Design, synthesis and biological evaluation of 3,4-diaryl-1,2,5-oxadiazole-2/5-oxides as highly potent inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.ejmech.2019.05.036

Structure-activity relationships for rigid analogues of combretastatin A-4 (CA-4) were investigated, leading to the discovery of a series of 3,4-diaryl-1,2,5-oxadiazole-N-oxides. Among them, 7n′ and 7n′′ showed remarkable antiproliferative activities agai

Full text of DOI:10.1016/j.ejmech.2019.05.036

Accepted Manuscript
Design, synthesis and biological evaluation of 3,4-diaryl-1,2,5-Oxadiazole-2/5-Oxides
as highly potent inhibitors of tubulin polymerization
Yilang Hong, Yinglin Zhao, Lei Yang, Minghuan Gao, Long Li, Shuai Man, Zhan
Wang, Qi Guan, Kai Bao, Daiying Zuo, Yingliang Wu, Weige Zhang
PII:
S0223-5234(19)30446-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.036
EJMECH 11346
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2019
Revised Date: 1 May 2019
Accepted Date: 12 May 2019
Please cite this article as: Y. Hong, Y. Zhao, L. Yang, M. Gao, L. Li, S. Man, Z. Wang, Q. Guan, K. Bao,
D. Zuo, Y. Wu, W. Zhang, Design, synthesis and biological evaluation of 3,4-diaryl-1,2,5-Oxadiazole-2/5-
Oxides as highly potent inhibitors of tubulin polymerization, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.036.
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Graphical abstract

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Synthesis and Biological
Design,
Evaluation
of
3,4-Diaryl-1,2,5-Oxadiazole-2/5-Oxides as Highly Potent Inhibitors of
Tubulin Polymerization
Yilang Honga, Yinglin Zhaoa, Lei Yangb, Minghuan Gaoc, Long Lia, Shuai Manc, Zhan
Wangd, Qi Guanb*, Kai Baoa, Daiying Zuoc, Yingliang Wuc*, Weige Zhanga*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
China
b
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua
Road, Shenhe District, Shenyang 110016, China
c Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, China
d
Pharmaceutical Department, The First Affiliated Hospital of Zhengzhou University, 1
Jianshedong Road, Erqi District, Zhengzhou 450052, China
Corresponding authors:
Tel: +86 24 43520220 (Q. Guan); +86 24 43520220 (W. Zhang); +86 24 23986278 (Y. Wu).
E-mail addresses: guanqi@syphu.edu.cn (Q. Guan), zhangweige2000@sina.com (W. Zhang),
yingliang_1016@163.com (Y. Wu).
Abstract
Structure-activity relationships for rigid analogues of combretastatin A-4 (CA-4) were
investigated, leading to the discovery of a series of 3,4-diaryl-1,2,5-oxadiazole-N-oxides. Among
them, 7n′ and 7n′′ showed remarkable antiproliferative activities against three cancer cell lines in
nanomolar concentrations. Interestingly, 7n′ inhibited tubulin polymerization much more
efficiently than CA-4. Cellular mechanism investigation elucidated 7n′ disrupted the cellular
microtubule structure, arrested cell cycle at G2/M phase and induces apoptosis. Molecular
modeling study revealed 1,2,5-oxadiazole-N-oxide ring could increase a hydrogen bond
interaction with the binding site. These results provide impetus and further guidance for the
development of new CA-4 analogues.
Keywords:
Synthesis,
anti-proliferative
activity,
tubulin,
combretastatin
A-4,
1,2,5-Oxadiazole-N-Oxides, molecular modelling.

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1. Introduction
Microtubules (MTs) are mainly composed of α,β-tubulin heterodimers and play important
roles in a series of cellular processes such as regulation of motility, cell signaling, formation and
maintenance of cell shape, secretion, and intracellular transport [1-5]. Because of the important
roles of MTs for the life cycle of the cell, the discovery and development of small molecules
which target on tubulin to hamper the dynamic of tubulin polymerization has been an established
strategy for anticancer therapy.
Combretastatin A-4 (CA-4, 1, Fig. 1), isolated from the bark of the South African tree
Combretum caffrum, binds to the colchicine binding site of tubulin to block microtubule assembly
causing cell death in the tumor [6,7]. CA-4 is a fascinating lead molecule in the design of
colchicine binding site inhibitors, due to its strong potency and relatively simple chemical
structure for optimization. Despite the great potential of CA-4, it presents chemical instability,
prone to the inactive trans-form, and shows a short biological half-life [8]. Therefore, a large
amount of heterocyclic ring bridging CA-4 analogous has been prepared to facilitate a cis-locked
configuration and provide optimal conformational geometry for interaction with the target [9-14].
Figure 1. Combretastatin A-4 and some its analogues.
Interestingly, we noted that many conformationally restricted analogues of CA-4, such as
compounds 2–4 [10-12], had one or two carbonyl group in the heterocyclic rigid ring scaffold
structure. This enlightens us that the carboxyl group on the heterocyclic ring may be helpful for
promoting the interaction with tubulin. And our previous research has indicated the potential of
the N-hydroxy maleimide skeleton (5) as a template for tubulin inhibitor [13]. In 2005, Tron and
co-workers reported the CA-4 analogue (6) with potent antiproliferative and antitubulin effects
[14]. In continuation of our efforts to search for novel tubulin inhibitors, we herein synthesized a
variety of 3,4-diaryl-1,2,5-oxadiazole-N-oxides analogues (7a–n, 8a, 8j and 8n) using furoxan
skeleton in place of the furazan ring of compound 6 and examined their antiproliferative and

