A novel 8-nitro quinoline-thiosemicarbazone analogues induces G1/S & G2/M phase cell cycle arrest and apoptosis through ROS mediated mitochondrial pathway

Article abstract of DOI:10.1016/j.bioorg.2020.103709

A series of novel 8-nitro quinoline-based thiosemicarbazone analogues were synthesized and characterized by various spectroscopic and single crystal X-ray analyses. The potent antitumor effects of synthesized compounds towards the cancer cells were evaluated by MTT assay. Amongst, the compound 3a exhibited the highest inhibitory activity and the compounds 3f and 3b were also showed significant activity. The molecular mechanistic studies of cell death have demonstrated that the treated potent compound 3a induced G1/S & G2/M phase cell cycle arrest and induced apoptosis via mitochondrial dysfunction and increased the production of cytotoxic ROS levels. The RT-PCR gene expression analysis revealed that the cell death induced by activation of caspase-3 dependent intrinsic apoptotic signaling pathway. Further, the molecular binding affinity of compounds with estrogen receptor alpha was calculated by molecular docking studies. Thus, novel 8-nitro quinoline-thiosemicarbazone analogues provide a unique tool for breast cancer therapeutic tactics.

Full text of DOI:10.1016/j.bioorg.2020.103709

Journal Pre-proofs
A Novel 8-Nitro Quinoline-Thiosemicarbazone Analogues Induces G1/S &
G2/M Phase Cell Cycle Arrest and Apoptosis through ROS Mediated Mito-
chondrial Pathway
Selvaraj Shyamsivappan, Raju Vivek, Arjunan Saravanan, Thangaraj
Arasakumar, Thangaraj Suresh, Shunmuganarayanan Athimoolam, Palathurai
Subramaniam Mohan
PII:
S0045-2068(20)30185-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103709
YBIOO 103709
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 January 2020
25 February 2020
26 February 2020
Please cite this article as: S. Shyamsivappan, R. Vivek, A. Saravanan, T. Arasakumar, T. Suresh, S. Athimoolam,
P. Subramaniam Mohan, A Novel 8-Nitro Quinoline-Thiosemicarbazone Analogues Induces G1/S & G2/M
Phase Cell Cycle Arrest and Apoptosis through ROS Mediated Mitochondrial Pathway, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.103709
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A Novel 8-Nitro Quinoline-Thiosemicarbazone Analogues Induces G1/S & G2/M Phase
Cell Cycle Arrest and Apoptosis through ROS Mediated Mitochondrial Pathway
Selvaraj Shyamsivappana, Raju Vivekb, Arjunan Saravananc, Thangaraj Arasakumara, Thangaraj
Suresh a, Shunmuganarayanan Athimoolamd, Palathurai Subramaniam Mohana,*
aSchool of Chemical Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India.
bCancer Research Program (CRP), Bio-Nano Therapeutics Research Laboratory, School of Life
Sciences, Department of Zoology, Bharathiar University, Coimbatore-641 046, India.
cDRDO-BU CLS, Bharathiar University campus, Coimbatore, Tamil Nadu, India.
dDepartment of Physics, Anna University, Nagercoil, Tamil Nadu, India
Abstract
A series of novel 8-nitro quinoline-based thiosemicarbazone analogues were synthesized
and characterized by various spectroscopic and single crystal X-ray analyses. The potent
antitumor effects of synthesized compounds towards the cancer cells were evaluated by MTT
assay. Amongst, the compound 3a exhibited the highest inhibitory activity and the compounds 3f
and 3b were also showed significant activity. The molecular mechanistic studies of cell death
have demonstrated that the treated potent compound 3a induced G1/S & G2/M phase cell cycle
arrest and induced apoptosis via mitochondrial dysfunction and increased the production of
cytotoxic ROS levels. The RT-PCR gene expression analysis revealed that the cell death induced
by activation of caspase-3 dependent intrinsic apoptotic signaling pathway. Further, the
molecular binding affinity of compounds with estrogen receptor alpha was calculated by
molecular docking studies. Thus, novel 8-nitro quinoline-thiosemicarbazone analogues provide a
unique tool for breast cancer therapeutic tactics.
1 | P a g e

