Design, synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule destabilizers

Article abstract of DOI:10.1080/14756366.2020.1759582

A series of 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as microtubule destabilizers were designed, synthesised and evaluated for anticancer activity. Based on bioisosterism, we introduced the tetrazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. The key intermediates ethyl 1-aryl-1H-tetrazole-5-carboxylates 10 can be simply and efficiently prepared via a microwave-assisted continuous operation process. Among the compounds synthesised, compound 6–31 showed noteworthy potency against SGC-7901, A549 and HeLa cell lines. In mechanism studies, compound 6–31 inhibited tubulin polymerisation and disorganised microtubule in SGC-7901 cells by binding to tubulin. Moreover, compound 6–31 arrested SGC-7901cells in G2/M phase. This study provided a new perspective for development of antitumor agents that target tubulin.

Full text of DOI:10.1080/14756366.2020.1759582

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation of 1-
Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as
novel microtubule destabilizers
Chao Wang, Yuelin Li, Zi Liu, Zeyu Wang, Zihan Liu, Shuai Man, Yujing Zhang,
Kai Bao, Yingliang Wu, Qi Guan, Daiying Zuo & Weige Zhang
To cite this article: Chao Wang, Yuelin Li, Zi Liu, Zeyu Wang, Zihan Liu, Shuai Man, Yujing
Zhang, Kai Bao, Yingliang Wu, Qi Guan, Daiying Zuo & Weige Zhang (2021) Design, synthesis and
biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule
destabilizers, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 549-560, DOI:
10.1080/14756366.2020.1759582
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 549–560
https://doi.org/10.1080/14756366.2020.1759582
ORIGINAL ARTICLE
Design, synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-
carbonyl)-1H-tetrazols as novel microtubule destabilizers
Chao Wanga , Yuelin Lia, Zi Liub, Zeyu Wangc, Zihan Liua, Shuai Manb, Yujing Zhanga, Kai Baoc, Yingliang Wub,
Qi Guana , Daiying Zuob and Weige Zhanga
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, China;
c
bDepartment of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China; Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang, China
ABSTRACT
ARTICLE HISTORY
Received 9 February 2020
Revised 6 April 2020
Accepted 18 April 2020
A series of 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as microtubule destabilizers were designed,
synthesised and evaluated for anticancer activity. Based on bioisosterism, we introduced the tetrazole moi-
ety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. The key intermediates ethyl
1-aryl-1H-tetrazole-5-carboxylates 10 can be simply and efficiently prepared via a microwave-assisted con-
tinuous operation process. Among the compounds synthesised, compound 6–31 showed noteworthy
potency against SGC-7901, A549 and HeLa cell lines. In mechanism studies, compound 6–31 inhibited
tubulin polymerisation and disorganised microtubule in SGC-7901 cells by binding to tubulin. Moreover,
compound 6–31 arrested SGC-7901cells in G2/M phase. This study provided a new perspective for devel-
opment of antitumor agents that target tubulin.
KEYWORDS
Tetrazole; microwave;
antiproliferative activity;
microtubule destabilizer;
molecular docking
1. Introduction
(substituted phenyl), B-ring (five-membered heterocycle, such as
pyrazole, oxazole and 1,2,3-triazole), C-ring (piperazine), D-ring
(substituted phenyl), and a carbonyl linkage between B-ring and
C-ring. Bioisosterism plays a major role in the search for analogues
of an active drug molecule. Application of bioisosterism instead of
the B-ring on the structure of XRP44X is one of the strategies
often used in design of the XRP44X analogues6. The modification
of B-ring has led to varied cytotoxic activity.
In the last decade, there has been great interest in compounds
containing the 1H-tetrazol scaffold because of its unique chemical
structure and broad spectrum of biological properties including
anticancer activity. For example, the 1-(3,4,5-Trimethoxyphenyl)-5-
(4-ethoxyphenyl)-1H-tetrazole was designed as tubulin inhibitor,
which showed low IC50 values at nanomolar level8.
Microtubule, is considered an important target for anticancer drug
discovery, playing a crucial role in a wide range of fundamental
cell functions including the shape maintenance, intracellular trans-
port and cell division1. Microtubule-targeted agents, according to
the mechanism of interfering with microtubule dynamics, have
been classified into microtubule stabilisers (taxanes and epothi-
lones) and microtubule destabilizers (alkaloids and colchicine (1))2.
Given the extensively successful clinical use of vinca alkaloids, the
microtubule destabilizers have aroused great interest among
medicinal chemists3. Over the past decades, a great many out-
standing microtubule destabilizers have been reported, such as
combretastatin A-4 (CA-4, 2)4 and XRP44X (3)5 (Figure 1).
XRP44X, an arylpyrazole derivative, was developed by Wasylyk
et al. as a novel microtubule destabilizer, which prominently inhib-
ited the polymerisation process of tubulin into microtubules by
As a part of our continuing effort on the development of novel
antitumor agents, we designed a series of novel XRP44X ana-
interacting with the colchicine-binding site of tubulin and dis- logues by introducing an 1H-tetrazol, hydrogen-bonding accept-
played potent cytotoxic activities against a wide variety of human
cancer cell lines at low nanomolar concentrations. A series of ary-
loxazole derivatives have been synthesised and were found to be
potent inhibitors of tubulin polymerisation by Pae and co-workers.
Among them, compound 4 showed excellent antiproliferative
in vitro and effectively reduced tumour growth in vivo using a
tumour xenograft model6. Our groups discovered a series of aryl-
triazole derivatives as a result of structural modifications of the
lead compound XRP44X7. The most potent compound 5 exhibited
excellent antiproliferative activity via disrupting cytoskeleton.
As shown in Figure 1, XRP44X and its analogues molecules can
ors, as B-ring of XRP44X (Figure 2). Herein, we described the
detailed synthetic routes, antiproliferative, tubulin polymerisation,
analysis of immunofluorescence staining and cell cycle analysis of
these compounds.
2. Result and discussion
2.1. Chemistry
The general synthetic approach for the preparation of 1-aryl-5-(4-
arylpiperazine-1-carbonyl)-1H-tetrazol (6) was illustrated in
be divided into three major structural elements i.e., A-ring Scheme 1. First of all, the substituted aromatic amines (7) were
CONTACT Qi Guan
guanqi@syphu.edu.cn; Weige Zhang
zhangweige2000@sina.com
Key Laboratory of Structure-Based Drug Design and Discovery,
zuodaiying@163.com Department of Pharmacology, Shenyang
Ministry of Education, Shenyang Pharmaceutical University, Shenyang, China; Daiying Zuo
Pharmaceutical University, Shenyang, China
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

550
C. WANG ET AL.
Figure 1. Chemical structures of some microtubule destabilizers.
Figure 2. Design of the target compounds.
reacted with ethyl oxalate in dichloromethane to afford corre- microwave conditions. As shown in Table 1, the optimised condi-
temperature9,10
.
tion was confirmed to be microwave irradiation at 130 ꢀC for
sponding
intermediates
(8)
at
room
20 min (entry 8). Accordingly, all intermediates
obtained smoothly.
9
were
Subsequently, 8 were reacted with triphenylphosphine and carbon
tetrachloride to give corresponding (E)-ethyl 2-chloro-2-(arylimi-
no)acetates (9) via Appel reaction under microwave irradiation.
Without further purification, compounds 9 reacted with sodium
azide in acetonitrile to get the key intermediates ethyl 1-aryl-1H-
2.2. Biological evaluation
tetrazole-5-carboxylates (10) in
a continuous operation pro-
2.2.1. In vitro antiproliferative activity
cess11–13. Finally, the key intermediates 10 reacted with corre-
sponding arylpiperazines to afford the target compounds (6) in
the presence of trimethylaluminium14.
All the target compounds have been evaluated for in vitro the
antiproliferative activities against different three human cancer
cell lines (gastric adenocarcinoma SGC-7901 cells, lung adenocar-
cinoma A549 cells and cervical carcinoma HeLa cells) using MTT
assay with colchicine and CA-4 as references. To examine the
more detailed structure-activity relationships, modifications
towards A-ring and D-ring were performed.
Careful observation of Table 2 revealed that introduction of
substituent into the ortho-position of A-ring could remarkably
enhance the antiproliferative effect, such as 6–28 ꢁ 6–30.
The overall preference order of substituent at the ortho-position
of A-ring is as follows: 2-methyl > 2-fluoro > 2-chloro > H.
Under conventional heating conduction, the reaction of 8 with
PPh3 in CCl4 to generate (E)-ethyl 2-chloro-2-(arylimino)acetates
(9) suffered from long reaction time and low yields (Table 1,
entries 1–5). Microwave irradiation offered many advantages, such
as rate enhancements and higher yields, over conventional heat-
ing and had become a popular technique that was widely used in
organic synthesis today15–18. When microwave irradiation was
incorporated into synthesis of 9–8, we found that the reaction
rate was greatly improved. We systematically screened the influ-
ence of reaction temperature and time on the yield of 9–8 under Furthermore, in the case of D-ring 3,5-dimethoxyphenyl fused

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
551
Scheme 1. Reagents and conditions: (a) EtO2CCOCl, Et3N, DCM, rt., 1 h; (b) Ph3P, CCl4, 130ꢀC, MW, 20 min; (c) NaN3, MeCN, rt., overnight; (d) arylpiperazines, AlMe3
(1.0 M solution in heptane), DCM, rt., overnight.
