Discovery of novel vinyl sulfone derivatives as anti-tumor agents with microtubule polymerization inhibitory and vascular disrupting activities

Article abstract of DOI:10.1016/j.ejmech.2018.08.074

Vinyl sulfone or sulfoxide moieties were firstly introduced to the structure of chalcone compound by replacing the carbonyl group to afford a series of novel compounds as potential anti-tubulin agents. All of the target compounds were evaluated for their anti-proliferative activity. Among them, compound 12m showed the most potent activity against a panel of cancer cell lines with IC₅₀ values ranging from 0.128 to 0.606 μM. Further mechanism studies demonstrated that compound 12m caused G2/M phase arrest, induced cell apoptosis and disrupted the intracellular microtubule network. Moreover, compound 12m reduced the cell migration and disrupted the capillary-like tube formation in human umbilical vein endothelial cell (HUVEC) assays. Importantly, compound 12m significantly and dose dependently inhibited tumor growth in H22 liver cancer allograft mouse model, which is more potent than control compound CA-4, suggesting that 12m deserves further research as a potential anti-tubulin agent targeting colchicine binding site on tubulin.

Full text of DOI:10.1016/j.ejmech.2018.08.074

Accepted Manuscript
Discovery of novel vinyl sulfone derivatives as anti-tumor agents with microtubule
polymerization inhibitory and vascular disrupting activities
Wenlong Li, Ying Yin, Hong Yao, Wen Shuai, Honghao Sun, Shengtao Xu, Jie Liu,
Hequan Yao, Zheying Zhu, Jinyi Xu
PII:
S0223-5234(18)30746-3
10.1016/j.ejmech.2018.08.074
EJMECH 10685
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2018
Revised Date: 7 August 2018
Accepted Date: 26 August 2018
Please cite this article as: W. Li, Y. Yin, H. Yao, W. Shuai, H. Sun, S. Xu, J. Liu, H. Yao, Z. Zhu, J.
Xu, Discovery of novel vinyl sulfone derivatives as anti-tumor agents with microtubule polymerization
inhibitory and vascular disrupting activities, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.08.074.
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Graphic abstract

ACCEPTED MANUSCRIPT
Discovery of Novel Vinyl Sulfone Derivatives as Anti-tumor
Agents with Microtubule Polymerization Inhibitory and
Vascular Disrupting Activities
Wenlong Lia, Ying Yina, Hong Yaoa, Wen Shuaia, Honghao Suna, Shengtao Xua, *, Jie
Liub,c, Hequan Yaoa, Zheying Zhud, Jinyi Xua, *
a
State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China
b
Department of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, P. R. China
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and
School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
d
Division of Molecular Therapeutics & Formulation, School of Pharmacy, The University of
Nottingham, University Park Campus, Nottingham NG7 2RD, UK
*Corresponding Author:
E-mail addresses: cpuxst@163.com (S. Xu), jinyixu@china.com (J. Xu).
Abstract
Vinyl sulfone or sulfoxide moieties were firstly introduced to the structure of
chalcone compound by replacing the carbonyl group to afford a series of novel
compounds as potential anti-tubulin agents. All of the target compounds were
evaluated for their anti-proliferative activity. Among them, compound 12m showed
the most potent activity against a panel of cancer cell lines with IC50 values ranging
from 0.128 to 0.606 µM. Further mechanism studies demonstrated that compound
12m caused G2/M phase arrest, induced cell apoptosis and disrupted the intracellular
microtubule network. Moreover, compound 12m reduced the cell migration and
disrupted the capillary-like tube formation in human umbilical vein endothelial cell
(HUVEC) assays. Importantly, compound 12m significantly and dose dependently
inhibited tumor growth in H22 liver cancer allograft mouse model, which is more
potent than control compound CA-4, suggesting that 12m deserves further research as

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a potential anti-tubulin agent targeting colchicine binding site on tubulin.
Key words: tubulin inhibitors; vinyl sulfone; colchicine binding site; anti-tumor
activity
1. Introduction
Microtubules are formed by the lateral aggregation of protofilaments, each of
which is composed of a chain of stable α- and β-tubulin dimers [1]. They are not
simple equilibrium polymers, instead they show complex polymerization dynamics
which are crucial to their cellular functions. The vital roles in cell proliferation,
trafficking, signaling and migration in eukaryotic cells make microtubules an
important target for cancer therapy [2]. Microtubule targeting agents (MTAs) that bind
to the taxane or vinca alkaloid binding site on tubulin, such as paclitaxel and
vinblastine, have been widely used in clinic for the treatment of cancers [3]. However,
the extremely structural complexity, low aqueous solubility as well as multidrug
resistance (MDR) and dose-limiting toxicity of these taxane and vinca alkaloid
binding sites inhibitors hampered their clinical applications. Nevertheless, MTAs that
target colchicine binding site on tubulin are generally characterized with simpler
skeletons, such as colchicine (1), combretastatin A-4 (2, CA-4) and their analogues 3
and 4 (Fig.1), which have the potential to overcome these limitations. Interest in these
colchicine binding site inhibitors (CBSIs) has been further raised by the discovery that
tubulin inhibitors especially those bind at colchicine binding site can target existing
vessels in the tumor tissues, thereby provoking a rapid collapse of the tumor
vasculature [4, 5]. Thus, CBSIs endowed with both antimitotic and vascular
disrupting profiles are continuing to attract pharmaceutical interest [6-8], which
provides a novel promising treatment for cancer therapy.
Fig. 1. Structures of the representative tubulin polymerization inhibitors and vascular disrupting
agents (VDAs).
In our prior review about the privileged structures of CBSIs, α,β-unsaturated ketone
structure that is mostly found in chalcones was considered as a privileged bridge in
the development of CBSIs [9]. Representative anti-tubulin chalcones are compound 3
and 4 which were reported by Ducki’s group in 1998, both showed remarkable
anti-proliferative activities [10]. The introduction of methyl on α position of carbonyl

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led to a dramatically improved cytotoxicity due to the more preferential s-trans
conformation adopted by 4, while s-cis conformation by 3 led to less potent activity.
Comprehensive structure-activity relationships (SARs) studies were conducted with a
conclusion that compound 4 bearing 2-methyl substituted α,β-unsaturated ketone as
the bridge was the most active [11, 12].
Vinyl sulfone has long been known for their synthetic utility in organic chemistry,
easily participating in 1,4-addition reactions and cycloaddition reactions [13].
Recently, this moiety has also been shown to exhibit a wide range of biological
activities including cysteine protease inhibition [14], neuroprotection [15, 16], c-FLIP
inhibition [17] and anti-tumor effects [18-20]. Thus, inspired by the structural
similarity of vinyl sulfone or sulfoxide to vinyl carbonyl as Michael acceptors, we
introduced vinyl sulfone or sulfoxide moieties to the structure of compound 3 to
afford a series of novel compounds as potential anti-tubulin agents (Fig. 2).
Considering the vital effect of methyl substituted at α position of α,β-unsaturated
ketone of compound 4, various groups were introduced to the α position of vinyl
sulfones or sulfoxides to explore the SARs. Herein, we report the synthesis, in vitro
and in vivo anti-tumor activity and mechanism studies of a series of novel vinyl
sulfone and sulfoxide derivatives as anti-tubulin agents targeting colchicine binding
site on tubulin.
Fig. 2. The design of vinyl sulfone and sulfoxide derivatives as tubulin inhibitors.
2. Results and discussion
2.1 Chemistry
The synthetic routes for the vinyl sulfones and sulfoxides are outlined in
Schemes 1-2. Intermediate 3,4,5-trimethoxybenzothiol 7 was prepared by diazo
reaction of commercially available 3,4,5-trimethoxyaniline with potassium ethyl
xanthate, following hydrolyzation under basic conditions. The key intermediate
phosphate 9a was obtained by
a
nucleophilic reaction of
7
with
diethoxyphosphorylmethyl 4-methylbenzenesulfonate 8 in the presence of K2CO3 in
acetonitrile. Subsequently, various groups were introduced to the α position of
phosphonate, chlorine-substituted 9b was afforded by treating 9a with N-chlorine

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succinimide (NCS) in carbon tetrachloride, and 9b was further converted to 9c in
refluxing methanol. Deprotonation of α-proton of phosphonate in 9a was carried out
by using n-butyllithium in THF under -78 °C, and the resulting carbanion was reacted
with methyl iodide to give 9d. Then phosphonates 9a-d were further selectively
oxidized to afford corresponding sulfones 10a-d and sulfoxides 11a-d by controlling
the equivalents of m-chloroperbenzoic acid (m-CPBA). Fluoro-substituted sulfone 10e
was obtained by treating 10a with NaH and equivalent selectfluor in THF. Finally, all
prepared phosphonates with sulfone or sulfoxide group were undergone wittig-horner
reactions with various aromatic aldehydes to produce the corresponding vinyl
sulfones 12a-q and vinyl sulfoxides 13a-d. Cyan-substituted vinyl sulfone 12r and
vinyl sulfoxide 13e were synthesized through another route. Intermediate 7 was
reacted with bromoacetonitrile under basic condition, and the furnished 14 was
oxidized to sulfone 15 and sulfoxide 16, which were further undergone Knoevenagel
reactions with acetyl protected isovanillin, and further deprotection of acetyl group
produced target vinyl sulfone 12r and vinyl sulfoxide 13e, respectively. Altogether,
twenty three compounds were finally synthesized as shown in Fig. 3.
Scheme 1. Reagents and conditions: (a) (i) NaNO2, 10% HCl, CH3OH, 0°C; (ii) potassium ethyl
xanthate, 65 °C, 55%; (b) 10% NaOH, CH3OH, rt, 94%; (c) K2CO3, CH3CN, rt, 63%; (d) NCS,
CCl4, rt, 72%; (e) CH3OH, reflux, 90%; (f) (i) n-BuLi, THF, -78°C; (ii) CH3I, -78°C - 45 °C,
48%.

