Thiophenol-formaldehyde triazole causes apoptosis induction in ovary cancer cells and prevents tumor growth formation in mice model

Article abstract of DOI:10.1016/j.ejmech.2019.03.033

In the present study a library of thiophenol-formaldehyde-triazole (TFT) derivatives was synthesized and screened against CAOV3, CAOV4 and ES-2 ovary cancer cell lines. Initial screening revealed that five-compounds 5a, 5b, 5j, 5h and 5i inhibited the viability of tested cell lines. Analysis of apoptosis revealed that increase in compound 5a (most active) concentration from 0.25 to 2.0 μM enhanced apoptotic cell proportion. Transwell assay showed reduction in invasive potential of CAOV3 cells on treatment with compound 5a. In wound healing assay increasing the concentration of compound 5a from 0.5 to 2.0 μM caused a significant (P < 0.05) decrease in the migration potential. Western blotting showed that compound 5a treatment markedly decreased the level of matrix metalloproteinase (MMP)-2 and ?9 in CAOV3 cells. Treatment of CAOV3 cells with compound 5a caused a marked decrease in Focal Adhesion Kinase (FAK) activation. Tumor growth was inhibited in the compound 5a treated mice markedly than those of untreated group. The tumor metastasis to liver, intestine, spleen and peritoneal cavity was markedly decreased in mice treated with 10 mg/kg dose of compound 5a. Examination of Von Willebrand factor (vWF) expression in liver, intestinal and pulmonary lesions showed a marked decrease in the compound 5a-treated mice. The infiltration of macrophages in the metastatic lesions showed a significant decrease in compound 5a-treated mice. In conclusion, the compound 5a inhibited ovary cancer cell viability and induced apoptosis through decrease in expression of vWF and metalloproteinase, suppression of FAK activation and decrease in infiltration of macrophages. The compound 5a therefore can be investigated further for the treatment of ovary cancer.

Full text of DOI:10.1016/j.ejmech.2019.03.033

European Journal of Medicinal Chemistry 172 (2019) 62e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Thiophenol-formaldehyde triazole causes apoptosis induction in
ovary cancer cells and prevents tumor growth formation in mice
model
,
*
Yan Jia a, Lihui Si a, Ruixin Lin b, Hongjuan Jin c, Wenwen Jian a, Qing Yu a, Shuli Yang a
a Department of Gynaecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China
b Department of Hepatopancreatobiliary Surgery, The Second Hospital of Jilin University, Changchun, Jilin, 130041, China
c Department of Plastic and Aesthetic Surgery, The First Hospital of Jilin University, Changchun, Jilin, 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study a library of thiophenol-formaldehyde-triazole (TFT) derivatives was synthesized and
screened against CAOV3, CAOV4 and ES-2 ovary cancer cell lines. Initial screening revealed that five-
compounds 5a, 5b, 5j, 5h and 5i inhibited the viability of tested cell lines. Analysis of apoptosis
revealed that increase in compound 5a (most active) concentration from 0.25 to 2.0 mM enhanced
apoptotic cell proportion. Transwell assay showed reduction in invasive potential of CAOV3 cells on
Received 2 January 2019
Received in revised form
13 March 2019
Accepted 14 March 2019
Available online 20 March 2019
treatment with compound 5a. In wound healing assay increasing the concentration of compound 5a
from 0.5 to 2.0 mM caused a significant (P < 0.05) decrease in the migration potential. Western blotting
Keywords:
showed that compound 5a treatment markedly decreased the level of matrix metalloproteinase (MMP)-
2 and ꢀ9 in CAOV3 cells. Treatment of CAOV3 cells with compound 5a caused a marked decrease in Focal
Adhesion Kinase (FAK) activation. Tumor growth was inhibited in the compound 5a treated mice
markedly than those of untreated group. The tumor metastasis to liver, intestine, spleen and peritoneal
cavity was markedly decreased in mice treated with 10 mg/kg dose of compound 5a. Examination of Von
Willebrand factor (vWF) expression in liver, intestinal and pulmonary lesions showed a marked decrease
in the compound 5a-treated mice. The infiltration of macrophages in the metastatic lesions showed a
significant decrease in compound 5a-treated mice. In conclusion, the compound 5a inhibited ovary
cancer cell viability and induced apoptosis through decrease in expression of vWF and metalloproteinase,
suppression of FAK activation and decrease in infiltration of macrophages. The compound 5a therefore
can be investigated further for the treatment of ovary cancer.
Macrophages
Pharmacophore
Heterocyclic
Metastasis
Migration
© 2019 Published by Elsevier Masson SAS.
1. Introduction
the distant organs demands for the development of novel and
effective treatment strategies. Studies are being continuously per-
Ovarian cancer is the most common cause of mortality among
gynaecologic cancers and the fifth leading cause of deaths in fe-
males because of cancer [1]. The 5-year survival rate of the females
suffering from ovarian cancer is very low which stresses for the
development of an effective treatment strategy for the ovary cancer
[2]. Difficulty in the detection of ovary cancer at an early stage and
dearth of available effective treatment makes ovary cancer a life-
threatening disease in females [3]. In ovarian cancer patients tu-
mor metastasis to the peritoneal tissues takes place at an advanced
stage [4]. Inhibition of ovarian cancer growth and its metastasis to
formed to investigate the mechanism of ovarian cancer for devel-
opment of efficient treatment strategies [5e8].
Triazole moiety comprises of a heterocyclic aromatic five-
membered ring and constitutes an important pharmacophore of
several bioactive molecules (Fig. 1) [9]. The molecules containing
triazole scaffold possess several biological activities like anti-tumor,
anti-inflammatory, etc. [9]. A large number of compounds bearing
triazole moiety have been identified as the biologically active
molecules [10]. Screening of the 5-mercapto-1,2,4-triazole de-
rivatives has led to the identification various molecules possessing
anticancer activity against various types of carcinoma cell lines
[11e13]. Molecular docking results demonstrate that 1,2,4-triazole
compounds inhibit proliferation of cancer cells through apoptosis
* Corresponding author.
E-mail address: SusannPdgesc@yahoo.com (S. Yang).
https://doi.org/10.1016/j.ejmech.2019.03.033
0223-5234/© 2019 Published by Elsevier Masson SAS.

Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
63
2.1.2. General procedure for propargylation
The solution of compound 3 (500 mg, 1.0 mmol) in THF was
stirred with NaH (600 mg, 1.0 mmol) and propargyl bromide
(458 mL, 4.0 mmol) for 1 h at room temperature. The completion of
the reaction was monitored by TLC. After 1 h, the reaction mixture
was purified by the column chromatography using 2:98 ethyl ac-
etate/hexane as the solvent system.
2.1.3. Procedure for preparation of azides
Fig. 1. Structure of triazole possessing anticancer activity.
Aromatic Azides. To a solution of 2,5-dimethyl aniline (500 mg,
4.13 mmol) in 1,4-dioxane at ꢀ15 ꢁC, 2 M sulphuric acid (10.3 mL,
20.6 mmol) was added in small instalments while stirring [24].
After 5 min, 3 M aqueous sodium nitrite solution (2.75 mL,
8.26 mmol) was added drop wise followed by careful addition of
3 M sodium azide solution (4.13 mL,12.39 mmol). After the addition
was over, the reaction mixture was allowed to attain room tem-
perature and extracted with diethyl ether (3 ꢂ 50 mL). The com-
bined organic layer was washed with saturated sodium bicarbonate
solution, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to yield aromatic azide that was used in the
next reaction without purification.