Introduction
Breast cancer and cervical cancer are the most common reasons for cancer-related deaths
for women.[1, 2] Currently, chemotherapy and radiotherapy are the important prime option for
cancer treatment. However, the toxicity and side effects associated with chemotherapeutics
minimize their efficacy. Due to the limited successes of the drugs that drives a strong desire for
the synthesis of new anticancer agents with targeted mechanisms. Estrogen Receptor alpha
(ERα) is a well-known hormone expressed in about 70 percentage of breast cancer cells in
clinical samples.[3] ERα promotes the growth of breast cancer cells by increasing the level of
oncogenic proteins and accelerates G1/S cell cycle phase transition.[4] Hence, targeting ERα is
one of the most challenging objectives for exploring a new therapeutics for ERα positive breast
cancer treatment.
In this therapeutic context, thiosemicarbazone analogues represent one of the most
promising classes of compounds that have a variety of molecular actions with potential
anticancer applications.[5-8] Initially, the anticancer effects of thiosemicarbazones were
portrayed due to their inhibition property of ribonucleotide reductase which catalyzes the rate-
restricting step of DNA synthesis.[9] 2-formyl pyridine thiosemicarbazone is a nitrogen-
containing heterocyclic thiosemicarbazone which was the first demonstrated thiosemicarbazone
based anticancer compound and consequently developed cancer drug was 3-aminopyridine-2-
carbaldehyde thiosemicarbazone (3-AP or Triapine), and entered stage I and stage II clinical trial
progressed in patients.[10-13] However, these drugs have not been active against advanced
tumors. Few sorts of thiosemicarbazone derivatives, for example, DpT, DpC, Dp44Mt have also
shown intense anticancer activity.[14-16]
On the other hand, the nitrogen-containing quinoline heterocycles have been found in
several class of natural and synthetic products that also demonstrates pronounced anticancer
activity through diversity of cell death mechanisms, such as inhibition of microtubule
polymerization,[17] inhibition of topoisomerase1,[18] inhibition of epidermal growth factor
receptor (EGFR),[19] inhibition of dehydrogenase 1A1 (ALDH1A1),[20] cell cycle arrest in the
G2 phase,[21] inhibition of Ataxia Telangiectasia Mutated (ATM) kinase,[22] inhibition of
haspin kinase,[23] inhibition of tubulin polymerization,[24] inhibition of Heme Oxygenase-
1,[25] and inhibition of tyrosine kinases[26], etc. Because of these interesting therapeutic
attributes of quinoline analogues, recently we have unfolded the one set of 2,3 dihydro 8nitro-4-
quinolone hybrids in the form of essential alkaloids.[27] Hence, more attractive tactics are to be
evolved for designing a novel quinoline incorporated thiosemicarbazone analogues which may
stand a chance for exhibiting the potential activity towards tumors.
Herein, we report a synthesis of novel bioactive 8-nitro quinoline based
thiosemicarbazone analogues and their potent inhibitory efficacy towards breast cancer (MCF-7)
and cervical cancer (HeLa) cells. Based on the MTT cytotoxic results, the structure-activity
relationship (SAR) studies were carried out for synthesized compounds. Among the tested
compounds the compound 3a exhibited the highest inhibitory activity, as well as the compounds
3f, and 3b were also showed significant inhibitory activity against the breast cancer cells.
2 | P a g e

