Synthesis and biological evaluation of vanadium complexes as novel anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.04.073

A class of vanadium complexes were prepared and investigated for their antiproliferative effects by MTT assay. The structure-activity relationship was extensively studied through the ligand variation. The results showed that the synthetic vanadium complexes demonstrated moderate to good antiproliferative activities against the four cancer cell lines including MGC803, EC109, MCF7 and HepG2, respectively. Of note was that most of the complexes showed preferential growth inhibitory activity to some degree toward gastric cancer line MGC803. Among them, complex 19 exhibited the most and broad-spectrum proliferative inhibition against the tested cell lines. In addition, mechanism studies illustrated that complex 19 could prevent the colony formation, migration and EMT process, as well as induce apoptosis of MGC803 cells. Furthermore, Western blot experiments revealed that the expression of apoptosis-related proteins changed, including up-regulation of Bax, PARP and caspase-3/9, as well as down-regulation of Bcl-2.

Full text of DOI:10.1016/j.ejmech.2019.04.073

Accepted Manuscript
Synthesis and biological evaluation of vanadium complexes as novel anti-tumor
agents
Ling-Pan Lu, Feng-Zhi Suo, Ya-Li Feng, Li-Li Song, Ying Li, Yang-Jie Li, Kai-Ti Wang
PII:
S0223-5234(19)30401-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.073
EJMECH 11308
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 December 2018
Revised Date: 17 April 2019
Accepted Date: 29 April 2019
Please cite this article as: L.-P. Lu, F.-Z. Suo, Y.-L. Feng, L.-L. Song, Y. Li, Y.-J. Li, K.-T. Wang,
Synthesis and biological evaluation of vanadium complexes as novel anti-tumor agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.073.
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Graphic abstract

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Synthesis and biological evaluation of vanadium complexes as
novel anti-tumor agents
Ling-Pan Lu a, Feng-Zhi Suo c, Ya-Li Feng a, Li-Li Song a, Ying Li d, Yang-Jie Li a,
Kai-Ti Wang b,*
a School of Pharmaceutical Sciences, Zhengzhou University of Industrial Technology,
Zhengzhou,451199, PR China
b School of Materials Science and Engineering, Chongqing University of Technology,
Chongqing 400054, PR China
c School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR
China
d
Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450001, PR China
*Corresponding Author
E-mail address: ktwang@cqut.edu.cn
ABSTRACT: A class of vanadium complexes were prepared and investigated for
their antiproliferative effects by MTT assay. The structure-activity relationship was
extensively studied through the ligand variation. The results showed that the synthetic
vanadium complexes demonstrated moderate to good antiproliferative activities
against the four cancer cell lines including MGC803, EC109, MCF7 and HepG2,
respectively. Of note was that most of the complexes showed preferential growth
inhibitory activity to some degree toward gastric cancer line MGC803. Among them,
complex 19 exhibited the most and broad-spectrum proliferative inhibition against the
tested cell lines. In addition, mechanism studies illustrated that complex 19 could
prevent the colony formation, migration and EMT process, as well as induce
apoptosis of MGC803 cells. Furthermore, western blot experiments revealed that the
expression of apoptosis-related proteins changed, including up-regulation of Bax,
PARP and caspase-3/9, as well as down-regulation of Bcl-2.

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Keywords: Vanadium complex; Antiproliferative effects; Migration; Apoptosis
1. INTRODUCTION
Vanadium, an essential and required trace element for human body, plays an
important role in biochemical processes [1, 2]. Vanadium complexes possess a broad
range of pharmacological properties such as anti-diabetes, anti-parasitism, anti-HIV,
anti-tuberculosis and anti-cancer activities [3-7], and therefore spur extensive research
interest in medicinal chemistry [5]. With the intensive study of medicinal
metallochemsitry, various bioactive vanadium complexes have been increasingly
developed. As shown in Fig. 1, vanadyl-hypocrellin A complex could not only
improve the water solubility of hypocrellin, but also increase its photostability,
affinity and anti-cancer activity [8]. In addition, vanadium complexes were widely
studied to exhibit insulin-mimetic effects and antidiabetic activities probably by a
nonspecific inhibition of phosphotyrosine phosphatase [9, 10]. For example,
dioxidovanadium (V) complex B was reported as a potent and selective protein
tyrosine phosphatase 1B (PTP1B) inhibitor, exhibiting anti-diabetic activity as well as
low cytotoxicity in vitro [11]. And vanadyl complex C with schiff base as organic
ligands could not only reduce the blood glucose level and improve the impaired
glucose tolerance in a diabetic rat model, but also show good lipophilic properties and
low cytotoxicity on Caco-2 cell line [9]. Moreover, vanadocene dichloride D (Fig. 1),
characterized by two cyclopentadienyl rings and both chlorine atoms coordinating to
the vanadium center in a “bent-sandwich” structure [12], was identified to have
antitumor effects on a wide spectrum of cancer cells such as testicular cancer,
leukemia, breast cancer, glioblastoma and colon cancer [13-16]. A recent research
showed that vanadocene dichloride could selectively inhibit the proliferation of Hela
cells over the normal cells, due to the inactivation of serine-threonine protein kinase
Aurora B [12]. Moreover, water soluble complex E, prepared by coordination of
antibiotic cefuroxime with vanadyl sulfate in aqueous solution, was reported to exert
manifold effects including antimicrobial activity, non-cytotoxicity and
immunosuppressive activity [17].

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As a continuation of our works [18], in order to find bioactive vanadium complexes
with potent and specific anticancer activities, we herein reported the synthesis of a
library of novel vanadium complexes, their antiproliferative activities as well as
preliminary mechanisms of inducing cell apoptosis.
O O
O
O
O
O
O
O
N N
O
V
N
O
N
O
O
V
N
O
N
O
O
V
O
HO
OH
OH
A
n
B
C
O
S
O
O
NH2
O
N
O
N
N
Cl
Cl
O
H
V
O
O
V O
OSHO3H
D
E
Fig.1. Biologically active vanadium complexes reported.
2. Results and discussion
2.1 Chemistry
The general synthetic route of the targeted complexes is depicted in Scheme 1. The
indene derivatives 2a-i were prepared by reacting indanone analogue 1 and ethyl
formate in diethyl ether in the presence of potassium tert-butoxide.
2-(Phenylthio)aniline 3 was prepared by reaction of thiophenol with 2-iodoaniline
catalyzed by a combination of cuprous chloride with cyclohexane-1,2-diamine in
water [19]. For the synthesis of 2-phenoxyaniline 5, it was generated by heating
iodobenzene, 2-aminophenol and cesium carbonate in the presence copper powder as
catalyst [20]. In addition, 2-chloroaniline reacted with triphenylphosphine catalyzed
by anhydrous nickel chloride to produce 8, which was subsequently treated with
sodium naphthalenide at low temperature to afford diphenylphosphanyl aniline 9. And
compound 11 was obtained through oxidation of aniline 9 with 30% hydrogen