Aliphatic Azides. A mixture of octanol (4.6 mL, 3.8 mmol), tri-
phenylphosphine (1.2 g, 4.6 mmol), iodine (1.16 g, 4.6 mmol), and
imidazole (0.26 g, 3.84 mmol) was thoroughly mixed in a round
bottom flask [24]. To this mixture was added sodium azide (0.99 g,
15.3 mmol) in DMSO and stirred at room temperature for 1 h. The
reaction mixture was extracted with diethyl ether (3 ꢂ 30 mL),
washed with brine, and concentrated under reduced pressure to
yield crude aliphatic azide that was used in the next reaction
without further purification.
induction by acting as inhibitors for methionine aminopeptidase
type II [14]. The 1,2,4-triazole derivatives are synthesized and
assessed for anti-cancer activity using docking simulation [15]. The
in vitro screening of the triazole derivatives have shown significant
activity against several types of cancer cells like ovarian, pulmonary
and melanoma [15]. The triazole derivatives synthesized from
various molecules also lead to the development of novel com-
pounds possessing antimicrobial activity [16,17]. The present study
was aimed to synthesize a series of thiophenol-formaldehyde tri-
azoles (TFT) and to investigate the synthesized molecules against
the ovary cancer cell lines. The study demonstrated that among the
synthesized library compound 5a is the most active against ovarian
cancer cells. The effect of compound 5a was also analysed on tumor
metastasis in mice model of ovary cancer. The study demonstrated
inhibition of ovary cancer cell viability through apoptosis induction
and suppression of tumor metastasis in mice by down-regulation of
vWF expression.
1,2,3-Triazole derivatives have been synthesized since long and
explored mainly for their anti-tumor potential [18]. The 2-
methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3- triazol-5-yl)an-
iline have been shown to inhibit tubulin polymerization through
interaction with b-tubulin via H-bonding with the amino acids [19].
Another compound, 3-(4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-
2.1.4. General procedure for synthesis of triazole compounds (5a-
5u)
The compound 4 (300 mg, 1.0 mmol) was dissolved in 5 ml of
H20:tBuOH (2:1) mixture. To this solution was added CuSO4
(20 mol%) and alkyl or aryl azide (1.5 mmol) and the mixture was
subjected to sonication at 45 ꢁC for 1e2 h. The progress of the re-
action was monitored by TLC. The products obtained were char-
acterized by 1H NMR, 13C NMR, and HRMS techniques; 2-(((1-(2-
fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
yl)benzo [d]isoxazole was found to significantly inhibit the prolif-
eration of MV4-11 cells at an IC50 of 2 mM [20]. The 4-[phenyl-1-(1-
phenyl-ethyl)]-1H-1,2,3-triazole displayed promising cytotoxic ac-
tivity against HL 60 cells [21]. The preparation of 1,2,3-triazole de-
rivatives of flavonoid, quinolone and oxadiazole lead to the
identification of compounds which inhibit growth of leukemia K-
562 and melanoma SK-MEL-5 cell lines [22]. The 1,2,3-triazole
derivatization of betulinic acid lead to the identification of two
compounds, 3{1N(2-cyanophenyl)-1H1,2,3-triazol-4yl}methyloxy
betulinic acid and 3{1N(5- hydroxy-naphth-1yl)-1H-1,2,3-triazol-
4yl}methyloxy betulinic acid with significant anti-proliferation
activity against leukemia cells HL-60 [23].
(5a). 1H NMR (400 MHz, (CD3)2SO)
7.20 (1H, d, J ¼ 8.0 Hz), 7.12e7.03 (6H, m), 4.68 (2H, s), 4.66 (2H, s),
4.24 (1H,s); 13C NMR (101 MHz, (CD3)2SO)
159.57, 157.47, 151.99,
d 8.01 (1H, s), 7.53.7.55 (1H, m),
d
133.75, 130.12, 128.05, 127.96, 127.54, 125.75, 125.70, 122.06, 122.02,
118.01, 117.80, 70.59, 60.04; HRMS (ESI) m/z calcd. for C16H14FN3OS
[M]þ: 315.0842, found: 315.0842; yield: 89%; melting point:
203e205 ꢁC.
2-(((1-(2,4-difluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
2. Materials and methods
methyl)benzenethiol (5b). 1H NMR (400 MHz, (CD3)2SO)
d 8.03 (1H,
s), 7.58.7.54 (1H, m), 7.21e7.19 (1H, m), 7.09e7.01 (4H, m),
2.1. Chemistry
6.91e6.83 (1H, m), 4.68 (2H, s), 4.66 (2H, s), 3.29 (1H,s); 13C NMR
(101 MHz, (CD3)2SO) d 161.80, 160.27, 159.70, 151.99, 133.75, 130.12,
2.1.1. General
129.54, 127.96, 123.19, 122.98, 114.69, 114.66, 105.95, 105.52, 70.59,
60.04; HRMS (ESI) m/z calcd. for C16H13F2N3OS [M]þ: 333.0747,
found: 333.0747; yield: 92%; melting point: 206e209 ꢁC.
1H and 13C NMR spectra of all the triazoles synthesized was
recorded in (CD3)2SO solvent using a Bruker AC-300P spectrometer.
The tetramethylsilane (TMS) was used as the internal standard. The
2-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
values of chemical shift (
d
values) and coupling constants (J values)
benzenethiol (5c). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s), 7.43
(2H, d, J ¼ 8.0 HZ), 7.25 (2H, d, J ¼ 8.0 HZ), 7.19e7.02 (4H, m), 4.66
(4H, s), 3.15 (1H,s); 13C NMR (101 MHz, (CD3)2SO)
151.53, 135.45,
are presented in ppm and Hz, respectively. The Bruker Daltonics
electro spray ionization apparatus was used for the measurement
of HRMS of all the compounds synthesized. Thin layer chroma-
tography was performed using commercially available silica gel
plates GF254 aluminium plates. The chemicals used for experi-
ments and solvents were obtained from Sigma-Aldrich (Sigma, St.
Louis, MO, USA). Purification of the compounds was achieved using
column chromatography on silica gel (Qualigens, 60e120 mesh).
d
133.75, 132.19, 130.12, 129.54, 127.96, 127.27, 127.07, 121.80, 121.05,
120.54, 70.59, 60.04; HRMS (ESI) m/z calcd. for C16H14BrN3OS [M]þ:
375.0041, found: 375.0041; yield: 90%; melting point: 204e206 ꢁC.
2-(((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
benzenethiol (5d). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s),
7.48e7.46 (2H, m), 7.22e6.99 (6H, m), 6.65 (4H, s), 4.66 (4H, s), 4.17

64
Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
(1H, s); 13C NMR (101 MHz, (CD3)2SO)
d
151.99, 137.93, 133.75,
(ESI) m/z calcd. for C16H15N3OS [M]þ: 297.0936, found: 297.0936;
yield: 88%; melting point: 212e214 ꢁC.
133.34, 132.10, 130.12, 129.54, 129.03, 127.96, 127.27, 127.07, 126.24,
124.71, 122.03, 121.05, 114.90, 70.59, 60.04; HRMS (ESI) m/z calcd.
for C16H14BrN3OS [M]þ: 375.0041, found: 375.0041; yield: 92%;
melting point: 212 ꢁC.