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antitubulin effects (Fig. 2). We also have investigated the role of the N-oxide group and the
difference of the two furoxan regioisomers.
Figure 2. Design of novel 3,4-diaryl-1,2,5-oxadiazole-2/5-oxides as potential antitubulin and anticancer agents.
2. Results and discussion
2.1 Chemistry
The preparation of the target furoxans was illustrated in Scheme 1. A survey of the literature
revealed that 3,4-diaryl-1,2,5-oxadiazole-2/5-oxides were, in general, prepared by a classical
reaction of bisoxime with sodium hypochlorite [15]. The bisoximes 19a, 19b, 19f–j, 20a and 20j
were prepared as described in our previous study [16] and 19c–e were newly synthesized and
identified.
Scheme 1. Reagents and conditions: (i) Dimethyl phosphite, NaOMe, MeOH, 50 °C; (ii) TsOH cat.,
3,4-dihydro-2H-pyran (THP), anhydrous toluene, under N2 atmosphere, 50 °C; (iii) (a) n-BuLi, anhydrous THF,
-55 °C; (b) Substituted benzaldehydes, anhydrous THF, -55~0 °C; (c) HCl aq. (12 M), MeOH, rt.; (iv)
Selenium(IV) oxide, DMSO, microwave, 800 W, 2 min; (v) NH2OH·HCl in Py/EtOH, reflux; (vi) NaClO, MeOH,
rt.; (vii) TiCl4, anhydrous dichloromethane, -20 °C.
Among all of the target furoxans, the ratio of two regioisomers is almost 1:1. The structures
1
of the regioisomers were confirmed on the basis of H-NMR spectroscopy (see experimental

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section) [17]. As for 7n′, its structure could be assigned on the basis of the knowledge that the
N-oxide group exerts a shielding influence on the 1H resonance of the adjacent
3,4,5-trimethoxyphenyl moiety, and comparison with 7n′′ by 1H-NMR spectroscopy (Fig. 3).
Figure 3. Comparison of 1H-NMR spectroscopy of 7n′ and 7n′′.
2.2. Biological evaluation

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2.2.1. In vitro antiproliferative activity
Initially, the mixture was tested for its effects on viability in three human cell lines
(SGC-7901, A549 and HT-1080 cell lines) due to the two regioisomers (ratio almost 1:1) could not
be separated by silica gel column chromatography. As summarized in Table 1, most compounds
showed moderate to excellent antiproliferative activities, although they were not as potent as CA-4.
The 3,4,5-trimethoxyphenyl moiety as ring A, resembling the trimethoxyaryl ring of colchicine, is
optimal for bioactivity (7a vs 8a, 7n vs 8n). Furthermore, the presence of 4-methoxyphenyl (7a)
or 3-hydroxy-4-methoxyphenyl moiety (7n) as ring B presents very good antiproliferative activity.
The other modifications of the ring B result in decreased bioactivity.
Table 1. In vitro antiproliferative activities of the target mixtures as measured with MTT test in
three human tumour cell lines
IC50 (µM ± SD)a
Compound
A549
0.17 ± 0.02
1.58 ± 0.06
15.51 ± 1.02
>30
SGC-7901
0.078 ± 0.006
0.36 ± 0.01
13.53 ± 0.54
>30
HT-1080
0.14 ± 0.01
0.50 ± 0.02
>30
7ab
7bb
7cb
7db
7eb
7fb
7gb
7hb
7ib
>30
0.12 ± 0.005
1.39 ± 0.06
>30
0.15 ± 0.007
0.44 ± 0.02
>30
1.08 ± 0.04
0.79 ± 0.03
>30
>30
19.48 ± 0.93
2.57 ± 0.12
>30
>30
>30
>30
7jb
>30
>30
7kb
7lb
7mb
7nb
8ab
8jb
28.34 ± 1.38
>30
15.22 ± 0.70
12.68 ± 0.61
29.04 ± 1.43
0.103 ± 0.009
>30
>30
>30
>30
>30
0.119 ± 0.011
>30
0.112 ± 0.007
>30
>30
>30
>30
8nb
1ac
3.35 ± 0.14
5.06 ± 0.22
10.14 ± 0.50
0.019 ± 0.002
0.045 ± 0.004
0.007 ± 0.001
a IC50: Concentration of the compound (µM) producing 50% cell growth inhibition after 72 h of drug exposure, as
determined by the MTT assay. Each experiment was carried out in triplicate.
b The furoxan regioisomers were tested as a mixture.
c Used as a positive control.
It was therefore of interest to separate and determine the biological activities of the two
regioisomers to ascertain the contribution of the individual isomer for the antitubulin and

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antiproliferative properties of the furoxans. The individual isomer (7n′ and 7n′′) and regioisomer
mixture (7n) were selected to evaluate their antiproliferative activity. The data (Table 2) indicate
that 7n′ and 7n′′ has potent antiproliferative effects against HT-1080 cell line, with IC50 of 84 ± 5
and 142 ± 12 nM. The 7n′ was slightly more active than the regioisomer mixture 7n, while 7n′′
was slightly less active. In the HT-1080 cell line pair, 7n′ was over 9-fold more potent than the
lead compound (6).
Table 2. In vitro antiproliferative activities of 7n, 7n′ and 7n′′ as measured with MTT test in three
human tumour cell lines
IC50 (µM ± SD)a
Compound
A549
SGC-7901
HT-1080
0.107 ± 0.004
0.091 ± 0.008
0.112 ± 0.007
0.325 ± 0.023
0.097 ± 0.005
0.079 ± 0.004
0.109 ± 0.006
0.706 ± 0.031
0.027 ± 0.004
0.102 ± 0.007
0.084 ± 0.005
0.142 ± 0.012
0.514 ± 0.027
0.006 ± 0.001
7n
7n′
7n′′
6b
1ab
0.013
± 0.001
a IC50: Concentration of the compound (µM) producing 50% cell growth inhibition after 72 h of drug exposure, as
determined by the MTT assay. Each experiment was carried out in triplicate.
b Used as a positive control.
In order to evaluate selectivity of compounds to cancer cell lines and non-cancer cell lines,
we further investigated the effect of 7n′ with high antiproliferative activity toward human
umbilical vein endothelial cells (Table 3). The selectivity indexes (SI) for A549 cell line,
SGC-7901 cell line and HT-1080 cell line were calculated. The cytotoxicity assay showed that the
compound of 7n′ have a higher selectivity than that of positive control drug CA-4.
IC50 (µM ± SD)a
Compound
SIb
SIc
SId
HUVECs
50.88
1.92
58.61
0.93
55.12
4.17
4.63 ± 0.002
7n′
CA-4
0.025
± 0.001
Table 3. In vitro antiproliferative activities of 7n′ in Human umbilical vein endothelial cells.
a IC50: Concentration of the compound (µM) producing 50% cell growth inhibition after 72 h of drug exposure, as
determined by the MTT assay. Each experiment was carried out in triplicate.
b SI = IC50(HUVECs)/IC50(A549). c SI = IC50(HUVECs)/IC50(SGC-7901). d SI = IC50(HUVECs)/IC50(HT-1080).
2.2.2. Effect on tubulin polymerization
Studies were conducted to determine if the individual isomer (7n′ and 7n′′) inhibited the
polymerization of purified bovine tubulin, as would be predicted from the effects in cells (Fig. 4).
CA-4 (a microtubule-destabilizing agent) and taxol (a microtubule-stabilizing agent) were used as
controls. The IC50 values of their ability to inhibit tubulin polymerization have been tabulated in

