Design, synthesis and biological evaluation of a novel tubulin inhibitor 7a3 targeting the colchicine binding site

Article abstract of DOI:10.1016/j.ejmech.2018.05.010

Tubulin inhibitors that target the colchicine binding site continue to emerge as promising anticancer agents. In this study, based on the anti-proliferative activities, a novel tubulin inhibitor 7a3 targeting the colchicine binding site was designed, synthesized, and optimized from a series of novel cis-restricted pyrazole analogues of combretastatin A-4. The structure-activity relationships (SARs) of these newly synthesized compounds are summarized indicating that the methyl substituent at the N1 position and deamination were significantly important for the anti-proliferative efficacy. The optimized compound 7a3 exhibited the ability to arrest the cell cycle in the G2/M phase, induce cell apoptosis, and inhibit cell migration in tumour cells. The results of the immunofluorescence analysis using confocal microscopy and the tubulin polymerization assay revealed that tubulin assembly was disrupted by 7a3 in vitro. Furthermore, the targeting identification of 7a3 was illuminated by solving the crystal structure of 7a3 in complex with tubulin at a resolution of 3.2 ? (PDB code 5Z4U), which confirmed the result of molecular docking and further demonstrated that 7a3 binds to the site of colchicine. Moreover, the pharmacokinetic analysis in mouse plasma showed that 7a3 rapidly reached a peak concentration at 0.25 h after intraperitoneal administration, and the T_1/2, C_max, and AUC_0-inf were 1.67 ± 0.28 h, 882 ± 71 ng mL^-1, and 1166 ± 129 h ng·mL^-1, respectively, after a single-dose administration analysed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In addition, the in vivo study indicated that 7a3 significantly inhibited the tumour growth of the SK-OV-3 xenograft in a nude mouse model. In conclusion, our study proved 7a3 to be a potential microtubule-targeting drug for cancer therapy. The SARs and mechanism of action studies of 7a3 based on the X-ray co-crystal structure provided insights into the next-generation tubulin inhibitors for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2018.05.010

Accepted Manuscript
Design, synthesis and biological evaluation of a novel tubulin inhibitor 7a3 targeting
the colchicine binding site
Qinhuai Lai, Yuxi Wang, Ruixue Wang, Weirong Lai, Liangze Tang, Yiran Tao, Yu
Liu, Ruirui Zhang, Luyi Huang, Haotian Xiang, Shaoxue Zeng, Lantu Gou, Hao Chen,
Yuqin Yao, Jinliang Yang
PII:
S0223-5234(18)30414-8
10.1016/j.ejmech.2018.05.010
EJMECH 10420
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2018
Revised Date: 20 April 2018
Accepted Date: 7 May 2018
Please cite this article as: Q. Lai, Y. Wang, R. Wang, W. Lai, L. Tang, Y. Tao, Y. Liu, R. Zhang,
L. Huang, H. Xiang, S. Zeng, L. Gou, H. Chen, Y. Yao, J. Yang, Design, synthesis and biological
evaluation of a novel tubulin inhibitor 7a3 targeting the colchicine binding site, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.010.
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ACCEPTED MANUSCRIPT
Graphical abstract
Based on the anti-proliferative activities, a novel tubulin inhibitor 7a3 targeting
the colchicine binding site was designed, synthesized, and optimized from a series of
novel cis-restricted pyrazole analogues of combretastatin A-4. The targeting
identification of 7a3 was illuminated by solving the crystal structure of 7a3 in
complex with tubulin at a resolution of 3.2 Å (PDB code 5Z4U). Moreover, the
anti-tumor activity and pharmacokinetic analysis in vivo were performed to
demonstrate that 7a3 is a potential microtubule-targeting drug for cancer therapy.

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of a novel tubulin
inhibitor 7a3 targeting the colchicine binding site
a,b
b
b
b
b
b
Qinhuai Lai , Yuxi Wang , Ruixue Wang , Weirong Lai , Liangze Tang , Yiran Tao ,
b
b
b
c
c
b
Yu Liu , Ruirui Zhang , Luyi Huang , Haotian Xiang , Shaoxue Zeng , Lantu Gou ,
b
a,b
b,d
Hao Chen *, Yuqin Yao *, Jinliang Yang
a
Research Center for Public Health and Preventive Medicine/Research Center for Occupational
Respiratory Diseases, West China School of Public Health and Healthy Food Evaluation Research
Center /No.4 West China Teaching Hospital, Sichuan University, Chengdu, P.R. China;
b
State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu, P.R. China;
c
Ophthalmic Laboratories & Department of Ophthalmology, Translational Neuroscience Center,
West China Hospital, Sichuan University, Chengdu, P.R. China;
d
Guangdong Zhongsheng Pharmaceutical Co., Ltd., China
*Correspondence to professor Yuqin Yao, Research Center for Public Health and Preventive
Medicine/Research Center for Occupational Respiratory Diseases, West China School of Public
Health/No.4 West China Teaching Hospital, Sichuan University, Chengdu, Sichuan, 610041,
No.16 People's South Road, Chengdu, 610041, P.R. China. Tel: + 86 28 85501580; fax: +86 28
8
5501580; e-mail: yuqin_yao@scu.edu.cn (professor Yuqin Yao); and professor Hao Chen, State
Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University,
Chengdu, P.R. China, e-mail: hchen55@uthsc.edu (professor Hao Chen
)
Abbreviations: ABC, ATP-binding cassette; P-gp, P-glycoprotein; MRP, multidrug resistant
proteins; CA-4, combretastatin A-4; SARs, structure-activity relationships; NBS,
N-bromosuccinimide; DME, 1,2-dimethoxyethane; CCK-8, cell counting kit-8; DMEM, dulbecco
minimum essential medium; FBS, fetal bovine serum; PI, propidium iodide; PK,
pharmacokinetics.

ACCEPTED MANUSCRIPT
Abstract
Tubulin inhibitors that target the colchicine binding site continue to emerge as promising
anticancer agents. In this study, based on the anti-proliferative activities, a novel tubulin inhibitor
7
a3 targeting the colchicine binding site was designed, synthesized, and optimized from a series of
novel cis-restricted pyrazole analogues of combretastatin A-4. The structure-activity relationships
SARs) of these newly synthesized compounds are summarized indicating that the methyl
(
substituent at the N1 position and deamination were significantly important for the
anti-proliferative efficacy. The optimized compound 7a3 exhibited the ability to arrest the cell
cycle in the G2/M phase, induce cell apoptosis, and inhibit cell migration in tumour cells. The
results of the immunofluorescence analysis using confocal microscopy and the tubulin
polymerization assay revealed that tubulin assembly was disrupted by 7a3 in vitro. Furthermore,
the targeting identification of 7a3 was illuminated by solving the crystal structure of 7a3 in
complex with tubulin at a resolution of 3.2 Å (PDB code 5Z4U), which confirmed the result of
molecular docking and further demonstrated that 7a3 binds to the site of colchicine. Moreover, the
pharmacokinetic analysis in mouse plasma showed that 7a3 rapidly reached a peak concentration
at 0.25 h after intraperitoneal administration, and the T , Cmax, and AUC0-inf were 1.67 ± 0.28 h,
1/2
-1
-1
8
82 ± 71 ng·mL , and 1166 ± 129 h·ng·mL , respectively, after a single-dose administration
analysed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In addition, the in
vivo study indicated that 7a3 significantly inhibited the tumour growth of the SK-OV-3 xenograft
in a nude mouse model. In conclusion, our study proved 7a3 to be a potential
microtubule-targeting drug for cancer therapy. The SARs and mechanism of action studies of 7a3
based on the X-ray co-crystal structure provided insights into the next-generation tubulin
inhibitors for cancer therapy.
Keywords: Tubulin inhibitor, Colchicine binding site, Combretastatin A-4, Pyrazole
analogue, Anti-tumour, Co-crystal structure

