Cell cycle inhibition, apoptosis, and molecular docking studies of the novel anticancer bioactive 1,2,4-triazole derivatives

Article abstract of DOI:10.1007/s11224-019-01453-3

Several 3-alkylsulfanyl-1,2,4-triazole derivatives were synthesized and their relevant structures confirmed based on their elemental analysis and nuclear magnetic resonance. The anticancer activity of all the derivatives was evaluated for A549, MCF7, and SKOV3 cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay wherein compound 5e demonstrated significant anti-proliferative activities against all cell lines whereas 5b and 5e showed efficient anti-proliferative actions in SKOV3 cell line having half maximal inhibitory concentration (IC₅₀) values of 0.81 and 0.53 μM, respectively. Furthermore, compound 5e was found to drive remarkable cell cycle arrest at the G2/M phase for SKOV3 cell lines in a concentration-dependent behavior. Molecular docking studies performed with these derivatives validated them as appropriate candidates for further studies of their potential anticancer activity.

Full text of DOI:10.1007/s11224-019-01453-3

Structural Chemistry
https://doi.org/10.1007/s11224-019-01453-3
ORIGINAL RESEARCH
Cell cycle inhibition, apoptosis, and molecular docking studies
of the novel anticancer bioactive 1,2,4-triazole derivatives
Javad Ghanaat1 & Mohammad A. Khalilzadeh1 & Daryoush Zareyee1 & Mohammadreza Shokouhimehr2
&
Rajender S. Varma3
Received: 24 September 2019 /Accepted: 7 November 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Several 3-alkylsulfanyl-1,2,4-triazole derivatives were synthesized and their relevant structures confirmed based on their ele-
mental analysis and nuclear magnetic resonance. The anticancer activity of all the derivatives was evaluated for A549, MCF7,
and SKOV3 cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay wherein compound 5e dem-
onstrated significant anti-proliferative activities against all cell lines whereas 5b and 5e showed efficient anti-proliferative actions
in SKOV3 cell line having half maximal inhibitory concentration (IC50) values of 0.81 and 0.53 μM, respectively. Furthermore,
compound 5e was found to drive remarkable cell cycle arrest at the G2/M phase for SKOV3 cell lines in a concentration-
dependent behavior. Molecular docking studies performed with these derivatives validated them as appropriate candidates for
further studies of their potential anticancer activity.
.
.
.
Keywords 1,2,4-Triazoles Anticancer activity Molecular docking Anticancer agents
Introduction
compounds is one of the most persuaded objectives of
present-day medicinal chemistry [3–6].
Cancer has remained as a critical health issue around the world
and is the second fatal cause after cardiovascular diseases. It
remains a very defiant pathology causing annual mortality
worldwide [1]. Presently available chemotherapy treatments
have several restrictions (e.g., serious incongruous effects in-
cluding resistance to treatment). Consequently, the explora-
tion for selective antitumor drugs is obligatory [2]. The prog-
ress in discovery of potentially active antineoplastic
Heterocyclic compounds with 1,2,4-triazolo ring structure
substituted with nitrogen and sulfur display a broad spectrum
of activities e.g. anticancer, antimicrobial, antifungal, antituber-
cular, analgesic, anti-inflammatory, and anticonvulsant [7–15].
Additionally, 1,2,4-triazoles are noted for having central ner-
vous system performing drugs with inhibitory effect on en-
zymes such as cholinesterase [7, 16]. In addition, it is demon-
strated that triazole derivatives exhibit antioxidative activity via
free radical scavenging by impeding lipid peroxidation [17]. Li
and colleagues synthesized complex encompassing 1,2,4 tri-
azole ring with prospective anticancer properties [18]; tethered
1,2,4 triazole entities demonstrated potential action against
breast cancer cell lines [19], whereas thiadiazine segment be-
stows anticancer efficiency as a heterocyclic ring compound
[20]. In addition, thiadiazine derivatives were realized to pres-
ent a selective cytotoxic effect toward tumor cells and have
been used as lead compounds [21]. Triazolothiadiazine com-
pound has also been introduced as a prominent medicine
against liver cancer. Previous studies have shown the
trimethoxyphenyl moiety can be an effective pharmacophore for
the anticancer treatment similar to natural occurring anticancer
compounds e.g. colchicine, combretastatin, podophyllotoxin,
and poly-methoxychalcone [22]. Indeed, the following 1,2,4
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-019-01453-3) contains supplementary
material, which is available to authorized users.
* Mohammad A. Khalilzadeh
mkhalil3@ncsu.edu
1
Department of Chemistry, Qaemshahr Branch, Islamic Azad
University, Qaemshahr, Iran
2
Department of Materials Science and Engineering, Research Institute
of Advanced Materials, Seoul National University, Seoul, Republic
of Korea
3
Regional Centre of Advanced Technologies and Materials, Faculty of
Science, Palacky University in Olomouc, Šlechtitelů 27, 783
71 Olomouc, Czech Republic

