Synthesis and anti-colon cancer activity of 1,2,4-triazole derivatives with aliphatic S-substituents

Article abstract of DOI:10.13005/ojc/350109

A series of new 3-mercapto-1,2,4-triazole derivatives have been synthesized and characterized by FT-IR, ¹H NMR & ¹³C NMR spectrophotometric techniques. The target compounds are designed as analogues for the anti-cancer agent Combretastatin A-4 with different aliphatic side substituents.The anticancer activity of the synthesized compounds was studied against colon cancer cell line SW480 using the crystal violet cytotoxicity assay. The results showed that all the synthesized compounds have growth inhibitive effect on the cancer cells with different inhibition levels. Compound 5a with -SMe group was found to be the most active one with 77.4% cell growth inhibition and 10 μM IC₅₀ value, it was also found to have relatively low cytotoxicity when tested against MDCK normal cells. The antioxidant activity of the synthesized compounds was tested using DPPH assay against ascorbic acid as a reference antioxidant agent at 50μM. Compound 4 showed highest antioxidant activity with DPPH radical scavenging capacity of 71%.

Full text of DOI:10.13005/ojc/350109

ISSN: 0970-020 X
CODEN: OJCHEG
2019, Vol. 35, No.(1):
Pg. 77-84
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis and Anti-Colon Cancer Activity of 1,2,4-Triazole
Derivatives with Aliphatic S-Substituents
SADIq AL-MANSURY1, ASIM A. BALAkIT2*, FATIN FADHEL ALkAzAzz3,
kAISER N. MADLUM4 and RANA A. GHALEB4
1Biochemistry & Pharmacology Department, Veterinary Medicine College,
Al-Qasim Green University, Babylon, Iraq.
2College of Pharmacy, University of Babylon, Babylon, Iraq.
3College of Science, Department of Chemistry, Al-Mustansiyriah University, Baghdad, Iraq.
4Department of Human Anatomy, College of Medicine, University of Babylon, Babylon, Iraq.
Corresponding author E-mail: asim_alsalehi@hotmail.com
http://dx.doi.org/10.13005/ojc/350109
Received: November 16, 2018; Accepted: January 27, 2019)
ABSTRACT
A series of new 3-mercapto-1,2,4-triazole derivatives have been synthesized and
characterized by FT-IR, 1H NMR & 13C NMR spectrophotometric techniques. The target
compounds are designed as analogues for the anti-cancer agent Combretastatin A-4 with
different aliphatic side substituents.The anticancer activity of the synthesized compounds
was studied against colon cancer cell line SW480 using the crystal violet cytotoxicity assay.
The results showed that all the synthesized compounds have growth inhibitive effect on
the cancer cells with different inhibition levels. Compound 5a with -SMe group was found
to be the most active one with 77.4% cell growth inhibition and 10 µM IC50 value, it was
also found to have relatively low cytotoxicity when tested against MDCK normal cells.
The antioxidant activity of the synthesized compounds was tested using DPPH assay
against ascorbic acid as a reference antioxidant agent at 50µM. Compound 4 showed
highest antioxidant activity with DPPH radical scavenging capacity of 71%.
keywords: Triazole, Colon cancer, Anticancer, Antioxidants, SW480.
INTRODUCTION
deaths.2 The design and synthesis of anticancer
agents is an interesting research area in which
scientists are endeavoring to develop synthetic
or natural compounds that can contribute in the
treatment of cancer and help patients to be cured
of this life-threatening disease.3 There is a very
Cancer is considered as the second
lethal disease worldwide1, colon cancer is the third
most common type of cancer around the world
and one of the leading reasons of cancer-related
This is an
Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018

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broad spectrum of natural compounds which are
considered as anticancer agents4 among them
combretastatin A 4 (CA-4) which is natural product
isolated from the South African tree; Combretum
caffrum Kuntze (Combretaceae).5 The anticancer
activity of CA-4 is attributed to the inhibition of
tubulin polymerization by binding to the colchicine
binding site.6 The simplicity of the structure of
CA-4 and its remarkable anticancer efficacy make
its structure a fundamental part of wide range of
synthetic anticancer agents which are considered
as combretastatin A-4 analogues.7-9 Based on the
structure-activity relationship (SAR) studies, it is
reported the CA-4 analogues must have fundamental
structural requirements for the optimal activity, the
molecule must have 3,4,5-trimethoxyphenyl and
4-methoxyphenyl groups attached to two adjacent
atoms (bridge), the two groups must be in cis-olefin
configuration.10, 11
group, the new compounds are designed to meet
the structural requirements of the highest expected
anticancer activity of CA-4 analogues to be tested
as anticancer agents against colon cancer cell line
SW480.The antioxidant capacity of the synthesized
compounds is also studied.