ACCEPTED MANUSCRIPT
1
. Introduction
Microtubules are crucial elements of the cytoskeleton and consisting of α- and
β-tubulin heterodimers [1, 2]. Microtubules play essential roles in a wide range of
cellular functions, such as division, signalling, motility, maintenance of cell shape,
and intracellular vesicle transport, among others [3, 4]. Therefore, they have been
represented as an attractive target in anti-tumour drug discovery [5, 6]. The assembly
and disassembly of the α- and β-tubulin heterodimers are dynamic equilibrium
processes. Numerous molecules that disrupt the equilibrium have been developed as
tubulin inhibitors [7, 8]. Most of these exert their effect by binding to one of the three
well-identified sites on β-tubulin. For example, paclitaxel binds to the taxane site as a
microtubule stabilizing agent, and vincristine binds to the vinca alkaloid site as a
destabilizing agent; these compounds are both proven cancer therapies used in the
clinic. However, their clinical efficacy is often limited due to the expression of
ATP-binding cassette (ABC) transporters in tumour cells, including P-glycoprotein
(P-gp), multidrug resistant protein (MRP), and breast cancer resistant protein (BCRP),
which have been found to be the mechanisms for resistance to these drugs [9-11].
Extensive research has indicated that tubulin inhibitors targeting the colchicine
binding site have been considered as candidates that can overcome the limitations of
drug resistance and improve their clinical benefit [12, 13].
Among the potent tubulin inhibitors binding to the colchicine site, combretastatin
A-4 (CA-4) is typical and well described (Fig. 1A). CA-4 was first isolated from the
bark of the South African tree Combretum caffrum by Pettit in 1998 [14]. By binding
strongly at the colchicine site of β-tubulin and arresting the cell cycle in the G2/M
phase [15, 16], CA-4 exhibits a powerful cytotoxicity against a large number of
cancer cell lines, including multidrug resistant cancer cells [17, 18]. However, its poor
water solubility and bioavailability has prevented its development as an antitumour
drug [19]. The disodium phosphate salt of CA-4 (CA-4P, Fig. 1A) and the serinamide
derivatives (AVE8062, Fig. 1A) were synthesized to improve performance and have
shown promising results in advanced clinical trials [20-23], which has stimulated

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more interest in developing CA-4 analogues as tubulin inhibitors targeting the
colchicine site.
Previous structure-activity relationships (SARs) studies have been carried out in
the past decades that have indicated that a cis-configuration of the olefinic bridge and
the 3,4,5-trimethoxy substitution on A-ring were indispensable for biological activity,
while the B-ring offered better prospects for modification [17, 24-26]. However, the
cis-configuration of CA-4 is readily converted to the trans-isomer during metabolism,
resulting in a dramatic decrease in antitumour activity [27]. Many cis-restricted
analogues of CA-4 have been evaluated after the replacement of the olefinic double
bond with five-membered heterocycles, such as pyrazole, thiazole, tetrazole, and
imidazole, etc., which have proven to have effective biological activity [28-33].
In our efforts to discover new tubulin inhibitors that target the colchicine binding
site, we developed a series of novel 1,4-substituted-3-(3’,4’,5’-trimethoxyphenyl)-5-
aminopyrazole analogues of CA-4 (Fig. 1B and Scheme 1). The trimethoxyphenyl
moiety was maintained as an A-ring, and modifications were focused on the B-ring
and pyrazole heterocycle. We introduced methyl, tert-butyl, 4-fluorophenyl, and
4
4
-methoxyphenyl at the N1 position. Additionally, the B-ring was modified with
-methoxyl, 3-fluoro-4-methoxyl, 4-ethoxyl, and 4-methylthiol. Compounds 6a1-6d4
were obtained and evaluated for their anti-proliferative activities. Compounds 6a3 and
a4 demonstrated a more potent activity than the others, which indicated that the
6
methyl substitution at the N1 position was advantageous for activity and inspired us to
focus on the further exploration of these analogues with N1-methylation.
Therefore, a simple deaminization procedure was performed to generate the
compounds 7a1-7a4 (Fig. 1B and Scheme 2) to estimate the potential effects of amino
groups on anti-proliferative activity. In addition, by moving the A-ring of compounds
7
a1-7a4 from the C3 to C5 positions, the corresponding compounds 12a1-12a4 were
produced to further explore whether different substitution positions of the A-ring on
the pyrazole heterocycle were crucially important for anti-proliferative efficacy (Fig.
1
B and Scheme 3). Here, we report the design, synthesis, and biological evaluation of
these novel analogues. The optimized compound 7a3 was identified to interact with

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the colchicine binding site by solving the co-crystal structure in complex with tubulin.
Our studies provide insights to develop next-generation tubulin inhibitors that target
the colchicine binding site for cancer therapy.
2
. Results and discussion
Fig. 1. (A) Chemical structures of CA-4, CA-4P, AVE8602, and colchicine. (B) tThe design of
novel tubulin inhibitors based on CA-4. (C) Proposed binding mode of 7a3 in the colchicine site
of β-tubulin in crystal structure 1SA0, and 7a3 (yellow) shares the similar binding mode with
CA-4 (slivery, middle) and colchicine (brillant blue, right).
2
.1. Chemistry
The 1,4-disubstituted-3-(3’,4’,5’-trimethoxyphenyl)-5-amino pyrazole analogues
a1−6d4 were synthesized by the general five-step synthesis outlined in Scheme 1.
6

