Discovery of 5,6-diaryl-1,2,4-triazines hybrids as potential apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2017.07.011

A series of 5,6-diaryl-1,2,4-triazines hybrids bearing a 1,2,3-triazole linker were synthesized by molecular hybridization strategy and evaluated for antiproliferative activity against three selected cancer cell lines (MGC-803, EC-109 and PC-3). The first structure-activity relationship (SAR) for these 5,6-diaryl-1,2,4-triazines is explored in this report with evaluation of 15 variants of the structural class. Among these chemical derivatives, 3-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-diphenyl-1,2,4-triazine (11E) showed the more potent inhibitory effect against three cell lines than 5-Fu. Cellular mechanism studies in MGC-803 cells elucidated 11E inhibited colony formation and arrested cell cycle at G2/M phase. Furthermore, compound 11E caused morphological changes, decreased mitochondrial membrane potential, and induced apoptosis through the apoptosis-related proteins in MGC-803 cells. It was the first time, to our knowledge, that 5,6-diaryl-1,2,4-triazines bearing a 1,2,3-triazole linker were used as potential apoptosis inducers.

Full text of DOI:10.1016/j.ejmech.2017.07.011

Accepted Manuscript
Discovery of 5,6-diaryl-1,2,4-triazines hybrids as potential apoptosis inducers
Dong-Jun Fu, Jian Song, Yu-Hui Hou, Ruo-Han Zhao, Jia-Huan Li, Ruo-Wang Mao,
Jia-Jia Yang, Ping Li, Xiao-Lin Zi, Zhong-Hua Li, Qing-Qing Zhang, Fei-Yan Wang,
Sai-Yang Zhang, Yan-Bing Zhang, Hong-Min Liu
PII:
S0223-5234(17)30533-0
10.1016/j.ejmech.2017.07.011
EJMECH 9574
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 April 2017
Revised Date: 4 July 2017
Accepted Date: 7 July 2017
Please cite this article as: D.-J. Fu, J. Song, Y.-H. Hou, R.-H. Zhao, J.-H. Li, R.-W. Mao, J.-J. Yang, P.
Li, X.-L. Zi, Z.-H. Li, Q.-Q. Zhang, F.-Y. Wang, S.-Y. Zhang, Y.-B. Zhang, H.-M. Liu, Discovery of 5,6-
diaryl-1,2,4-triazines hybrids as potential apoptosis inducers, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.07.011.
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Graphical abstract

ACCEPTED MANUSCRIPT
Discovery of 5,6-diaryl-1,2,4-triazines hybrids as
potential apoptosis inducers
Dong-Jun Fu1a, Jian Song1a, Yu-Hui Houa, Ruo-Han Zhaoa, Jia-Huan Lia, Ruo-Wang
Maoa, Jia-Jia Yanga, Ping Lia, Xiao-Lin Zic, Zhong-Hua Lia, Qing-Qing Zhanga, Fei-
Yan Wanga, Sai-Yang Zhang*b, Yan-Bing Zhang*a, Hong-Min Liua*
aNew Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou 450001, China; Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan
Province; Key Laboratory of Technology of Drug Preparation (Zhengzhou University), Ministry of Education;
Key Laboratory of Henan Province for Drug Quality and Evaluation.
bSchool of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China.
cPathology and Laboratory Medicine, University of California, Irvine, Orange, CA 92868, USA.
1These authors contributed equally to this work.
*Correspondence: celeron16@163.com (S.-Y. Zhang), zhangyb@zzu.edu.cn (Y.-B. Zhang), liuhm@zzu.edu.cn (H.-
M. Liu)
Abstract: A series of 5,6-diaryl-1,2,4-triazines hybrids bearing a 1,2,3-
triazole linker were synthesized by molecular hybridization strategy and
evaluated for antiproliferative activity against three selected cancer cell
lines (MGC-803, EC-109 and PC-3). The first structure-activity
relationship (SAR) for these 5,6-diaryl-1,2,4-triazines is explored in this
report with evaluation of 15 variants of the structural class. Among these
chemical
derivatives,
3-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-
yl)methyl)thio)-5,6-diphenyl-1,2,4-triazine (11E) showed the more potent
inhibitory effect against three cell lines than 5-Fu. Cellular mechanism
studies in MGC-803 cells elucidated 11E inhibited colony formation and
arrested cell cycle at G2/M phase. Furthermore, compound 11E caused
morphological changes, decreased mitochondrial membrane potential,
and induced apoptosis through the apoptosis-related proteins in MGC-803
cells. It was the first time, to our knowledge, that 5,6-diaryl-1,2,4-
triazines bearing a 1,2,3-triazole linker were used as potential apoptosis
inducers.
Keywords: 5,6-diaryl-1,2,4-triazines; molecular hybridization strategy;
morphological changes; apoptosis; apoptosis-related proteins.
1. Introduction
5,6-Diaryl-1,2,4-triazine, an important heterocyclic skeleton, has been
reported to exhibit a variety of biological activities [1-7]. 2-((5,6-
diphenyl-1,2,4-triazin-3-yl)thio)-N-arylacetamide derivative 1 with strong
electron-withdrawing nitro group on the arylacetamide moiety exhibited
potent α-glucosidase inhibitory activity (IC50 = 12.46 ± 0.13 µM) [8]. 5,6-
diaryl-1,2,4-triazine bearing 3-morpholinoethylamine moiety 2 displayed
a promising antithrombotic profile in vivo, which demonstrated less
ulcerogenicity in rats as compared to aspirin [9]. 1,2,4-triazine derivative
3 was synthesized and screened for inhibition of cyclooxygenases (COX-

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1 and COX-2) with anti-oxidant activity based on a cellular assay using
human whole blood and lipoxygenase [10]. However, few studies
investigated 5,6-diaryl-1,2,4-triazine moiety as a promising anticancer
scaffold (Fig. 1).
Fig. 1. Bioactive 5,6-diaryl-1,2,4-triazine derivatives.
1,2,3-triazole, a privileged scaffold in drug discovery, displayed a wide
array of biological activities as antibacterial, anti-fungal, anti-HIV, anti-
allergic, anti-tubercular and anti-inflammatory agents [11-14]. Recently,
our group have reported four series of 1,2,3-triazole derivatives as
potential antitumor agents (Fig. 2): 1,2,3-triazole-chalcone hybrid 4
inhibited the proliferation of SK-N-SH cancer cells by inducing apoptosis
and arresting the cell cycle at the G1 phase [15]; the novel 1,2,3-triazole-
chalcone 5 bearing a 4,5-dihydrothiazole showed the potent activity with
an IC50 value of 8.16 µM against neuroendocrine cancer cells [16]; 1,2,3-
triazole-dithiocarbamate based selective lysine specific demethylase 1
inactivator 6 inhibited gastric cancer cell apoptosis, invasion, and
migration [17]; the steroidal hybrid 7 arrested cell cycle at G2/M phase,
induced apoptosis accompanied with decrease of mitochondrial
membrane potential [18].
Fig. 2. 1,2,3-triazole derivatives as antiproliferative agents.
Molecular hybridization strategy is a useful concept in drug design and
development based on the combination of pharmacophoric moieties of
different bioactive substances to produce a new hybrid with improved
affinity and efficacy, when compared to the parent drugs [19, 20]. These