2-(((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzene-
thiol (5m). 1H NMR (400 MHz, (CD3)2SO)
d 8.03 (1H, s), 7.43 (1H, d,
J ¼ 4.0 Hz), 7.32e7.34 (1H, m), 7.23e6.88 (6H, m), 4.70 (2H, s), 4.65
2-(((1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
(2H, s), 3.29, (1H, s), 2.35 (3H, s); 13C NMR (101 MHz, (CD3)2SO)
benzenethiol (5e). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s), 7.81
(1H, s), 7.45e7.47 (1H, m), 7.23e7.02 (6H, m), 4.69 (2H, s), 4.65 (2H,
s), 3.28 (1H, s); 13C NMR (101 MHz, (CD3)2SO)
151.53, 139.27,
d 151.53, 139.18,138.72,133.75, 130.12,129.54, 128.41,127.96,127.27,
127.07, 126.49, 124.15, 121.05, 116.19, 70.59, 60.04, 21.20; HRMS
(ESI) m/z calcd. for C17H17N3OS [M]þ: 311.1092, found: 311.1092;
yield 86%; melting point 209e211 ꢁC.
d
133.75, 130.50, 130.12, 129.54, 128.07, 127.96, 127.27, 127.07, 122.91,
122.86, 121.05, 118.29, 70.59, 60.04; HRMS (ESI) m/z calcd. for
2-(((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
C
16H14BrN3OS [M]þ: 375.0041, found: 375.0041; yield: 88%;
benzenethiol (5n). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s),
8.90e8.81 (2H, m), 7.53e7.40 (2H, m), 7.22e7.04 (4H, m), 4.69 (2H,
s), 4.67 (2H, s), 4.01, (1H, s); 13C NMR (101 MHz, (CD3)2SO)
151.99,
melting point: 217 ꢁC.
2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
d
benzenethiol (5f). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s), 7.32
(2H, d, J ¼ 8.0 Hz), 7.26 (2H, d, J ¼ 8.0 Hz), 7.19e7.02 (4H, m), 4.66
(4H, s), 4.15 (1H, s); 13C NMR (101 MHz, (CD3)2SO)
151.53, 136.01,
141.93, 133.75, 133.19, 132.74, 130.12, 129.54, 128.04, 128.01, 127.96,
127.27, 127.07, 126.18, 122.03, 70.59, 60.04; HRMS (ESI) m/z calcd.
for C16H14N4O3S [M]þ: 342.0787, found: 342.0787; yield 89%;
melting point 203e205 ꢁC.
d
133.75, 132.65, 130.12, 129.54, 129.01, 127.96, 127.27, 127.07, 122.93,
121.05, 70.59, 60.04; HRMS (ESI) m/z calcd. for C16H14ClN3OS [M]þ:
331.0546, found: 331.0546; yield: 90%; melting point: 206e209 ꢁC
2-(((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)
2-(((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
benzenethiol (5o). 1H NMR (400 MHz, (CD3)2SO)
d 8.33 (1H, s), 8.03
(1H, s), 7.81.7.79 (1H, m), 7.52e7.50 (2H, m), 7.23e7.02 (4H, m), 4.70
methyl)benzenethiol (5g). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H,
s), 7.40 (1H, s), 7.28e7.02 (6H, m), 4.69 (2H, s), 4.65 (2H, s), 3.28 (1H,
s), 2.35 (3H, s), 2.33 (3H, s); 13C NMR (101 MHz, (CD3)2SO)
151.53,
(2H, s), 4.67 (2H, s), 3.29, (1H, s); 13C NMR (101 MHz, (CD3)2SO)
d
151.53, 147.64, 137.68, 133.75, 131.09, 130.12, 129.54, 127.96, 127.27,
d
127.07, 121.48, 121.05, 119.90, 70.59, 60.04; HRMS (ESI) m/z calcd.
for C16H14N4O3S [M]þ: 342.0787, found: 342.0787; yield: 90%;
melting point: 205e208 ꢁC.
140.54, 138.13, 134.35, 133.75, 132.06, 130.12, 129.54, 127.96, 127.27,
127.07, 126.12, 125.44, 121.05, 70.59, 60.04, 20.37, 19.44; HRMS (ESI)
m/z calcd. for C18H19N3OS [M]þ: 325.1249, found: 325.1249; yield:
86%; melting point: 218 ꢁC.
2-(((1-(2,4-dinitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
benzenethiol (5p). 1H NMR (400 MHz, (CD3)2SO)
d 8.70 (1H, s), 8.30
2-(((1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)
(1H, d, J ¼ 8.0 Hz), 8.12e8.03 (2H, m), 7.19e7.21 (2H, m), 7.07e7.05
methoxy)methyl)benzenethiol (5h). 1H NMR (400 MHz, (CD3)2SO)
(2H, m), 4.70 (2H, s), 4.67 (2H, s), 4.01, (1H, s); 13C NMR (101 MHz,
d
8.03 (1H, s), 7.91e7.80 (2H, m), 7.48 (1H, m), 7.28e7.05 (5H, m),
(CD3)2SO) d 151.99, 147.09, 145.59, 135.00, 133.75, 130.70, 130.12,
4.65 (4H, s), 3.15 (1H, s); 13C NMR (101 MHz, (CD3)2SO)
d
151.99,
129.54, 127.96, 127.60, 127.27, 127.07, 124.70, 122.03, 70.59, 60.04;
HRMS (ESI) m/z calcd. for C16H13N5O5S [M]þ: 387.0637, found:
387.0637; yield: 92%; melting point: 205e208 ꢁC.
136.03, 135.98, 132.86, 130.12, 129.54, 128.94, 127.96, 127.27, 127.21,
127.14, 126.85, 124.75, 122.03, 121.12, 70.59, 60.04; HRMS (ESI) m/z
calcd. for C17H14F3N3OS [M]þ: 365.0810, found: 365.0810; yield:
89%; melting point: 207e208 ꢁC.
2-(((1-pentyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
(5q). 1H NMR (400 MHz, (CD3)2SO)
d 7.99 (1H, s), 7.23 (1H, d,
2-(((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)
J ¼ 4.0 Hz), 7.13e7.06 (3H, m), 4.66 (2H, s), 4.56 (2H, s), 4.20e4.03
methoxy)methyl)benzenethiol (5i). 1H NMR (400 MHz, (CD3)2SO)
d
(2H, m), 3.56 (1H, s), 1.82e1.78 (2H, m), 1.42e1.34 (4H, m),
8.03 (1H, s), 7.75 (2H, d, J ¼ 8.0 Hz), 7.66 (2H, d, J ¼ 8.0 Hz),
7.64e7.05 (4H, m), 4.65 (2H, s), 4.56 (2H, s), 3.40 (1H, s); 13C NMR
(101 MHz, (CD3)2SO) 151.53, 142.39, 133.75, 130.12, 129.54, 127.96,
1.01e0.97 (3H, m); 13C NMR (101 MHz, (CD3)2SO)
d 142.45, 133.75,
130.12, 129.54, 127.96, 127.27, 127.07, 124.44, 70.59, 60.04, 51.72,
29.80, 26.85, 22.93, 14.01; HRMS (ESI) m/z calcd. for C15H21N3OS
[M]þ: 291.1405, found: 291.1405; yield: 87%; melting point:
209e211 ꢁC.
d
126.91, 125.51, 123.81, 121.76, 121.70, 121.64, 121.59, 121.32, 121.05,
70.59, 60.04; HRMS (ESI) m/z calcd. for C17H14F3N3OS [M]þ:
365.0810, found: 365.0810; yield: 91%; melting point: 209 ꢁC.