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peroxide. Subsequently, various imine ligands were readily obtained by condensation
reaction of the obtained aniline derivatives with indene derivative 2 at room
temperature. Finally, the desired vanadium complexes 14-27 were produced by
treating ligands with NaH in THF, followed by adding to the solution of VCl3(THF)3
to proceed coordination reaction (Scheme 2). Additionaly, crystals of complexes 16
and 22 suitable for X-ray cyrstal analysis were grown from the chilled THF and
hexane mixture solution, and the data were appended in supporting information (Fig.
S1 and Table S1/S2).
Scheme 1. Synthesis of ligand block.

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Scheme 2. Synthesis of vanadium complex library.
2.2 Evaluation of biological activity
2.2.1 Antiproliferative activity
Complexes 14-27 were prepared and investigated for their antiproliferative effects
on several cancer cell lines including MGC803 (human gastric cancer cell line),
EC109 (human esophageal cancer cell line), MCF7 (human breast cancer cell line)
and HepG2 (human hepatocellular cancer cell line), by using the MTT assay. The
clinical anticancer drug 5-fluorouracil (5-Fu) was employed as the reference drug [21].
Besides, vanadocene dichloride (D) was also evaluated for its inhibitory activity in
order to make comparison with the synthetic vanadium complexes in this work. The
antiproliferative data are summarized in Tables 1-2.

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Initially, complexes 14-21 were screened toward the tested cancer cell lines, with
the aim to explore the impact of indannoe structure on the cell proliferation inhibitory
ablities. As shown in Table 1, complex 14 showed good and preferred growth
inhibition against the gastric cancer cell line MGC803 with single digit IC50 value, in
comparison to the moderate inhibition against EC109, MCF7 and HepG2 cell lines.
And complex 15 with phenyl substitution (R) generally displayed slightly improved
antiproliferative activities, compared to that of complex 14 with hydrogen atom
substitution. Interestingly, the introduction of 3,5-dimethyl phenyl to R position,
generating complex 16, led to evidently improved cell proliferation inhibition toward
all test cancer lines, however, its analogues complex 17 and 18 generally displayed
decreased inhibitory activities (IC50s > 32 µM) as well as selective inhibition toward
MGC803 cell line (IC50 = 7.29 and 15.90 µM, respectively). Intriguingly, the ring
expansion from 5- to 6-membered ring of indanone to give complex 19, exerted
significantly improved potent and broad proliferation inhibitory activities against all
test cell lines, with IC50 values ranging from 2.69 to 7.21 µM. However, 7-membered
analogue complex 20 presented sharply decreased inhibitory abilities, in comparison
to that of complex 15 and 19. Furthermore, the replacement of thiophenol fragment
with methylpropanethiol (complex 21) led to a generally decreased activity compared
with its counterpart complex 19. The evaluation of this series of complexes featured
by thiolphenol as one of the ligands revealed that the phenyl group for R position and
a six-membered ring indanone were favorable to elicit growth inhibition of the test
cancer cells, highlighting the structural requirements of this new chemotype metallo
compounds for the tumor suppression. Next, complexes 22-25 were produced in order
to investigate the effect of additional ligand variations on the antiproliferative activity.
As shown in Table 2, complex 22-25 were found to be less active than complex 19,
suggesting that phenol group as ligand part seemed to be disfavored to the activity. It
should be noted that complex 22-25 all showed selective inhibitory activities toward
MGC803 cell line, consistent with that of aforementioned thiophenol series. Finally,
complex 26 and 27 with triphenylphosphine (oxide) as ligand part were prepared and
tested for their activities. The results showed that complex 26 was evidently less

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active than its oxide analogue complex 27, indicating the importance of the properties
of coordination atom to the vanadium metal center.
Table 1. Antiproliferative activity of complex 14-21 against the tested cancer cell
lines.
IC50 (µM)a
Complex
R
n
MGC803
4.79±0.57
EC109
MCF7
HepG2
14
15
16
17
18
19
20
H
Ph
1
1
1
1
1
2
3
16.81±1.19 16.42±1.21 17.96±1.25
12.29±1.09 12.06±1.08 14.74±1.16
3.62±0.55
10.99±1.04
7.29±0.86
15.90±1.20
2.69±0.56
6.48±0.81
3,5-diMe-Ph
3,5-diMeO-Ph
3,5-diCF3-Ph
Ph
4.00±0.60
>32
2.89±0.46
>32
5.00±0.70
>32
>32
>32
>32
7.21±0.85
13.04±1.11
4.52±0.65
>32
5.50±0.74
>32
Ph
21
D
-
-
-
-
-
-
4.48±0.65
>32
9.00±0.95
>32
7.22±0.85
>32
11.24±1.05
>32
5-Fu
6.92±0.88
7.15±1.25
9.08±0.94
11.21±1.54
aInhibitory activity was assayed by exposure for 72 h to substance and expressed as concentration required to
inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent
experiments.
Table 2. Antiproliferative activity of complex 22-27 against the tested cancer cell
lines.
IC50 (µM)a
No.
Structure
MGC803
EC109
MCF7
HepG2

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22
23
24
25
26
6.24±0.79
9.42±0.97
5.07±0.70
4.95±0.69
15.74±1.19
19.16±1.28
10.50±1.02
11.11±1.04
>32
>32
22.92±1.36
>32
9.77±0.99
8.05±0.90
>32
9.53±0.97
>32
14.21±1.15
13.16±1.11
>32
27
3.13±0.50
8.33±0.72
7.35±0.86
6.50±1.08
-
>32
>32
>32
>32
D
5-Fu
-
6.92±0.88
7.15±1.25
9.08±0.94
11.21±1.54
a Inhibitory activity was assayed by exposure for 72 h to substance and expressed as concentration required to
inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent
experiments.
2.2.2 Cell clonogenicity and migration
The most active as well as broad-spectrum proliferative inhibition of complex 19
spurred us to have an preliminary understanding of its effect on cell colony formation
inhibition. This assay measured the ability of tumor cells to grow and form foci in a
way that was not restricted by growth contact inhibition, which was characteristic in
normal untransformed cells, and therefore clonogenicity was commonly employed as
an indirect evaluation of tumor cells undergoing neoplastic transformation [22, 23].
As shown in Fig. 2A, fewer and smaller colonies cells were observed after treatment
of MGC803 with complex 19, and the colony inhibition rate reached around high 90%
at 2 µM. Additionally, the effect of complex 19 on the migration ability of MGC803

