Molecular diversity of trimethoxyphenyl-1,2,3-triazole hybrids as novel colchicine site tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.01.033

Structurally diverse trimethoxyphenyl-1,2,3-triazole hybrids were designed, synthesized and evaluated for their antiproliferative activity against three cancer cell lines (PC3, MGC803 and HepG2). Among them, trimethoxyphenyl-1,2,3-triazole containing the

Full text of DOI:10.1016/j.ejmech.2019.01.033

Accepted Manuscript
Molecular diversity of trimethoxyphenyl-1,2,3-triazole hybrids as novel colchicine site
tubulin polymerization inhibitors
Dong-Jun Fu, Ping Li, Bo-Wen Wu, Xin-Xin Cui, Cheng-Bin Zhao, Sai-Yang Zhang
PII:
S0223-5234(19)30043-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.033
EJMECH 11040
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 November 2018
Revised Date: 28 November 2018
Accepted Date: 14 January 2019
Please cite this article as: D.-J. Fu, P. Li, B.-W. Wu, X.-X. Cui, C.-B. Zhao, S.-Y. Zhang, Molecular
diversity of trimethoxyphenyl-1,2,3-triazole hybrids as novel colchicine site tubulin polymerization
inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.01.033.
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Graphic abstract

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Molecular diversity of trimethoxyphenyl-1,2,3-triazole hybrids
as novel colchicine site tubulin polymerization inhibitors
Dong-Jun Fua, d, Ping Lid, Bo-Wen Wud, Xin-Xin Cuid, Cheng-Bin Zhaoe, Sai-Yang Zhanga, b, c, d*
aSchool of Basic Medical Science, Zhengzhou University, Zhengzhou, 450001, China
bThe Academy of Medical Science, Zhengzhou University, Zhengzhou, 450001, China
cHenan Institutes of Advanced Technology, Zhengzhou University, Zhengzhou, 450001, China
dSchool of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research
and Safety Evaluation, Zhengzhou University, Zhengzhou 450001, China
eThe First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
*Corresponding author, E-mail addresses: saiyangz@zzu.edu.cn (S.-Y. Zhang)
Abstract
Structurally diverse trimethoxyphenyl-1,2,3-triazole hybrids were designed,
synthesized and evaluated for their antiproliferative activity against three cancer cell
lines (PC3, MGC803 and HepG2). Among them, trimethoxyphenyl-1,2,3-triazole
containing the coumarin fragement 19c displayed better antiproliferative activity
results with IC50 values from 0.13µM to 1.74µM than anticancer drug colchicine.
Compound 19c could inhibit MGC803 cell growth and colony formation, induce
G2/M phase arrest by down expression of CDK1, and promote apoptosis by
regulating DR5 and Bcl-2 family. Moreover, 19c strongly inhibited tubulin
polymerization by interacting with the colchicine site.
Keywords
Trimethoxyphenyl-1,2,3-triazole, Coumarin, Colchicine site tubulin inhibitor, Cell
cycle, Apoptosis
1. Introduction
Microtubules (composed of α-tubulin and β-tubulin heterodimers) play a key role in
various cellular processes, including cellular shape maintenance, spindle formation,
and intracellular transportation [1, 2]. Microtubule was regarded as an attractive target
for chemotherapeutic agents to treat cancer [3, 4]. Tubulin targeting agents bind at
four binding sites: vinca alkaloids, taxanes, colchicine and laulimalide sites [5].
Although some tubulin inhibitors that bind to vinca alkaloids and taxanes binding
sites have been approved by FDA, there are no FDA approved colchicine site tubulin
inhibitors currently [6].
Recently, variety of colchicine site tubulin inhibitors containing a trimethoxyphenyl
(TMP) moiety were designed for the treatment of cancer. Cis-restricted pyrazole
analogue 1 (Fig. 1) disrupted microtubule assembly, inhibited migration and
suppressed tumor growth of the SK-OV-3ꢀ xenograft model [7]. β-Lactam-azide
derivative 2 as colchicine site tubulin inhibitors from our group displayed potent
anticancer activity against MGC-803 cells with an IC50 value of 106
nM by induction
of G2/M arrest and apoptosis [8] 3-(3′,4′,5′-Trimethoxyanilino)benzo[b]furan 3
.
bound to the colchicine site of tubulin and exhibited potent vascular disrupting
properties both in vitro and in vivo [9]. 2-Amino-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl

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thiazole 4 as microtubule targeting agent showed potent inhibitory activity in
multidrug resistant cancer cells and induced cell death by apoptotic pathway [10].
Indole derivative 5 acting at colchicine binding site was significantly effective against
P-glycoprotein (P-gp) mediated multiple drug resistance (MDR) and taxol
resistance [11]. 2-Aminoimidazole-carbonyl analog 6 bound to tubulin could increase
reactive oxygen species level and induce apoptosis via poly(ADP-ribose)
polymerase-cleavage in HeLa cells [12]. Therefore, tubulin inhibitors binding to
colchicine site bear the trimethoxyphenyl (TMP) moiety which may be essential for
interaction with tubulin.
Fig. 1. Colchicine site tubulin inhibitors containing a trimethoxyphenyl (TMP) moiety
1,2,3-Triazole was usually used as a potentially pharmacological linker and
fragement to design novel anticancer hybrids in medicinal chemistry [13-15]. HDAC8
inhibitor 7 (Fig. 2) exhibited selective acetylation of cohesin and exerted
growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells [16].
2-Aminophenyl-(1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone 8 as an
orally anticancer agent could suppress the transcriptional functions of estrogen-related
receptor α with an IC50 of 0.021 µM in a cell-based reporter gene assay [17].
1,4-Substituted triazole 9 as a novel androgen receptor antagonist showed potent
antitumor activity against prostate cancer cells and induced 60% tumor growth
inhibition of the CW22Rv1 xenograft model [18]. In addition, 1,2,3-triazole displayed
potentially antitubulin effect in medicinal chemistry to design novel tubulin
polymerization inhibitors. Sulfanilamide derivative 10 from our group displayed
potent tubulin polymerization inhibitory activity in vitro with an IC50 of 2.41 M [19].
Combretatriazole 11 as cytotoxic and antitubulin agent was discovered by a column
chromatography-free
parallel
solution-phase
synthesis
[20].
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide 12 induced M-phase arrest in
HeLa cells at 5 µM and inhibited tubulin polymerization in vitro at 10 µM [21].

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Therefore, 1,2,3-triazole unit was used as a potentially antitubulin linker to design
target molecules in this work.
Fig. 2. Anticancer 1,2,3-triazoles 7~9 and tubulin polymerization inhibitors 10~12.
Based on the above findings, antitubulin trimethoxyphenyl and antitubulin
1,2,3-triazole yielded a novel scaffold that has three parts (Fig. 3): (i) a
trimethoxyphenyl (TMP) scaffold as an antitubulin fragment; (ii) a 1,2,3-triazole as an
antitubulin and anticancer linker to combine different antiproliferative skeletons; (iii)
anticancer
unit:
2H-chromen-2-one,
4-methyl-2H-chromen-2-one,
5,6-diphenyl-1,2,4-triazine, benzo[d]thiazole, 4,5-dihydrothiazole, benzo[d]oxazole,
10H-phenothiazine, 1,3,4-thiadiazole, 1H-tetrazole, 4-methyl-2H-chromen-2-one,
sulfanilamide and 1H-indole [22-27]. Importantly, antiproliferative mechanisms and
antitubulin mechanisms of target compounds were described clearly.

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Fig. 3. Structure-based drug design for colchicine site tubulin inhibitors.
2. Results and discussion
2.1. Chemistry
Alkyne intermediates 13a~13o used in this work were synthesized firstly in the
high yields and structures of alkyne intermediates 13a~13o were described in the
Scheme 1. Hydroxy derivatives, sulfhydryl derivatives and amino derivatives were
reacted with propargyl bromide in the prescnce of sodium hydroxide to obtain alkyne
intermediates 13a~13m. In addition, benzene sulfonyl chloride derivatives were
reacted with propargylamine to form alkyne intermediates 13n~13o in the prescnce of
triethylamine.