Further, we evaluated the molecular mechanistic studies by flow cytometric and RT-PCR
analysis and the results revealed that compounds induced G1/S and G2/M cell cycle arrest, and
intrinsic apoptotic signaling activation of apoptotic marker caspase-3. Furthermore, the
molecular binding affinities of compounds with ERα were studied by molecular docking. The
results demonstrated that the compounds effectively inhibit the growth of breast cancer MCF-7
cells.
Results and Discussion
Synthesis and Characterization of Novel Quinoline Based Thiosemicarbazone Analogues
Initially, The starting precursor C6 substituted 2,3-dihydro-8-nitro-4-quinolones 2a-c
were synthesized from 2-nitro anilines and 3-bromopropionic acid (Scheme 1). Then we had
prepared the substituted thiosemicarbazide from hydrazine and substituted isothiocyanates via
nucleophilic addition reaction. Condensation of the reactants thiosemicarbazides (1a-d) with 2,3-
dihydro-8-nitro-4-quinolone moieties (2a-c) afforded the quinoline-based thiosemicarbazone
analogues (3a-l) in good yields (Scheme 2).
O
R1
R1
10 % aq KOH
aq Na2CO3
O
O
PPA
OH
+
N
Br
OH
NH2
N
H
H
NO2
2a-c
NO2
NO2
Scheme 1. Synthesis of 2,3-dihydro-8-nitro-4-quinolones 2a-c
R
HN
S
NH
O
N
R1
R1
NH2NH2
RNCS
H
H
DCM
N
N
CH3COOH
H2N
R
RT 2H
N
N
H
MeOH, Reflux
S
H
NO2
2a-c
NO2
3a-l
1a-d
= H, CH3, CH2CH3, C6H5
R
1 = H, Cl, CH3
R
Scheme 2. Synthesis of Quinoline-Thiosemicarbazones 3a-l
The structures of the synthesized compounds were confirmed by IR, 1H NMR, 13C NMR
spectroscopic techniques. The proposed compounds were compatible with analyzed data. The IR
spectrum of the compound 3c showed characteristic bands at 3370 cm-1, 2926 cm-1 for NH, 1713
cm-1 for C=S and 1625 cm-1 for C=N. Further, the absence of the band between 2700 cm-1 to
1
2500 cm-1 showed the disappearance of the carbonyl group of the quinolone moiety. In the H
NMR spectrum, the compound 3c showed signals at δ 3.48-3.44 as multiplet and 2.81 as triplet
for aliphatic CH2 protons of quinoline ring, δ 3.66-3.59 as multiplet attributed for aliphatic CH2,
δ 1.16 as triplet for CH3, broad singlets at δ 10.27 for N-NH and δ 8.39 for Q-NH, and aromatic
3 | P a g e

13
protons were found at δ 8.64 to 6.70 ppm respectively. Similarly, the C NMR spectra of 3c
showed disappearance of the carbonyl group and carbon assignments for thionyl C=S at δ
177.46, Quinoline C=N at δ 143.81 and aliphatic carbons at δ 38.51, 38.40, 23.43, & 14.51 and
aromatic carbons 143.21, 132.71, 132.14, 127.58, and 121.55. The structure of the compound
was further confirmed by X-ray diffraction and elemental analysis.
X-ray Crystallographic Studies
Figure 1. ORTEP diagram of compounds 3a, 3c, and 3i
Molecular geometry and crystal packing of compound 3i
The asymmetric part of the unit cell contains two molecules of the title compound (3i).
The compound crystallized in the centrosymmetric triclinic lattice with the two asymmetric units
(i.e., four molecules) in the unit cell. The NO2 groups are oriented almost parallel to the attached
benzene ring in one of the units [1.8(3)] and slightly deviated from the benzene plane in another
molecular unit [16.3(2)]. Both the molecular units in the asymmetric parts are oriented almost
parallel to each other. The heterocyclic ring adopts an envelope conformation; the flap atom C8 /
C18 deviates by 0.595(3) / 0.597(3) Å from the plane through the remaining ring atoms.
Intermolecular interactions play a significant role in the crystal packing of many organic
molecular assemblies. Especially, hydrogen bonds are the key factor that connects the molecules
through ring and chain formations. It is interesting and much useful to dismantle the molecular
aggregations and designate the hydrogen-bonded assembly with graph-set notations.[28] Both
molecules in the asymmetric part feature intramolecular N-H…O hydrogen bond which forms self-
associated S(6) motif. Crystal packing features classical N-H…O, C-H…Cl, and N-H…S
hydrogen bonds and non-classical C-H…O, C-H…Cl, and C-H…S interactions. One of the C-
H…X interaction is observed to be bifurcated or three-centered interactions [C18-H18
A...O1#3/S1#4]. The intricate hydrogen bonding network has many ring and chain hydrogen bond
motifs, viz., a self associated S(6) motifs formed through intramolecular N-H…O hydrogen bonds,
2
a dimeric ring R2 (8) motif formed through intermolecular N-H…S hydrogen bonds, etc. These
2
2
ring R2 (8) motifs are placed adjacently leading to two parallel chain C2 (6) motif extending along
4 | P a g e

