Molecular docking studies, biological evaluation and synthesis of novel 3-mercapto-1,2,4-triazole derivatives

Article abstract of DOI:10.1007/s11030-020-10050-0

Abstract: Synthesis of bioactive heterocyclic compounds having effective biological activity is an essential research area for wide-ranging applications. In this study, a conventional methodology has been developed for the synthesis of a series of new 3-mercapto-1,2,4-triazole derivatives 4a–f. The purity and structure of the synthesized molecules were confirmed by ¹H NMR, ¹³C NMR and elemental analysis. In addition, the prepared compounds were screened for their anti-proliferative activity against three human cancer cell lines including A549 (lung cancer), MCF7 (breast cancer) and SKOV3 (ovarian cancer) using MTT reduction assay. All the tested compounds demonstrated remarkable cytotoxic activity with IC₅₀ values ranging from 3.02 to 15.37?μM. The heterocyclic compound bearing 3,4,5-trimethoxy moiety was found to be the most effective among the series displaying an IC₅₀ of 3.02?μM specifically against the ovarian carcinoma cancer cell line (SKOV3). Moreover, Annexin V-FITC/propidium iodide staining assay indicated that this compound can induce apoptosis in SKOV3 cells. Furthermore, cell cycle assay showed a significant cell cycle arrest at the G2/M phase in a dose-dependent manner for this compound. The molecular docking results was showed binding modes of potent compound 4d perfectly corroborated the suggestion of binding to the colchicine site. The entire results conclude that 3-mercapto-1,2,4-triazole derivatives can be synthesized by a green method for biological and pharmacological applications. Graphic abstract: New analogs of 3-mercapto-1,2,4-triazole potential derivatives for anti-proliferative activity were synthesized. Cytotoxic activity of all synthesized compounds was evaluated against tree human cancer cell lines: lung (A549), breast (MCF7) and ovarian (SKOV3).[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-020-10050-0

Molecular Diversity
https://doi.org/10.1007/s11030-020-10050-0
ORIGINAL ARTICLE
Molecular docking studies, biological evaluation and synthesis
of novel 3‑mercapto‑1,2,4‑triazole derivatives
Javad Ghanaat1 · Mohammad A. Khalilzadeh1 · Daryoush Zareyee1
Received: 6 July 2019 / Accepted: 7 February 2020
© Springer Nature Switzerland AG 2020
Abstract
Synthesis of bioactive heterocyclic compounds having efective biological activity is an essential research area for wide-
ranging applications. In this study, a conventional methodology has been developed for the synthesis of a series of new
3-mercapto-1,2,4-triazole derivatives 4a–f. The purity and structure of the synthesized molecules were confrmed by 1H
NMR, 13C NMR and elemental analysis. In addition, the prepared compounds were screened for their anti-proliferative
activity against three human cancer cell lines including A549 (lung cancer), MCF7 (breast cancer) and SKOV3 (ovarian
cancer) using MTT reduction assay. All the tested compounds demonstrated remarkable cytotoxic activity with IC50 values
ranging from 3.02 to 15.37 µM. The heterocyclic compound bearing 3,4,5-trimethoxy moiety was found to be the most efec-
tive among the series displaying an IC50 of 3.02 µM specifcally against the ovarian carcinoma cancer cell line (SKOV3).
Moreover, Annexin V-FITC/propidium iodide staining assay indicated that this compound can induce apoptosis in SKOV3
cells. Furthermore, cell cycle assay showed a signifcant cell cycle arrest at the G2/M phase in a dose-dependent manner for
this compound. The molecular docking results was showed binding modes of potent compound 4d perfectly corroborated
the suggestion of binding to the colchicine site. The entire results conclude that 3-mercapto-1,2,4-triazole derivatives can
be synthesized by a green method for biological and pharmacological applications.
Graphic abstract
New analogs of 3-mercapto-1,2,4-triazole potential derivatives for anti-proliferative activity were synthesized. Cytotoxic
activity of all synthesized compounds was evaluated against tree human cancer cell lines: lung (A549), breast (MCF7) and
ovarian (SKOV3).