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Table 3. We found that both 7n′ and 7n′′, like the CA-4, exhibit strong the antitubulin
polymerization activity that corresponds well with their antiproliferative effects (Fig. 4). 7n′ (IC50
= 0.367 ± 0.015 µM) was nearly 2-fold more potent for the inhibition of tubulin polymerization
than the CA-4 (IC50 = 0.643 ± 0.021 µM). A similar but less pronounced effect was seen for the
isomer 7n′′ (1.205 ± 0.103 µM). These results indicate that the position of the N-oxide group is
crucial for activity in the furoxan scaffold.
Table 4. In vitro tubulin polymerization inhibitory activities of 7n, 7n′ and 7n′′.
Compound
IC50 (µM ± SD)
0.367 ± 0.015
1.205 ± 0.103
0.643 ± 0.021
7n′
7n′′
CA-4
Fig. 4. Cell-free tubulin polymerization assay of 7n′ and 7n′′. The negative and positive controls are taxol and
CA-4, respectively.
2.2.3. Immunofluorescence staining assays
To clearly examine the microtubule network of HT-1080 cells changes induced by 7n′, a
cellular immunofluorescence staining assay was carried out. The results indicated that the
untreated cells displayed well-organized microtubule structures while 7n′ and CA-4 caused a
severe destruction of the microtubule system (Fig. 5). We also observed some multinucleated cells.
These characteristics were similar to those of the typical tubulin inhibitors and positive control
drug CA-4 [18].

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Figure. 5. Immunostaining of tubulin assembly in HT-1080 cells. HT-1080 cells were treated with either 7n′ (100
nM) or CA-4 (1, 18 nM) for 24 h. Microtubules and unassembled tubulin are shown in green, and nuclei, stained
with DAPI are shown in blue.
2.2.4. Cell cycle analysis.
Tubulin inhibitors usually arrest cell cycle at G2/M phase, thereby prevents the growth and
proliferation of cancer cells. To understand whether the antiproliferative effect of 7n′ on HT-1080
cells is on account of cell cycle arrest, cell cycle analysis of 7n′ was determined by a flow
cytometry analysis at different concentrations and time (Fig. 6). The analysis results indicated that
HT-1080 cells treated with 84 nM of 7n′ showed a significant growth arrest at the G2/M phase
after 24 h compared to control cells (46% and 3%, respectively). After 72 h, the percentage of
apoptotic cells, as indicated by the Sub-G1 peak, was a significant increase for 7n′ compared to the
control (68% and 2%, respectively). In addition, a dose-dependent cell accumulation in the G2/M
phase was also evidently noted after treatment of HT-1080 cells with 42 nM, 84 nM and 168 nM
of 7n′ at 24 h. These results for 7n′ are as same as typical tubulin-targeting antitumor agents,
which characteristically cause G2/M arrest followed by apoptosis [19].

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Fig. 6. (A) Effects of 7n′ on cell cycle distribution in HT-1080 cells determined by flow cytometry analysis; (B)
The time-dependent of cell accumulation in the G2/M phase and apoptosis in the Sub-G1 were evidently noted after
treatment of HT-1080 cells with 84 nM of 7n′ at 6 h, 12 h, 24 h, 48 h and 72 h; (C) The dose-dependent of cell
accumulation in the G2/M phase and apoptosis in the Sub-G1 were evidently noted after treatment of HT-1080 cells
with 42 nM, 84 nM and 168 nM of 7n′ at 24 h.
2.2.5. Molecular docking studies.
To further explore of the difference of effects on inhibition of tubulin polymerization between
the regioisomers 7n′ and 7n′′, a virtual molecular modeling study was conducted using
CDOCKER program in Discovery Studio 3.0 software [20], It is worth mentioning that both 7n′
and 7n′′ occupied the co-crystallized ligand (CA-4) binding site (PDB ID: 5LYJ) of α,β-tubulin