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After methyl esterification of 3,4,5-trimethoxybenzoic acid 1, followed by treatment
with sodium hydride in refluxing acetonitrile-tetrahydrofuran [34], the resulting
compound 3 was reacted with the appropriate hydrazine derivatives R -NHNH ,
1
2
which led to the generation of the pyrazole heterocyclic compounds 4a-4d [35].
Subsequently, the reaction of bromo-substitution with N-bromosuccinimide (NBS)
yielded the key intermediates 5a-5d with good yields. Through the Suzuki reaction
with several phenylboronic acids in the presence of Cs CO and PdCl (PPh ) in
2
3
2
3 2
1
,2-dimethoxyethane (DME) at 80 °C overnight, we successfully accomplished the
synthesis of analogues 6a1-6d4. Analogues 6a1-6a4 were easy to deaminate, and the
resulting analogues 7a1-7a4 were obtained (Scheme 2) [36]. The synthetic procedure
for the modification of moving the A-ring from the C3 to C5 positions on the pyrazole
heterocycle is shown in Scheme 3. Starting from a Suzuki reaction with the
bromobenzene 8, (1-methyl-1H-pyrazol-5-yl)boronic acid 9, Na CO , and Pd(PPh )
2
3
3 4
in a mixed solution of THF-H O (2:1) at 70 °C for 12 h, compound 10 was
2
synthesized and a bromine was then introduced at the C4 position. Finally, analogues
1
2a1-12a4 were obtained by Suzuki reactions with the intermediate 11 and the
appropriate phenylboronic acids.
Scheme 1. Synthesis of compounds 6a1-6d4. Reagents and conditions: (a) concentrated sulfuric
acid, CH OH, 65 °C, 24 h, 100%; (b) CH CN-THF (1:1), NaH, 80 °C, 5 h, 77%; (c) R -NHNH ,
3
3
1
2
AcOH-EtOH (1:1), 100 °C, 12 h, 78-87%; (d) N-bromosuccinimide, DMF, 0 °C, 3 h, 84-89%; (e)

ACCEPTED MANUSCRIPT
R PhB(OH) , Cs CO PdCl (PPh ) , DME, 80 °C, 12 h, 31-47%.
2
2
2
3,
2
3 2
O
O
O
O
O
N
O
N
H
a
N
CH3
N CH3
R
NH2
R
6
6
6
6
a1, R =p-OCH3
7a1, R =p-OCH3
a2, R =p-OCH -m-F
7a2, R =p-OCH3-m-F
7a3, R =p-OCH2CH3
7a4, R =p-SCH3
3
a3, R =p-OCH CH3
2
a4, R =p-SCH3
Scheme 2. Synthesis of compounds 7a1-7a4. Reagents and conditions: (a) butyl nitrite, THF,
5 °C, 3 h, 82-90%.
6
N
Br
N CH3
N
N CH3
a
+
O
O
O
O
B
O
HO
OH
O
8
9
10
N
N
N CH3
N CH3
Br
R
b
c
O
O
O
O
O
O
11
1
1
1
1
2a1, R= p-OCH3
2a2, R= p-OCH -m-F
3
2a3, R= p-OCH CH
2
3
2a4, R= p-SCH3
Scheme 3. Synthesis of compounds 12a1-12a4. Reagents and conditions: (a) Na CO , Pd(PPh ) ,
2
3
3 4
THF-H O (2:1), 70 °C, 12 h, 95%; (b) N-bromosuccinimide, DMF, 0 °C, 3 h, 86%; (c) ArB(OH) ,
2
2
Na CO , Pd(PPh ) , THF-H O (2:1), 70 °C, 12 h, 60-63%.
2
3
3 4
2
2
.2. Activities of anti-proliferation in vitro
The newly synthesized analogues were screened for their anti-proliferative
activities against SK-OV-3 (human ovarian cancer cell) and MDA-MB-231 (human
breast cancer cell) with CA-4 as positive control using a CCK-8 assay. The results are
summarized in Table 1 and show that most of the tested compounds exhibited

ACCEPTED MANUSCRIPT
moderate to potent anti-proliferative efficacy compared with CA-4, which exhibited
strong cytotoxicity with IC values of 5.9 nM and 4.5 nM against the two tumour
5
0
cells. Among them, compounds 7a1-7a4 and 12a1-12a4 displayed considerable
efficacy with IC values ranging from 0.017 to 0.307 μM, which indicated the
5
0
importance of the modifications in the pyrazole heterocycle and B-ring. In addition,
compounds 7a2, 7a3, 12a2, and 12a3 were further evaluated for their
anti-proliferative activities in four additional cancer cell lines, HeLa (human epithelial
cervical cancer cell), A549 (non-small-cell lung cancer cell), CT26 (mouse colon
cancer cell), and MCF-7 (human breast cancer cell). All of them possessed potent
efficacy with IC values ranging from 28.8 to 187.3 nM, slightly less active than
5
0
CA4 with IC values ranging from 14.2 to 94.3 nM (Table 2 and Supplementary Fig.
5
0
1
). Among these tested compounds, 7a3 exerted the best anti-proliferative activity and
was comparable to that of reported five-membered heterocycles analogues of CA-4
28-33]. Based on the activity of anti-proliferation in vitro, 7a3 was selected for
further study.
[
2
.3. Structure-activity relationships study
According to the anti-proliferative activity evaluations of these compounds
mentioned above, the preliminary structure-activity relationships (SARs) of the
pyrazole analogues of CA-4 were investigated.
Generally, compounds 6a1-6d4 displayed moderate activities compared with
CA-4, with IC values in the micromolar range. Notably, of the 16 compounds, 6a3
5
0
and 6a4 with methyl substituted at the N1 position possessed much higher potency,
showing IC values of 0.338-0.438 μMꢀand 0.490-0.748 μM against SK-OV-3and
5
0
MDA-MB-231 cells, respectively. The replacement of the methyl group with larger
bulky groups led to a dramatic decrease in activity for some compounds (6a4
compared with 6b4, 6c4, and 6d4). However, the activities of the compounds with the
additional introduction of 4-ethyoxyl at the B-ring were only slightly decreased (6a3
compared with 6b3, 6c3, and 6d3). Compounds with the substitution of 4-methoxyl or

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3
-fluoro-4-methoxyl on the B-ring showed minimal activities, and there was little
change when methyl was transformed to other substituents (6a1-6a2 compared with
b1-6b2, 6c1-6c2, and 6d1-6d2). In summary, these results revealed the crucial
6
contributions of the methyl at N1 and the 4-ethyoxyl or 4-methylthiol on the B-ring
for anti-proliferative efficacy (6a3 and 6a4).
Compounds 7a1-7a4 exhibited stronger activities than those bearing an amino
group on the pyrazole heterocycle, with IC50 values of 0.017-0.155 μM against
SK-OV-3 cells and 0.031-0.307 μM against MDA-MB-231 cells (compared with
6
a1-6a4), especially for compounds 7a1 and 7a2, which displayed an approximately
83-887-fold increase in potency comparted with compounds 6a1 and 6a2. These
1
amazing results indicated that the removal of the amino was critically important for
anti-proliferative efficacy. The effects on anti-proliferative activity of different
substitution positions of the A-ring on the pyrazole heterocycle were also evaluated.
As reported in Table 1, the compounds 12a1-12a4, with the A-ring at the C5 position,
exhibited comparable anti-proliferative efficacy to compounds 7a1-7a4, with the
A-ring at the C3 position, which demonstrated the minor effect on activity of changes
in the A-ring substitution positions.