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above interesting findings and our continuous quest to identify more
potent anticancer agents led to the molecular hybridization of 5,6-diaryl-
1,2,4-triazine and bioactive scaffolds by a 1,2,3-triazole linker to
integrate them in one molecular platform to generate a new hybrid with a
potential antiproliferative activity.
As shown in Fig. 3, a molecular hybridization strategy based on the
structures of a 5,6-diaryl-1,2,4-triazine and a 1,2,3-triazole yielded a
scaffold which has three parts: (i) a 5,6-diaryl-1,2,4-triazine scaffold as a
central core, (ii) a 1,2,3-triazole as a linker to combine different
antiproliferative active skeletons, and (iii) a variety of reported
antiproliferative skeletons (benzyl moiety, chalcone, formononetin,
coumarin, sulfanilamide) [21-23]. To the best of our knowledge, there
have been no literature reports regarding 5,6-diaryl-1,2,4-triazine hybrids
bearing a 1,2,3-triazole linker as antiproliferative agents so far. These
findings also encouraged us to investigate the potential synergistic effect
of 1,2,3-triazole and 5,6-diaryl-1,2,4-triazine scaffolds.
Fig. 3. Rational molecular hybridization design.
2. Results and discussion
2.1. Chemistry
To explore the potential synergistic effect of 1,2,3-triazole and 5,6-
diaryl-1,2,4-triazine scaffolds, 5,6-diaryl-1,2,4-triazine derivatives
without triazole moiety 10A-10C were synthesized in Scheme 1.

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Condensation of commercially available compound 8 with
o
thiosemicarbazide in acetic acid at 120 C for 3.5 h provided the 5,6-
diphenyl-1,2,4-triazine-3-thiol 9 [24], which was then reacted with
different bromides (1-bromopropan-2-one, 3-bromoprop-1-yne, and 3-
bromopropanoic acid) in the presence of triethylamine as base to obtain
compound 10A-10C.
O
O
N
N
N
N
b
a
N
R1
S
N
SH
9
10A-10C
8
N
N
N
N
N
N
O
N
N
S
N
S
OH
S
O
10C
10A
10B
Scheme 1. Reagents and conditions: (a) AcOH, thiosemicarbazide, reflux,
3.5 h; (b) Et3N, bromides (1-bromopropan-2-one or 3-bromoprop-1-yne,
or 3-bromopropanoic acid), Acetone, reflux.
In order to obtain the target 5,6-diaryl-1,2,4-triazines hybrids bearing a
1,2,3-triazole linker, various azide derivatives 11a-11l were used to
synthesize the target compounds by click reaction. The structures of azide
derivatives 11a-11l in this work were shown in Scheme 2. The synthetic
methods and structure confirmation of azide derivatives 11a-11l were
described in the Supporting Information.

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Br
Cl
N3
N3
N3
N3
11a
11b
11f
11c
11d
O
F
O
N3
N3
N3
O
11g
11e
O
NH2
N3
O
O
S
N3
O
N3
O
O
11j
11i
11h
O
O
N3
O
O
O
N3
O
11k
11l
Scheme 2. Azide derivatives 11a-11l in this work.
As shown in Scheme 3, 5,6-diaryl-1,2,4-triazine-triazole derivatives
11A-11L were achieved by reaction of compound 10B with various
azides [25, 26] using sodium ascorbate and CuSO4.5H2O in THF at room
temperature. The structures of all the new synthesized compounds 10A-
1
10C and 11A-11L were characterized by H NMR 、 13C NMR and
HRMS.

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N
N
N
N
N
a
N
S
S
R2
N
11A-11LN
N
10B
Cl
Br
N
N
N
N
N
N
S
S
N
N
N
N
N
N
11B
11A
N
N
N
N
N
S
N
N
N
N
N
N
N
11D
11C
O
F
N
N
N
N
S
N
N
S
N
N
N
N
N
N
11E
O
11F
O
S
NH2
N
N
N
N
O
N
O
N
S
N
N
N
S
N
N
N
11H
11G
N
N
N
N
N
S
N
N
O
N
O
S
N
N
N
N
O
O
11J
11I
N
N
N
N
N
N
O
N
N
S
N
S
O
N
O
O
N
O
O
N
11L
11K
Scheme 3. Reagents and conditions: (a) Sodium ascorbate, CuSO4.5H2O,
azides 11a-11l, THF/H2O, r.t.
2.2. Antiproliferative activity
In continuation with our efforts toward the identification of novel
derivatives with anticancer potential, we evaluated the antiproliferative
activity of 5,6-diaryl-1,2,4-triazine derivatives 10A-10C and 5,6-diaryl-

