accumulation
would damage cellular proteins, lipids and DNA to suppress cell growth. To evaluate
whether E24 induce to accumulate ROS in cells, DCFH-DA stain method was performed
by flow cytometry. As positive group, the peaks of treated cells moved right to higher
signal direction. It showed that E24 also resulted in intracellular ROS accumulation in
HeLa cells.
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2.8. Compound E24 disrupt microtubules dynamics leading to mitotic catastrophe
Microtubules (MTs) play a crucial role in various fundamental cellular processes, mainly
including forming cytoskeleton and mitotic spindle. Interfering the tubulin-microtubule
dynamics contributes to some changes of cells in morphology, which could be examined
by normal optical or fluorescence microscopy. To picture the direct alternations of cells,
immunofluorescence staining was employed with Cy3-labeled tubulin antibody and
DAPI. As shown in Fig. 7, the untreated MT displayed orderly organized thin fibers.
After 12 h treatment of 0.020 µM E24, cells arrest in mitotic phase significantly increased
and displayed abnormal even multipolar spindle. The microtubule fibers break down,
turning to shorter and rarer like being treated with colchicine. In contrast, paclitaxel
dramatically enhanced microtubule polymerization, with an increase in the density of

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cellular microtubules and the formation of long thick microtubule bundles. Multinuclear
giant cells, resulting from mitotic catastrophe, also could be observed in high dose group
of E24. Results above implied that E24 prevented tubulin from polymerizing to
ultimately cause cell death.
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2.9. Metabolic stability and pharmacokinetic study
To provide guidance of drug-like properties for further animal model, metabolic stability
and pharmacokinetics model of E24 were performed. To evaluate the metabolic ability,
E24 was in vitro incubated with liver microsomes from three species respectively at
37 °C for an indicated time in NADPH regenerating system, terminated by ice-cold acetic
ether and determined by LC-MS/MS. It was indicated that E24 was relatively stable.
There still remained 31.1% of E24 in human liver microsomes after 2 h incubation (Fig.
8A). As known widely, a short half-life time (t1/2) of compounds would lead to low
bioavailability and frequent administration. The t1/2 of E24 was evidently improved,
while the mean was 170.73 min, 123.48 min and 37.02 min for human, monkey and rat,
respectively.
The plasma concentration-time profile of E24 is shown in Fig. 8B, while the
corresponding pharmacokinetic parameters calculated using non-compartment model
analysis are listed in Table 3. The plasma concentration of E24 declined remarkably after
intravenous injection. The t1/2 was 0.901 ± 0.203 h, the MRT(0-∞) was 1.091 ± 0.217 h, the
AUC(0-∞) was 413.674 ± 105.64 ng/ml*h and the Vz was 5.031 ± 1.817 L/kg, revealing
quick elimination rate in blood and short residence time of E24 in vivo.

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2.10. Compound E24 suppressed tumor growth in vivo
To test the potent activity of E24 in vivo, HeLa cells xenograft model was established.
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placed to 6 groups: control (no treatment), vehicle, CA-4P, and E24 with three doses. It
was represented in Fig. 9A that vehicle group did not significantly differ from untreated-
control group. Compared with the rapid tumor growth of these two groups, treatment of
E24 suppressed tumor growth in dose-dependent fashion, and effect of 20 mg/kg E24
equaled to that of CA-4P group. The average relative inhibition rate of high dose reached
72.9% by the end of the monitoring period, while treatment with CA-4P led to a 79.4%
tumor inhibition. The weight of excised tumors was also collected (Fig. 9B) and it
suggested the potent activity of E24. The average tumor weight of each group was 1.096
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0.079 g, 1.014 ± 0.116 g, 0.187 ± 0.061g, 0.812 ± 0.092 g, 0.631 ± 0.045 g and 0.266 ±
0.051 g, respectively. Furthermore, in comparison with the control and vehicle groups,
E24 caused neither an obvious loss of body weight (Fig. 9C) nor the weight change of
main organs (Fig. 9D), which indicated that compound E24 displayed no signs of toxicity
in these mice, especially in high dose. All of these data suggested that compound E24
exhibited obvious antitumor efficacy in HeLa xenografts via the suppression of
proliferation.
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In conclusion, a series of novel compounds (E01-E28) were designed, synthesized and
evaluated as potential inhibitors of tubulin polymerization. Compound E24 displayed the
most potent activity against a wide variety of cancer cell lines, (GI ranging 0.05~0.98
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µM) and tubulin polymerization inhibition (IC50 = 1.49 µM) with reference drug CA-4(P)
(GI50 ranging 0.019~0.32 µM, IC50 = 2.18 µM). Its activity against HeLa cells was
improved one magnitude, compared with previous compound 7 (GI50 0.21 µM). The
mechanism of action studies proved that E24 disturbed the dynamics of tubulin
catastrophe and rescue, which triggered cells cycle arrest in G2/M phase followed by
mitotic catastrophe. After 12 h treatment of 0.020 µM E24, cells arrest in mitotic phase
significantly increased and displayed abnormal even multipolar spindle. It also suggested
E24 induced PARP cleavage up-regulation and intracellular ROS accumulation, leading
to cell apoptosis. Compared with untreated control, the total percentage of apoptotic cells
raised up to about 2-5 folds in E24 treatment groups. The molecular docking revealed
binding model of E24 with tubulin (PDB ID: 1SA0). The docking poses was convincing,
further validated by performing molecular dynamics simulation. It was found that
compound E24 could tightly bind into tubulin heterodimers with β Lys 254 and β Cys
241 of tubulin in the docking pose. The half time (t ) displayed species differences in
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three microsomes. The plasma elimination half-life (t1/2), peak plasma concentration
(Cmax), mean retention time (MRT), the area under the curve (AUC0- ) and distribution
volume (V ) of E24 after intravenous administration were 0.90 ± 0.22 h, 594.50 ± 97.23
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ng/mL, 1.09 ± 0.22 h, 413.67 ± 105.64 ng/mL*h and 5.03 ± 1.82 L/kg, respectively. In
HeLa-xenografts, compound E24 exhibited obvious antitumor efficacy via the
suppression of tumor growth without weight loss of body or organs. The average relative
inhibition rate of high dose group reached 72.9% by the end of the monitoring period. As
a novel antimitotic agent, compound E24 exhibits potential pharmacokinetic and anti-
tumor activity in vivo and deserves further development.