a-axis of the unit cell. These set of chain motifs are separated along b-axis length leading to ring
4
R4 (24) motifs, which are arranged in cascade along the same a-axis of the unit cell (Figure. 3).
Table 1. Hydrogen bonds geometries in compound 3i
D-H…A (Å, °)
N(2)-H(2)…O(1)
d(D-H) (Å) d (H…A) (Å) d(D…A) (Å) <(DHA)(°)
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.97
0.93
0.97
0.97
0.97
2.01
2.06
2.66
2.56
2.89
2.58
2.62
2.29
2.94
2.97
2.61
2.87
2.5
2.626(3)
2.653(2)
3.476(2)
3.338(2)
3.315(2)
3.336(2)
3.469(2)
3.045(3)
3.581(2)
3.897(2)
3.387(3)
3.552(2)
3.440(3)
128
126
159
150
113
147
168
147
125
174
138
128
165
N(7)-H(7)…O(3)
N(4)-H(4A)…S(2)#1
N(10)-H(10B)...S(1)#1
N(10)-H(10A)...Cl(2)#2
N(5)-H(5B)...S(2)#4
N(9)-H(9)...S(1)#4
N(10)-H(10A)...O(2)#6
C(8)-H(8A)...S(2)#1
C(14)-H(14)...Cl(2)#2
C(18)-H(18A)...O(1)#3
C(18)-H(18A)…S(1)#4
C(18)-H(18B)...O(2)#5
Equivalent position: #1-x+1,-y+1,-z ;#2 -x+1,-y+2,-z ; #3 x,y,z-1; #4 –x,
-y+1,-z ; #5 –x,-y+1, -z+1; #6 -x+1, -y+1,-z+1;
Figure 2. Packing diagram of compound 3i viewed down along a-axis of the unit cell. H-bonds
are shown as dashed lines.
5 | P a g e