Struct Chem
triazole compounds are generally utilized in medicines:
anastrozole, alprazolam (tranquilizer), benatradin (diuretic), estaz-
olam (sedative, tranquilizer), etoperidone (antidepressant), flucon-
azole, itraconazole, letrozole, nefazodone (antidepressant), ribavi-
rin, rilmazafone (hypnotic, anxiolytic), trapidil (hypotensive),
trazodon (anxiolytic, antidepressant), vorozole (antineoplastics),
terconazole (the powerful azole antifungal agents), etc. [23].
Continuing our investigations on the preparation of newer
heterocyclic compounds with active biological properties on
human cancerous cell lines, we have synthesized compounds,
5a–f, in 68–88% yields (Scheme 1) [24–28]. The anti-
proliferative effects of these latest 3-alkylsulfanyl-1,2,4-tri-
azole derivatives on three human cancerous cell lines includ-
ing MCF7 (breast cancer), A549 (lung cancer), and SKOV3
(ovarian cancer) were evaluated with varying concentrations
of compounds 5a–f. The efficiency of these synthesize com-
pounds was assessed on cell cycles for 24 h using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) reduction assay. Molecular docking studies and stain-
ing assay of Annexin-V-FITC/propidium iodide were also
accomplished.
coupling constants were measured in Hertz. The melting
points were obtained using a capillary tube by a Stuart
Scientific apparatus. The analytical analyses for C, H, N,
and chlorine were within 0.4% of the theoretical values.
Human breast cancer MCF-7 and colon cancer HCT-15 cell
lines were supplied from the American Type Cell Culture
Collection (ATCC) and maintained in the standard medium
and grown as a monolayer in DMEM containing 10% FBS,
2 mM glutamine, 100 units/mL penicillin, and 100 g/mL
streptomycin.
General procedure for the preparation of target compounds
5a–f
A mixture of intermediates 4-(4-methoxyphenyl)-5-4-alkyl-
4H-[1,2,4]triazole-3-thiols, 5a–f, (1.0 mmol), different
substituted phenacyl bromides (1.0 mmol), and K2CO3 (0.15
g, 1.1 mmol) in 5.0 mL of anhydrous acetone was stirred for
7 h at room temperature. After completion of the reaction, the
solvent was evaporated to afford the crude products followed
by recrystallization affording the pursued compounds in 65–
88% yields as monitored by thin-layer chromatography. The
1
13C NMR and H NMR spectra of the compounds 5a–f are
shown in the supplementary information.
Experimental
Chemistry
Biology
All reagents and solvents were purchased from commercial
MTT assay
suppliers and used without purification. The 1H NMR and 13
C
NMR spectra were recorded using a Bruker BioSpin spec-
trometer at 500, 400, 250, and 125 MHz. Tetramethylsilane
(TMS) was used as the internal standard. Chemical shifts are
reported in parts per million (ppm) downfield from TMS and
The in vitro anti-proliferative activities of prepared com-
pounds and etoposide as standard drug were accomplished
against three different human cancer cell lines including
adenocarcinomic human alveolar basal epithelial cells
Scheme 1 Procedure for the
preparation of 5a–f compounds.
Reagents and conditions: (a)
NH2NH2.H2O, ethanol, reflux;
(b) 4-methoxyphenyl
isothiocyanate, ethanol, reflux; (c)
1 M NaOH, reflux, 1 M HCl; (d)
K2CO3, acetone, substituted
phenacyl bromides