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Table 1. The in vitro anti-proliferative activities of compounds 6a1-6d4, 7a1-7a4, and 12a1-12a4
against SK-OV-3 and MDA-MB-231 cell lines with CA-4 reference.
a
IC50 ± SEM (ꢁM)
Compounds
R1
R2
SKOV3
16.30 ± 0.77
31.78 ± 2.06
0.338 ± 0.083
0.438 ± 0.084
40.29 ± 1.82
16.93 ± 1.05
1.12 ± 0.16
24.25 ± 3.29
28.92 ± 1.24
33.21 ± 2.26
2.83 ± 0.12
7.37 ± 3.03
19.03 ± 2.35
14.66 ± 2.80
1.21 ± 0.14
13.97 ± 0.77
MDA-MB-231
31.12 ± 1.05
36.38 ± 1.12
0.490 ± 0.158
0.748 ± 0.240
31.29 ± 0.52
29.49 ± 1.54
2.57 ± 0.69
6
6
6
6
6
6
6
6
a1
a2
a3
a4
b1
b2
b3
b4
CH
3
3
3
3
p-OCH
3
CH
CH
CH
p-OCH
p-OCH
p-SCH
p-OCH
p-OCH -m-F
p-OCH CH
p-SCH
p-OCH
p-OCH -m-F
p-OCH CH
p-SCH
p-OCH
-Phenyl p-OCH -m-F
-Phenyl p-OCH CH
-Phenyl p-SCH
-m-F
3
CH
2
3
3
Bu-t
Bu-t
3
3
Bu-t
2
3
Bu-t
20.80 ± 3.73
33.69 ± 3.46
24.59 ± 1.14
1.66 ± 0.13
3
6
6
6
6
c1
c2
c3
c4
p-F-Phenyl
p-F-Phenyl
p-F-Phenyl
p-F-Phenyl
3
3
2
3
22.93 ± 1.17
15.90 ± 2.93
18.38 ± 0.70
2.01 ± 0.18
3
6d1
6d2
6d3
6d4
p-OCH
3
3
3
3
-Phenyl
3
p-OCH
p-OCH
p-OCH
3
2
3
7.05 ± 1.75
3
7a1
7a2
7a3
7a4
CH
CH
CH
CH
3
3
3
3
p-OCH
p-OCH -m-F
p-OCH CH
p-SCH
3
0.089 ± 0.011
0.055 ± 0.003
0.017 ± 0.003
0.155 ± 0.027
0.166 ± 0.049
0.041 ± 0.006
0.031 ± 0.002
0.307 ± 0.016
3
2
3
3
12a1
12a2
12a3
12a4
CH
CH
CH
CH
3
3
3
3
p-OCH
p-OCH -m-F
p-OCH CH
p-SCH
3
0.069 ± 0.002
0.066 ± 0.006
0.043 ± 0.004
0.052 ± 0.004
0.074 ± 0.002
0.057 ± 0.013
0.072 ± 0.004
0.041 ± 0.003
3
2
3
3
CA-4
0.0059 ± 0.0014
0.0045 ± 0.0002
a
The IC was defined as the compound concentration required to inhibit cell proliferation by 50%.
5
0
Data are expressed as the mean ± SEM from at least three independent experiments.

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Table 2. The in vitro anti-proliferative activities of compounds (7a2, 7a3, 12a2 and 12a3) against
several tumour cell lines with CA-4 as a positive reference.
a
IC50 ± SEM (nM)
Tumour Cell lines
7
a2
7a3
12a2
66.1 ± 5.6
12a3
42.7 ± 3.4
71.6 ± 4.5
CA-4
5.9 ± 1.4
4.5 ± 0.2
SK-OV-3
MDA-MB-231
HeLa
55.3 ± 3.4
40.6 ± 5.8
28.8 ± 1.7
141.1 ± 41.2
58.7 ± 3.4
37.2 ± 11.2
16.7 ± 3.0
31.4 ± 0.7
32.8 ± 2.9
67.0 ± 0.8
58.0 ± 2.4
35.4 ± 5.6
57.3 ± 7.1
111.9 ± 6.0
187.3 ± 38.1
150.6 ± 34.7
112.8 ± 4.9
93.1 ± 14.6 14.2 ± 2.4
99.0 ± 16.5 94.3 ± 16.1
165.2 ± 22.8 45.1 ± 13.2
A549
CT26
MCF-7
39.9 ± 7.8
39.1 ± 11.5
a
The IC50 is defined as the compound concentration required to inhibit cell proliferation by 50%.
Data are expressed as the mean ± SEM from three independent experiments.
2
.4. Molecular modelling
The molecular docking simulations on β-tubulin were performed using a
previously reported procedure. The binding mode observed for 7a3 was very similar
to that for CA-4 and colchicine in the co-crystallized tubulin structure
(DAMA-colchicine, PDB code 1SA0) (Fig. 1C). The trimethoxyphenyl group of 7a3
was placed in proximity to Val238, Ile378, and Leu248, and occupied the same
position as the corresponding moiety of colchicine and CA-4. Furthermore, the
pyrazole was placed towards the external part of the pocket, which does not seem to
establish some specific interactions with tubulin. This observation was further
confirmed by the X-ray co-crystal structure of 7a3 in complex with β-tubulin (Fig. 5)
2
.5 Analysis of cell cycle effects induced by 7a3
The effects of 24 h treatment with different concentrations of 7a3 on the cell
cycle in SK-OV-3 cells were determined by flow cytometry, and untreated cells were
used as a negative control. 7a3 was found to be effective in arresting the cell cycle,
resulting in the accumulation of cells in the G2/M phase. Regarding the untreated

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cells, the percentage of cells in the G0/G1 phase was 67.79%, while only 14.12%
were in the G2/M phase. However, after incubation with 7a3 at the concentrations of
4
6
0, 80, and 160 nM for 24 h, the percentages increased to 24.59%, 34.65%, and
4.79%, respectively (Fig. 2A and Fig. 2C). These results show that compound 7a3
significantly induced cellular mitotic arrest in the G2/M phase, which was consistent
with other well-known tubulin inhibitors.
2
.6. 7a3 induced cellular apoptosis
To characterize the mode of cell death induced by 7a3, a biparametric
cytofluorimetric analysis was performed using propidium iodide (PI), which stains
DNA and is permeable only to dead cells and fluorescent immunolabelling of the
protein annexin-V, which binds to phosphatidylserine (PS) in a highly selective
manner [37]. Dual staining for annexin-V with PI permits discrimination between live
-
-
+
-
cells (annexin-V /PI ), early apoptotic cells (annexin-V /PI ), late apoptotic cells
+
+
-
+
(
annexin-V /PI ), and necrotic cells (annexin-V /PI ) [38]. SK-OV-3 cells treated
with 7a3 at the concentrations of 40, 80, and 160 nM for 48 h showed an
accumulation of apoptotic cells (annexin-V positive cells) from 4.90% (untreated
control) to 8.00 %, 23.72 %, and 56.82 %, respectively (Fig. 2B and Fig. 2D). These
results indicated that compound 7a3 possibly exerted the anti-proliferative effect
through the induction of cellular apoptosis.