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4.1. Materials
All chemical solvents and reagents used in current study were analytical grade and
purchased at Nanjing Chemical Reagent CO., LTD (Nanjing, China) or Aladdin
(Shanghai, China). The reactions were monitored by thin layer chromatography (TLC),
which was performed on the glass-backed silica gel sheets (silica gel 60 GF254) and
detected using UV light (254 nm or 365 nm). Melting points were determined on an XT4
MP apparatus (Taike Corp., Beijing, China). NMR spectra were collected on a Bruker
DPX 400 model spectrometer in DMSO-d . ESI-MS spectra were recorded on a Mariner
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System 5304 Mass spectrometer. Elemental analyses were performed on a CHN-O-Rapid
instrument. All compounds gave satisfactory chemical analyses (±0.4%). All types of
cancer cell lines used in this work were obtained from State Key Laboratory of
Pharmaceutical Biotechnology, Nanjing University. Cell cycle Assay Kit and Apoptosis
Assay Kit were purchased from KeyGEN BioTech (Nanjing, China). GAPDH and PARP
antibodies and ECL Substrate Kit were provided by WanleiBio (Shenyang, China).
Crystal violet staining solution, RIPA buffer, PMSF, BCA Protein Assay Kit, DAPI, β-
tubulin monoclonal antibody, Cy3-labeled rabbit anti-mouse secondary antibody and
ROS Assay Kit were purchased from Beyotime BioTech (Nanjing, China). SPF female
and male Sprague-Dawley rats and BALB/c nude mice were purchased from the Model
Animal Research Center of Nanjing University (Nanjing, China). Microsomes were
provided by RILD BioTech (Shanghai, China).

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4.2. Chemistry
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20
21
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23
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25
4.2.1. Synthesis of chalcones (C01-C16)
Substituted phenyl formaldehyde A (2.0 mmol) and acetophenone B (2.0 mmol) were
dissolved in ethanol (20 mL) and KOH (6.0 mmol, 336 mg) were added. After stirred at
room temperature overnight, the reaction mixture was filtered and washed with water and
cold ethanol. The crude product was purified by recrystallization from ethanol and
dichloromethane to give C01-C16 with yields of 62.0-95.4 %.
3
3
3
3
3
3
26
27
28
29
30
31
4.2.2. Synthesis of 3,5-diphenyl-4,5-dihydro-1H-pyrazole (D01-D16)
A mixture of C (1.0 mmol) and hydrazine hydrate (2.0 mmol) was dissolved in i-PrOH (5
mL) and refluxed for 4 h. The reaction mixture was cooled and kept at -20 °C overnight.
Then the solid obtained was filtered off and washed with petroleum ether and taken for
the next step immediately. Following this method, we got crude compounds D01-D16.
3
3
3
3
3
3
3
3
32
33
34
35
36
37
38
39
4.2.3. Synthesis of 3,5-diphenyl-4,5-dihydro-1H-pyrazole-1-carboxylate (E01-E28)
To a stirred solution of K CO (1.0 mmol) in methanol (5 mL), D (1.0 mmol) was added
2
3
at room temperature with the protection of N . After complete dissolution of D, the
2
mixture was moved into ice bath and chloroformate (1.0 mmol) was added dropwise with
stirring. After stirred more 2 h at room temperature, the mixture was filtered and the
solvent was removed. The crude product was purified by recrystallization from ethanol
and dichloromethane to give E01-E28 with yields of 35.7-85.2 %.