Table 1. The optimisation of conditions for the preparation of compound 9–8.
polymerisation inhibitor (CA-4) and microtubule stabilising agent
(Paclitaxel) were used as the positive and negative controls,
respectively. As shown in Figure 3, compound 6–31 inhibited
tubulin assembly in a concentration-dependent manner. In con-
trast, paclitaxel could raise the proportion of tubulin polymerisa-
tion in comparison with the untreated cells. The results suggested
that compound 6–31 interferes with the microtubule polymerisa-
tion in a similar way as CA-4.
Entry
Heating
Oil bath
Oil bath
Oil bath
Oil bath
Oil bath
MW
MW
MW
MW
MW
Temp (8C)
Reflux
Reflux
Reflux
Reflux
Reflux
120
130
130
130
140
Time (min)
Yield (%)
Trace
1a
2a
3a
4a
5a
6b
7b
8b
9b
10b
60
360
600
720
960
20
10
20
30
20
10c
30c
41c
52d
45c
50d
67d
62d
60d
2.2.3. Analysis of immunofluorescence staining
To further confirm the influence of inhibition of tubulin polymer-
isation in cells, immunofluorescence staining was carried out. SGC-
7901 cell lines were treated for 24 h with CA-4 and compound
6–31, at their respective 2-fold IC50 concentrations. As given in
Figure 4, the microtubule network without drug treatment dis-
plays normal arrangement and organisation in control cells.
Whereas SGC-7901 cells were treated with CA-4 and compound
6–31 and the results demonstrated that microtubules were
destroyed and wrapped around the nucleus in comparison with
the control. These results suggest that compound 6–31 inhibits
microtubule assembly and disrupts cytoskeleton similarly to CA-4.
aReaction conditions: ethyl 2-((3-bromophenyl)amino)-2-oxoacetate (1 mmol),
Ph3P (1.5 mmol), CCl4 (5 ml).
bReaction conditions: ethyl 2-((3-bromophenyl)amino)-2-oxoacetate (1 mmol),
Ph3P (1.5 mmol), CCl4 (5 ml), MW.
cThe yields were determined by HPLC.
dIsolated yields.
compounds showed significant anticancer activities. Compound
6–31 was found to be the most potent compound among all the
target compounds with IC50 value of 0.090–0.650 lM against the
three cancer cell lines.
2.2.4. Cell cycle analysis
It is well known that most tubulin inhibitors induce cell cycle
arrest in the G2/M phase7. Thus, the effect of compound 6–31on
the cell cycle of SGC-7901 cells was analysed by flow cytometry.
SGC-7901 cells were incubated with CA-4 or compound 6–31, at
their respective two-fold IC50 concentrations, and the proportion
of tested cells at different cell cycle phases was analysed by flow
cytometry after 0, 12, 24, 36, 48, and 72 h of treatment,
2.2.2. Effect on tubulin polymerisation
In order to examine whether the compounds interact with tubulin,
we chose the most active compound 6–31 to evaluate for its
inhibition of tubulin polymerisation in vitro. Microtubule respectively.

552
C. WANG ET AL.
Table 2. The antiproliferative activities of all target compounds
(IC50 SD, lM)a
Compound
R1
R2
SGC-7901
A549
HeLa
6–1
6–2
6–3
6–4
6–5
6–6
6–7
6–8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>30
28.3 2.1
>30
13.8 0.8
27.8 2.0
15.5 1.2
>30
16.1 1.4
>30
>30
9.92 0.46
>30
>30
>30
>30
18.2 1.8
22.1 1.6
19.3 1.7
>30
20.6 1.8
>30
>30
>30
3.34 0.23
3.11 0.19
>30
26.1 2.3
0.360 0.017
28.7 3.2
1.01 0.05
27.6 2.7
10.5 0.9
10.6 0.47
>30
3-fluoro
2-chloro
3-chloro
3-methyl
3-methoxy
4-methoxy
>30
8.22 0.23
25.3 1.9
13.2 1.0
>30
15.0 1.2
>30
3-trifluoromethyl
3,4-dimethyl
2,6-dimethyl
3,4-dimethoxy
2,5-dimethoxy
3,5-dimethoxy
3-methyl
3-methoxy
3,5-dimethoxy
3-methoxy
3,5-dimethoxy
3-methoxy
3,5-dimethoxy
3-methyl
3-methoxy
4-methoxy
3,4-dimethyl
3,5-dimethoxy
3-methoxy
3,5-dimethoxy
3,5-dimethoxy
3,5-dimethoxy
3,5-dimethoxy
3,5-dimethoxy
3,5-dimethoxy
6–9
6–10
6–11
6–12
6–13
6–14
6–15
6–16
6–17
6–18
6–19
6–20
6–21
6–22
6–23
6–24
6–25
6–26
6–27
6–28
6–29
6–30
6–31
6–32
CA–4b
Colchicineb
>30
>30
27.3 1.9
3.86 0.27
3.10 0.25
5.80 0.30
10.0 1.1
2.17 0.20
20.9 1.5
6.71 0.19
>30
2.47 0.21
1.68 0.12
17.2 1.4
11.3 0.9
0.504 0.021
29.8 3.0
4.2 0.24
>30
12.2 1.6
4.59 0.18
11.8 0.50
>30
24.3 1.6
1.08 0.13
>30
1.51 0.09
2.53 0.11
4.73 0.14
>30
2-fluoro
2-fluoro
2-fluoro
3-fluoro
3-fluoro
4-fluoro
4-fluoro
2-chloro
2-chloro
2-chloro
2-chloro
2-chloro
3-chloro
3-chloro
2-bromo
3-bromo
4-bromo
2-methyl
3-methyl
>30
10.5 0.41
11.7 0.49
>30
>30
3.12 0.15
>30
7.77 0.17
>30
>30
22.0 1.5
0.580 0.023
29.4 0.6
1.85 0.13
5.67 0.19
7.48 0.18
>30
0.268 0.012
0.812 0.034
0.034 0.004
0.066 0.005
>30
29.4 3.1
0.650 0.017
2.34 0.16
0.070 0.007
0.075 0.011
0.090 0.008
1.44 0.09
0.036 0.002
0.096 0.009
aIC50: the half maximal inhibitory concentration.
bUsed as positive controls.
Figure 3. The effect of compound 6–31 on tubulin polymerisation. The tubulin had been pre-incubated for 1 min with 6–31 at 2.5 mM, 5 mM, 10 mM and 20 mM, CA-4
at 5 mM, Paclitaxel at 5 mM or vehicle DMSO at room temperature before GTP was added to start the tubulin polymerisation reactions. The reaction was monitored
at 37ꢀC.
As shown in Figure 5, cells treated with either CA-4 or com- dependent manner. After treatment with compound 6–31 for
pound 6–31 were arrested at the G2/M phase. In addition, 12 h, the percentage of cells in the G2/M phase increased to
whereas CA-4 or 6–31 induced a sharply decrease of G1 cell 63.0% from 22.2%. After 72 h, there were significantly increased
population, and an increase of G2/M cell population in time- numbers of cells in sub-G1 after exposure to CA-4 and compound

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
553
Figure 4. Effects of 6–31 and CA-4, at their respective two-fold IC50 concentrations, on the cellular microtubule network and microtubule reassemble by immunofluor-
escence. SGC-7901 cells were treated with 6–31 or CA-4 for 24h, and then direct microscopy detection of the fixed and stained cell was performed. The cellular micro-
tubules were stained with anti-a-tubulin-FITC specific antibodies (green). DNA was stained by 4,6-diamidino-2-phenylindole (DAPI, blue). (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article).
6–31, respectively, indicating the induction of apoptosis. The cell www.swissadme.ch/index.php) for their adaptability with Lipinski’s
cycle analysis suggests that compound 6–31 arrests SGC-7901 cell rule of five19. As shown in Table 3, XRP44X and compound 6–31
in G2/M phase followed by cellular apoptosis.
conform well to the Lipinski’s rule of five. Compared with XRP44X,
compound 6–31 has a lower value of lipid-water partition coefficient.
This data indicates that compound 6–31 may have better water solu-
bility than XRP44X. In addition, compound 6–31 exists six hydrogen
bond receptors, much more than XRP44X, which helps to reduce the
binding energy between the compound and the action site.
2.3. Molecular modelling
To further understand the binding interactions, molecular docking of
the most active compound 6–31 was carried out with tubulin crystal
structure (PDB code: 3HKC) using the CDOCKER programme of
Discovery Studio 3.0 software. As shown in Figure 6, XRP44X and
compound 6–31 are located at the same position with a similar con-
formation in the binding pocket. Meanwhile, the hydrogen bond
exists between the carbonyl group of XRP44X or compound 6–31
with amino acid residue Alab317. Moreover, it is worth noting that
the residue of Asnb258 forms a hydrogen bond with the 2 N of 1H-
tetrazole of compound 6–31 and the residue of Lysb352 forms a
hydrogen bond with the 4 N of 1H-tetrazole of compound 6–31. It
suggests that the 1H-tetrazole derivatives can not only maintain the
right conformation, but also nicely nestle in the active site. The 1H-
tetrazole moiety and unique active site interactions set the stage for
structure-based design of more potent derivatives.
3. Conclusion
In summary, we had designed and synthesised a series of XRP44X
derivatives having a 1H-tetrazole B-ring as the hydrogen-bonding
acceptors and found that these compounds showed good growth
inhibition activities against a range of human cancer cells. Among
them, compound 6–31, represented the most active compound
with IC50 values of 0.090–0.650 lM against three cancer cell lines.