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cells was also investigated by transwell assay. The results revealed that complex 19
markedly inhibited the migration of MGC803 cells dose-dependently (Fig. 2B).
Meanwhile, western blot results showed that the expression of mesenchymal cell
marker N-cadherin protein decreased, while epithelial cell biomarker E-cadherin
increased, indicating that complex 19 possibly prevented the migration of MGC803
via the inhibition of EMT process (epithelial-mesenchymal transition) (Fig. 3).
Fig. 2. (A) Representative images of MGC803 cell colonies after treatment with indicated
concentrations of complex 19 for 9 d; (B) Migration inhibition of MGC803 by complex 19 at
indicated doses via transwell assay. The results shown were representative of three independent
experiments.

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Fig. 3. Expression of E-Cadherin and N-Cadherin when cells were treated with complex 19 at
indicated concentrations. **P<0.01 was considered statistically highly significant. Data are mean
± SD. All experiments were carried out at least three times.
2.2.3 Cell apoptosis and mechanism involved
Next, complex 19 was assessed for its ability to induce apoptosis of MGC803 cells.
Cell apoptosis is also known as programmed cell death, accompanying
characteristic morphological changes and specific biochemical features [24]. Initially,
the Hochest 33258 staining was performed to examine the cell morphological changes
[25]. Over a period of 24 h incubation with complex 19 at indicated concentrations,
characteristic cell morphologic changes such as chromatin shrinkage, rounding and
formation of apoptotic bodies were observed and presented a dose-response feature
(Fig. 4A), suggesting that the apoptosis of MGC803 cells could be induced by
complex 19 concentration-dependently. In order to further examine the effect of
complex 19 on the apoptosis of MGC803 cells, Annexin V-FITC/PI double staining
method was used for the apoptosis analysis. As shown in Fig. 4B, incubation of
MGC803 cells with complex 19 led to a dose-dependent apoptosis increase, with the
percentage of apoptosis cells of 12.0%, 17.7% and 36.6%, respectively, compared to
the control (0.9%).

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Fig. 4. Complex 19 induced apoptosis of MGC803 cells. (A) Apoptosis analysis with Hoechst
33258 staining; (B) Apoptosis of MGC803 cell line induced by complex 19 for 24 h using
Annexin V-FITC/PI double staining and flow-cytometry calculation. The experiments were
performed three times, and a representative experiment is shown.
To further investigate the underlying apoptotic mechanism of MGC803 cells
induced by complex 19, the western blot analysis was performed to investigate the
expression change of apoptosis related proteins. As shown in Fig. 5A, it was observed
that the expression of anti-apoptotic protein Bcl-2 decreased in a dose-dependent
manner after treatment of MGC803 cells with complex 19 (Fig. 5B), while the
expression of pro-apoptotic protein Bax was elevated accordingly (Fig. 5C). Besides,
the accumulation of Bax was involved in activating the caspases, subsequently
promoting the release of cytochrome and other pro-apoptotic factors from the
mitochodria [26, 27], so that the expressions of both caspase-3 and caspase-9 were
also examined. As shown in Fig. 5D and 5E, caspase-3/-9 were both upregulated

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evidently, indicating the apoptosis occurring via the caspase-mediated pathway.
Additionally, poly ADP-ribose polymerase (PARP) involved in DNA repair and gene
integrity monitoring, was an important substrate of caspase-3, and PARP cleavage
was considered as a hallmark of cell apoptosis [28]. And it was found that the cleaved
PARP was clearly upregulated concentration-dependently (Fig. 5F). Taken together,
all these results demonstrated that complex 19 could induce the apoptosis of MGC803
cells possibly via the mediation of intrinsic apoptotic pathway.
Fig. 5. Expression changes of apoptosis-related proteins induced by complex 19. (A) Complex 19
induced expression changes of Bax, Bcl-2 and caspase family members in MGC803 cells; (B-F)
Statistical analysis of expression levels of Bax, Bcl-2 and cleaved-caspased PARP/9/3.
3. Conclusions
In conclusion, the present study reveals a novel class of vanadium complexes as
metal-based proliferative inhibitors. The antiproliferative results demonstrates that the
synthetic vanadium complexes generally exerted moderate to good activities toward
the tested cancer cell lines. Of note was that complex 19 exhibited the best as well as

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wide-spectrum activity toward all the tested cancer cell lines, with the IC50 values
ranging from 2.7 to 7.2 µM. In consideration of its good inhibition toward human
gastric cancer cell line MGC803, complex 19 was further evaluated for its cellular
activities, and the results indicated that complex 19 could remarkably inhibit the cell
clonogenicity, migration, and EMT process of MGC803 cells. Also, complex 19 could
induce the apoptosis of MGC803 via an innate apoptotic pathway. It was anticipated
that complex 19 can serve as a template for developing new metallo agents for
treatment of anticancer especially for gastric cancers.
4. Experimental section
4.1 General
All reagents and solvents were purchased from commercially available sources, and
dry solvents were treated from an MBraun solvent purification system. The NMR
spectra were recorded on Bruker-400 MHz for 1H NMR, and 100 MHz for 13C NMR
spectrometer. Elemental analyses were recorded on an elemental Vario EL
spectrometer. It should be noted that the targeted vanadium complexes were not
characterized by NMR due to their paramagnetic nature of the vanadium center, but
by the infrared spectra analysis.
4.2 General procedure for the synthesis of ligands and targeted complexes.
4.2.1 The synthesis of intermediate 2a-h
To a slurry of tBuOK (0.90 g, 1.5 eq) in 10 mL anhydrous diethyl ether was added
7-phenyl-1-indanone (1.0 g, 5 mmol, 1 eq) and 0.8 mL ethyl formate (2.0 eq) at 0 °C,
and a large amount of white precipitate appeared immediately. The mixture was kept
stirring at 0°C for 30 min, followed by warming to room temperature and stirred
overnight. The reaction mixture was acidified with Formic acid in ethanol to adjust
pH < 7, and the resultant solution was directly used for the next step without further
purification to react with amine derivatives for preparation of imine ligands.
4.2.2 The synthesis of ligand 4a and complex 14
Ligand 4a: A Schlenk flask was charged with CuCl (4.8 mg, 0.048 mmol),