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Scheme 2. Reagents and conditions: (a) m-CPBA (2.2 e.q), DCM, rt, 55-90%; (b) m-CPBA (0.9
e.q), DCM, 0°C, 58-82%; (c) NaH, acetyl protected aldehydes, THF, N2, 0°C; when R3 or R4 = OH
(i) NaH, aldehydes, THF, N2, 0°C; (i) 10% NaOH, CH3OH, rt, 28-87%; (d) NaH, selectfluor, THF;
(e) bromoacetonitrile, K2CO3, CH3CN, rt, 91.5%; (f) (i) aldehydes, piperidine (cat.), AcOH (cat.),
toluene, reflux; (ii) 10% NaOH, CH3OH, rt, 32-34%.
O
O
H3CO
H3CO
S
R4
R3
H3CO
H3CO
S
R4
R3
O
R1
R1
OCH3
12a-r
R2
OCH3
13a-e
R2
12a R1, R2, R3, R4 = H;
12c R1, R2, R4 = H, R3 = N(CH3)2; 12d R1, R2, R4 = H, R3 = CH3;
12b R1, R2, R4 = H, R3 = F;
13a
R1, R2 = H, R3 = OCH3, R4 = OH;
13b R1, R2 = H, R3 = OCH3, R4 = NH2;
13c R1 = Cl, R2 = H, R3 = OCH3, R4 = OH;
13d
12e R1, R2, R4 = H, R3 = NO2;
12g R1, R2 = H, R3, R4 = OCH3;
12i R1, R2, R4 = H, R3 = OH;
12k R1, R2 = H, R3 = OCH3, R4 = F; 12l R1, R2 = H, R3 = OCH3, R4 = OH;
12m R1, R2 = H, R3 = OCH3, R4 = NH2;
12f R1, R2, R4 = H, R3 = OCH3;
12h R1 = H, R2, R3, R4 = OCH3;
12j R1, R2 = H, R3, R4 = OH;
R1 = CH3, R2 = H, R3 = OCH3, R4 = OH;
13e R1 = CN, R2 = H, R3 = OCH3, R4 = OH;
12n R1 = Cl, R2 = H, R3 = OCH3, R4 = OH;
12o R1 = OCH3, R2 = H, R3 = OCH3, R4 = OH;
12p R1 = CH3, R2 = H, R3 = OCH3, R4 = OH;
12q R1 = F, R2 = H, R3 = OCH3, R4 = OH;
12r R1 = CN, R2 = H, R3 = OCH3, R4 = OH;
Fig. 3. The structures of vinyl sulfones 12a-r and vinyl sulfoxides 13a-e.
2.2 In vitro anti-proliferative activity
Given that ring B of classical CBSIs plays a vital effect on determining their
binding affinities to tubulin, we firstly investigated the impact of this moiety on

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activity. Thus, twelve compounds 12a-l with varying substitutions on ring B were
preliminarily screened for their anti-proliferative activities in human leukemia cell
lines (K562) by the MTT assay. The results (see Supporting Information Fig. S1)
indicated that compounds 12j and 12l displayed comparable anti-proliferative
activities to the positive control CA-4 at a concentration of 1 µM. However,
compound 12j was inactive at the concentration of 0.1 µM, and the inhibitive rate of
12l also decreased to 35.3%. Thus, compound 12l bearing isovanillin as ring B was
chosen for next round modifications.
Subsequently, eleven compounds 12m-r and 13a-e were further synthesized, and
their anti-proliferative activities against three human cancer cell lines including
human leukemia cell lines (K562), human hepatocellular carcinoma cells (HepG2)
and human lung adenocarcinoma cells (A549), were evaluated by the MTT assay
using compound 3 and CA-4 as the positive control. The cytotoxicity data of
compounds 12l-r and 13a-e were listed in Table 1, and the results showed that most of
the newly synthesized compounds displayed potent activities against three cancer cell
lines. Vinyl sulfone 12l and vinyl sulfoxide 13a, whose R1 were H exhibited more
potent activity than the compounds with other substitutions as R1. Replacing R1 with
methyl group was not favorable for activity improvement, which is much different
from the result that chalcone compound 4 was 20 folds increase in cytotoxicity
towards 3 [10]. When R1 was replaced with cyan group, both 12r and 13e showed
much less potent activity. Interestingly, replacing hydroxyl with amino group (12m)
led to slight improvements in anti-proliferative activities against K562, HepG2 and
A549 cell lines with the IC50 values of 0.131, 0.217 and 0.606 µM, respectively.
2.3 In vitro tubulin polymerization inhibitory assay
To elucidate whether the synthesized compounds target the tubulin-microtubule
system, the in vitro tubulin polymerization inhibition activities of 12l, 12m, 13a and
13c were evaluated due to their good anti-proliferative activities. The IC50 values
listed in Table 2 showed that these compounds are potent tubulin polymerization
inhibitors. Compound 13c (IC50 = 3.83 µM) was the most potent compound in
inhibiting tubulin polymerization which was comparable to compound 3 (IC50 = 3.69
µM) and slightly less potent than CA-4 (IC50 = 2.17 µM). Compound 12m that
displayed most potent anti-proliferative activity in the cytotoxic assay also showed
potent activity in tubulin polymerization inhibition with an IC50 value of 4.25 µM
.
Therefore, taking considerations of the potent activities of compound 12m showing
both in the in vitro anti-proliferative assay and tubulin polymerization inhibition assay,
12m was selected for further biological studies.

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Table 1
Anti-proliferative activities of compounds against three human cancer cell linesa
IC50 values (µM)b
Compd.
K562
HepG2
A549
0.219±0.019 0.267±0.010 2.282±0.170
12l
12m
12n
12o
12p
12q
12r
13a
13b
13c
13d
13e
3
0.128±0.003 0.217±0.015 0.606±0.084
>1
>1
>5
0.190±0.011 0.599±0.055 2.885±0.044
0.580±0.031 0.710±0.102 2.734±0.153
0.473±0.010 0.591±0.006 1.312±0.147
>1
>1
>5
0.258±0.008 0.307±0.038 1.455±0.291
0.746±0.038 0.753±0.022 2.354±0.158
0.183±0.009 0.243±0.042 1.204±0.254
>1
>1
>1
>1
>5
>5
0.060±0.009 0.102±0.013 0.387±0.026
0.021±0.001 0.012±0.001 0.037±0.001
CA-4
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by the
MTT assay as described in the Experimental Section.
b IC50 values are indicated as the mean ± SD (standard error) of at least three independent experiments.
Table 2
Effects of the selected compounds on tubulin polymerization inhibition.
Compd.
IC50 values
(µM)a
12l
12m
13a
13c
3
CA-4
8.23±0.92 4.25±0.75 7.18±0.88 3.83±0.42 3.69±0.13 2.17±0.02
a IC50 values are indicated as the mean ± SD (standard error) of three independent experiments.
2.4 Anti-microtubule effects in K562 cells
Immunofluorescent assay was performed to investigate the effect of compound
12m on microtubule networks. As shown in Fig. 4, K562 cells without drug treatment
exhibited normal filamentous microtubules arrays. However, after exposure to 12m
(0.05 µM, 0.10 µM, 0.20 µM) or CA-4 (0.005 µM, 0.010 µM, 0.020 µM) for 24 h, the
microtubule networks in cytosol were disrupted; these results indicated that 12m can
induce a dose-dependent collapse of the microtubule networks.