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41
42
43
44
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46
47
4.2.4. Methyl
3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E01)
1
White powder. Yield: 62.7%. m.p. 122-124 °C. H-NMR (DMSO-d , 600 MHz) δ 7.76 -
6
7.74 (m, 2H), 7.48 - 7.42 (m, 3H), 6.50 (s, 2H), 5.39 (dd, J = 11.9, 5.4 Hz, 1H), 3.84 (dd,
J = 17.9, 11.9 Hz, 1H), 3.73 (s, 6H), 3.67 (s, 3H), 3.63 (s, 3H), 3.17 (dd, J = 17.9, 5.5 Hz,
+
1H). MS (ESI) m/z: 371.05 (C H N O , [M+H] ). Anal. Calcd. for C H N O : C,
2
0
22
2
5
20 22
2
5
64.85; H, 5.99; O, 21.60; Found: C, 64.84; H, 6.00; O, 21.62.
3
3
3
3
3
3
3
3
3
48
49
50
51
52
53
54
55
56
4.2.5. Ethyl 3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxylate
(E02)
1
White powder. Yield: 66.1%. m.p. 138-140 °C. H-NMR (DMSO-d , 600 MHz) δ 7.81 -
6
7.70 (m, 2H), 7.51 - 7.41 (m, 3H), 6.50 (s, 2H), 5.39 (dd, J = 11.9, 5.5 Hz, 1H), 4.17 -
4.00 (m, 2H), 3.84 (dd, J = 17.9, 11.9 Hz, 1H), 3.73 (s, 6H), 3.63 (s, 3H), 3.18 (dd, J =
+
17.9, 5.5 Hz, 1H), 1.20 - 1.07 (m, 3H). MS (ESI) m/z: 385.17 (C H N O , [M+H] ).
21
24
2
5
Anal. Calcd. for C H N O : C, 65.61; H, 6.29; O, 20.81; Found: C, 65.64; H, 6.28; O,
2
1
24
2
5
20.80.
3
3
3
3
3
3
57
58
59
60
61
62
4.2.6. Propyl
3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E03)
1
White powder. Yield: 53.8%. m.p. 141-143 °C. H-NMR (DMSO-d , 600 MHz) δ 7.76 -
6
7.75 (m, 2H), 7.50 - 7.41 (m, 3H), 6.50 (s, 2H), 5.39 (dd, J = 11.9, 5.6 Hz, 1H), 4.06 -
3.96 (m, 2H), 3.85 (dd, J = 17.8, 11.9 Hz, 1H), 3.73 (s, 6H), 3.63 (s, 3H), 3.19 (dd, J =
+
17.9, 5.6 Hz, 1H), 1.51 (s, 2H), 0.75 (s, 3H). MS (ESI): 399.18 (C22
H
26
N
2
O
5
, [M+H] ).

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3
3
63
64
65
Anal. Calcd. for C22
20.07.
26 2 5
H N O : C, 66.32; H, 6.58; O, 20.08; Found: C, 66.31; H, 6.59; O,
3
3
3
3
3
3
3
3
66
67
68
69
70
71
72
73
4.2.7. Benzyl
3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E04)
1
White powder. Yield: 59.4%. m.p. 143-145 °C. H-NMR (DMSO-d
6
, 600 MHz) δ 7.77 -
7.75 (m, 2H), 7.49 - 7.42 (m, 3H), 7.30 (br, m, 5H), 6.49 (s, 2H), 5.44 (dd, J = 11.9, 5.5
Hz, 1H), 5.19 - 5.09 (m, 2H), 3.86 (dd, J = 17.9, 11.9 Hz, 1H), 3.68 (s, 6H), 3.64 (s, 3H),
+
3.20 (dd, J = 17.9, 5.5 Hz, 1H). MS (ESI): 447.18 (C H N O , [M+H] ). Anal. Calcd.
26
26
2
5
for C H N O : C, 69.94; H, 5.87; O, 17.92; Found: C, 69.95; H, 5.88; O, 17.91.
2
6
26
2
5
3
3
3
3
3
3
3
3
74
75
76
77
78
79
80
81
4.2.8. Methyl 3-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E05)
1
White powder. Yield: 61.2%. m.p. 164-166 °C. H-NMR (DMSO-d , 600 MHz) δ 7.39 -
6
7.30 (m, 2H), 7.26 (s, 1H), 7.04 - 7.02 (m, 1H), 6.49 (s, 2H), 5.39 (dd, J = 11.9, 5.4 Hz,
1H), 3.87 - 3.78 (m, 4H), 3.73 (s, 6H), 3.67 (s, 3H), 3.63 (s, 3H), 3.18 (dd, J = 17.9, 5.5
+
Hz, 1H). MS (ESI): 401.16 (C21
H
24
N
2
O
6
, [M+H] ). Anal. Calcd. for C21
H
24
N
2
O
6
: C,
62.99; H, 6.04; O, 23.97; Found: C, 63.00; H, 6.05; O, 23.98.
3
3
3
3
82
83
84
85
4.2.9. Ethyl 3-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E06)
1
White powder. Yield: 51.9%. m.p. 161-163 °C. H-NMR (DMSO-d , 600 MHz) δ 7.40 -
6
7.29 (m, 2H), 7.25 (s, 1H), 7.04 - 7.00 (m, 1H), 6.49 (s, 2H), 5.38 (dd, J = 11.9, 5.5 Hz,

