Development of 1,3,4-oxadiazole thione based novel anticancer agents: Design, synthesis and in-vitro studies

Article abstract of DOI:10.1016/j.biopha.2017.08.110

A series of new 1,3,4-oxadiazole-2(3H)-thione analogues (3a to 3o) have been designed, synthesized and evaluated for their anticancer activity. Four different cancerous cell lines viz. HeLa (cervical), U-87 (glioblastoma), Panc (pancreatic) and MCF-7 (breast) were used to assess the potency of the synthesized compounds as anticancer agents. Among them 3i and 3j showed promising cytotoxicity against HeLa cell line. Further, 3i and 3j successfully inhibited cell cycle progression and displayed cell death in HeLa cells via apoptosis as visualized by Annexin V APC and DNA fragmentation assay. 3i and 3j induced caspase-3 activation, PARP cleavage, increase in expression of proapoptotic protein Bax and decrease in the expression of antiapoptotic protein Bcl-2. Also, 3i and 3j induced overexpression of p21 and decreased expression of cyclin B1 indicating the arrest of cells in G₂-M phase of the cell cycle. Therefore, new lead compounds are being suggested having anticancer activity through cell cycle inhibition and apoptosis.

Full text of DOI:10.1016/j.biopha.2017.08.110

Biomedicine&Pharmacotherapy95(2017)721–730
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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Original article
Development of 1,3,4-oxadiazole thione based novel anticancer agents:
Design, synthesis and in-vitro studies
Nalini Yadava, Parveen Kumarb, Aruna Chhikarab, Madhu Chopraa,⁎
a
Laboratory of Anti-cancer Drug Development, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
Department of Chemistry, Dyal Singh College, University of Delhi, New Delhi 110003, India
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oxadiazole thiones
Cytotoxicity
G2-M phase arrest
Apoptosis
A series of new 1,3,4-oxadiazole-2(3H)-thione analogues (3a to 3o) have been designed, synthesized and
evaluated for their anticancer activity. Four different cancerous cell lines viz. HeLa (cervical), U-87 (glio-
blastoma), Panc (pancreatic) and MCF-7 (breast) were used to assess the potency of the synthesized compounds
as anticancer agents. Among them 3i and 3j showed promising cytotoxicity against HeLa cell line. Further, 3i
and 3j successfully inhibited cell cycle progression and displayed cell death in HeLa cells via apoptosis as vi-
sualized by Annexin V APC and DNA fragmentation assay. 3i and 3j induced caspase-3 activation, PARP clea-
vage, increase in expression of proapoptotic protein Bax and decrease in the expression of antiapoptotic protein
Bcl-2. Also, 3i and 3j induced overexpression of p21 and decreased expression of cyclin B1 indicating the arrest
of cells in G2-M phase of the cell cycle. Therefore, new lead compounds are being suggested having anticancer
activity through cell cycle inhibition and apoptosis.
1. Introduction
excellent anticancer potency and lower side effects. [6,7]
Oxadiazole are an important class of heterocyclic aromatic com-
Cancer continues to be major health issue in most parts of the world
and is the prominent cause of death world-wide [1]. According to
World Health Organization, cancer is the second leading cause of death
globally and was responsible for 8.8 million deaths in 2015. Around
70% of deaths from cancer occur in low-income and underdeveloped/
developing countries [2]. According to a report from National Cancer
Institute, estimated number of cancer deaths in children and teenagers
was 1960 in 2014 whereas 15,780 of them were diagnosed with cancer.
An individual’s risk of developing cancer at any point in his life-time
depends on various factors viz. age, genetics, and exposure to risk fac-
tors which include some potentially avoidable lifestyle habits [3] such
as smoking, alcohol, improper and unhealthy diet, insufficient physical
activity, obesity, and certain infections which account for a high per-
centage of cancer cases all over the world [4].
Cancer treatment has been the primary goal of pharmaceutical in-
dustries and R & D wings in research centers over the last many decades
[5]. Among the various treatment regimens available today, che-
motherapy is most commonly used for treating various kinds of cancers
worldwide. Serious side effects of the present drug entities, drug re-
sistance and failure of anti-tumor drugs are the main obstacles in suc-
cessful development of anticancer drugs. Therefore, there is a crucial
need for the development of molecules which are endowed with
pounds with broad spectrum of biological activities such as anti-
bacterial [8], antimycobacterial [9], antifungal [10], anti-HIV [11],
anti-inflammatory and analgesic [12–14], hypoglycemic [15], antic-
onvulsant [16] and anticancer properties [17,18] besides other biolo-
gical properties such as genotoxic [19] and lipid peroxidation in-
hibitory activities [20]. 1,3,4-oxadiazole is commonly used moiety for
pharmacophore development and has been thoroughly investigated
because of its good metabolic profile and hydrogen bonding ability
within the receptor site. 1,3,4-oxadiazoles are extremely good bioisos-
teres of esters and amides, and the presence of azole (eN]CeO) group
increases the lipophilicity which facilitates its transportation through
cell membranes to reach the target site and show various biological
activities [21]. Besides these activities, oxadiazoles (Fig. 1) have also
been seen to display excellent anticancer activities in various in vitro
and in vivo models. For example, one of the 2, 3-dihydro-1,3,4-ox-
adiazole-2-thione derivatives, compound A having oxadiazole thione
nucleus attached with the phenyl ring showed 73.9% of tumor inhibi-
tion on EAC animal models [22]. Another 1,3,4-oxadiazole derivative
with an amide group, B having oxadiazole moiety showed potent an-
ticancer activity on 4 cancerous cell lines HepG2 (12.4 μg/mL), WI-38
(17.3 μg/mL), VERO (15.8 μg/mL) and MCF-7 (25.80 μg/mL) [23]. In
addition one more 3-(1,3,4-oxadiazol) containing compound C having
⁎
Corresponding author.
E-mail addresses: mchopradu@gmail.com, mchopra@acbr.du.ac.in (M. Chopra).
http://dx.doi.org/10.1016/j.biopha.2017.08.110
Received 26 July 2017; Received in revised form 21 August 2017; Accepted 24 August 2017
0753-3322/©2017ElsevierMassonSAS.Allrightsreserved.

N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
Fig. 1. Design strategy of new derivatives of ox-
adiazole thiones.
oxadiazole moeity showed excellent antiproliferative effect against full
panel of 60 cell lines [24,25]. One of the novel 5-(30-indolyl)-2-(sub-
stituted)-1,3,4-oxadiazole containing 4-pyridyl as the −R group sub-
stitution, D showed excellent cytotoxicity activity against PC3 prostatic
cancer cell line [17]. In another series of 2-aminomethyl-5-(quinolin-2-
yl)-1,3,4-oxadiazole-2(3H)-thione quinolone derivatives, one of the
analogues containing (4-Chlorophenyl) amino moiety E displayed very
good anticancer activity against HepG2 cell line [26].