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nucleus
microtubule
merged
nucleus
microtubule
B)
merged
A)
control
control
0.05 µM
0.10 µM
0.005 µM
0.010 µM
0.020 µM
0.20 µM
Fig. 4. Effects of compound 12m (A) and CA-4 (B) on the cellular microtubule networks visualized by
immunofluorescence. K562 cells were treated with vehicle control 0.1% DMSO, 12m (0.05 µM, 0.10 µM, 0.20
µM), CA-4 (0.005 µM, 0.010 µM, 0.020 µM). Then, the cells were fixed and stained with anti-α-tubulin−FITC
antibody (green), Alexa Fluor 488 dye and counterstained with DAPI (blue).
2.5 Cell cycle analysis
Most microtubule polymerization inhibitors disrupt cell mitosis and exert cell cycle
arrest effects [21]. Thus, the effect of 12m on cell cycle progression was examined
using propidiumiodide (PI) staining by flow cytometry analysis in K562 cells. As
depicted in Fig. 5, compound 12m caused a concentration-dependent G2/M arrest in
K562 cells. When treated with 12m at 0.05, 0.10, and 0.20 µM for 48 h, the
percentages of cells arrested at the G2/M phase were 13.05%, 16.15%, and 21.99%,
respectively, while that in the CA-4 treated groups were 13.84%, 22.59% and 24.95%
at 0.005, 0.010, and 0.020 µM, respectively. These results demonstrated that
compound 12m can induce the cell cycle arrest at the G2/M phase.
Fig. 5．Compound 12m (A) and CA-4 (B) induced G2/M arrest in K562 cells. K562 cells were incubated with

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varying concentrations of 12m (0, 0.05, 0.1, and 0.2 µM) or CA-4 (0, 0.005, 0.010, and 0.020 µM) for 48 h. Cells
were harvested and stained with PI and then analyzed by flow cytometry. The percentages of cells in different
phases of cell cycle were analyzed by ModFit 4.1.
2.6 Cell apoptosis analysis
Most microtubule polymerization inhibitors can up-regulate the expression of
pro-apoptotic proteins and down-regulate the expression of antiapoptotic proteins,
thus inducing cell apoptosis [22]. To assess whether compound 12m would induce
cell apoptosis, 12m treated K562 cells were stained with annexin V-FITC and
propidium iodide (PI) and analyzed by flow cytometry. As shown in Fig. 6, compound
12m induced K562 cell apoptosis in a dose-dependent manner. The percentage of
apoptotic cells after the 48 h treatment was only 1.46% in the control group. However,
the total numbers of early (Annexin -V+/PI−) and late (Annexin-V+/PI+) apoptotic
cells increased to 17.88%, 22.65% and 49.29% after treatment with 12m at 0.05, 0.10,
0.20 µM for 48 h, respectively (Fig. 6A). Similarly, after treatment with CA-4 at 0.005,
0.010, 0.020 µM for 48 h, the total numbers of early and late apoptotic cells increased
to 25.28%, 53.05% and 63.05%, respectively (Fig. 6B). These results confirmed that
compound 12m and CA-4 effectively induced cell apoptosis in K562 cells in a
dose-dependent manner (Fig. 6C and Fig. 6D).

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Fig. 6. (A) Compound 12m induced apoptosis in K562 cells. K562 cells were incubated with varying
concentrations of 12m (0, 0.05, 0.1, and 0.2 µM). After 48 h of incubation, cells were collected and stained with
Annexin-V/PI, followed by flow cytometric analysis. The percentages of cells in each stage of cell apoptosis were
quantified by flow cytometry: (upper left quadrant) necrosis cells; (upper right quadrant) late-apoptotic cells;
(bottom left quadrant) live cells; and (bottom right quadrant) early apoptotic cells. (B) CA-4 induced apoptosis in
K562 cells. (C) Histograms display the percentage of cell distribution after treatment with 12m. (D) Histograms
display the percentage of cell distribution after treatment with CA-4.
2.7 In vitro evaluation of anti-vascular activity
Most CBSIs possess vascular disrupting activity, which is thought to disrupt
microtubule dynamics to induce endothelial cell shape change. To evaluate the
anti-vascular activity of compound 12m, we used HUVEC culture assay to assess the
ability of 12m to inhibit HUVEC migration which is the key step to generate new
blood vessels. As shown in Fig. 7A, the untreated cells migrated to fill the area that
was initially scraped after 24 h. In contrast, compound 12m and CA-4 significantly
inhibited the HUVEC migration in a dose-dependent manner.
Then we evaluated the ability of compound 12m in a tube formation assay. After
being seeded on Matrigel, HUVECs form the capillary-like tubules with multicentric

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junctions. After exposure to 12m (0.05 µM, 0.10 µM, 0.20 µM) or CA-4 (0.005 µM,
0.010 µM, 0.020 µM) for 6 h, the capillary-like tubes were interrupted in different
levels (Fig. 7B). These results showed that compound 12m can effectively inhibit the
tube formation of HUVECs.
Fig. 7. Effects on the HUVECs migration and tube formation. (A) Scratches were created with sterile 200 µL
pipette and images were captured using phase contrast microscopy at 0 h and 24 h after treatment with 12m (0.05
µM, 0.10 µM, 0.20 µM) or CA-4 (0.005 µM, 0.010 µM, 0.020 µM). (B) Images depicting the formation of HUVEC
capillary-like tubular network by treatment with 12m (0.05 µM, 0.10 µM, 0.20 µM) or CA-4 (0.005 µM, 0.010 µM,
0.020 µM) for 6h.
2.8 In vivo anti-tumor activity of 12m
To evaluate the in vivo anti-tumor activity of 12m, liver cancer allograft mouse
model was established by subcutaneous inoculation of H22 cells into the right flank of
mice. The tumor size and the body weights of mice were monitored and recorded
every 2 days. Paclitaxel (PTX) was selected as the positive control. As shown in Fig.
8, the reduction in tumor weight reached 74.60% at a dose of 8 mg/kg/2 day (i.v.) of
PTX at 21 days after initiation of treatment as compared to vehicle. Treatment with
12m at doses of 15 and 30 mg/kg/day (i.v.) resulted in tumor inhibitory rates of 53.4%
and 65.4%, respectively, which is more potent than CA-4 treatment groups (inhibitory
rates of 45.0% and 56.7% at doses of 15 and 30 mg/kg/day, respectively) (Fig. 8b).
Notably, 12m did not significantly affected body weight even at the dose up to 30
mg/kg, while treatment with PTX at a dose of 8 mg/kg/2 day led to a significant
decrease of body weight (Fig. 8a). Thus, compound 12m was efficacious and safe in
inhibiting tumor growth with a dose-dependent manner in vivo and deserved further
evaluation.

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Fig. 8. a) Body weight changes of mice during treatment. (b) 12m treatment resulted in significantly lower tumor
weight compared with controls.
2.9 Docking study
To illustrate the binding mode of the most active compound 12m with tubulin,
docking study was performed by using the DOCK program in the Discovery Studio
3.0 software with the tubulin crystal structure (PDB: 1SA0). Resembling the binding
mode of colchicine with tubulin (Fig. 9A), the trimethoxyphenyl moiety of 12m was
positioned in the binding cavity buried in tubulin while the Met259 and Thr314
residues hold the tail of 12m (3-amino-4-methoyl phenyl moiety) (Fig. 9C). The
introduction of methyl group on α position of carbonyl in chalcone compound 3 led to
a dramatically improved cytotoxicity of compound 4, which partly due to the more
preferential s-trans conformation adopted by 4 [10]. Besides, the methyl located at
α-position of carbonyl in compound 4 increased the hydrophobic interaction with the
residue Lys254 (Fig. 9B). However, methyl group substituted at α-position of sulfone
in compound 12p didn’t led to a significant improvement of activity, we speculated
that the trimethoxyphenyl moiety of 12p cannot be oriented into the deep cavity due
to the hydrophobic interaction of methyl group with the residue Lys254 (Fig. 9C).

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Fig. 9. Proposed binding models for representative compounds binding with tubulin (PDB code: 1SA0) (A)
colchicine (shown in orange); (B) compound 3 (shown in cyan) and 4 (shown in yellow); (C) 12m (shown in pink)
and 12p (shown in white).
3. Conclusions
In summary, a series of novel vinyl sulfone and sulfoxide derivatives have been
designed, synthesized, and evaluated as tubulin polymerization inhibitors by replacing
the carbonyl group of chalcone compound 3. Preliminary anti-proliferative screening
of twelve new compounds validated 12l as a high cytotoxic compound. Following
optimization of this lead resulted in the further synthesis and biological evaluations of
eleven derivatives. Among them, compound 12m showed the most potent activity
against three cancer cell lines with IC50 values ranging from 0.128 to 0.606 µM. It was
found that substitutions at α position of vinyl sulfones or sulfoxides were not
favorable for the improvement of activity, which is unlike chalcone compound 3.
Besides, compound 12m also displayed moderate inhibitory activity in tubulin
assembly (IC50 = 4.25 µM). Furthermore, mechanism studies demonstrated that 12m
caused cell cycle arrest in the G2/M phase and induced cell apoptosis in K562 cells.
And the immunofluorescent assay indicated that 12m can effectively disrupt
microtubule networks in a dose-dependent manner. The wound healing and tube
formation assays further identified 12m as a novel tubulin polymerization inhibitor
with potent vascular disrupting activity. Moreover, molecular modelling studies
demonstrated that 12m interacted with tubulin at the colchicine binding site. Finally,