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86
87
88
89
1H), 4.15 - 4.06 (m, 2H), 3.84 - 3.77 (m, 4H), 3.73 (s, 6H), 3.63 (s, 3H), 3.19 (dd, J =
+
18.0, 5.4 Hz, 1H), 1.13 (s, 3H). MS (ESI): 415.18 C H N O , [M+H] ). Anal. Calcd. for
2
2
26
2
6
C H N O : C, 63.76; H, 6.32; O, 23.16; Found: C, 63.77; H, 6.33; O, 23.15.
22
26
2
6
3
3
3
3
3
3
3
3
3
90
91
92
93
94
95
96
97
98
4.2.10. Propyl
3-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E07)
1
White powder. Yield: 54.2%. m.p. 172-174 °C. H-NMR (DMSO-d , 600 MHz) δ 7.41 -
6
7.30 (m, 2H), 7.26 (s, 1H), 7.04 - 7.02 (m, 1H), 6.50 (s, 2H), 5.39 (dd, J = 11.9, 5.6 Hz,
1H), 4.05 - 3.98 (m, 2H), 3.86 - 3.78 (m, 4H), 3.73 (s, 6H), 3.63 (s, 3H), 3.19 (dd, J =
+
17.9, 5.5 Hz, 1H), 1.50 (s, 2H), 0.74 (s, 3H). MS (ESI): 429.19 (C H N O , [M+H] ).
2
3
28
2
6
Anal. Calcd. for C H N O : C, 64.47; H, 6.59; O, 22.40; Found: C, 64.48; H, 6.58; O,
2
3
28
2
6
22.42.
3
4
4
4
4
4
4
4
99
00
01
02
03
04
05
06
4.2.11. Benzyl
3-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E08)
White powder. Yield: 57.3%. m.p. 129-131 °C. H-NMR (DMSO-d
1
6
, 600 MHz) δ 7.41 -
7.07 (br, m, 8H), 7.04 - 7.02 (m, 1H), 6.47 (s, 2H), 5.44 (dd, J = 11.9, 5.5 Hz, 1H), 5.18 -
5.11 (br, m, 2H), 3.84 (dd, J = 18.0, 12.0 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 6H), 3.64 (s, 3H),
+
3.21 (dd, J = 18.0, 5.5 Hz, 1H). MS (ESI): 477.19 (C H N O , [M+H] ). Anal. Calcd.
27
28
2
6
for C H N O : C, 68.05; H, 5.92; O, 20.14; Found: C, 68.03; H, 5.91; O, 20.12.
2
7
28
2
6

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4
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08
09
10
11
12
13
14
4.2.12. Methyl
3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E09)
1
White powder. Yield: 85.2%. m.p. 165-167 °C. H-NMR (DMSO-d , 600 MHz) δ 7.69 (d,
6
J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.48 (s, 2H), 5.36 (dd, J = 11.8, 5.3 Hz, 1H),
3.83 - 3.76 (m, 4H), 3.73 (s, 6H), 3.66 (s, 3H), 3.63 (s, 3H), 3.13 (dd, J = 17.8, 5.3 Hz,
+
1H). MS (ESI): 401.16 (C H N O , [M+H] ). Anal. Calcd. for C H N O : C, 62.99; H,
21
24
2
6
21 24
2
6
6.04; O, 23.97; Found: C, 63.00; H, 6.05; O, 23.98.
4
4
4
4
4
4
4
4
15
16
17
18
19
20
21
22
4.2.13. Ethyl 3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E10)
1
White powder. Yield: 81.6%. m.p. 111-113 °C. H-NMR (DMSO-d , 600 MHz) δ 7.69 (d,
6
J = 8.8 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 6.48 (s, 2H), 5.35 (dd, J = 11.8, 5.3 Hz, 1H),
4.13 - 4.05 (m, 2H), 3.83 - 3.75 (m, 4H), 3.73 (s, 6H), 3.63 (s, 3H), 3.14 (dd, J = 17.8, 5.3
+
Hz, 1H), 1.14 (s, 3H). MS (ESI): 415.18 (C H N O , [M+H] ). Anal. Calcd. for
22
26
2
6
C H N O : C, 63.76; H, 6.32; O, 23.16; Found: C, 63.77; H, 6.33; O, 23.15.
22
26
2
6
4
4
4
4
4
4
4
23
24
25
26
27
28
29
4.2.14. Propyl
3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E11)
1
White powder. Yield: 80.3%. m.p. 105-107 °C. H-NMR (DMSO-d , 600 MHz) δ 7.69 (d,
6
J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.49 (s, 2H), 5.36 (dd, J = 11.8, 5.4 Hz, 1H),
4.06 - 3.93 (m, 2H), 3.83 - 3.76 (m, 4H), 3.72 (s, 6H), 3.62 (s, 3H), 3.15 (dd, J = 17.8, 5.4
+
Hz, 1H), 1.50 (s, 2H), 0.74 (s, 3H). MS (ESI): 429.19 (C H N O , [M+H] ). Anal.
20
22
2
5
22 2 5
Calcd. for C20H N O : C, 64.47; H, 6.59; O, 22.40; Found: C, 64.48; H, 6.58; O, 22.42.