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity
All the newly synthesized compounds 3a to 3o were evaluated for
their antiproliferative activity on 4 cancerous cell lines viz HeLa (cer-
vical), U-87 (glioblastoma), Panc (pancreatic) and MCF-7 (breast) using
MTT growth inhibition assay [27]. Doxorubicin (DOX) was used as
positive control. The results are shown in Table 1.
During an ongoing programme to develop new, potent and less toxic
anticancer compounds and inspired with the diverse biological prop-
erties of oxadiazoles and their role in anticancer drug development, we
synthesized a series of new 1,3,4-oxadiazole thione derivatives as po-
tent anticancer agents. The 1,3,4-oxadiazole thione core pharmaco-
phore was extended by linking substituted phenyl ring via methylene to
study the effect of ‘R’ group in generating anticancer activity.
Synthesized compounds were studied for their cytotoxicity and
Structural Activity Relationship (SAR) on 4 cancerous cell lines viz.
HeLa, MCF-7, U87 and Panc. The possible mechanism of cytotoxicity
action of the most active compounds was further studied through via-
bility staining assays, DNA fragmentation, cell cycle and western blot
analysis of key proteins involved in apoptotic pathways as well as cell
cycle signaling pathways.
As exhibited in Table 1, compounds 3i and 3j bearing nitro and
fluoro at the para position of benzene ring respectively, displayed
highest activity against HeLa cervical cancer cell line with a half
maximal (50%) inhibitory concentration (IC50) value of 7.92 μM and
7.37 μM, respectively after 48 h. 3i and 3j showed better anti-
proliferative activity as compared to earlier reported 5-(Dec-9-enyl)-
(3H)-1,3,4-oxadiazol-2-thione and 3-(4-[5-Mercapto-1,3,4-oxadiazole-
2-yl] phenylimino)-5-bromo-indolin-2-one which showed IC50 value of
11.90
1.3 μM and 10.64 μM respectively on HeLa cells [28,29].
DOX showed an IC50 value of 1.25 μM against HeLa cell line. SAR
studies revealed substitution of methyl groups at the para position (3a)
or both at the meta and para positions (3b) did not increase the potency
of the compound and showed 50% inhibitory activity above 27 μM for
HeLa, U87 and Panc, owing to electron donating nature of methyl
groups. Iodo group at para position (3c) displayed good activity on
Panc (21.91
and HeLa. Similarly, bromo substitution at the para position (3d) dis-
played good activity against HeLa, Panc and U87 (18.08
in comparison to its substitution at the ortho position (3e) which dis-
played less activity (> 28 μM) against all the cell lines. Substitution of
the chloro group at the para position (3f) displayed satisfactory activity
1.41 μM), whereas it was slightly less active for U87
2. Results and discussion
1.32 μM)
2.1. Chemistry
In the present study, fifteen new mannich bases of 1,3,4-oxadiazole
thiones have been designed and synthesized in order to screen for their
anticancer activity. The synthesis of compounds 3a to 3o followed the
general synthetic pathway outlined in Scheme 1. Firstly, 4-(tert-butyl)
benzohydrazide was reacted with carbon disulphide in the presence of
ethanol at 0 °C for 20 min and then refluxed for 4 h until the evolution
of H2S ceased. Retrieved intermediate 5-(4-tert-butylphenyl)-1,3,4-ox-
adiazole-2(3H)-thione was purified by column chromatography and
further reacted with different amines in the presence of formaldehyde
in anhydrous ethanol at room temperature to yield mannich bases of
1,3,4-oxadiazole thiones 3a to 3o which were recrystallized from an-
hydrous ethanol and characterized with 1H NMR, 13C NMR, IR and
HRMS.
against HeLa (23.44
1.20 μM), U87 (22.44
1.25 μM) and Panc
(22.80
2.58 μM) while its dual substitution at ortho and meta po-
sitions (3g) lead to reduction in its cytotoxic activity (> 25 μM) on all
the treated cell lines. Ortho nitro substitution (3h) was ineffective until
a dose of 50 μM on all the tested cell lines, while its para counterpart
(3i) showed very impressive activity against HeLa (7.92
and quite good activity against U87 (17.78
(20.83
0.57 μM)
1.92 μM) and Panc
1.15 μM), therefore suggesting that electronegative group is
favoured at p-position. Substitution of fluoro group at the ortho position
(3k) displayed less growth inhibitory activity (> 26 μM) against all the
four cell lines whereas its substitution on para position (3j) showed
remarkable growth inhibitory activity against HeLa (7.37
and good activity (< 20 μM) against U-87 (19.6 1.82 μM) and Panc
(12.17 2.47 μM). Above study concludes that halogen or nitro group
0.50 μM),
722

N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
Scheme 1. General scheme for the synth-
esis of compounds 3a-3o. Reagents and
Conditions: (A) CS2, KOH, ethanol, 0 °C,
4 h. (B) Amines, HCHO 40%, RT, 4–6 h.
substitution at the para position of benzene ring increases its activity
which might be because of inductive effect of halogens which deacti-
vates the ring. Substitution with carboxylic group at the para position
Table 1 and we have also calculated the selectivity index of most active
compounds and DOX in Table 2. The degree of selectivity (selectivity
index, SI) of the most active compounds (3i and 3j) and DOX has been
presented as the ratio of IC50 of the compounds on the normal cell line
vs IC50 of same compounds on cancerous cell lines. A SI value inferior to
2 may indicate a general toxicity of an extract or a pure compound [30].
In our study we found SI superior to 2 (Table 2) for 3i (SI = 14.53 for
HeLa), 3j (SI = 15.57 for HeLa) and DOX (SI = 62.28 for HeLa) on all
the tested cell lines which suggested that the compounds exhibited
selectivity against cancer cell lines.
(3l) showed satisfactory activity only against Panc (25.64
1.95 μM)
and was inactive against all other cell lines tested which could be due to
highly polar nature of this compound. Substitution of ethoxy group
(3m) in the benzene ring also did not increase its potency and IC50
obtained for this compound was more than 30 μM because of electron
donating nature of lone pairs present on the oxygen of ethoxy group. In
last two compounds phenyl groups were replaced with deactivating
groups pyridine (3n) and pyrimidine (3o) rings respectively. The pre-
sence of second nitrogen is advantageous in pyrimidine containing
2.2.2. Cellular morphological study
compound 3o (IC50 13.50
ison to pyridine containing compound 3n (IC50 32.05
2.17 μM) against HeLa cells in compar-
Based on the promising results of 3i and 3j, these compounds were
further tested for their mechanism of apoptotic activity on HeLa cells.
Apoptosis is an active, ATP-dependent process and is characterized by
hallmarks such as membrane blebbing, nuclear (essentially, chromatin)
condensation, cell shrinkage, internucleosomal DNA fragmentation and
protein cleavage [31]. To determine the effect of these compounds on
morphology of cells, HeLa cells were treated with the IC50 concentra-
tions of 3i and 3j for 24 and 48 h and the cells were observed under a
phase contrast microscope. The control cells showed regular polygonal
or oval shape. Cells underwent remarkable morphological changes such
as shrinking, showed membrane blebbing in addition to a few round
and floating cells, along with few elongated cells (Fig. S1 in Supple-
mentary file). The number of viable HeLa cells was significantly
2.92 μM).