2-(((1-(2,4,6-trichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)
2-(((1-heptyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
(5r). 1H NMR (400 MHz, (CD3)2SO)
d 7.99 (1H, s), 7.23e7.06 (4H, m),
methyl)benzenethiol) (5j). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H,
s), 7.43 (2H, s), 7.21e7.19 (1H, m), 7.08e7.01 (3H, m), 4.70 (2H, s),
4.65 (2H, s), 3.29 (1H, s); 13C NMR (101 MHz, (CD3)2SO)
150.75,
4.66 (4H, s), 4.13e4.02 (2H, m), 3.29 (1H, s), 1.82e1.76 (2H, m),
1.36e1.31 (8H, m), 1.09e0.97 (3H, m); 13C NMR (101 MHz,
d
(CD3)2SO) d 142.45, 133.75, 130.12, 129.54, 127.96, 127.27, 127.07,
138.70, 135.84, 135.10, 133.75, 130.12, 129.85, 129.54, 127.96, 127.27,
124.44, 70.59, 60.04, 51.72, 31.64, 29.06, 27.55, 26.96, 22.93, 14.01;
HRMS (ESI) m/z calcd. for C17H25N3OS [M]þ: 319.1718, found:
319.1718; yield: 86%; melting point: 214 ꢁC.
127.07, 119.63, 70.59, 60.04; HRMS (ESI) m/z calcd. for
C
16H12Cl3N3OS [M]þ: 398.9767, found: 398.9767; yield: 92%;
melting point 206 ꢁC.
2-(((1-nonyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
2-(((1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
(5s). 1H NMR (400 MHz, (CD3)2SO)
d 7.99 (1H, s), 7.24e7.26 (1H, m),
benzenethiol (5k). 1H NMR (400 MHz, (CD3)2SO)
d
8.28 (1H, d,
7.13e7.07 (3H, m), 5.07 (1H, s), 4.65 (2H, s), 4.56 (2H, s), 4.20e4.01
(2H, m), 1.83e1.79 (2H, m), 1.46e1.31 (12H, m), 1.09e0.97 (3H, m);
J ¼ 4.0 Hz), 8.03 (1H, s), 8.50e8.52 (1H, m), 7.62e7.01 (9H, m), 4.72
(2H, s), 4.66 (2H, s), 3.31 (1H, s); 13C NMR (101 MHz, (CD3)2SO)
13C NMR (101 MHz, (CD3)2SO)
d 142.45, 133.75, 130.12, 129.54,
d
150.80, 136.80, 134.10, 133.75, 130.12, 129.54, 128.31, 127.96,
127.96, 127.27, 127.07, 124.44, 70.59, 60.04, 51.72, 31.64, 29.06,
127.29, 127.28, 127.07, 126.86, 126.40, 125.69, 124.22, 123.60, 122.06,
112.84, 70.59, 60.04; HRMS (ESI) m/z calcd. for C20H17N3OS [M]þ:
347.1092, found: 347.1092; yield: 90%; melting point: 219 ꢁC.
2-(((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzene-
28.95, 27.55, 26.96, 22.93, 14.01; HRMS (ESI) m/z calcd. for
C
19H29N3OS [M]þ: 347.2031, found: 347.2031; yield: 85%; melting
point: 210e212 ꢁC.
2-(((1-decyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
thiol (5l). 1H NMR (400 MHz, (CD3)2SO)
d
8.03 (1H, s), 7.35e7.37 (2H,
m), 7.21e6.93 (7H, m), 4.66 (4H, s), 34.13 (1H, s); 13C NMR
(101 MHz, (CD3)2SO) 151.53, 137.08, 133.75, 130.12, 129.54, 129.27,
127.96, 127.60, 127.27, 127.07, 121.38, 121.05, 70.59, 60.04; HRMS
(5t). 1H NMR (400 MHz, (CD3)2SO)
d 7.99 (1H, s), 7.24e7.22 (1H, m),
7.11e7.04 (3H, m), 4.67 (2H, s), 4.65 (2H, s), 4.24e3.95 (2H, m), 3.30
(1H, s), 1.84e1.79 (2H, m), 1.46e1.32 (14H, m), 1.09e0.97 (3H, m);
d
13C NMR (101 MHz, (CD3)2SO)
d 142.45, 133.75, 130.12, 129.54,

Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
65
127.96, 127.27, 127.07, 124.44, 70.59, 60.04, 51.72, 31.64, 29.06,
was assessed by Transwell assay [26]. Briefly, the cells were incu-
bated with 0.25, 0.5, 1.0, 1.5 and 2.0 M concentrations of compound
28.95, 27.55, 26.96, 22.93, 14.01; HRMS (ESI) m/z calcd. for
m
C
20H31N3OS [M]þ: 361.2188, found: 361.2188; yield: 92%; melting
5a for 8 h. The cells following incubation were harvested and sub-
sequently seeded onto the upper chambers at 2 ꢂ 105 cells/ml den-
sity in the serum-free DMEM. The lower chambers contained DMEM
mixed with fetal bovine serum (10%). The cell culture was performed
for 24 h and subsequently the chambers were inverted for staining
the cells with hematoxylin. The proportion of the cells migrated to
lower chamber was calculated for the assessment of invasion po-
tential. The cell counting was carried out randomly in five fields and
images were captured at x400 magnification.
point: 216 ꢁC.
2-(((1-dodecyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzenethiol
(5u). 1H NMR (400 MHz, (CD3)2SO)
d 7.84 (1H, s), 7.25e7.27 (1H, m),
7.11e7.04 (3H, m), 4.64 (2H, s), 4.60 (2H, s), 4.43 (1H, s), 4.18e3.98
(2H, m), 1.78e1.74 (2H, m), 1.48e1.32 (18H, m), 1.02e0.99 (3H, m);
13C NMR (101 MHz, (CD3)2SO)
d 142.45,133.75,130.12,129.54,127.96,
127.27, 127.07, 124.44, 70.59, 60.04, 51.72, 31.64, 29.06, 28.95, 27.55,
26.96, 22.93, 14.01; HRMS (ESI) m/z calcd. for C22H35N3OS [M]þ:
389.2501, found: 389.2501; yield: 91%; melting point: 228 ꢁC.
2.2.6. Migration assay
2.2. Biology
CAOV3 cell migration potential after incubation with compound
5a was determined by wound-healing assay. The cells were incu-
2.2.1. Cell culture
bated with 0.25, 0.5, 1.0, 1.5 and 2.0 mM concentrations of com-
The human ovarian cancer cell lines CAOV3, CAOV4 and ES-2
were obtained from the Shanghai Institute of Biochemistry and
Cellular Biology of the Chinese Academy of Sciences (Shanghai,
China). Dulbecco's modified Eagle's medium (DMEM; Gibco Life
Technologies, Carlsbad, CA, USA) mixed with 10% fetal bovine
serum was used for culturing the cell lines. The medium was sup-
plemented with penicillin (100 U/ml) and streptomycin (100 U/ml)
and the cells were cultured at 37 ꢁC under humidified atmosphere
containing 5% CO2.
pound 5a for 48 h at 2 ꢂ 105 density per well in 6-well plates [27].