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35
36
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38
4.2.15. Benzyl
3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E12)
White powder. Yield: 78.4%. m.p. 161-163 °C. H-NMR (DMSO-d
1
6
, 600 MHz) δ 7.69 (d,
J = 8.8 Hz, 2H), 7.29 (br, s, 5H), 7.00 (d, J = 8.8 Hz, 2H), 6.47 (s, 2H), 5.41 (dd, J = 11.8,
5.4 Hz, 1H), 5.17 - 5.09 (m, 2H), 3.85 - 3.77 (m, 4H), 3.67 (s, 6H), 3.64 (s, 3H), 3.16 (dd,
+
J = 17.8, 5.3 Hz, 1H). MS (ESI): 477.19 (C H N O , [M+H] ). Anal. Calcd. for
27
28
2
6
C H N O : C, 68.05; H, 5.92; O, 20.14; Found: C, 68.03; H, 5.91; O, 20.12.
27
28
2
6
4
4
4
4
4
4
4
4
4
39
40
41
42
43
44
45
46
47
4.2.16. Methyl 3-(3-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E13)
1
White powder. Yield: 77.1%. m.p. 137-139 °C. H-NMR (DMSO-d , 600 MHz) δ 9.63 (s,
6
1H), 7.25 - 7.20 (m, 2H), 7.12 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.48 (s, 2H),
5.37 (dd, J = 11.8, 5.4 Hz, 1H), 3.79 (dd, J = 17.8, 11.9 Hz, 1H), 3.73 (s, 6H), 3.66 (s,
3H), 3.63 (s, 3H), 3.10 (dd, J = 17.8, 5.3 Hz, 1H). MS (ESI): 387.00 (C H N O ,
2
0
22
2
6
+
[M+H] ). Anal. Calcd. for C20
H
22
N
2
O
6
: C, 62.17; H, 5.74; O, 24.84; Found: C, 62.18; H,
5.73; O, 24.83.
4
4
4
4
4
48
49
50
51
52
4.2.17. Ethyl 3-(3-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E14)
1
White powder. Yield: 73.3%. m.p. 186-188 °C. H-NMR (DMSO-d
6
, 600 MHz) δ 9.63 (s,
1H), 7.25 - 7.21 (m, 2H), 7.12 (d, J = 7.7 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.48 (s, 2H),
5.36 (dd, J = 11.9, 5.5 Hz, 1H), 4.17 - 4.00 (m, 2H), 3.79 (dd, J = 17.8, 11.9 Hz, 1H),

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55
56
3.73 (s, 6H), 3.63 (s, 3H), 3.11 (dd, J = 17.8, 5.4 Hz, 1H), 1.14 (s, 3H). MS (ESI): 401.16
+
(C H N O , [M+H] ). Anal. Calcd. for C H N O : C, 62.99; H, 6.04; O, 23.97; Found:
2
1
24
2
6
21 24
2
6
C, 62.97; H, 6.05; O, 23.98.
4
4
4
4
4
4
4
4
4
57
58
59
60
61
62
63
64
65
4.2.18. Propyl 3-(3-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E15)
1
White powder. Yield: 65.9%. m.p. 108-110 °C. H-NMR (DMSO-d , 600 MHz) δ 9.63 (s,
6
1H), 7.24 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 1.7 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 6.85 (d, J
= 7.6 Hz, 1H), 6.49 (s, 2H), 5.36 (dd, J = 11.9, 5.6 Hz, 1H), 4.04 - 4.01 (m, 2H), 3.79 (dd,
J = 17.8, 11.9 Hz, 1H), 3.73 (s, 6H), 3.63 (s, 3H), 3.11 (dd, J = 17.8, 5.6 Hz, 1H), 1.50 (s,
+
2H), 0.75 (s, 3H). MS (ESI): 415.18 (C H N O , [M+H] ). Anal. Calcd. for C H N O :
2
2
26
2
6
22 26
2
6
C, 63.76; H, 6.32; O, 23.16; Found: C, 63.77; H, 6.31; O, 23.17.
4
4
4
4
4
4
4
4
66
67
68
69
70
71
72
73
4.2.19. Benzyl 3-(3-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E16)
1
White powder. Yield: 61.4%. m.p. 165-167 °C. H-NMR (DMSO-d
6
, 600 MHz) δ 9.64 (s,
1H), 7.48 - 6.95 (br, m, 8H), 6.85 (d, J = 7.6 Hz, 1H), 6.47 (s, 2H), 5.41 (dd, J = 11.8, 5.5
Hz, 1H), 5.17 - 5.15 (m, 2H), 3.81 (dd, J = 17.9, 11.9 Hz, 1H), 3.67 (s, 6H), 3.64 (s, 3H),
+
3.13 (dd, J = 17.9, 5.4 Hz, 1H). MS (ESI): 463.18 (C H N O , [M+H] ). Anal. Calcd.
26
26
2
6
for C H N O : C, 67.52; H, 5.67; O, 20.76; Found: C, 67.50; H, 5.68; O, 20.77.
2
6
26
2
6