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the in vivo anti-tumor activity of 12m was validated in H22 liver cancer allograft
mouse model with the tumor growth inhibitory activity more potent than CA-4.
Altogether, vinyl sulfone derivative 12m may represent a novel class of anti-tubulin
agent with potent anti-vascular and anti-tumor activity and deserves further
investigation.
4. Experimental
4.1 Chemistry
4.1.1.General
Most chemicals and solvents were purchased from commercial sources. Further
1
purification and drying by standard methods were employed when necessary. H
NMR and 13C NMR spectra were recorded on Bruker-300 spectrometers in the
indicated solvents (TMS as internal standard). Data are reported as follows: chemical
shift in ppm (d), multiplicity (s =singlet, d =doublet, t =triplet, q =quartet, brs = broad
singlet, m =multiplet), coupling constant (Hz), and integration. High Resolution Mass
measurement was performed on Agilent QTOF 6520 mass spectrometer with electron
spray ionization (ESI) as the ion source. Purity of all tested compounds was ≥ 95%, as
estimated by HPLC analysis. Flash column chromatography was carried out using
commercially available silica gel (200-300 mesh) under pressure.
4.1.2. Synthesis of intermediates 9a-d
4.1.2.1. 3,4,5-Trimethoxybenzenethiol 7. 3,4,5-Trimethoxybenzenethiol 7 was
synthesized according to literature report [21]. 3,4,5-trimethoxyaniline 5 (2.0 g, 10.9
mmol) was dissolved into the mixture of methanol (5 mL) and concentrated HCl (5
mL), sodium nitrite (1g, 14.4 mmol) dissolved with H2O (5 mL) was added slowly at
0 °C. The temperature was kept below 5 °C and the reaction mixture was stirred until
it became a clear solution, after which the reaction solution was added slowly to a
solution of potassium ethyl xanthate (3.4 g, 21.2 mmol) in H2O (20 mL) at 65 °C. The
mixture was stirred at 65 °C for 30 min and cooled to room temperature. The resulting
mixture was extracted with EtOAc (3 × 50 mL), and the combined organic extracts
were then washed with brine (100 mL), dried over Na2SO4 and concentrated in vacuo.
The crude product was purified by flash column chromatography (silica gel, PE/EA
15:1) affording xanthate 6 in 55.8% yield (1.73 g). Then, to a solution of xanthate 6 (1
g, 3.5 mmol) in ethanol (20 mL), a solution of sodium hydroxide (3 M, 20 mL) was
added and the reaction was heated to 65 °C. The reaction was stirred at 65 °C for 3 h
and cooled to room temperature. The mixture was acidified to pH 5 by the addition of
10% aqueous HCl and extracted with EtOAc (3 × 100 mL). The combined organic
layers were then washed with brine (200 mL), dried over Na2SO4 and concentrated in

ACCEPTED MANUSCRIPT
vacuo. The crude 3,4,5-trimethoxybenzenethiol 7 (640 mg, 94%) was used directly
for the next step without further purification.
4.1.2.2. Intermediate 9a. To a solution of 7 (5.6 g, 28 mmol) in MeCN (50 mL),
potassium carbonate (4.6 g, 33.6 mmol) was added. After stirring for 10 min,
diethoxyphosphorylmethyl 4-methylbenzenesulfonate 8 was added into the mixture,
and the reaction was stirred at 50 °C for 2 h. The reaction mixture was then extracted
with EtOAc (3 × 100 mL). The combined organic layers were then washed with brine
(100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography using CH2Cl2/MeOH (200/1,V/V) as an
1
eluent to afford the product 9a as yellow oil (600 mg, 63%). H NMR (300 MHz,
CDCl3) δ 6.77 (s, 2H), 4.21-4.11 (m, 4H), 3.86 (s, 6H), 3.83 (s, 3H), 3.19 (d, J = 13.8
Hz, 2H), 1.33 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 152.85, 137.04,
129.75 (d, J = 5.3 Hz), 107.44, 62.25 (d, J = 6.8 Hz), 60.38, 55.73, 29.20 (d, J = 147.8
Hz), 15.91 (d, J = 6.0 Hz); ESI-MS m/z 350.1 [M+Na]+ 373.1.
4.1.2.3. Intermediate 9b. To a solution of 9a (1.9 g, 5.43 mmol) in CCl4 (25 mL),
NCS (863 mg, 6.52 mmol) was added. The mixture was stirred at room temperature
for 2 hours. The insoluble materials were filtered off and the filtrate was concentrated
in vacuo. The residue was purified by flash column chromatography using
CH2Cl2/MeOH (200/1,V/V) as an eluent to afford the product 9b as colorless oil (1.52
1
g, 72%). H NMR (300 MHz, CDCl3) δ 6.85 (s, 2H), 5.23 (d, J = 12.3 Hz, 1H), 4.30
(p, J = 7.1 Hz, 4H), 3.86 (s, 6H), 3.84 (s, 3H), 1.39 (t, J = 7.1 Hz, 6H); 13C NMR (75
MHz, CDCl3) δ 152.93, 138.65, 126.07 (d, J = 10.5 Hz), 110.06, 64.11 (t, J = 6.0 Hz),
61.15 (d, J = 147.8 Hz), 60.37, 55.78, 15.93 (d, J = 6.0 Hz); ESI-MS m/z 384.1
[M-Cl]+ 349.1, [M+Na]+ 407.0.
4.1.2.4. Intermediate 9c. A solution of 9b (500 mg, 1.42 mmol) in MeOH was
refluxed for 30 min. The reaction mixture was concentrated in vacuo to afford the
1
intermediate 9c as yellow oil (488 mg, 90%). H NMR (300 MHz, CDCl3) δ 6.83 (s,
2H), 4.93 (d, J = 10.5 Hz, 1H), 4.32-4.09 (m, 4H), 3.83 (s, 6H), 3.82 (s, 3H), 3.55 (s,
3H), 1.34 (td, J = 7.0, 2.9 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 152.71, 137.78,
128.25 (d, J = 7.5 Hz), 109.75, 86.35 (d, J = 179.3 Hz), 63.08 (m), 60.34, 56.57 (d, J
= 10.5 Hz), 55.71, 15.95 (d, J = 6.0 Hz); ESI-MS m/z 380.1 [M+Na]+ 403.1.
4.1.2.5. Intermediate 9d. To a solution of 9a (1 g, 2.86 mmol) in anhydrous THF, a
solution of n-BuLi in n-hexane (2.5 M, 1.5 mL, 3.4 mmol) was added dropwise at
-78 °C under N2 atmosphere. After stirring for 30 min, a solution of CH3I (212 µL, 3.1
mmol) in anhydrous THF was added, then the reaction was warmed up to room
temperature and monitored by TLC. After reaction finished, the mixture was extracted
with CH2Cl2 (3 × 50 mL). The combined organic layers were then washed with brine
(50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography using CH2Cl2/MeOH (200/1,V/V) as an

ACCEPTED MANUSCRIPT
1
eluent to afford the product 9d as yellow oil (500 mg, 48%). H NMR (300 MHz,
CDCl3) δ 6.83 (s, 2H), 4.19 (td, J = 7.3, 5.0 Hz, 4H), 3.87 (s, 6H), 3.84 (s, 3H), 3.25
(dd, J = 16.1, 7.4 Hz, 3H), 1.53 (dd, J = 17.1, 7.4 Hz, 3H), 1.34 (t, J = 7.1 Hz, 6H);
13C NMR (75 MHz, CDCl3) δ 152.64, 137.54, 127.88 (d, J = 6.1 Hz), 109.75, 62.30
(dd, J = 12.4, 7.0 Hz), 60.34, 55.71, 38.95 (d, J = 141.8 Hz), 16.01 (d, J = 2.3 Hz),
15.93 (d, J = 1.5 Hz); ESI-MS m/z 364.1 [M+Na]+ 387.1.
4.1.3. Synthesis of intermediates 10a-e.
4.1.3.1. General procedures for synthesis of intermediates 10a-d. To a solution of
9a-d (1 mmol) in CH2Cl2, m-CPBA (75%, 2.2 mmol) was added. After reaction
completed, the mixture was extracted with CH2Cl2 (3 × 50 mL). The combined
organic layers were then washed with saturated NaHCO3, brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by flash column
chromatography to afford the products 10a-d in 55-90% yields.
4.1.3.1.1 Intermediate 10a. Colorless oil, yield 86.7%. 1H NMR (300 MHz, CDCl3) δ
7.21 (s, 2H), 4.27-4.08 (m, 4H), 3.91 (s, 6H), 3.89 (s, 3H), 3.76 (d, J = 17.0 Hz, 2H),
1.30 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 152.80, 142.12, 134.14, 104.96,
62.84 (d, J = 6.0 Hz), 60.44, 56.00, 53.5 (d, J = 136.5 Hz), 15.75 (d, J = 6.0 Hz);
ESI-MS m/z 382.1 [M+Na]+ 405.1.
4.1.3.1.2 Intermediate 10b. Colorless oil, yield 55.4%. 1H NMR (300 MHz, CDCl3) δ
7.23 (s, 2H), 4.92 (d, J = 12.9 Hz, 1H), 4.36-4.23 (m, 4H), 3.92 (s, 4H), 3.92 (s, 6H),
13
1.36 (td, J = 7.0, 3.2 Hz, 6H); C NMR (75 MHz, CDCl3) δ 152.58, 142.91, 129.73,
106.98, 65.88 (d, J = 155.3 Hz), 64.72 (m), 60.52, 56.03, 15.80 (d, J = 6.0 Hz);
ESI-MS m/z 416.0 [M+Na]+ 439.0.
4.1.3.1.3 Intermediate 10c. Colorless oil, yield 78.4%. 1H NMR (300 MHz, CDCl3) δ
7.18 (s, 2H), 4.48 (d, J = 12.4 Hz, 1H), 4.16-4.14 (m, 4H), 3.86 (s, 6H), 3.85 (s, 3H),
3.57 (s, 3H), 1.25 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 152.53, 142.42, 130.90,
106.60, 93.38 (d, J = 165.8 Hz), 63.63 (dd, J = 6.7, 3.5 Hz), 62.83 (d, J = 6.6 Hz),
60.41, 55.95, 15.78 (dd, J = 5.9, 3.1 Hz); ESI-MS m/z 412.1 [M+Na]+ 435.1.
4.1.3.1.4 Intermediate 10d. Colorless oil, yield 90.3%. 1H NMR (300 MHz, CDCl3) δ
7.21 (s, 2H), 4.26-4.15 (m, 4H), 3.93 (s, 6H), 3.92 (s, 3H), 3.62 (dd, J = 19.0, 7.4 Hz,
13
1H), 1.56 (dd, J = 15.8, 7.4 Hz, 3H), 1.33 (t, J = 7.0 Hz, 6H); C NMR (75 MHz,
CDCl3) δ 152.62, 142.06, 132.12, 105.99, 62.84 (d, J = 6.0 Hz), 59.8 (d, J = 137.3
Hz), 55.98, 15.79 (dd, J = 6.2, 2.3 Hz), 11.01 (d, J = 4.1 Hz); ESI-MS m/z 396.1
[M+Na]+ 419.1.
4.1.3.2. Intermediate 10e. To a solution of 10a (500 mg, 1.31 mmol) in anhydrous
THF, a solution of selectfluor (510 mg, 1.44 mmol) in DMF was added dropwise at
0°C. After reaction completed, the mixture was extracted with CH2Cl2 (3 × 50 mL).