MATERIAL AND METHODS
General
Chemicals and reagents were purchased
from commercial sources and used without any
further purification.1H (400 MHz) and 13C NMR (100
MHz) spectra were recorded in CDCl3 or DMSO-
d6 on a Bruker Bio Spin 400 MHz spectrometer.
Chemical shifts d (ppm) are reported relative to
tetramethylsilane (TMS).IR spectra were recorded on
a Tensor II, Bruker-Optics FT-IR spectrophotometer
as KBr discs. Melting points were measured using
Stuart SMP10 apparatus. Column chromatography
was carried out using silica gel 200 – 300 Mesh.
3,4,5 Trimethoxybenzohydrazide was synthesized
according to literature procedure.27
A great attention has been devoted to the
1,2,4-triazol derivatives because of their biological
activities, particularly sulfur-containing ones found
to have a broad-spectrum of biological activities
such as herbicidal12, anticancer13, antidepressant14,
antimicrobial15, antifungal16, analgesic/anti-
inflammatory17, antiviral18, and antioxidant.19 Many
studies have shown that the pharmacological
activities of 1,2,4-triazole and their derivatives due
to existence of the1,2,4-triazole nucleus.20 Some
of compounds with triazole ring are reported as
potent anticancer CA-4 analogues.21 The presence
of S-linkers in the structure improve some drug-
like parameters such as decreasing lipophilicity,
increasing water solubility, and offering good
hydrogen bond acceptors.22
Synthesis: 4-(4-Methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol (4):
4-Methoxyphenyl isothiocyanate (1.65
gm,10 mmol) was added to a solution of 3,4,5-
trimethoxybenzohydrazide(2.263g,10mmol)inethanol
(30ml),themixturewasthensubjectedtorefluxconditions
for4hthenwascooledatroomtemperature.Theformed
solidwascollectedbyfiltrationandwashedwithethanoland
derided.Then the solid product was dissolved in 20 ml of
2 N NaOH and refluxed for 4 hours.The mixture was left
to cool to room temperature and then filtrated;the filtrate
wasacidifiedwith10%hydrochloricacid.Theparticipated
solid was filtrated off, washed with cold water.
Antioxidants are compounds that have the
ability to reduce the oxidative stress by inhibiting
the oxidation process leading to preventing the
cell death resulting from generated free radicals.23
The excessive release of free radicals is intimately
related to cancer disease24, thereby antioxidants
can contribute in fighting this deadly disease.
There is huge number of both natural and synthetic
compounds that are investigated for their antioxidant
capacity.25, 26
light-yellow solid; yield, 81%; m.p. 253-
255°C; FT-IR:3091-2931(C-H, aromatic), 2829(C-H,
aliphatic), 2754 (SH), 1606 (C=N), 1591 (C=C); 1H
NMR (400 MHz, DMSO-d6) d:3.57 (s, 6H, 2xOCH3),
3.65 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.61 (s, 2H,
Aromatic), 7.07 (d, J = 8.8 Hz, 2H, Aromatic), 7.30
(d, J = 8.8 Hz, 2H Aromatic), 14.07 (br, 1H, SH).
13C NMR (100-MHz, DMSOd6) d: 56.0, 56.2 (2C),
60.5, 106.28 (2C), 115.0 (2C), 121.3, 127.8, 130.5
(2C), 139.4, 150.7, 153.0 (2C), 160.1, 169.3.
In the present work we report the design
and synthesis of new 1,2,4-triazole derivatives
with aliphatic substituents attached to the sulfur

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BAlAKIT et al., Orient. J. Chem., Vol. 35(1), 77-84 (2019)
General procedure for the synthesis of target
compounds (5a-j)
7.7 Hz, 2H, Aromatic). 13C NMR (100 MHz, CDCl3)
d: 14.7, 26.8, 55.69-55.91(3C), 60.88, 105.3 (2C),
115.9 (2C), 121.9, 127.0, 128.7(2C) 138.8, 144.5,
153.0 (2C), 154.6, 160.5.