Moreover, compound 6–31 could disrupt microtubule network in
living cancer cells, arrest cell cycle at G2/M phase and induce
apoptosis in a dose- and time-dependent manner. Docking studies
suggest that compound 6–31 may be a potential tubulin inhibitor.
A hydrogen bond was present between Alab317 with the carbonyl
group of compound 6–31. Another two hydrogen bonds were
also observed between Asnb258 and Lysb352 with the 1H-tetra-
zole (B-ring). In addition, the prediction of drug-like properties
2.4. Prediction of drug-like properties
Furthermore, to explore the drug-like properties of the target
compounds, some physicochemical properties of XRP44X and studies shows that compound 6–31 has better pharmacokinetic
compound 6–31 were predicted using free online website (http:// properties than XRP44X. All of these results indicate compound

554
C. WANG ET AL.
Figure 5. The effect of CA-4 and compound 6–31 on cell cycle. SGC-7901 cells lines treated with CA-4 and compound 6–31, at their respective two-fold IC50 concen-
trations, for 0, 12, 24, 36, 48 and 72h.
Figure 6. (A) Superimposition of XRP44X (yellow) and 6–31 (red) in colchicine-binding site; (B) interaction of XRP44X (yellow) with tubulin (green dotted lines repre-
sent H-bond); (C) Interactions of 6–31 (red) with tubulin (green dotted lines represent H-bond).
Table 3. Prediction of physicochemical propertiesa of 3 and 6–31.
TMS as the internal reference on a Bruker AVANCE 400 or 600 (1H
at 400 or 600 MHz, 13 C at 150 MHz). Mass spectra (MS) were meas-
ured on an Agilent 1100-sl mass spectrometer with an electro-
spray ionisation source from Agilent Co. Ltd. High resolution
accurate mass determinations (HRMS) for all of the final target
compounds were obtained on a Bruker Micromass Time of Flight
mass spectrometer equipped with electrospray ionisation (ESI).
TLC analysis was used for determining the extent of reactions
under UV light (wavelength: 365 nm and 254 nm). Melting point
was measured (uncorrected) on hot-stage microscope (Beijing
Taike, X-4). The microwave reactions were carried out in a single
mode cavity microwave synthesiser (CEM Corporation, NC, USA).
Compound
miLogP
TPSA
natoms
MW
nON
nOHNH
nrotb
Standard
3
6–31
<5
3.48
2.29
<140
41.37
85.61
<500
380.87
408.45
<10
2
6
<5
0
0
ꢂ10
48
54
4
6
amiLogP: molinspiration predicted Log P; TPSA: topological polar surface area;
natoms: No. of atoms; MW: molecular weight; nON: No. of hydrogen bond accept-
ors; nOHNH: No. of hydrogen bond donors; nrotb: No. of rotatable bonds.
6–31 as a promising microtubule destabilizer for further investiga-
tion in anticancer drug development.
4. Experimental
4.1. Chemistry
4.1.2. General synthetic procedures for arylpiperazines
4.1.1. Materials and methods
A solution of arylamine (1 mmol), bis(2- chloroethyl)amine hydro-
chloride (1.1 mmol) and K2CO3 (3 mmol) in n-BuOH were stirred at
All of reagents and solvents were purchased from chemical com-
pany. 1H NMR and 13 C NMR spectra were tested in CDCl3 with irradiated in a microwave reactor for 30 min at 150 ꢀC. The

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
555
reaction mixture was cooled to room temperature and dissolved 4.1.5.1. Ethyl 1-phenyl-1H-tetrazole-5-carboxylate (10–1). Light yel-
in methanol (4 ml), followed by the addition of diethyl ether low Solid; yield: 76%; 1H NMR (600 MHz, CDCl3): d ¼ 7.70 (3H, m),
(150 ml). The precipitate formed was recovered by filtration and 7.60 (2H, m), 4.55 (2H, q), 1.47 (3H, t, J ¼ 6.8 Hz). ESI-MS: m/
washed with diethyl ether to obtain arylpiperazine as HCl salt. The z ¼ 219.1 [M þ H]þ.
HCl salt was used for the next reaction without further
purification20,21
.
4.1.5.2. Ethyl 1-(2-fluorophenyl)-1H-tetrazole-5-carboxylate (10–2).
Yellow Solid; yield: 66%; 1H NMR (600 MHz, CDCl3): d ¼ 7.63 (1H,
m), 7.57 (1H, m), 7.40 (1H, t, J ¼ 7.2 Hz), 7.34 (1H, m), 4.46 (2H, q),
1.37 (3H, t, J ¼ 6.8 Hz). ESI-MS: m/z ¼ 237.1 [M þ H]þ.
4.1.3. General synthetic procedures for ethyl 2-oxo-2-(arylamino)a-
cetates (8)
To a solution of substituted aniline (10 mmol) and triethylamine
(1 ml, 10 mmol) in DCM was added ethyl chlorooxoacetate
(10 mmol) in DCM. The reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was poured into water
and extracted with DCM (50 ml ꢃ 3). The combined organic layer
was washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated to yield the crude product. The crude product was
purified by column chromatography (n-hexane/EtOAc ¼ 1:2) on
silica gel to afford pure products. For example:
4.1.5.3. Ethyl 1–(3-fluorophenyl)-1H-tetrazole-5-carboxylate (10–3).
Light yellow Solid; yield: 71%; 1H NMR (600 MHz, CDCl3): d ¼ 7.57
(1H, m), 7.34 (2H, m), 7.29 (1H, m), 4.47 (2H, q), 1.40 (3H, t,
J ¼ 8.1 Hz). ESI-MS: m/z ¼ 237.0 [M þ H]þ.
4.1.5.4. Ethyl 1-(4-fluorophenyl)-1H-tetrazole-5-carboxylate (10–4).
Light yellow Solid; yield: 77%; 1H NMR (600 MHz, CDCl3): d ¼ 7.51
(2H, m), 7.27 (2H, m), 4.56 (2H, q), 1.40 (3H, t, J ¼ 7.1 Hz). ESI-MS:
m/z ¼ 237.1 [M þ H]þ.
4.1.3.1. Ethyl 2-oxo-2-(phenylamino)acetate (8–1). White Solid;
yield: 91%; MS (ESI) m/z 194.1 [M þ H]þ.
4.1.5.5. Ethyl 1-(2-chlorophenyl)-1H-tetrazole-5-carboxylate (10–5).
Yellow Solid; yield: 70%; 1H NMR (600 MHz, CDCl3): d ¼ 7.61 (2H,
m), 7.51 (2H, m), 4.42 (2H, q), 1.34 (3H, t, J ¼ 7.1 Hz). ESI-MS: m/
z ¼ 253.0 [M þ H]þ.
4.1.3.2. Ethyl 2-((3-bromophenyl)amino)-2-oxoacetate (8–8). White
Solid; yield: 92%; MS (ESI) m/z 272.0 [M þ H]þ.
4.1.5.6. Ethyl 1-(3-chlorophenyl)-1H-tetrazole-5-carboxylate (10–6).
Yellow Solid; yield: 73%; 1H NMR (600 MHz, CDCl3): d ¼ 7.60 (1H,
m), 7.54 (2H, m), 7.42 (1H, m), 4.47 (2H, q), 1.40 (3H, t, J ¼ 7.5 Hz).
ESI-MS: m/z ¼ 253.0 [M þ H]þ.
4.1.4. General synthetic procedures for (E)-ethyl 2-chloro-2-(aryli-
mino)acetates (9)
A solution of triphenylphosphine (1.5 mmol) and ethyl 2-oxo-2-
(arylamino)acetate (1 mmol) in CCl4 (5 ml) were stirred at irradiated
in a microwave reactor for 20 min at 130 ꢀC. The reaction mixtures
were cooled to room temperature and the precipitate was filtered
off. The filtrate was concentrated to yield the crude product. The
crude product was purified by column chromatography (n-hex-
4.1.5.7. Ethyl 1-(2-bromophenyl)-1H-tetrazole-5-carboxylate (10–7).
1
Yellow Solid; yield: 76%; H NMR (600 MHz, CDCl3): d ¼ 7.64 (1H, d,
J ¼ 7.8 Hz), 7.35 (1H, t, J ¼ 7.4 Hz), 7.11 (1H, d, J ¼ 8.4 Hz), 6.92 (1H,
d, J ¼ 7.8 Hz), 4.48 (2H, q), 1.45 (3H, t, J ¼ 7.4 Hz). ESI-MS: m/
z ¼ 297.0 [M þ H]þ.
ane/EtOAc
For example:
¼
4:1) on silica gel to afford pure products.
4.1.5.8. Ethyl 1-(3-bromophenyl)-1H-tetrazole-5-carboxylate (10–8).
Light yellow Solid; yield: 62%; 1H NMR (600 MHz, CDCl3): d ¼ 7.76
(1H, m), 7.70 (1H, m), 7.46 (2H, m), 4.47 (2H, q), 1.40 (3H, t,
J ¼ 7.4 Hz). ESI-MS: m/z ¼ 297.0 [M þ H]þ.
4.1.4.1. (E)-Ethyl 2-((3-bromophenyl)imino)-2-chloroacetate (9–8).
Light yellow oil; yield: 67%; 1H NMR (600 MHz, CDCl3): d ¼ 7.65
(1H, s), 7.63 (1H, d, J ¼ 1.4 Hz), 7.22 (1H, t, J ¼ 2.9 Hz), 7.16 (1H, t,
J ¼ 2.0 Hz), 4.40 (2H, q, J ¼ 7.2 Hz), 1.38 (3H, t, J ¼ 7.1 Hz). ESI-MS:
m/z ¼ 290.5 [M þ H]þ.