2
2
4
Figure. 3 A view of chain and ring motifs [S(6), R2 (8), C2 (6) and R4 (24)] between the
molecules formed through classical hydrogen bonds. H-bonds are shown as dashed lines.
In Vitro Cytotoxic Activity of 8-Nitroquinoline-Thiosemicarbazone Analogues
There have been periodical reports which revealed that nitroquinolines have significant
anticancer activity; recently our research group reported new 8-nitroquinoline based derivatives
that showed excellent anticancer against human breast cancer and cervical cancer cells.[27, 29]
Further, thiosemicarbazone compounds also exhibited promising anticancer activity against
breast and cervical cancer cells with an optimized molecular mechanism of action.[30-33] These
reports suggest that 8-nitroquinoline based compounds may inhibit the growth of MCF-7 and
HeLa cells. Hence, to examine the In vitro cytotoxic impacts of newly synthesized 8-
nitroquinoline based thiosemicarbazone compounds 3a-l, we have selected specified cancer cell
lines of MCF-7 and HeLa cells for comparative studies. The non-tumorigenic epithelial breast
cell line MCF10A was used to determine the toxicity of the compounds.
The synthesized compounds exposed non-significant growth inhibitory activity against
MCF10A cells up to 100 µM concentrations. Hence, all the synthesized compounds were
screened by MTT (3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide) assay against
the breast cancer MCF-7 and cervical cancer HeLa cell lines at below 100 µM concentrations.
The familiar anticancer drug Doxorubicin (Dox) was selected as a positive control to compare
the antitumor potency. The results were demonstrated by the IC50 values, which correspond to
the required concentration of compounds to inhibit the 50% of cell viability. The IC50 values of
the newly synthesized compounds and the positive control Dox against the selected cell lines of
MCF-7 and HeLa were summarized in Table 2.
The MTT antitumor activity results indicated that all the tested compounds inhibit the
growth of tested cancer cell lines MCF-7 (IC50 < 26.24±0.76) and HeLa (IC50 < 33.82±0.86).
Specifically, the parent unsubstituted compound 3a exhibited highly significant (p<0.01)
inhibition activity against the breast cancer MCF-7 cells compared with HeLa cells with the IC50
value of 8.14±0.63 and 17.21±0.78 μM/mL-1. The electron-donating methyl group substituted in
6 | P a g e

the amino group of thiosemicarbazide and C6 position of quinoline ring compound 3f also
showed significant activity (p<0.05) with MCF-7 (12.23±1.12) and HeLa (18.13±0.67) cells. The
parent unsubstituted quinoline with methyl thiosemicarbazide compound 3b also showed
respectable activity against the MCF-7 (15.35±1.19) and HeLa (19.14±1.53) cells. The electron-
withdrawing chlorine substituted at the C6 position of quinoline compounds 3i, 3j, 3k, 3l showed
very less activity with IC50 > 21.25±0.51. The other compounds 3c, 3d, 3e, 3g, 3h have shown
moderate activity against both the tested cell lines. However, the compounds did not show any
toxicity against the normal MCF10A cells up to 100 µM concentration which revealed that the
compounds are nontoxic towards the normal cells.
Table 2. MTT cytotoxicity assay of 3a-l against MCF-7 & HeLa cells
with IC50 concentrations (µM)
IC50 of (µM) IC50 of (µM)
MCF-7 Cells HeLa Cells
R1
S. No
R
Compounds
1
H
H
H
3a
3b
3c
8.14±0.63
15.35±1.19
18.45±1.43
19.27±1.31
20.16±0.94
12.23±1.12
22.41±1.67
24.84±0.84
21.25±0.51
22.92±1.22
25.48±0.21
26.24±0.76
14.18±1.36
17.21±0.78
19.14±1.53
21.46±1.27
22.38±0.64
24.52±0.74
18.13±0.67
29.16±1.12
32.31±1.21
25.26±0.73
30.61±1.24
32.27±0.46
33.82±0.86
16.14±1.24
CH3
2
CH2CH3
C6H5
3
H
4
H
3d
3e
CH3
CH3
5
H
CH3
6
3f
CH2CH3 CH3
7
3g
3h
3i
C6H5
CH3
Cl
8
9
H
CH3
10
Cl
3j
CH2CH3
C6H5
11
Cl
3k
3l
12
Cl
Control
DOX
Structure-Activity Relationship (SAR)
Based on MTT cytotoxicity results of the synthesized quinoline-based thiosemicarbazone
analogues, we have investigated the impacts of the substitutions on the C6 position of the
quinoline ring (R) and substitution on the amino group of thiosemicarbazide (R1). From the
cytotoxic results Table 2, it was very clear that the compounds 3a, 3f, and 3b are more active
than the other reported compounds. The compound 3a has IC50 values of 8.14±0.63 and
17.21±0.78 μM/mL-1 for MCF-7 and HeLa cells, which is lesser than the reference compound
Dox for MCF-7 cells, and has a close value for HeLa cells. From this result, we inferred that the
parent compound 3a showed reasonable inhibition activity than the R, R1 substituted compounds.
On the investigation of R1 substitution, the hydrogen substituted compounds 3a-d (MCF-
7 IC50 8.14±0.63 < 19.27±1.31 and HeLa IC50 17±0.8 < 22±0.4) are more potent than the
7 | P a g e