Struct Chem
(A549), human ovarian carcinoma cells (SKOV3), and breast
cancer cells (MCF7). Approximately 1 × 104 cells were seed-
ed in micro culture 96-well plate in RPMI supplemented with
10% fetal bovine solution (FBS) and incubated for 24 h at 37
°C in CO2 incubator. Then cells were exposed with 100 μL of
compounds which were diluted in RPMI to favorable concen-
trations (0.5, 1, 2.5, 5, and 10 μM). After incubation (48 h), 10
μL of MTT (5 mg/mL) was added to each well followed by
4-h incubation. Subsequently, after safe removal of superna-
tant, the formazan crystals were dissolved in DMSO (200 μL).
Then, the absorbance was recorded at 490-nm wavelength
using microplate reader (Bioteck) [29].
were prepared in DMSO and 10 μL aliquot of each sample
was added to the 190 μL erythrocytes in a 96-well plate,
making final concentrations of 1, 10, and 50 μg/mL, in tripli-
cate. Triton X-100 (2% v/v) and DMSO were contained as
positive and negative controls, respectively. The plate was
incubated for 1 h at 37 °C. Then, the cell suspensions were
centrifuged. Each supernatant (100 μL) was collected and the
absorbance was measured at 451 nm. The percentage of he-
molysis was calculated as follows:
ÂÀ
Á À
ÁÃ
%Hemolysis ¼
A
sample–Ablank = Apositive control  100
Molecular docking studies
Apoptosis assay (Annexin-V-FITC)
Molecular docking studies were carried out for the promising
compound 5e which displayed considerable cytotoxic effect
against three different human cancer cell lines. Flexible-ligand
docking procedure was performed by AutoDock 4software
(AutoDockTools 1.5.6) [37, 38]. The crystal structure was
taken from the protein data bank (PDB_ID: 4O2B tubulin-
colchicine complex). Chain D was selected for docking stud-
ies. The requisite hydrogen atoms Kollman united atom
charges and solvation parameters were included using the
AutoDock Tools. The ligands were docked in the active site
region utilizing the Lamarckian genetic algorithm. This region
was specified through a grid map with 40, 40, and 40 grid
points (X, Y, Z dimensions, respectively) that entirely covers
the space initially occupied by the crystallographic ligand.
Centroids selected were X = 17.0192, Y = 65.9939, and Z =
43.3901. We applied the default grid spacing (0.375 Å) and
accomplished 30 docking runs. The simple root-mean-square
deviation value of the ligands in the re-docked and crystallo-
graphic conformations was 0.67.
To investigate the influence of compounds 5e on apoptosis,
SKOV3 cells (1 × 106) were seeded in a six-well plate and
after 24-h incubation, cells were treated with 0.5 and 1 μM
concentration of compound 5e and after 48 h incubation
were trypsinized and washed with cold phosphate-
buffered saline (PBS) and centrifuged at 3000 rpm.
Then, the cells were suspended in binding buffer 5 μL
of Annexin-V-FITC at room temperature for 15 min and
then PI solution; the flow cytometer used was a FACScan
(Becton Dick-inson). The percentage of apoptotic cells
could be calculated using the excitation and emission set-
tings of 488 nm and 535 nm and that of PI using 488 nm
and 610 nm [30].
Cell cycle analysis
SKOV3 cells (1 × 106) were first seeded in a six-well plate.
Then, the medium was treated with compound 5e with con-
centrations of 0.5, 1, and 5 μM and incubated for 48 h. In
addition, vehicle alone containing DMSO (10 μM) was
utilized as negative control. The cells were harvested by
trypsinization, washed with PBS, and fixed with 70% eth-
anol after 48 h of treatment. Finally, the cells were stained
with propidium iodide (50 μg/mL) in buffer solution com-
prising 0.1% Trition X-100 and 0.2 mg/mL RNase. Cell
cycle analysis was carried out using flow cytometer
(Partec PAS).
Result and Discussion
Chemistry
The transformation sequence utilized for the synthesis of
the lead compounds is presented in Scheme 1. The essen-
tial starting materials, methyl ester derivatives (1) on
reacting with hydrazine hydrate (NH2NH2.H2O), gave ac-
id hydrazides (2) in 80% yield. The reaction of different
acid hydrazides (2) with 4-methoxyphenyl isothiocyanate
in refluxing ethanol afforded the corresponding interme-
diate carbothioamide salts (3). These salts then underwent
ring closure in the presence of NaOH to deliver the rele-
vant substituted 3-mercapto-1,2,4-triazole derivatives (4)
in refluxing water and then acidification with HCl 1 M to
pH = 5–6. Then, the 1,2,4-triazole derivatives (4) were
substituted by diverse commercially available phenacyl
Hemolysis assay
The hemolytic activity of the chosen compound 5e and
etoposide was studied using a procedure explained by Carter
et al. [31–36]. Fresh whole human blood was collected into
dipotassium ethylenediaminetetraacetic acid–coated
Vacutainer tubes and centrifuged for 5 min. The red blood cell
pellets were washed with saline and PBS solutions twice.
Then, the washed erythrocytes were re-dispersed in the PBS
solution. Different concentrations of the selected compounds