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2-iodoaniline (0.12 mL, 1.05 mmol), trans-1,2-diaminocyclo- hexane (0.26 mL, 2.21
mmol), water (6.3 mL) and thiophenol (0.06 mL, 0.57 mmol). The flask was sealed
under a positive pressure of argon and the resulting violet solution was heated
overnight at 120oC. The product was extracted from the aqueous layer with
dichloromethane, dried and concentrated in vacuo. The crude mixture was then
purified by flash chromatography (10 % CH2Cl2 in hexane) to give
2-(phenylthio)aniline 3 (102.1 mg, 97 %) as a colorless liquid. Then, the obtained 3 (1
eq) was added in the solution of 2a, and the resultant reaction mixture was stirred over
24h at room temperature. Ligand 4a was obtained by filtration without further
1
purification, H NMR (400 MHz, CDCl3): δ 11.93, 8.04 (d, JHH = 11.8, 13.5 Hz, 1H,
OH), 7.85, 7.82 (d, JHH = 11.8, 13.5 Hz, 1H, Ar-H), 7.61 (dd, JHH = 7.6, 1.4 Hz, 1H,
Ar-H), 7.56 (dd, JHH = 7.7, 1.5 Hz, 1H, Ar-H), 7.53–7.42 (m, 4H, Ar-H), 7.41–7.32 (m,
3H, Ar-H), 7.29–7.19 (m, 3H, Ar-H), 7.19–7.10 (m, 2H, Ar-H), 7.02 (dtd, JHH = 18.8,
7.6, 1.1 Hz, 1H, N=CH), 3.65, 3.41 (s, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ
193.28 (C-OH), 148.73 (N=CH), 146.95 (N-Ar), 141.24, 140.60 (S-Ar), 140.20,
137.48, 137.17, 136.38, 135.89, 135.38, 131.61, 130.47, 129.39, 129.24, 128.98,
127.27, 127.06, 126.42, 125.82, 125.68, 123.42, 123.12, 122.75, 121.16, 118.52,
113.58, 112.97, 112.74 (Ar), 109.60 (N=CH-C), 28.42 (CH2); Anal. Calcd. for
C22H19ONS: C, 76.49; H, 5.54; N, 4.05; S, 9.28. Found: C, 76.51; H, 5.53; N, 4.05; S,
9.26.
Complex 14: To a suspension of NaH (60%, 1.2 eq) in dried THF was added ligand
4a (1eq), and the resultant mixture was kept stirring over 4 h at room temperature,
which was then added dropwise to a solution of VCl3(THF)3 (1.2 eq) in dried THF
over 30 min. The reaction was stirred for another 16 h at room temperature, and
treated by filtration to remove sodium chloride. The filtrate was concentrated in cacuo,
followed by recrystallization in THF/hexane (v/v = 1/3) to give the desired complex
14. FT-IR (KBr pellets): ν/cm-1 3056, 2973, 2874, 1638, 1591, 1561, 1529, 1467,
1446, 1401, 1296, 1271, 1198, 1184, 1166, 1147, 1093, 1024, 1013, 967, 944, 918,
864, 755, 733, 689, 577, 540, 519, 493, 464, 446; Anal. Calcd. for C26H24NO2SVCl2:
C, 58.22; H, 4.51; N, 2.61; S, 5.98. Found: C, 58.21; H, 4.51; N, 2.62; S, 5.95.

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Similar experimental procedures were adapted for the preparation of list of complexes
shown in Scheme 1.
4.2.3 The synthesis of ligand 4b and complex 15
Ligand 4b: 1H NMR (400 MHz, CDCl3): δ 11.77, 7.95 (d, JHH = 11.6, 13.5 Hz, 1H,
OH), 7.63-7.32 (m, 9H, Ar-H), 7.31-7.23 (m, 5H, Ar-H), 7.22-7.10 (m, 3H, Ar-H),
7.00 (dtd, JHH = 16.3, 7.5, 1.2 Hz, 1H, N=CH), 3.66, 3.44 (s, 2H, CH2); 13C NMR
(100 MHz, CDCl3) δ 191.53 (C-OH), 148.95 (N=CH), 147.29 (N-Ar), 141.36, 141.00
(S-Ar), 140.27, 139.90, 137.52, 137.40, 136.46, 136.14, 135.15, 131.49, 130.69,
129.39, 128.57, 128.37, 127.70, 126.46, 126.40, 126.26, 125.37, 123.66, 121.46,
120.37, 112.42 (Ar), 108.65 (N=CH-C), 29.30 (CH2); Anal. Calcd. for C28H21ONS: C,
80.16; H, 5.05; N, 3.34; S, 7.64. Found: C, 80.13; H, 5.03; N, 3.35; S, 7.66.
Complex 15: FT-IR (KBr pellets): ν 3078, 3052, 3035, 2970, 2885, 1594, 1561,
1489, 1469, 1442, 1425, 1393, 1297, 1277, 1259, 1197, 1165, 1089, 1076, 1036, 1016,
960, 924, 897, 859, 799, 788, 759, 743, 698, 591, 553, 526, 506, 491, 466, 453, 433
cm-1; Anal. Calcd. for C32H28NO2SVCl2: C, 62.75; H, 4.61; N, 2.29; S, 5.24. Found: C,
62.77; H, 4.58; N, 2.29; S, 5.28.
4.2.4 The synthesis of ligand 4c and complex 16
Ligand 4c: To a Schlenk flask with positive pressure of argon was charged with
CuCl (4.8 mg, 0.048 mmol), 2-iodoaniline (0.12 mL, 1.05 mmol),
trans-1,2-diaminocyclo-hexane (0.26 mL, 2.21 mmol), 6.3 mL water and thiophenol
o
(0.06 mL, 0.57 mmol) and the resultant mixture was brought to 120 C and stirred
overnight. The reaction misture was treated by extraction with DCM for three times,
and then the organic phase was dried and concentrated in vacuo. The crude product
was then subjected to flash chromatography (10 % CH2Cl2 in hexane) to give
2-(phenylthio)aniline 3 (102 mg, 97 %) as a colorless liquid [19]. Then, compound 3
(1 eq) was added in the solution of 2c, and the reaction was stirred over 24 h at rt.
Compound 4c was readily obtained by filtration, 1H NMR (400 MHz, CDCl3): δ 11.77,
7.95 (d, JHH = 11.7, 13.5 Hz, 1H, OH), 7.63-7.07 (m, 15H, Ar-H), 7.06-6.95 (m, 1H,
N=CH), 3.65, 3.43 (s, 2H, CH2), 2.40, 2.37 (s, 6H, CH3); 13C NMR (100 MHz, CDCl3)