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4
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75
76
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79
80
81
82
4.2.20. Methyl 3,5-diphenyl-4,5-dihydro-1H-pyrazole-1-carboxylate (E17)
1
White powder. Yield: 81.6%. m.p. 137-138 °C. H-NMR (DMSO-d , 600 MHz) δ 7.77 -
6
7.74 (m, 2H), 7.53 - 7.40 (m, 3H), 7.35 (t, J = 7.3 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H), 7.23 -
7.15 (m, 2H), 5.46 (dd, J = 11.9, 4.8 Hz, 1H), 3.88 (dd, J = 17.9, 11.9 Hz, 1H), 3.64 (s,
1
3
3H), 3.14 (dd, J = 18.0, 4.9 Hz, 1H). C-NMR (DMSO-d , 151 MHz) δ 154.20, 153.08,
6
143.10, 131.62, 130.64, 129.24, 127.89, 127.09, 125.91, 61.43, 53.05, 42.72. MS (ESI):
+
281.12 (C17
H
16
N
2
O
2
, [M+H] ). Anal. Calcd. for C17
H
16
N
2
2
O : C, 72.84; H, 5.75; O, 11.41;
Found: C, 72.83; H, 5.76; O, 11.42.
4
4
4
4
4
4
4
4
4
4
83
84
85
86
87
88
89
90
91
92
4.2.21. Methyl
5-(3-hydroxy-4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboxylate (E18)
1
White powder. Yield: 55.3%. m.p. 167-169 °C. H-NMR (DMSO-d
6
, 600 MHz) δ 9.01 (s,
1H), 7.76 - 7.74 (m, 2H), 7.52 - 7.39 (m, 3H), 6.85 (d, J = 8.8 Hz, 1H), 6.65 - 6.56 (m,
2H), 5.31 (dd, J = 11.8, 4.4 Hz, 1H), 3.80 (dd, J = 17.8, 11.9 Hz, 1H), 3.72 (s, 3H), 3.64
1
3
(s, 3H), 3.09 (dd, J = 17.9, 4.4 Hz, 1H). C-NMR (DMSO-d , 151 MHz) δ 154.19,
6
152.98, 147.48, 147.21, 135.78, 131.72, 130.59, 129.24, 127.04, 116.70, 112.87, 112.80,
+
61.02, 56.09, 52.98, 42.66. MS (ESI): 327.00 (C H N O , [M+H] ). Anal. Calcd. for
18
18
2
4
C H N O : C, 66.25; H, 5.56; O, 19.61; Found: C, 66.23; H, 5.57; O, 19.62.
18
18
2
4
4
4
4
4
93
94
95
96
4.2.22. Methyl
5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxylate
(E19)
1
White powder. Yield: 42.9%. m.p. 115-117 °C. H-NMR (DMSO-d , 600 MHz) δ 7.75
6
(dd, J = 6.7, 3.0 Hz, 2H), 7.48 - 7.42 (m, 3H), 7.13 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7

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Hz, 2H), 5.39 (dd, J = 11.8, 4.8 Hz, 1H), 3.84 (dd, J = 17.9, 11.8 Hz, 1H), 3.72 (s, 3H),
+
3.63 (s, 3H), 3.12 (dd, J = 17.9, 4.8 Hz, 1H). MS (ESI): 311.13 (C H N O , [M+H] ).
1
8
18
2
3
Anal. Calcd. for C H N O : C, 69.66; H, 5.85; O, 15.47; Found: C, 69.60; H,5.89; O,
1
8
18
2
3
15.49.
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5
5
5
5
5
5
5
5
02
03
04
05
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09
10
4.2.23. Methyl 5-(3,4-dimethoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxylate
(E20)
1
White powder. Yield: 35.7%. m.p. 110-111 °C. H-NMR (DMSO-d , 600 MHz) δ 7.77 -
6
7.73 (m, 2H), 7.47 - 7.44 (m, 3H), 6.90 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.69
(dd, J = 8.3, 2.0 Hz, 1H), 5.39 (dd, J = 11.8, 5.0 Hz, 1H), 3.83 (dd, J = 17.9, 11.8 Hz, 1H),
3.72 (d, J = 3.3 Hz, 6H), 3.64 (s, 3H), 3.15 (dd, J = 17.9, 5.0 Hz, 1H). MS (ESI): 341.14
+
(C19
H
20
N
2
O
4
, [M+H] ). Anal. Calcd. for C19
H
20
N
2
O
4
: C, 67.05; H, 5.92; O, 18.80; Found:
C, 67.03; H,5.93; O, 18.81.
5
5
5
5
5
5
5
5
11
12
13
14
15
16
17
18
4.2.24. Methyl
3-phenyl-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E21)
1
White powder. Yield: 46.1%. m.p. 116-118 °C. H-NMR (DMSO-d
6
, 600 MHz) δ 7.74 -
7.74 (m, 2H), 7.48 - 7.38 (m, 3H), 6.72 (s, 1H), 6.56 (s, 1H), 5.50 (dd, J = 12.0, 5.1 Hz,
1H), 3.82 - 3.72 (m, 7H), 3.63 (d, J = 11.6 Hz, 6H), 3.02 (dd, J = 17.6, 5.1 Hz, 1H). MS
+
(ESI): 371.15 (C H N O , [M+H] ). Anal. Calcd. for C H N O : C, 64.85; H, 5.99; O,
2
0
22
2
5
20 22
2
5
21.60; Found: C, 64.88; H,5.97; O, 21.59.