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1,2,4-triazine-triazole hybrids 11A-11L using the MTT assay. 1,2,3-
triazole derivatives have reported as potent antiproliferative agents
against PC-3 cells, MGC-803 cells and EC-109 cells [27]. All the target
compounds in this work contained a 1,2,3-triazole moiety. Therefore,
these three cancer cell lines (MGC-803, EC-109, PC-3) were selected to
evaluate their antiproliferative activity. When some 1,2,3-triazole
derivatives were reported as potent antiproliferative agents, 5-fluorouracil
(5-FU) was selected as the control [27]. Therefore, 5-Fu was also used as
the reference drug in this MTT assay.
The antiproliferative activity results against all three cancer cells for
the candidate compounds were shown in Table 1. In order to investigate
the effect of the inhibitory activity of 1,2,3-triazole, non-1,2,3-triazole-
5,6-diphenyl-1,2,4-triazines 10A-10C and 1,2,3-triazole-5,6-diphenyl-
1,2,4-triazines 11A-11L were examined for their antiproliferative activity.
Removal of the 1,2,3-triazole was clearly detrimental for the inhibitory
activity against the three cancer cell lines, as shown by compounds 10A-
10C. The introduction of 1,2,3-triazole scaffold resulted in an
improvement of inhibitory activity. Especially, compound 11E showed
the more potent inhibitory effect against three cell lines than 5-Fu. This
result suggested that 1,2,3-triazole moiety may play a synergistic role in
determining activity.
In order to complete the structure activity relationship study, a series of
5,6-diphenyl-1,2,4-triazine-1,2,3-triazole hybrids were prepared and
evaluated for their antiproliferative activity. With the exception of
compounds 11A-11F, all the compounds bearing a 1,2,3-triazole moiety
exhibited potential antiproliferative activity. To determine whether the
substituents on the phenyl ring have an effect on the inhibitory activity,
compounds with various groups on the phenyl ring were compared with
activity. We found that the substitution on the phenyl ring was important
for the activity showing an over 4-fold activity loss, when the fluoro
group was replaced with methyl group (compounds 11E vs. 11C). In
addition, the size of halogen group on the phenyl ring may affected the
antiproliferative activity. Replacement of the bromine atom of compound
11B and chlorine atom of compound 11A with a fluorine atom 11E led to
an increase of the activity.
Furthermore, compounds with a chalcone scaffold (11G), a
sulfanilamide scaffold (11H), a naphthyl scaffold (11I), a formononetin
scaffold (11J) and coumarin scaffolds (11K-11L) were synthesized to
explore the effect of bioactive scaffolds for their antiproliferative activity.
Compound bearing a chalcone scaffold 11G displayed antiproliferative
activity against MGC-803 cells with an IC50 value of 36.67 µM. However,
compounds possessing a sulfanilamide group 11H or a naphthyl group

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11I were unactive against three cancer cell lines. We also found that the
type of coumarin ring affected the activity comparing compound 11K
with compound 11L. In addition, there is an interesting discovery that
5,6-diphenyl-1,2,4-triazine-1,2,3-triazole hybrid bearing a formononetin
moiety 11J exhibited more potent cytotoxicity against PC-3 cancer cells
than 5-Fu. However, compound 11J displayed no significant
antiproliferative activity against MGC-803 cells and EC-109 cells
indicating that compound 11J had good selectivity among different
cancer cells.
Compound 11E displayed the potent antiproliferative activity against
MGC-803 cells with an IC50 value of 7.59 µM. Compound 11E was
further examined for possible cytotoxicity against GES-1 (normal human
gastric epithelial cell line). We found that compound 11E exhibited weak
cytotoxicity against GES-1 (31.71±1.24 µM). The results indicated that
compound 11E had good selectivity between the selected cancer cell line
(MGC-803) and a normal cell line (GES-1).
Table 1. Antiproliferative activity of target derivatives.
Compound
IC50 (µM)a
EC-109
>100
>100
>100
24.24±1.16
>100
12.42±0.04
11.05±1.56
19.12±1.11
28.41±0.11
>100
MGC-803
>100
>100
>100
13.23±0.99
>100
34.75±0.51
11.35±0.97
7.59±0.28
22.71±2.86
36.67±4.69
>100
PC-3
>100
>100
10A
10B
10C
11A
11B
11C
11D
11E
11F
11G
11H
11I
>100
14.66±0.09
22.00±3.39
22.63±2.02
15.60±1.09
10.63±0.28
27.51±0.39
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10.23±0.77
92.34±8.27
57.77±4.36
12.87±1.20
11J
11K
11L
5-FU
9.79±0.17
56.20±5.48
a Antiproliferative activity was assayed by exposure for 48 h to substances
and expressed as concentration required to inhibit tumor cell proliferation
by 50% (IC50). Data are presented as the means±SDs from the dose-
response curves of three independent experiments.
2.3. Clone assays
Based on the most excellent activity against MGC-803 cells,

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compound 11E was chosen to perform colony formation to investigate
whether 11E could inhibit MGC-803 cells proliferation (Fig. 4). The
assay measures the ability of MGC-803 cells to grow and form foci,
which represents an indirect estimation of neoplastic transformation [28].
As shown in Fig. 4, MGC-803 cells treated with 11E exhibited fewer and
smaller colonies compared to the control, which indicated that compound
11E could significantly inhibit the proliferation of MGC-803 cells in a
concentration-dependent manner.
Fig. 4. Representative images of MGC-803 cells colonies after treatment
11E with various concentrations for a week.
2.4. 11E induces G2/M phase arrest
Targeting the cell cycle of tumor cells has been recognized as a
promising strategy for cancer therapy [29]. In this study, compound 11E
was chosen to investigate the effect of our designed derivatives on the
cell cycle of MGC-803 cells. After treating MGC-803 cells with
compound 11E at different concentrations (0 µM, 6 µM, 8 µM) for 48 h,
cells were then fixed and stained with PI for flow cytometry analysis. As
shown in Fig. 5, compound 11E concentration dependently arrested cell
cycle at G2/M phase.

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Fig. 5. Effects of 11E on MGC-803 cell cycle progress for 48 h.
2.5. Morphological changes of MGC-803 cells induced by compound 11E
Morphological changes of cancer cells are always associated with the
growth inhibition induced by cytotoxic agents [30]. Inspired by the good
inhibition of compound 11E against MGC-803 cells, we then investigated
whether compound 11E was able to induce morphological changes. After
being incubated with 11E for 48 h at different concentrations (0 µM, 6
µM, 8µM, 10 µM), the morphological changes of MGC-803 cells were
recorded using an inverted microscope. As shown in Fig. 6A, significant
changes of cell morphology such as rounding up and cell debris were
observed, especially at high concentrations. After staining with Hoechst
33258, remarkable nuclear changes including the chromatin condensation,
nuclear fragmentation and condensation were also observed (Fig. 6B).
Fig. 6. (A)The morphological changes of cells were observed under an
inverted microscope (magnification,×400).(B) MGC-803 cells were
incubated with indicated concentrations of 1035 for 48 hours and then
stained with DAPI. The stained nuclei were then observed under a
fluorescentmicroscope (magnification,×400).