ACCEPTED MANUSCRIPT
The combined organic layers were then washed with saturated NaHCO3, brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by flash
column chromatography using CH2Cl2/MeOH (150/1,V/V) as an eluent to afford the
intermediate 10e as yellow oil (270 mg, 51.6%). 1H NMR (300 MHz, CDCl3) δ 7.25
(s, 2H), 5.44 (dd, J = 45.6, 6.4 Hz, 1H), 4.41-4.28 (m, 4H), 3.97 (s, 3H), 3.96 (s, 6H),
1.41 (t, J = 7.1 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 152.88, 143.01, 129.53, 106.30,
95.25 (d, J = 156.8 Hz), 64.50 (dd, J = 15.5, 6.7 Hz), 60.54, 56.05, 15.84 (d, J = 5.9
Hz); ESI-MS m/z 400.1 [M+Na]+ 423.1.
4.1.4. General procedures of intermediates 11a-d.
To a solution of 9a-d (1 mmol) in CH2Cl2, m-CPBA (75%, 0.95 mmol) were added
slowly at 0 °C. After reaction completed, the mixture was extracted with CH2Cl2 (3 ×
50 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by flash
column chromatography to afford the products 11a-d in 58-82% yields.
1
4.1.4.1 Intermediate 11a. Colorless oil, yield 82.3%. H NMR (300 MHz, CDCl3) δ
6.93 (s, 2H), 4.22-4.02 (m, 4H), 3.87 (s, 6H), 3.82 (s, 3H), 3.30 (dt, J = 29.9, 14.6 Hz,
13
2H), 1.29 (dt, J = 14.8, 7.1 Hz, 6H); C NMR (75 MHz, CDCl3) δ 153.49, 139.77,
138.92 (d, J = 8.4 Hz), 100.23, 62.36 (dd, J = 17.6, 6.4 Hz), 60.35, 55.87, 54.82 (d, J
= 137.3 Hz), 15.79 (t, J = 5.7 Hz); ESI-MS m/z 366.1 [M+Na]+ 389.1.
1
4.1.4.2 Intermediate 11b. Colorless oil, yield 58.2%. Mixture of α and β isomers. H
NMR (300 MHz, CDCl3) δ 7.08 (s, 0.7 H), 6.92 (s, 1.3H), 4.79 (d, J = 12.0 Hz,
0.35H), 4.55 (d, J = 12.0 Hz, 0.65H), 4.41-4.34 (m, 1.4H), 4.32-4.21 (m, 2.6H), 3.94
(s, 2.1H), 3.93 (s, 3.9H), 3.92 (s, 1H), 3.91 (s, 2.1H), 1.47-1.31 (m, 6H); ESI-MS m/z
400.1 [M+Na]+ 423.0.
1
4.1.4.3 Intermediate 11c. Colorless oil, yield 75.4%. Mixture of α and β isomers. H
NMR (300 MHz, CDCl3) δ 7.06 (s, 1.5H), 7.01 (s, 0.4H), 4.44 (d, J = 7.5 Hz, 0.8H),
4.28-4.11 (m, 4H), 3.91 (s, 6H), 3.88 (s, 3H), 3.54 (s, 0.8H), 3.47 (s, 2.2H), 1.37-1.25
(m, 6H); ESI-MS m/z 396.1 [M+Na]+ 419.1.
1
4.1.4.4 Intermediate 11d. Colorless oil, yield 75.8%. Mixture of α and β isomers. H
NMR (300 MHz, CDCl3) δ 7.02 (s, 1.4H), 6.84 (s, 0.6H), 4.29-4.14 (m, 2.8H),
4.14-4.08 (m, 1.2H), 3.91 (s, 4.2H), 3.91 (s, 2.1H), 3.89 (s, 1.8H), 3.88 (s, 0.9H), 3.38
-3.29 (m, 0.7H), 2.97-2.86 (m, 0.3H), 1.39-1.19 (m, 9H); ESI-MS m/z 380.1 [M+Na]+
403.1.
4.1.5. General procedures for synthesis of compounds 12a-k, 12m. To a solution of
10a (0.2 mmol) in anhydrous THF, NaH (60%, 0.24 mmol) was added at 0 °C under
N2 atmosphere. After stirring for 15 min, aromatic aldehyde (0.24 mmol) was added
into the mixture and the reaction was stirred for another 1 h. The reaction mixture was

ACCEPTED MANUSCRIPT
then extracted with EtOAc (3 × 20 mL). The combined organic layers were then
washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by flash column chromatography using PE/EA
(5/1,V/V) as an eluent to afford the compounds 12a-k, 12m.
4.1.5.1. (E)-1,2,3-trimethoxy-5-(styrylsulfonyl)benzene (12a). White solid, yield
1
76.7%, m.p. 122-124 °C. H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 15.4 Hz, 1H),
7.45-7.17 (m, 5H), 7.17 (s, 2H), 6.87 (d, J = 15.4 Hz, 1H), 3.92 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 153.63, 141.86, 135.25, 132.44, 131.16, 129.09, 128.55, 127.47,
105.04, 60.93, 56.51; HR-MS (ESI) m/z: calcd for C17H18O5S [M+H]+ 335.0948,
found 335.0945.
4.1.5.2. (E)-5-((4-fluorostyryl)sulfonyl)-1,2,3-trimethoxybenzene (12b). White solid,
yield 65.2%, m.p. 130-132 °C.
1H NMR (300 MHz, CDCl3) δ 7.61 (d, J = 15.4 Hz,
1H), 7.16 (s, 2H), 7.12-7.06 (m, 2H), 6.83-6.77 (d, J = 15.4 Hz, 1H), 5.32-4.90 (m,
13
1H), 3.91 (s, J = 5.3 Hz, 9H); C NMR (75 MHz, CDCl3) δ 165.42 (d, J = 33.8 Hz)
153.15, 141.92, 140.02, 134.62, 130.08 (d, J = 6.8 Hz), 128.16, 126.72 (d, J = 2.3 Hz),
115.83 (d, J = 22.5 Hz), 104.51, 60.44, 56.01; HR-MS (ESI) m/z: calcd for
C17H17FO5S [M+H]+ 353.0853, found 353.0855.
4.1.5.3. (E)-N,N-dimethyl-4-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)aniline (12c).
Yellow solid, yield 65.3%, m.p. 175-177 °C.
1H NMR (300 MHz, CDCl3) δ 7.56 (d, J
= 15.2 Hz, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.15 (s, 2H), 6.64 (d, J = 8.8 Hz, 2H), 6.58
(d, J = 15.2 Hz, 1H), 3.91 (s, 6H), 3.88 (s, 3H), 3.01 (s, 6H); 13C NMR (75 MHz,
CDCl3) δ 153.49, 152.28, 142.75, 141.85, 136.59, 130.37, 120.81, 119.25, 111.73,
104,68, 60.91, 56.47, 40.03; HR-MS (ESI) m/z: calcd for C19H23NO5S [M+H]+
378.1370, found 378.1366.
4.1.5.4. (E)-1,2,3-trimethoxy-5-((4-methylstyryl)sulfonyl)benzene (12d). White solid,
1
yield 85.3%, m.p. 120-121 °C. H NMR (300 MHz, CDCl3) δ 7.62 (d, J = 15.4 Hz,
1H), 7.39 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 7.16 (s, 2H), 6.81 (d, J = 15.4
Hz, 1H), 3.91 (s, 6H), 3.90 (s, 3H), 2.37 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 153.10,
141.76, 141.45, 141.33, 134.98, 129.31, 129.18, 128.07, 125.72, 104.45, 60.43, 56.00,
20.98; HR-MS (ESI) m/z: calcd for C18H20O5S [M+H]+ 349.1104, found 349.1103.
4.1.5.5. (E)-1,2,3-trimethoxy-5-((4-nitrostyryl)sulfonyl)benzene (12e). Yellow solid,
yield 67.3%, m.p. 178-180 °C. 1H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 8.6 Hz, 2H),
7.63-7.58 (m, 3H), 7.09 (s, 2H), 6.95 (d, J = 15.5 Hz, 1H), 3.85 (s, 6H), 3.84 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 153.76, 148.99, 142.83, 138.58, 138.46, 134.11, 131.83,
129.22, 124.26, 105.25, 60.98, 56.55; HR-MS (ESI) m/z: calcd for C17H17NO7S
[M+NH4]+ 397.1064, found 397.1067.
4.1.5.6. (E)-1,2,3-trimethoxy-5-((4-methoxystyryl)sulfonyl)benzene (12f). White solid,
1
yield 95.7%, m.p. 138-140 °C. H NMR (300 MHz, CDCl3) δ 7.60 (d, J = 15.3 Hz,
1H), 7.44 (d, J = 8.5 Hz, 2H), 7.16 (s, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 15.3