4.1.5.9. Ethyl 1-(4-bromophenyl)-1H-tetrazole-5-carboxylate (10–9).
Light yellow Solid; yield: 69%; 1H NMR (600 MHz, CDCl3): d ¼ 7.72
(2H, d, J ¼ 8.5 Hz), 7.40 (2H, d, J ¼ 8.5 Hz), 4.46 (2H, q), 1.40 (3H, t,
J ¼ 7.81 Hz). ESI-MS: m/z ¼ 297.0 [M þ H]þ.
4.1.5. General synthetic procedures for ethyl 1-aryl-1H-tetrazole-5-
carboxylates (10)
4.1.5.10. Ethyl 1-(o-tolyl)-1H-tetrazole-5-carboxylate (10–10).
Yellow Solid; yield: 67%; 1H NMR (600 MHz, CDCl3): d ¼ 7.52 (1H,
m), 7.42 (1H, d, J ¼ 7.4 Hz), 7.39 (1H, t, J ¼ 7.7 Hz), 7.25 (1H, dd,
J ¼ 1.2 Hz, J ¼ 7.7 Hz), 4.40 (2H, q), 2.05 (1H, s), 1.32 (3H, t,
J ¼ 6.8 Hz). ESI-MS: m/z ¼ 233.1 [M þ H]þ.
Because of the instability of intermediates 9, we described a
microwave-assisted continuous operation process method rather
than stepwise operation process for the conversion of 8–10. A
solution of triphenylphosphine (1.5 mmol) and ethyl 2-oxo-2-(aryla-
mino)acetate 8 (1 mmol) in CCl4 (5 ml) were stirred at irradiated in
a microwave reactor for 20 min at 130 ꢀC. The reaction mixtures
were cooled to room temperature and the precipitate was filtered
off. The filtrate was evaporated and dissolved in CH3CN. Sodium
azide (1.2 mmol) was added at room temperature under N2 for
16 h. The solvent was then removed under reduced pressure and
extracted with ethyl acetate (50 ml ꢃ 3). The combined organic
layer was washed with water and brine and then dried over
Na2SO4, filtered and concentrated to yield the crude product. The
crude product was purified by column chromatography (n-hex-
4.1.5.11. Ethyl 1-(m-tolyl)-1H-tetrazole-5-carboxylate (10–11).
Yellow Solid; yield: 75%; 1H NMR (600 MHz, CDCl3): d ¼ 7.45 (1H, t,
J¼ 8.4 Hz), 7.41 (1H, d, J¼ 8.1 Hz), 7.28 (2H, t, J¼ 11.8 Hz), 4.45 (2H,
q), 2.46 (3H, s), 1.37 (3H, t, J¼ 7.3 Hz). ESI-MS: m/z ¼ 233.1 [M þ H]þ.
4.1.6. General synthetic procedures for 1-aryl-5–(4-arylpiperazine-
1-carbonyl)-1H-tetrazols (6)
ane/EtOAc
For example:
¼
3:1) on silica gel to afford pure products. To a solution of arylpiperazine (0.1 mmol) in anhydrous DCM was
added trimethylaluminum (0.5 ml, 1 M in heptane). The reaction

556
C. WANG ET AL.
was stirred at room temperature under N2 for 15 min. A solution (600 MHz, CDCl3): d ¼ 7.57 (5H, d, J ¼ 7.5 Hz), 7.20 (1H, t, J ¼ 8.4 Hz),
of an
appropriate ethyl 1-aryl-1H-tetrazole-5-carboxylate 6.50 (2H, m), 6.44 (1H, s), 3.92 (2H, t, J ¼ 4.8 Hz), 3.7 (3H, s), 3.69
(0.1 mmol) in anhydrous DCM was added and the reaction was (2H, t, J ¼ 5.1 Hz), 3.24 (2H, t, J ¼ 4.9 Hz), 3.14 (2H, t, J ¼ 4.8 Hz). 13 C
stirred at room temperature under N2 for 16 h. The reaction was NMR (150 MHz, CDCl3): d ¼ 159.6, 154.6, 150.8, 146.6, 132.7, 129.7,
quenched with 5 ml of 1 M HCl and diluted with DCM. The com- 129.0, 128.8 (2 C), 123.0 (2 C), 108.5, 104.6, 102.4, 54.2, 48.8, 48.3,
bined organic layer was washed with water and brine and 45.9, 41.5; HRMS calcd for C19H20N6NaO2 [M þ Na]þ 387.1545,
then dried over Na2SO4, filtered and concentrated to yield the found 387.1578.
crude product. The crude product was purified by column chro-
matography (n-hexane/EtOAc ¼ 1:1) on silica gel to afford
4.1.6.7. 1-Phenyl-5-(4-(4-methoxyphenyl)piperazine-1-carbonyl)-1H-
1
pure products.
tetrazol (6–7). White Solid; yield: 71%; Mp: 152.7–154.2 ꢀC; H NMR
(600 MHz, CDCl3): d ¼ 7.51 (5H, m), 6.79 (4H, t, J ¼ 13.5 Hz) , 3.86
(2H,s), 3.71 (3H, s), 3.62 (2H,s), 3.05 (2H, s), 2.95 (2H,s). 13 C NMR
4.1.6.1.
1-Phenyl-5–(4-phenylpiperazine-1-carbonyl)-1H-tetrazol
(6–1). White Solid; yield: 45%; Mp: 140.0–141.8 ꢀC; 1H NMR (150 MHz, CDCl3): d ¼ 154.6, 153.7, 146.7, 143.7, 132.7, 129.6, 128.8
(600 MHz, CDCl3): d ¼ 7.50 (5H, d, J ¼ 10.8 Hz), 7.22 (2H, t, (2 C), 122.9 (2 C), 118.2 (2 C), 113.6 (2 C), 54.5, 50.3, 49.9, 46.1, 41.6;
J ¼ 7.6 Hz), 6.85 (3H, m), 3.86 (2H, s), 3.63 (2H, s), 3.17 (2H, s), 3.07 HRMS
calcd
for
C19H20N6NaO2
[M þ Na]þ
387.1545,
(2H, s). 13 C NMR (150 MHz, CDCl3): d ¼ 154.6, 149.4, 146.6, 132.7, found 387.1574.
129.6, 128.8 (2 C), 128.3 (2 C), 122.9 (2 C), 120.1, 115.9 (2 C), 48.9,
48.4, 45.9, 41.5; HRMS calcd for C18H18N6NaO [M þ Na]þ 357.1440,
4.1.6.8. 1-Phenyl-5-(4-(3-trifluoromethyl)piperazine-1-carbonyl)-1H-
tetrazol (6–8). Light yellow Solid; yield: 60%; Mp: 77.7–79.2 ꢀC; 1H
NMR (600 MHz, CDCl3): d ¼ 7.59 (5H, m), 7.39 (1H, t, J ¼ 7.8 Hz),
7.16 (1H, d, J ¼ 7.8 Hz), 7.11 (1H, s), 7.06 (1H, d, J ¼ 8.8 Hz), 3.95
found 357.1477.
4.1.6.2.
1-Phenyl-5-(4-(3-fluorophenyl)piperazine-1-carbonyl)-1H-
tetrazol (6–2). Light yellow Solid; yield: 49%; Mp: 135.9–137.8 ꢀC; (2H, t, J ¼ 5.0 Hz), 3.76 (2H, t, J ¼ 5.0 Hz), 3.30 (2H, t, J ¼ 5.4 Hz),
1H NMR (600 MHz, CDCl3): d ¼ 7.50 (5H, d), 7.14 (1H, q, J ¼ 7.8 Hz), 3.22 (2H, t, J ¼ 5.4 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 154.6, 149.6,
6.58 (1H, q, J ¼ 8.5 Hz), 6.51 (2H, m,), 3.84 (2H, t, J ¼ 4.7 Hz), 3.64 146.5, 132.7, 129.7, 128.8, 128.8 (2 C), 128.7, 123.0 (2 C), 118.7,
(2H, t, J ¼ 4.4 Hz), 3.17 (2H, t, J ¼ 5.0 Hz), 3.08 (2H, t, J ¼ 4.7 Hz). 13 C 116.3 (m), 114.2, 112.1 (m), 48.4, 47.9, 45.8, 41.4; HRMS calcd for
NMR (150 MHz, CDCl3): d ¼ 162.7 (d, J ¼ 237.7 Hz), 154.6, 151.0 (d, C19H17F3N6NaO [M þ Na]þ 425.1314, found 425.1311.
J ¼ 9.2 Hz), 146.6, 132.7, 129.7, 129.4 (d, J ¼ 10.2 Hz), 128.8 (2 C),
123.0 (2 C), 110.9 (d, J ¼ 2.5 Hz), 106.2 (d, J ¼ 20.4 Hz), 102.6 (d,
4.1.6.9. 1-Phenyl-5-(4-(3,4-dimethylphenyl)piperazine-1-carbonyl)-
1H-tetrazol (6–9). Pink Solid; yield: 57%; Mp: 138.6–140.1 ꢀC; 1H
NMR (600 MHz, CDCl3): d ¼ 7.57 (5H, m), 7.04 (1H, d, J ¼ 8.1 Hz) ,
6.73 (1H, s), 6.66 (1H, d, J ¼ 8.4 Hz) , 3.29 (2H, t, J ¼ 4.7 Hz), 3.68
(2H, s), 3.18 (2H, t, J ¼ 5.4 Hz), 3.07 (2H, t, J ¼ 4.7 Hz), 2.23 (3H, s),
J ¼ 24.0 Hz), 48.3, 47.8, 45.7, 41.3; HRMS calcd for C18H17FN6NaO
[M þ Na]þ 375.1346, found 375.1378.