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(Fig. 7D). As with the binding mode of CA-4 (Fig. 7A), 7n′ forms three essential hydrogen bonds
with βCys241, αThr179 and αVal181, respectively. Meanwhile, it becomes apparent that 7n′
maintains two important interactions with αAsn101 and αVal181 (Fig. 7C), which does not appear
in the binding mode of 7n′′ (Fig. 7B). This may account for the fall in tubulin polymerization
inhibitory activity observed for 7n′′.
Figure. 7. Docking pose of (A) CA-4 (crystal structure in green), (B) 7n′′ (in blue), (C) 7n′′ (in yellow), and (D),
with α-(in purple) and β-(in gray) heterodimers of tubulin. Hydrogen bonds are shown using green dashed lines.
3. Conclusion
In this work, a structure-based medicinal chemistry optimization was conducted on the
tubulin inhibitor 6, and a series of new compounds was designed and synthesized by incorporating
the 1,2-diarylalkene bridge of CA-4 into a 1,2,5-oxadiazole-2/5-oxides. The structures of
regioisomers were identified by NMR spectroscopy. Notably, 7n′ displayed the more potent
antiproliferative activity than the lead compound 6 against three different cancer cell lines. The
effect of 7n′ on tubulin polymerization was investigated using fluorescence-based tubulin
polymerization assays, immunofluorescence analysis of microtubule network, and a molecular
docking study. Interestingly, 7n′ exerted stronger inhibitory effect on tubulin polymerization than
CA-4. Like CA-4, 7n′ also effectively caused G2/M arrest and induced cell apoptosis. The results

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indicated the importance of the furoxan as bridging skeleton in the structural optimization of CA-4
and the N-oxide group on the heterocyclic ring may be helpful for promoting the interaction with
tubulin.
4. Experimental section
General: All chemicals and reagents of analytical grade were commercially available and
were used without further purification unless otherwise indicated. 1H-NMR and 13C-NMR spectra
were measured in CDCl3 or d6-DMSO with TMS as the internal reference on a Bruker AVANCE
13
spectrometer operating at 400 MHz or 600 MHz (1H at 400 or 600 MHz, C at 100 MHz). Mass
spectra were recorded with an Agilent 1100-sl mass spectrometer with an electrospray ionization
interface operated in the positive ion mode at Agilent Co., Ltd. Thin layer chromatography was
performed using the glass-backed silica gel sheets fluorescent indicator visualizing with UV light
at 254 nm. Column chromatography was performed using 200-300 mesh silica gel obtained from
Qingdao PUKE eluting with ethyl acetate and petroleum ether.
4.1. Synthesis
4.1.1. General synthetic procedures for dioxime 19a-k, 20a and 20j.
The synthesis of the key intermediates 19 and 20 was achieved in five steps from
commercially available benzaldehyde 9 or 10 as shown in Scheme 1, which has been illustrated in
detail in our previous study [15].
4.1.1.1. 1-(3,4,5-trimethoxyphenyl)-2-(naphthalen-2-yl) ethanone (15f).
1
White solid; yield: 73%; M.p.: 68-70 °C; H-NMR (400 MHz, CDCl3): 7.80 (3H, m, Ph-H), 7.72
(1H, s, Ph-H), 7.45 (2H, m, Ph-H), 7.39 (1H, d, Ph-H), 7.30 (2H, s, Ph-H), 4.40 (2H, s, COCH2),
3.89 (3H, s, OCH3), 3.86 (6H, s, OCH3); MS (ESI) m/z: 337.2 [M+H]+, 359.1 [M+Na]+.
4.1.1.2. 1-(3,4-methylenedioxyphenyl)-2-[3-(benzyloxy)-4-methoxyphenyl]ethanone (16j).
White solid; yield: 79%; M.p.: 98-101 °C; 1H-NMR (400 MHz, CDCl3): 7.56 (1H, dd, Ph-H), 7.46
(2H, s, Ph-H), 7.42 (1H, s, Ph-H), 7.35 (2H, t, Ph-H), 7.29 (1H, m, Ph-H), 6.83 (4H, m, Ph-H),
6.01 (2H, s, OCH2O), 5.11 (2H, s, OCH2), 4.09 (2H, s, COCH2), 3.85 (3H, s, OCH3); MS (ESI)
m/z: 377.3 [M+H]+, 399.3 [M+Na]+.
4.1.1.3. 1-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl) ethane-1,2-dione (17f).
Pale yellow solid; yield: 71%; M.p.: 129-131 °C; 1H-NMR (400 MHz, CDCl3): 8.41 (1H, s, Ph-H),
8.10 (1H, dd, Ph-H), 7.94 (3H, m, Ph-H), 7.65 (1H, m, Ph-H), 7.57(1H, m, Ph-H), 7.28 (2H, s,

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OCH3), 3.96 (3H, s, OCH3), 3.89 (6H, s, OCH3); MS (ESI) m/z: 351.2 [M+H]+, 373.3 [M+Na]+.
4.1.2. General synthetic procedures for 3,4-diphenyl-1,2,5-oxadiazole-2-oxides
Dioxime (1 mmol) was dissolved in methanol (10 mL). The 20% sodium hypochlorite
aqueous solution (2 mmol) was added dropwise into the above mixture at -20 °C for 1 h. And then,
the resulting mixture was filtered and dried. The crude product was purified by column
chromatography (n-hexane : EtOAc= 10:1 as eluent) on silica gel to afford pure products. For
example:
4.1.2.1. 3-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7a′) and
4-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7a′′) regioisomers
(ratio 1:1.35).
White solid; yield: 93%; (7a′) 1H-NMR (400 Hz, CDCl3): δ 3.74 (6H, s), 3.83 (3H, s), 3.89 (3H, s),
6.76 (2H, s), 6.97 (2H, d, J=8.74), 7.50 (2H, d, J=8.74); (7a′′) 1H-NMR (400 Hz, CDCl3): δ 3.71
(6H, s), 3.84 (3H, s), 3.88 (3H, s), 6.80 (2H, s), 6.97 (2H, d), 7.48 (2H, d); MS (ESI) m/z: 359.2
[M+H]+, 381.2 [M+Na]+。
4.1.2.2. 3-(4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7b′) and
4-(4-ethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7b′′) regioisomers (ratio
1:1.4).
1
White solid; yield: 93%; (7b′) H-NMR (400 Hz, CDCl3): δ 1.43 (3H, s), 3.74 (6H, s), 3.90 (3H,
s), 4.07 (2H, m), 6.76 (2H, s), 6.96 (2H, d, J=8.20), 7.50 (2H, d, J=8.20); (7b′′) 1H-NMR (400 Hz,
CDCl3): δ 1.43 (3H, s), 3.72 (6H, s), 3.89 (3H, s), 4.07 (2H, m), 6.80 (2H, s), 6.96 (2H, d, J=8.20),
7.49 (2H, d, J=8.20); MS (ESI) m/z: 373.2 [M+H]+, 395.2 [M+Na]+.
4.1.2.3. 3-(4-chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7c′) and
4-(4-chlorophenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7c′′) regioisomers (ratio
1.1:1).
White solid; yield: 91%; (7c′) 1H-NMR (400 Hz, CDCl3): δ3.75 (6H, s), 3.91 (3H, s), 6.73 (2H, s),
1
7.46 (2H, m), 7.53 (2H, m); (7c′′) H-NMR (400 Hz, CDCl3): δ 3.72 (6H, s), 3.89 (3H, s), 6.71
(2H, s), 7.46 (2H, m), 7.53 (2H, m); MS (ESI) m/z: 363.2 [M+H]+, 385.2 [M+Na]+.
4.1.2.4.
3-(4-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide
(7d′)
and
4-(4-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7d′′) regioisomers (ratio
1:1.5).