electron-donating methyl group, and electron-withdrawing chlorine substituted compounds 3e-l.
When, compared to the methyl and chlorine substituted compounds, the methyl-substituted
compounds 3e-h (MCF-7 IC50 12.23±1.12 < 24.84±0.84 and HeLa IC50 18.13±0.67 <
32.31±1.21) showed more potential inhibition activity than the chlorine substituted compounds
3i-l (MCF-7 IC50 21.25±0.51 < 26.24±0.76 and HeLa IC50 25.26±0.73 < 33.82±0.86) against the
tested cells. From the above observations, we conclude that the potential activity order of R1
substitution was H > CH3> Cl.
The cytotoxicity impacts of the R substitutions of the synthesized compounds showed
that the hydrogen substituted compounds 3a, 3e, and 3i (MCF-7 IC50 8.14±0.63 < 21.25±0.51
and HeLa IC50 17.21±0.78 < 25.26±0.73) showed more activity than the methyl, ethyl, and
phenyl substituted compounds. It is observed that among the electron-donating group substituted
compounds, the methyl group substituted compounds 3b, 3f, and 3j showed more activity than
the ethyl and phenyl substituted compounds. Based on the MTT results of the compounds the
effective potent compounds, namely 3a, 3f, and 3b were chosen for further experiments.
Compounds Induced Morphological Changes and Apoptotic Activity
Morphological changes of the compounds treated cancer cells were detected by phase-
contrast microscopy. The selected breast cancer MCF-7 and cervical HeLa cancer cells were
treated with the compounds 3a, 3f, and 3b for 24 hours. After treatment, the cells were appeared
to be rounded, detached from neighbor cells, shrunken, and with a completely disintegrated
membrane whereas, the untreated control cells were showed normal and healthy morphology
(Figure 4a). The results demonstrated that the synthesized compounds were significantly
increased the rate of apoptotic cell death in the tested MCF-7 and HeLa cells.
The apoptosis induced by the compounds were further studied by acridine orange/ethidium
bromide (AO/EB) dual staining method. Acridine orange is a green fluorescent cell-permeable dye,
which is taken up by nuclear DNA of both live and dead cells, while ethidium bromide is a
fluorescent dye, which has taken up only by nuclear-damaged DNA and membrane integrity lost
cells such as late apoptotic and dead cells and would emit orange to red fluorescence.[34] The
selected MCF-7 and HeLa cells were treated with the compounds 3a, 3f, and 3b for 24 hours and
then the cells were stained with AO/EB mixture and observed under the fluorescence microscopy.
From the results, it was observed that the compounds 3a treated cells were appeared to be densely
red and slightly orange color fluorescence with extensive nuclear margination accompanied by
chromatin condensation and fragmentation which indicates the apoptotic and necrotic cells (Figure
4b). The compounds 3b and 3f treated cells were showed orange with slightly red fluorescence
which revealed as of early and late apoptotic cells, whereas the control untreated MCF-7 and HeLa
cells appeared as bright greenish which remain unaltered. The compounds showed high apoptotic
activity against the MCF-7 cells.
The nuclear damages induced by compounds 3a, 3b, and 3f on MCF-7 and HeLa cells
were detected by DAPI staining method, and the nuclear morphological changes were observed
under fluorescence microscopy. DAPI (4’,6-diamidino-2-phenylindole) is a known blue
fluorescent nuclear stain, which binds with dead cells but it strongly binds to the adenine-
8 | P a g e