Keywords Mercapto-triazole derivatives · Molecular docking studies · Anti-proliferative activity · Cell cycle arrest
* Mohammad A. Khalilzadeh
mkhalil3@ncsu.edu
Extended author information available on the last page of the article
Vol.:(0123456789)
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Molecular Diversity
exposing active biological properties on human cancerous
cell lines [27–31], the triazole nucleus has gained much
attention in recent years, due to its metabolic stability,
good solubility and capability for hydrogen bonding [32]
like para-methyl derivative of trimethoxyphenyl-based
5-amino-1,2,4-triazoles as potent antimitotic agents with
IC50 values of 0.21 and 3.2 nM against CEM and HeLa
cells, respectively [33]; we have synthesized compounds
4a–f with 68–86% yields (Scheme 1). We have also inves-
tigated the anti-proliferative efects of new analog 3-mer-
capto-1,2,4-triazoles on three human cancer cell lines:
A549 (lung cancer), MCF7 (breast cancer) and SKOV3
(ovarian cancer) with diferent concentrations of com-
pounds 4a–f. The efect of these synthesized compounds
was evaluated on cell cycles for 24 h using MTT reduction
assay. Molecular docking studies and Annexin V-FITC/
propidium iodide staining assay were also investigated.
Introduction
The existence, number and position of nitrogen in the het-
erocyclic compounds play important roles in many chemi-
cal properties and application efectiveness. In general,
the heterocyclic compounds are part of synthetic chemi-
cals, natural products and bioactive pharmaceuticals that
improve the quality of life [1–3]. 1,2,4-Triazoles and
N-bridged heterocyclic derivatives show various biologi-
cal activities [4, 5] and have been utilized in medicinal
chemistry with incorporation into a wide-ranging diversity
of medicinally interesting drug candidates [6–8]. Although
many triazoles compounds are commercially available, the
1,2,4-triazole nucleus and N-bridged heterocyclic deriva-
tives having novel advantages can be developed by the
advance modern heterocyclic chemistry methods. Today,
a variety of biological activities have been reported pos-
sessing various types of biological activity such as anti-
infammatories, antifungal, antiviral, antimicrobial agents,
anti-leishmanial, antihypertensive and anti-migraine activ-
ities [9–15]. In the market, there are many drugs contain-
ing the 1,2,4-triazole moieties, e.g., triazolam, alprazolam,
etizolam, furacilin, ribavirin, hexaconazole, triadimefon,
myclobutanil, rizatriptan, propiconazole, futrimazole, etc.
[16–19]. Importantly, sulfur-containing heterocyclic com-
pounds are very essential for practical medical applica-
tions. In addition, a recent literature survey reveals that the
presence of the mercapto- and thione-substituted 1,2,4-tri-
azole moieties is an important structural feature of a wide
range of synthetic medicines [20–22]. Furthermore, many
synthesized 1,2,4-triazole ring moieties containing sulfur
have been pragmatically applied for their anticancer, anti-
fungal, anti-mycobacterial, anti-tubercular, diuretic and
hypoglycemic properties [23–25] through a large number
of their derivatives. Owing to their wide variety of bio-
logical activities and important chemotherapeutics attrib-
utes, substituted 1,2,4-triazole moieties such as letrozole,
vorozole and anastrozole (Fig. 1) have been used for the
treatment of breast cancer [26]. In the continuation of our
research on the synthesis of new heterocyclic compounds
Experimental section
Chemicals and instrumentation
All starting materials and reagents were obtained from
Sigma-Aldrich, and Merck Company. commercial solvents
were used without any pretreatment or purifcation. The
1H NMR and 13C NMR spectra were recorded using tetra-
methylsilane (TMS) as the internal standard on a Bruker
BioSpin spectrometer at 400 and 100 MHz, respectively.