To the solution of 4 (0.373 gm,1 mmol) in
20 ml of 50% aqueous 2-propanol, NaOH (0.04 g,
1.5 mmol) was added, the mixture was stirred for
5 min then appropriate alkyl halide (1.1 mmol) was
added to. The reaction mixture was refluxed for
8 hours. The reaction progress was monitored by
TlC, after completion the 2-propanol was evaporated
under reduced pressure and the product was
extracted by chloroform, solvent was evaporated to
give the crude solid product which was washed with
hexane and then purified by column chromatography.
The melting points, eluents and Rf values of the
synthesized compounds are shown in Table 1.
3-(Butylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5c)
Yellow crystal, yield 78%;FT-IR:3001 (C-H,
aromatic), 2958 (C-H, aliphatic), 1609 (C=N), 1588
(C=C); 1H NMR (400 MHz, CDCl3) 0.91 (t, J = 7.3
Hz, 3H, SCH2CH2CH2CH3), 1.43 (sext, J = 7.3 Hz,
2H, SCH2CH2 CH2CH3), 1.7 (p, J = 7.4 Hz, 2H, SCH2
CH2CH2 CH3), 3.2 (t, J = 7.3 Hz, 2H, SCH2 CH2CH2
CH3), 3.64 (s, 6H, 2 x OCH3), 3.83 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 6.69 (s, 2H, Aromatic), 7.08
(d, J = 8.8 Hz, 2H, Aromatic), 7.19 (d, J = 8.8 Hz,
2H, Aromatic). 13C NMR (100 MHz, CDCl3) d: 13.5,
21.8, 31.35, 32.2, 55.68-55.9 (3C), 60.2, 105.3 (2C),
115.0(2C), 121.9, 127.1, 128.8 (2C), 139.1, 153,
153.5(2C), 154.6, 160.5.
Table 1: Melting points, eluents and Rf values of 4, 5a-j
Compound m. p.°C
Eluent(n-hexane: Rf (n-hexane: ethyl
ethyl acetate)
acetate; 1.5 : 2.5)
4
253-255
102-103
80-81
--
0.54
0.07
0.14
0.285
0.357
0.297
0.214
0.2
3-(Hexylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5d)
White solid, yield 87%; FT-IR: 3110 (C-H,
5a
5b
5c
5d
5e
5f
1: 2
1: 2
104-106
106-108
94-96
1: 1.5
1: 1.5
1: 1
aromatic), 2836 (C-H, aliphatic), 1608 (C=N),
1
1588 (C=C); H NMR (400 MHz, CDCl3) d: 0.87
112-113
93-95
1: 1.5
1: 1.5
1: 1.5
1: 1
(t, J = 6.7 Hz, 3H, S(CH2)5CH3), 1.30 (sext, J =
4.5 Hz, 2H, S(CH2)4CH2CH3), 1.42 (p, J = 7.2 Hz,
4H, S(CH2)2CH2CH2CH2CH3), 1.64 (p, J =7.5 Hz,
2H, SCH2CH2(CH2)3CH3), 3.26 (t, J = 7.6 Hz 2H,
SCH2(CH2)4CH3), 3.64 (s, 6H, 2 x OCH3), 3.8 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 6.69 (s, 2H, Aromatic),
7.08 (d, J = 8.5 Hz, 2H, Aromatic), 7.2 (d, J = 8.9
Hz, 2H, Aromatic). 13C NMR (100 MHz, CDCl3) d:
13.9, 22.5, 28.4, 29.3, 31.2, 32.2, 55.6, 55.9(2C),
60.8, 105.33(2C), 115.03(2C), 121.8, 127.1, 128.8
(2C), 139.1, 153 (2C), 153.5, 154.6, 160.5.