Overall, MTT results indicate that polar electronegative groups at para
position increase the cytotoxicity except in case for carboxylic group
which is highly polar. All the oxadiazole thione derivatives reported in
this work have been tested on U87 MG (Glioma) and Panc cell lines
(pancreatic) for the first time and showed good to moderate overall
activity on these cell lines also.
Moreover, all the compounds tested showed less inhibitory effect
against breast cancer cell line MCF-7 in comparison to other three cell
lines. All the compounds did not show any antiproliferative effect
against non-cancerous HEK (Human Embryonic Kidney) cells until
100 μM. The result for noncancerous cell line also has been included in
Table 1
In vitro anti-proliferative activities of the novel synthesized 1,3,4-oxadiazole thione derivatives (3a-3o)a on four human cancer cell lines and one normal cell line.
Compounds
HeLa (IC50) μM
U87 (IC50) μM
Panc (IC50) μM
MCF7 (IC50) μM
HEK (IC50) μM
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
DOX
28.95
30.51
27.00
23.60
28.29
23.44
31.70
> 50
7.92
2.01
1.35
1.09
0.86
2.00
1.20
1.99
28.40
28.36
25.83
18.08
29.47
22.44
27.86
> 50
17.78
19.6
31.18
33.31
31.63
31.76
26.20
1.58
0.95
1.52
1.08
1.32
1.51
1.25
1.34
27.17
27.27
21.91
22.21
28.83
22.80
25.92
40.35
20.83
12.17
26.10
25.64
29.91
29.10
23.70
1.828
1.31
2.02
1.41
1.25
0.63
2.58
1.21
2.08
1.15
2.47
2.93
1.95
3.08
2.07
2.42
0.31
41.76
31.16
39.36
40.79
37.79
40.07
35.27
> 50
35.23
43.08
46.76
45.85
> 50
41.71
31.35
1.36
1.32
2.55
1.77
3.11
2.28
2.27
2.36
185.5
171.7
147.4
137.1
147.2
162
152.7
> 200
115.1
114.8
158.5
168.2
169.9
145.2
140
0.69
0.88
0.87
0.40
0.86
0.46
0.89
0.57
0.50
4.19
3.08
1.79
2.92
2.17
1.92
1.82
2.22
2.33
1.27
2.45
0.68
0.75
0.73
0.75
0.77
0.55
7.37
27.61
31.08
33.17
32.05
13.50
1.25
2.04
2.02
1.39
2.54
2.09
2.73
1.49
0.31
0.63
0.46
0.39
0.29
77.86
a
The data represented the mean of three experiments in triplicate and were expressed as mean
cells was observed. Doxorubicin was used as positive control.
SD. The IC50 value was represented as the concentration at which 50% survival of
723

N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
Table 2
Selectivity index, SI (IC50 of a compound on normal cell line/IC50 of the same compound on cancerous cell lines) calculated for 3i, 3j and DOX on four human cancerous cell lines.
IC50 (HEK) (μM) IC50 (HeLa) (μM) IC50 (U87) (μM) IC50 (Panc) (μM) IC50 (MCF7) (μM)
3i
SI (3i)
3j
SI (3j)
DOX
SI (DOX)
115.1
114.8
77.86
0.68
0.75
0.46
7.92
14.53
7.37
15.57
1.25
62.28
0.57
0.50
0.39
17.78
6.47
19.6
5.85
1.58
49.27
1.92
1.82
0.29
20.83
5.52
12.17
9.43
1.82
42.78
1.15
2.47
35.23
3.26
43.08
2.66
1.36
57.25
2.22
2.33
0.31
0.31
reduced after 24 and 48 h of treatment with 3i and 3j as compared to
untreated cells. Cellular debris was seen in plates treated with 3i, 3j and
DOX after 48 h of treatment. These morphological changes in HeLa
cells after treatment with 3i and 3j clearly showed that cells had un-
dergone stress, stopped multiplying and were showing signs of apop-
totic cell death which was more prominent after 48 h of treatment.
condensed or fragmented chromatin and were stained yellowish green
or orange.
After 48 h, most of the cells treated with 3i, 3j and DOX stained
orange or red with fragmented chromatin showing the cells in late
apoptotic phase in comparison to untreated cells where a very few
number of cells stained red. The staining results with AO/EB showed a
time dependent increase in the number of cells showing red stained
chromatin at their one end which is a hallmark of apoptosis and few of
the cells stained totally red, showing necrotic mode of cell death.
2.2.3. Cell migration study
Migration of cancer cells is a fundamental step in tumor progression
and metastasis, and the extent of migration ability depends upon the
metastatic potential of cancer cells [32]. Migration was determined by
enumerating total cells intruding the wound area in control and treated
dishes after 24 and 48 h. Photographs were captured at different time
intervals i.e. 0, 24 and 48 h. As shown in Fig. S2A in Supplementary
file, the number of invasive cervical cancer cells penetrating the wound
area was significantly reduced after treatment with 3i, 3j and DOX as
compared to the untreated control group. The number of cells migrated
to the wound induced region have been shown in graphical form for
each compound in Fig. S2B in Supplementary file. Results of cell mi-
gration assay evidently indicate that treatment with 3i and 3j might
suppress the migration of HeLa cells.
2.2.4.2. Annexin-V (APC)/propidium iodide dual staining assay. The
apoptotic effect of 3i and 3j was further checked by Annexin V (AV)/
Propidium iodide (PI) dual staining assay [35]. AV/PI staining helps to
examine the occurrence of phosphatidylserine externalization and
facilitates the detection of live cells. Our results after staining the
treated cells with AV/PI showed that, the percentage of total apoptotic
cells significantly increased after 24 and 48 h when treated with these
compounds (Fig. 3). The percentage of cells in late apoptotic phase was
found to be 11.98%, 6.35% and 12.76% in case of treatment with 3i, 3j
and DOX respectively as compared to 0.53% in untreated control cells
after 24 h treatment. The percentage apoptotic cells increased to
52.19%, 51.43% and 57.58% in 3i, 3j and DOX treated cells
respectively as compared to 0.94% cells in untreated control cells
after 48 h (Table 3). These results confirm a time dependent apoptosis
inducing activity of 3i and 3j in cervical cancer cells.
2.2.4. Apoptotic studies
2.2.4.1. Acridine
orange/ethidium
bromide
(AO/EB)
staining
assay. Viability stains determine the membrane integrity of a cell
based on the uptake or exclusion of a dye from the cell [33]. The
ethidium bromide/acridine orange stain (EB/AO stain) is a viability
stain that detects apoptotic cells. Ethidium bromide is a dye that is only
able to pass through the membrane of a dead or dying cell and makes
the cell fluoresce red. Acridine orange is membrane-permeable dye that
will stain all cells in the sample and makes the cell fluoresce green [34].