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1
White solid; yield: 92%; (7d′) H-NMR (400 Hz, CDCl3): δ 3.76 (6H, s), 3.92 (3H, s), 6.69 (2H,
s), 7.82 (2H, m), 8.33 (2H, m); (7d′′) 1H-NMR (400 Hz, CDCl3): δ 3.73 (6H, s), 3.91 (3H, s), 6.77
(2H, s), 7.73 (2H, m), 8.23 (2H, m); MS (ESI) m/z: 374.4 [M+H]+, 396.2 [M+Na]+, 372.0 [M–H]–,
407.9 [M+Cl]–.
4.1.2.5. 3-(3-fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7e′)
and 4-(3-fluoro-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7e′′)
regioisomers (ratio 1.1:2).
White solid; yield: 92%; (7e′) 1H-NMR (400 Hz, CDCl3): δ 3.77 (6H, s), 3.92 (3H, s), 3.94 (3H, s),
6.74 (2H, s), 7.04 (1H, d, J=8.54), 7.33 (1H, d, J=2.2), 7.40 (1H, dd, J=8.54, J=2.2); (7e′′)
1H-NMR (400 Hz, CDCl3): δ 3.75 (6H, s), 3.90 (3H, s), 3.95 (3H, s), 6.77 (2H, s), 7.02 (1H, d,
J=8.54), 7.33 (1H, d, J=2.2), 7.40 (1H, dd, J=8.54, J=2.2); MS (ESI) m/z: 377.2 [M+H]+, 399.2
[M+Na]+, 415.2 [M+K]+.
4.1.2.6. 3-(naphthalen-2-yl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7f′) and
4-(naphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7f′′) regioisomers
(ratio 1:1.1).
White solid; yield: 91%; (7f′) 1H-NMR (400 Hz, CDCl3) δ 3.64 (6H, s), 3.90 (3H, s), 6.77 (2H, s),
7.53 (3H, m), 7.88(3H, m), 8.22 (1H, s); (7f′′) 1H-NMR (400 Hz, CDCl3) δ 3.61 (6H, s), 3.88 (3H,
s), 6.82 (2H, s), 7.53 (3H, m), 7.88 (3H, m), 8.17 (1H, s); MS (ESI) m/z: 379.2 [M+H]+, 401.2
[M+Na]+, 417.2 [M+K]+.
4.1.2.7. 3-(4-(benzyloxy)phenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7g′) and
4-(4-(benzyloxy) phenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7g′′) regioisomers
(ratio 1:1).
White solid; yield: 93%; (7g′) 1H-NMR (400 Hz, CDCl3): δ 3.71 (6H, s), 3.90 (3H, s), 5.11 (2H, s),
1
6.75 (2H, s), 7.04 (2H, d, J=8.40), 7.37 (5H, m), 7.51 (2H, d, J=8.40); (7g′′) H-NMR (400 Hz,
CDCl3): δ 3.69 (6H, s), 3.88 (3H, s), 5.12 (2H, s), 6.78 (2H, s), 7.04 (2H, d, J=8.40), 7.37 (5H, m),
7.49 (2H, d, J=8.40); MS (ESI) m/z: 435.3 [M+H]+, 457.3 [M+Na]+, 473.2 [M+K]+.
4.1.2.8. 3-(4-(benzyloxy)-3-bromophenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-
oxide (7h′) and 4-(4-(benzyloxy)-3-bromophenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-
oxide (7h′′) regioisomers (ratio 1:1.4).
1
White solid; yield: 93%; (7h′) H-NMR (400 Hz, CDCl3): δ 3.73 (6H, s), 3.91 (3H, s), 5.22 (2H,