Chemical shifts are reported in parts per million (ppm)
downfeld from tetramethylsilane, and coupling constants
were measured in hertz. The melting points were deter-
mined in a capillary tube using a Stuart Scientifc appara-
tus and are uncorrected. The IR spectra were recorded by
employing an FT-IR PerkinElmer spectrometer using KBr
disks. The high-resolution mass spectra were obtained
using an HP 5937 mass-selective detector (Agilent tech-
nologies). The analytical results for C, H, N and chlorine
were within 0.4% of the theoretical values.
Fig. 1 Chemical structures of
N
N
C
C
some breast cancer drugs
N
N
N
N
N
N
N
N
N
Cl
C
N
N
C
N
N
N
Vorozole
Letrozole
Anastrozole
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Molecular Diversity
O
O
O
H
N
H
N
NH2
O
a
N
H
N
H
b
S
R1
R1
O
R1
(3)
(2)
(1)
R1= H, 4-Me, 4-OMe,3,4,5-(OMe)3
4-OH, 4-NO2
c
N
N
N
SH
R1
O
(4a-f)
Scheme 1 General procedure for synthesis of target compounds 4a–f. Reagent and conditions: (a) NH2NH2·H2O (80%), ethanol, refux; (b)
4-methoxy phenyl isothiocyanate, ethanol, refux; (c) 1 M NaOH, refux, and then 1 M HCl
General synthetic procedure for preparation of compounds
4a–f
153.10, 166.60, 167.37. Anal. Calcd. for C15H13N3OS:C,
63.58; H, 4.62; N, 14.83. Found: C, 63.83; H, 4.60; N, 14.88.
A mixture of diferent methyl benzoate (1 mmol) and
hydrazine hydrate (0.75 g, 15 mmol, 80%) in 7 mL of
ethanol was heated under refux for 6 h. Excess ethanol
was distilled and the contents were allowed to cool. The
solid obtained product was fltered, washed with water and
dried to aford the corresponding benzohydrazide, which
was used for the next reaction. Then, a solution of diferent
benzohydrazides (1 mmol) and 4-methoxy phenyl isothio-
cyanate (1 mmol, 0.165 g) in ethanol (10 mL) was heated
at refux for 5 h and cooled down to room temperature
gradually. The suspension was fltered, and the solid was
washed with ethanol and dried to give the intermediate
hydrazine carbothioamide as a white solid product. 1 M
NaOH (10 mL) was added to this solid, and the mixture
was heated at refux for 1 h. The resulting solution was
cooled at room temperature and acidifed to pH 5–6 with
1 M HCl. The precipitate was fltered, washed with water
and dried to obtain the title compounds as white solid.
4‑(4‑Methoxyphenyl)‑5‑p‑tolyl‑4H‑1,2,4‑triazole‑3‑thiol
(4b) Yield: 82%; m.p.; 122–124 °C; H NMR (400 MHz,
1
DMSO-d6): δ 3.05 (S, 3H, Me), 3.79 (S, 3H, OMe), 6.73 (d,
2H, J=8.4 Hz, H-3′ and H-5′), 6.99 (d, 2H, J=8.8 Hz, H-3
and H-5), 7.19 (d, 2H, J=6.4 Hz, H-2′ and H-6′), 7.40 (d,
2H, J=7.6 Hz, H-2 and H-6). 13C NMR (100 MHz, DMSO-
d6): δ 22.76, 55.67, 114.76, 122.66, 128.90, 129.47, 134.40,
138.08, 148.39, 157.31. Anal. Calcd. for C16H15N3OS:C,
64.62; H, 5.08; N, 14.13. Found: C, 64.36; H, 5.100; N,
14.07.
4,5‑Bis(4‑methoxyphenyl)‑4H‑1,2,4‑triazole‑3‑thiol
1
(4c) Yield: 68%; m.p.; 128–130 °C; H NMR (400 MHz,
DMSO-d6): δ 3.70 (S, 3H, OMe), 3.78 (S, 3H, OMe), 6.80
(d, 2H, J=8.8 Hz, H-3′ and H-5′), 6.93 (dd, 2H, J=4.4
and 2.0 Hz, H-2′ and H-6′), 7.02 (d, 2H, J=6.8 Hz, H-3
and H-5), 7.13 (d, 2H, J=8.8 Hz, H-2 and H-6). 13C NMR
(100 MHz, DMSO-d6): δ 55.52, 55.70, 114.05, 114.17,
128.78, 130.16, 150.36, 158.59, 159.30, 169.13. Anal.