HeLa cells were labeled with AO/EB at 24 and 48 h after treatment with
3i, 3j and DOX and were examined under a fluorescent microscope.
Fig. 2 illustrates that after 24 h of treatment, most untreated cells were
showing pale green chromatin with organized nuclear structure
whereas in 3i, 3j and DOX treated cells, a few cells were showing
2.2.4.3. DNA fragmentation analysis. In apoptotic cells, activated
nuclease enzymes cleave the intact DNA in to shorter
mononucleosomal fragments. Fragmentation of DNA results in
a
characteristic DNA ladder like pattern on agarose gel electrophoresis
[36]. Thus, DNA fragmentation assay was carried out to further confirm
the apoptosis inducing effect of these compounds on HeLa cells. A
typical DNA smeared ladder pattern was observed in 3i, 3j and DOX
treated lanes. In contrast, there was little or no degradation of DNA
from control cells even after 48 h further confirming the apoptotic
mode of cell death induced by compounds 3i and 3j in HeLa cells
Fig. 2. Acridine Orange/Ethidium Bromide Dual
Staining Assay. HeLa cells were treated with IC50
concentration of 3i, 3j and DOX and stained with
AO/EtBr after 24 and 48 h and images were captured
with a fluorescence microscope. Blue colored arrows
indicate apoptotic cells.
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N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
Fig. 3. A and B. Flow cytometric quadrant dot plot of apoptotic HeLa cells after 24 and 48 h treatment respectively with 3i, 3j and DOX. Apoptosis of HeLa cancer cells as detected by
Annexin-V/PI dual staining method.
(Fig. 4A).
and 3j treated cells (Fig. 4B). Expression levels of pro-apoptotic protein
Bax increased significantly in the treated lanes. The overall bax/bcl-2
ratio was getting elevated which is critical for the induction of
apoptosis and determines whether cells will undergo apoptosis [41,42].
2.2.4.4. Western blot analysis. To further elucidate the detailed
mechanism of cell death caused by these compounds on HeLa cells
the expression levels of caspase 3, PARP, Bax and Bcl-2 which are the
key regulators of apoptosis were determined. Induction of apoptosis is
accompanied by the activation of several caspases within the same cell
[37]. Caspase-3 is an executioner caspase which is activated by both
extrinsic (death ligand) or intrinsic (mitochondrial) pathways and is
involved in the mass proteolysis which ultimately leads to apoptosis
[38]. HeLa cells were treated with DMSO, 3i and 3j and the protein was
extracted. Our findings revealed that, treated cells showed a decrease in
the expression levels of procaspase 3 as compared to control cells and
showed expression of cleaved caspase-3 which was absent in control
cells indicating that caspase 3 was getting activated after treatment
with 3i and 3j (Fig. 4B). Caspase-3 is required for the cleavage of ICAD/
DFF-45 (inhibitor of caspase-activated DNase) and leads to apoptotic
chromatin condensation and DNA fragmentation which concludes in
apoptotic cell death [39]. Activated caspase-3 further leads to cleavage
of PARP which is an important phenomenon of apoptosis as it prevents
the depletion of NAD and ATP which are needed for the later stages of
apoptotic process [40]. Presence of cleaved fragment of PARP
corresponding to 89 kDa was seen in 3i and 3j treated cells whereas
there was no expression of cleaved fragment of PARP in untreated cells
(Fig. 4B). These results indicate that 3i and 3j lead to cell death in HeLa
by caspase dependent apoptotic pathway which further leads to DNA
fragmentation, chromatin condensation and PARP cleavage. One of the
most widely studied negative regulators of apoptosis is the proto-
oncogene Bcl-2 whose expression was significantly downregulated in 3i
2.2.5. Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect
either by arresting the cell cycle or by induction of apoptosis or a
combined effect of both cell cycle arrest and apoptosis [43]. To eluci-
date the anti-apoptotic mechanism of 3i and 3j further, compounds
were analyzed to anticipate the phase of cell cycle at which the cells
were being arrested. The treatment of HeLa cells with IC50 dose of 3i, 3j
and DOX resulted in a decrease in the percentage of cells in the G1 and S
phase and a significant increase in the percentage of cells in G2 phase.
As summarized in table in Fig. 4, the percentage of cells in G1 phase in
untreated cells was 59.30% which reduced to 44.91% in 3i treated
cells, 48.21% in 3j treated cells and to 7.01% in DOX treated cells. The
population of cells significantly increased in G2-M phase i.e. as com-
pared to 20.74% cells in G2-M phase in untreated control cells, there
were approximately 37.25%, 37.87% and 69.66% cells in 3i, 3j and
DOX treated cells, respectively. These results clearly state that the
treatment considerably increased the population of cells in G2 phase
after 24 h of treatment as compared to untreated control (Fig. 4D).
2.2.6. Western blot analysis of cell cycle related proteins
Next, we aimed to check the expression of cell cycle related proteins
viz. p21 and cyclin B1 after treatment with 3i and 3j in HeLa cells. p21
is known to prevent proliferation of cells by inhibiting transition be-
tween G1/S and G2/M phase of the cell cycle as it directly inhibits the
Table 3
Quantitative apoptosis assay of HeLa cells using Annexin-V/PI dual staining method by FACS.a
Compounds
Time (h)
Viable Cells (Q1) %
Early Apoptotic Cells (Q2) %
Late Apoptotic Cells (Q3) %
Necrotic Cells (Q4) %
Apoptotic Cells (Q2 + Q3) %
Control
DOX
3i
24
24
24
24
48
48
48
48
97.55
43.30
43.09
48.88
89.45
22.79
26.39
1.19
0.44
0.01
0.53
0.12
1.48
0.51
0.39
1.43
0.81
5.63
7.62
8.22
0.16
0.97
0.13
0.85***
0.48***
0.39***
0.51
43.43
44.54
43.34
8.80
14.00
13.80
14.17
0.52***
12.76
11.98
6.35
2.46***
0.16**
0.14**
0.34ns
0.05
56.19
56.52
49.69
9.74
71.58
65.99
65.60
2.90***
1.64***
0.72***
1.16**
0.22
2.09***
3j
0.92***
1.92***
Control
DOX
3i
1.49
0.94
1.65
0.98***
0.96***
0.70**
0.95**
1.21**
57.58
52.19
51.43
0.96***
0.34***
1.97***
0.78**
0.24***
1.79***
0.91***
0.80***
2.88***
3j
26.18 0.49***
a
The percentage of viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells are presented as mean
S.D. (n = 3). ***P < 0.001, **P < 0.01 vs. Control of each
quadrant for 24 or 48 h. ns = not significant difference as compared to control.