Struct Chem
bromides using potassium carbonate (K2CO3) as a base in
dry acetone solution to produce the target compounds
(5a–f) in moderate to good yields, ranging 65~88%.
documented compound, 6a was deployed as a control and
the ensuing outcomes are outlined in Table 1 as half maximal
inhibitory concentration (IC50, μM) [40, 41]. It was motivat-
ing to note that almost all the newly synthesized compounds
exhibited strong anti-proliferative activities toward the exam-
ined cell lines. In particular, analogue 5e displayed higher
anti-proliferative action against A549, MCF-7, and SKOV3
cell lines than the formerly identified active compound 6a;
most promising compounds 5b and 5e displayed 4- and 6-
fold improvement compared with active compound 6a and
etoposide in impeding SKOV3 cell regeneration with IC50
values 0.81 and 0.53 μM, respectively. Figure 1 demonstrates
the incubation of the SKOV3, A549, and MCF-7 cells with
varying concentrations of 5e after 48 h treatment.
Biology
Cytotoxicity assay
The prepared compounds 5a–f were investigated for their
anti-proliferative action against three human cancer cell lines:
lung adenocarcinoma epithelial cells (A549), human ovarian
carcinoma cells (SKOV3), breast cancer cells (MCF7), and
normal cell line (L929) utilizing MTT assay [39]. In addition,
for the comparison study with etoposide and the earlier
Table 1 Cytotoxic studies of compounds 5a–f against cancer cells (A549, MCF-7 SKOV3, and normal L929) (IC50, μM)
O
N
N
N
N
SH
S
O
O
N
N
R2
R1
O
O
O
5a-f
6a
Compound
R1
IC50 SD values against cancer cell lines (µM)a for 48 h
R2
H
A-549
MCF-7
SKOV3
L929
95.67
>100
>100
>100
83.90
>100
>100
_
5a
5b
5c
H
H
H
3.24 0.04
2.27 0.21
0.98 0.23
5.18 0.61
2.82 0.41
0.78 0.26
3.34 0.01
4.65 0.23
2.89 0.32
2.25 0.27
0.81 0.21
3.12 0.03
2.10 0.08
0.53 0.31
3.45 0.34
3.02 0.24
3.74 0.29
3,4,5-(OMe)3 1.87 0.24
4-Cl
H
4.15 0.46
1.11 0.31
5d 3,4,5-(OMe)3
5e 3,4,5-(OMe)3 3,4,5-(OMe)3 0.62 0.06
5f 3,4,5-(OMe)3 4-Cl
4.82 0.28
4.32 0.34
2.56 0.23
6a
Etoposide
a IC50 values are shown as average values of the three independent experiments performed in quadruplicates; coefficients of variation being < 10%