5g
5h
5i
90-92
0.33
0.19
0.13
80-82
5j
109-110
1: 2
4-(4-Methoxyphenyl)-3-(methylthio)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5a)
Yellow solid, yield, 84%; FT-IR: 3090-2928
(C-H, aromatic), 2829 (C-H, aliphatic), 1607 (C=N),
1585 (C=C); 1H NMR (400 MHz, CDCl3) d: 2.7 (s, 3H,
SCH3), 3.63 (s, 6H, 2 x OCH3), 3.82 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 6.69 (s, 2H, Aromatic), 7.08
(d, J = 8.9 Hz, 2H, Aromatic), 7.20 (d, J = 8.9 Hz, 2H,
Aromatic). 13C NMR (100 MHz, CDCl3) d: 14.64, 55.69,
55.93(2C), 60.8, 105.37 (2C), 115.18 (2C), 121.3,
126.9(2C), 128.7, 139.8, 153 (2C), 154.1, 154.8, 160.6
3-(Isobutylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5e)
White solid, yield 78%; FT-IR: 3003
(C-H, aromatic), 2867 (C-H, aliphatic), 1606
1
(C=N), 1588 (C=C); H NMR (400 MHz, CDCl3)
3-(Ethylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5b)
Yellow solid, yield 82%; FT-IR: 3091-2931
d: 1.01 (d, J = 6.6 Hz, 6H, SCH2CH(CH3)2), 2.02
(m, 1H, SCH2CH(CH3)2), 3.18 (d, J = 6.8 Hz, 2H,
SCH2CH(CH3)2), 3.6 (6H, 2 x OCH3), 3.83 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 6.69 (s, 2H Aromatic),
7.08 (d, J = 8.7 Hz, 2H, Aromatic), 7.19 (d, J = 8.7
Hz, 2H, ArH B). 13C NMR (100 MHz, CDCl3) d: 21.8
(2C), 26.45, 40.9, 55.68 (3C), 60.87, 105.3(2C),
115.03 (2C), 121.9, 127.1, 128.8 (2C), 139.1, 153.0
(2C), 153.7, 154.58, 160.5.
(C-H, aromatic), 2829 (C-H, aliphatic), 1607 (C=N),
1
1587 (C=C); H NMR (400 MHz, CDCl3) d: 1.43
(t, J = 7.4 Hz, 3H, SCH2CH3), 3.3 (q, J = 7.3 Hz,
2H, SCH2CH3), 3.64 (s, 6H, 2 x OCH3), 3.82 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.69 (s, 2H, Aromatic),
7.03 (d, J = 8.9 Hz, d, 2H, Aromatic), 7.21 (d, J =

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BAlAKIT et al., Orient. J. Chem., Vol. 35(1), 77-84 (2019)
3-(Allylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5f)
2H, Aromatic), 7.29 (d, J = 8.77 Hz, 2H, Aromatic).
13C NMR (100 MHz, DMSO-d6) d: 48.03, 49.07,
56.03, 56.18, 60.6, 61.7, 68.8, 105.6 (2C), 115.13
(2C), 121.5, 127.1, 129.9 (2C), 139.2, 144.35,
153.1(2C), 153.8, 159.89.
Yellow solid, yield 87%; FT-IR: 3091 (C-H,
aromatic), 2838 (C-H, aliphatic), 1607 (C=N),
1
1585 (C=C); H NMR (400 MHz, CDCl3) d: 3.63
(s, 6H, 2 x OCH3), 3.82 (s, 3H, OCH3), 3. 85
(s, 3H, OCH3), 3.87 (d, J=7.1 Hz, 2H, SCH2CH=CH2),
5.12 (d, J=9.97 Hz, 1H, SCH2CH=CH2), 5.23
(d, J=9.2 1H, SCH2CH=CH2), 5.94 (q, J=7.4 Hz,
1H, SCH2CH=CH2), 6.69 (s, 2H, Aromatic), 7.08
(d, J = 8.8 Hz, 2H, Aromatic), 7.19 (d, J = 8.7 Hz,
2H, Aromatic). 13C NMR (100 MHz, CDCl3) d: 35.38,
55.7-55.917 (3C), 60.89, 105.33(2C), 115.03(2C),
119.0, 121.9, 127.1, 128.8 (2C), 132.65, 139.1,
152.7, 153 (2C), 154.7, 160.5.
Methyl2-((4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)
acetate (5j)
Brown solid, yield 83%, FT-IR: 3069-2935
(C-H, aromatic), 2961 (C-H, aliphatic), 1735 (-C=O),
1
1610 (C=N), 1587 (C=C); H NMR (400 MHz,
CDCl3) d: 3.6 (s, 6H, 2 x OCH3), 3.77 (s, 3H, OCH3,
ester), 3.82 (s, 3H, OCH3), 3. 86 (s, 3H, OCH3), 4.15
s, 2H,-SCH2COOCH3, 6.69 (s, 2H, Aromatic), 7.02 (d,
J = 6.9 Hz, 2H, Aromatic), 7.22 (d, J = 7,2 Hz, 2H,
Aromatic); 13C NMR (100 MHz, CDCl3) d: 34.2, 52.9,
55.7- 56.9(3C), 60.9, 105.32 (2C), 115.22(2C), 121.5,
126.6, 128.7(2C), 139.2, 151.4, 153.8 (2C), 154.9,
160.7, 168.8 (C=O).