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s), 6.73 (2H, s), 6.98 (1H, dd, J=8.85, J=1.82), 7.41 (5H, m), 7.45 (1H, m), 7.87 (1H, d, J=1.82);
(7h′′) 1H-NMR (400 Hz, CDCl3): δ 3.71 (6H, s), 3.89 (3H, s), 5.23 (2H, s), 6.77 (2H, s), 6.89 (1H,
d, J=8.85), 6.98 (1H, dd, J=8.85, J=1.82), 7.41 (5H, m), 7.87 (1H, d, J=1.82); MS (ESI) m/z:
515.2 [M+H]+, 537.2 [M+Na]+, 571.1 [M+K]+.
4.1.2.9. 3-(3-(benzyloxy)phenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7i′) and
4-(3-(benzyloxy)phenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7i′′) regioisomers
(ratio 1:1).
White solid; yield: 92%; (7i′) 1H-NMR (400 Hz, CDCl3): δ 3.71 (6H, s), 3.90 (3H, s), 5.02 (2H, s),
1
6.73 (2H, s), 7.09 (2H, m), 7.22 (1H, m), 7.32 (1H, m), 7.37 (5H, m); (7i′′) H-NMR (400 Hz,
CDCl3): δ 3.68 (6H, s), 3.88 (3H, s), 5.05 (2H, s), 6.78 (2H, s), 7.09 (2H, m), 7.22 (1H, m), 7.32
(1H, m), 7.37 (5H, m); MS (ESI) m/z: 435.3 [M+H]+, 457.3 [M+Na]+, 473.2 [M+K]+.
4.1.2.10. 3-(3-(benzyloxy)-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2- oxide
(7j′) and 4-(3-(benzyloxy)-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5oxadiazole-2-
oxide (7j′′) regioisomers (ratio 1:1.4).
White solid; yield: 94%; (7j′) 1H-NMR (400 Hz, CDCl3): δ 3.74 (6H, s), 3.91 (3H, s), 3.91 (3H, s),
5.03 (2H, s), 6.75 (2H, s), 6.93 (1H, d), 7.11 (2H, dd), 7.17 (1H, s), 7.32 (5H, m); (7j′′) 1H-NMR
(400 Hz, CDCl3): δ3.72 (6H, s), 3.89 (3H, s), 3.92 (3H, s), 5.07 (2H, s), 6.72 (2H, s), 6.94 (1H, d),
7.15 (2H, dd), 7.17 (1H, s), 7.32 (5H, m); MS (ESI) m/z: 487.3 [M+Na]+, 503.2 [M+K]+.
4.1.2.11. 4-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-1,2,5-oxadiazole-2-oxide (8a′) and
3-(benzo[d][1,3]dioxol-5-yl)-4-(4-methoxyphenyl)-1,2,5-oxadiazole-2-oxide (8a′′) regioisomers
(ratio 1.1:1).
1
White solid; yield: 92%; (8a′) H-NMR (400 Hz, CDCl3): δ 3.84 (3H, s), 6.03 (2H, s), 6.84 (1H,
1
m), 6.94 (2H, d, J=7.48), 6.99 (1H, s), 7.02 (1H, m), 7.48 (2H, m); (8a′′) H-NMR (400 Hz,
CDCl3): δ 3.85 (3H, s), 6.02 (2H, s), 6.84 (1H, m), 6.94 (2H, d, J=7.48), 6.99 (1H, s), 7.02 (1H,
m), 7.46 (2H, m); MS (ESI) m/z: 313.1 [M+H]+, 335.1 [M+Na]+, 646.6 [2M+Na]+.
4.1.2.12.4-(benzo[d][1,3]dioxol-5-yl)-3-(3-(benzyloxy)-4-methoxyphenyl)-1,2,5-oxadiazole-2-oxid
e (8j′) and 3-(benzo[d][1,3]dioxol-5-yl)-4-(3-(benzyloxy)-4-methoxyphenyl)-1,2,5-oxadiazole-2-
oxide (8j′′) regioisomers (ratio 1:1.2).
White solid; yield: 93%; (8j′) 1H-NMR (400 Hz, CDCl3): δ 3.91 (3H, s), 5.02 (2H, s), 6.03 (2H, s),
1
6.81 (1H, s), 6.92 (1H, m), 6.98 (2H, m), 7.12 (2H, m), 7.34 (5H, m); (8j′′) H-NMR (400 Hz,

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CDCl3): δ 3.92 (3H, s), 5.05 (2H, s), 6.02 (2H, s), 6.83 (1H, s), 6.92 (1H, m), 6.98 (2H, m), 7.12
(2H, m), 7.34 (5H, m); MS (ESI) m/z: 419.2 [M+H]+, 441.2 [M+Na]+, 457.2 [M+K]+.
4.1.3. General synthetic procedures for 7n, 7m, 7k, 7l and 8n.
A solution of 7j (2.71 mmol) in dry dichloromethane (100 mL) was treated with TiCl4 under
nitrogen atmosphere. The reaction mixture was stirred at -20 °C for 1h, worked up by dilution of
dichloromethane, washed with water and brain. The combined organic layer was dried over
Na2SO4. The removal of the solvent give the crude product, which on chromatographic
purification on silica gel with n-hexane : EtOAc = 3:1 as eluent.
4.1.3.1. 3-(4-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7k′) and
4-(4-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7k′′) regioisomers
(ratio 1:1).
White solid; yield: 93%; (7k′) 1H-NMR (400 Hz, CDCl3): δ 3.73 (6H, s), 3.91 (3H, s), 5.70 (H, s),
6.76 (2H, s), 6.92 (2H, d, J=8.06), 7.43 (2H, d, J=8.06); (7k′′) 1H-NMR (400 Hz, CDCl3): δ 3.71
(6H, s), 3.89 (3H, s), 5.70 (H, s), 6.80 (2H, s), 6.92 (2H, d, J=8.06), 7.41 (2H, d, J=8.06); MS (ESI)
m/z: 345.2 [M+H]+, 367.2 [M+Na]+, 383.2 [M+K]+, 342.9 [M–H]–.
4.1.3.2. 3-(3-bromo-4-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7l′)
and 4-(3-bromo-4-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7l′′)
regioisomers (ratio 1:1).
White solid; yield: 92%; (7l′) 1H-NMR (400 Hz, CDCl3): δ 3.77 (6H, s), 3.92 (3H, s), 6.12 (H, s),
6.75 (2H, s), 7.08 (1H, d, J=8.85), 7.39 (1H, d, J=8.40), 7.84 (1H, d, J=1.64); (7l′′) 1H-NMR (400
Hz, CDCl3): δ 3.75 (6H, s), 3.90 (3H, s), 6.12 (H, s), 6.79 (2H, s), 7.08 (1H, d, J=8.85), 7.39 (1H,
d, J=8.40), 7.80 (1H, d, J=1.64); MS (ESI) m/z: 422.9 [M+H]+.
4.1.3.3. 3-(3-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7m′) and
4-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7m′′) regioisomers
(ratio 1:1).
White solid; yield: 90%; (7m′) 1H-NMR (400 Hz, CDCl3): δ 3.71 (6H, s), 3.89 (3H, s), 6.76 (2H,
s), 6.98 (1H, d, J=6.79), 7.03 (1H, d, J=8.27), 7.15 (1H, s), 7.30 (1H, d, J=6.79); (7m′′) 1H-NMR
(400 Hz, CDCl3): δ 3.69 (6H, s), 3.87 (3H, s), 6.82 (2H, s), 6.98 (1H, d, J=6.79), 7.03 (1H, d,
J=8.27), 7.10 (1H, s), 7.30 (1H, d, J = 6.79); MS (ESI) m/z: 345.2 [M+H]+, 367.2 [M+Na]+, 342.9
[M–H]–.