Struct Chem
Fig. 1 a Growth inhibition by 5e on SKOV3 cells. b Growth inhibition by 5e on MCF7cells. c Inhibition growth of 5e on A549 cells
Furthermore, analogues 5b and 5e did not have any effect on
the normal cells L929, suggesting that these compounds can
be selective toward cancer cells.
Cytotoxicity values against different cancer cell lines were
expressed with various concentrations (μg/mL) of compounds
causing 50% inhibition of the cell growth (IC50) as shown in
Fig. 2 Flow cytometric study of SKOV3 cancer cells treated with compound 5e. Cells were stained using Annexin-V-PI and quantified by flow
cytometry

Struct Chem
Table 1. All tested compounds presented considerable cytotox-
ic activity against cell lines, especially compounds 5b and 5e
with IC50 values of 0.53–1.87 μM. The best IC50 values were
obtained for SKOV3 cell line. A survey of the structures and
activities in Table 1 revealed that the IC50 values of compounds
with different substituents are related to their substituents. In
addition, the effect of substituent on the cytotoxic activity de-
pends on the type of tested cell line. For example, the compar-
ison of unsubstituted compound 5a and 3,4,5-(OMe)3 deriva-
tive, 5b, reveals that the introduction of methoxy group im-
proves the activity against all three cell lines.
cells apoptosis [30]. SKOV3 cells were treated with 0.5 and
1 μM concentrations of compound for 48 h. Subsequently, the
cells were stained with Annexin-V and PI utilizing the apo-
ptosis detection kit (Annexin-V-FITC). Figure 2 shows that
compound 5e could potently induce apoptosis in SKOV3 cells
in a concentration-dependent behavior. Results uncovered that
rise in the percentage of apoptosis and apoptotic cell percent-
age was 24.2 and 19.1% at 0.5 and 1 μM, respectively. The
negative control (untreated cells) and positive control
(etoposide-treated cells) demonstrated 3.9 and 19.6 % of ap-
optosis, respectively.
Annexin-V-FITC flow cytometric study of apoptosis
Analysis of cell cycle arrest
The flow cytometry assay using propidium iodide (PI) solu-
tion and protein Annexin-V with SKOV3 cells was carried out
to evaluate the capability of compound 5e to induce cancer
Cell cycle analysis was performed in human ovarian carcino-
ma cells (SKOV3) cell line to elucidate the effect of com-
pound 5e on cell cycle arrest. The cells were exposed to
Fig. 3 Cell cycle analysis of SKOV3 treated with compound 5e. Cells were exposed to 0.5, 1, and 5 μM concentrations for 48 h and subsequently
harvested for cell cycle examination

Struct Chem
Fig. 4 Hemolytic activity of compound 5e and etoposide against human
erythrocytes at the concentrations of 1, 10, and 50 μg/mL. The negative
control (CT) consisted of erythrocytes treated with dimethyl sulfoxide
(DMSO) and the positive control consisted of erythrocytes treated with
Triton X-100 (TX, 2% v/v)
compound 5e for 48 h at the concentrations of 0.5, 1, and 5
μM. Figure 3 shows that compound 5e resulted in 49.8, 60.6,
and 62.8% of cell cycle arrest under the S-phase, under the
concentrations of 0.5, 1, and 5 μM, respectively, while the
untreated cells had only 13% cell cycle arrest under the S-
phase. The acquired results demonstrated that compound 5e
significantly inhibits the entry to G2/M phase and induces S-
phase arrest.
Toxicity of compound 5e against erythrocytes
Toxicity of compound 5e was studied against human erythro-
cytes by determining its hemolytic activity to evaluate the
safety profile of the designed compounds. This compound
was examined at various concentrations of 1, 10, and 50 μg/
mL, in comparison with positive control Triton X-100 and
standard anticancer agent etoposide. As depicted in Fig. 4,
Fig. 5 3D and 2D representation of binding mode of compound 5e (a and b) with tubulin (PDBID: 4O2B)