3-(Isopropylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5g)
White solid, yield 83%; FT-IR: 2999 (C-H,
aromatic), 2961 (C-H, aliphatic), 1607 (C=N), 1586
1
(C=C); H NMR (400 MHz, CDCl3) d: 1.4 (dd, J =
6.7 Hz, 6H, CH(CH3)2), 3.6 (s, 6H, 2 x OCH3), 3.82
(s, 3H, OCH3), 3. 86 (s, 3H, OCH3), 3.93 (sep, J = 6.6
Hz, 1H, CH(CH3)2, 6.69 (s, 2H, Aromatic), 7.0 (d, J= 8.9
Hz, 2H, Aromatic), 7.16 (d, J = 8.8 Hz, 2H, Aromatic).
13C NMR (100 MHz, CDCl3) d: 23.5 (2C), 38.6, 55.68-
55.9(3C), 60.8, 105.3(2C), 115.03(2C), 121.9, 127.2,
128.9 (2C), 139.1, 152.3, 153 (2C), 154.4, 160.4.
Culturing of SW480 and MDCk Cell Lines
The stock human colon cancer (SW480)
and Madin-Darby Canine Kidney (MDCK) cell lines
were obtained from the Cancer Research lab. and
Cell Culture at the College of Medicine, University
of Babylon. The cells were grown as monolayer
and maintained as an exponentially growth phase
in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% heat inactivated fetal
bovine serum (FBS) and 1% penicillin-streptomycin
(GIBCO®) in tissue culture flask and incubated at
37oC in humidified atmosphere containing 5% CO2.
The cells were maintained by the replacement of
fresh medium.
3-(Cyclohexylthio)-4-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4H-1,2,4-triazole (5h)
White solid, yield 81%; FT-IR: 2936 (C-H,
aromatic), 2848 (C-H, aliphatic), 1606 (C=N), 1589
1
(C=C); H NMR (400 MHz, CDCl3) d: 1.2- 2.13
(m, 11H, cyclohexyl), 3.63 (s, 6H, 2 x OCH3), 3.81 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 6.68(s, 2H, Aromatic),
7.0 (d, J = 7.7 Hz, 2H, Aromatic), 7.17 (d, J = 7.9 Hz,
2H, Aromatic). 13C NMR (100 MHz, CDCl3) d: 25.57,
25.8, 33.4, 33.47, 46.48, 55.68, 55.8-55.9 (3C), 60.89,
105.33(2C), 115.0 (2C), 121.9, 127.1, 128.8 (2C),
139.1, 152.6, 152.9 (2C), 154.4, 160.4.
Anticancer activity
The test compounds were dissolved in
acetone at concentration of 10 mM and diluted
with DMEM to get the desired concentrations for
the treatments of cell lines. Mixture of acetone:
DMEM (200 µl, with the same ratios used in the test
samples) was added to the negative control samples,
the added volumes of acetone have no any effect
on the cell growth when compared with the positive
control samples, accordingly a normalized control
was depended in the study.
4-(4-Methoxyphenyl)-3-((oxiran-2-ylmethyl)thio)-
5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole (5i)
light yellow solid, yield 79%; FT-IR: 2936
(C-H, aromatic), 2844 (C-H, aliphatic), 1601 (C=N),
1585 (C=C); 1H NMR (400 MHz, DMSO-d6) d: 3.57
(s, 6H, 2 x OCH3), 3.65 (s, 3H, OCH3), 3. 79 (s,
3H, OCH3), 3.8 (q, J = 6.5 Hz, 2H, CH2 ring), 3.9
(q, J = 5.2 Hz, 1H, CH ring), 4.16 (q, J = 5.3 Hz, 2H,
SCH2-), 6.57 (s, 2H, Aromatic), 7.08 (d, J = 8.8 Hz,
Overnight cultures of the cells grown
in 25 cm2 tissue culture flask were examined
under inverted microscope. Cells were detached
using trypsin, washed twice by adding 5 ml of

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PBS and centrifugation at 1500 rpm for 5 minutes.