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2.6. Apoptosis detection by flow cytometry
The effect of compound 11E on the apoptosis was investigated using
the propidium iodide (PI) and Annexin V-FITC biparametric
cytofluorimetric analysis. After treatment with different concentrations of
compound 11E (0 µM, 6 µM, 8µM, 10 µM) for 48 h, MGC-803 cells
were stained with PI and FITC, and then analyzed by the flow cytometry
[31]. As illustrated in Fig. 7, compound 11E induced apoptosis of MGC-
803 cells in a concentration-dependent manner. Specifically, the
percentage of apoptotic cells was about 3.3% for the control group. When
treated with high concentration (10 µM) of compound 11E, around 54.8%
of apoptosis rate was observed. While the late apoptosis rate of MGC-803
cells was not changed significantly with increasing concentrations.
Fig. 7. Compound 11E induced apoptosis of MGC-803 cells in a
concentration-dependent manner.
2.7. 11E decreased the membrane potential (∆Ψ) of the mitochondria.
The decrease of mitochondrial membrane potential through multiple
mechanisms including deficiency of oxidizable substrates for the
blockage of respiration, the mitochondria, or uncoupling of the inner
membrane, followed by cytochrome c release from the mitochondria and
expression changes of apoptosis-related proteins (e.g. Bax, Bcl-2,
caspase3/9, parp, etc) have been observed during apoptosis process. The
remarkable apoptosis induced by compound 11E promoted us to explore
whether this compound had an effect on the mitochondrial membrane
potential. MGC-803 cells were incubated at the indicated concentrations
(0 µM, 6 µM, 8µM, 10 µM) for 48 h, then stained and analyzed by flow
cytometry. As shown in Fig. 8, compound 11E caused the mitochondrial

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membrane potential decrease in a concentration-dependent manner. The
percentage of cells at 10 µM was 42.9%, significantly higher than that of
the control group (4.1%).
Fig. 8. MGC-803 cells were treated with vehicle control and various
concentrations of 11E (0µM, 6µM, 8µM, and 10µM) for 48 hours. The
mitochondria membrane potential was measured by JC-1 dye retention
using flow cytometry.
2.8. 11E affected apoptosis-related protein expression.
Proteins in Bcl-2 family are key regulators of apoptosis and
overexpression of the prominent pro-survival Bcl-2 family members like
Bcl-2 and Bcl-xL is a common feature responsible for deregulation of
apoptosis in human adenocarcinomas, which make tumor cells resistant to
conventional cancer therapeutic agents and thereby resulting in hindrance
to cancer cell death [32, 33]. Caspases are cysteine-dependent, aspartate-
directed proteases that have been shown to play an important part in the
apoptotic cascade of events and caspase 3 and caspase 9 are one of the
most important effectors [34]. Poly (ADP ribose) polymerase (PARP),

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the first caspase substrate identified, is a particularly interesting death
substrate, and its cleavage is now recognized as a hallmark of apoptosis
[35].
To further investigate the mechanism of apoptosis, western blot
analysis was performed in MGC-803 cells by treatment with compound
11E (0µM, 6µM, 8µM, and 10µM) for 48 h. As shown in Fig. 9,
treatment of MGC-803 cells with compound 11E resulted in increased
expression of Bax, Cleaved-Caspase 9, Cleaved-Caspase 3, Cleaved
PARP and DR4 in a concentration-dependent manner. Meanwhile, the
expression of apoptotic-related proteins (Bcl-xl and Bcl-2) decreased
accordingly. Based on these results, 1,2,3-triazole-5,6-diphenyl-1,2,4-
triazine 11E was a potent apoptosis inducer.
Fig. 9 Expression changes of apoptosis related proteins. (A) compound
11E induced expression changes of apoptosis related proteins in MGC-
803 cells; (B-H) Statistical analysis of protein expression levels. The
results shown were representative of three independent experiments. **: p
< 0.01 verse control , ***: p < 0.001 verse control, ****: p < 0.0001
verse control.
3. Conclusions
Following our previous work, we designed a series of novel 1,2,3-
triazole-5,6-diphenyl-1,2,3-triazole derivatives based on molecular
hybridization strategy. Majority of hybrids possessed moderate to good
growth inhibition against the tested cancer cells. Particularly, compound

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11E exhibited more potent antiproliferative activity than well-known
antitumor drug 5-Fu against all three cancer cell lines. The preliminary
SAR illustrated that 1,2,3-triazole as a reported antitumor scaffold could
play potential synergistic effect for 1,2,3-triazole-5,6-diphenyl skeleton.
These hybrids in this work might serve as bioactive fragments and lead
compounds for developing more potent cytotoxic agents.
The mechanism investigation showed that compound 11E can inhibit
the colony formation, halted cell cycle progression at the G2/M phase,
induce morphological changes and lead to apoptosis. Further mechanisms
of apoptosis indicated that 1,2,3-triazole-5,6-diphenyl-1,2,3-triazole 11E
induced MGC-803 cells apoptosis probably through mitochondrial
pathway by the decrease of mitochondrial membrane potential and
regulation the expression of apoptosis-related proteins. In summary,
novel 5,6-diphenyl-1,2,3-triazole derivative bearing a 1,2,3-triazole
moiety 11E was a potential apoptosis inducer, which can be used as a
lead compound for the development of potent cancer chemotherapeutic
agents in the drug discovery process.
4. Experimental section
4.1. General
Reagents and solvents were used without special treatment. Melting
points were determined on an X-5 micromelting apparatus and are
1
13
uncorrected. H NMR and C NMR spectra were recorded on a Bruker
400 MHz and 100 MHz spectrometer, respectively. High resolution mass
spectra (HRMS) of all derivatives were recorded on a Waters Micromass
Q-T of Micromass spectrometer by electrospray ionization (ESI).
4.2. General procedure for the synthesis of compounds 10A-10C
5,6-Diphenyl-1,2,4-triazine-3-thiol 9 was prepared by a reported
method [1]. To a solution of 9 (0.5 mmol, 1.0 eq) and bromide (0.5 mmol,
1.0 eq) in acetone (10 mL) was added Et3N (0.75 mmol, 1.5 eq), the
mixture was refluxed for about 3~4 h. Upon completion, EtOAc and H2O
were added. The aqueous layer was extracted with EtOAc for several
times; the combined organic layers were washed with H2O for several
times to remove the acetone, and then washed with brine, dried over
MgSO4 and evaporated to give the products. The residue was purified
with column chromatography (hexane: EtOAc = 7:1) to obtain analogue
10A-10C.
4.2.1. 1-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)propan-2-one (10A)