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4.2.25. Methyl
3-phenyl-5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E22)
1
White powder. Yield: 50.4%. m.p. 148-150 °C. H-NMR (DMSO-d , 600 MHz) δ 7.73 -
6
7.71 (m, 2H), 7.47 - 7.38 (m, 3H), 6.22 (s, 2H), 5.81 (dd, J = 13.0, 6.2 Hz, 1H), 3.76 (s,
6H), 3.63 - 3.38 (m, 7H), 3.01 (dd, J = 17.2, 6.1 Hz, 1H). MS (ESI): 371.15 (C H N O ,
2
0
22
2
5
+
[M+H] ). Anal. Calcd. for C H N O : C, 64.85; H, 5.99; O, 21.60; Found: C, 64.89;
2
0
22
2
5
H,5.99; O, 21.58.
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5
5
5
5
5
5
5
5
5
5
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35
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4.2.26. Methyl 3-(3-ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E23)
1
White powder. Yield: 48.7%. m.p. 137-138 °C. H-NMR (DMSO-d , 600 MHz) δ 7.39 -
6
7.27 (m, 2H), 7.24 (s, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.49 (s, 2H), 5.39 (dd, J = 11.9, 5.4
Hz, 1H), 4.06 (q, J = 6.9 Hz, 2H), 3.82 (dd, J = 18.0, 11.9 Hz, 1H), 3.73 (s, 6H), 3.67 (s,
1
3
3H), 3.63 (s, 3H), 3.18 (dd, J = 18.0, 5.4 Hz, 1H), 1.33 (t, J = 6.9 Hz, 3H). C-NMR
(DMSO-d , 151 MHz) δ 159.13, 154.29, 153.55, 153.26, 138.81, 137.05, 133.00, 130.33,
6
119.48, 117.06, 112.40, 103.00, 63.65, 61.74, 60.38, 56.32, 53.12, 42.84, 15.08. MS
+
(ESI): 415.18 (C H N O , [M+H] ). Anal. Calcd. for C H N O : C, 63.76; H, 6.32; O,
2
0
22
2
6
22 26
2
6
23.16; Found: C, 63.75; H, 6.31; O, 23.15.
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5
5
5
38
39
40
41
4.2.27. Methyl
3-(m-tolyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-
carboxylate (E24)
1
White powder. Yield: 61.9%. m.p. 126-128 °C. H-NMR (DMSO-d , 600 MHz) δ 7.59 (s,
6
1H), 7.52 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 6.48 (s,

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2H), 5.39 (dd, J = 11.8, 5.3 Hz, 1H), 3.82 (dd, J = 17.9, 11.9 Hz, 1H), 3.73 (s, 6H), 3.67
1
3
(s, 3H), 3.63 (s, 3H), 3.15 (dd, J = 17.9, 5.3 Hz, 1H), 2.34 (s, 3H). C-NMR (DMSO-d ,
6
151 MHz) δ 154.38, 153.56, 153.26, 138.85, 138.47, 137.05, 131.59, 131.28, 129.11,
127.50, 124.38, 102.93, 61.63, 60.39, 56.31, 53.10, 42.83, 21.34. MS (ESI): 385.15
+
(C H N O , [M+H] ). Anal. Calcd. for C H N O : C, 65.61; H, 6.29; O, 20.81; Found:
2
1
24
2
5
21 24
2
5
C, 65.63; H, 6.28; O, 20.82.
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5
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5
5
5
5
5
5
5
5
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4.2.28. Methyl 3-(3-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E25)
1
White powder. Yield: 47.2%. m.p. 163-165 °C. H-NMR (DMSO-d , 600 MHz) δ 7.90 (t,
6
J = 1.7 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.9 Hz,
1H), 6.50 (s, 2H), 5.40 (dd, J = 11.9, 5.5 Hz, 1H), 3.83 (dd, J = 18.0, 12.0 Hz, 1H), 3.74
1
3
6
(s, 6H), 3.68 (s, 3H), 3.63 (s, 3H), 3.20 (dd, J = 18.0, 5.6 Hz, 1H). C-NMR (DMSO-d ,
151 MHz) δ 153.55, 153.21, 153.14, 138.61, 137.09, 134.03, 133.17, 131.40, 129.46,
126.10, 122.54, 103.08, 61.97, 60.39, 56.33, 53.21, 42.56. MS (ESI): 405.11
+
(C H ClN O , [M+H] ). Anal. Calcd. for C H ClN O : C, 59.34; H, 5.23; O, 19.76;
2
0
21
2
5
20 21
2
5
Found: C, 59.38; H, 5.21; O, 19.74.
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5
5
5
60
61
62
63
64
4.2.29. Methyl 3,5-bis(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxylate
(E26)
1
White powder. Yield: 83.7%. m.p. 216-218 °C. H-NMR (DMSO-d , 600 MHz) δ 7.00 (s,
6
2H), 6.48 (s, 2H), 5.40 (dd, J = 11.9, 5.3 Hz, 1H), 3.86 - 3.77 (m, 7H), 3.74 (s, 6H), 3.69
1
3
6
(s, 3H), 3.67 (s, 3H), 3.63 (s, 3H), 3.23 (dd, J = 18.0, 5.3 Hz, 1H). C-NMR (DMSO-d ,