Calcd. for C16H15N3O2S:C, 61.32; H, 4.82; N, 13.41. Found:
C, 61.07; H, 4.83; N, 134.46.
4‑(4‑Methoxyphenyl)‑5‑phenyl‑4H‑1,2,4‑triazole‑3‑thiol
1
(4a) Yield: 86%; m.p.; 110–112 °C; H NMR (400 MHz,
DMSO-d6): δ 3.79 (S, 3H, OMe), 7.00 (dd, 2H, J=4.8 and
2.0 Hz, H-3′ and H-5′), 7.23 (dd, 2H, J=4.8 and 2.0 Hz, H-2′
and H-6′), 7.30–7.35 (m, 3H, H-3, H-4 and H-5), 7.35–7.42
(m, 2H, H-2 and H-6). 13C NMR (100 MHz, DMSO-d6):
δ 56.37, 107.77, 119.48, 120.12, 129.55, 132.37, 134.10,
5‑(3,4,5‑Trimethoxyphenyl)‑4‑(4‑methoxyphenyl)‑4H‑1,2,
1
4‑triazole‑3‑thiol (4d) Yield: 76%; m.p; 134–136 °C; H
NMR (400 MHz, DMSO-d6): 3.73 (S, 3H, OMe), 3.83
(S, 6H, OMe), 7.23 (S, 2H, H-2 and H-6). 7.27 (d, 2H,
1 3

Molecular Diversity
J=8.0 Hz, H-3 and H-5), 7.35 (d, 2H, J=6.00 Hz, H-2
and H-6). 13C NMR (100 MHz, DMSO-d6): δ 55.67, 56.37,
60.56, 106.97, 112.92, 118.38, 124.34, 126.35, 141.80,
153.10, 167.37. Anal. Calcd. for C18H19N3O4S: C, 57.89;
H, 5.13; N, 11.25. Found: C, 58.12; H, 5.15; N, 11.20.
the medium was replaced with complete medium containing
compounds 4d at 25, 50 and 100 µM concentrations. After
48 h of drug treatment, cells from the supernatant were har-
vested by trypsinization, washed with PBS at 3000 rpm and
stained with 5 mL of Annexin V-FITC (Sigma Chemical Co)
in PBS at room temperature for 15 min and then PI solution
(Keygen Biotech, China) for another additional 10 min. Flow
cytometry was performed using a FACScan (Becton–Dickin-
son) equipped with a single 488 nm argon laser. The percent-
age of apoptotic cells was calculated using the EXPO32 ADC
analysis software.
4‑(5‑Mercapto‑4‑(4‑methoxyphenyl)‑4H‑1,2,4‑triazole‑3‑yl)
phenol (4e) Yield: 73%; m.p.; 121–123 °C; 1H NMR
(400 MHz, DMSO-d6): δ 3.78 (S, 3H, OMe), 6.73 (d, 2H,
J=8.4 Hz, H-3 and H-5), 6.93 (dd, 2H, J=4.4 and 2.4 Hz,
H-3′ and H-5′), 7.02 (d, 2H, J=6.8 Hz, H-2′ and H-6′), 7.19
(d, 2H, J=6.8 Hz, H-2 and H-6). 13C NMR (100 MHz,
DMSO-d6): δ 56.37, 107.77,119.48, 102.12, 129.55,
132.37, 134.10, 153.10, 166.60, 167.37. Anal. Calcd. for
C15H13N3O2S: C, 60.18; H, 4.38; N, 14.04; O, 10.69. Found:
C, 59.93; H, 4.39; N, 13.98.
Cell cycle analysis
SKOV3 cells were seeded in 6-well plates (3×105 cells/well),
incubated in the presence or absence of compound 4d at the
25, 50 and 100 µM concentrations for 24 h, harvested by cen-
trifugation and then fxed in ice-cold 70% ethanol overnight.