The cells were scratched using plastic scraper and the cellular
monolayer was washed to clear it from the detached cells. The
adherent cells were subjected to incubation for 24 h under an at-
mosphere of 5% CO2 at 37 ꢁC. The migration potential of CAOV3 cells
was recorded by monitoring the distance travelled by the cells in
the scraped region.
2.2.7. Western blot analysis
CAOV3 cells were subjected incubation with 0.25, 0.5, 1.0, 1.5
2.2.2. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
bromide) assay
Changes in proliferation of CAOV3 cells after incubation with
compound 5a were analysed by MTT assay [20]. Briefly, CAOV3 cells
were dispersed at 1.5 ꢂ 106 cells/ml density into the 96-well plates.
The cells were subjected to incubation with 0.25, 0.5, 0.75, 1.0, 1.5,
and 2.0 mM concentrations of compound 5a for 48 h. The cells were
collected, washed with PBS and subsequently lysed by treatment
with RIPA (Roche, Shanghai, China) for 45 min under ice-cold
conditions assay [27]. The cellular lysates were subjected to
centrifugation for 20 min at 14,000 rpm at 4 ꢁC. The concentration
of proteins was analysed by the bicinchoninic acid (BCA) and sep-
aration was achieved on SDS-PAGE gel (10%). The proteins were
transferred subsequently to polyvinylidene difluoride membranes.
Incubation of the membranes was then performed overnight with
primary antibodies at 4 ꢁC. After incubation, membranes were
washed using tris-buffered saline and Tween-20 three times. Then,
the membrane incubation was carried out at room temperature
with horseradish peroxidase-labelled goat anti-rabbit IgG second-
ary antibody for 1 h. Detection of the protein bands was performed
using chemiluminescence and autoradiography (ChemiDoc XRS;
Bio-Rad Laboratories, Inc., Hercules, CA, USA). The Quantity One
software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA,
USA) was used for densitometry determination. The primary anti-
bodies used were against MMP-2 (dilution 1:1000; catalog number
#4022), MMP-9 (dilution 1:500; catalog number #3852), p-FAK
(dilution 1:500; catalog number #3283), FAK (dilution 1:500; cat-
1.75 and 2.0
m
M concentrations of compound 5a for 48 h in an
incubator under 5% CO2 at 37 ꢁC. Following incubation, 20
m
l of MTT
solution (5 mg/ml) was put into each well of the plate and incu-
bated for 4 h. From each well supernatant was decanted and
dimethyl sulfoxide (150 ml) was added. Cell viability was deter-
mined by measuring the absorbance of each well at 490 nm. The
reading were measured three times for each of the well.
2.2.3. DNA fragmentation assay
Into T-75 flasks, CAOV3 cells were cultured for 12 h at
2 ꢂ 105 cells per flask density [25]. The medium was then replaced
with fresh medium containing 0.25, 0.5, 1.0, 1.5 and 2.0 mM con-
centrations of compound 5a and incubated in it for 48 h. The
genomic DNA was prepared using QIAamp DNA Mini kit (Qiagen)
according to the guidelines mentioned in the user manual. Agarose
gel (1.8%) was used for the DNA sample electrophoresis over 2 h at
50 V. The gels were stained with ethidium bromide (Sigma-Aldrich)
and subjected to visualization under ultraviolet (UV) trans-
illuminator (Wealtech Corp., Reno, NV, USA).
alog number #3285) and b-actin (dilution 1:1000; catalog number
#4967) (Cell Signalling, Shanghai, China).
2.2.8. Animals
Twenty 5-week old female BALB/c nude mice were purchased
from the Chinese Academy of Science. The mice were acclimatized
one week before the actual experiment to the laboratory conditions
and were housed under standard laboratory conditions. All the
mice were provided free access to food and water ad libitum. The
experimental procedures were performed according to the guide-
lines issued by the Animal Ethics Committee, School of Medicine,
Shanghai Jiaotong University, China.
2.2.4. Cell cycle analysis
In 6-well plates CAOV3 cells at a density of 3 ꢂ 106 cells per well
were added and cultured for 24 h [25]. The compound 5a at 0.25,
0.5, 1.0, 1.5 and 2.0 mM concentrations was put into the plates and
cells were incubated for 48 h. After compound 5a treatment the
cells were washed with PBS, collected and subjected to overnight
fixing in ethyl alcohol (70%) at 4 ꢁC. Then tris-hydrochloride buffer
(pH 7.6) mixed with 1% RNase A was added to the cells followed by
propidium iodide staining. Cell cycle distribution of CAOV3 cells
was assessed by examining the DNA contents using flow cytometry.
2.2.9. Preparation of ovarian cancer mice model
The solid viable tumor mass was established by inoculation of
CAOV3 cells at a concentration of 2 ꢂ 106 in 100
ml volume of PBS on
the dorsal side of nude mice [28]. From the mice tumor was extracted
under anesthesia by laparotomy and then dissected into small pieces
2.2.5. Invasion assay
CAOV3 cell invasion potential after incubation with compound 5a

66
Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
of 1 mm3 dimension. The tumor was implanted into the right ovary of
mice orthotopically through surgical suture under sterilized condi-
tions. The ovary was placed to its original site carefully and the wall of
abdominal cavity was stitched using 3-0 silk suture. The mice were
then assigned randomly into three groups of 5-each. The mice in the
two treatment groups were intraperitoneally administered com-
pound 5a dissolved in DMSO at 5 mg/kg and 10 mg/kg doses after 1 h
synthesized using click chemistry procedure and evaluated against
ovarian cancer cells. For this purpose, thiophenol was subjected to
MBH-reaction using DABCO (1,4-diazabicyclo[2.2.2]octane) as base
to obtain a-hydroxymethyl thiophenol (3) in 89% yield. The com-
pound 3 was subsequently propargylated using NaH under anhy-
drous conditions to afford the alkyne derivative (4) in 86% yield
(Scheme 1).
of tumor implantation. Mice in the untreated group received equal of
normal saline. The mice were sacrificed on day 21 after tumor im-
plantation to extract liver, lungs, intestine and peritoneal tissues for
analysis of tumor metastasis. The extractedtumor samples werefixed
in formalin (10%) and then embedded in paraffin wax. The tissue
The aromatic azides required were synthesized from the ani-
lines by diazotization reaction with sodium nitrite under acidic
conditions and subsequently reacted in situ with sodium azide
(Scheme 2).
All the aliphatic azides were synthesized by the reaction of
aliphatic alcohol with NaN3, Ph3P and I2 in the presence of imid-
azole as base (Scheme 3).
samples were sliced into 2 mm sections and then subjected to H&E for
histopathological examination.
The alkyne derivative (4) was subjected to 1,3-dipolar cycload-
dition reaction with the synthesized aromatic and aliphatic azides.
The reaction was performed using CuSO4$5H2O and sodium
ascorbate in the presence of t-BuOH/H2O (2:1; v/v) solvent (Scheme
1). The reaction lead to the formation of 1,4-substituted-triazole
compounds (5aꢀ5u) in very good yield under sonication at a
temperature of 45 ꢁC in 1e2 h (Scheme 1). The products obtained
were characterized by 1H NMR, 13C NMR, and HRMS techniques. In
1H NMR, cyclization of azides to form triazoles was confirmed by
2.2.10. Immunohistochemical analysis
The pulmonary, intestinal, peritoneal and liver tissues sections
after extraction and PBS washing were embedded in paraffin wax.