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Scheme 1. Reagents and conditions: (a) NaOH, acetone, reflux; (b) Et3N, CH2Cl2, r.t.
In order to synthesize trimethoxyphenyl-1,2,3-triazole derivatives, nucleophilic
substitution of 3,4,5-trimethoxyaniline 14 with 1-(chloromethyl)-4-methoxybenzene
15
in
acetone
at
reflux
condition
for
5
hours
provided
3,4,5-trimethoxy-N-(4-methoxybenzyl)aniline 16. The intermediate 16 was reacted
with chloroacetyl chloride in the presence of potassium carbonate to form
2-chloro-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide 17, which was

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subjected
to
azidation
reaction
with
sodium
azide
to
afford
2-azido-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide 18.
As shown in Scheme 2, trimethoxyphenyl-1,2,3-triazole derivatives 19a~19o were
achieved by click reaction of compound 18 with various alkyne intermediates
13a~13o using sodium ascorbate and CuSO4.5H2O at room temperature. The
structures of all trimethoxyphenyl-1,2,3-triazole derivatives 19a~19o were
characterized using spectral methods, and all spectral data corroborated the assumed
structures.
Scheme 1. Reagents and conditions: (a) NaOH, acetone, reflux; (b) K2CO3,
chloroacetyl chloride, CH2Cl2, r.t; (c) NaN3, acetone, reflux; (d) Sodium ascorbate,
CuSO4.5H2O, alkyne intermediates 13a~13o, acetone/H2O = 1/1, r.t.
2.2. Antiproliferative activity
To discovery novel antitumor agents and potent colchicine site tubulin inhibitors,
we evaluated the antiproliferative activity of all trimethoxyphenyl-1,2,3-triazole
derivatives 19a~19o against three cancer cell lines PC3 (prostate cancer cells),
MGC803 (gastric cancer cells) and HepG2 (liver cancer cells) using the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.
Based on previous references, colchicine as an well-known colchicine site tubulin
inhibitor was selected as the reference drug in this work .
The antiproliferative activity results of candidate analogues against all three cancer
cell lines were shown in Table 1. The replacement of the alkynyl group of compounds
13b~13c by the trimethoxyphenyl-1,2,3-triazole fragement of compounds 19b~19c
resulted in a powerful improvement of antiproliferative activity. Especially,
trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c displayed the better

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antiproliferative activity with IC50 values from 0.13µM to 1.74µM than control drug
colchicine.
These
antiproliferative
activity
results
indicate
that
trimethoxyphenyl-1,2,3-triazole fragement might display potent anticancer activity in
vitro and could be used as the leading unit to design more similar derivatives.
In
order
to
obtain
potent
antiproliferative
compounds,
trimethoxyphenyl-1,2,3-triazole hybrids attaching bioactive units (2H-chromen-2-one,
5,6-diphenyl-1,2,4-triazine, benzo[d]thiazole, 4,5-dihydrothiazole, benzo[d]oxazole,
10H-phenothiazine, 1,3,4-thiadiazole, 1H-tetrazole, 4-methyl-2H-chromen-2-one,
sulfanilamide and 1H-indole) were designed and evaluated for their antiproliferative
activity based on above finding. By this way, the molecular deversity of
trimethoxyphenyl-1,2,3-triazoles
were
explored.
Changing
the
4-hydroxy-2H-chromen-2-one unit (19a) to a 7-hydroxy-2H-chromen-2-one unit (19b)
led to a significant improvement of the activity against all the tested cell lines,
indicating that the location of substituent group attaching 1,2,3-triazole was important
for inhibitory activity. Among all trimethoxyphenyl-1,2,3-triazole hybrids,
trimethoxyphenyl-1,2,3-triazole-phenothiazine hybrids 19k and 19l showed the weak
antiproliferative activity with IC50 values of >80µM. This inhibitory activity results
displayed that phenothiazine significantly decreased the anticancer activity in vitro for
trimethoxyphenyl-1,2,3-triazole
fragement.
For
trimethoxyphenyl-1,2,3-triazole-thiazole hybrids 19e and 19f, phenyl ring attaching
dihydrothiazole was important for the activity showing an over 3-fold activity
increase against MGC803 cell line, when the dihydrothiazole group was replaced with
benzo[d]thiazole group (compounds 19e vs. 19f). Replacement of the methyl of
trimethoxyphenyl-1,2,3-triazole-sulfanilamide hybrid 19n with a bromine atom of
trimethoxyphenyl-1,2,3-triazole-sulfanilamide hybrid 19o led to an increase of the
activity against MGC803 and HepG2 cells, indicating the substituent group on phenyl
ring affected the inhibitory activity for trimethoxyphenyl-1,2,3-triazole-sulfanilamide
hybrids. The molecular deversity and structure activity relationship studies revealed
that at least ten units (coumarin, phenoxy goup, diaryl-1,2,4-triazine, benzo[d]thiazole,
4,5-dihydrothiazole, benzo[d]oxazole, 1,3,4-thiadiazole, 1H-tetrazole, sulfanilamide
and 1H-indole) were effective for trimethoxyphenyl-1,2,3-triazole fragement to
generate new antiproliferative hybrids.
Table 1: Antiproliferative activity of the synthetic derivatives 19a~19o.
O
O
N
R
N
N
O
N
O
O
19a~19o
IC50 (µM)
MGC803
>80
Compounda
R
-
PC3
>80
HepG2
>80
13b

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-
>80
>80
>80
13c
19a
5.14±0.33
3.66±0.29
6.25±3.56
19b
19c
0.86±0.18
0.34±0.04
0.18±0.03
0.13±0.01
1.30±0.12
1.74±0.54
45.26±2.43
28.45±1.19
15.96±0.94
5.05±1.36
12.54±1.00
0.48±0.03
1.61±0.20
1.85±0.99
20.53±3.54
49.19±4.37
25.01±4.40
15.63±3.16
19d
19e
19f
19g
19.63±2.06
1.52±0.03
21.83±3.82
19h
16.51±2.05
10.87±1.65
5.00±0.52
6.57±0.20
38.55±4.05
28.32±0.85
19i
19j
>80
>80
>80
>80
>80
>80
19k
19l

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2.39±0.37
42.57±1.09
40.09±0.95
30.54±2.98
19m
19n
12.44±2.51
1.58±0.11
12.48±0.66
0.59 ± 0.05
0.43±0.01
7.37±2.23
4.60±0.37
19o
-
0.27 ± 0.01
Colchicine
a Antiproliferative activity was assayed by exposure for 48 hours.
2.3. Compound 19c inhibits MGC803 cells growth and colony
formation
Based on the antiproliferative activity results of all trimethoxyphenyl-1,2,3-triazole
derivatives, the most potent trimethoxyphenyl-1,2,3-triazole- coumarin hybrid 19c
was prioritized to perform further experiments for evaluating antiproliferative
mechanisms against MGC803 cells (Fig. 4). From the colony formation results in Fig.
4A, MGC803 cells treated with trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c
(15nM, 30nM, and 60nM) exhibited fewer and smaller colonies compared with
control, which indicated that compound 19c could significantly inhibit the
proliferation of MGC803 cells in a concentration-dependent manner. As shown in Fig.
4, cell viability of MGC803 cells treated with hybrid 19c at 60nM, 90nM and 120nM
were 62.63%, 31.82% and 16.02% respectively. These results suggested that 19c
could significantly inhibit growth of MGC803 cells.
Fig. 4. (A) Representative images of MGC803 cells colonies after treatment 19c with
various concentrations for 7 days. (B-C) The effect of 19c in reducing cell viabilities
of MGC803 cells. The cells were treated with the indicated concentrations of 19c for
48 hours. (D) Number of colonies.**: p < 0.01 verse control, ***: p < 0.001 verse
control. Three separate experiments were carried out, error bars were shown in the
figure.

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2.4. Compound 19c induces G2/M phase arrest
The effects of selected compound 19c on cell cycle phase arrest in MGC803 cells
were evaluated by flow cytometry. After treating MGC803 cells with compound 19c
at different concentrations (30nM, 60nM and 90nM), MGC803 cells were then fixed
and stained with PI for flow cytometry analysis [28]. As shown in Fig. 5, compound
19c could prolong the G2/M phase, shorten the S phase and G1 phase compared to the
control. Especially, compound 19c at 90 nM increased to 74.02% of G2/M cell cycle
distribution. The above results showed that compound 19c caused cell cycle arrest at
G2/M phase against MGC803 cells in a concentration-dependent manner.
Cyclin-dependent kinases (CDKs) belong to a family of serine/threonine protein
kinases which play a pivotal role in the regulation of the eukaryotic cell division,
control of gene transcription and other processes of the cell cycle [29]. Inhibition of
CDKs is believed to present unique opportunities for anticancer drug discovery [30].
In addition, CDK1 activation at the onset of mitosis results from the concurrent
inhibition of Wee1 and Myt1 and stimulation of the phosphatase CDC25C [31]. Thus,
we tested the expression level of CDK1 and CDC25C in MGC803 cells by western
blot. As shown in Fig. 5C, compound 19c decreased the expression level of CDK1 in
a concentration dependent manner, which led to cell cycle arrest in G2/M phase. But
there is not a upregulation on p-CDK1 and CDC25C markers. Further results showed
that the downregulation of CDK1 can not be rescued by the proteasome inhibitor
MG132 (Fig. 5D). To detect whether 19c influenced the activity that CDK1 bound to
Cyclin B1, an immunoprecipitation assay was performed. In Fig. 5E, the binding
activity has no significant change.