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δ 192.60 (C-OH), 149.98 (N=CH), 148.24 (N-Ar), 142.64, 142.10 (S-Ar), 141.66,
141.29, 138.49, 137.52, 137.29, 136.76, 136.05, 135.86, 132.43, 131.66, 130.49,
129.62, 129.43, 128.82, 127.67, 127.30, 126.43, 124.53, 122.46, 121.42, 113.51 (Ar),
109.79 (N=CH-C), 30.37 (CH2), 21.48, 21.44 (CH3); Anal. Calcd. for C30H25ONS: C,
80.50; H, 5.63; N, 3.13; S, 7.16. Found: C, 80.52; H, 5.62; N, 3.12; S, 7.14.
Complex 16: FT-IR (KBr pellets): ν 3054, 2962, 2910, 2886, 1595, 1561, 1476,
1447, 1405, 1389, 1286, 1271, 1192, 1159, 1110, 1067, 1036, 1018, 967, 926, 866,
857, 815, 788, 750, 692, 605, 580, 558, 537, 524, 490, 461, 450, 419 cm-1; Anal.
Calcd. for C34H32NO2SVCl2: C, 63.75; H, 5.04; N, 2.19; S, 5.01. Found: C, 63.77; H,
5.00; N, 2.18; S, 5.04.
4.2.5 The synthesis of ligand 4d and complex 17
Ligand 4d: 1H NMR (400 MHz, CDCl3): δ 11.87, 7.95 (d, JHH = 11.8, 13.4 Hz, 1H,
OH), 7.63-7.08 (m, 12H, Ar-H), 7.00 (dtd, JHH = 8.7, 7.5, 1.2 Hz, 1H, N=CH), 6.73,
6.63 (d, JHH = 2.3, 2.3Hz, 2H, Ar-H), 6.53, 6.50 (t, JHH = 2.3, 2.3Hz, 1H, Ar-H), 3.81
13
(d, JHH = 1.4Hz, 6H, CH3), 3.65, 3.43 (s, 2H, CH2); C NMR (100 MHz, CDCl3) δ
192.41 (C-OH), 150.09 (N=CH), 148.29 (N-Ar), 142.58, 142.01 (S-Ar), 141.15,
140.77, 140.40, 137.34, 136.54, 136.19, 135.83, 135.48, 132.47, 131.97. 131.72,
130.51, 129.47, 129.16, 128.89, 127.31, 126.38, 124.92, 122.55, 121.30, 113.38 (Ar),
108.24 (N=CH-C), 55.44 (OCH3), 30.35 (CH2); Anal. Calcd. for C30H25O3NS: C,
75.13; H, 5.25; N, 2.92; S, 6.69. Found: C, 75.15; H, 5.26; N, 2.93; S, 6.67.
Complex 17: FT-IR (KBr pellets): ν 3058, 2961, 2879, 2837, 164, 1595, 1563, 1446,
1417, 1283, 1268, 1206, 1156, 1061, 1047, 1021, 999, 967, 927, 868, 814, 792, 748,
690, 603, 578, 556, 545, 494, 450 cm-1; Anal. Calcd. for C34H32NO4SVCl2: C, 60.72;
H, 4.80; N, 2.08; S, 4.77. Found: C, 60.73; H, 4.76; N, 2.09; S, 4.78.
4.2.6 The synthesis of ligand 4e and complex 18
1
Ligand 4e: H NMR (400 MHz, CDCl3): δ 11.79 (d, JHH = 12.0 Hz, 1H, OH),
8.06-7.86 (m, 3H, Ar-H), 7.67-7.50 (m, 3H, Ar-H), 7.49-7.34 (m, 2H, Ar-H),
7.32-7.07 (m, 7H, Ar-H),7.03 (dtd, JHH = 11.0, 7.5, 1.2 Hz, 1H, N=CH), 3.68, 3.46 (s,
13
2H, CH2); C NMR (100 MHz, CDCl3) δ 191.89 (C-OH), 150.26 (N=CH), 148.56