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Fig. 2. (A) Cell cycle distribution of SK-OV-3 cells after treatment with the indicated
concentrations of 7a3 for 24 h; cell cycles were arrested in G2/M phase. (B) Cell apoptosis assay
showing that 7a3 could induce apoptosis of SK-OV-3 cells after incubation for 48 h in a
concentration-dependent manner. (C) Statistic histograms showing that 7a3 induced cell cycle
arrest in the G2/M phase. (D) Statistic histograms showing that 7a3 induced cell apoptosis. Cells
were treated with 40, 80, and 160 nM of 7a3 and stained with PI to analyse the cell cycle or were
stained with Annexin V-FITC/PI to measure cell apoptosis. Data are represented as the mean ±
SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 indicates
significantly different from the control by the t-test. Data are expressed as the mean ± SEM from
three independent experiments.
2
.7. 7a3 inhibited the migration of SK-OV-3 cells
Scratch wound and transwell migration experiments were performed

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respectively to determine the inhibition of the migratory potential of SK-OV-3 cells by
7
a3 with CA-4 serving as reference. In the scratch wound assay, the inhibitory effect
of treatment with 7a3 was comparable to that with CA-4, and there were significant
differences between the control and drug treated groups (7a3 and CA-4). Compared
with the control group, 7a3 or CA-4 significantly suppressed the cell migration at the
concentration of 80 nM or 160 nM, (Fig. 3A and Fig. 3B). The wound closure
percentages were 79.5 ± 2.8, 44.3 ± 4.0, and 32.1 ± 0.9 after 24 h in the control group,
8
0 nM, and 160 nM of 7a3-treated group, respectively. And the percentages were 41.9
2.9 and 29.2 ± 2.1 in 80 nM and 160 nM of CA4-treated group, respectively. (P <
.01, Fig. 3B). In the transwell migration assay, a significant inhibition of migration
±
0
was observed in the treated groups, and the inhibitory effect of treatment with 7a3 was
similar with that of CA-4 at the concentrations of 25 nM, 50 nM, and 100 nM
respectively ( Fig. 3C and Fig. 3D). The results showed that the mean numbers of
migrated cells after 24 h were 50.7 ± 2.5, 27.3 ± 2.1, 21.7 ± 2.5, and 18.3 ± 2.1 in the
control group, 25 nM, 50 nM, and 100 nM of 7a3-treated group, respectively. And the
mean numbers of migrated cells after 24 h were 49.3 ± 2.1, 26.3 ± 1.2, 20.7 ± 3.9, and
1
8.3 ± 2.9 in the corresponding concentrations of CA-4, respectively (P < 0.01, Fig.
3
D). The scratch wound and transwell migration assay results indicated that cancer
cell migration was significantly decreased by 7a3.

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Fig. 3. (A) The results of scratch wound assay. Representative images of wound closure after
treatment with vehicle (control), 7a3 (80 and 160 nM), and CA-4 (80 and 160 nM) for 0, 12, and
2
2
4 h. Magnification, × 40. (B) Wound closure percentages in the different groups after treatment for
4 h. (C) Results of the transwell migration assay. Representative images of the stained migrated
cells after incubation with vehicle (control), 7a3, and CA-4 at the concentrations of 25, 50, and
00 nM for 24 h. Magnification, × 200. (D) Number of the stained migrated cells in the different
1
groups. **P < 0.01, and ***P < 0.001 compared with the control group by the t-test. Data are
expressed as the mean ± SEM from three independent experiments.
2
.8.
I
nhibition of tubulin polymerization
To investigate the inhibitory activity of 7a3 by its interactions with microtubule
system, the tubulin polymerization inhibition activities were evaluated of 7a3 at
different concentrations (2.5, 10, and 40 µM) with CA-4 (10 µM) serving as positive
control. As shown in Fig. 4A, 7a3 inhibited tubulin polymerization in a
concentration-dependent manner, and the inhibitory activity of 7a3 at 10 µM was
comparable to that of CA-4 at 10 µM). 30.5%, 60.4%, and 85.3% of tubulin
polymerization were observed in the treatment group of 7a3 at the concentrations of

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2
.5, 10, and 40 µM, respectively, compared to the vehicle group (DMSO). These
results corresponded well with the anti-proliferative potency, which indicated that the
growth of cancer cells was inhibited by 7a3 through the inhibition of tubulin
polymerization.
2
.9. Immunofluorescence analysis
To investigate whether the biological activity of 7a3 was derived from an
interaction with tubulin, we further examined the effect of 7a3 on the cellular
microtubule network. SK-OV-3 cells were treated with DMSO (control) or 7a3 at the
concentrations of 10, 100, and 1000 nM for 24 h. After incubation, the cells were
fixed in methanol, stained with FITC-conjugated (green) anti-β-tubulin antibody,
counterstained with DAPI (blue) and then analysed by confocal microscopy. As
shown in Fig. 4B, the DMSO-treated cells (control) exhibited a normal arrangement
and organization, with microtubules extending from the central regions to the cell
periphery. In contrast, after 24 h of exposure to 7a3, the microtubule polymerization
and spindle formation became disordered, and the cells were arrested in mitosis with
abnormal mitotic spindles, which demonstrated that 7a3 strongly inhibited tubulin
assembly in SK-OV-3 cells.

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Fig. 4. (A) Effects of 7a3 on in vitro tubulin polymerization. Polymerization of tubulin at 37 °C in
the presence of 7a3 (2.5, 10, and 40 µM), CA-4 (10 µM), and DMSO (1%, v/v) were monitored
continuously by measuring the absorbance at 340 nm every 1 min for 30 min. (B)
Immunofluorescence assays showed the effects of 7a3 on the organization of the microtubule
network of SK-OV-3 cells. After treatment with DMSO (control) or 7a3 at the indicated
concentrations (10, 100, and 1000 nM) for 24 h, cells were stained with FITC-conjugated
anti-β-tubulin antibody and DAPI. Microtubules and unassembled tubulin are shown in green, and
DNA is shown in blue.
2
.10. X-ray crystal structure of T2R-TTL complexed with 7a3.

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To further understand the binding mechanism of 7a3 and to provide available
indications for structural optimization, X-ray crystallography was used to elucidate the
interactions between 7a3 and tubulin. We solved the soaked-crystal structure of the
T2R-TTL (consisting of a/β-tubulin, the stathmin-like protein RB3, and tubulin
tyrosine ligase) with 7a3 at a resolution of 3.2 Å (PDB code 5Z4U). The details of the
data collection and structure refinement statistics are provided in Supplementary Table
1
. As expected, 7a3 occupies the colchicine binding site on tubulin (Fig. 5B). All the
interactions between 7a3 and tubulin at the colchicine binding site are hydrophobic
interactions with the side chain of the residues in the β-subunit, including L240, A248,
K252, N256, V316, K350, and A352 (no hydrogen bonds were found), which provides
us with a new possible modification for further structural optimization (Fig. 5C).
Colchicine targets the β-subunit of tubulin and prevents it from adopting a
straight conformation, thus inhibiting microtubule assembly. Consistently, the binding
of 7a3 also blocked the curve-to-straight conformational change of tubulin by the
steric clashes between 7a3 and the surrounding secondary structure elements (Fig.
5
D). In addition, we found that 7a3 occupied the same position as colchicine (PDB
code 4o2b) and CA-4 (PDB code 5LYJ) and overlapped with them quite well (Fig.
E).These findings indicated that the inhibitory mechanism of 7a3 against tubulin was
achieved through binding to the colchicine site.
5