After that, ethanol was removed and the cells were re-sus-
pended in ice-cold PBS, treated with RNAse A (Keygen Bio-
tech, China) at 37 °C for 30 min and then incubated with the
DNA staining solution propidium iodide (PI, Keygen Biotech,
China) at 4 °C for 30 min. Approximately, 10,000 events were
detected by fow cytometry (Beckman Coulter, Epics XL) at
488 nm. The data regarding the number of cells in diferent
phases of the cell cycle were analyzed using EXPO32 ADC
analysis software.
4‑(4‑Methoxyphenyl)‑5‑(4‑nitrophenyl)‑4H‑1,2,4‑tria‑
1
zole‑3‑thiol (4f) Yield: 82%; m.p.; 142–144 °C; H NMR
(400 MHz, DMSO-d6): δ 3.75 (S, 3H, OMe), 6.90 (d, 2H,
J=8.8 Hz, H-3′ and H-5′), 7.27 (d, 2H, J=8.0 Hz, H-2 and
H-6), 8.17 (d, 2H, J=8.8 Hz, H-2′ and H-6′), 8.35 (dd, 2H,
J=5.2 and 2.0 Hz, H-3 and H-5). 13C NMR (100 MHz,
DMSO-d6): δ 55.67, 109.77, 113.74, 120.06, 123.86,
128.90, 129.87, 132.40, 139.06, 141.19, 149.79, 157.30.
Anal. Calcd. for C15H12N4O3S: C, 54.87; H, 3.68; N, 17.06;
O, 14.62. Found: C, 54.65; H, 3.69; N, 16.99.
Biology
Molecular docking studies
In vitro MTT assay
In order to determine the basic structure of the biologi-
cal results discussed above, the binding mode of promising
compounds 4d to the colchicine-binding site of tubulin was
investigated using AutoDock software (version 4.2). The 3D
structures for the compounds were built and optimized by
ChemBioDraw Ultra 16.0 and ChemBio3D Ultra 16.0 soft-
ware. These optimized 3D structures are utilized for docking.
The β-subunit of the target (chain D) was taken into account,
and the rest was deleted. The AutoDock protocol [35] was
followed to prepare the fnal receptor pdbqt fle. A grid map
with 40, 40 and 40 grid points (X, Y, Z dimensions, resp.) and
centroid: X=17.0192, Y=65.9939, Z=43.3901 was selected.
The best scoring pose of each complex as judged by the Auto-
Dock was chosen and visually analyzed for interactions. The
3D, 2D poses of most active compound 4d taken using dis-
covery studio 2016.
The cytotoxic efects of all newly synthesized compounds
4a–f were evaluated by using MTT assay on three human
cancer cell lines (MCF-7, A549 and SKOV3). 1×104 cells
per well were seeded in 96-well culture plates in culture
medium supplemented with 10% FBS and incubated for 24 h
at 37 °C in a CO2 incubator. After incubation, cells were
treated with 100 µL of test compounds and were diluted to
the desired concentrations in culture. After 48 h of incuba-
tion, 10 µL MTT (5 mg/mL) was added to each well and
the plates were further incubated until a purple precipitate
was visible (4 h). Then, the medium was carefully removed,
the precipitates were dissolved in 200 µL of DMSO, and
the absorbance of the solution at 490 nm was measured.
IC50 values, at which cell growth was inhibited by 50% were
calculated with GraphPad Prism Software version 7.0 [34].
Annexin V‑FITC for apoptosis
The apoptotic efect of compound 4d was further evaluated
by Annexin V-FITC/PI assay. SKOV3 cells were cultured in
6-well plates (1×106 cells/well) and incubated for 24 h. Then,
1 3

Molecular Diversity
Table 1 Cytotoxic activities of compounds 4a–f against cancer cells
Results and discussion
Chemistry and cytotoxicity assay
(A549, MCF-7 and SKOV3)
N
N
SH
N
Pursuant to a survey of the literature, the substituted
1,2,4-triazole ring is found to be an interesting group of
heterocyclic compounds that are highly regarded by con-
temporary medicinal chemists owing to their appreciable
anticancer efects on diferent types of cancer cell lines
[36, 37]. Furthermore, the introduction of proper hetero-
cyclic compounds would be a good strategy to overcome
some going problems such as metabolic instability, low
oral bioavailability, poor aqueous solubility and undesired
side efects [38]. The structure–activity relationship stud-
ies indicated that the presence of a 3,4,5-trimethoxyphenyl
ring-A is one of the essentials for potent anticancer activ-
ity [39]. The steric and electronic efects of diferent elec-
tron-donating alkoxy groups on the benzene moiety were
chosen to improve the cytotoxicity and physicochemical
properties of these compounds.