725

N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
Fig. 4. A. Agarose gel electrophoresis of DNA extracted from HeLa cells after treatment with IC50 concentration of 3i, 3j and doxorubicin for 24 and 48 h. M: DNA ladder; L1: Control
(48 h); L2: Doxorubicin (48 h); L3: 3i (24 h); L4: 3i (48 h); L5: 3j (24 h); L6: 3j (48 h). B. Alteration in the levels of various apoptotic markers viz. Caspase-3, PARP, Bcl-2 and Bax after
treatment with IC50 concentration of 3i and 3j for 48 h. C. Change in the levels of proteins p21 and cyclin B1 involved in G2-M phase of the cell cycle after treatment with IC50
concentration of 3i and 3j for 48 h. D. Alteration in the percentage of cells in different phases of the cell cycle after treatment with 3i, 3j and DOX after a period of 24 h. Quantification of
cells in different phases of cell cycle in tabular form after treatment with 3i, 3j and DOX. Data are presented as mean value
SD. **P < 0.01 for G0 phase of treated vs. control cells;
***P < 0.001 for G1 phase of treated vs. control cells; §§§P < 0.001 for G2 phase of treated vs. control cells; ns = not significant difference for treated vs. respective controls.
cyclins and CDKs [44]. As illustrated in Fig. 4C, expression levels of p21
were getting significantly increased in the cells after treatment as
compared to control cells. Expression level of cyclin B1 almost dimin-
ished in the cells treated with 3i or 3j. Cyclin B1 is the regulatory
subunit of CDK1 (cdc2), and reduction in the expression level of cyclin
B1 can arrest cells in the G2 phase of the cell cycle and trigger cell death
by preventing the chromosomes from condensing and aligning [45].
Thus, this study clearly indicates that HeLa cells were getting arrested
in G2-M phase of the cell cycle after 3i and 3j treatment and undergoing
apoptosis through caspase dependant pathway.
phosphatidyl serine externalization, fragmentation of DNA and increase
in the bax/bcl2 ratio along with the cleavage of PARP and procaspase-3
proteins. Compounds 3i and 3j also profoundly arrested cells in G2-M
phase of the cell cycle in flow cytometry analysis after staining with
Propidium Iodide. Western blot analysis of p21 revealed that 3i and 3j
significantly increased its expression and downregulated the expression
of cyclin B1 which is evocative of the potency of 3i and 3j to arrest cells
in G2-M phase by targeting these cell cycle regulators. Thus, promising
compounds 3i and 3j may be considered as a suitable lead for further
development of anticancer drugs in future.
3. Conclusion
4. Experimental
In the present work, fifteen new 5-(4-(tert-butyl)phenyl)-1,3,4-ox-
adiazole-2(3H)-thiones have been designed and synthesized by an ef-
ficient and convenient synthetic strategy and have been evaluated for
their in vitro anticancer activity using various assays. Two compounds
3i and 3j showed best apoptotic activity on HeLa cervical cancer cells
which was better than those of earlier reported oxadiazole derivatives
in the literature on the same cell line. None of the synthesized deriva-
tives were showing cytotoxicity against Human Embryonic Kidney
(HEK) cells indicating their selectivity against tumor cells. In addition,
in our study, the mechanistic details of apoptotic activity was further
confirmed through morphological changes induced in the cells after
treatment, decrease in the migration of cells in the induced wound area,
increased staining of cells with ethidium bromide, induction of
4.1. Chemistry
All the chemicals and reagents were of analytical grade and were
purchased from TCI, Sigma-Aldrich, Spectrochem and SD Fine
Chemicals Pvt. Ltd. India, and used without further purification. The
reactions were monitored using analytical thin layer chromatography
(TLC) with silica gel 60–120 (Merck) and F254 pre-coated plates
(0.25 mm) thickness. Spot on the TLC plates were visualized using ul-
traviolet light both at short (254 nm) and long wavelength (365 nm) UV
light. 1H NMR and 13C NMR spectra were recorded on Jeol 400 MHz
nuclear magnetic resonance spectrometers. Chemical shifts are reported
in δ (TMS δ, 0.00) or with the solvent reference relative to TMS em-
ployed as the internal standard (CDCl3, δ 7.26; DMSO-d6, δ 3.33) and
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N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
multiplicities of NMR signals are designated as s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet, for unresolved lines). IR spectra
of the compounds were recorded on Perkin Elmer FTIR spectrometer
(Perkin-Elmer FT-IR Model 9 Spectrophotometer, Singapore). Melting
points were obtained on a Kofler apparatus and are uncorrected. HRMS
data were collected on Agilent 6520 Q-TOF ESI-HRMS and APCI-HRMS
instrument.
138.18, 143.87, 156.45, 159.79 176.52. IR (KBr): 3381.89, 2960.93,
2870.21, 1618.66, 1591.73, 1505.26, 1424.47, 1454.24, 1367.78,
1245.88, 1190.60, 1146.66, 1094.22, 1007.76, 815.00, 756.88, 698.77.
HRMS calculated for C19H20IN3OS [M+Na]+ 488.0270, found [M
+Na]+ 489.1379
4.1.7. 3-(((4-bromophenyl)amino)methyl)-5-(4-(tert-butyl)phenyl)-1,3,4-
oxadiazole-2(3H)-thione (3d)
4.1.1. General procedure for the synthesis of compound 5-(4-tert-
butylphenyl)-1,3,4-oxadiazole-2(3H)-thione (2)
White shiny crystalline solid, yield: 73%, mp: 166–168 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.34 (s, 9H), 5.16 (t, J = 8 Hz, 1H, NH), 5.50 (d,
J = 8 Hz, 2H), 6.81 (d, J = 12 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 7.49 (d,
J = 8 Hz, 2H), 7.81 (d, J = 8 Hz, 2H). 13C (400 MHz, CDCl3) δ: 31.16,
35.31, 58.53, 112.05, 116.05, 119.44, 126.30, 126.49, 132.34, 143.23,
To a solution of 4-(tert-butyl)benzohydrazide (1) (1.92 g, 0.01 mol)
in 25 mL of ethanol at 0 °C, carbon disulphide (2 mL) and potassium
hydroxide (0.6 g, 0.01 mol) were added and reaction was refluxed until
the evolution of H2S gas ceased (4 h). Excess solvents were evaporated
under reduced pressure and the residue obtained was dissolved in water
and then acidified with dilute hydrochloric acid (10%) to pH 5. The
precipitate obtained was filtered off, dried and purified by column
chromatography on silica gel (eluent: methanol/chloroform = 2: 98) to
give compound 2.
156.48, 159.82, 176.55. IR (KBr):
ν ) 3316.69, 2962.34,
(cm−1
2904.03, 2870.21, 1665.43, 1618.66, 1593.14, 1510.93, 1455.66,
1372.35, 1319.59, 1250.13, 1189.19, 1149.50, 1088.55, 1010.60,
817.83, 759.72, 697.35, 643.49. HRMS calculated for C19H20BrN3OS
[M+K]+ 456.1493, found [M+K]+ 456.1350.
4.1.8. 3-(((2-bromophenyl)amino)methyl)-5-(4-(tert-butyl)phenyl)-1,3,4-
oxadiazole-2(3H)-thione (3e)
4.1.2. General procedure for the synthesis of compounds (3a-3o)
To a solution of the intermediate (2.0 mmol) (2) in ethanol (20 mL),
2.0 mmol of different amines and 10 mmol of HCHO were added and
stirred at room temperature for 4–6 h. The residue obtained was filtered
and washed with cold ethanol and petroleum ether. The filtrate ob-
tained was dried and crystallized from anhydrous ethanol or acetone to
get the compounds 3a to 3o.