ACCEPTED MANUSCRIPT
Hz, 1H), 3.91 (s, 6H), 3.89 (s, 3H), 3.83 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 162.09,
153.58, 142.24, 141.68, 135.74, 130.35, 125.03, 124.61, 114.54, 104.87, 60.92, 56.49,
55.43; HR-MS (ESI) m/z: calcd for C18H20O6S [M+H]+ 365.1053, found 365.1053.
4.1.5.7. (E)-5-((3,4-dimethoxystyryl)sulfonyl)-1,2,3-trimethoxybenzene (12g). White
solid, yield 82.4%, m.p. 145-147 °C. 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J = 15.3
Hz, 1H ), 7.17 (s, 2H), 7.10 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.87 (d, J = 8.3 Hz, 1H),
6.73 (d, J = 15.3 Hz, 1H), 3.92 (s, 6H), 3.90 (s, 3H), 3.89 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 153.09, 151.41, 148.90, 145.67, 141.45, 135.15, 124.76, 124.32, 122.91,
110.66, 109.63, 104.43, 60.43, 56.00, 55.52, 55.47; HR-MS (ESI) m/z: calcd for
C19H22O7S [M+H]+ 395.1159, found 395.1165.
4.1.5.8.
(E)-1,2,3-trimethoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)benzene
(12h). White solid, yield 90.9%, m.p. 151-152 °C. 1H NMR (300 MHz, CDCl3) δ 7.56
(d, J = 15.3 Hz, 1H), 7.17 (s, 2H), 6.79 (d, J = 15.3 Hz, 1H), 6.72 (s, 2H), 3.92 (s, 6H),
3.90 (s, 3H), 3.87 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 153.12, 153.06, 141.86,
141.44, 140.47, 134.81, 127.22, 125.94, 105.45, 104.51, 60.46, 56.01, 55.75; HR-MS
(ESI) m/z: calcd for C20H24O8S [M+H]+ 425.1265, found 425.1269.
4.1.5.9. (E)-4-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)phenol (12i). Green solid,
1
yield 89.0%, m.p. 155-157 °C. H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 7.55
(d, J = 7.9 Hz, 2H), 7.49 (d, J = 15.6 Hz, 1H), 7.31 (d, J = 15.6 Hz, 1H), 7.15 (s, 1H),
6.79 (d, J = 7.6 Hz, 2H), 3.85 (s, 6H), 3.72 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ
160.83, 153.66, 142.12, 141.80, 136.67, 131.45, 124.26, 123.90, 116.32, 104.85,
60.67, 56.81; HR-MS (ESI) m/z: calcd for C20H24O8S [M+H]+ 351.0902, found
351.0905.
4.1.5.10. (E)-4-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)benzene-1,2-diol (12j).
Green solid, yield 88.5%, m.p. 186-188 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.73 (s,
1H), 7.15 (s, 1H), 7.42 (d, J = 15.3 Hz, 1H), 7.26 (d, J = 15.2 Hz, 1H), 7.18 (s, 2H),
7.08 (s, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 3.88 (s, 6H), 3.75 (s,
3H); 13C NMR (75 MHz, DMSO-d6) δ 153.14, 148.95, 145.62, 142.00, 141.29,
136.24, 123.96, 123.83, 122.05, 115.68, 115.48, 104.35, 60.16, 56.31; HR-MS (ESI)
m/z: calcd for C17H18O7S [M+H]+ 367.0846, found 367.0845.
4.1.5.11. (E)-5-((3-fluoro-4-methoxystyryl)sulfonyl)-1,2,3-trimethoxybenzene (12k).
White solid, yield 95.9%, m.p. 170-172 °C. 1H NMR (300 MHz, CDCl3) δ 7.54 (d, J
= 15.2 Hz, 1H), 7.25-7.22 (m, 2H), 7.16 (s, 2H), 6.96 (d, J = 8.3 Hz, 1H), 6.73 (d, J =
15.3 Hz, 1H), 3.92 (s, 12H); 13C NMR (75 MHz, CDCl3) δ 154.02, 153.62, 150.52 (d,
J = 12.9 Hz), 140.51, 135.32, 126.21, 126.11 (d, J = 3.0 Hz), 125.51 (d, J = 6.8 Hz),
124.16 (d, J = 39.8 Hz), 115.15 (d, J = 18.8 Hz), 113.32, 104.96, 60.92, 56.50, 56.28;
HR-MS (ESI) m/z: calcd for C18H19FO6S [M+H]+ 383.0959, found 383.0963.
4.1.5.12. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)aniline (12m).
Yellow solid, yield 60%, m.p. 104-106 °C. 1H NMR (300 MHz, CDCl3) δ 7.52 (d, J =

ACCEPTED MANUSCRIPT
15.3 Hz, 1H), 7.14 (s, 2H), 6.89 (dd, J = 8.2, 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H),
6.76 (d, J = 8.3 Hz, 1H), 6.65 (d, J = 15.3 Hz, 1H), 3.87-3.91 (m, 12H); 13C NMR (75
MHz, CDCl3) δ 153.01, 149.43, 142.01, 141.41, 136.31, 135.38, 124.78, 123.52,
120.38, 112.64, 109.62, 104.15, 60.47, 55.96, 55.11; HR-MS (ESI) m/z: calcd for
C18H21NO6S [M+H]+ 380.1162, found 380.1167.
4.1.6 General procedures for synthesis of compounds 12l, 12n-q, 13a-d.
To a solution of 10a-d (0.2 mmol) in anhydrous THF, NaH (60%, 0.24 mmol)
was added at 0 °C under N2 atmosphere. After stirring for 15 min, acetyl protected
isovanillin (0.24 mmol) was added into the mixture and the reaction was stirred for
another 1 h. The reaction mixture was then extracted with EtOAc (3 × 20 mL). The
combined organic layers were then washed with saturated brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was dissolved in MeOH (5 mL), 5%
sodium hydroxide (5 mL) was added. After stirring for 30 min at room temperature,
the reaction mixture was acidified by the addition of 10% aqueous HCl, and then
diluted with EtOAc, washed with water, and brine successively, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by flash column
chromatography using PE/EA (4/1,V/V) as an eluent to afford the compounds 12l,
12n-q, 13a-d.
4.1.6.1. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)phenol (12l).
White solid, yield 87.0%,
m.p. 127-129 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.20 (s,
1H), 7.45 (d, J = 15.3 Hz, 1H), 7.33 (d, J = 15.3 Hz, 1H), 7.17 (s, 2H), 7.15-7.09 (m,
13
2H), 6.97 (d, J = 8.3 Hz, 1H), 3.86 (s, 6H), 3.81 (s, 3H), 3.73 (s, 3H); C NMR (75
MHz, DMSO-d6) δ 153.65, 151.02, 147.18, 142.12, 141.84, 136.52, 125.73, 125.62,
122.47, 115.33, 112.40, 104.92, 60.67, 56.82, 56.16; HR-MS (ESI) m/z: calcd for
C18H20O7S [M+H]+ 381.1003, found 381.1004.
4.1.6.2. (Z)-2-methoxy-5-(2-chloro-2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)phenol
(12n). Yellow oil, yield 44.2%.
1H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H), 7.45 (d, J
= 1.8 Hz, 1H), 7.25-7.22 (m, 2H), 7.11 (s, 2H), 6.81 (d, J = 8.5 Hz,1H), 3.85 (s, 3H),
3.85 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 152.87, 148.52, 145.11, 142.22, 133.90,
131.43, 128.28, 124.10, 124.06, 115.36, 109.97, 105.72, 60.51, 56.03, 55.52; HR-MS
(ESI) m/z: calcd for C18H19ClO7S [M+H]+ 415.0613, found 415.0623.
4.1.6.3.(E)-2-methoxy-5-(2-methoxy-2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)phenol
1
(12o). Yellow oil, yield 57.1%. H NMR (300 MHz, CDCl3) δ 7.32 (d, J = 2.0 Hz,
1H), 7.18 (s, 2H), 7.13 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.11 (s, 1H), 6.86 (d, J = 8.4 Hz,
1H), 5.77 (s, 1H), 3.92 (s, 9H), 3.90 (s, 3H), 3.89 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 152.89, 151.05, 147.53, 145.26, 141.83, 133.19, 124.22, 122.86, 122.03, 114.86,
110.06, 105.01, 61.34, 60.47, 55.97, 55.46; HR-MS (ESI) m/z: calcd for C19H22O8S
[M+H]+ 411.1108, found 411.1111.