ACCEPTED MANUSCRIPT
(N-Ar), 142.29, 141.75 (S-Ar), 140.32, 137.71, 137.52, 137.38, 136.90, 136.48,
135.61, 132.78, 132.57, 132.01, 131.67, 130.68 (Ar), 130.41 (CF3), 129.44, 129.32,
128.96, 128.83, 127.30, 126.34, 126.19, 122.88, 113.35 (Ar), 109.24 (N=CH-C),
30.42 (CH2); 19F NMR (376 MHz, CDCl3): δ -62.56, -62.64 (s, 6F, CF3); Anal. Calcd.
for C30H19ONSF6: C, 64.86; H, 3.45; N, 2.52; S, 5.77. Found: C, 64.85; H, 3.47; N,
2.54; S, 5.76.
Complex 18: FT-IR (KBr pellets): ν 3062, 2977, 2906, 2886, 1661, 1597, 1561,
1496, 1445, 1404, 1380, 1277, 1183, 1139, 1107, 1092, 1022, 984, 944, 899, 867, 808,
787, 750, 736, 710, 692, 681, 605, 583, 554, 532, 492, 449, 412 cm-1; Anal. Calcd. for
C34H26NO2SF6VCl2: C, 54.56; H, 3.50; N, 1.87; S, 4.28. Found: C, 54.56; H, 3.48; N,
1.88; S, 4.31.
4.2.7 The synthesis of ligand 4g and complex 19
Ligand 4g: 1H NMR (400 MHz, CDCl3): δ 12.00, 8.05 (d, JHH = 11.8, 13.6 Hz, 1H,
OH), 7.60-7.56 (m, 1H, Ar-H), 7.47-7.29 (m, 7H, Ar-H), 7.25-7.05 (m, 9H, Ar-H),
7.01-6.93 (m, 1H, N=CH), 2.93-2.86 (m, 2H, CH2), 2.66-2.60 (m, 2H, CH2); 13C
NMR (100 MHz, CDCl3) δ 186.75 (C-OH), 142.50 (N=CH), 142.47, 142.34 (S-Ar),
141.92 (N-Ar), 137.84, 136.51, 134.46, 132.45, 129.53, 129.50, 129.41, 128.62,
128.30, 127.73, 127.59, 127.11, 126.54, 126.12, 125.28, 125.13, 121.22, 120.43,
112.77 (Ar), 107.09 (N=CH-C), 30.20, 27.34 (CH2); Anal. Calcd. for C29H23ONS: C,
80.34; H, 5.35; N, 3.23; S, 7.40. Found: C, 80.37; H, 5.37; N, 3.21; S, 7.39.
Complex 19: FT-IR (KBr pellets): ν 3056, 2946, 2094, 2879, 1644, 1583, 1550,
1481, 1463, 1445, 1416, 1380, 1329, 1281, 1267, 1231, 1212, 1180, 1154, 1092, 1071,
1027, 1016, 998, 951, 871, 856, 831, 802, 783, 758, 747, 732, 718, 696, 685, 649, 587,
554, 514, 467, 449 cm-1; Anal. Calcd. for C33H30NO2SVCl2: C, 63.26; H, 4.83; N,
2.24; S, 5.12. Found: C, 63.28; H, 4.81; N, 2.22, S, 5.07.
4.2.8 The synthesis of ligand 4h and complex 20
Ligand 4h: 1H NMR (400 MHz, CDCl3): δ 12.07, 8.10 (d, JHH = 11.2, 13.8 Hz, 1H,
OH), 7.57 (dd, JHH = 25.2, 7.6 Hz, 1H, Ar-H), 7.44-7.05 (m, 16H, Ar-H), 6.98 (m, 1H,
N=CH), 2.70-2.62 (m, 2H, CH2), 2.50-2.19 (m, 2H, CH2), 1.87, 1.63 (t, JHH = 6.6 Hz,

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13
2H, CH2); C NMR (100 MHz, CDCl3) δ 197.44 (C-OH), 142.81 (N=CH), 141.49,
140.88, 139.90, 139.64, 138.31, 137.10, 135.61, 130.41, 129.61, 129.52, 129.12,
128.87, 128.02, 127.28, 126.62, 126.40, 122.77, 121.98, 114.15, 110.29 (Ar), 30.85,
29.86, 27.95 (CH2); Anal. Calcd. for C30H25ONS: C, 80.50; H, 5.63; N, 3.13; S, 7.16.
Found: C, 80.47; H, 5.61; N, 3.11; S, 7.19.
Complex 20: FT-IR (KBr pellets): ν 3058, 2932, 2857, 1639, 1579, 1552, 1478,
1463, 1445, 1425, 1369, 1340, 1303, 1270, 1241, 1223, 1209, 1276, 1154, 1092, 1063,
1041, 1023, 1005, 991, 954, 918, 867, 852, 812, 783, 762, 751, 703, 689, 638, 605,
551, 533, 511, 456 cm-1; Anal. Calcd. for C34H32NO2SVCl2: C, 63.75; H, 5.04; N,
2.19; S, 5.01. Found: C, 63.77; H, 5.01; N, 2.18; S, 5.03.
4.2.9 The synthesis of ligand 4i and complex 21
Ligand 4i: 1H NMR (400 MHz, CDCl3): δ 12.47, 8.10 (d, JHH = 12.2, 14.0 Hz, 1H,
OH), 7.46 (dd, JHH = 7.6, 1.5 Hz, 1H, Ar-H), 7.43-7.29 (m, 6H, Ar-H), 7.28-7.14 (m,
5H, Ar-H), 6.90 (m, 1H, N=CH), 3.02, 2.94 (t, JHH = 6.6 Hz, 2H, CH2), 2.79, 2.70 (t,
13
JHH = 6.6 Hz, 2H, CH2); 1.31, 1.15 (s, 9H, C(CH3)3); C NMR (100 MHz, CDCl3) δ
187.77 (C-OH), 144.58 (N=CH), 143.52 (N-Ar), 143.41 (S-Ar), 140.07, 138.22,
130.60, 130.45, 130.21, 128.66, 127.67, 127.06, 126.07, 121.44, 120.31, 112.07 (Ar),
107.72 (N=CH-C), 48.22 (S-C-(CH3)3), 31.17 (CH2), 30.64 (C-(CH3)3), 28.30 (CH2);
Anal. Calcd. for C27H27ONS: C, 78.41; H, 6.58; N, 3.39; S, 7.75. Found: C, 78.42; H,
6.56; N, 3.41; S, 7.70.
Complex 21: FT-IR (KBr pellets): ν 3053, 2965, 2943, 2896, 2877, 2863, 2841,
1641, 1585, 1549, 1458, 1447, 1432, 1414, 1378, 1367, 1327, 1298, 1283, 1229, 1215,
1187, 1164, 1091, 1036, 1015, 946, 861, 804, 775, 760, 717, 702, 651, 629, 586, 553,
520, 484, 462, 448, 422 cm-1; Anal. Calcd. for C31H34NO2SVCl2: C, 61.39; H, 5.65; N,
2.31; S, 5.29. Found: C, 61.37; H, 5.68; N, 2.33; S, 5.32.
4.2.10 The synthesis of ligand 6 and complex 22
1
Ligand 6: H NMR (400 MHz, CDCl3): δ 11.68, 8.11 (d, JHH = 9.8, 13.9 Hz, 1H,
OH), 7.55-6.99 (m, 17H, Ar-H), 6.99-6.80 (m, 1H, N=CH), 3.69; 3.01-2.82;
2.71-2.58;1.21 (q, JHH = 7.0 Hz; m; m; t, JHH = 7.0 Hz; 4H, CH2); 13C NMR (100 MHz,