R1
O
Compound
*IC50 SD values against cancer cell
lines (µM)
R1
A-549
MCF-7
SKOV3
4a
4b
4c
4d
4e
4f
H
4-Me
9.18 0.13 12.45 0.21 10.48 37
12.26 0.28 11.87 0.26 9.13 0.29
8.90 0.17 9.25 0.42 6.28 0.31
4.32 0.12 4.65 0.32 3.02 027
6.16 0.18 7.61 0.12 5.87 0.12
12.87 0.45 13.46 0.17 15.37 0.28
4-OMe
3,4,5-(OMe)3
4-OH
4-NO2
In this study, the heterocyclic compounds were syn-
thesized by employing the sequence outlined following
the procedure depicted in Scheme 1: frstly, the methyl
ester derivatives (1) converted to the corresponding acid
hydrazides (2) by treating hydrazine hydrate 80% in almost
quantitative yield. After cooling the mixture to room
temperature, the solid hydrazide product was washed
with water and then dried. The reaction of diferent acid
hydrazides (2) with 4-methoxy phenyl isothiocyanate in
the presence of sodium hydroxide as a base in refuxing
water followed by acidifcation with HCl 1 M to pH = 5–6
resulted in carbothioamide salts (3) which then underwent
intramolecular ring closure to generate the corresponding
substituted 3-mercapto-1,2,4-triazole derivatives 4a–f.
The in vitro anti-proliferative activity of synthesized
compounds against three human cancer cell lines includ-
ing adeno carcinomic human alveolar basal epithelial cells
(A549), breast cancer cells (MCF7) and human ovarian
carcinoma cells (SKOV3) was evaluated by using stand-
ard MTT assays according to the literature method, and
etoposide was chosen as a standard drug [40]. Cytotoxicity
values against diferent cancer cell lines expressed as the
compound concentration (µM) that causes 50% inhibition
of cell growth (IC50) are shown in Table 1 and Fig. 2.
All the tested compounds presented considerable cyto-
toxic activity against cell lines, in particular, compounds
4d and 4e with IC50 values of 3.02–5.87 µM. The best IC50
values were obtained against SKOV3 cell line. In addi-
tion, most compounds showed superior growth inhibitory
activity against all tested cell lines to that of reference
drug etoposide. A survey on the structures and activities
in Table 1 revealed that the IC50 values of compounds with
Etoposide
2.99 0.16 3.89 0.1
4.74 0.14
*IC50 values are indicated as the mean of three independent experi-
ments
Fig. 2 a Inhibition growth of 4d on SKOV3 cells. b Cytotoxicity
activities of compounds 4a–f and etoposide against SKOV3 cell
1 3

Molecular Diversity
Fig. 3 Compound 4d induced
the apoptosis of SKOV3 cells.
The percentages of cells in each
stage of apoptosis were quan-
titated by fow cytometry. (B1,
upper left quadrant), necrotic
cells; (B2, upper right quadrant)
late apoptotic cells; (B3, bottom
left quadrant) live cells; and
(B4, bottom right quadrant)
early apoptotic cells
diferent substituents are related to their substituent and
also the efect of the substituent on the cytotoxic activity
depends on the type of tested cell line. For example, the
comparison of un-substituted compound 4a and 4-methyl
derivative 4b reveals that the introduction of 4-methyl
group improves the activity against MCF7 and SKOV3
cells while diminishes the activity toward A549 cells.