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Fig. 5. (A-B) Effects of 19c against MGC803 cell cycle progress for 48 hours. (C)
Cell cycle related proteins expression levels of 19c treated MGC803 cells. (D) CDK1
levels of MGC803 cells treated with/without MG132 (5µM). (E) The activity CDK1
bind to Cyclin B1 after 19c treatment. Three separate experiments were carried out,
error bars were shown in the figure.
2.5. Compound 19c induces apoptosis and regulates the expression
of apoptosis-related proteins
To investigate whether trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c affect
apoptosis, apoptosis assay was explored using our reported method [32]. After
treatment with compound 19c at different concentrations (0 nM, 30nM, 60nM and
90nM) for 48 hours, MGC803 cells were stained with the propidium iodide (PI) and
Annexin V-FITC, and then analyzed by the flow cytometry. As illustrated in Fig.
6A-B, the percentage of apoptotic cells was about 4.8% for the control group. When
treated with 90nM 19c, around 61.8% of apoptosis rate was observed. These results
exhibited that trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c induced apoptosis
of MGC803 cells in a concentration-dependent manner.
Apoptosis as a physiological form of programmed cell death is important for tissue
homeostasis by elimination of harmful cells, which is often correlated with the
regulation of the Bcl-xl, DR5, X-linked inhibitor of apoptosis protein (XIAP),
poly(ADP-ribose)polymerase (PARP) and Bax proteins [33-35]. As shown in Fig. 6C,
the protein levels of Bcl-xl and XIAP were decreased in concentration-dependent
manners after treatment of compound 19c (0 nM, 30nM, 60nM and 90nM). In
addition, the expresion level of cleaved-PARP, Bax and DR5 were increased.

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Fig. 6. (A-B) 19c induced apoptosis against MGC803. (C) Expression changes of
apoptosis-related proteins against MGC803 cells treated with 19c for 48 hours. Three
separate experiments were carried out, error bars were shown in the figure.
2.6. Compound 19c inhibits tubulin polymerization targeting the
colchicine binding site
The
tubulin
polymerization
inhibition
activity
in
vitro
of
trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c was evaluated . When tubulin
was incubated with the tested compound 19c (1µM, 2µM and 4µM), the increased
tendency of the fluorescence intensity was obviously slowed down. The IC50 value of
compound 19c was 2.164µM against tubulin (Fig. 7A). It revealed
trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c was a novel tubulin
polymerization inhibitor.
In the N,N’-ethylenebis(iodoacetamide) (EBI) assay, preincubation of compound
19c at 4uM prevented the formation of EBI: β-tubulin adduct resulting in the decrease
of the adduct band comparing with DMSO, EBI treatment and 0.4uM 19c treatment,
which was consistent with the effect of colchicine at 10µM. These results (Fig. 7B)
indicated that trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c directly bind to
the cochicine-binding site of β-tubulin.

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Fig. 7. (A) Tubulin polymerization inhibitory activity of 19c (1µM, 2µM and 4µM)
and colchicine (2µM). (B) EBI competition assay on MGC803 cells.
2.7. Immunofluorescence of compound 19c
To investigat the effects to microtubules, trimethoxyphenyl-1,2,3-triazole-coumarin
hybrid 19c was selected to do immunofluorescence assay by staining tubulin [36]. As
shown in Fig. 8, 60 nM 19c moderately depolymerized interphase microtubules
whereas 90 nM 19c induced much stronger depolymerization effects. These
immunofluorescence experiments indicated that compound 19c was a novel tubulin
polymerization inhibitor.
Fig. 8. Compound 19c showed strong depolymerizing effects on the interphase

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microtubule network.
2.8. Docking study of compound 19c
Based on the above experiments, trimethoxyphenyl-1,2,3-triazole-coumarin hybrid
19c has been identified as a novel tubulin polymerization inhibitor binding to
colchicine site. In the current work, molecular docking methodologies were also used
to
explore
any
molecular
interaction
exist
between
trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c and residues lies in the active
site cativity of tubulin. We have used Autodock as an automated tool to perform
docking and selected PDB code 1SA0.
As shown in Fig. 9, 3,4,5-trimethoxyphenyl ring, 4-methoxyphenyl ring, and amide
group formed three hydrogen bonds with residues ASN329, TYR224 and LYS352,
respectively. This finding illustrated the importance of 3,4,5-trimethoxyphenyl
scaffold for tubulin polymerization inhibitors. In addition, 1,2,3-triazole and oxygen
atom attaching to 4-methyl-2H-chromen-2-one formed two hydrogen bonds with
residues VAL353 and ILE355 respectively. These results explained that
trimethoxyphenyl-1,2,3-triazole-coumarin hybrids 19b~19c were more potent than
coumarins 13b~13c without the 1,2,3-triazole unit for the antiproliferative activity.

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Fig. 9. Molecular docking studies of compound 19c (PDB code:1SA0). (A)
compound 19c binds to β-tubulin (surface). (B) The complex formed between α,
β-tubulin and compound 19c. (C) Hydrogen bonds observed between compound 19c
and residues.
3. Conclusions
In summary, the molecular diversity of trimethoxyphenyl-1,2,3-triazole hybrids as
potential
antiproliferative
agents
was
explored.
Among
them,
trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c exhibited more potent
anticancer activity in vitro than anticancer drug colchicine. Molecular mechanisms
illustrated that compound 19c could induce MGC803 cells apoptosis, the inhibition of
colony formation, and G2/M phase arrest. From the tubulin polymerization assay in
vitro and EBI assay, compound 19c was a novel tubulin polymerization inhibitor
binding to colchicine site.

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4. Experiment section
4.1 Chemistry
Reagents and solvents were used according to our previous refs [32].
4.2 General procedure to synthesize alkyne intermediates
13a~13m and target compounds 19a~19o
To a solution of hydroxy compounds, sulfhydryl compounds or amino compounds
(1.0 eq) and propargyl bromide (1.2 eq) in acetone (20 ml) was added NaOH (1.5 eq),
the mixture was refluxed for about 6 hours. Upon completion, the residue was
purified with column chromatography (hexane:EtOAc = 7:1) to obtain analogue
13a~13m. Sulfonyl chlorides (1.0 eq) and propynylamine (1.2 eq) in CH2Cl2 (20 ml)
was added Et3N (1.5 eq), the mixture was stirred at room temperature for about 6 h.
Upon completion, the residue was purified with column chromatography
(hexane:EtOAc = 9:1) to obtain analogue 13n~13o.
Compound 17 (1.0eq) [5] and NaN3 were dissolved in acetone and refluxed for 5 h
to obtain intermediate 18. Upon completion, the residue was purified with column
chromatography (hexane:EtOAc = 6:1) to obtain analogue 18. Compound 18 (1.0eq),
alkyne derivatives 13a~13o (1.0eq), CuSO4.5H2O (0.2eq) and sodium ascorbate
(0.1eq) were dissolved in acetone/H2O (5 ml/5 ml) to stir for 12 h at room temperature.
Upon completion, the residue was purified with column chromatography
(hexane:EtOAc = 10:1) to obtain analogue 19a~19o.
4.2.1. 4-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (13a)
Yellow solid, 83% yield, m.p.:197~199 oC. 1H NMR (400 MHz, CDCl3) δ 7.74 (dd, J
= 7.9, 1.2 Hz, 1H), 7.58 – 7.36 (m, 1H), 7.35 – 7.03 (m, 2H), 5.76 (s, 1H), 4.80 (d, J =
2.4 Hz, 2H), 2.62 (t, J = 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 163.22, 161.41,
152.28, 131.54, 122.98, 122.04, 115.71, 114.33, 90.66, 76.90, 74.73, 55.85. HRMS
(ESI) calcd for C18H14N4O5S Na[M + Na]+: 421.0585, found: 421.0589.
4.2.2. 7-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (13b)
Yellow solid, 82% yield, m.p.:134~136 oC. 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J =
9.5 Hz, 1H), 7.41 – 7.25 (m, 1H), 6.82 (dd, J = 7.2, 2.2 Hz, 2H), 6.18 (d, J = 9.5 Hz,
1H), 4.68 (d, J = 2.4 Hz, 2H), 2.52 (t, J = 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ
159.95, 159.48, 154.56, 142.32, 127.86, 112.55, 112.14, 111.96, 101.07, 76.39, 75.59,
55.18. HRMS (ESI) calcd for C18H14N4O5SNa[M + Na]+: 421.0582, found: 421.0585.
4.2.3. 4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (13c)
Yellow solid, 87% yield, m.p.:149~150 oC. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J =
8.4 Hz, 1H), 6.93 – 6.73 (m, 2H), 6.06 (d, J = 0.8 Hz, 1H), 4.68 (d, J = 2.4 Hz, 2H),
2.51 (s, 1H), 2.32 (d, J = 0.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 160.02, 159.31,
153.94, 151.47, 124.64, 113.18, 111.60, 111.30, 101.09, 76.45, 75.52, 55.15, 17.64.
HRMS (ESI) calcd for C19H16N4O5SNa[M + Na]+: 435.0738, found: 435.0739。