The samples were cut into thin 2 mm sections and subjected to in-
cubation with antibodies against vWF (Dako, Australia) [27]. The
samples after washing were incubated with horseradish peroxidase
(HRP)-conjugated rabbit anti-rat secondary antibody (Dako). The
sections were then stained with 3,30 diaminobenzidine (DAB) and
subsequently subjected to hematoxylin counterstaining. The image
examination software Image Pro Plus version 4.5 software (Diag-
nostic Instruments, USA) was used for the immunohistochemical
quantification of the digitized images.
the resonance of H-atom of triazole ring at
d 8.0 Hz in addition to,
chemical shift of other protons in aromatic and aliphatic region. The
structure was further supported by 13C NMR and HRMs, which
showed all the expected carbon signals corresponding to the tri-
azolyl derivatives.
2.2.11. Statistical analysis
All the experiments were repeated three times. The data pre-
sented are the means standard SD. Student's t-test and one-way
ANOVA were used for the data analysis. The P < 0.05 was taken to
indicate statistically significant differences. SPSS v17 software (SPSS,
Inc., Chicago, IL, USA) was used for statistical analysis of the data.
3.2. Biology
Initial screening of the synthesized compounds (5a-5u) against
CAOV3, CAOV4 and ES-2 ovarian cancer cells revealed that five-
compounds 5a, 5b, 5j, 5h and 5i significantly inhibited viability.
However, among the five compounds 5a was found to be most
active against the tested ovarian cancer cell lines. The inhibitory
effect of compound 5a on ovarian cancer cells was investigated in
detail. Compound 5a significantly inhibited the viability of CAOV3,
3. Results and discussion
3.1. Chemistry
CAOV4 and ES-2 cells in
(Fig. 2). The cell viability was measured on treatment with 0.25, 0.5,
0.75, 1.0, 1.5, 1.75 and 2.0 M concentrations of compound 5a. The
a concentration-dependent manner
Considering the biological importance of triazolyl derivatives, a
series of thiophenol-formaldehyde-triazolyl (TFT) compounds was
m

Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
67
Scheme 1. Synthesis of thiophenol-formaldehyde-triazolyl (TFT) compounds.
Library of the synthesized compounds. Isolated yield of the compounds 86e94%.
Scheme 2. Preparation of aromatic triazoles.
Scheme 3. Preparation of aliphatic triazoles.
Fig. 3. Compound 5a treatment leads to nuclear fragmentation in CAOV3 cells. (A) The
cells after compound 5a-treatment were analysed for nuclear morphology using
Hoechst 33342 staining by a fluorescent microscope.
viability was reduced significantly (P < 0.05) at 48 h on treatment
with compound 5a from 0.5
and ES-2 cell viability was reduced to 27, 26 and 29%, respectively
on incubation with 2.0 M concentration of compound 5a for 48 h.
At 0.25 M concentration, compound 5a did not change the
mM concentration. The CAOV3, CAOV4
m
m
in the untreated cells. The population of CAOV3 cells in the S phase
viability of CAOV3, CAOV4 and ES-2 cells significantly.
decreased to 11.48% on exposure to 2.0
pound 5a in comparison to 18.89% in control cultures. In G2/M
phase compound 5a exposure at 2.0 M concentration reduced
CAOV3 cell population to 18.08% compared to 22.68% in the un-
treated cultures.
mM concentration of com-
In CAOV3 cells compound 5a treatment for 48 h was followed by
the analysis of cell apoptosis by staining with Hoechst 33342
(Fig. 3). Increase in the compound 5a concentration from 0.25 to
m
2.0
mM enhanced apoptotic cell proportion significantly (P < 0.05).
At 2.0
m
M concentration of compound 5a the presence of charac-
Carcinoma development and its progression is associated with
the invasion and migration potential of the cells [29,30]. Detached
cancer cells use the membrane receptors to penetrate different
microenvironments for metastasis [29,30]. In the present study the
invasive potential of CAOV3 cells was reduced significantly
(P < 0.05) on treatment with compound 5a in concentration based
manner (Fig. 5). The cells after 48 h of incubation with 0.25, 0.5, 1.0,
teristic apoptotic nuclei was evident in CAOV3 cell cultures. The
apoptotic nuclei were not seen in CAOV3 cells cultured for 48 h in
the absence of compound 5a. The percentage of apoptotic nuclei
was recorded as 1.26, 8.67, 21.54, 45.89 and 62.53%, respectively on
treatment with 0.25, 0.5, 1.0, 1.5 and 2.0 mM concentrations of
compound 5a for 48 h.
CAOV3 cells after exposure to 0.25, 0.5, 1.0, 1.5 and 2.0 mM
1.5 and 2.0
Transwell assay. The invasive potential of CAOV3 cells didn't show
any significant change on treatment with 0.25 M concentration of
compound 5a. Incubation with 0.5, 1.0, 1.5 and 2.0 M concentra-
tions of compound 5a decreased cell invasion to 84.54, 67.76, 39.14
and 8.62%, respectively.
mM concentrations of compound 5a were examined by
concentrations of compound 5a for 48 h were analysed by flow
cytometry (Fig. 4). A significant increase in the percentage of
CAOV3 cells was observed in G0/G1 phase on exposure to com-
pound 5a. The percentage of CAOV3 cells in G2/M and S phases
decreased with the increase in concentration of compound 5a.
m
m
Exposure to 2.0
mM concentration of compound 5a increased per-
These findings proved that compound 5a also inhibits tumor
metastasis in vivo in mice. To understand the mechanism of
centage of CAOV3 cells in G0/G1 to 69.54% in comparison to 57.77%
Fig. 2. Effect of compound 5a on CAOV3, CAOV4 and ES-2 cell viability. The cells were incubated with compound 5a for 48 h. Viability of the cells was assessed by MTT assay at 48 h
*P < 0.05, **P < 0.02 and ***P < 0.01 compared to 0 M compound 5a concentration.
m

68
Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
compound 5a mediated suppression of tumor metastasis the
expression of pathways and molecules associated with the cell
invasion and migration was investigated. The cell metastasis is
commonly regulated by the matrix metalloproteinases, TIMPs and
FAK [31,32]. Activation of FAK plays an important role in the
migration and proliferation of tumor cells through alteration of
cell-ECM interactions [32,33]. The CAOV3 cell migration after 48 h
of treatment with 0.25, 0.5, 1.0, 1.5 and 2.0
compound 5a was determined by wound healing assay (Fig. 6).
Increasing the concentration of compound 5a from 0.5 to 2.0
mM concentrations of
m
M
led to a significant (P < 0.05) decrease in the migration potential of
CAOV3 cells. The cell migration was decreased from 89.65 to 6.43%
with the increase in compound 5a concentration from 0.5 to
2.0
mM.
Metastasis of the tumor cells is facilitated by the matrix met-
alloproteinases through break down of basement membrane and
components of extracellular matrix [29,31,34]. The metastasis po-
tential of tumor cells is related to the expression of matrix
matelloproteinase-9 [31]. In the present study the effect of 0.25, 0.5,
Fig. 4. Cell cycle arrest in CAOV3 cells in G0/G1 phase by compound 5a. The cells after
exposure to compound 5a were examined by flow cytometry. The presented values are
mean SD of three separate experiments.
Fig. 5. Effect of compound 5a on invasion of CAOV3 cells. The cell incubation with
compound 5a was followed by assessment of invasion potential by Transwell method.
The images were captured at x100 magnification.