Interestingly, the highest inhibition was obtained with
3,4,5-trimethoxy derivative 4d against all tested cell lines,
displaying IC50 values in the range of 3.02–4.65 µM. Its
activity was better than that of etoposide against SKOV3
cell line. Comparison of the anti-proliferative activities
of 4d with other derivatives obviously indicates that the
existence of 3,4,5-trimethoxyphenyl moiety plays a vital
role in presenting improved anti-proliferative activity. A
simple structure–activity relationship also indicated that
the anti-proliferative activity was related to the substituted
groups R1. The strong inhibitory potential for 4d and 4e
derivatives could be attributed to the electron-donating
3,4,5-trimethoxy and hydroxyl group present at para posi-
tion of the aryloxy ring. A slight decrease in activity was
observed when these groups was replaced by a methyl or
electron-withdrawing group 4-NO2 in compounds 4a and
4f. For example, regarding the IC50 value on SKOV3 cell
compound 4d (3.02 µM) with the electron-donating group,
3,4,5-trimethoxy 4e (5.87 µM) and the hydroxyl group at
R1 compared to un-substituted derivative 4a (10.48 µM),
4-methyl derivative 4b (9.13 µM) and 4-NO2 derivative 4f
(15.37 µM) having electron-withdrawing group can donate
an electron at the para position of phenyl ring strongly
improving cytotoxicity of these compounds. In contrast,
withdrawing the group led to a reduction in the potency of
these compounds to inhibit cancer cells.
Hany et al. have reported a series of novel compounds
carrying 1,2,4-triazole a scafold as potent anti-tumor
agents. 4-Me and 4-(OMe)3 derivatives with IC50 values
of 1.5 and 1.2 µM showed potent cytotoxic activity against
PaCa-2 and HT-29 cell lines [41]. In the other study, the
cytotoxicity activity of a series of novel S-substituted
1H-3-R-5-mercapto-1,2,4-triazoles was tested on the
HT-29 colorectal cancer cell line and results revealed that
4-methyl derivative (R = 4-Me) with IC50 values of 87 µM
had the highest activity against tested cell line [42]. A
series of 3-[3-[4-(substituted)-1-cyclicamine]propyl]thio-
5-substituted[1,2,4]triazoles derivatives were designed and
synthesized by Murty and coworkers. The cytotoxicity
1 3

Molecular Diversity
Fig. 4 Cell cycle arrest efect of
4d. SKOV3 cells were treated
with 4d at the indicated concen-
trations for 24 h
assay revealed that 3-Me and 3-Cl derivatives were more
potent than other synthesized compounds against HL-60
cells [43]. As shown by our results (Table 1), derivatives
with 4-OMe, 4-OH and 4-(OMe)3 substitutions also exhib-
ited the highest level of cytotoxicity with an IC50 value of
3.02–9.25 µM against all tested cell lines.
Analysis of cell cycle arrest
To evaluate the cell cycle arrest efect of compound 4d,
SKOV3 cells were treated with DMSO (0.01%) at various
concentrations (25, 50 and 100 µM) for 24 h. According to
the results shown in Fig. 4, compound 4d resulted in signif-
cant cell cycle arrest at the G2/M phase in a dose-dependent
manner. As shown in Fig. 4, in the compound 4d-treated
group, 30% of the cells were at the G2/M phase 24 h after
the treatment, whereas for the DMSO-treated group, 19% of
the cells were in this phase at the same time after treatment.
The percentage of cells at the G2/M phase correspondingly
increased to 43% and 72%, respectively, after increasing the
concentration of 4d to 50 and 100 µM for 24 h.
Annexin V‑FITC fow cytometric analysis
of apoptosis
To clarify the possibility of phosphatidylserine externaliza-
tion and investigate the consequence of apoptosis or non-
specifc necrosis, we evaluated the apoptotic efect on 4d
compound without any additive and with DMSO addition
(0.01%) separately, using control cells, by Annexin V-FITC/
PI dual staining assay [44]. SKOV3 cells were treated with
the aforementioned compounds at 25, 50 and 100 µM for
48 h. As shown in Fig. 3, dose-dependent apoptosis occurred
in SKOV3 cells treated with compound 4d with diferent
concentrations. The results demonstrated increasing percent-
ages of apoptotic cells with increasing doses. Compound 4d
exhibited 19.32, 35.6 and 41.37% apoptosis at 25, 50 and
100 µM concentrations for 48 h, respectively.