ACCEPTED MANUSCRIPT
4.2.4. 2-(Prop-2-yn-1-ylthio)benzo[d]thiazole (13e)
Yellow solid, yield: 80%; m.p.: 50~52 °C. 1H NMR (400 MHz, CDCl3) δ 7.64 – 7.45
(m, 1H), 7.44 – 7.29 (m, 1H), 7.27 – 7.06 (m, 2H), 3.99 (d, J = 2.6 Hz, 2H), 2.23 (t, J
= 2.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 161.95, 150.97, 140.71, 123.37, 123.11,
117.64, 108.96, 76.88, 71.42, 19.65. HRMS (ESI) calcd for C10H8NS2 [M + H]+:
206.0099, found: 206.0097.
4.2.5. 2-(Prop-2-yn-1-ylthio)-4,5-dihydrothiazole (13f)
1
Colourless oil, yield: 89%. H NMR (400 MHz, DMSO) δ 4.16 (t, J = 8.0 Hz, 2H),
3.94 (d, J = 2.6 Hz, 2H), 3.48 (t, J = 8.0 Hz, 2H), 3.21 (t, J = 2.6 Hz, 1H). 13C NMR
(100 MHz, DMSO) δ 161.81, 79.50, 74.13, 63.92, 35.48, 20.09. HRMS (ESI) calcd
for C6H8NS2 [M + H]+: 158.0098, found: 158.0099.
4.2.6. 2-(Prop-2-yn-1-ylthio)benzo[d]oxazole (13g)
Yellow solid, yield: 86%; m.p.: 51~53 °C. 1H NMR (400 MHz, CDCl3) δ 7.56 (dd, J
= 8.0, 1.0 Hz, 1H), 7.38 (dd, J = 7.3, 1.2 Hz, 1H), 7.30 – 7.09 (m, 2H), 4.01 (d, J = 2.6
13
Hz, 2H), 2.23 (t, J = 2.6 Hz, 1H). C NMR (100 MHz, DMSO) δ 157.79, 146.80,
136.54, 119.21, 118.95, 113.47, 104.79, 72.68, 67.21, 15.47. HRMS (m/z): Calcd.
C10H8NOS, [M+H]+m/z: 190.0327, found: 190.0329.
4.2.7. 5,6-diphenyl-3-(prop-2-yn-1-ylthio)-1,2,4-triazine (13h)
o
1
Yellow solid, yield: 63%, m.p.:97~99 C. H NMR (400 MHz, Acetone-d6) δ 7.65 –
7.36 (m, 10H), 4.19 (d, J = 2.6 Hz, 2H), 2.77 (t, J = 2.6 Hz, 1H). 13C NMR (100 MHz,
Acetone-d6) δ 169.89, 156.57, 155.45, 136.63, 136.35, 131.68, 130.83, 130.35, 130.16,
129.32, 129.25, 80.13, 72.65, 19.47. HRMS (ESI): Calcd. C18H14N3S, [M+H]+m/z:
304.0909, found: 304.0913.
4.2.8. 2-(Prop-2-yn-1-ylthio)-1,3,4-thiadiazole (13i)
1
Colourless oil, 70% yield. H NMR (400 MHz, DMSO) δ 9.59 (dd, J = 3.5, 1.6 Hz,
13
1H), 4.23 (dd, J = 2.2, 1.6 Hz, 2H), 3.25 (ddd, J = 7.9, 4.9, 2.6 Hz, 1H). C NMR
(100 MHz, DMSO) δ 163.98, 154.52, 78.98, 74.77, 22.21. HRMS (ESI) calcd for
C5H5N2S2 [M + H]+: 156.9897, found: 156.9900.
4.2.9. 1-Methyl-5-(prop-2-yn-1-ylthio)-1H-tetrazole (13j)
White solid, yield: 84%, m.p.: 61~62 °C. 1H NMR (400 MHz, DMSO) δ 4.12 (d, J =
13
2.6 Hz, 2H), 3.99 (s, 3H), 3.26 (t, J = 2.6 Hz, 1H). C NMR (100 MHz, DMSO) δ
152.29, 78.87, 74.93, 33.81, 21.58. HRMS (m/z): Calcd. C5H7N4S, [M+H]+m/z:
155.0391, found: 155.0397.
4.2.10. 10-(Prop-2-yn-1-yl)-10H-phenothiazine (13k)
White solid, 92% yield, m.p.:90~92 oC. 1H NMR (400 MHz, CDCl3) δ 7.20 – 6.99 (m,
6H), 6.87 (td, J = 7.6, 1.5 Hz, 2H), 4.43 (d, J = 2.4 Hz, 2H), 2.37 (t, J = 2.4 Hz, 1H).
13C NMR (100 MHz, CDCl3) δ 143.08, 126.40, 126.00, 122.32, 121.93, 113.74, 78.14,
73.39, 37.48. HRMS (m/z): Calcd. C15H12NS, [M+H]+m/z: 238.0690, found:
238.0696.
4.2.11. 10-(Prop-2-yn-1-yl)-2-(trifluoromethyl)-10H-phenothiazine (13l)

ACCEPTED MANUSCRIPT
White solid, 90% yield, m.p.:85~86 oC. 1H NMR (400 MHz, DMSO) δ 7.41 – 7.27 (m,
4H), 7.22 (d, J = 1.8 Hz, 1H), 7.20 (s, 1H), 7.04 (td, J = 7.5, 1.0 Hz, 1H), 4.74 (d, J =
2.3 Hz, 2H), 3.54 (t, J = 2.3 Hz, 1H). 13C NMR (100 MHz, DMSO) δ 144.00, 142.79,
128.34, 128.08, 128.02, 127.80, 127.49, 126.99, 123.62, 120.98, 119.55, 115.43,
111.17, 78.84, 77.29, 37.55. HRMS (m/z): Calcd. C16H11F3NS, [M+H]+m/z: 306.0564,
found: 306.0567.
4.2.12. 1-(Prop-2-yn-1-yl)-1H-indole (13m)
White solid, 70% yield, m.p.:67~68 oC. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 7.9
Hz, 1H), 7.31 (dd, J = 8.2, 0.5 Hz, 1H), 7.21 – 7.13 (m, 1H), 7.11 (d, J = 3.2 Hz, 1H),
7.10 – 7.02 (m, 1H), 6.53 – 6.39 (m, 1H), 4.76 (d, J = 2.5 Hz, 2H), 2.30 (t, J = 2.5 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 134.72, 127.83, 126.19, 120.83, 120.07, 118.83,
108.27, 101.04, 76.69, 72.47, 34.71. HRMS (m/z): Calcd. C11H10N, [M+H]+m/z:
156.0813, found: 156.0817.
4.2.13. 4-Methyl-N-(prop-2-yn-1-yl)benzenesulfonamide (13n)
o
White soild, yield：83%, m.p.: 75-76 C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J =
8.1 Hz, 2H), 7.23 (t, J = 10.6 Hz, 2H), 4.66 (s, 1H), 3.76 (dd, J = 5.9, 2.4 Hz, 2H),
2.36 (s, 3H), 2.03 (t, J = 2.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 142.85, 135.51,
128.70, 126.39, 76.94 , 71.98, 31.85 , 20.54. HRMS (m/z): Calcd. C10H12NO2S,
[M+H]+m/z: 210.0589, found: 210.0592.
4.2.14. 4-Bromo-N-(prop-2-yn-1-yl)benzenesulfonamide (13o)
White solid, 75% yield, m.p.:102~103 oC. 1H NMR (400 MHz, CDCl3) δ 7.78 – 7.61
(m, 2H), 7.63 – 7.39 (m, 2H), 5.24 (t, J = 6.0 Hz, 1H), 3.78 (dd, J = 6.1, 2.5 Hz, 2H),
13
2.03 (t, J = 2.5 Hz, 1H). C NMR (100 MHz, CDCl3) δ 144.75, 136.94, 133.79,
131.67, 83.72, 78.62, 37.15. HRMS (m/z): Calcd. C9H9BrNO2S, [M+H]+m/z:
273.9537, found: 273.9539.
4.2.15. 2-Azido-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (18)
White solid, 86% yield, m.p.:95~96 oC. 1H NMR (400 MHz, CDCl3) δ 7.08 (d, J = 8.6
Hz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 6.03 (s, 2H), 4.71 (s, 2H), 3.77 (s, 3H), 3.71 (s, 3H),
3.64 (s, 6H), 3.58 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 166.12, 158.22, 152.71,
137.12, 134.70, 129.70, 128.04, 112.75, 104.50, 59.94, 55.18, 54.26, 51.66, 49.71.
HRMS (m/z): Calcd. C19H23N4O5, [M+H]+m/z: 387.1668, found: 387.1672.
4.2.16.N-(4-methoxybenzyl)-2-(4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-1H-1,2,3-tri
azol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19a)
White solid, 84% yield, m.p.:181~182 oC. 1H NMR (400 MHz, DMSO) δ 8.28 (s, 1H),
7.74 (d, J = 7.1 Hz, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.35 (t, J =
7.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.61 (s, 2H), 6.20 (s,
1H), 5.46 (s, 2H), 5.20 (s, 2H), 4.80 (s, 2H), 3.72 (s, 9H), 3.67 (s, 3H). 13C NMR (100
MHz, DMSO) δ 165.15, 164.33, 161.52, 158.54, 153.10, 152.75, 140.50, 137.15,
135.45, 132.80, 129.83, 128.90, 126.90, 124.21, 122.78, 116.47, 115.07, 113.63,
106.08, 91.32, 62.74, 60.01, 56.00, 55.02, 51.88, 51.16. HRMS (m/z): Calcd.
C31H31N4O8, [M+H]+m/z: 587.2142, found: 587.2147.
4.2.17.N-(4-methoxybenzyl)-2-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-tri
azol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19b)