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4.1.6.4. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfonyl)prop-1-en-1-yl)phenol
1
(12p). White solid, yield 37.5%, m.p. 145-147 °C. H NMR (300 MHz, DMSO-d6) δ
9.21 (s, 1H), 7.53(s, 1H), 7.06 (s, 2H), 6.96 (s, 3H), 3.81 (s, 6H), 3.76 (s, 3H), 3.70 (s,
3H), 2.04 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 153.68, 149.56, 146.94, 142.00,
137.32, 134.57, 134.51,126.59, 122.85, 117.11, 112.54, 105.63, 60.68, 56.87, 56.08,
13.64; HR-MS (ESI) m/z: calcd for C19H22O7S [M+H]+ 395.1159, found 395.1158.
4.1.6.5. (E)-2-methoxy-5-(2-fluoro-2-((3,4,5-trimethoxyphenyl)sulfonyl)vinyl)phenol
(12q). White solid, yield 27.6%, m.p. 120-122 °C. 1H NMR (300 MHz, CDCl3) δ 7.22
(d, J = 1.9 Hz, 1H), 7.20 (s, 2H), 7.10 (dd, J = 8.3, 1.8 Hz, 1H), 6.92 (d, J = 42 Hz,
1H), 6.87 (s, 1H), 5.66 (s, 1H), 3.93 (s, 6H), 3.92 (s, 6H); 13C NMR (75 MHz, CDCl3)
δ 153.08, 147.87, 145.24, 131.50, 123.12, 123.03, 122.41, 115.42, 115.31, 114.74,
110.09, 105.25, 60.54, 56.04, 55.97, 55.49; HR-MS (ESI) m/z: calcd for C18H19FO7S
[M+NH4]+ 416.1174, found 416.1177.
4.1.6.6. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfinyl)vinyl)phenol (13a).
1
White solid, yield 28.6%, m.p. 130-132 °C. H NMR (300 MHz, CDCl3) δ 7.25 (d, J
= 15.8 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.97 (dd, J = 8.3, 1.8 Hz, 1H), 6.90 (s, 2H),
6.83 (d, J = 8.3 Hz, 1H), 6.68 (d, J = 15.5 Hz, 1H), 6.04 (s, 1H), 3.90 (s, 9H), 3.87 (s,
13
3H); C NMR (75 MHz, CDCl3) δ 153.59, 147.91, 145.50, 139.51, 138.35, 136.66,
130.20, 126.66, 120.78, 112.54, 110.13, 100.84, 60.42, 55.88, 55.51; HR-MS (ESI)
m/z: calcd for C18H20O6S [M+H]+ 365.1053, found 365.1054.
4.1.6.7. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfinyl)vinyl)aniline (13b).
1
White solid, yield 35.4%, m.p. 100-102 °C. H NMR (300 MHz, CDCl3) δ 7.23 (d, J
= 15.6 Hz, 1H), 6.89 (s, 2H), 6.88-6.83 (m, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.64 (d, J =
15.5 Hz, 1H), 3.90 (s, 6H), 3.87 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 153.55, 148.53,
139.36, 138.70, 137.49, 136.09, 129.58, 126.17, 119.18, 112.40, 109.64, 100.78,
60.43, 55.88, 55.09; HR-MS (ESI) m/z: calcd for C18H21NO5S [M+H]+ 364.1213,
found 364.1212.
4.1.6.8. (Z)-2-methoxy-5-(2-chloro-2-((3,4,5-trimethoxyphenyl)sulfinyl)vinyl) phenol
(13c). Yellow oil, yield 55.6%. 1H NMR (300 MHz, CDCl3) δ 7.24 (s, 1H), 7.15 (d, J
= 2.0 Hz, 1H), 7.04 (dd, J = 2.1 Hz, 8.5Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.82 (s, 2H),
13
5.96 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H),3.86 (s, 6H); C NMR (75 MHz, CDCl3) δ
153.36, 147.36, 145.37, 139.82, 137.85, 137.69, 135.50, 125.27, 121.87, 114.99,
110.20, 101.05, 60.47, 55.65, 55.53; HR-MS (ESI) m/z: calcd for C18H19ClO6S
[M+H]+ 390.0669, found 399.0660.
4.1.6.8. (E)-2-methoxy-5-(2-((3,4,5-trimethoxyphenyl)sulfinyl)prop-1-en-1-yl)phenol
(13d). White solid, yield 40.6%, m.p. 135-137 °C. Mixture of α and β isomers (1:1),
1H NMR (300 MHz, CDCl3) δ 7.26 (s, 0.5H), 7.10 (d, J = 2.0 Hz, 0.5H), 7.04 (d, J =
2.0 Hz, 0.5H), 7.03 (dd, J = 8.4, 1.9 Hz, 0.5H), 6.95 (dd, J = 8.4, 1.9 Hz, 0.5H), 6.90
(s, 0.5H), 6.89 (s, 1H), 6.86 (s, 1H), 6.74 (s, 1H), 6.20 (s, 0.5H), 6.10 (s, 0.5H), 3.91

ACCEPTED MANUSCRIPT
(s, 3H), 3.88 (s, 4.5H), 3.86 (s, 1.5H), 3.83 (s, 3H), 1.93 (d, J = 1.2 Hz, 1.5H), 1.91 (d,
J = 1.3 Hz, 1.5H); HR-MS (ESI) m/z: calcd for C19H22O6S [M+H]+ 379.1210, found
379.1210.
4.1.7. Synthesis of target compounds 12r and 13e.
4.1.7.1. Intermediate 14. To a solution of 7 (750 mg, 3.75 mmol) in MeCN, potassium
carbonate (621 mg, 4.5 mmol) was added. After stirring for 10 min, bromoacetonitrile
(313 µL, 4.5 mmol) was added into the mixture, then the reaction was stirred at room
temperature for 1 h. the mixture was extracted with CH2Cl2 (3 × 50 mL). The
combined organic layers were then washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to afford the crude product 14 as gray solid (820 mg,
91.5%), which was used directly for the next step without further purification, m.p.
1
109-111 °C. H NMR (300 MHz, CDCl3) δ 6.83 (s, 2H), 3.88 (s, 6H), 3.85 (s, 3H),
13
3.57 (s, 2H); C NMR (75 MHz, CDCl3) δ 153.04, 138.55, 125.95, 116.48, 110.05,
55.82, 43.04, 21.80; ESI-MS m/z 239.1 [M+Na]+ 262.0.
4.1.7.2. Intermediate 15. To a solution of 14 (600 mg, 2.51 mmol) in CH2Cl2,
m-CPBA (75%, 1.27 g, 5.52 mmol) was added. The reaction mixture was stirred for 1
h, and then was extracted with CH2Cl2 (3 × 50 mL). The combined organic layers
were then washed with saturated NaHCO3, brine, dried over anhydrous Na2SO4, and
concentrated in vacuo to afford the crude product 15 as gray solid (620 mg, 91.2%),
which was used directly for the next step without further purification, m.p.
1
139-141 °C. H NMR (300 MHz, CDCl3) δ 7.26 (s, 2H), 4.15 (s, 2H), 3.99 (s, 3H),
13
3.98 (s, 6H); C NMR (75 MHz, CDCl3) δ 153.20, 143.25, 130.33, 110.27, 105.51,
60.61, 56.12, 45.39; ESI-MS m/z 271.1 [M-H]- 270.1.
4.1.7.3. Intermediate 16. To a solution of 14 (600 mg, 2.51 mmol) in CH2Cl2,
m-CPBA (75%, 262 mg, 2.13 mmol) was added slowly at 0 °C. The reaction mixture
was stirred for 1 h, and then was extracted with CH2Cl2 (3 × 50 mL). The combined
organic layers were then washed with saturated NaHCO3, brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to afford the crude product 16 as gray solid (364
mg, 56.9%), which was used directly for the next step without further purification,
m.p. 126-128 °C.
1H NMR (300 MHz, CDCl3) δ 6.98 (s, 2H), 3.94 (s, 6H), 3.91 (s,
3H), 3.76 (dd, J = 38.6, 15.7 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 153.76, 140.79,
135.32, 110.84, 100.37, 60.48, 55.99, 44.37; ESI-MS m/z 255.1 [M+Na]+ 278.0.
4.1.7.4.
(E)-2-((3,4,5-trimethoxyphenyl)sulfonyl)-3-(3-hydroxy-4-methoxyphenyl)
acrylonitrile (12r). To a solution of 15 (80 mg, 0.3 mmol) and acetyl protected
isovanillin (63 mg, 0.33 mmol) in toluene, piperidine (cat, 5 µL) and AcOH (cat, 5
µL) was added, and the mixture was refluxed and monitored by TLC. After reaction
completed, toluene was removed in vacuo and the residue was extracted with EtOAc
(3 × 50 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to afford crude product 17 or 18. Then,