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4.1.3.4. 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7n′)
and 4-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7n′′)
regioisomers (ratio 1:1.4).
White solid; yield: 92 %; (7n′) 1H-NMR (400 Hz, CDCl3) δ 3.75 (6H, s), 3.91 (3H, s), 3.94 (3H, s),
6.77 (2H, s), 6.92 (1H, d, J=8.40), 7.11 (1H, dd, J=8.40, J=2.14), 7.17 (1H, d, J=2.14); (7n′′)
1H-NMR (400 Hz, CDCl3) δ 3.73 (6H, s), 3.90 (3H, s), 3.95 (3H, s), 6.81 (2H, s), 6.91 (1H, d,
J=8.40), 7.07 (1H, dd, J=8.40, J=2.12), 7.17 (1H, d, J=2.12); MS (ESI) m/z: 375.2 [M+H]+, 397.2
[M+Na]+, 413.4 [M+K]+, 373.0 [M–H]–.
4.1.3.5. 4-(benzo[d][1,3]dioxol-5-yl)-3-(3-hydroxy-4-methoxyphenyl)-1,2,5-oxadiazole -2-oxide
(8n′) and 3-(benzo[d] [1,3]dioxol-5-yl)-4-(3-hydroxy-4-methoxyphenyl)-1,2,5-oxadiazole -2-oxide
(8n′′) regioisomers (ratio 1.3:1).
White solid; yield:93%; (8n′) 1H-NMR (400 Hz, CDCl3): δ 3.94 (3H, s), 5.77 (H, s), 6.04 (2H, s),
1
6.84 (1H, d, J=3.12), 6.91 (1H, d, J=8.22), 7.00 (1H, m), 7.06 (3H, m); (8n′′) H-NMR (400 Hz,
CDCl3): δ 3.95 (3H, s), 5.77 (H, s), 6.03 (2H, s), 6.86 (1H, d, J=3.12), 6.90 (1H, d, J=8.22), 7.00
(1H, m), 7.06 (3H, m); MS (ESI) m/z: 329.1 [M+H]+, 351.1 [M+Na]+, 326.9 [M–H]– ; 13C-NMR
(100 MHz, CDCl3): δ 56.1, 60.9, 106.2, 120.7, 125.6, 127.1, 127.6, 127.7, 127.7, 129.0, 130.2,
136.8, 138.5, 147.8, 153.2.
4.2. Separation and purification of 7n′ and 7n′′.
Recycling Preparative HPLC was run on a LC-9103 (Japan Analytical Industry), equipped
with a high pressure pump, an manual sampler, an UV detector, and an online degasser. System
control and data acquisition were performed using a MultiChro ver 5.0.0.0 (Yullin Technology).
The chromatographic separation was carried out on a Sunfire-C18 OBDTM Prep Column (150 × 10
mm). The mobile phase was composed of aqueous solution containing methyl alcohol and water
(v/v = 3:2). The flow rate was set at 7 mL/min, the injection volume was 2.5 mL and the column
temperature was maintained at 35 °C. The detect wavelength was determined at 245 nm. This
separation yielded 21.2 mg of 7n′ and 24.4 mg of 7n′′ from 50 mg of the mixture. The structures
of 7n′ and 7n′′ were identified by 1H-NMR and 13C-NMR.
3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7n′).
1
White solid, H-NMR (400 Hz, CDCl3) δ 3.75 (s, 6H), 3.91 (s, 3H), 3.94 (s, 3H), 6.77 (s, 2H),
6.92 (d, 1H, J=8.40), 7.11 (dd, 1H, J = 8.40, J=2.14), 7.17 (d, 1H, J=2.14); 13C-NMR (100 MHz,

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CDCl3): δ C 155.9, 153.6, 148.2, 145.9, 140.2, 121.8, 121.6, 114.9, 114.1, 110.7 (×2), 105.6 (×2),
61.0, 56.2, 56.1, 50.9; MS (ESI) m/z: 375.2 [M+H]+, 397.2 [M+Na]+, 413.4 [M+K]+, 373.0 [M–
H]–.
4-(3-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,5-oxadiazole-2-oxide (7n′′).
1
White solid, H-NMR (400 Hz, CDCl3) δ 3.73 (s, 6H), 3.90 (s, 3H), 3.95 (s, 3H), 6.81 (s, 2H),
6.91 (d, 1H, J=8.40), 7.07 (dd, 1H, J=8.40, J=2.12), 7.17 (d, 1H, J=2.12); 13C-NMR (100 MHz,
CDCl3): δ C 155.8, 153.4, 148.7, 145.9, 139.7, 121.1 (×2), 119.7, 117.9, 114.5, 110.6 (×2), 106.2
(×2), 61.0, 56.2, 56.1, 29.7; MS (ESI) m/z: 375.2 [M+H]+, 397.2 [M+Na]+, 413.4 [M+K]+, 373.0
[M–H]–.
4.3. Biochemistry methods
4.3.1. Antiproliferative activity assay
Gastric adenocarcinoma cells (SGC-7901), lung adenocarcinoma cells (A549) and
fibrosarcoma cells (HT-1080) cultured in RPMI 1640/10% (V/V) fetal bovine serum, in 5% CO2
water saturated atmosphere at 37 °C. SGC-7901, A549 and HT-1080 cells suspensions were
prepared and dispensed into 96-well plates giving 4000-5000 cells/well respectively. The
antiproliferative activity in vitro of CA-4 and all the target compounds were measured using the
MTT assay. All assays were conducted in triplicate to determine the average of the reports. The
plates were sent back to the incubator for 24 hours to allow the cells to reattach. These compounds
were initially prepared at 20 µg/mL in DMSO. Triplicate wells were treated with media and
various concentrations of compounds from 10 µg/mL to 0.003 µg/mL, and reincubated for a
further 72 h. After cells were treated with drug for 72 h, the culture medium was then removed and
the cells washed with 100 mL phosphate buffered saline (PBS). MTT solution (20.0 µL/well) in
PBS was added. After 4 h incubation, the solution in the wells was poured out and 150 µL
dimethyl sulfoxide was added to each well. Plates were read in a microplate reader (MK3, Thermo,
Germany). The absorbance value of control cells (without compound addition) was defined as 100%
cell viability. The concentration required for 50% inhibition of cell viability (IC50) was defined as
a 50% reduction in the concentration of untreated absorbance in the MTT method. Each
experiment was carried out in triplicate. The results are shown in Table 1 and Table 2.
4.3.2. Tubulin polymerisation study
The effects of 7n′, 7n′′ and CA-4 on the polymerization of tubulin in vitro was determined