Struct Chem
only a limited hemolysis occurred following exposure to com-
pound 5e at a higher concentration of 50 μg/mL, while Triton
X-100 produced 100% hemolysis. Compound 5e and
etoposide showed insignificant hemolytic activity at the con-
centrations of 1 and 10 μg/mL compared with negative con-
trol. Consequently, the compound 5e presented minimum tox-
icity towards human erythrocytes.
phenyl rings exhibited the highest anti-proliferative activity 6-
fold more potent than our previous compound 6a with IC50
value 0.53 μM on human ovarian carcinoma cells (SKOV3).
Furthermore, scrutiny of these results revealed that insertion
of second phenyl group particularly with electron donner sub-
stitution such as 4-methoxy and 3,4,5-trimethoxyphenyl in
comparison with our previous study could significantly im-
prove cytotoxic effects of newly synthesized compounds.
Moreover, Annexin-V-FITC assay verified that compounds
5e induced apoptosis of cells in a concentration-dependent
behavior and arrest cell cycle progression in the G2/M phase
via cytoskeletal disruption and microtubule depolymerization.
Toxicity assays against human erythrocytes showed the effi-
cient therapeutic window of these prototype compounds sim-
ilar to standard drug etoposide. The molecular modelling in-
vestigation performed on the colchicine binding site of tubulin
indicated that such compounds bind on the colchicine binding
position of the tubulin. Overall, the current study demonstrates
that these newly synthesized rigid analogs of 4H-1,2,4-
triazoles may function as lead compounds for further insight
into new developments of anticancer agents.
Molecular modeling for the binding of compound 5e
to tubulin
Molecular docking studies were performed to further investi-
gate the binding ability of the promising compounds 5e to the
colchicine binding site of α,β-tubulin employing the
AutoDock software (version 4.2) and the visualization was
done using the discovery studio 2016. The coordinates of
the protein structure co-crystallized with colchicine were ac-
quired from the Protein Data Bank (PDB ID: 4O2B) [42].
Colchicine was first docked into the pocket of the tubulin as
the representative and susceptible compound in this study.
Accordingly, the presence of 3,4,5-trimethoxy phenyl and ni-
trogen groups in the ring of the synthesized compounds may
intensify the attachment of these compounds to the relevant
receptor and provide a proper orientation toward the hydrogen
bond formation [43, 44]. Docking pose shown in Fig. 5a, b
indicates that the 3,4,5-trimethoxy phenyl moiety in com-
pound 5e was interred in the hydrophobic pocket of the col-
chicine binding site in the β chain and surrounded by amino
acid residues such βVal238, βLeu242, βAsp251, βThr240,
βLeu252, βAsn349, βGly237, and βThr239 located in hy-
drophobic pocket and 1,2,4-triazole moiety is buried in the
amino acid residues including βAla250, βLys254, and
βLeu248. Molecular docking studies indicate the forming
aPi-Sigma between the 4-methoxyphenyl ring and βLys352
and βasn258. Moreover, as shown in 2D representation of
compound 5e (Fig. 5b), the formation of Pi-Alkyl bond be-
tween the 3,4,5-trimethoxyphenyl ring of compound 5e and
amino acid residues βAla354, βIle318 and βLeu255 also
between the triazole ring and βLeu248 and βAla250, was
detected. Moreover, a hydrogen bond between carbonyl group
and amino acid residues βAla317 was formed.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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