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0
Light yellow solid; Mp:102~104 C; Yield: 35%. 1H NMR (400 MHz, Acetone-
d6) δ 7.75 – 7.27 (m, 10H), 4.32 (s, 2H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 201.49, 170.52, 156.47, 155.24, 136.66, 136.40, 131.60, 130.75, 130.33, 130.10,
129.28, 129.23, 41.54, 29.11. HR-MS (ESI): Calcd. C18H16N3OS, [M+H]+m/z:
322.1018, found: 322.1014.
4.2.2. 5,6-diphenyl-3-(prop-2-yn-1-ylthio)-1,2,4-triazine (10B)
0
1
Yellow solid; Mp:96~99 C; Yield: 63%. H NMR (400 MHz, Acetone-d6) δ
13
7.65 – 7.36 (m, 10H), 4.19 (d, J = 2.6 Hz, 2H), 2.77 (t, J = 2.6 Hz, 1H). C NMR
(100 MHz, Acetone-d6) δ 169.89, 156.57, 155.45, 136.63, 136.35, 131.68, 130.83,
130.35, 130.16, 129.32, 129.25, 80.13, 72.65, 19.47. HR-MS (ESI): Calcd. C18H14N3S,
[M+H]+m/z: 304.0909, found: 304.0908.
4.2.3. 4-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)butanoic acid (10C)
N
N
O
N
S
OH
Light yellow solid; Mp:172~174 0C; Yield: 34%. 1H NMR (400 MHz, Acetone-d6)
13
δ 7.56 – 7.13 (m, 10H), 3.44 (dd, J = 9.4, 4.5 Hz, 2H), 2.80 (t, J = 6.9 Hz, 2H). C
NMR (100 MHz, Acetone-d6) δ 173.04, 171.06, 156.54, 155.20, 136.80, 136.55,
131.58, 130.76, 130.35, 130.09, 129.31, 129.26, 34.39, 26.42. HR-MS (ESI): Calcd.
C18H16N3O2S, [M+H]+m/z: 338.0965, found: 338.0963.
4.3. General procedure for the synthesis of compounds 11A-11L.
Compound 10B (1 mmol), azide derivatives (1 mmol), CuSO4.5H2O
(0.2 mmol) and sodium ascorbate (0.1 mmol) were dissolved in THF/H2O
(5 ml/5 ml) to stir for 8 h at room temperature. Upon completion, the
precipitated product was filtered off and washed with ethanol to afford
the crude product, which was purified with column chromatography
(hexane: EtOAc = 9:1) to obtain analogue 11A-11L.

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4.3.1. 3-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenyl-1,2,4-triazine (11A)
Light yellow solid; Mp:120~122 0C; Yield: 52%. 1H NMR (400 MHz, Acetone-d6) δ
13
7.97 (s, 1H), 7.60 – 7.28 (m, 14H), 5.58 (s, 2H), 4.66 (s, 2H). C NMR (100 MHz,
Acetone-d6) δ 170.68, 156.46, 155.21, 144.89, 136.72, 136.40, 135.91, 134.53, 131.64,
130.77, 130.65, 130.34, 130.15, 129.73, 129.34, 129.27, 124.06, 53.41, 26.05. HR-
MS (ESI): Calcd. C25H20ClN6S, [M+H]+m/z: 471.1160, found: 471.1159.
4.3.2. 3-(((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenyl-1,2,4-triazine (11B)
Light yellow solid; Mp:132~134 0C; Yield: 41%. 1H NMR (400 MHz, Acetone-d6) δ
7.97 (s, 1H), 7.60 – 7.32 (m, 12H), 7.24 (d, J = 8.4 Hz, 2H), 5.57 (s, 2H), 4.66 (s, 2H).
13C NMR (100 MHz, Acetone-d6) δ 170.68, 156.47, 155.21, 144.91, 136.71, 136.40,
136.38, 132.73, 131.64, 130.94, 130.77, 130.34, 130.15, 129.35, 129.27, 124.08,
122.65, 53.46, 26.04. HR-MS (ESI): Calcd. C25H20BrN6S, [M+H]+m/z: 515.0657,
found: 515.0654.
4.3.3. 3-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenyl-1,2,4-triazine (11C)
Light yellow solid; Mp:119~121 0C; Yield: 64%. 1H NMR (400 MHz, Acetone-d6) δ
7.90 (s, 1H), 7.60 – 7.32 (m, 10H), 7.15 (dd, J = 22.4, 8.0 Hz, 4H), 5.50 (s, 2H), 4.65
(s, 2H), 2.27 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ 170.73, 156.46, 155.18,
144.69, 138.82, 136.73, 136.42, 133.95, 131.62, 130.77, 130.34, 130.27, 130.14,
129.33, 129.27, 128.92, 123.81, 54.03, 26.09, 21.11. HR-MS (ESI): Calcd. C26H23N6S,
[M+H]+m/z: 451.1708, found: 451.1705.

ACCEPTED MANUSCRIPT
4.3.4. 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-5,6-diphenyl-1,2,4-
triazine (11D)
0
Light yellow solid; Mp:130~132 C; Yield: 72%. 1H NMR (400 MHz, CDCl3) δ
7.50 (s, 1H), 7.47 – 7.20 (m, 13H), 7.12 (dd, J = 6.6, 2.9 Hz, 2H), 5.38 (s, 2H), 4.57 (s,
13
2H). C NMR (100 MHz, CDCl3) δ 168.68, 154.50, 152.85, 143.41, 133.98, 133.76,
133.38, 129.79, 128.61, 128.31, 128.10, 127.85, 127.47, 127.44, 127.31, 126.79,
121.52, 52.94, 24.38. HR-MS (ESI): Calcd. C25H21N6S, [M+H]+m/z: 437.1549, found:
437.1548.
4.3.5. 3-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenyl-1,2,4-triazine (11E)
Light yellow solid; Mp:126~128 0C; Yield: 64%. 1H NMR (400 MHz, CDCl3) δ 7.50 (s,
1H), 7.41 (dd, J = 12.7, 5.1 Hz, 4H), 7.37 – 7.32 (m, 2H), 7.31 – 7.27 (m, 2H), 7.24 (d,
J = 7.8 Hz, 2H), 7.11 (dd, J = 8.6, 5.2 Hz, 2H), 6.90 (dd, J = 11.9, 5.3 Hz, 2H), 5.33 (s,
2H), 4.56 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 168.80, 162.97, 160.50, 154.67,
153.05, 143.71, 134.13, 130.01, 129.44, 128.91, 128.77, 128.52, 128.25, 127.65,
127.50, 121.64, 115.11, 52.33, 24.52. HR-MS (ESI): Calcd. C25H20FN6S, [M+H]+m/z:
455.1456, found: 455.1454.
4.3.6. 3-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-
diphenyl-1,2,4-triazine (11F)