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by using a fluorescence-based tubulin polymerization assay kit (Cytoskeleton-Cat. # BK011P)
according to the manufacturer's agreement. In brief, 7n′ and 7n′′ with series concentrations (0.33
µM, 1.11 µM, 3.33 µM) were incubated with purified bovine tubulin and buffer containing 20%
glycerol, 2 mM MgCl2, 80 mM PIPES, 1 mM GTP and 0.5 mM EGTA at 37 °C. Then, the
assembly of tubulin was monitored kinetically using a fluorescent plate reader. The IC50 value was
defined as the concentration of product which inhibited the extent of polymerization by 50%. Each
experiment was carried out in triplicate. The IC50 values were calculated using SPSS software.
4.3.3. Immunofluorescence staining
Immunofluorescence studies were performed to detect the microtubule associated tubulin
protein after exposure to CA-4 and the selected 7n′. HT-1080 cells were seeded in a 24-well plate
at 1×104 cells each well and grown for 24 h. Cells were treated with the vehicle, the CA-4 (18 nM)
or 7n′ (100 nM) for 12 h. The control and treated cells were fixed with 4% formaldehyde in PBS
for 30 minutes -20 °C, twice with PBS and permeabilised with 0.1% (v/v) Triton X-100 in PBS for
5 minutes. The cells were then blocked in PBS for 10 minutes with 5% bovine serum albumin
(BSA). The primary α-tubulin antibody (Santa Cruz, CA) was diluted (1: 100) with 2% BSA in
PBS, and the plates were incubated overnight at 4 °C. The cells were washed by PBS to remove
the unbound primary antibody, and then the cells were incubated with the FITC-conjugated
anti-mouse secondary antibody which diluted (1: 1000) with 2% BSA in PBS for 3 hours at 37 °C.
The cells were washed by PBS to remove the unbound secondary antibody, and the nucleus were
stained by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). And then, immunofluorescence
was detected using a fluorescence microscope (Olympus, Tokyo, Japan).
4.3.4. Cell cycle analysis
It is well known that microtubulin inhibitors such as colchicine and CA-4 block cell cycle
distribution in G2/M phase. Thus, the flow cytometry analysis was used to study the effect of 7n′
on the cell cycle progression of the HT-1080 cells. The HT-1080 cells were treated with 7n′ (0.5, 1,
2 fold IC50) for 24 h, with 7n′ (1 fold IC50) for 6 h, 12 h, 24 h, 48 h and 72 h, and the untreated
cells was used as a control. The samples were then analysed by FACScan flow cytometry
(BectoneDickinson, Franklin Lakes, NJ, USA).
4.4. Molecular docking studies.
The molecular docking studies were performed by using the CDOCKER protocol in

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Discovery Studio 3.0. The crystal structure of tubulin complexed with CA-4 (PDB: 5LYJ) was
obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb). The proteins were then
prepared using the the CHARMm force field for energy minimization under default settings. The
CA-4, 7n′ and 7n′′ were drawn with Chemdraw and minimized using the CHARMm force field in
Discovery Studio 3.0. To assess the CDOCKER protocol performance, the co-crystallized ligand
CA-4 was firstly docked and the binding mode was investigated. For the synthesized target
compounds, the docked structures revealed that 7n′ and 7n′′ have the similar binding features as
CA-4 and colchicine binding site. Statistical analysis. All data were expressed as the mean ±
standard deviation and analyzed with SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).
Analysis was conducted via an ANOVA method. P<0.05 was considered to indicate a statistically
significant difference.
Acknowledgements
We gratefully acknowledge the National Natural Science Foundation of China (81673293 and
81602969), Project funded by China Postdoctoral Science Foundation (2018M641715). Plan for
Development of Young Scholars of Shenyang pharmaceutical university (ZQN2018002), Doctoral
Research Funding of Liaoning Province (201601144), Education Fund Item of Liaoning Province
(201610163L12), and Science and Technology Project of Shenyang City (17-231-1-41) for the
generous financial support. This work was also supported by the Program for Innovative Research
Team of the Ministry of Education.

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Highlights
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New 1,2,5-oxadiazole-2/5-oxides derivatives were designed and synthesized as potent
combretastatin derivatives.
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7n′ exhibited superior potency compared to the lead compound 6.
7n′ exerted stronger inhibitory effect on tubulin polymerization than CA-4.
The IC50 of 7n′ was over 185.2-fold than the CA-4 in the human umbilical vein endothelial
cells.
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Docking study revealed the furoxan as bridging skeleton may be helpful for promoting the
interaction with tubulin.