ACCEPTED MANUSCRIPT
White solid, 89% yield, m.p.:172~173 oC. 1H NMR (400 MHz, DMSO) δ 8.18 (s, 1H),
8.00 (d, J = 9.5 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 14.5, 5.3 Hz, 3H),
7.04 (dd, J = 8.6, 2.3 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 6.60 (s, 2H), 6.31 (d, J = 9.5
Hz, 1H), 5.30 (s, 2H), 5.18 (s, 2H), 4.80 (s, 2H), 3.73 (s, 9H), 3.67 (s, 3H). 13C NMR
(100 MHz, DMSO) δ 165.18, 161.14, 160.24, 158.54, 155.29, 153.10, 144.27, 141.50,
137.15, 135.48, 129.82, 129.49, 128.91, 126.49, 113.62, 112.90, 112.62, 112.54,
106.07, 101.49, 61.59, 60.00, 55.99, 55.01, 51.91, 51.11. HRMS (m/z): Calcd.
C31H31N4O8, [M+H]+m/z: 587.2142, found: 587.2149.
4.2.18.N-(4-methoxybenzyl)-2-(4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1H
-1,2,3-triazol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19c)
o
1
White solid, 91% yield, m.p.:92~93 C. H NMR (400 MHz, DMSO) δ 8.17 (s, 1H),
7.70 (d, J = 8.8 Hz, 1H), 7.16 (dd, J = 10.3, 5.5 Hz, 3H), 7.05 (dd, J = 8.8, 2.5 Hz,
1H), 6.87 (d, J = 8.6 Hz, 2H), 6.60 (s, 2H), 6.23 (d, J = 1.1 Hz, 1H), 5.30 (s, 2H), 5.18
13
(s, 2H), 4.79 (s, 2H), 3.72 (s, 9H), 3.67 (s, 3H), 2.40 (d, J = 0.8 Hz, 3H). C NMR
(100 MHz, DMSO) δ 165.18, 161.04, 160.10, 158.53, 154.65, 153.39, 153.10, 141.53,
137.15, 135.47, 129.82, 128.91, 126.48, 113.62, 113.31, 112.61, 111.24, 106.07,
101.50, 61.57, 60.00, 55.99, 55.01, 51.90, 51.10, 18.10. HRMS (m/z): Calcd.
C32H33N4O8, [M+H]+m/z: 601.2298, found: 601.2299.
4.2.19.N-(4-methoxybenzyl)-2-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)-N-(3,4,5-tri
methoxyphenyl)acetamide (19d)
White solid, 83% yield, m.p.:145~146 oC. 1H NMR (400 MHz, DMSO) δ 8.12 (s, 1H),
7.31 (dd, J = 8.4, 7.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 7.9 Hz, 2H), 6.95
(t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.60 (s, 2H), 5.16 (d, J = 4.4 Hz, 4H),
4.80 (s, 2H), 3.72 (s, 9H), 3.67 (s, 3H). 13C NMR (100 MHz, DMSO) δ 165.21,
158.54, 158.06, 153.11, 142.32, 137.15, 135.50, 129.83, 129.48, 128.92, 126.07,
120.76, 114.58, 113.62, 106.07, 60.89, 60.00, 55.98, 55.01, 51.90, 51.06. HRMS
(m/z): Calcd. C28H31N4O6, [M+H]+m/z: 519.2244, found: 519.2247.
4.2.20.2-(4-((Benzo[d]thiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxybe
nzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19e)
White solid, 80% yield, m.p.:138~139 oC. 1H NMR (400 MHz, DMSO) δ 8.02 (d, J =
10.0 Hz, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.56 – 7.44 (m, 1H), 7.43 – 7.32 (m, 1H), 7.13
(d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.56 (s, 2H), 5.12 (s, 2H), 4.77 (s, 2H),
13
4.73 (s, 2H), 3.71 (d, J = 5.5 Hz, 9H), 3.66 (s, 3H). C NMR (100 MHz, DMSO) δ
165.89, 165.14, 158.52, 153.07, 152.58, 141.92, 137.11, 135.45, 134.70, 129.82,
128.91, 126.36, 125.36, 124.52, 121.82, 121.23, 113.61, 106.04, 59.99, 55.97, 55.01,
51.86, 51.11, 27.35. HRMS (m/z): Calcd. C29H30N5O5S2, [M+H]+m/z: 592.1688,
found: 592.1692.
4.2.21.2-(4-(((4,5-Dihydrothiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-metho
xybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19f)
White solid, 80% yield, m.p.:136~137 oC. 1H NMR (400 MHz, DMSO) δ 7.91 (s, 1H),
7.15 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.58 (s, 2H), 5.12 (s, 2H), 4.78 (s,
2H), 4.43 (s, 2H), 4.18 (t, J = 8.0 Hz, 2H), 3.72 (s, 9H), 3.67 (s, 3H), 3.47 (t, J = 8.0
13
Hz, 2H). C NMR (100 MHz, DMSO) δ 165.17, 162.59, 158.53, 153.08, 142.22,
137.13, 135.48, 129.83, 128.92, 125.19, 113.61, 106.06, 63.94, 60.00, 55.98, 55.01,
51.88, 51.06, 35.33, 26.56. HRMS (m/z): Calcd. C25H30N5O5S2, [M+H]+m/z:
544.1688, found: 544.1689.