ACCEPTED MANUSCRIPT
Fig. 5. X-ray analysis of the T2R-TTL-7a3 complex. (A) Docked pose of 7a3 (orange), CA-4
(magenta), and colchicine (cyan) overlapped with DAMA-colchicine (green) in the tubulin
binding site. (B) Cartoon representation of the overall structure of the T2R-TTL complex with 7a3.
The α-units of the T2R-TTL complex are shown in black; the β-units are shown in grey; TTL is
shown in green and RB3 is shown in blue. GTP, GDP, and 7a3 are shown by magenta, cyan, and
yellow spheres, respectively. (C) Detailed interactions of 7a3 (yellow sticks) at the colchicine
binding site. Specific residues contacting 7a3 are shown by grey sticks. Oxygen and nitrogen atoms
are coloured red and blue, respectively. (D) Interference of 7a3 with the tubulin straight
conformation. The close-up view of the 7a3 binding site from the superimposition of the
tubulin-TPI1 complex (coloured as in Fig. 5B) and tubulins, as found in straight protofilaments
(PDB ID 1JFF, pink), shows that 7a3 binding is not compatible with the straight conformation. The
7
a3 molecule is shown in yellow sphere representation. Conformational changes upon binding of

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7
a3 are labelled. (E) Close-up view of the conformational changes at the colchicine-binding site
between 7a3, colchicine (PDB 4O2B), and CA-4 (PDB code 5LYJ).
2
.11. Pharmacokinetic study of 7a3
The pharmacokinetic study of 7a3 was carried out in male C57 mice that were
administered a single dose of 50 mg/kg by intraperitoneal injection. Blood samples
were collected from the submaxillary vein of the mice at different time points (0 min,
5
min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) after dosing. The
pharmacokinetics (PK) of 7a3 in mice plasma were analysed by LC/MS/MS. Three
quasi-molecular and fragment ions were selected for qualitative analysis of 7a3
(
369.05/339.00 collision energy (CE): 25 V, 369.05/336.00 CE: 22 V, 369.05/306.80
CE: 28 V and IS (m/z 317.05/285.90 CE: 19 V, 317.05/284.80 CE: 12 V,
17.05/165.10 CE: 40 V) (Fig. 6C, Supplementary Fig. 2B and 2C). The MRM mode
3
was used to monitor both quasi-molecular/fragment ions of 7a3 and IS for the 7a3
concentration quantified in mice plasma.
The retention time of 7a3 and IS (CA-4) were 3.113 min and 2.825 min,
respectively (Supplementary Fig. 2A). The calibration curve of 7a3 in mouse plasma
was constructed by plotting peak area ratios of IS using the weight linear regression.
The method showed good linearity over the range from 0.01 to 500 ng/mL with a
2
correlation coefficient r > 0.999 (Y = 0.0123349X+0.0247413, r = 0.9993726)
(Supplementary Fig. 2A). The main PK parameters of 7a3 in mouse plasma were
examined by non-compartmental analysis. As summarized in Table 3, 7a3 reached the
-1
peak concentration (Cmax = 882 ± 71 ng·mL ) at 0.25 h and then dropped rapidly at 4
h, and it was no longer detectable at 12 h (Fig. 6D). 7a3 showed an estimated
elimination half-life of 1.65 h (t1/2 = 1.65 h), and the area under the plasma
concentration – time curve AUC0-24 h and AUC0-inf were 1139 ± 121 and 1166 ± 129
-1
h·ng·mL , respectively.

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Table 3. Pharmacokinetic parameters for 7a3 in plasma (50 mg/kg, intraperitoneal administration,
single-dose)
a
PK Parameters
Parameters Values
1.67 ± 0.28
0.423 ± 0.0756
0.25
t1/2 (h)
-1
K (h )
el
tmax (h)
-
1
Cmax (ng·mL )
882 ±71
-
1
AUC0-24h (h·ng·mL )
1139 ± 121
1166 ±129
1498 ± 481
1785 ± 762
1.51 ±0.54
-
1
AUC0-inf (h·ng·mL )
-
1
AUMC0-24h (h·h·ng·mL )
-
1
AUMC0-inf (h·h·ng·mL )
MRT (h)
a
Parameters: t , terminal exponential half-life; K , represents the fraction of drug eliminated per
1
/2
el
unit of time; tmax, time the maximum concentration occurred; Cmax, maximum plasma
concentration; AUC, area under the plasma concentration–time curve; AUMC, area under the first
moment curve; MRT, mean residence time.
2
.12. 7a3 induced anti-tumour activity in vivo
To evaluate the in vivo therapeutic efficacy of 7a3, a human ovarian
adenocarcinoma xenograft model was established by subcutaneous injection of
SK-OV-3 cells in the backs of six-week-old Balb/c nude mice. Once the tumours
3
reached a size of ∼200 mm , the mice were randomly assigned to three groups. In two
of the groups, 7a3 or CA-4, dissolved in 5% (v/v) DMSO/olive oil, was injected
intraperitoneally at a dose of 50 mg/kg. Both drugs and the vehicle were administered
three times as the arrows indicate (Fig. 6A). Treatment with 7a3 resulted in a
significant reduction in tumour growth compared with that observed in the vehicle
group by the end of the observation (62.8%, P < 0.05, Fig. 6A). No obvious body
weight loss occurred during the entire treatment period, suggesting that the
administration of 7a3 was well tolerated (Fig. 6B). The vital organs, including the
heart, liver, spleen, lung and kidney, were collected and further analysed by H&E
staining. No observable pathologic changes were observed in these organs from the
nude mice (Fig. 6E), which indicated that there were no overt toxicities when treated
with 7a3.

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Fig. 6. (A) 7a3 inhibited tumour growth in an SK-OV-3 xenograft model. Tumour-bearing nude

ACCEPTED MANUSCRIPT
mice were administered vehicle, 7a3 (50 mg/kg) or CA4 (50 mg/kg). Injections were given
intraperitoneally every two days three times (indicated by arrows). Data are presented as the mean
±
SEM of the tumour volume and body weight at each time point for six animals per group. *P <
0
.05 compared with the control group by t-test. (B) Body weight of the mice recorded on the
indicated days after treatments. (C) Pharmacokinetic study of 7a3 in mouse plasma analysed by
UHPLC-ESI-MS/MS. Representative MRM chromatograms of 7a3 (tR = 3.113 min; m/z
3
69.05/339.00, 369.05/336.00, 369.05/306.80) and IS (CA-4, t = 2.825 min, 317.05/285.90,
R
3
17.05/284.80, and 317.05/284.81). (D) Drug concentration-time curve of 7a3 in plasma after the
administration of a single intraperitoneal dose of 50 mg/kg. The blood samples were collected at 0
min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h (E) Representative images of
.
H&E-stained vital organs. No obvious alternation was observed in the organs examined, including
heart, liver, spleen, lung, and kidney, after treatment with 7a3. Magnification, × 400.
3
. Conclusions
In this study, a series of novel pyrazole analogues were designed, synthesized,
and evaluated for their anti-proliferative activities in the ovarian cancer cell line
SK-OV-3 and the breast cancer cell line MDA-MB-231, and most of the tested
compounds exhibited moderate to potent activities compared with CA-4. The SARs
studies revealed that compounds with the methyl substituent at the N1 position and
without amino groups exhibited excellent anti-proliferative activities. Furthermore,
transformation of the A-ring from the C3 to the C5 position on the pyrazole
heterocycle led to minor changes in activities. The selected compounds 7a2, 7a3,
1
2a2, and 12a3 were further identified to possess a high potency in four additional
cancer cells (HeLa, A549, CT26, and MCF-7). Among these compounds, 7a3
exhibited the best biological activity and was used in further biological studies. As an
analogue of the proven microtubule inhibitors CA-4 and colchicine, 7a3 exhibited the
typical cellular effects of microtubule interacting agents and was shown to arrest the
cell cycle in the G2/M phase and induce apoptosis in SK-OV-3 cells. Moreover, the
scratch wound and transwell migration assay indicated that 7a3 induced a significant