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151 MHz) δ 154.32, 153.59, 153.45, 153.19, 139.69, 138.98, 137.03, 127.11, 104.49,
102.82, 61.70, 60.58, 60.39, 56.46, 56.35, 53.11, 43.02. MS (ESI): 461.18 (C H N O ,
2
3
28
2
8
+
[M+H] ). Anal. Calcd. for C H N O : C, 59.99; H, 6.13; O, 27.80; Found: C, 59.96; H,
2
3
28
2
8
6.14; O, 27.82.
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5
5
5
5
5
5
5
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74
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4.2.30. Methyl 3-(4-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-
1-carboxylate (E27)
1
White powder. Yield: 45.8%. m.p. 168-169 °C. H-NMR (DMSO-d , 600 MHz) δ 9.95 (s,
6
1H), 7.58 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.47 (s, 2H), 5.34 (dd, J = 11.7,
5.2 Hz, 1H), 3.76 (dd, J = 17.7, 11.8 Hz, 1H), 3.73 (s, 6H), 3.65 (s, 3H), 3.63 (s, 3H),
+
3.09 (dd, J = 17.7, 5.2 Hz, 1H). MS (ESI): 387.15 (C H N O , [M+H] ). Anal. Calcd.
20
22
2
6
for C20
H
22
N
2
O
6
: C, 62.17; H, 5.74; O, 24.84; Found: C, 62.15; H,5.75; O, 24.85.
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5
5
5
5
5
5
5
78
79
80
81
82
83
84
85
4.2.31. Methyl
3-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carboxylate (E28)
White powder. Yield: 81.3%. m.p. 143-145 °C. H-NMR (DMSO-d
1
6
, 600 MHz) δ 7.31 (d,
J = 1.9 Hz, 1H), 7.24 (dd, J = 8.3, 2.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.48 (s, 2H),
5.37 (dd, J = 11.8, 5.2 Hz, 1H), 3.83 - 3.76 (m, 7H), 3.73 (s, 6H), 3.66 (s, 3H), 3.63 (s,
+
3H), 3.16 (dd, J = 17.8, 5.3 Hz, 1H). MS (ESI): 431.17 (C H N O , [M+H] ). Anal.
2
2
26
2
7
Calcd. for C H N O : C, 61.39; H, 6.09; O, 26.02; Found: C, 61.35; H,6.11; O, 26.04.
2
2
26
2
7

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4.2.32. Crystal structure determination
Crystal structure determination of compound E04 was carried out at 293 K on a Bruker
D8 VENTURE PHOTON diffractometer equipped with graphite-monochromated Mo Kα
2
(λ = 0.71073 Å) radiation. The structure was solved by direct methods and refined on F
by full-matrix least-squares methods using SHELXTL (Bruker, German). All the non-
hydrogen atoms were refined with anisotropic thermal parameters. All the hydrogen
atoms were placed in geometrically idealized positions and constrained to ride on the
parent atoms.
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4.3. Biology
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4.3.1. Cell culture
Human liver carcinoma cell line HepG2, human non-small-cell-lung cancer cell line
A549, human colon cancer cell line HT29, human epithelial cervical cancer cell line
HeLa, human breast cancer cell line MDA-MB-231 and human ovarian carcinoma cell
line A2780 were cultivated in DMEM medium containing 100 units/mL of penicillin, 100
µg/mL of streptomycin and 10% (v/v) FBS and incubated at 37 °C in a humidified
2
atmosphere containing 5% CO .
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4.3.2. Anti-proliferation assay
The anti-proliferative and cytotoxic effect of compounds were evaluated by employing
MTT assay. Cells in logarithmic phase were harvested and plated into 96-well plates
(5,000 cells/well). Then cells were treated with different concentrations of compounds.
After incubation of 48h, 10 µL MTT dye (5 mg/mL) was added to each well, followed by

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incubating for more 4 h. Then, the suspension was discarded and 150 µL DMSO was
added to each well. After 10 min shaking on shaking table, plates were read in Infinite®
M200 Pro Multimode Microplate Reader (Tecan, Switzerland) at the wavelength of 570
nm (reference wavelength 650 nm). Three replicate wells were used for each
concentration and each assay was carried out at least three times. The values GI were
5
0
calculated with Prism 7.0 (GraphPad Software, USA). The GI50 was defined as the
compound concentration required to inhibit cell proliferation by 50%, in comparison with
cell treated with the maximum amount of DMSO (0.1%) and considered as 100%
viability.
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4.3.3. Colony formation assay
1,000 HeLa cells in exponential growth was seeded into each well of 6-well plate. After
incubation for 12 h, cells were treated with test compounds for 2 weeks and the culture
medium containing compounds was changed every 3 days. Then the cells were stained by
1 mL crystal violet staining solution for 30 min and washed twice with fresh PBS. The
stained colonies were counted and compared with the control cells.
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4.3.4. Tubulin polymerization inhibition assay
OD340 Method was performed to evaluate the effect of the compounds on tubulin
assembly in vitro.[31] Porcine brain tubulin was purified as described previously[32]
with modification. Test compounds in a range of concentrations were placed into 96-well
plates (10 µL/well). A solution of tubulin in G-PEM buffer was prepared freshly with ice
bath and added into plates (90 µL/well). Thus, in single well, it contains 3.6 mg/mL