ACCEPTED MANUSCRIPT
0
Light yellow solid; Mp:108~111 C; Yield: 65%. 1H NMR (400 MHz, CDCl3) δ
7.46 (s, 1H), 7.45 – 7.20 (m, 10H), 7.08 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H),
5.30 (s, 2H), 4.56 (s, 2H), 3.69 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 168.90, 158.83,
154.65, 153.02, 143.46, 134.18, 133.96, 129.96, 128.79, 128.56, 128.48, 128.28,
127.62, 127.49, 125.55, 121.45, 113.41, 54.28, 52.67, 24.57. HR-MS (ESI): Calcd.
C26H23N6OS, [M+H]+m/z: 467.1657, found: 467.1654.
4.3.7. (E)-3-(4-(4-(((5,6-diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-
1,2,3-triazol-1-yl)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (11G)
0
Light yellow solid; Mp:162~164 C; Yield: 61%. 1H NMR (400 MHz, CDCl3) δ
8.09 (s, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 15.6 Hz, 1H), 7.56 – 7.23 (m, 15H),
13
6.90 (d, J = 8.8 Hz, 2H), 4.67 (s, 2H), 3.80 (s, 3H). C NMR (100 MHz, CDCl3) δ
187.09, 162.66, 140.89, 136.50, 135.90, 134.06, 133.96, 130.32, 130.09, 129.92,
129.68, 129.24, 128.80, 128.55, 128.28, 127.67, 127.64, 127.58, 127.52, 122.62,
120.64, 120.11, 118.38, 112.94, 54.52, 24.45. HR-MS (ESI): Calcd. C34H27N6O2S,
[M+H]+m/z: 583.1919, found: 583.1916.
4.3.8. 4-(4-(((5,6-diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)benzenesulfonamide (11H)
0
Light yellow solid; Mp:236~238 C; Yield: 56%. 1H NMR (400 MHz, DMSO-d6) δ
8.85 (s, 1H), 8.06 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.58 – 7.33 (m, 12H),
4.77 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 168.94, 155.63, 154.18, 144.64,
143.71, 138.39, 135.08, 134.87, 130.74, 129.60, 129.25, 129.19, 128.36, 128.33,
127.37, 122.02, 120.12, 24.63. HR-MS (ESI): Calcd. C24H20N7O2S2, [M+H]+m/z:
502.1127, found: 502.1120.
4.3.9. 3-(((1-(naphthalen-2-ylmethyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-
5,6-diphenyl-1,2,4-triazine (11I)

ACCEPTED MANUSCRIPT
0
Light yellow solid; Mp:136~138 C; Yield: 69%. 1H NMR (400 MHz, CDCl3) δ
7.80 – 7.63 (m, 3H), 7.60 (s, 1H), 7.53 (s, 1H), 7.52 – 7.23 (m, 10H), 7.20 (dt, J =
13
11.5, 3.8 Hz, 3H), 5.54 (s, 2H), 4.57 (s, 2H). C NMR (100 MHz, CDCl3) δ 168.85,
154.68, 153.02, 143.70, 134.15, 133.91, 132.15, 132.12, 130.90, 129.94, 128.75,
128.47, 128.27, 128.07, 127.61, 127.45, 126.92, 126.73, 126.29, 125.66, 125.64,
124.24, 121.80, 53.32, 24.57. HR-MS (ESI): Calcd. C29H23N6S, [M+H]+m/z:
487.1709, found: 487.1705.
4.3.10. 7-(3-(4-(((5,6-diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)propoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (11J)
0
Light yellow solid; Mp:162~164 C; Yield: 70%. 1H NMR (400 MHz, CDCl3) δ
8.10 (d, J = 8.9 Hz, 1H), 7.81 (s, 1H), 7.63 (s, 1H), 7.42 (dd, J = 14.5, 7.9 Hz, 6H),
7.29 (ddt, J = 21.4, 15.3, 7.7 Hz, 6H), 6.94 – 6.81 (m, 3H), 6.70 (d, J = 2.2 Hz, 1H),
4.59 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 5.7 Hz, 2H), 3.76 (s, 3H), 2.41 –
2.24 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 174.73, 168.86, 161.60, 158.56, 156.76,
154.78, 153.11, 151.09, 143.32, 134.10, 133.88, 130.03, 129.09, 128.79, 128.54,
128.24, 127.66, 127.51, 126.88, 123.85, 123.13, 122.24, 117.66, 113.57, 112.93,
99.73, 63.80, 54.32, 45.93, 28.60, 24.52. HR-MS (ESI): Calcd. C37H31N6O4S,
[M+H]+m/z: 655.2128, found: 655.2127.
4.3.11. 7-(3-(4-(((5,6-diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)propoxy)-2H-chromen-2-one (11K)
0
Light yellow solid; Mp:127~129 C; Yield: 68%. 1H NMR (400 MHz, CDCl3) δ
7.63 (s, 1H), 7.53 (d, J = 9.5 Hz, 1H), 7.48 – 7.23 (m, 11H), 6.75 – 6.63 (m, 2H), 6.17
(d, J = 9.5 Hz, 1H), 4.60 (s, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.94 (t, J = 5.7 Hz, 2H),

ACCEPTED MANUSCRIPT
2.33 (p, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 168.88, 160.48, 160.01,
154.76, 153.11, 143.39, 142.25, 134.13, 133.91, 130.05, 128.81, 128.55, 128.25,
127.90, 127.67, 127.52, 123.97, 122.17, 112.41, 111.88, 111.47, 100.63, 63.80, 46.00,
28.64, 24.52. HR-MS (ESI): Calcd. C30H25N6O3S, [M+H]+m/z: 549.1712, found:
549.1709.
4.3.12. 4-(3-(4-(((5,6-diphenyl-1,2,4-triazin-3-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)propoxy)-2H-chromen-2-one (11L)
0
Light yellow solid; Mp:131~132 C; Yield: 70%. 1H NMR (400 MHz, DMSO) δ
8.17 (s, 1H), 7.67 (ddd, J = 15.6, 8.3, 4.1 Hz, 2H), 7.54 – 7.28 (m, 12H), 5.85 (s, 1H),
4.63 (s, 2H), 4.58 (t, J = 6.7 Hz, 2H), 4.22 (t, J = 5.7 Hz, 2H), 2.43 – 2.26 (m, 2H).
13C NMR (100 MHz, DMSO) δ 169.24, 164.70, 161.53, 155.54, 154.09, 152.68,
142.89, 135.14, 134.89, 132.71, 130.80, 129.64, 129.31, 129.22, 128.43, 128.36,
124.05, 123.77, 122.98, 116.33, 115.05, 90.54, 66.78, 46.69, 28.67, 24.89. HR-MS
(ESI): Calcd. C30H25N6O3S, [M+H]+m/z: 549.1715, found: 549.1709.
4.4. Biological testing.
The MTT assay, Clonogenicity assay, analysis of cell cycle distribution,
Hoechst 33258 staining, detection of apoptosis and mitochondrial
membrane potential were carried out following our previously reported
method [36]. The Western blot analysis was performed according to the
methods previously published [37]. Statistical analysis was performed
according to the our reported methods [22]. Therefore, no details are
given here.
Acknowledgment
This work was supported by the National Natural Sciences Foundations
of China (No. 81673322, 81273393, 81430085, 21372206, and
81172937), Ph.D. Educational Award from Ministry of Education (No.
20134101130001).
References
1. H. Irannejad, M. Amini, F. Khodagholi, N. Ansari, S.K. Tusi, M. Sharifzadeh, A. Shafiee,
Synthesis and in vitro evaluation of novel 1,2,4-triazine derivatives as neuroprotective agents,
Bioorg. Med. Chem. 18 (2010) 4224-4230.
2. G. Wang, Z. Peng, J. Wang, X. Li, J. Li, Synthesis, in vitro evaluation and molecular
docking studies of novel triazine-triazole derivatives as potential α-glucosidase inhibitors, Eur.
J. Med. Chem. 125 (2017) 423-429.