Fig. 7. Effect of compound 5a on MMPs and FAK expression. The protein expression in
CAOV3 cells after incubation with compound 5a was assessed by western blotting. The
protein expression was compared to b-actin as loading control.
Fig. 6. Effect of compound 5a on migration of CAOV3 cells. The cell incubation with compound 5a for 48 h was followed by assessment of migration potential by wound healing
assay. The images were captured at x100 magnification.

Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
69
Fig. 8. Compound 5a inhibits vWF expression in tumor metastasis lesions. The vWF expression was examined in the lesions extracted from liver, lungs, intestinal and peritoneal
tissues.
1.0, 1.5 and 2.0
m
M concentrations of compound 5a on the expres-
The vWF expression was reduced to ~7, ~8, 6, and ~5%, respectively
in liver, lungs, intestinal and peritoneal lesions of 10 mg/kg treat-
ment group compared to the untreated mice. In the 5 mg/kg
treatment group the vWF expression was higher than those of
10 mg/kg treatment mice group.
Microphages also release invasive proteases and adhesion
related factors to promote dissemination of the tumors [38,39]. The
infiltration of macrophages in the metastatic lesions of liver, in-
testinal and pulmonary tissues showed a significant decrease in the
compound 5a-treated mice compared to the corresponding lesion
from untreated mice (Fig. 9). The suppression of macrophage
infiltration was markedly higher in the mice treated with 10 mg/kg
doses of compound 5a compared to 5 mg/kg treatment group. The
macrophage infiltration in the lesions of liver, pulmonary tissues
intestinal and peritoneum was ~8, ~9, ~7 and ~5% in mice treated
with 10 mg/kg doses of compound 5a.
sion of MMP-2 and -9 in CAOV3 cells was determined by Western
blot assay (Fig. 7). Compound 5a treatment markedly decreased the
level of MMP-2 and -9 in CAOV3 cells from 0.5
No difference in the MMP-2 and -9 expression in untreated and
0.25 M compound 5a treatment CAOV3 cells cultures was recor-
ded. Treatment of CAOV3 cells with compound 5a caused a marked
decrease in FAK activation from 0.5 M concentration at 48 h. With
mM concentration.
m
m
the enhancement of compound 5a concentration from 0.5 to 2.0 mM
a remarkable decrease was recorded in p-FAK expression.
Metastasis of the malignant tumor cells also depends on the
vWF expression which determines neovascularisation [35]. In the
tumor microenvironment microphages promote angiogenesis of
the cells by secreting matric proteases [36,37]. In the ovary tumor
mice model effect of compound 5a on tumor metastasis was ana-
lysed after 20 days. The tumor growth was inhibited in the com-
pound 5a treated mice markedly than those of untreated mice.
However, the tumor growth was slightly larger in the 5 mg/kg
compound 5a treatment group than the 10 mg/kg groups. The size
of tumor extracted from negative control, untreated, 5 mg/kg and
10 mg/kg compound 5a treatment groups was measured as 0,
1956 367, 1258 298 and 213.34 mm3, respectively on day 20.
Metastasis of tumor was observed to liver, intestine, spleen and
peritoneal cavity in all the mice of untreated group. However, the
tumor metastasis was decreased in mice treated with 10 mg/kg
compound 5a. Examination of vWF expression by immunohisto-
chemistry in liver, intestinal, pulmonary and peritoneal lesions
showed a marked decrease in compound 5a-treated mice (Fig. 8).
4. Conclusion
In summary, compound 5a inhibited ovarian cancer cell prolif-
eration, caused onset of apoptosis and decreased metastasis partly
through targeting FAK activation and down-regulation of MMP
expression. In vivo compound 5a suppressed tumor development
and metastasis by suppression of macrophage infiltration and vWF
expression. Thus, compound 5a needs to be investigated further for
the development of ovarian cancer treatment.
Acknowledgements
This study was supported by the Natural science Fund of
Science and Technology Department in Jilin (No. 20180101010JC).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.03.033.
References
[1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics,
CA Cancer J. Clin. 58 (2008) 71e96.
[2] M. Raki, D.T. Rein, A. Kanerva, A. Hemminki, Gene transfer approaches for
gynecological diseases, Mol. Ther. 14 (2006) 154e163.
[3] E.R. Bertone-Johnson, Epidemiology of ovarian cancer: a status report, Lancet
365 (2005) 101e102.
Fig. 9. Compound 5a suppresses macrophage infiltration into metastatic tumor.
Immunohistostaining was used to determine macrophage infiltration in tumor
metastasis lesions from liver, intestinal and pulmonary tissues.
[4] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, Cancer statistics, 2009, Ca -

70
Y. Jia et al. / European Journal of Medicinal Chemistry 172 (2019) 62e70
Cancer J. Clin. 59 (2009) 225e249.
Basappa, B.K.S. Rangappa, Synthesis of 1,2-benzisoxazole tethered 1,2,3-
triazoles that exhibit anticancer activity in acute myeloid leukemia cell lines
by inhibiting histone deacetylases, and inducing p21 and tubulin acetylation,
Bioorg. Med. Chem. 23 (2015) 6157e6165.
[5] S.I. Kim, J.W. Lee, M. Lee, H.S. Kim, H.H. Chung, J.W. Kim, N.H. Park, Y.S. Song,
J.S. Seo, Genomic landscape of ovarian clear cell carcinoma via whole exome
sequencing, Gynecol. Oncol. 148 (2018) 375e382.
[6] R. Murakami, N. Matsumura, J.B. Brown, K. Higasa, T. Tsutsumi, M. Kamada,
H. Abou-Taleb, Y. Hosoe, S. Kitamura, K. Yamaguchi, et al., Exome sequencing
landscape analysis in ovarian clear cell carcinoma shed light on key chro-
mosomal regions and mutation gene networks, Am. J. Pathol. 187 (2017)
2246e2258.
[7] M.K. McConechy, J. Ding, J. Senz, W. Yang, N. Melnyk, A.A. Tone, L.M. Prentice,
K.C. Wiegand, J.N. McAlpine, S.P. Shah, et al., Ovarian and endometrial endo-
metrioid carcinomas have distinct CTNNB1 and PTEN mutation profiles, Mod.
Pathol. 27 (2014) 128e134.
[21] S.N.P. Bathula, R. Vadla, Bioactivity of 1,4-disubstituted 1,2,3-triazoles as
cytotoxic agents against the various human cell lines, Asian J. Pharm. Clin. Res.
4 (2011) 66e67.
[22] N. Pokhodylo, O. Shyyka, V. Matiychuk, Synthesis of 1,2,3-triazole derivatives
and evaluation of their anticancer activity, Sci. Pharm. 81 (2013) 663e676.
[23] R. Majeed, P.L. Sangwan, P.K. Chinthakindi, I. Khan, N.A. Dangroo, N. Thota,
A. Hamid, P.R. Sharma, A.K. Saxena, S. Koul, Synthesis of 3-O-propargylated
betulinic acid and its 1,2,3-triazoles as potential apoptotic agents, Eur. J. Med.
Chem. 63 (2013) 782e792.
[8] T. Katoh, M. Yasuda, K. Hasegawa, E. Kozawa, J. Maniwa, H. Sasano, Estrogen-
producing endometrioid adenocarcinoma resembling sex cord-stromal tumor
of the ovary: a review of four postmenopausal cases, Diagn. Pathol. 7 (2012)
164.