ACCEPTED MANUSCRIPT
4.2.22.2-(4-((Benzo[d]oxazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxybe
nzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19g)
White solid, 76% yield, m.p.:152~153 oC. 1H NMR (400 MHz, DMSO) δ 8.05 (s, 1H),
7.79 – 7.55 (m, 2H), 7.51 – 7.24 (m, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.5 Hz,
2H), 6.58 (s, 2H), 5.13 (s, 2H), 4.78 (s, 2H), 4.72 (s, 2H), 3.72 (d, J = 3.7 Hz, 9H),
3.66 (s, 3H). 13C NMR (100 MHz, DMSO) δ 165.14, 163.68, 158.52, 153.08, 151.35,
141.82, 141.24, 137.13, 135.45, 129.82, 128.91, 125.32, 124.63, 124.33, 118.34,
113.60, 110.25, 106.05, 59.99, 55.97, 55.00, 51.88, 51.12, 26.45. HRMS (m/z): Calcd.
C29H30N5O6S, [M+H]+m/z: 576.1917, found: 576.1919.
4.2.23.2-(4-(((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4
-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19h)
o
1
White solid, 82% yield, m.p.:82~83 C. H NMR (400 MHz, DMSO) δ 7.99 (s, 1H),
7.56 – 7.31 (m, 10H), 7.13 (d, J = 8.2 Hz, 2H), 6.85 (d, J = 8.3 Hz, 2H), 6.56 (s, 2H),
13
5.11 (s, 2H), 4.77 (s, 2H), 4.68 (s, 2H), 3.71 (d, J = 3.6 Hz, 9H), 3.66 (s, 3H). C
NMR (100 MHz, DMSO) δ 169.26, 165.17, 158.52, 155.66, 154.15, 153.07, 142.47,
137.11, 135.47, 135.16, 134.90, 130.80, 129.81, 129.71, 129.32, 129.24, 128.91,
128.43, 128.37, 125.16, 113.60, 106.04, 60.00, 55.97, 55.01, 51.86, 51.08, 24.88.
HRMS (m/z): Calcd. C37H36N7O5S, [M+H]+m/z: 690.2499, found: 690.2500.
4.2.24.2-(4-(((1,3,4-Thiadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxy
benzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19i)
White solid, 91% yield, m.p.:128~129 oC. 1H NMR (400 MHz, DMSO) δ 9.55 (s, 1H),
8.00 (s, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.58 (s, 2H), 5.12 (s,
2H), 4.78 (s, 2H), 4.67 (s, 2H), 3.72 (d, J = 2.9 Hz, 9H), 3.66 (s, 3H). 13C NMR (100
MHz, DMSO) δ 165.13, 164.84, 158.52, 154.25, 153.07, 141.75, 137.12, 135.45,
129.81, 128.91, 125.45, 113.62, 106.07, 60.00, 55.98, 55.02, 51.86, 51.09, 28.28.
HRMS (m/z): Calcd. C24H27N6O5S2, [M+H]+m/z: 543.1484, found: 543.1488.
4.2.25.N-(4-methoxybenzyl)-2-(4-(((1-methyl-1H-tetrazol-5-yl)thio)methyl)-1H-1,2,3-t
riazol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19j)
White solid, 62% yield, m.p.:150~151 oC. 1H NMR (400 MHz, DMSO) δ 7.96 (s, 1H),
7.14 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.58 (s, 2H), 5.12 (s, 2H), 4.78 (s,
2H), 4.60 (s, 2H), 3.88 (s, 3H), 3.72 (d, J = 2.4 Hz, 9H), 3.66 (s, 3H). 13C NMR (100
MHz, DMSO) δ 165.14, 158.53, 153.08, 153.00, 141.72, 137.13, 135.44, 129.81,
128.91, 125.37, 113.61, 106.06, 60.00, 55.98, 55.01, 51.87, 51.09, 33.56, 27.65.
HRMS (m/z): Calcd. C24H29N8O5S, [M+H]+m/z: 541.1982, found: 541.1987.
4.2.26.2-(4-((10H-phenothiazin-10-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxybe
nzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19k)
White solid, 70% yield, m.p.:139~140 oC. 1H NMR (400 MHz, DMSO) δ 7.82 (s, 1H),
7.11 (dd, J = 9.9, 7.7 Hz, 6H), 6.93 (d, J = 7.8 Hz, 4H), 6.85 (d, J = 8.5 Hz, 2H), 6.55
(s, 2H), 5.16 (s, 2H), 5.09 (s, 2H), 4.76 (s, 2H), 3.71 (s, 3H), 3.69 (s, 6H), 3.65 (s, 3H).
13C NMR (100 MHz, DMSO) δ 165.15, 158.51, 153.05, 143.96, 142.98, 137.11,
135.44, 129.79, 128.93, 127.51, 126.74, 124.84, 122.63, 122.29, 115.54, 113.60,
106.06, 59.98, 55.95, 55.00, 51.80, 51.09, 43.91. HRMS (m/z): Calcd. C34H34N5O5S,
[M+H]+m/z: 624.2281, found: 624.2286.
4.2.27.N-(4-methoxybenzyl)-2-(4-((2-(trifluoromethyl)-10H-phenothiazin-10-yl)methyl

ACCEPTED MANUSCRIPT
)-1H-1,2,3-triazol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19l)
White solid, 91% yield, m.p.:152~153 oC. 1H NMR (400 MHz, DMSO) δ 7.91 (s, 1H),
7.34 (d, J = 7.8 Hz, 1H), 7.24 (d, J = 10.4 Hz, 2H), 7.19 – 7.05 (m, 4H), 6.98 (dd, J =
12.1, 5.0 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.56 (s, 2H), 5.24 (s, 2H), 5.11 (s, 2H),
4.76 (s, 2H), 3.71 (s, 3H), 3.70 (s, 6H), 3.65 (s, 3H). 13C NMR (100 MHz, DMSO) δ
165.08, 158.51, 153.05, 144.54, 143.30, 142.49, 137.12, 135.44, 129.81, 128.94,
128.30, 127.94, 127.30, 126.92, 125.38, 124.93, 123.31, 122.67, 121.32, 119.13,
116.01, 113.58, 111.92, 106.06, 59.97, 55.94, 55.00, 51.80, 51.10, 43.87. HRMS
(m/z): Calcd. C35H33F3N5O5S, [M+H]+m/z: 692.2154, found: 692.2159.
4.2.28.2-(4-((1H-indol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxybenzyl)-N-(3,
4,5-trimethoxyphenyl)acetamide (19m)
White solid, 88% yield, m.p.:184~185 oC. 1H NMR (400 MHz, DMSO) δ 7.87 (s, 1H),
7.56 (dd, J = 16.2, 8.0 Hz, 2H), 7.44 (d, J = 3.1 Hz, 1H), 7.12 (t, J = 7.7 Hz, 3H), 7.01
(t, J = 7.4 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.55 (s, 2H), 6.44 (d, J = 3.0 Hz, 1H),
13
5.48 (s, 2H), 5.07 (s, 2H), 4.75 (s, 2H), 3.71 (s, 3H), 3.68 (s, 6H), 3.64 (s, 3H). C
NMR (100 MHz, DMSO) δ 165.14, 158.51, 153.04, 143.29, 137.09, 135.52, 135.43,
129.79, 128.90, 128.58, 128.19, , 124.70, 121.08, 120.36, 119.08, 113.60, 110.04,
106.06, 100.91, 59.99, 55.94, 55.01, 51.82, 51.00, 40.79. HRMS (m/z): Calcd.
C30H32N5O5, [M+H]+m/z: 542.2403, found: 542.2407.
4.2.29.N-(4-methoxybenzyl)-2-(4-(((4-methylphenyl)sulfonamido)methyl)-1H-1,2,3-tri
azol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (19n)
White solid, 79% yield, m.p.:67~68 oC. 1H NMR (400 MHz, DMSO) δ 8.11 (t, J = 6.1
Hz, 1H), 7.78 (s, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.14 (d, J =
8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.59 (s, 2H), 5.05 (s, 2H), 4.78 (s, 2H), 4.02 (d,
J = 6.1 Hz, 2H), 3.72 (s, 9H), 3.67 (s, 3H), 2.35 (s, 3H). 13C NMR (100MHz, DMSO)
δ 165.13, 158.53, 153.09, 143.22, 142.55, 137.52, 137.13, 135.52, 129.82, 129.54,
128.93, 126.57, 124.83, 113.62, 106.08, 60.00, 55.99, 55.02, 51.85, 50.87, 38.06,
20.88. HRMS (m/z): Calcd. C29H34N5O7S, [M+H]+m/z: 596.2179, found: 596.2183.
4.2.30.2-(4-(((4-Bromophenyl)sulfonamido)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-metho
xybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (19o)
White solid, 73% yield, m.p.:106~107 oC. 1H NMR (400 MHz, DMSO) δ 8.32 (t, J =
6.1 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.15 (d, J
= 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.59 (s, 2H), 5.05 (s, 2H), 4.78 (s, 2H), 4.09
(d, J = 6.0 Hz, 2H), 3.72 (s, 9H), 3.67 (s, 3H). 13C NMR (100 MHz, DMSO) δ 165.10,
158.55, 153.11, 142.92, 139.87, 137.18, 135.51, 132.13, 129.84, 128.93, 128.56,
126.02, 124.90, 113.63, 106.11, 60.01, 56.01, 55.03, 51.88, 50.89, 37.99. HRMS
(m/z): Calcd. C28H31BrN5O7S, [M+H]+m/z: 660.1128, found: 660.1129.
4.3 Biology
4.3.1 General method
Cell culture, colony formation, flow cytometry analysis, western blot,
immunostaining assay, EBI assay and tubulin polymerization assay in vitro were
proceeded according to our reported papers in European journal of medicinal
chemistry, Molecules and Scientific reports [5, 8, 19].