Off-white shiny crystalline solid, yield: 75%, mp: 109–111 °C. 1H
NMR (400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.59 (d, J = 8 Hz, 2H), 5.71 (t,
J = 8 Hz, 1H, NH), 6.67-6.71 (m, 1H, Ar),7.22 (d, J = 4 Hz, 2H,
Ar),7.43 (d, J = 8 Hz, 1H, Ar),7.49 (d, J = 8 Hz, 2H, Ar),7.82 (d,
J = 8 Hz, 2H, Ar). 13C (400 MHz, CDCl3) δ: 31.16, 35.31, 58.02,
110.74, 113.35, 119.50, 120.67, 126.28, 126.52, 128.77, 132.96,
141.53, 156.44, 159.85, 176.54. IR (KBr): 3405.99, 2962.34, 2904.23,
2871.63, 1598.81, 1510.93, 1423.06, 1408.88, 1250.13, 1149.50,
1097.06, 1019.10, 841.93, 751.21, 701.60, 659.08. HRMS calculated
for C19H20BrN3OS [M+K]+ 456.1493, found [M+K]+ 456.1458.
4.1.3. 5-(4-tert-butylphenyl)-1,3,4-oxadiazole-2(3H)-thione (2)
1H NMR (400 MHz, CDCl3) (δ): White solid, yield: 85%. 1H NMR
(400 MHz, CDCl3) (δ): 1.34 (s, 9H), 7.51 (d, J = 8 Hz, 2H), 7.865 (d,
= 12J = 12 Hz, 2H), 11.36 (s, 1H).
4.1.9. 5-(4-(tert-butyl)phenyl)-3-(((4-chlorophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3f)
4.1.4. 5-(4-(tert-butyl)phenyl)-3-((4-tolylamino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3a)
White solid, yield: 76%, mp: 181–184 °C. 1H NMR (400 MHz,
CDCl3) (δ): 1.33 (s, 9H), 5.16 (t, J = 8 Hz, 1H, NH), 5.50 (d, J = 8 Hz,
2H), 6.86 (d, J = 8 Hz, 2H), 7.17 (d, J = 8 Hz, 2H), 7.49 (d, J = 12 Hz,
2H), 7.81 (d, J = 8 Hz, 2H). 13C (400 MHz, CDCl3) δ: 31.15, 35.31,
58.67, 115.60, 119.45, 124.86, 126.49, 129.45, 142.76, 156.48,
159.81, 176.55. IR (KBr): 3318.11, 2966.60, 2902.81, 2871.63,
1620.07, 1608.75, 1598.81, 1519.14, 1496.76, 1431.56, 1374.86,
1329.51, 1264.31, 1087.13, 1034.69, 1012.01, 826.33, 762.55, 698.77,
653.41. HRMS calculated for C19H20ClN3OS [M]+ 373.1015, found
[M]+ 373.0523.
White solid, yield: 51.3%, mp: 144–146 °C. 1H NMR (400 MHz,
CDCl3) δ: 1.33 (s, 9H), 2.23 (s, 3H), 5.09 (t, J = 8 Hz, 1H, NH), 5.51 (d,
J = 8 Hz, 2H), 6.84 (d, J = 8 Hz, 2H, Ar), 7.02 (d, J = 8 Hz, 2H, Ar),
7.48 (d, J = 8 Hz, 2H, Ar), 7.80 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz,
CDCl3) δ: 20.53, 31.13, 35.26, 59.12, 114.46, 119.60, 126.20, 126.43,
129.31, 130.02, 141.63, 156.25, 159.65, 176.48. IR (KBr): 3397.48,
2963.76, 2871.63, 1618.66, 1522.27, 1424.47, 1369.20, 1328.09,
1248.72, 1148.08, 1089.97, 1012.01, 813.58, 761.13, 701.60, 650.58.
HRMS calculated for C20H23N3OS [M+Na]+ 376.1460, found [M
+Na]+ 376.0417.
4.1.10. 5-(4-(tert-butyl)phenyl)-3-(((2,3-dichlorophenyl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (3g)
4.1.5. 5-(4-(tert-butyl)phenyl)-3-(((3,4-dimethyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3b)
White shiny crystalline solid, yield: 71.3%, mp: 164–166 °C. 1H
NMR (400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.58 (d, J = 8 Hz, 2H, CH2),
5.82 (t, J = 8 Hz, 1H, NH), 6.91 (d, J = 8 Hz, 1H, Ar), 7.08-7.16 (m,
2H, Ar), 7.49 (d, J = 8 Hz, 2H, Ar), 7.81 (d, J = 8 Hz, 2H, Ar). 13C
(400 MHz, CDCl3) δ: 31.33, 35.29, 57.73, 111.14, 118.64, 119.36
120.84, 126.28, 126.51, 128 133.42 142.18, 156.53, 159.92, 176.54.
IR (KBr): 3424.41, 2962.34, 2870.21, 1618.66, 1588.89, 1505.26,
1423.06, 1369.20, 1304.00, 1255.80, 1201.94, 1116.90, 1040.36,
1012.01, 840.51, 761.13, 700.19, 659.08. HRMS calculated for
White solid, yield: 75.7%, mp: 156–158 °C. 1H NMR (400 MHz,
CDCl3) δ: 1.33 (s, 9H), 2.13 (s, 3H), 2.20 (s, 3H), 5.03 (t, J = 8 Hz, 1H,
NH), 5.51 (d, J = 8 Hz, 2H), 6.70 (d, J = 8 Hz, 1H), 6.75 (s, 1H), 6.97
(d, J = 8 Hz, 1H), 7.48 (d, J = 8 Hz, 2H), 7.80 (d, J = 8 Hz, 2H). 13C
(400 MHz, CDCl3) δ: 18.82, 20.05, 31.13, 35.25, 59.07, 111.65,
116.14, 119.65, 126.19, 126.41, 128.05, 130.48, 137.74, 141.94,
156.22, 159.64, 176.51. IR (KBr): 3431.98, 2969.13, 2370.69,
1627.02, 1525.10, 1383.88, 1272.74, 1160.12, 1118.06, 1089.66,
1042.13, 952.87, 833.58, 764.38. HRMS calculated for C21H25N3OS [M
+K]+ 406.2701, found [M+K]+ 406.2113.
C
19H19Cl2N3OS [M+Na]+ 430.0524, found [M+Na]+ 430.0589.