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CDCl3) δ 187.89 (C-OH), 145.03 (N=CH), 144.81 (N-Ar), 143.58 (S-Ar), 143.33,
143.21, 139.97, 134.57, 133.66, 133.32, 132.55, 130.70, 130.50, 130.43, 130.06,
129.58, 128.58, 128.28, 127.74, 127.27, 127.15, 126.52, 126.21, 124.66, 123.97,
123.01, 122.48, 122.02, 120.62, 118.67, 118.56, 117.78, 114.93, 113.83, 111.43 (Ar),
107.58 (N=CH-C), 31.28, 29.41, 28.23, 22.58 (CH2); Anal. Calcd. for C29H23O2N: C,
83.43; H, 5.55; N, 3.35. Found: C, 83.41; H, 5.56; N, 3.37.
Complex 22: FT-IR (KBr pellets): ν 3061, 2978, 2905, 1595, 1578, 1552, 1485,
1462, 1451, 1440, 1413, 1370, 1354, 1331, 1299, 1279, 1248, 1228, 1209, 1191, 1177,
1162, 1104, 1089, 1067, 1042, 1020, 1009, 944, 922, 878, 868, 849, 831, 813, 798,
780, 758, 748, 726, 708, 697, 660, 624, 588, 540, 508, 493, 479, 460, 439, 410 cm-1;
Anal. Calcd. for C33H30NO3VCl2: C, 64.93; H, 4.95; N, 2.29. Found: C, 64.95; H,
4.92; N, 2.26.
4.2.11 The synthesis of ligand 7b and complex 23
1
Ligand 7b: H NMR (400 MHz, CDCl3): δ 11.45 (d, JHH = 11.6 Hz,1H, O-H),
7.41-7.22 (m, 11H, Ar-H), 7.17 (d, JHH = 7.5 Hz, 1H, Ar-H), 7.00 (d, JHH = 7.8 Hz, 2H,
Ar-H), 6.97 (d, JHH = 7.4 Hz, 1H, N=C-H), 2.95 (m, 2H, CH2), 2.68 (m, 2H, CH2). 13C
NMR (100 MHz, CDCl3): δ 188.01 (N=CH), 143.76 (C=C-OH), 143.17, 140.97,
133.63, 130.60, 129.53, 128.45, 127.24, 126.43, 122.70, 116.06 (-Ar), 106.76
(C=C-CH=N), 31.38, 28.25 (CH2). Anal. Calcd. for C23H19NO: C, 84.89; H, 5.89; N,
4.30. Found: C, 84.92; H, 5.88; N, 4.31.
Complex 23: FT-IR (KBr pellets): ν/cm-1 3054, 3033, 2973, 2952, 2932, 2906,
2876, 2850, 1638, 1579, 1555, 1496, 1465, 1441, 1413, 1368, 1351, 1326, 1302, 1281,
1243, 1219, 1205, 1174, 1150, 1070, 1028, 1011, 963, 917, 883, 855, 831, 803, 782,
758, 706, 671, 661, 619, 592, 564, 543, 526, 505, 484, 439, 408. Anal. Calcd. for
C31H34NO3VCl2: C, 63.06; H, 5.80; N, 2.37. Found: C, 63.07; H, 5.74; N, 2.32.
4.2.12 The synthesis of ligand 7c and complex 25
1
Ligand 7c: H NMR (400 MHz, CDCl3): δ 11.39 (d, JHH = 11.1 Hz, 1H, OH, ),
7.42-7.23 (m, 8H, Ar-H), 7.19 (d, JHH = 7.5 Hz, 1H, N=CH), 2.97 (m, 2H, CH2), 2.66
13
(m, 2H, CH2). C NMR (100 MHz, CDCl3): δ 189.57 (N=CH), 143.97 (C=C-OH),

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143.67, 142.84, 141.55, 133.03, 131.26, 130.69, 128.48, 127.91, 127.35, 126.61 (Ar),
109.91 (C=C-CH=N), 31.02, 28.14 (CH2). 19F NMR (376 MHz, CDCl3): δ -154.82
(2F, m-Ar-F), -162.58 (2F, o-Ar-F), -164.70 (1F, p-Ar-F). Anal. Calcd. for
C23H14F5NO: C, 66.51; H, 3.40; N, 3.37. Found: C, 66.53; H, 3.42; N, 3.37.
Complex 25: FT-IR (KBr pellets): ν/cm-1 3058, 3034, 2973, 2955, 2927, 2911, 2867,
2838, 1589, 1557, 1464, 1446, 1433, 1363, 1334, 1297, 1263, 1235, 1225, 1194, 1179,
1166, 1153, 1110, 1093, 1058, 1037, 1013, 965, 933, 916, 877, 857, 829, 806, 784,
773, 758, 704, 678, 667, 648, 620, 600, 594, 577, 567, 549, 512, 503, 480, 460, 447,
423. Anal. Calcd. for C31H29F5NO3VCl2: C, 54.72; H, 4.30; N, 2.06. Found: C, 54.71;
H, 4.25; N, 2.08.
4.2.13 The synthesis of ligand 10 and complex 26
Ligand 10: 1H NMR (400 MHz, CDCl3): δ 12.07-11.67 (m, 1H, OH), 7.56-7.06 (m,
22H, Ar-H), 6.97-6.82 (m, 1H, N=CH), 2.98-2.79; 2.67-2.53; 2.48-2.39 (m, 4H, CH2);
13C NMR (100 MHz, CDCl3) δ 186.59 (C-OH), 143.41 (N=CH), 143.20 (N-Ar),
142.49 (S-Ar), 142.46, 142.40, 139.88, 134.19, 134.01, 133.92, 133.40, 133.20,
132.88, 132.72, 132.52, 132.50, 130.01, 129.56, 129.40, 129.34, 128.98, 128.29,
127.89, 127.81, 127.66, 127.43, 127.36, 127.18, 126.63, 126.54, 126.05, 125.93,
125.41, 125.08, 121.74, 114.17, 114.15 (Ar), 100.52 (N=CH-C), 30.24, 28.31, 27.16,
21.39 (CH2); 31P (162 MHz, CDCl3) δ -19.98, -22.52; Anal. Calcd. for C35H28ONP: C,
82.49; H, 5.54; N, 2.75. Found: C, 82.51; H, 5.59; N, 2.76.
Complex 26: FT-IR (KBr pellets): ν 3056, 3025, 2952, 2879, 1584, 1550, 1481,
1462, 1451, 1438, 1417, 1378, 1350, 1312, 1295, 1280, 1252, 1230, 1215, 1181, 1159,
1123, 1091, 1065, 1027, 1016, 1001, 956, 868, 857, 829, 803, 784, 772, 754, 745, 735,
716, 699, 694, 670, 651, 621, 591, 574, 563, 546, 521, 507, 495, 482, 452, 433, 418
cm-1; Anal. Calcd. for C39H35NO2PVCl2: C, 66.68; H, 5.02; N, 1.99. Found: C, 63.67;
H, 5.05; N, 1.99.
4.2.14 The synthesis of ligand 12 and complex 27
Ligand 12: 1H NMR (400 MHz, CDCl3): δ 10.81, 8.24 (d, JHH = 13.1, 13.2 Hz, 1H,
OH), 7.71-7.54 (m, 6H, Ar-H), 7.54-7.19 (m, 15H, Ar-H), 7.15 (d, 1H, JHH = 7.4 Hz,