Statistical Analysis
These exponentially growing cells were seeded in
96-well plates (5000 cells/well) and incubated at
37°C for 24 h for attachment. Then, the media was
removed and 200 µl of fresh medium containing
the test compound at different concentrations were
added to each well in four replicates and incubated at
37°Cfor48hours.Thewellswerethenwashedbyadding
200 µl of PBS for each well, left for 5 min and removed
by sterile pasture pipet. This process was repeated
twice. The wells were treated with 100 µl of crystal
violate solution for 10 minutes.The dye was discarded
and the plates were washed with distilled water three
times.The plates were left to dry at room temperature,
thentheabsorbancewasmeasuredat570nmbyusing
plate reader (Human). The inhibition percentage was
calculated using the following formula:27, 3
Data were processed using SigmaPlot
version 12.0 software. a Student's t-test was used
to determine the statistical significance for unpaired
observations.The P values less than 0.01 considered
as statistically significant.
RESULTS AND DISCUSSION
Synthesis
The target compounds (4, 5a-j) were
synthesized via three steps synthetic route (Scheme
1). The starting materials have been chosen to
get the structural requirements that make the new
compounds as CA-4 analogues. The structure
of compound 4 is the simplest in the series, it is
considered as the precursor for the synthesis of
compounds 5a-j, the modification of the structure
is done by the attaching the aliphatic groups to
the –S atom (-S linker). In the first step, 3,4,5
trimethoxybenzohydrazide (1) was reacted with
4-methoxyphenyl isothiocyanate (2) to give the
intermediate (3) which was isolated and then treated
with NaOH to achieve the intramolecular cyclization
giving the 1,2,4-triazole derivative 4. In the last step
the hydrogen atom of the –SH group of compound
4 was substituted by different aliphatic substituents
upon treatment with the appropriate alkyl halide
in basic medium. Relatively good isolated yields
(78 - 84 %) were obtained after purification by column
chromatography.
%inhibition=[(Abs control-Abs samples)/(Abs control )]*100
Determination of IC50 Value
The IC50 of 5a was determined by
treating the cancer cell line (SW480) with different
concentrations of 5a to find out the concentration
that results a 50% cell growth inhibition. The IC50
value was calculated by using SigmaPlot version
12 software.
Antioxidant activity
The DPPH radical scavenging protocol was
applied to assess the scavenging activity of synthetized
compounds. Solutions of the test compounds (0.005
M in DMSO) were prepared, then, 50 µM solutions
were prepared by dilution with methanol.Ascorbic acid
(50 µM) was also prepared in the same manner. In
test tube, 3 ml of the test compound were mixed with
50 µl of DPPH (0.004 M in methanol), the mixtures
were incubated in dark place at 25°C for 30 min
ascorbic acid was used as standard antioxidant. The
absorbance was recorded at 518 nm.The percentage
of scavenging activity of the test compounds was
calculated according to the following formula:
O
NCS
O
H
N
H
N
MeO
MeO
NH2
MeO
MeO
N
H
N
H
+
S
OMe
OMe
1
OMe
2
OMe
3
5a
R = -CH3
5b R = -CH2CH3
5c R = -CH2(CH2)2CH3
5d R = -CH2(CH2)4CH3
R = -CH2-CH-(CH3)2
5f R = -CH2-CH=CH2
5g R = -CH(CH3)2
5h R = -cyclohexyl
N
N
N
N
MeO
MeO
R
5e
S
SH
N
N
MeO
MeO
MeO
MeO
O
5i R =
O
OMe
OMe
5a-j
4
O
5j
R =
% Scavenging of DPPH= [(Ac-As)/Ac]X 100
Scheme 1. Synthesis of 4, 5a-j
Where Ac is the absorbance of control
(no sample or standard added to the DPPH solution)
and As is the absorbance of the test.The tests were
done in triplicate.
Anticancer Activity
To evaluate the anticancer activity of
compound 4 and study the effect of structure
modification on the activity of compounds 5a-j,

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BAlAKIT et al., Orient. J. Chem., Vol. 35(1), 77-84 (2019)
human colon cancer cell lines (SW480) were treated
with two different concentrations (10 µM and 50 µM) of
compounds4,5a-jfor48hthecellgrowthinhibitionwas
determined using crystal violet cytotoxicity assay, Fig.1
shows the effect of the compound on the cells viability.