ACCEPTED MANUSCRIPT
3. E. Plebanek, F. Chevrier, V. Roy, T. Garenne, F. Lecaille, D. Warszycki, A.J. Bojarski, G.
Lalmanach, L.A. Agrofoglio, Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine
compounds targeting cysteine cathepsins K and S, Eur. J. Med. Chem. 121 (2016) 12-20.
4. P. Singla, V. Luxami, K. Paul, Synthesis and in vitro evaluation of novel triazine analogues
as anticancer agents and their interaction studies with bovine serum albumin, Eur. J. Med.
Chem. 117 (2016) 59-69.
5. S. Federico, A. Ciancetta, N. Porta, S. Redenti, G. Pastorin, B. Cacciari, K.N. Klotz, S.
Moro, G. Spalluto, 5,7-Disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazines as pharmacological
tools to explore the antagonist selectivity profiles toward adenosine receptors, Eur. J. Med.
Chem. 108 (2016) 529-541.
6. Z. Zheng, J.-A. Pinson, S.J. Mountford, S. Orive, S.M. Schoenwaelder, D. Shackleford, A.
Powell, E.M. Nelson, J.R. Hamilton, S.P. Jackson, I.G. Jennings, P.E. Thompson, Discovery
and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922), Eur. J. Med. Chem. 122
(2016) 339-351.
7. H. Bera, S. Chigurupati, Recent discovery of non-nucleobase thymidine phosphorylase
inhibitors targeting cancer, European Journal of Medicinal Chemistry. 124 (2016) 992-1003.
8. G. Wang, X. Li, J. Wang, Z. Xie, L. Li, M. Chen, S. Chen, Y. Peng, Synthesis, molecular
docking and α-glucosidase inhibition of 2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-N-
arylacetamides, Bioorg. Med. Chem. Lett. 27 (2017) 1115-1118.
9. R.S. Tamboli, R. Giridhar, H.P. Gandhi, A.M. Kanhed, H.M. Mande, M.R. Yadav, Design,
green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-
morpholinoethylamine moiety as potential antithrombotic agents, J. Enzym. Inhib. Med.
Chem. 31 (2016) 704-713.
10. M. Khoshneviszadeh, O. Shahraki, M. Khoshneviszadeh, A. Foroumadi, O. Firuzi, N.
Edraki, H. Nadri, A. Moradi, A. Shafiee, R. Miri, Structure-based design, synthesis,
molecular docking study and biological evaluation of 1,2,4-triazine derivatives acting as
COX/15-LOX inhibitors with anti-oxidant activities, J. Enzym. Inhib. Med. Chem. 31 (2016)
1602-1611.
11. D. Dheer, V. Singh, R. Shankar, Medicinal attributes of 1,2,3-triazoles: Current
developments, Bioorg. Chem. 71 (2017) 30-54.
12. R.-M. Silvana, M. Andrijana, Recent Trends in 1,2,3-Triazolo-Nucleosides as Promising
Anti-Infective and Anticancer Agents, Curr. Med. Chem. 22 (2015) 1462-1499.
13. R.S. Keri, S.A. Patil, S. Budagumpi, B.M. Nagaraja, Triazole: A Promising
Antitubercular Agent, Chem. Biol. Drug Des. 86 (2015) 410-423.
14. J. Yubo, K. Chunxiang, Recent Advances in the Synthesis of 1-Monosubstituted 1,2,3-
Triazoles, Mini-Rev. Med. Chem. 13 (2013) 713-719.
15. D.-J. Fu, S.-Y. Zhang, Y.-C. Liu, X.-X. Yue, J.-J. Liu, J. Song, R.-H. Zhao, F. Li, H.-H.
Sun, Y.-B. Zhang, H.-M. Liu, Design, synthesis and antiproliferative activity studies of 1,2,3-
triazole-chalcones, MedChemComm. 7 (2016) 1664-1671.
16. D.J. Fu, J. Song, R.H. Zhao, Y.C. Liu, Y.B. Zhang, H.M. Liu, Synthesis of novel
antiproliferative 1,2,3-triazole hybrids using the molecular hybridisation approach, J. Chem.
Res. (2016)
17. Y.C. Zheng, Y.C. Duan, J.L. Ma, R.M. Xu, X. Zi, W.L. Lv, M.M. Wang, X.W. Ye, S.
Zhu, D. Mobley, Triazole-dithiocarbamate based selective lysine specific demethylase 1
(LSD1) inactivators inhibit gastric cancer cell growth, invasion, and migration, J. Med. Chem.
56 (2013) 8543-8560.
18. D.J. Fu, L. Zhang, J. Song, R.W. Mao, R.H. Zhao, Y.C. Liu, Y.H. Hou, J.H. Li, J.J. Yang,
C.Y. Jin, Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth
and migration of PC-3 cells via MAPK/Wnt signaling pathways, Eur. J. Med. Chem. 127
(2016) 87.
19. V.-J. Claudio, D. Amanda, B. Vanderlan da Silva, J.B. Eliezer, F. Carlos Alberto
Manssour, Molecular Hybridization: A Useful Tool in the Design of New Drug Prototypes,
Curr. Med. Chem. 14 (2007) 1829-1852.