4.1.6.3. 1-Phenyl-5-(4-(2-chlorophenyl)piperazine-1-carbonyl)-1H-
tetrazol (6–3). Light yellow Solid; yield: 60%; Mp: 76.7–78.4 ꢀC; 1H 2.19 (2H, s). 13 C NMR (150 MHz, CDCl3): d ¼ 154.6, 147.6, 146.7,
NMR (600 MHz, CDCl3): d ¼ 7.59 (5H, m), 7.01 (4H, m), 3.96 (2H, t, 136.5, 129.7, 129.3, 128.8 (2 C), 128.7, 122.9 (2 C), 117.9, 114.3,
J ¼ 4.2 Hz), 3.68 (2H, t, J ¼ 4.7 Hz), 3.10 (2H, t, J ¼ 5.0 Hz), 3.02 (2H, 113.6, 49.3, 49.0, 46.1, 41.6, 19.1, 17.8; HRMS calcd for
t, J ¼ 4.9 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 154.8, 147.0, 146.7, C20H22N6NaO [M þ Na]þ 385.1753, found 385.1788.
129.7, 129.7, 128.8 (2 C), 128.6, 126.7, 123.7, 122.87 (2 C), 119.6,
114.3, 50.3, 49.8, 46.4, 41.9; HRMS calcd for C18H17ClN6NaO
4.1.6.10. 1-Phenyl-5-(4-(2,6-dimethylphenyl)piperazine-1-carbonyl)-
1H-tetrazol (6–10). Light yellow Solid; yield: 49%; Mp: 71.7–73.2 ꢀC;
1H NMR (600 MHz, CDCl3): d ¼ 7.55 (5H, m), 7.08 (2H, d, J ¼ 8.1 Hz),
6.83 (1H, t, J ¼ 8.1 Hz), 3.74 (2H, s), 3.60 (2H, t, J ¼ 4.0 Hz), 2.90 (2H,
[M þ Na]þ 391.1050, found 391.1064.
4.1.6.4. 1-Phenyl-5-(4-(3-chlorophenyl)piperazine-1-carbonyl)-1H-
tetrazol (6–4). Light yellow Solid; yield: 58%; Mp: 102.5–104.0 ꢀC; t, J ¼ 5.4 Hz), 2.85 (2H, t, J ¼ 4.7 Hz), 1.65 (6H, s). 13 C NMR
1H NMR (600 MHz, CDCl3): d ¼ 7.58 (5H, m), 7.19 (1H, t, J ¼ 8.1 Hz), (150 MHz, CDCl3): d ¼ 169.2, 159.7, 150.7, 131.0, 130.7, 129.3 (2 C),
6.87 (2H, t, J ¼ 8.5 Hz), 6.77 (1H, dd, J ¼ 2.0 Hz, J ¼ 8.1 Hz), 3.91 (2H, 126.9 (2 C), 124.4 (2 C), 119.9 (2 C), 115.3, 52.0, 51.6, 46.9, 42.0, 17.4
t, J ¼ 5.0 Hz), 3.71 (2H, t, J ¼ 5.0 Hz), 3.25 (2H, t, J ¼ 4.8 Hz), 3.16 (2 C); HRMS calcd for C20H22N6NaO [M þ Na]þ 385.1753,
(2H, t, J ¼ 5.4 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 154.6, 150.5, found 385.1788.
146.6, 134.1, 132.6, 129.7, 129.3, 128.8 (2 C), 123.0 (2 C), 119.7,
115.7, 113.7, 48.4, 47.8, 45.7, 41.3; HRMS calcd for C18H17ClN6NaO
4.1.6.11.
1-Phenyl-5-(4-(3,4-dimethoxyphenyl)piperazine-1-car-
[M þ Na]þ 391.1050, found 391.1073.
bonyl)-1H-tetrazol (6–11). Light yellow Solid; yield: 53%; Mp:
101.6–102.6 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.59 (5H, d,
J ¼ 10.8 Hz), 7.80 (1H, d, J ¼ 9.5 Hz), 6.55 (1H, s), 6.44 (1H, d,
4.1.6.5. 1-Phenyl-5-(4-(3-methylphenyl)piperazine-1-carbonyl)-1H-
1
tetrazol (6–5). White Solid; yield: 61%; Mp: 135.2–137.1 ꢀC; H NMR J ¼ 8.1 Hz), 3.94 (2H, t, J ¼ 6.1 Hz), 3.87 (3H, s), 3.84 (3H, s), 3.70
(600 MHz, CDCl3): d ¼ 7.49 (5H, s), 7.10 (1H, s), 6.66 (3H, s), 3.85 (2H, t, J ¼ 5.4 Hz), 3.14 (2H, t, J ¼ 5.1 Hz), 3.05 (2H, t, J ¼ 5.5 Hz). 13 C
(2H, s), 3.61 (2H, s), 3.15 (2H, s), 3.05 (2H, s), 2.25 (3H, s). 13 C NMR NMR (150 MHz, CDCl3): d ¼ 154.6, 148.5, 146.6, 144.2, 143.5, 132.7,
(150 MHz, CDCl3): d ¼ 154.6, 149.5, 146.7, 138.1, 132.7, 129.7, 128.8 129.7, 128.8 (2 C), 122.9 (2 C), 110.7, 107.9, 102.6, 55.2, 54.8, 50.4,
(2 C), 128.1, 122.9 (2 C), 120.9, 116.8, 113.0, 48.9, 48.5, 46.0, 41.5, 50.1, 46.2, 41.7; HRMS calcd for C20H22N6NaO3 [M þ Na]þ 417.1651,
20.7; HRMS calcd for C19H20N6NaO [M þ Na]þ 371.1596, found 417.1661.
found 371.1633.
4.1.6.12.
1-Phenyl-5-(4-(2,5-dimethoxyphenyl)piperazine-1-car-
4.1.6.6. 1-Phenyl-5-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-1H-
bonyl)-1H-tetrazol (6–12). Light yellow Solid; yield: 51%; Mp:
1
tetrazol (6–6). White Solid; yield: 67%; Mp: 144.3–146.2 ꢀC; H NMR 78.9–80.8 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.58 (5H, m), 6.79 (1H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
557
d, J ¼ 9.1 Hz), 6.53 (1H, dd, J ¼ 2.5 Hz, J ¼ 9.4 Hz), 6.45 (1H, d, (d, J ¼ 20.9 Hz), 111.0 (d, J ¼ 27.5 Hz),108.5, 104.7, 102.4, 54.2, 48.8,
48.3, 46.0, 41.6; HRMS calcd for C19H19FN6NaO2 [M þ Na]þ
J ¼ 3.2 Hz), 3.94 (2H, t, J ¼ 5.0 Hz), 3.82 (3H, s), 3.76 (3H, s), 3.66
(2H, t, J ¼ 4.7 Hz), 3.10 (2H, t, J ¼ 5.0 Hz), 2.99 (2H, t, J ¼ 5.0 Hz). 13 C
NMR (150 MHz, CDCl3): d ¼ 154.7, 153.0, 146.8, 145.4, 139.9, 132.7,
129.6, 128.8 (2 C), 122.8 (2 C), 110.9, 105.6, 105.2, 54.8, 54.6, 49.7,
49.1, 46.3, 41.7; HRMS calcd for C20H22N6NaO3 [M þ Na]þ 417.1651,
found 417.1693.
405.1451, found 405.1500.
4.1.6.18. 1-(3-Fluorophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–18). Light yellow Solid; yield: 52%; Mp:
48.7–50.1 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.54 (1H, m), 7.40 (2H,
m), 7.28 (1H, m), 6.08 (3H, s), 3.94 (2H, t, J ¼ 5.3 Hz), 3.78 (8H, s),
3.26 (2H, t, J ¼ 5.3 Hz), 3.20 (2H, t, J ¼ 5.3 Hz). 13 C NMR (150 MHz,
CDCl3): d ¼ 162.6 (d, J ¼ 255.9 Hz), 161.6 (2 C), 155.3, 152.3, 147.6,
134.8 (d, J ¼ 10.2 Hz), 131.2 (d, J ¼ 9.6 Hz), 119.8 (d, J ¼ 3.0 Hz),
117.9 (d, J ¼ 20.8 Hz), 112.1 (d, J ¼ 26.5 Hz), 95.9 (2 C), 92.8, 55.3
(2 C), 49.9, 49.4, 46.9, 42.5; HRMS calcd for C20H22FN6O3 [M þ H]þ
413.1737, found 413.1759.
4.1.6.13.
1-Phenyl-5-(4-(3,5-dimethoxyphenyl)piperazine-1-car-
bonyl)-1H-tetrazol (6–13). Light yellow Solid; yield: 61%; Mp:
121.3–123.2 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.57 (5H, m), 6.07
(3H, m), 3.91 (2H, t, J ¼ 4.9 Hz), 3.77 (6H, s), 3.69 (2H, t, J ¼ 4.7 Hz),
3.23 (2H, t, J ¼ 4.9 Hz), 3.13 (2H, t, J ¼ 5.0 Hz). 13 C NMR (150 MHz,
CDCl3): d ¼ 160.5 (2 C), 154.6, 151.3, 146.6, 132.6, 129.7, 128.8 (2 C),
122.9 (2 C), 94.8 (2 C), 91.6, 54.3 (2 C), 48.8, 48.3, 45.8, 41.4; HRMS
calcd for C20H23N6O3 [M þ H]þ 395.1832, found 395.1859.