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the crude was dissolved in MeOH (5 mL), 5% sodium hydroxide (5 mL) was added.
After stirring for 30 min at room temperature, the reaction mixture was acidified by
the addition of 10% aqueous HCl, and then diluted with EtOAc, washed with water,
and brine successively, dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography using PE/EA (3/2,V/V) as an
1
eluent to afford the compound 12r as yellow solid (30 mg, 31.1%). H NMR (300
MHz, CDCl3) δ 7.95 (s, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.38 (dd, J = 8.5, 2.1 Hz, 1H),
7.12 (s, 2H), 6.85 (d, J = 8.5 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 6H), 3.84 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 153.13, 151.54, 150.31, 145.83, 142.50, 132.16, 125.71,
123.07, 115.47, 113.28, 110.73, 110.41, 105.17, 60.53, 56.06, 55.74; HR-MS (ESI)
m/z: calcd for C19H19NO7S [M+NH4]+ 423.1220, found 423.1223.
4.1.7.5.
(E)-2-((3,4,5-trimethoxyphenyl)sulfinyl)-3-(3-hydroxy-4-methoxyphenyl)
acrylonitrile (13e). Compound 13e was prepared by the same method as compound
12r. Yellow solid, yield 29.0%, m.p. 185-187 °C. 1H NMR (300 MHz, CDCl3) δ 7.69
(s, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.43 (dd, J = 8.5, 2.0 Hz, 1H), 6.96 (s, 2H), 6.91 (d,
J = 8.5 Hz, 1H), 3.95 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
153.64, 149.94, 145.54, 144.05, 140.60, 135.70, 124.37, 123.90, 115.03, 114.23,
113.19, 110.25, 101.19, 60.48, 55.97, 55.65; HR-MS (ESI) m/z: calcd for
C19H19NO6S [M+H]+ 390.1011, found 390.1012.
4.2 Pharmacology
4.2.1 In vitro anti-proliferative assay
K562, HepG2 and A549 cells were purchased from Nanjing KeyGen Biotech Co.
Ltd. (Nanjing, China). The cytotoxicity of the test compounds was determined using
the MTT assay. Briefly, the cell lines were incubated at 37 °C in a humidified 5% CO2
incubator for 24 h in 96-microwell plates. After medium removal, 100 mL of culture
medium with 0.1% DMSO containing the test compounds at different concentrations
was added to each well and incubated at 37 °C for another 72 h. The MTT (5 mg/mL
in PBS) was added and incubated for another 4 h, the optical density was detected
with a microplate reader at 490 nm. The IC50 values were calculated according to the
dose-dependent curves. All the experiments were repeated in at least three
independent experiments.
4.2.2 In vitro tubulin polymerization inhibitory assay
An amount of 2 mg/mL tubulin (Cytoskeleton) was resuspended in PEM buffer
containing 80 mM piperazine-N,N’-bis(2-ethanesulfonic acid) sequisodium salt
PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2, and 15% glycerol. Then the mixture
was preincubated with compounds or vehicle DMSO on ice. PEG containing GTP
was added to the final concentration of 3 mg/mL before detecting the tubulin

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polymerization reaction. After 30 min, the absorbance of different concentrations was
detected by a spectrophotometer at 340 nm at 37 °C. The area under the curve was
used to determine the concentration that inhibited tubulin polymerization by 50%
(IC50), which was calculated with GraphPad Prism Software version 5.02.
4.2.3 Immunofluorescence Staining
K562 cells were seeded into 6-well plates and then treated with vehicle control
0.1% DMSO, 12m (0.05 µM, 0.1 µM, 0.2 µM). The cells were fixed with 4%
paraformaldehyde and then penetrated with PBS for three times. After blocking for 20
min by adding 50-100 µL goat serum albumin at room temperature, cells were
incubated with a monoclonal antibody (anti-α-tubulin) at 37 °C for 2 h. Then the cells
were washed three times by PBS following staining by fluorescence antibody and
labeling of nuclei by 4,6-diamidino-2-phenylindole (DAPI). Cells were finally
visualized using a fluorescence microscope (OLYMPUS, Japan).
4.2.4 Cell cycle analysis
K562 cells were seeded into 6-well plates and incubated at 37 °C in a humidified
5% CO2 incubator for 24 h, and then treated with or without 12m at indicated
concentrations for another 72 h. The collected cells were fixed by adding 70% ethanol
at 4 °C for 12 h. Subsequently, the cells were resuspended in PBS containing 100 mL
RNase A and 400 mL of propidium iodide for 30 min. The DNA content of the cells
was measured using a FACS Calibur flow cytometer (Bectone Dickinson, San Jose,
CA, USA).
4.2.5 Cell apoptosis analysis
After treatment with or without 12m at indicated concentrations for 72 h, the cells
were washed twice in PBS, centrifuged and resuspended in 500 mL AnnexinV
binding buffer. The cells were then harvested, washed and stained with 5 mL Annexin
V-APC and 5 mL 7-AAD in the darkness for 15 min. Apoptosis was analyzed using a
FACS Calibur flow cytometer (Bectone Dickinson, San Jose, CA, USA).
4.2.6 Wound healing assay
K562 cells were grown in 6-well plates for 24 h. Scratches were made in
confluent monolayers using 200 µL pipette tip. Then, wounds were washed twice with
PBS to remove non-adherent cell debris. The media containing different
concentrations (0, 0.05, 0.1, 0.2 µM) of the compound 12m were added to the
petridishes. Cells which migrated across the wound area were photographed using
phase contrast microscopy at 0 h and 24 h. The migration distance of cells migrated in
to the wound area was measured manually.

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4.2.7 Tube formation assay
EC Matrigel matrix was thawed at 4 °C overnight, and HUVECs suspended in
DMEM were seeded in 96-well culture plates at a cell density of 50,000 cells/well
after polymerization of the Matrigel at 37 °C for 30 min. They were then treated with
20 µL different concentrations of compound 12m or vehicle for 6 h at 37 °C. Then,
the morphological changes of the cells and tubes formed were observed and
photographed under inverted microscope (OLYMPUS, Japan).
4.2.8 In vivo anti-tumor evaluation
Five-week-old male Institute of Cancer Research (ICR) mice were purchased
from Shanghai SLAC Laboratory Animals Co. Ltd. A total of 1 × 106 H22 cells were
subcutaneously inoculated into the right flank of ICR mice according to protocols of
tumor transplant research, to initiate tumor growth. After incubation for one day, mice
were weighted and at random divided into four groups of six animals. The groups
treated with 12m and CA-4 were administered 15, 30 mg/kg in a vehicle of 10%
DMF/2% Tween 80/88% saline, respectively. The positive control group was treated
with PTX (8 mg/kg) every 2 days by intravenous injection. The negative control
group received a vehicle of 10% DMF/2% Tween 80/88% saline through intravenous
injection. Treatments of 12m and CA-4 were done at a frequency of intravenous
injection one dose per day for a total 21 consecutive days while the positive group
was treated with PTX one dose per two days. The mice were sacrificed after the
treatments and the tumors were excised and weighed. The inhibition rate was
calculated as follows: Tumor inhibitory ratio (%) = (1-average tumor weight of
treated group/average tumor weight of control group) × 100%.
4.3 Molecular modeling
In our study, the X-ray structure of the DAMA-colchicine-α,β-tubulin complex
was downloaded from the Protein Data Bank (PDB code 1SA0). The protein was
prepared by removal of the stathmin-like domain, subunits C and D, water molecules
and colchicine using Discovery Studio modules. The docking procedure was
performed by employing DOCK program in Discovery Studio 3.0 software, and the
structural image was obtained using PyMOL software.
Acknowledgments
The authors acknowledge the National Natural Science Foundation of China (No.
81673306, 81703348), and The Open Project of State Key Laboratory of Natural
Medicines, China Pharmaceutical University (No. SKLNMKF 201710) for financial
support.

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Highlights:
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A series of novel vinyl sulfone and sulfoxide derivatives were synthesized.
12m showed the most potent in vitro anti-proliferative activity.
12m was identified as a tubulin polymerization inhibitor with potent vascular disrupting
activity.
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12m exhibited tumor growth inhibition in H22 liver cancer allograft mouse model.