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inhibition of cancer cell migration. Furthermore, the immunofluorescence analysis
using confocal microscopy and the tubulin polymerization assay indicated that
microtubule polymerization and spindle formation were disordered by 7a3. In
addition, the co-crystal structure of tubulin in complex with 7a3 at a resolution of 3.2
Å (PDB code 5Z4U) was elucidated by X-ray crystallography, which confirmed the
result of molecular docking and further demonstrated that 7a3 was a tubulin inhibitor
that bound to the colchicine site. The pharmacokinetic analysis in mouse plasma
showed that 7a3 rapidly reached a peak concentration at 0.25 h after intraperitoneal
-1
administration, and the T , Cmax, and AUC0-inf were 1.67 ± 0.28 h, 882 ± 71 ng·mL ,
1
/2
-1
and 1166 ±129 h·ng·mL , respectively. Importantly, the therapeutic efficacy of 7a3
was demonstrated in a human ovarian adenocarcinoma xenograft model. A significant
inhibition of tumour growth was observed without overt toxicities after treatment with
7
a3. In conclusion, our results demonstrated that compound 7a3 is a promising novel
tubulin inhibitor for cancer treatment. The SARs and mechanism research based on
the co-crystal structure of tubulin in complex with small molecules provides great
insight for the development of novel tubulin inhibitors for cancer therapy.
4
. Experimental section
4
.1. General chemistry
All the chemicals and reagents were commercially available without further
purification. TLC was performed on 0.20 mm silica gel 60 F254 plates (Qingdao
1
13
Haiyang Chemical, China). H NMR and C NMR spectra were recorded on a Bruker
Avance 400 spectrometer (Bruker Company, Germany), using TMS as an internal
standard and DMSO-d or CDCl as solvents. The chemical shifts are given as parts
6
3
per million (ppm) relative to TMS. The mass spectral data were collected on a Q-TOF
Premier mass spectrometer (Micromass, Manchester, UK). The melting points were
obtained on a Buchi-Tottoli apparatus and were uncorrected. The purity of each
compound was determined by reverse-phase HPLC (XBridge column, C18, 4.6 mm ×

ACCEPTED MANUSCRIPT
1
50 mm, 5 µm) using a Water 2695 series LC system. The results of HPLC analysis
are presented in Supplementary Materials (Section 4), and the purity of each
compoundꢀ(≥ 95%) was confirmed by monitoring at 254 nm.
4
.2. Methyl 3,4,5-trimethoxybenzoate (2)
Concentrated sulfuric acid (10 mL) was added dropwise at ambient temperature
to a stirring solution of compound 1 (10.0 g, 47.2 mmol) in CH OH (100 mL), then
3
refluxed at 65 °C for 24 h and monitored by TLC. After completion, the reaction
mixture was concentrated under reduced pressure, ethyl acetate (100 mL) was then
added, and the pH was adjusted to 7.0 with saturated NaHCO aqueous solution. The
3
organic layer was separated, and the remaining water phase was extracted with ethyl
acetate (80 mL) two times. The combined organic layer was washed with saturated
brines and dried over anhydrous Na SO . The solvent was removed under reduced
2
4
pressure to give compound 2 without further purification. Yield 100%, 10.8 g. White
1
solid, mp 76-77 °C, literature 82-83 °C [39]. H NMR (400 MHz, Chloroform-d) δ
1
3
7
.23 (s, 2H), 3.84 (s, 12H). C NMR (101 MHz, Chloroform-d) δ 166.7, 152.9, 142.2,
+
1
25.1, 106.8, 60.9, 56.2, 52.2. HRMS (ESI) calcd for C H O [M+Na] 249.0739,
1
1
14
5
found 249.0734.
4
.3. 3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (3)
NaH (60%, 1.80 g, 44.2 mmol) was added to a mixed solution of compound 2
10.0 g, 44.2 mmol) in dry CH CN and THF (v: v = 1:1, 160 mL), and the mixture
(
3
was refluxed at 80ꢀ°C and monitored by TLC. The solvent was removed under
reduced pressure after 5 h, then the mixture was poured into ice water (100 mL), and
the pH was adjusted to 2.0 with 2 M HCl. The mixture was extracted three times with
ethyl acetate (100 mL × 3), and the combined organic layer was washed with
saturated brines, dried with Na SO , and filtered, and the solvent was then removed
2
4
under reduced pressure to give a yellow solid, which was purified by column
chromatography on silica gel to give the title compound 3. Yield 77%, 8.0 g. Pale
1
yellow solid, mp 134-136 °C, literature 138-139 °C [40]. H NMR (400 MHz,

ACCEPTED MANUSCRIPT
1
3
DMSO-d ) δ 7.24 (s, 2H), 4.77 (s, 2H), 3.86 (s, 6H), 3.76 (s, 3H). C NMR (101
6
MHz, Chloroform-d) δ 171.0, 153.0, 142.9, 124.3, 107.4, 61.0, 56.3, 45.8, 29.7.
+
HRMS (ESI) calcd for C H NO [M] 235.0845, found 235.0845.
1
2
13
4
4
.4. General procedure for the synthesis of compounds 4a-4d.
The appropriate hydrazine derivative R -NHNH (17.0 mmol) was added to a
1
2
mixed solution of compound 3 (2.00 g, 8.51 mmol) in AcOH (25 mL) and EtOH (25
mL), and the mixture was heated under reflux at 100ꢀ°C overnight (12 h). After
completion, the solvent was evaporated under reduced pressure, and the resulting
residue was dissolved in ethyl ether (50 mL); then, the organic phase was washed
with saturated aq NaHCO (50 mL × 3) and brine (50 mL) and was dried over Na SO .
3
2
4
The reaction mixture was filtered, and the filtrate was removed in vacuo. The crude
residue was then purified by silica gel column chromatography to obtain compounds
4
a-4d.
4
.4.1. 1-methyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (4a)
Compound 4a was obtained from 3 and methylhydrazine according to general
1
procedure. Yield 86.6%, 1.94g. Pale yellow solid, mp 168-169 °C. H NMR (400
MHz, DMSO-d ) δ 6.91 (s, 2H), 5.69 (s, 1H), 5.21 (s, 2H), 3.80 (s, 6H), 3.65 (s, 3H),
6
1
3
3
.55 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 153.3, 149.7, 145.6, 137.7, 129.6,
+
1
02.6, 88.6, 60.9, 56.2, 34.6. HRMS (ESI) calcd for C H N O [M+H] 264.1348,
1
3
17
3
3
found 264.1344.
4
.4.2. 1-(tert-butyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (4b)
Compound 4b was obtained from 3 and tert-butylhydrazine according to general
1
procedure. Yield 85.8%, 2.23 g. Pale yellow solid, mp 95-97 °C. H NMR (400 MHz,
DMSO-d ) δ 6.90 (s, 2H), 5.77 (s, 1H), 4.91 (s, 2H), 3.80 (s, 6H), 3.66 (s, 3H), 1.57 (s,
6
1
3
9
9
3
H). C NMR (101 MHz, Chloroform-d) δ 153.3, 153.3, 129.3, 113.9, 106.2, 102.8,
+
1.3, 61.1, 56.5, 56.1, 29.4, 29.2. HRMS (ESI) calcd for C H N O [M+H]
1
6
23
3
3
06.1818, found 306.1821