4.1.6.19. 1-(4-Fluorophenyl)-5-(4-(3-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–19). White Solid; yield: 81%; Mp:
117.4–118.7 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.59 (2H, m), 7.25
(2H, m), 7.19 (1H, t, J ¼ 8.4 Hz), 6.52 (1H, dd, J ¼ 2.3 Hz, J ¼ 8.4 Hz),
6.48 (1H, dd, J ¼ 2.0 Hz, J ¼ 8.4 Hz), 6.45 (1H, s), 3.91 (2H, t,
J ¼ 5.4 Hz), 3.78 (5H, m), 3.25 (2H, t, J ¼ 5.4 Hz), 3.20 (3H, t,
J ¼ 5.0 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 163.5 (d, J ¼ 245.7 Hz),
160.7, 155.3, 151.8, 147.7, 130.1, 129.8 (d, J ¼ 2.5 Hz), 126.5 (d,
J ¼ 8.1 Hz) (2 C), 116.9 (d, J ¼ 23.5 Hz) (2 C), 109.5, 105.7, 103.4,
55.3, 49.9, 49.3, 47.1, 42.6; HRMS calcd for C19H19FN6NaO2
[M þ Na]þ 405.1451, found 405.1482.
4.1.6.14. 1-(2-Fluorophenyl)-5-(4-(3-methylphenyl)piperazine-1-car-
bonyl)-1H-tetrazol (6–14). Light yellow Solid; yield: 69%; Mp:
102.5–104.0 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.70 (1H, m), 7.56
(1H, m), 7.39 (1H, t, J ¼ 8.1 Hz), 7.28 (1H, t, J ¼ 9.5 Hz), 7.19 (1H, t,
J ¼ 8.1 Hz), 6.77 (3H, t, J ¼ 6.7 Hz), 4.03 (2H, t, J ¼ 4.7 Hz), 3.92 (2H,
t, J ¼ 4.7 Hz), 3.27 (4H, t, J ¼ 5.1 Hz), 2.34 (3H, s). 13 C NMR
(150 MHz, CDCl3): d ¼ 153.9, 153.9 (d, J ¼ 245.7 Hz), 149.6, 147.6,
131.3 (d, J ¼ 8.1 Hz), 128.1, 125.9, 124.4 (d, J ¼ 3.5 Hz), 123.1 (d,
J ¼ 7.1 Hz) 120.8, 116.7, 115.7 (d, J ¼ 19.4 Hz), 114.7 (d, J ¼ 18.4 Hz),
112.9, 48.9, 48.5, 46.2, 41.5, 20.7; HRMS calcd for C19H19FN6NaO
[M þ Na]þ 389.1502, found 389.1531.
4.1.6.20. 1-(4-Fluorophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–20). Brown Solid; yield: 45%; Mp:
48.1–49.4 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.59 (2H, m), 7.25 (2H,
m), 6.08 (3H, s), 3.91 (2H, t, J ¼ 4.7 Hz), 3.78 (8H, s), 3.25 (2H, t,
J ¼ 5.0 Hz), 3.20 (2H, t, J ¼ 4.4 Hz). 13 C NMR (150 MHz, CDCl3):
d ¼ 162.5 (d, J ¼ 247.7 Hz), 160.6 (2 C), 154.3, 151.3, 146.7, 128.8 (d,
J ¼ 4.6 Hz), 125.4 (d, J ¼ 9.2 Hz) (2 C), 115.5 (d, J ¼ 23.5 Hz) (2 C),
94.8 (2 C), 91.7, 54.3 (2 C), 48.9, 48.4, 45.9, 41.5; HRMS calcd for
C20H22FN6O3 [M þ H]þ 413.1737, found 413.1774.
4.1.6.15. 1-(2-Fluorophenyl)-5-(4-(3-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–15). Light yellow Solid; yield: 52%; Mp:
105.9–107.8 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.70 (1H, m), 7.56
(1H, m), 7.38 (1H, t, J ¼ 7.6 Hz), 7.28 (1H, t, J ¼ 9.8 Hz), 7.21 (1H, t,
J ¼ 8.7 Hz), 6.56 (1H, m), 6.49 (2H, d, J ¼ 6.9 Hz), 4.02 (2H, t,
J ¼ 5.0 Hz), 3.91 (2H, t, J ¼ 4.7 Hz), 3.80 (3H, s), 3.28 (4H, t,
J ¼ 5.0 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 159.6, 153.9, 153.9 (d,
J ¼ 251.7 Hz), 150.9, 147.6, 131.4 (d, J ¼ 8.6 Hz), 129.0, 125.9, 124.4
(d, J ¼ 3.8 Hz), 121.3 (d, J ¼ 12.5 Hz), 115.7 (d, J ¼ 18.1 Hz), 108.5,
104.5, 102.3, 54.2, 48.7, 48.2, 46.1, 41.4; HRMS calcd for
C19H19FN6NaO2 [M þ Na]þ 405.1451, found 405.1493.
4.1.6.21. 1-(2-Chlorophenyl)-5-(4-(3-methylphenyl)piperazine-1-car-
bonyl)-1H-tetrazol (6–21). Light yellow Solid; yield: 72%; Mp:
107.0–109.3 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.56 (4H, m), 7.19
(1H, t, J ¼ 7.4 Hz), 6.76 (3H, m), 4.09 (2H, t, J ¼ 4.75 Hz), 3.87 (2H, t,
J ¼ 5.4 Hz), 3.27 (2H, t, J ¼ 5.4 Hz), 3.24 (2H, t, J ¼ 5.4 Hz), 2.33 (3H,
s). 13 C NMR (150 MHz, CDCl3): d ¼ 153.6, 149.6, 148.0, 138.1, 131.1,
129.4, 129.4, 128.8, 128.1, 127.5, 127.1, 120.8, 116.7, 112.9, 48.9,
48.4, 46.2, 41.5, 20.7; HRMS calcd for C19H19ClN6NaO [M þ Na]þ
405.1207, found 405.1246.
4.1.6.16. 1-(2-Fluorophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–16). Yellow Solid; yield: 69%; Mp:
52.1–53.2 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.70 (1H, m), 7.56 (1H,
m), 7.39 (1H, t, J ¼ 7.8 Hz), 7.29 (1H, m), 6.11 (2H, d, J ¼ 2.0 Hz),
6.08 (1H, t, J ¼ 2.0 Hz), 4.02 (2H, t, J ¼ 5.2 Hz), 3.90 (2H, t,
J ¼ 4.6 Hz), 3.79 (6H, s), 3.27 (4H, t, J ¼ 5.2 Hz). 13 C NMR (150 MHz,
CDCl3): d ¼ 160.6 (2 C), 153.9, 153.9 (d, J ¼ 253.8 Hz), 151.5, 147.6,
131.4 (d, J ¼ 7.6 Hz), 126.0, 124.4 (d, J ¼ 3.5 Hz), 121.3 (d,
J ¼ 12.2 Hz), 115.6 (d, J ¼ 19.3 Hz), 94.8 (2 C), 91.5, 54.3 (2 C), 48.7,
48.2, 42.0, 41.4; HRMS calcd for C20H22FN6O3 [M þ H]þ 413.1737,
found 413.1768.
4.1.6.22. 1-(2-Chlorophenyl)-5-(4-(3-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–22). Light yellow Solid; yield: 74%; Mp:
1
99.8–101.5 ꢀC; H NMR (600 MHz, CDCl3): d ¼ 7.56 (4H, m), 7.21 (1H,
t, J ¼ 9.1 Hz), 6.55 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 6.48 (2H, m), 4.09
(2H, t, J ¼ 5.6 Hz), 3.87 (2H, t, J ¼ 4.7 Hz), 3.80 (3H, s), 3.29 (2H, t,
J ¼ 5.2 Hz), 3.25 (2H, t, J ¼ 4.7 Hz). 13 C NMR (150 MHz, CDCl3):
d ¼ 159.6, 153.6, 150.9, 148.0, 131.1, 131.0, 129.4, 129.0, 128.8,
127.5, 127.1, 108.4, 104.5, 102.3, 54.2, 48.8, 48.2, 46.1, 41.4; HRMS
calcd for C19H19ClN6NaO2 [M þ Na]þ 421.1156, found 421.1199.