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4.3.2 Cell culture
PC-3 (human prostate cancer), MGC803 (human gastric cancer) and HepG2 (human hepatoma)
cells were cultured at 37 °C in an atmosphere containing 5% CO2, using RPMI-1640 medium with
10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin.
4.3.3 MTT assay
Cells were seeded in 96-well plates at a density of 5 x 103 per well for 24 hours, then treated with
different concentrations as shown in the figures. After 48 hours, 20 µL MTT solution was added to
each well, and incubated at 37 °C for 4 hours. 150 µL DMSO was added to each well to dissolve
the formazan after discarding the supernatant, the absorbance was determined at 490 nm. Data are
calculated with SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA).
4.3.4 Colony formation assay
MGC803 cells were seeded in a 6-well plate and incubated at 37 °C in 5% CO2 for 24 h, then
treated with different concentrations of 19c. After 7 days, remove the culture medium, wash the
cells with PBS twice, fix with 4% paraformaldehyde and stain with 0.1% crystal violet. The cells`
image were captured with camera, and the number of colonies were quantified using Image J
software (National Institutes of Health).
4.3.5 Cell cycle distribution assay
MGC803 cells were seeded in 6-well culture plate, then treated with certain concentration of
compound for 48 hours. Then cells were harvested and fixed at 4 °C with 70% ethanol overnight.
The fixed cells were washed and resuspended using PBS with 10 mg/mL RNaseA and 50 mg/mL
PI incubated for 20 minutes in the dark. Then, samples were analyzed for DNA content with flow
cytometry. (Becton, Dickinson and Company, NJ)
4.3.6 Cell apoptosis assay
MGC803 cells were seeded in 6-well culture plate and treated with certain concentration of
compound for 48 hours. Then cells were harvested and suspended in binding buffer containing
Annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/mL). After that, samples were incubated for 20 min
in dark and analyzed with flow cytometry (Becton, Dickinson and Company, NJ)
4.3.7 Immunoprecipitation
MGC803 cells were seeded in 6-well culture plate and treated with certain concentration of
compound for 48 hours. Then cells were harvested and lysed. Cell extracts were used to detect the
level of CDK1. Then the concentration of cell extracts were adjusted to have the same level of
CDK1. The anti-CDK1 were added into adjusted cell extracts for 30 minutes, then 20µL agarose
beads were added and co-incubated at 4°C overnight. Beads were washed 3 times after that, and
denatured with loading buffer. Western blotting was performed to detect CDK1 and Cyclin B1.
4.3.8 Immunostaining and microscopy
Cell slices were placed on the bottom of a 24-well plate. MGC803 cells were seeded on the slices
and incubated overnight. Then cells were treated with certain concentration of 19c. After 48 hours,
slices were fixed by 4% paraformaldehyde for 15 min after washed by PBS for 3 times. 0.5%

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Triton-X-100 was added and shaked for 20 min. 0.1% BSA was used to block for 30 min and then
removed. The slices were added β-tubulin antibody (1:100) and incubated overnight at 4 °C. Then
slices were washed by PBST 3 times, bind with secondary antibody with FITC signal (1:500) in a
dark. DAPI was used to stain for 5 min and then removed. After that, images were captured by
Laser scanning confocal microscope (Nikon, Japan).
4.3.9 In vitro tubulin polymerization assay
An amount of 5.6 mg/ml tubulin was resuspended in PEM buffer (containing 80 mmol/L PIPES
(pH 6.9), 1 mmol/L EGTA, 0.5 mmol/L MgCl2, 1 mmol/L ATP, 10.2% (v/v) glycerol) and then
was preincubated with compound 19c, colchicine or vehicle DMSO on ice. The reaction was
monitored by a spectrophotometer in absorbance at 420 nm at 37 °C every minute (excitation
wavelength is 340nm).
4.3.10 EBI competition assay
6-Well plates were seeded with MGC803 cells at 5 × 105 cells per well. Cells were first incubated
with compound 19c, colchicine or DMSO for 2 hours and afterward treated with EBI (100 µM)
for 2 hours. Then the cells were harvested and lysed. Cell extracts were used for Western blotting
analysis.
4.3.11 Western blot analysis
Treated and untreated cells were harvested and lysed. Protein lysates were denatured and resolved
by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were incubated with
appropriate antibodies at 4 °C for 8 hours after blocking with 5% skimmed milk. After conjugated
with secondary antibodies, the detection of proteins was carried out with an ECL kit.
4.3.12 Statistical analysis
Data from three independent experiments are presented as mean ± SD. IC50 values and Student’s
t-tests were calculated by SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA). **: p < 0.01 verse
control, ***: p < 0.001 verse control.
4.4 Docking studies
Trimethoxyphenyl-1,2,3-triazole-coumarin hybrid 19c was selected to do molecular
docking study. For the receptor preparation, the PDB code (1SA0) was downloaded
from the Protein Data Bank. The detailed method was shown in our previous paper
[5].
5. Declaration of interest
The authors declare that there is no conflict of interest regarding the publication of
this paper.
Acknowledgment
This work was supported by the National Natural Sciences Foundations of China (No.
81703541, 81673322) and China Postdoctoral Science Foundation. This work was
supported by MEDCHEMEXPRESS. Thanks to MCE Award for Scientists Promoting

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Biology and Medicine Research.
References
[1] J.-Y. Chang, H.-P. Hsieh, C.-Y. Chang, K.-S. Hsu, Y.-F. Chiang, C.-M. Chen, C.-C. Kuo, J.-P. Liou,
7-Aroyl-aminoindoline-1-sulfonamides as a Novel Class of Potent Antitubulin Agents, J. Med. Chem.
49 (2006) 6656-6659.
[2] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat Rev Cancer. 4 (2004)
253.
[3] C.-Y. Nien, Y.-C. Chen, C.-C. Kuo, H.-P. Hsieh, C.-Y. Chang, J.-S. Wu, S.-Y. Wu, J.-P. Liou, J.-Y.
Chang, 5-Amino-2-Aroylquinolines as Highly Potent Tubulin Polymerization Inhibitors, J. Med. Chem.
53 (2010) 2309-2313.
[4] I. Khelifi, T. Naret, D. Renko, A. Hamze, G. Bernadat, J. Bignon, C. Lenoir, J. Dubois, J.-D. Brion,
O. Provot, M. Alami, Design, synthesis and anticancer properties of IsoCombretaQuinolines as potent
tubulin assembly inhibitors, Eur. J. Med. Chem. 127 (2017) 1025-1034.
[5] D.-J. Fu, J.-J. Yang, P. Li, Y.-H. Hou, S.-N. Huang, M.A. Tippin, V. Pham, L. Song, X. Zi, W.-L.
Xue, L.-R. Zhang, S.-Y. Zhang, Bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment
exerting potent antiproliferative activity through microtubule destabilization, Eur. J. Med. Chem. 157
(2018) 50-61.
[6] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal, Recent developments in tubulin polymerization
inhibitors: An overview, Eur. J. Med. Chem. 87 (2014) 89-124.
[7] 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, Eur. J. Med. Chem. 156 (2018) 162-179.
[8] D.J. Fu, L. Fu, Y.C. Liu, J.W. Wang, Y.Q. Wang, B.K. Han, X.R. Li, C. Zhang, F. Li, J. Song,
Structure-Activity Relationship Studies of β-Lactam-azide Analogues as Orally Active Antitumor
Agents Targeting the Tubulin Colchicine Site, Sci Rep. 7 (2017) 12788.
[9] R. Romagnoli, P.G. Baraldi, M.K. Salvador, F. Prencipe, C. Lopez-Cara, S. Schiaffino Ortega, A.
Brancale, E. Hamel, I. Castagliuolo, S. Mitola, R. Ronca, R. Bortolozzi, E. Porcù, G. Basso, G. Viola,
Design, Synthesis, in Vitro, and in Vivo Anticancer and Antiangiogenic Activity of Novel
3-Arylaminobenzofuran Derivatives Targeting the Colchicine Site on Tubulin, J. Med. Chem. 58 (2015)
3209-3222.
[10] R. Romagnoli, P.G. Baraldi, A. Brancale, A. Ricci, E. Hamel, R. Bortolozzi, G. Basso, G. Viola,
Convergent Synthesis and Biological Evaluation of 2-Amino-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl
Thiazoles as Microtubule Targeting Agents, J. Med. Chem. 54 (2011) 5144-5153.
[11] D.-J. Hwang, J. Wang, W. Li, D.D. Miller, Structural Optimization of Indole Derivatives Acting at
Colchicine Binding Site as Potential Anticancer Agents, ACS Med. Chem. Lett. 6 (2015) 993-997.
[12] N. Hura, A.V. Sawant, A. Kumari, S.K. Guchhait, D. Panda, Combretastatin-Inspired Heterocycles
as Antitubulin Anticancer Agents, ACS Omega. 3 (2018) 9754-9769.
[13] D.J. Fu, J. Song, Y.H. Hou, R.H. Zhao, J.H. Li, R.W. Mao, J.J. Yang, P. Li, X.L. Zi, Z.H. Li,
Discovery of 5,6-diaryl-1,2,4-triazines hybrids as potential apoptosis inducers, Eur. J. Med. Chem. 138
(2017) 1076-1088.
[14] S.-Y. Zhang, D.-J. Fu, X.-X. Yue, Y.-C. Liu, J. Song, H.-H. Sun, H.-M. Liu, Y.-B. Zhang, Design,
Synthesis and Structure-Activity Relationships of Novel Chalcone-1,2,3-triazole-azole Derivates as
Antiproliferative Agents, Molecules. 21 (2016) 653.