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4
.4.3. 1-(4-fluorophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (4c)
Compound 4c was obtained from 3 and 4-fluorophenylhydrazine according to
1
general procedure. Yield 77.7%, 2.27 g. Pale yellow solid, mp 121-122 °C. H NMR
(400 MHz, DMSO-d ) δ 7.66 (dd, J = 8.9, 5.0 Hz, 2H), 7.34 (t, J = 8.8 Hz, 2H), 7.00
6
1
3
(
s, 2H), 5.92 (s, 1H), 5.40 (s, 2H), 3.82 (s, 6H), 3.68 (s, 3H). C NMR (101 MHz,
Chloroform-d) δ 163.0, 160.5, 153.4, 151.4, 146.0, 138.2, 134.4, 128.8, 126.4, 126.3,
+
116.5, 116.3, 103.0, 88.2, 60.9, 56.3. HRMS (ESI) calcd for C H FN O [M+H]
18 18 3 3
3
44.1410, found 344.1409.
4
.4.4. 1-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (4d)
Compound 4d was obtained from 3 and 4-methoxyphenylhydrazine according to
1
general procedure. Yield 79.8%, 2.41 g. Pale yellow solid, mp 176-177 °C. H NMR
(
400 MHz, DMSO-d ) δ 7.51 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 6.99 (s,
6
1
3
2
H), 5.89 (s, 1H), 5.26 (s, 2H), 3.82 (s, 6H), 3.81 (s, 3H), 3.67 (s, 3H). C NMR (101
MHz, Chloroform-d) δ159.2, 153.4, 151.0, 146.1, 131.0, 129.0, 126.3, 114.7, 103.0,
+
8
3
7.6, 60.9, 56.3, 55.6. HRMS (ESI) calcd for C H N O [M+H] 356.1610, found
1
9
21
3
4
56.1604.
4
.5. General procedure for the synthesis of compounds 5a-5d.
A solution of compounds 4a-4d (1.00 g, 1 equiv) in DMF (10 mL) was stirred in
an ice-salt bath, and a solution of NBS (1.1 equiv) in DMF (5 mL) was added
dropwise. The resulting mixture was stirred at 0 °C for 3 h and monitored by TLC.
After completion, the solvent was removed under reduced pressure with the use of an
oil pump, and ethyl ether (20 mL) was then added. The organic phase was washed
with water (20 mL) and brine (20 mL), dried over Na SO , filtered and concentrated.
2
4
The crude product was purified by silica gel column chromatography to obtain
compounds 5a-5d.
4
.5.1. 4-bromo-1-methyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (5a)
Compound 5a was obtained from 4a and bromine according to general procedure.

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1
Yield 89.2%, 1.16 g. Pale yellow solid, mp 168-169 °C. H NMR (400 MHz,
13
DMSO-d ) δ 7.06 (s, 2H), 5.48 (s, 2H), 3.80 (s, 6H), 3.68 (s, 3H), 3.63 (s, 3H). C
6
NMR (101 MHz, Chloroform-d) δ 153.1, 146.1, 143.7, 138.0, 128.01, 104.4, 104.1,
+
6
0.9, 56.1, 35.4. HRMS (ESI) calcd for C H BrN O [M+H] 342.0453, 344.0433,
1
3
16
3
3
found 342.0450, 344.0437.
4
.5.2. 4-bromo-1-(tert-butyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine (5b)
Compound 5b was obtained from 4b and bromine according to general
1
procedure. Yield 84.9%, 1.07 g. Pale yellow solid, mp 122-123 °C. H NMR (400
MHz, DMSO-d ) δ 7.04 (s, 2H), 5.03 (s, 2H), 3.80 (s, 6H), 3.68 (s, 3H), 1.58 (s, 9H).
6
1
3
C NMR (101 MHz, Chloroform-d) δ 153.1, 144.2, 143.0, 137.9, 128.4, 104.6, 60.9,
+
6
0.0, 56.1, 29.0. HRMS (ESI) calcd for C H BrN O [M+H] 384.0923, 386.0902,
1
6
22
3
3
found 384.0917, 386.0903.
4
.5.3. 4-bromo-1-(4-fluorophenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine
(5c)
Compound 5c was obtained from 4d and bromine according to general procedure.
1
Yield 83.7%, 1.03 g. Pale yellow solid, mp 140-142 °C. H NMR (400 MHz,
13
Chloroform-d) δ 7.63-7.57 (m, 2H), 7.23-7.16 (m, 4H), 3.92 (s, 6H), 3.88 (s, 3H). C
NMR (101 MHz, Chloroform-d) δ 163.2, 160.7, 153.2, 147.9, 143.80, 138.4, 134.4,
1
27.4, 126.0, 125.9, 116.7, 116.5, 104.8, 60.9, 56.2. HRMS (ESI) calcd for
+
C H BrFN O [M+H] 422.0516, 424.0495, found 422.0510, 424.0493.
18
17
3
3
4
.5.4. 4-bromo-1-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-amine
(5d)
Compound 5d was obtained from 4c and bromine according to general procedure.
1
Yield 85.4%, 1.05 g. Pale yellow solid, mp 162-164 °C. H NMR (400 MHz,
Chloroform-d) δ 7.50 (d, J = 8.9 Hz, 2H), 7.19 (s, 2H), 7.01 (d, J = 9.0 Hz, 2H), 3.92
13
(
s, 6H), 3.88 (s, 3H), 3.85 (s, 3H). C NMR (101 MHz, Chloroform-d) δ 159.4, 153.1,
1
47.43, 143.8, 128.2, 131.2, 127.7, 125.8, 114.8, 104.8, 75.9, 60.9, 56.2, 55.6. HRMS
+
(
ESI) calcd for C H BrN O [M+H] 434.0715, 436.0695, found 434.0713,
1
9
20
3
4