4.1.6.17. 1-(3-Fluorophenyl)-5-(4-(3-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–17). Light yellow Solid; yield: 64%; Mp:
124.3–126.0 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.54 (1H, m), 7.40
(2H, t, J ¼ 8.1 Hz), 7.28 (1H, t, J ¼ 8.8 Hz), 7.20 (1H, t, J ¼ 9.4 Hz),
6.53 (1H, d, J ¼ 8.3 Hz), 6.49 (1H, d, J ¼ 7.6 Hz), 6.45 (1H, s), 3.94
(2H, t, J ¼ 4.3 Hz), 3.79 (3H, s), 3.76 (2H, t, J ¼ 5.0 Hz), 3.27 (2H, t,
J ¼ 5.8 Hz), 3.20 (2H, t, J ¼ 4.7 Hz). 13 C NMR (150 MHz, CDCl3):
d ¼ 161.7 (d, J ¼ 245.2 Hz), 159.6, 154.2, 150.7, 146.5, 133.7 (d,
4.1.6.23. 1-(2-Chlorophenyl)-5-(4-(4-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–23). Yellow Solid; yield: 68%; Mp:
119.9–121.2 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.56 (4H, m), 6.92
J ¼ 9.2 Hz), 130.2 (d, J ¼ 8.6 Hz), 129.1, 118.7 (d, J ¼ 3.0 Hz), 116.8 (2H, d, J ¼ 8.8 Hz), 6.86 (2H, d, J ¼ 9.5 Hz), 4.08 (2H, t, J ¼ 4.7 Hz),

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3.87 (2H, t, J ¼ 4.7 Hz), 3.78 (3H, s), 3.16 (2H, t, J ¼ 5.09 Hz), 3.12 127.4–129.2 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.78 (1H, t,
(2H, t, J ¼ 5.0 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 153.6, 148.0 (2 C),
143.8, 131.1, 131.0, 129.4, 128.8, 127.5, 127.1, 118.1 (2 C), 113.5
(2 C), 54.5, 50.3, 49.7, 46.3, 41.6; HRMS calcd for C19H19ClN6NaO2
[M þ Na]þ 421.1156, found 421.1197.
J ¼ 1.8 Hz), 7.70 (1H, d, J ¼ 8.3 Hz), 7.54 (1H, d, J ¼ 7.9 Hz), 7.44 (1H,
t, J ¼ 8.3 Hz), 6.09 (3H, s), 3.95 (2H, s), 3.78 (8H, s), 3.27 (2H, t,
J ¼ 5.4 Hz), 3.21 (2H, t, J ¼ 5.4 Hz). 13 C NMR (150 MHz, CDCl3):
d ¼ 161.6 (2 C), 155.1, 152.2, 147.6, 134.7, 133.8, 131.0, 129.7, 127.3,
122.8, 95.9 (2 C), 92.8, 55.3 (2 C), 50.0, 49.5, 47.0, 42.6; HRMS calcd
for C20H21BrN6NaO3 [M þ Na]þ 495.0756, found 495.0784.
4.1.6.24. 1-(2-Chlorophenyl)-5-(4-(3,4-dimethylphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–24). Light yellow Solid; yield: 73%; Mp:
110.9–112.0 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.55 (4H, m), 7.05
(1H, d, J ¼ 8.8 Hz), 6.76 (1H, d, J ¼ 2.7 Hz), 6.70 (1H, dd, J ¼ 2.7 Hz,
J ¼ 8.4 Hz), 4.07 (2H, t, J ¼ 5.0 Hz), 3.87 (2H, t, J ¼ 4.7 Hz), 3.22 (2H,
t, J ¼ 5.4 Hz), 3.19 (2H, t, J ¼ 5.0 Hz), 2.24 (3H, s), 2.19 (3H, s). 13 C
NMR (150 MHz, CDCl3): d ¼ 153.6, 148.0, 147.8, 136.4, 131.1, 131.0,
129.4, 129.3, 128.8, 128.7, 127.5, 127.1, 117.8, 113.5, 49.4, 48.8,
46.2, 41.6, 19.2, 17.8; HRMS calcd for C20H21ClN6NaO [M þ Na]þ
419.1363, found 421.406.
4.1.6.30. 1-(4-Bromophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–30). Brown Solid; yield: 72%; Mp:
122.4–123.2 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.62 (2H, d,
J ¼ 8.6 Hz), 7.40 (2H, d, J ¼ 8.6 Hz), 6.01 (3H, s), 3.85 (2H, t,
J ¼ 5.7 Hz), 3.71 (8H, s), 3.19 (2H, t, J ¼ 5.3 Hz), 3.14 (2H, t,
J ¼ 5.2 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 160.5 (2 C), 154.6, 152.1,
146.5, 131.9 (2 C), 131.6 (2 C), 124.7, 116.0, 94.9 (2 C), 91.2, 54.3
(2 C), 48.9, 48.4, 46.0, 41.6; HRMS calcd for C20H21BrN6NaO3
[M þ Na]þ 495.0756, found 495.0804.
4.1.6.25. 1-(2-Chlorophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–25). Light yellow Solid; yield: 80%; Mp:
77.5–79.4 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.54 (4H, m), 6.09 (2H,
s), 6.08 (1H, s), 4.08 (2H, t, J ¼ 4.9 Hz), 3.86 (2H, t, J ¼ 4.6 Hz), 3.78
(6H, s), 3.27 (2H, t, J ¼ 4.6 Hz), 3.24 (2H, t, J ¼ 5.5 Hz). 13 C NMR
(150 MHz, CDCl3): d ¼ 161.6 (2 C), 154.7, 152.5, 149.0, 132.2, 132.1,
130.4, 129.8, 128.5, 128.2, 95.8 (2 C), 92.6, 55.3 (2 C), 49.8, 49.2,
47.1, 42.5; HRMS calcd for C20H21ClN6NaO3 [M þ Na]þ 451.1261,
found 451.1268.
4.1.6.31. 1-(2-Methylphenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–31). Yellow Solid; yield: 85%; Mp:
74.1–76.0 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.48 (1H, t, J ¼ 7.6 Hz),
7.40 (1H, d, J ¼ 7.8 Hz), 7.36 (1H, t, J ¼ 7.4 Hz), 7.26 (1H, s), 6.08
(3H, s), 3.95 (2H, t, J ¼ 5.7 Hz), 3.83 (2H, t, J ¼ 5.0 Hz), 3.77 (6H, s),
3.23 (2H, t, J ¼ 5.2 Hz), 3.20 (2H, t, J ¼ 5.2 Hz), 2.11 (3H, s). 13 C NMR
(150 MHz, CDCl3): d ¼ 160.5 (2 C), 153.7, 151.4, 147.7, 133.8, 132.1,
130.4, 130.2, 125.9, 125.5, 94.8 (2 C), 91.6, 54.3 (2 C), 48.9, 48.3,
45.9, 41.4, 16.5; HRMS calcd for C21H24N6NaO3 [M þ Na]þ 431.1808,
found 431.1849.
4.1.6.26. 1-(3-Chlorophenyl)-5-(4-(3-methoxyphenyl)piperazine-1-
carbonyl)-1H-tetrazol (6–26). White Solid; yield: 81%; Mp:
150.8–152.4 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.63 (1H, s), 7.54
(1H, m), 7.50 (2H, d, J ¼ 4.7 Hz), 7.20 (1H, t, J ¼ 8.9 Hz), 6.53 (1H, d,
J ¼ 8.4 Hz), 6.49 (1H, d, J ¼ 9.5 Hz), 6.45 (1H, s), 3.93 (2H, t,
J ¼ 5.4 Hz), 3.79 (5H, s), 3.27 (2H, t, J ¼ 5.0 Hz), 3.21(2H, t,
J ¼ 5.0 Hz). 13 C NMR (150 MHz, CDCl3): d ¼ 160.7, 155.2, 151,8,
147.6, 135.6, 134.6, 130.9, 130.8, 130.1, 124.5, 122.3, 109.6, 105.7,
103.5, 55.3, 49.9, 49.4, 47.1, 42.6; HRMS calcd for C19H19ClN6NaO2
[M þ Na]þ 421.1156, found 421.1188.
4.1.6.32. 1-(3-Methylphenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–32). Brown Solid; yield: 69%; Mp:
76.6–78.3 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.43 (2H, m), 7.34 (2H,
m), 6.09 (3H, s), 3.93 (2H, s), 3.78 (6H, s), 3.70 (2H, s), 3.24 (2H, t,
J ¼ 5.4 Hz), 3.15 (2H, s), 2.45 (3H, s). 13 C NMR (150 MHz, CDCl3):
d ¼ 160.6 (2 C), 154.7, 151.2, 146.6, 139.2, 132.6, 130.5, 128.5, 123.5,
119.9, 95.0 (2 C), 91.9, 54.3 (2 C), 49.0, 48.5, 45.7, 41.3, 20.4; HRMS
calcd for C21H24N6NaO3 [M þ Na]þ 431.1808, found 431.1854.
4.1.6.27. 1-(3-Chlorophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–27). Light yellow Solid; yield: 70%; Mp:
132.4–134.1 ꢀC; 1H NMR (600 MHz, CDCl3): d ¼ 7.63 (1H, s), 7.55
(1H, m), 7.50 (2H, m), 6.08 (3H, s), 3.93 (2H, t, J ¼ 5.2 Hz), 3.78 (8H,
s), 3.26 (2H, t, J ¼ 4.9 Hz), 3.20 (2H, t, J ¼ 5.2 Hz). 13 C NMR
(150 MHz, CDCl3): d ¼ 160.5 (2 C), 154.1, 151.3, 146.6, 134.5, 133.6,
129.9, 129.7, 123.5, 121.3, 94.8 (2 C), 91.7, 54.3 (2 C), 48.9, 48.4,
45.9, 41.5; HRMS calcd for C20H22ClN6O3 [M þ H]þ 429.1442,
found 429.1449.
4.2. Biological evaluation
4.2.1. Cell culture
The human gastric adenocarcinoma SGC-7901 cells, lung adeno-
carcinoma A549 cells and cervical carcinoma HeLa cells were cul-
tured in RPMI-1640 medium containing 10% FBS, 100 U/mL
streptomycin and 100 U/mL penicillin at 37 ꢀC in a humidified
atmosphere containing 5% CO2. All cell lines were purchased from
the American Type Culture Collection (ATCC, Manassas, VA).