[9] J.K. Sahu, S. Ganguly, A. Kaushik, Triazoles: a valuable insight into recent
developments and biological activities, Chin. J. Nat. Med. 11 (2013) 456e465.
[10] M. Olah, R. Rad, L. Ostopovici, A. Bora, N. Hadaruga, D. Hadaruga, R. Moldovan,
A. Fulias, M. Mractc, T.I. Oprea, WOMBAT and WOMBAT-PK: bioactivity da-
tabases for lead and drug discovery, in: S.L. Schreiber, T.M. Kapoor, G. Wess
(Eds.), Chemical Biology: from Small Molecules to Systems Biology and Drug
Design, vols. 1e3, Wiley-VCH Verlag GmbH, Weinheim, 2008, pp. 760e786.
[11] X. Li, X.Q. Li, H.M. Liu, X.Z. Zhou, Z.H. Shao, Synthesis and evaluation of
[24] S.G. Agalave, S.R. Maujan, V.S. Pore, Click chemistry: 1,2,3-triazoles as phar-
macophores, Chem. Asian J. 6 (2011) 2696e2718.
[25] J. Hwang-B, J.H. Park, I.S. Chung, Tumstatin induces apoptosis mediated by Fas
signaling pathway in oral squamous cell carcinoma SCC-VII cells, Oncol. Lett.
10 (2015) 1016e1022.
[26] B. Wu, K. Hu, S. Li, J. Zhu, L. Gu, H. Shen, B.D. Hambly, S. Bao, W. Di, Dihy-
droartiminisin inhibits the growth and metastasis of epithelial ovarian cancer,
Oncol. Rep. 27 (2012) 101e108.
[27] H.-Q. Huang, J. Tang, S.-T. Zhou, T. Yi, H.-L. Peng, G.-B. Shen, N. Xie, K. Huang,
T. Yang, J.-H. Wu, C.-H. Huang, Y.-Q. Wei, X. Zhao, Orlistat, a novel potent
antitumor agent for ovarian cancer: proteomic analysis of ovarian cancer cells
treated with Orlistat, Int. J. Oncol. 41 (2012) 523e532.
antitumor activities of novel chiral 1,2,4-triazole Schiff bases bearing
butenolide moiety, Org. Med. Chem. Lett. 2 (2012) 26.
[12] D. Chowrasia, C. Karthikeyan, L. Choure, S. Sahabjada, M. Gupta, M.d. Arshad,
P. Trivedi, Synthesis, characterization and anti-cancer activity of some fluo-
rinated 3,6-diaryl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazoles, Arab. J. Chem
(2013) 6e10.
g
-
[28] B. Wui, K. Hui, S. Li, J. Zhui, L. Gu, H. Shen, B.D. Hambly, S. Bao, W. Di, Dihy-
droartiminisin inhibits the growth and metastasis of epithelial ovarian cancer,
Oncol. Rep. 27 (2012) 101e108.
[29] J. Zhang, B. Wang, Arsenic trioxide (As(2)O(3)) inhibits peritoneal invasion of
ovarian carcinoma cells in vitro and in vivo, Gynecol. Oncol. 103 (2006)
199e206.
[13] P. Chand, J.A. Chesney, B.F. Clem, G.H. Tapolsky, S. Telang, J.O. Trent, Small-
Molecule Choline Kinase Inhibitors as Anti-cancer Therapeutics. Patent: US
2011/0257211 A1.
[14] Y.P. Hou, J. Sun, Z.H. Pang, P.C. Lv, D.D. Li, L. Yan, H.J. Zhang, E.X. Zheng, J. Zhao,
H.L. Zhu, Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-
benzodioxan fragment as a novel class of potent methionine aminopepti-
dase type II inhibitors, Bioorg. Med. Chem. 19 (2011) 5948e5954.
[15] V. Georgiyants, L. Perekhoda, N. Saidov, I. Kadamov, Docking studies and
biological evaluation of anti-cancer activity of new 1,2,4-triazole (4h) de-
rivatives, Scr. Sci. Pharm. 1 (2014) 46e53.
[16] B.S. Holla, B.K. Sarojini, B.S. Rao, P.M. Akberali, N.S. Kumari, V. Shetty, Syn-
thesis of some halogen-containing 1,2,4-triazolo-1,3,4-thiadiazines and their
antibacterial and anticancer screening studies - part I, Farmaco 56 (2001)
565e570.
[17] D. Gupta, D.K. Jain, Synthesis, antifungal and antibacterial activity of novel
1,2,4-triazole derivatives, J. Adv. Pharm. Technol. Res. 6 (2015) 141e146.
[18] D. Dheer, V. Singh, R. Shankar, Medicinal attributes of 1,2,3-triazoles: current
developments, Bioorg. Chem. 71 (2017) 30e54.
[30] S. Cattaruzza, R. Perris, Proteoglycan control of cell movement during wound
healing and cancer spreading, Matrix Biol. 24 (2005) 400e417.
[31] R. Hoekstra, F.A. Eskens, J. Verweij, Matrix metalloproteinase inhibitors: cur-
rent developments and future perspectives, Oncol. 6 (2001) 415e427.
[32] J.T. Parsons, K.H. Martin, J.K. Slack, J.M. Taylor, S.A. Weed, Focal adhesion ki-
nase: a regulator of focal adhesion dynamics and cell movement, Oncogene 19
(2000) 5606e5613.
[33] P.P. Provenzano, P.J. Keely, The role of focal adhesion kinase in tumor initia-
tion and progression, Cell Adhes. Migrat. 3 (2009) 347e350.
[34] L. Anfosso, T. Efferth, A. Albini, U. Pfeffer, Microarray expression profiles of
angiogenesis-related genes predict tumor cell response to artemisinins,
Pharmacogenomics J. 6 (2006) 269e278.
[35] N. Weidner, J. Folkman, F. Pozza, et al., Tumor angiogenesis: a new significant
and independent prognostic indicator in early-stage breast carcinoma, J. Natl.
Cancer Inst. 84 (1992) 1875e1887.
[36] L.M. Coussens, Z. Werb, Inflammation and cancer, Nature 420 (2002)
860e867.
[37] C.E. Lewis, J.W. Pollard, Distinct role of macrophages in different tumor mi-
croenvironments, Cancer Res. 66 (2006) 605e612.
[38] N. Jonjic, G. Peri, S. Bernasconi, et al., Expression of adhesion molecules and
chemotactic cytokines in cultured human mesothelial cells, J. Exp. Med. 176
(1992) 1165e1174.
[39] E. Wang, Y. Ngalame, M.C. Panelli, et al., Peritoneal and subperitoneal stroma
may facilitate regional spread of ovarian cancer, Clin. Cancer Res. 11 (2005)
113e122.
[19] K. Odlo, J. Hentzen, J.F.D. Chabert, S. Ducki, O.A.B.S.M. Gani, I. Sylte, M. Skrede,
V.N. Flørenes, T.V. Hansen, 1,5-Disubstituted 1,2,3-triazoles as cis-restricted
analogues of combretastatin A-4: synthesis, molecular modeling and evalu-
ation as cytotoxic agents and inhibitors of tubulin, Bioorg. Med. Chem. 16
(2008) 4829e4838.
[20] N. Ashwini, M. Garg, C.D. Mohan, J.E. Fuchs, S. Rangappa, S. Anusha,
T.R. Swaroop, K.S. Rakesh, D. Kanojia, V. Madan, A. Bender, H.P. Koeffler,