4.1.11. 5-(4-(tert-butyl)phenyl)-3-(((2-nitrophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3h)
4.1.6. 5-(4-(tert-butyl)phenyl)-3-(((4-iodophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3c)
Mustard yellow solid, yield: 42%, mp: 174–176 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.68 (d, J = 8 Hz, 2H, NH), 6.85 (t,
J = 8 Hz, 1H), 7.48-7.55 (m, 4H, Ar), 7.82 (d, J = 8 Hz, 2H, Ar), 8.20
(d, J = 8 Hz, 1H), 8.82 (d, J = 8 Hz, 1H, Ar). 13C (400 MHz, CDCl3) δ:
31.11, 35.29, 56.56, 115.24, 118.46, 119.24, 126.29, 126.53, 126.99,
133.92, 136.57, 142.26, 156.64, 160.10, 176.56. IR (KBr): 3381.89,
Grayish blue crystalline solid, yield: 52%, mp: 124–126 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.34 (s, 9H), 5.16 (t, J = 8 Hz, 1H, NH), 5.49 (d,
J = 8 Hz, 2H), 6.72 (d, J = 8 Hz, 2H, Ar), 7.47 (d, J = 8 Hz, 2H, Ar),
7.49 (d, J = 8 Hz, 2H, Ar), 7.81 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz,
CDCl3) δ: 31.12, 35.28, 58.31, 81.56 116.53, 126.26, 119.41, 126.46,
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Biomedicine&Pharmacotherapy95(2017)721–730
2960.93, 2870.21, 1614.40, 1577.55, 1506.68, 1423.06, 1340.85,
1244.46, 1167.93, 1095.64, 1060.20, 840.51, 748.38, 697.35, 627.90.
HRMS calculated for C19H20N4O3S [M+Na]+ 407.1154, found [M
+Na]+ 407.1151.
2873.05, 1618.66, 1515.19, 1421.64, 1367.78, 1234.54, 1146.66,
1091.39, 922.72, 826.33, 756.88, 703.43. HRMS calculated for
C
21H25N3O2S [M+1]+ 384.1746, found [M+1]+ 384.1741.
4.1.17. 5-(4-(tert-butyl)phenyl)-3-((pyridin-3-ylamino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3n)
4.1.12. 5-(4-(tert-butyl)phenyl)-3-(((4-nitrophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3i)
White crystalline solid, yield: 73%, mp: 162–164 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.23 (t, J = 8 Hz, 1H, NH), 5.55 (d,
J = 8 Hz, 2H), 7.13-7.16 (q, J = 4 Hz, 1H, Ar), 7.31-7.33 (m, 1H, Ar),
7.49 (d, J = 8 Hz, 2H, Ar), 7.81 (d, J = 8 Hz, 2H, Ar), 8.08 (d,
J = 4 Hz, 2H, Ar), 8.27 (d, J = 4 Hz, 2H, Ar). 13C (400 MHz, CDCl3) d:
31.11, 35.28, 58.15, 119.32, 120.51, 123.95, 126.27, 126.47, 137.37,
140.52, 141.31, 156.52, 159.85, 176.57. IR (KBr): 3254.33, 2960.93,
1617.24, 1590.31, 1370.61, 1423.06, 1262.89, 1097.06, 1013.43,
843.34, 758.30, 704.44. HRMS calculated for C18H20N4OS [M+1]+
341.1437, found [M+1]+ 341.1674.
Light brown solid, yield: 72%, mp: 174–176 °C. 1H NMR (400 MHz,
CDCl3) δ: 1.33 (s, 9H), 5.58 (d, J = 8 Hz, 2H), 5.79 (t, J = 8 Hz, 1H,
NH), 6.97 (d, J = 8 Hz, 2H, Ar), 7.50 (d, J = 8 Hz, 2H, Ar), 7.81 (d,
J = 8 Hz, 2H, Ar), 8.13 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz, CDCl3) δ:
31.10, 35.31, 57.32, 113.24, 119.17, 126.24, 126.34, 126.50, 140.54,
150.01, 156.74, 159.98, 176.65. IR (KBr): 3376.22, 2963.76, 2904.23,
2873.05, 1600.23, 1506.68, 1424.47, 1373.45, 1325.26, 1257.22,
1190.60, 1148.08, 1106.98, 1010.63, 840.51, 756.88, 697.35, 643.49.
HRMS calculated for C19H20N4O3S [M+Na]+ 407.1154, found [M
+Na]+ 407.1156.
4.1.18. 5-(4-(tert-butyl)phenyl)-3-((pyrimidin-2-ylamino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (3o)
4.1.13. 5-(4-(tert-butyl)phenyl)-3-(((4-fluorophenyl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (3j)
White Powder, yield: 78%, mp: 176–178 °C. 1H NMR (400 MHz,
CDCl3) δ: 1.33 (s, 9H), 5.83 (d, J = 8 Hz, 2H), 6.35 (t, J = 8 Hz, 1H),
6.71 (1, J = 4 Hz, 1H), 7.48 (d, J = 8 Hz, 2H), 7.83 (d, J = 12 Hz, 2H),
8.39 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz, CDCl3) δ: 31.12, 35.25,
55.69, 113.04, 119.56, 126.16, 126.49, 156.23, 158.37, 159.55,
161.05, 176.68. IR (KBr): 3251.49, 2963.76, 2871.63, 1587.47,
1530.78, 1415.97, 1373.45, 1262.89, 1141.00, 1038.94, 1010.60,
840.51, 759.72, 700.19. HRMS calculated for C17H19N5OS [M+1]+
342.1389, found [M+1]+ 342.1383.
White crystalline solid, yield: 65%, mp: 76–78 °C. 1H NMR
(400 MHz, CDCl3) (ppm): 1.33 (s, 9H), 5.09 (t, J = 8 Hz, 1H, NH),
5.49 (d, J = 8 Hz, 2H), 6.84-6.94 (m, 4H), 7.49 (d, J = 8 Hz, 2H), 7.81
(d, J = 8 Hz, 2H, Ar). 13C (400 MHz, CDCl3) δ: 31.12, 35.27, 59.32,
115.53, 115.60, 115.95, 116.17, 119.46, 126.25, 126.44, 140.23,
156.04, 156.41, 158.40, 159.76, 176.50. IR (KBr): 3367.72, 2963.76,
2905.65, 2871.63, 1615.82, 1513.77, 1424.47, 1367.78, 1309.66,
1251.55, 1223.20, 1146.66, 1092.80, 1012.01, 829.17, 759.72, 701.60,
649.16. HRMS calculated for C19H20FN3OS [M+Na]+ 380.1209, found
[M+Na]+ 380.1171.
4.2. Biological evaluation
4.1.14. 5-(4-(tert-butyl)phenyl)-3-(((2-fluorophenyl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (3k)
4.2.1. Cell proliferation assay
U87, HeLa, Panc and MCF-7 cells (3000 cells per well) were cul-
tured and seeded into 96-well plates. After overnight incubation, the
cells were treated with compounds 3a to 3o at different concentrations
(0.1 μM to 100 μM) at 37 °C in an atmosphere of 5% CO2 for 48 h.
Doxorubicin (DOX) was used as positive control. The concentration of
DMSO used to dissolve the fractions did not exceed 0.3%. Cell viability
was determined by using MTT assay (Mosmann et al., December 1983).