Molecular docking studies
Previous studies have shown that trimethoxyphenyl moi-
ety acts as a pharmacophoric group for the anticancer
effect of some natural anticancer compounds such as
colchicines, combretastatin, podophyllotoxin and poly-
methoxychalcone [45, 46]. Mechanistically, these com-
pounds were found to be tubulin inhibitors that bind to the
1 3

Molecular Diversity
presence of 3,4,5-trimethoxy phenyl and nitrogen 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 [49, 50]. As shown in Fig. 5a, trimethoxyphenyl
moiety of 4d is surrounded by several amino acid residues
such as βAla354, βIle378, βIle318, βVal238, βCys241,
βLeu242, βVal315, βAsp25, βThr314, βLeu248, βLeu255,
βAla317 and βAla316, located in a hydrophobic pocket.
1,2,4-Triazole moiety is buried in the amino acid residues
including βAla316, βLys254, βAla317 and βLeu255.
Molecular docking studies indicate the formation of two
Pi-Sigma between the trimethoxyphenyl ring and Leu255
and βLeu248. Moreover, as shown in 2D representation of
compound 4d (Fig. 5b), the formation of Pi-alkyl and alkyl
bonds between the p-methoxyphenyl ring of compound 4d,
and amino acid residues βLys352 between the trimethoxy-
phenyl ring, and Lys352 and Ala316 could be detected. A
sulfur-X interaction between p-methoxyphenyl ring and
βMet259 is also clarifed.
Conclusion
In this study, we designed and synthesized new analogs
of 3-mercapto-1,2,4-triazole potential derivatives for anti-
proliferative activity. Cytotoxic activity of the synthesized
compounds was evaluated against three human cancer cell
lines: lung (A549), breast (MCF7) and ovarian (SKOV3).
Anti-proliferative results showed that all tested compounds
potentially could inhibit the growth of cancerous cells, in
particular, in case of compounds 4d and 4e. Furthermore,
Annexin V-FITC assay suggested that compound 4d sig-
nifcantly induces apoptosis in SKOV3 in a concentration-
dependent manner and arrests cell cycle progression at
the G2/M phase due to microtubule depolymerization
and cytoskeletal disruption. Molecular docking study get
an obvious insight into the binding site of compound 4d
with tubulin indicating that the binding of this compound
to the colchicine binding site of beta-tubulin that caus-
ing improved anti-proliferative activity. The synthesized
3-mercapto-1,2,4-triazoles may fnd potential practical
applications as a new class of cytotoxic agents as the anti-
proliferative activity of these compounds could be also
useful.
Fig. 5 3D and 2D representation of binding mode of compound 4d (a
and b) with tubulin (PDBID: 4O2B)
colchicine-binding site, leading to microtubule depolym-
erization. Microtubules have critical roles in several cel-
lular processes, particularly in mitosis. Therefore, micro-
tubules have been considered as an attractive target for
anticancer drug discovery [47]. Molecular modeling was
performed to investigate promising binding modes for the
potential ligands toward tubulin polymerization into the
colchicine-binding site of tubulin by using the AutoDock
software (version 4.2). The visualization was carried out
using Discovery Studio 2016 software. The most active
compounds 4d were docked into the colchicine-binding
site of tubulin using the high-resolution crystal structure
of the tubulin–colchicine complex screening from protein
data bank (PDB entry: 4O2B) [48]. Colchicine was frst
docked into the pocket of the tubulin as the representative
and susceptible compound in this study. Accordingly, the
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Afliations
Javad Ghanaat1 · Mohammad A. Khalilzadeh1 · Daryoush Zareyee1
1
Department of Chemistry, Qaemshahr Branch, Islamic Azad
University, Qaemshahr, Iran
1 3