The cell growth inhibition was calculated,Table 2 shows
the obtained results, treatment of the cancer cell lines
(SW480) with compound 4 which has free –SH group
caused 6 and 10% cell growth inhibition at 10µM and
50µM respectively. Substitution of the H atom of
the –SH group by methyl (compound 5a) caused a
significant increase in the cytotoxic activity where the
values of cell growth inhibition were found to be 50.4
and 77.4% at 10µM and 50µM respectively, on the
other hand, the replacement of the H atom with primary
aliphatic straight chains (n-ethyl, n-butyl and n-hexyl
substituentsincompounds5b, 5cand5drespectively)
caused moderate improvement in the cytotoxic activity,
within the results related to compounds 5a-d it is
clear that increasing the chain length decreases the
activity, while no significant change in the cytotoxicity
levels was observed with the other compounds where
branched, olefinic, cyclic or ester groups are present as
S-substituents in the structures of compounds 5e-j.
The results indicated that 5a is the most
potent cytotoxic agent in the series, accordingly the
next step was to evaluate the IC50 of compound 5a
against the same cell line, the results showed that
10µM is the required concentration to cause 50%
cancer cell lines (SW480) growth inhibition after
incubation for 48 hours. Furthermore, to test the
cytotoxicity of 5a against normal cells, MDCK normal
cell lines were treated with 5a at 10 µM concentration
(IC50) for 48 h only 20% cell growth inhibition was
observed, see Fig. 1 and Table 2.
100
SW480+10 uM
SW480+50 uM
MDCK+10 uM
80
60
40
20
0
Antioxidant Activity
The synthesized compounds (4, 5a-j) were
subjected to DPPH assay to evaluate their free
radical scavenging activity (antioxidant activity).The
obtained results are shown in Table 3, compound 4
exhibited the highest antioxidant activity (71%) when
compared with ascorbic acid (80% as the reference
antioxidant), this could be attributed to the availability
of free –SH group that makes the compound capable
to accommodate the free radical and stabilize it by
delocalization. Compounds 5e, 5f, 5i and 5j showed
low to moderate antioxidant activity while the others
(5a-d, 5g and 5h) did not show any level of free
radical scavenging activity.
5
5
j
5
4
5
5
5
5
f
i
5
h
c
a
b
d
5
5
g
e
Compounds
Fig. 1. Normalized cells viability percentage of SW480
cancer cell line and MDCk normal cell line at two different
concentrations after incubation for 48 hours
Table 2: Cytotoxicity levels of 4, 5a-j against cancer and normal cells at
different concentrations for 48 hours
Compound
Percentage of cell
growth inhibition for SW480
IC50( µM) Percentage of cell growth inhibition
for MDCK normal cells at IC50
50 µM
10 µM
4
10
77.4
61.1
52.2
36.3
13
22
11
25
13
6
50.4
26.2
18
16
3
6.5
1
3
--
10
--
--
--
--
--
--
--
--
--
--
20
--
--
--
--
--
--
--
--
--
5a
5b
5c
5d
5e
5f
5g
5h
5i
3.4
3.7
5j
20

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BAlAKIT et al., Orient. J. Chem., Vol. 35(1), 77-84 (2019)
Table 3: Results of DPPH assay for
antioxidant activity
of 4, 5a-j was evaluated against colon cancer
line (SW480), compound 5a was found to be the
most potent one and showed a kind of selectivity
to cancer cells when subjected to evaluating its
cytotoxicity against MDCK normal cell lines at
10µM concentration (IC50). The results revealed 5a
is a promising anticancer agent to be subjected for
further studies.The antioxidant activity of 4, 5a-j was
studied, the order of the activities was found to be
4, 5i, 5e, 5j, 5f.
Compound
% Scavenging of DPPH
free radical at 50 µM*
ascorbic acid (standard)
80
71
24
10
45
18
4
5e
5f
5i
5j
*Compounds 5a-d, 5g and 5h did not show any antioxidant
activity.
ACkNOWLEDGMENT
CONCLUSION
The authors thank Al-Qasim Green
University, Al-Mustansiyriah University and the
University of Babylon for the financial support.
Series of 3-mercapto-1,2,4-triazole
derivatives have been designed as combretastatin
A-4 analogues with different aliphatic substituents
attached to the S atom (4, 5a-j). The target
compounds were synthesized and characterized by
different spectroscopic techniques. The cytotoxicity
Conflict of Interests
The authors declare that there is no conflict
of interests regarding the publication of this paper.
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