Following treatment, MTT dye (3-(4, 5 dimethylthiazol-2-yl)-2, 5-di-
phenyltetrazolium bromide) (Himedia, Mumbai, India) solution in PBS
(5 mg/mL) was added and incubated in a CO2 incubator for 4 h.
Formazan crystals formed were dissolved in DMSO and absorbance was
measured by using the microplate reader (Tecan, Genios-Pro) at
570 nm. Cell viability of treated cells was calculated in reference to the
vehicle control cells using the formula as viability (%) =
[100 * (Sample Abs)/(Control Abs)], where Abs is the absorbance value
at 570 nm.
White crystalline solid, yield: 76%, mp: 88–90 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.37 (t, 1H, NH), 5.57 (d, J = 8 Hz,
2H), 6.75-6.77 (m, 1H), 6.97-7.06 (m, 2H), 7.23 (t, J = 8 Hz, 1H, Ar),
7.49 (d, J = 8 Hz, 2H, Ar), 7.81 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz,
CDCl3) δ: 31.12, 35.27, 57.92, 114.16, 115.05, 115.24, 119.68, 119.75,
124.77, 124.80, 126.24, 126.47, 132.56, 132.67, 150.72, 153.10,
156.40, 159.80, 176.55. IR (KBr): 3411.66, 2963.76, 1620.07,
1520.86, 1454.24, 1370.61, 1254.39, 1194.86, 1094.22, 1040.36,
1013.43, 839.09, 751.21, 701.60. HRMS calculated for C19H20FN3OS
[M+Na]+ 380.1209, found [M+Na]+ 380.1183
4.1.15. 4-(((5-(4-(tert-butyl)phenyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)
methyl)amino)benzoic acid (3l)
White crystalline solid, yield: 42%, mp: 208–210 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.33 (s, 9H), 5.23 (t, J = 8 Hz, 1H, NH), 5.55 (d,
J = 8 Hz, 2H), 7.14 (d, J = 8 Hz, 1H), 7.32 (d, J = 8 Hz, 1H, Ar), 7.49
(d, J = 8 Hz, 2H, Ar), 7.81 (d, J = 8 Hz, 2H, Ar), 8.08 (d, J = 8 Hz, 1H,
Ar), 8.27 (d, J = 4 Hz, 1H, Ar). 13C (400 MHz, DMSO) δ: 31.25, 35.44,
57.75, 112.80, 119.70, 120.29, 126.43, 126.62, 126.93, 131.58,
150.19, 156.24, 159.51, 167.75, 176.06. IR (KBr): 3357.80, 2963.761,
1678.19, 1605.90, 1254.39, 1179.26, 1101.31, 841.93, 765.391,
698.77. HRMS calculated for C20H21N3O3S [M+1]+ 384.1383, found
[M+1]+ 384.3089.
4.2.2. Morphological analysis
Appropriate number of HeLa cells was seeded in to 6 well plates for
24 and 48 h treatment with 3i, 3j and DOX. The morphology of the cells
was observed in inverted phase contrast microscope (Nikon, Eclipse TE
2000, Japan) after 24 and 48 h of treatment at a focus of 10× and the
images were captured using NIS elements software.
4.1.16. 5-(4-(tert-butyl)phenyl)-3-(((4-ethoxyphenyl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (3m)
4.2.3. Cell migration study
HeLa cells were seeded into 6-well culture plates with serum-con-
taining medium and were cultured until the cell density reached ∼80%
confluency. After 24 h of incubation, an artificial homogeneous wound
was created by scratching the monolayer with a sterile 200 μL pipette
tip. After scratching, the cells were washed with serum-free medium
and treated with 3i, 3j and DOX. Images of the cells migrated into the
wound induced area were captured at a period of 0, 24 and 48 h using
NIS element software. The assay was performed in triplicates.
Bluish white solid powder, yield: 40%, mp: 78–80 °C. 1H NMR
(400 MHz, CDCl3) δ: 1.30 (s, 9H), 1.31 (t, J = 8 Hz, 3H), 3.88-3.93 (q,
2H), 4.92 (t, J = 8 Hz, 1H, NH), 5.45 (d, J = 8 Hz, 2H), 6.74 (d,
J = 8 Hz, 2H, Ar), 6.82 (d, J = 8 Hz, 2H, Ar), 7.45 (d, J = 8 Hz, 2H,
Ar), 7.77 (d, J = 8 Hz, 2H, Ar). 13C (400 MHz, CDCl3) δ: 15.02, 31.13,
35.26, 59.85, 63.94, 115.72, 115.90, 119.58, 126.21, 126.43, 137.66,
153.09, 156.27, 159.66, 176.45. IR (KBr): 3390.40, 2966.60, 2902.81,
728

N. Yadav et al.
Biomedicine&Pharmacotherapy95(2017)721–730
4.2.4. Acridine orange/ethidium bromide (AO/EB) staining assay
4.2.9. Statistical analysis
The values were recorded as mean
by using one way ANOVA test; differences below the 0.001 level
(P < 0.05) were considered as statistically significant.
HeLa cells were grown in 6 well plates (0.5 × 106 cells per well) for
24 h and were treated with and without IC50 concentration of com-
pounds 3i, 3j and DOX for 24 and 48 h. After treatment, cells were
washed twice with phosphate buffer saline (PBS) and fixed with 4%
paraformaldehyde at 4 °C for 10 min. Then, the cells were incubated
with 0.5 mL of PBS solution containing mixture of acridine orange and
ethidium bromide in dark for 10 min at 37 °C. The stained cells were
washed with PBS to remove the excess dye and the cells were observed
immediately under fluorescence microscope (Nikon, Eclipse, E 600).
SD. The data were analyzed
Conflict of interest
All authors declare no conflicts of interest.
Acknowledgements
Nalini Yadav would like to acknowledge Lady Tata Memorial Trust,
Mumbai, India for providing the fellowship for conducting the research
work. All the authors would like to acknowledge University Scientific
Instrumentation Centre, Delhi University for providing NMR and IR
facilities, Sophisticated Analytical Instrumentation Facility, Central
Drug Research Institute, Lucknow for providing HRMS facility.
4.2.5. Annexin-V (APC)/propidium iodide dual staining assay
HeLa cells at a density of 0.5 × 106 per plate were seeded overnight
and treated with compounds 3i, 3j and DOX at their IC50 concentration
and harvested at scheduled time points post-treatment and re-sus-
pended in 300 μL 1× binding buffer followed by incubation with 5 μL
Annexin V and 5 μL propidium iodide for 20 min in dark. The samples
were analyzed for Annexin V and PI binding by FACS calibur (Becton
Dickinson) using Cell Quest pro software. Data was collected using
logarithmic amplification of both the FL2 (PI) and FL4 (AV) channels.
Quadrants of the dot plots were analyzed using the Cell Quest pro
software. Unstained cells were used as control for auto-fluorescence.
Cells stained alone with Annexin V or PI were used to adjust the pho-
tomultiplier voltages settings to eliminate spectral overlap between the
FL2 and FL4 signals.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.biopha.2017.08.110.
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