ACCEPTED MANUSCRIPT
20. H.-L. Ng, S. Chen, E.-H. Chew, W.-K. Chui, Applying the designed multiple ligands
approach to inhibit dihydrofolate reductase and thioredoxin reductase for anti-proliferative
activity, Eur. J. Med. Chem. 115 (2016) 63-74.
21. D.-J. Fu, S.-Y. Zhang, Y.-C. Liu, L. Zhang, J.-J. Liu, J. Song, R.-H. Zhao, F. Li, H.-H.
Sun, H.-M. Liu, Y.-B. Zhang, Design, synthesis and antiproliferative activity studies of novel
dithiocarbamate–chalcone derivates, Bioorg. Med. Chem. Lett. 26 (2016) 3918-3922.
22. D.-J. Fu, L. Zhang, J. Song, R.-W. Mao, R.-H. Zhao, Y.-C. Liu, Y.-H. Hou, J.-H. Li, J.-J.
Yang, C.-Y. Jin, P. Li, X.-L. Zi, H.-M. Liu, S.-Y. Zhang, Y.-B. Zhang, Design and synthesis
of formononetin-dithiocarbamate hybrids that inhibit growth and migration of PC-3 cells via
MAPK/Wnt signaling pathways, Eur. J. Med. Chem. 127 (2017) 87-99.
23. A.Y. Guan, C.L. Liu, M. Li, Z.N. Li, M.X. Zhang, H. Zhang, Synthesis and bioactivity of
novel coumarin derivatives, Nat. Prod. Commun. 6 (2011) 1917-1920.
24. M. Khoshneviszadeh, M.H. Ghahremani, A. Foroumadi, R. Miri, O. Firuzi, A. Madadkar-
Sobhani, N. Edraki, M. Parsa, A. Shafiee, Design, synthesis and biological evaluation of
novel anti-cytokine 1,2,4-triazine derivatives, Bioorg. Med. Chem. 21 (2013) 6708-6717.
25. L.-Y. Ma, L.-P. Pang, B. Wang, M. Zhang, B. Hu, D.-Q. Xue, K.-P. Shao, B.-L. Zhang, Y.
Liu, E. Zhang, H.-M. Liu, Design and synthesis of novel 1,2,3-triazole-pyrimidine hybrids as
potential anticancer agents, Eur. J. Med. Chem. 86 (2014) 368-380.
26. S.Y. Zhang, D.J. Fu, X.X. Yue, Y.C. Liu, J. Song, H.H. Sun, H.M. Liu, Y.B. Zhang,
Design, Synthesis and Structure-Activity Relationships of Novel Chalcone-1,2,3-triazole-
azole Derivates as Antiproliferative Agents, Molecules. 21 (2016) 653.
27. B. Yu, X.-J. Shi, J.-l. Ren, X.-N. Sun, P.-P. Qi, Y. Fang, X.-W. Ye, M.-M. Wang, J.-W.
Wang, E. Zhang, D.-Q. Yu, H.-M. Liu, Efficient construction of novel D-ring modified
steroidal dienamides and their cytotoxic activities, Eur. J. Med. Chem. 66 (2013) 171-179.
28. I.N. Alimova, B. Liu, Z. Fan, S.M. Edgerton, T. Dillon, S.E. Lind, A.D. Thor, Metformin
inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro, Cell
Cycle. 8 (2009) 909.
29. G.I. Evan, K.H. Vousden, Proliferation, cell cycle and apoptosis in cancer, Nature. 411
(2001) 342-348.
30. Alexi I. Goranov, A. Gulati, N. Dephoure, T. Takahara, T. Maeda, Steven P. Gygi, S.
Manalis, A. Amon, Changes in Cell Morphology Are Coordinated with Cell Growth through
the TORC1 Pathway, Curr. Bio. 23 (2013) 1269-1279.
31. Z. Wen, Y. Zhang, X. Wang, X. Zeng, Z. Hu, Y. Liu, Y. Xie, G. Liang, J. Zhu, H. Luo, B.
Xu, Novel 3′,5′-diprenylated chalcones inhibited the proliferation of cancer cells in vitro by
inducing cell apoptosis and arresting cell cycle phase, Eur. J. Med. Chem. 133 (2017) 227-
239.
32. P. Sharma, T. Srinivasa Reddy, D. Thummuri, K.R. Senwar, N. Praveen Kumar, V.G.M.
Naidu, S.K. Bhargava, N. Shankaraiah, Synthesis and biological evaluation of new
benzimidazole-thiazolidinedione hybrids as potential cytotoxic and apoptosis inducing agents,
Eur. J. Med. Chem. 124 (2016) 608-621.
33. P.R. Kamath, D. Sunil, A.A. Ajees, K.S.R. Pai, S. Biswas, N′-((2-(6-bromo-2-oxo-2H-
chromen-3-yl)-1H-indol-3-yl)methylene)benzohydrazide as a probable Bcl-2/Bcl-xL inhibitor
with apoptotic and anti-metastatic potential, Eur. J. Med. Chem. 120 (2016) 134-147.
34. K.R. Senwar, T.S. Reddy, D. Thummuri, P. Sharma, V.G.M. Naidu, G. Srinivasulu, N.
Shankaraiah, Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3′-methoxy-4′-
(2-amino-2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumour agents, Eur. J.
Med. Chem. 118 (2016) 34-46.
35. J. Zhou, M. Ji, Z. Zhu, R. Cao, X. Chen, B. Xu, Discovery of 2-substituted 1H-
benzo[d]immidazole-4-carboxamide derivatives as novel poly(ADP-ribose)polymerase-1
inhibitors with in vivo anti-tumor activity, Eur. J. Med. Chem. 132 (2017) 26-41.
36. B. Yu, X.-J. Shi, Y.-F. Zheng, Y. Fang, E. Zhang, D.-Q. Yu, H.-M. Liu, A novel [1,2,4]
triazolo [1,5-a] pyrimidine-based phenyl-linked steroid dimer: Synthesis and its cytotoxic
activity, Eur. J. Med. Chem. 69 (2013) 323-330.

ACCEPTED MANUSCRIPT
37. B. Yu, S.-Q. Wang, P.-P. Qi, D.-X. Yang, K. Tang, H.-M. Liu, Design and synthesis of
isatin/triazole conjugates that induce apoptosis and inhibit migration of MGC-803 cells, Eur. J.
Med. Chem. 124 (2016) 350-360.

ACCEPTED MANUSCRIPT
Highlights
SAR for this 5,6-diaryl-1,2,4-triazine scaffold is explored.
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11E inhibited colony formation and arrested cell cycle at G2/M phase.
11E caused morphological changes and decreased mitochondrial membrane
potential.
5,6-Diaryl-1,2,4-triazine hybrids were discovered as novel apoptosis inducers.
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