4.1.6.28. 1-(2-Bromophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–28). Yellow Solid; yield: 80%; Mp:
1
71.1–72.4 ꢀC; H NMR (600 MHz, CDCl3): d ¼ 7.74 (1H, dd, J ¼ 7.9 Hz,
J ¼ 1.0 Hz), 7.56 (2H, m), 7.47 (1H, m), 6.09 (2H, s), 6.08 (1H, d,
J ¼ 2.0 Hz), 4.10 (2H, t, J ¼ 5.0 Hz), 3.86 (2H, t, J ¼ 5.4 Hz), 3.78 (6H,
s), 3.29 (2H, t, J ¼ 5.0 Hz), 3.24 (2H, t, J ¼ 5.4 Hz). 13 C NMR
(150 MHz, CDCl3): d ¼ 160.5 (2 C), 153.5, 151.5, 147.9, 132.6, 132.5,
131.4, 127.8, 127.7, 118.7, 94.7 (2 C), 91.5, 54.3 (2 C), 48.8, 48.1,
46.1, 41.4; HRMS calcd for C20H22BrN6O3 [M þ H]þ 473.0937,
found 473.0954.
4.2.2. In vitro antiproliferative activity
The antiproliferative activity assay in vitro was carried out referring
to the previously reported method22.
4.2.3. Effect on tubulin polymerisation
The tubulin polymerisation assay was performed by using the
commercial tubulin polymerisation assay kit (Cytoskeleton-
4.1.6.29. 1-(3-Bromophenyl)-5-(4-(3,5-dimethoxyphenyl)piperazine-
1-carbonyl)-1H-tetrazol (6–29). White Solid; yield: 52%; Mp: Cat.#BK011P) referred to the protocol of manufacturer23.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
559
4.2.4. Analysis of immunofluorescence staining
vascular-disrupting agents for cancer therapy. J Med Chem
2013;56:9008–18.
Immunofluorescence staining studies were investigated using the
reported method24,25
.
7. Wu Y, Feng D, Gao M, et al. Design and synthesis of 5-aryl-
4-(4-arylpiperazine-1-carbonyl)-2H-1,2,3-triazole derivatives as
colchicine binding site inhibitors. Sci Rep 2017;7:1–9.
8. Romagnoli R, Baraldi PG, Salvador MK, et al. Synthesis and
evaluation of 1,5-disubstituted tetrazoles as rigid analogues
of combretastatin A-4 with potent antiproliferative and anti-
tumor activity. J Med Chem 2012;55:475–88.
4.2.5. Cell cycle analysis
Cell cycle analysis assay was followed the procedure of rele-
vant report26.
9. Ibrahim YA, Al-Awadi NA, Al-Azemi TF, et al. Improved
microwave synthesis of unsymmetrical N,N’-diaryl-1,2-amino-
ethane and imidazolidinium salts as precursors of N-hetero-
cyclic carbenes. RSC Adv 2014;4:38869–76.
10. Pape F, Teichert JF. Tethered NHC ligands for stereoselective
alkyne semihydrogenations. Synthesis 2017;49:2470–2482.
11. Fevig JM, Pinto DJ, Han Q, et al. Synthesis and SAR of ben-
zamidine factor Xa inhibitors containing a vicinally-substi-
tuted heterocyclic core. Bioorg Med Chem Lett 2001;11:
641–5.
4.3. Molecular modelling
Molecular modelling studies. Molecular docking was carried out
by CDOCKER programme of Discovery Studio 3.0 software (PDB:
3HKC). The 3 D structure of 3HKC in docking study was down-
loaded from Protein Data Bank. The docking poses were selected
according to previous studies7.
Disclosure statement
No potential conflict of interest was reported by the author(s).
12. Jia ZJ, Wu Y, Huang W, et al. 1-(2-Naphthyl)-1H-pyrazole-5-
carboxylamides as potent factor Xa inhibitors. Part 3: design,
synthesis and SAR of orally bioavailable benzamidine-P4
inhibitors. Bioorg Med Chem Lett 2004;14:1229–34.
13. Zhang X, Cai C, Winters M, et al. Design, synthesis and
SAR of a novel series of heterocyclic phenylpropanoic
acids as GPR120 agonists. Bioorg Med Chem Lett 2017;27:
3272–8.
Funding
We gratefully acknowledge the National Natural Science
Foundation of China [81673293 and 81602969], Project funded by
China Postdoctoral Science Foundation [2018M641715], Liaoning
Revitalisation Talents Programme [XLYC1802072] and Plan for
Development of Young Scholars of Shenyang pharmaceutical uni-
versity [ZQN2018002] for the generous financial support. This
work was also supported by the Programme for Innovative
Research Team of the Ministry of Education.
14. Quan ML, Ellis CD, He MY, et al. Nonbenzamidine tetrazole
derivatives as factor Xa inhibitors. Bioorg Med Chem Lett
2003;13:369–73.
15. Chandrasekaran S, Ramanathan S, Basak T. Microwave
material processing-a review. AIChE 2012;58:330–63.
16. Guan Q, Feng D, Bai Z, et al. Microwave-assisted synthesis,
molecular docking and antiproliferative activity of (3/5-aryl-
1,2,4-oxadiazole-5/3-yl)(3,4,5-trimethoxyphenyl)methanone
oxime derivatives. MedChemComm 2015;6:1484–93.
17. Guan Q, Zuo D, Jiang N, et al. Microwave-assisted synthesis
and biological evaluation of 3,4-diaryl maleic anhydride/N-
substituted maleimide derivatives as combretastatin A-4
analogues. Bioorg Med Chem Lett 2015;25:631–4.
ORCID
Chao Wang
Qi Guan
http://orcid.org/0000-0002-1551-3592
http://orcid.org/0000-0002-5003-8723
References
18. Wen
Z,
Xu
J,
Wang
Z,
et
al.
3-(3,4,5-
1. Wang Q, Arnst KE, Wang Y, et al. Structure-guided design,
synthesis, and biological evaluation of (2-(1H-indol-3-yl)-1H-
imidazol-4-yl)(3,4,5-trimethoxyphenyl) methanone (ABI-231)
analogues targeting the colchicine binding site in tubulin. J
Med Chem 2019;62:6734–50.
2. Jiang L, Goto M, Zhu DQ, et al. Scaffold Hopping-driven
optimization of 4-(quinazolin-4-yl)-3,4-dihydroquinoxalin-
2(1H)-ones as novel tubulin inhibitors. ACS Med Chem Lett
2020;11:83–9.
3. Niu H, Strecker TE, Gerberich JL, et al. Design, structure
guided design, synthesis, and biological evaluation of novel
benzosuberene analogues as inhibitors of tubulin polymer-
ization. J Med Chem 2019;62:5594–615.
4. Xu Q, Bao K, Sun M, et al. Design, synthesis and structure
activity relationship of 3,6-diaryl-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazines as novel tubulin inhibitors. Sci Rep
2017;7:1–12.
Trimethoxyphenylselenyl)-1H-indoles and their selenoxides
as combretastatin A-4 analogs: microwave-assisted synthesis
and biological evaluation. Eur J Med Chem 2015;90:184–94.
19. Hu H, Dong Y, Li M, et al. Design, synthesis and biological
evaluation of novel thieno[3,2-d]pyrimidine and quinazoline
derivatives as potent antitumor agents. Bioorg Chem 2019;
90:1–11.
20. Juvale K, Wiese M. 4-substituted-2-phenylquinazolines as
inhibitors of BCRP. Bioorg Med Chem Lett 2012;22:
6766–9.
21. Paudel S, Acharya S, Yoon G, et al. Exploration of substituted
arylpiperazine-tetrazoles as promising dual norepinephrine
and dopamine reuptake inhibitors. Bioorg Med Chem 2016;
24:5546–55.
22. Feng D, Wu Y, Wang H, et al. Synthesis and antiproliferative
activity of 2-aryl-4-(3,4,5-trimethoxybenzoyl)-1,2,3-triazol deriva-
tives as microtubule-destabilizing agents. RSC Advances 2017;7:
29103–11.
5. Wasylyk C, Zheng H, Castell C, et al. Inhibition of the Ras-
Net (Elk-3) pathway by a novel pyrazole that affects microtu-
bules. Cancer Res 2008;68:1275–83.
6. Choi MJ, No ES, Thorat DA, et al. Synthesis and biological
evaluation of aryloxazole derivatives as antimitotic and
23. Sun M, Xu Q, Xu J, et al. Synthesis and bioevaluation of N,4-
diaryl-1,3-thiazole-2-amines as tubulin inhibitors with potent
antiproliferative activity. PLoS One 2017;12:1–15.

560
C. WANG ET AL.
24. Wang Z, Qi H, Shen Q, et al. 4,5-Diaryl-3H-1,2-dithiole-3-thio- 25. Wang Z, Yang Q, Bai Z, et al. Synthesis and biological evalu-
nes and related compounds as combretastatin A-4/oltipraz
hybrids: Synthesis, molecular modelling and evaluation as
ation of 2,3-diarylthiophene analogues of combretastatin A-
4. Med Chem Commun 2015;6:971–6.
antiproliferative agents and inhibitors of tubulin. Eur J Med 26. Xu Q, Sun M, Bai Z, et al. Design, synthesis and bioevalua-
Chem 2016;122:520–9.
tion of antitubulin agents carrying diaryl-5,5-fused-hetero-
cycle scaffold. Eur J Med Chem 2017;139:242–9.