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Ar-H), 6.98; 6.88 (dd, JHH = 13.7, 7.6 Hz; t, JHH = 6.8 Hz; 1H, N=CH), 3.74;
13
3.01-2.88; 2.88-2.74;1.85 (s; m; m; t, JHH = 6.3 Hz; 4H, CH2); C NMR (100 MHz,
CDCl3) δ 186.04 (C-OH), 147.08 (N=CH), 144.01 (N-Ar), 143.54 (S-Ar), 143.36,
136.20, 133.91, 133.44, 133.34, 132.73, 132.52, 132.50, 132.07, 131.97, 131.06,
130.74, 130.33, 128.82, 128.70, 128.21, 127.67, 127.62, 126.43, 120.81, 120.68,
115.28, 114.98, 114.91, 114.27 (Ar), 112.53 (N=CH-C), 67.96, 29.43, 25.60, 22.72
(CH2); 31P (162 MHz, CDCl3) δ 37.46, 37.13; Anal. Calcd. for C35H28O2NP: C, 79.98;
H, 5.37; N, 2.67. Found: C, 80.01; H, 5.39; N, 2.66.
Complex 27: FT-IR (KBr pellets): ν 3056, 3020, 2950, 2900, 1637, 1586, 1557,
1480, 1462, 1451, 1440, 1415, 1371, 1349, 1328, 1298, 1280, 1251, 1226, 1211, 1193,
1175, 1160, 1120, 1088, 1059, 1037, 1026, 1015, 997, 953, 920, 866, 859, 833, 804,
782, 757, 728, 717, 695, 670, 655, 690, 571, 561, 550, 528, 521, 510, 488, 473, 448,
430 cm-1; Anal. Calcd. for C39H35NO3PVCl2: C, 65.19; H, 4.91; N, 1.95. Found: C,
65.17; H, 4.92; N, 1.93.
4.3 Antiproliferative activity assays
Exponentially growing cells were added into 96-well plates at a concentration of
about 3,000 cells per well. Over a period of 24 h, the culture medium was discarded,
and fresh medium containing indicated concentrations of tested compounds was
added to each well. After 72 h incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) solution was added into each well and stood for
another 4 h. Then the medium was removed, followed by the addition of 150 µL
DMSO for each well. The plate was agitated on the plate shaker to make the dark blue
crystal dissolved. And MTT assays were performed and cell viability was assessed at
570nm by a microplate reader (Biotech, Shanghai, China).
4.4 Clonogenicity assay
MGC803 cells (about 1000 cells/well) were seeded in a 6-well plate and incubated
for 24 h, then the media were removed followed by adding the fresh media containing
indicated concentrations of complex 19. After 9 days of incubation, the cells were
washed with PBS for three times, and then fixed with 4% paraformaldehyde for

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30min. The cell colonies were then visualized by using 0.1% crystal violet staining.
The cells were then imaged, and the colony populaton were quantified by Image J
software (Developed by National Institutes of Health).
4.5 Hoechst 33258 staining
MGC803 cells were seeded in a 6-well plate (2×105/well), and incubated overnight
for adherent followed by treatment with complex 19 at indicated concentrations for 24
h. Hoechst 33258 was used for cell staining over 30 min in the dark. The cells were
observed under a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).
4.6 Transwell migration assay
100 mL medium containing 1% FBS, indicated concentrations of complex 19 with
10,000 MGC803 cells were added to each upper chambers of Transwell plate. Then
500 µL medium with 20% FBS was used as chemoattractant in the lower chambers.
After 72 h of incubation, both chambers were washed with PBS for three times,
followed by staining with Hoechst33258 (10 µg/mL) for 15 min. The migrated cells
were determined by using high content screening system (ArrayScan XTI, Thermo
Fisher Scientific).
4.7 Cell apoptosis assay
MGC803 cells were seeded into a 6-well plate (2×105/well) and incubated for 24 h.
Then the cells were treated with indicated concentrations of the tested complex 19 for
24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit
(Biovision) was used according to the manufacturer’s protocol. The cells were
analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific,
MA).
4.8 Western blot analysis
MGC803 cells were treated with different concentrations of complex 19 for 24 h,
the cells were collected, lysed in radio-immunoprecipitation assay (RIPA) buffer
contained protease inhibitor cocktail for 30 min, followed by centrifugation at 12,000

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o
rpm for 10 min at 4 C. After collecting the supernatant, the protein was determined
using a bicinchonininc acid assay kit (Beyotmie Biotechnology, Haimen, China).
o
Then cell lyses were boiled with loading buffer for 10 min at 100 C for SDS-
polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to
nitrocellulose (NC) membranes and then were blocked with 5% skim milk at room
temperature for 2 h, followed by incubation with primary antibodies at 4oC overnight.
Next, the membrane was washed with the secondary antibody (1: 5000) at room
temperature for 2 h. Finally, the blots were washed in TBST/TBS and the
immunoblots were visualized by enhanced chemiluminescence (ECL) and exposed on
Kodak radiographic film.
4.9 Statistical analysis
All experimental data in biology were expressed as means ± SD. Statistical
significance between two groups was evaluated by the Student’s t-test. P value less
than 0.05 was considered statistically significant.
Acknowledgement
This work was supported by Zhengzhou University of Industrial Technology Natural
Science Foundation (Project No.2018YB020) and Foundation of The Science and
Technology Department of Henan Province (Project No. 182102310169).
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ACCEPTED MANUSCRIPT
Highlights
ò A library of vanadium complexes showed moderate to good growth inhibition
against the tested cancer cells.
ò Complex 19 demonstrated the best as well as selective proliferation inhibition
toward MGC-803 cells.
ò Complex 19 inhibited the cell colony formation, migration and EMT process of
MGC803.
ò Complex 19 induced the apoptosis of MGC-803 cells, and led to the expression
changes of key proteins related to apoptosis.