ACCEPTED MANUSCRIPT
[15] D.-J. Fu, S.-Y. Zhang, Y.-C. Liu, X.-X. Yue, J.-J. Liu, J. Song, R.-H. Zhao, F. Li, H.-H. Sun, Y.-B.
Zhang, H.-M. Liu, Design, synthesis and antiproliferative activity studies of 1,2,3-triazole-chalcones,
MedChemComm. 7 (2016) 1664-1671.
[16] T. Suzuki, Y. Ota, M. Ri, M. Bando, A. Gotoh, Y. Itoh, H. Tsumoto, P.R. Tatum, T. Mizukami, H.
Nakagawa, S. Iida, R. Ueda, K. Shirahige, N. Miyata, Rapid Discovery of Highly Potent and Selective
Inhibitors of Histone Deacetylase 8 Using Click Chemistry to Generate Candidate Libraries, J. Med.
Chem. 55 (2012) 9562-9575.
[17] S. Xu, X. Zhuang, X. Pan, Z. Zhang, L. Duan, Y. Liu, L. Zhang, X. Ren, K. Ding,
1-Phenyl-4-benzoyl-1H-1,2,3-triazoles as Orally Bioavailable Transcriptional Function Suppressors of
Estrogen-Related Receptor α, J. Med. Chem. 56 (2013) 4631-4640.
[18] C. Ferroni, A. Pepe, Y.S. Kim, S. Lee, A. Guerrini, M.D. Parenti, A. Tesei, A. Zamagni, M. Cortesi,
N. Zaffaroni, M. De Cesare, G.L. Beretta, J.B. Trepel, S.V. Malhotra, G. Varchi, 1,4-Substituted
Triazoles as Nonsteroidal Anti-Androgens for Prostate Cancer Treatment, J. Med. Chem. 60 (2017)
3082-3093.
[19] D.J. Fu, J.F. Liu, R.H. Zhao, J.H. Li, S.Y. Zhang, Y.B. Zhang, Design and Antiproliferative
Evaluation of Novel Sulfanilamide Derivatives as Potential Tubulin Polymerization Inhibitors,
Molecules. 22 (2017) 1470.
[20] L. Cafici, T. Pirali, F. Condorelli, E. Del Grosso, A. Massarotti, G. Sorba, P.L. Canonico, G.C.
Tron, A.A. Genazzani, Solution-Phase Parallel Synthesis and Biological Evaluation of
Combretatriazoles, J. Comb. Chem. 10 (2008) 732-740.
[21] J.A. Stefely, R. Palchaudhuri, P.A. Miller, R.J. Peterson, G.C. Moraski, P.J. Hergenrother, M.J.
Miller, N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a New Scaffold that Provides Rapid
Access to Antimicrotubule Agents: Synthesis and Evaluation of Antiproliferative Activity Against
Select Cancer Cell Lines, J. Med. Chem. 53 (2010) 3389-3395.
[22] D.J. Fu, S.Y. Zhang, Y.C. Liu, L. Zhang, J.J. Liu, J. Song, R.H. Zhao, F. Li, H.H. Sun, H.M. Liu,
Design, synthesis and antiproliferative activity studies of novel dithiocarbamate-chalcone derivates,
Bioorg. Med. Chem. Lett. 26 (2016) 3918.
[23] D.J. Fu, L. Zhang, J. Song, R.W. Mao, R.H. Zhao, Y.C. Liu, Y.H. Hou, J.H. Li, J.J. Yang, C.Y. Jin,
Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of
PC-3cells via MAPK/Wnt signaling pathways, Eur. J. Med. Chem. 127 (2016) 87-99.
[24] D.J. Fu, Y.C. Liu, J.J. Yang, J. Zhang, C.D. Xiong, Z.S. Cao, X.X. Yin, W. Wei, Y.B. Zhang,
Design and synthesis of sulfonamide-1,2,3-triazole derivatives bearing a dithiocarbamate moiety as
antiproliferative agents, J. Chem. Res. 41 (2017) 523-525.
[25] H.M. Liu, Y.B. Zhang, Y.C. Liu, R.H. Zhao, J. Song, D.J. Fu, Synthesis of novel antiproliferative
1,2,3-triazole hybrids using the molecular hybridisation approach, J. Chem. Res. 40 (2016) 674-677.
[26] D.J. Fu, Y.H. Hou, S.Y. Zhang, Y.B. Zhang, Efficient click reaction towards novel sulfonamide
hybrids by molecular hybridization strategy as antiproliferative agents, J. Chem. Sci. 130 (2018) 6.
[27] D.J. Fu, R.H. Zhao, J.H. Li, J.J. Yang, R.W. Mao, B.W. Wu, P. Li, X.L. Zi, Q.Q. Zhang, H.J. Cai,
Molecular diversity of phenothiazines: design and synthesis of phenothiazine-dithiocarbamate hybrids
as potential cell cycle blockers, Mol. Divers.21 (2017) 933-942.
[28] Z. Wen, Y. Zhang, X. Wang, X. Zeng, Z. Hu, Y. Liu, Y. Xie, G. Liang, J. Zhu, H. Luo, B. Xu,
Novel 3′,5′-diprenylated chalcones inhibited the proliferation of cancer cells in vitro by inducing cell
apoptosis and arresting cell cycle phase, Eur. J. Med. Chem. 133 (2017) 227-239.
[29] V. Ganga Reddy, T. Srinivasa Reddy, V. Lakshma Nayak, B. Prasad, A.P. Reddy, A. Ravikumar, S.

ACCEPTED MANUSCRIPT
Taj,
A.
Kamal,
Design,
synthesis
and
biological
evaluation
of
N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2
inhibitors, Eur. J. Med. Chem. 122 (2016) 164-177.
[30] C. Zha, W. Deng, Y. Fu, S. Tang, X. Lan, Y. Ye, Y. Su, L. Jiang, Y. Chen, Y. Huang, J. Ding, M.
Geng, M. Huang, H. Wan, Design, synthesis and biological evaluation of tetrahydronaphthyridine
derivatives as bioavailable CDK4/6 inhibitors for cancer therapy, Eur. J. Med. Chem. 148 (2018)
140-153.
[31] C.G. Takizawa, D.O. Morgan, Control of mitosis by changes in the subcellular location of
cyclin-B1–Cdk1 and Cdc25C, Curr. Opin. Cell Biol. 12 (2000) 658-665.
[32] D.-J. Fu, J. Song, Y.-H. Hou, R.-H. Zhao, J.-H. Li, R.-W. Mao, J.-J. Yang, P. Li, X.-L. Zi, Z.-H. Li,
Q.-Q. Zhang, F.-Y. Wang, S.-Y. Zhang, Y.-B. Zhang, H.-M. Liu, Discovery of 5,6-diaryl-1,2,4-triazines
hybrids as potential apoptosis inducers, Eur. J. Med. Chem. 138 (2017) 1076-1088.
[33] M. Silva, D. Grillot, A. Benito, C. Richard, G. Nunez, J.L. Fernandez-Luna, Erythropoietin can
promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2, Blood. 88
(1996) 1576.
[34] S.R. Kumari, H. Mendoza-Alvarez, R. Alvarez-Gonzalez, Functional Interactions of p53 with
Poly(ADP-ribose) Polymerase (PARP) during Apoptosis following DNA Damage: Covalent
Poly(ADP-ribosyl)ation of p53 by Exogenous PARP and Noncovalent Binding of p53 to the
&lt;em&gt;M&lt;/em&gt;&lt;sub&gt;r&lt;/sub&gt; 85,000 Proteolytic Fragment, Cancer Res. 58
(1998) 5075.
[35] M. Holcik, C. Yeh, R.G. Korneluk, T. Chow, Translational upregulation of X-linked inhibitor of
apoptosis (XIAP) increases resistance to radiation induced cell death, Oncogene. 19 (2000) 4174.
[36] C. Jiménez, Y. Ellahioui, R. Álvarez, L. Aramburu, A. Riesco, M. González, A. Vicente, A.
Dahdouh, A. Ibn Mansour, C. Jiménez, D. Martín, R.G. Sarmiento, M. Medarde, E. Caballero, R.
Peláez, Exploring the size adaptability of the B ring binding zone of the colchicine site of tubulin with
para-nitrogen substituted isocombretastatins, Eur. J. Med. Chem. 100 (2015) 210-222.

ACCEPTED MANUSCRIPT
Highlights
.
.
.
.
Molecular diversity of trimethoxyphenyl-1,2,3-triazoles was explored.
19c inhibited colony formation against MGC803 cells at 30nM.
19c arrested cell cycle at G2/M phase and induced apoptosis.
19c was a novel colchicine site tubulin polymerization inhibitor.