thymine of double-stranded DNA in the nucleus and distinguishes chromatin condensation or
nuclear damage by producing stronger blue fluorescence.[35] The selected MCF-7 and HeLa
cells were treated with the compounds 3a, 3f, and 3b for 24 hours and then the cells were stained
with DAPI. After staining the treated MCF-7 cells were exhibited bright blue fluorescence than
the HeLa cancer cells, and highly condensed chromatins and nuclear fragmentations that occur in
the MCF-7 breast cancer cells (Figure 4c) whereas, the untreated control cells showed normal
morphology. The DAPI nuclear staining results revealed that the compounds 3a, 3f, and 3b
induced significant apoptotic nuclear fragments in breast cancer MCF-7 compared to HeLa
cancer cells.
The observed favorable potency of compounds 3a, 3f, and 3b towards the cancer cells
prompted us to examine the mechanism of ROS action. During the earlier investigation,
quinoline and thiosemicarbazone analogues served as good anticancer agents by effectively
inducing apoptosis in cancer cells through the increased production of ROS.[31, 36-38] Hence,
we investigated the intracellular ROS levels by the DCFH-DA (2’,7’-dichlorodihydrofluorescein
diacetate) assay. The selected MCF-7 and HeLa cancer cells were treated with effective
compounds of 3a, 3f, and 3b at IC50 concentration, and after treatment, the cells were further
stained with DCFH-DA for 30 minutes. The presence of intracellular ROS, the nonfluorescent
DCFH-DA is quickly oxidized into fluorescence dichlorofluorescein (DCF), which shows the
intense green fluorescence. Interestingly, the compounds treated cells displayed enhanced the
green fluorescence intensity which was almost similar to the hydrogen peroxide positive control
(Figure 4d). The increased level of ROS in the compounds treated MCF-7 cells was highly
significant when compared with HeLa cells. Further, the increased level of ROS was
quantitatively determined from the percentage of total cells with increased green fluorescence
intensity (Figure 4e). From the results, it was observed that the compounds significantly
increased the production intracellular ROS in the cancer cells.
The compound 3a induced G1/S and G2/M phase cell cycle arrest in MCF-7 cells
To investigate the mechanistic action of cell cycle arrest, we selected the efficient
compound of 3a against breast cancer MCF-7 cells and the cell cycle was evaluated by the flow
cytometry method. The MCF-7 cells were treated with 3a compound for 24 hours and the
untreated cells were used as control. After the treatment the cell cycle was analyzed, the results
of untreated control MCF-7 cells showed 54.92 % of G0-G1 Phase, 13.45% of G1/S phase and
31.25 of G2/M phase. However, the compound 3a treated MCF-7 cells were showed 22.28 % of
G0-G1Phase, 20.00% of G1/S phase and 54.05 of G2/M phase (Figure 4f). From the above cell
cycle study observed that the compound 3a treated MCF-7 cancer cells were significantly
increased in G1/S and G2/M phase, whereas the G0-G1 phase cells were decreased when
compared with the control cells. The increased percentage of cells in G1/S and G2/M phase
indicated that the compound 3a was significantly induced G1/S and G2/M phase cell cycle arrest
in MCF-7 breast cancer cells.
9 | P a g e

Figure 4. In vitro anticancer efficacy of 8-nitroquinoline based thiosemicarbazones 3a, 3f, and 3b
a) Morphological analysis of compounds treated MCF-7 and HeLa cells. b) Apoptosis detection
of the compounds treated MCF-7 and HeLa cells by acridine orange and ethidium bromide
(AO/EB) staining method c) Detection apoptotic nuclear damage of the compounds cells by
DAPI staining method. d) Detection of increased intracellular ROS production of compounds
cells by DCFH-DA staining method e) Quantification of ROS in compounds treated MCF-7 and
HeLa cells using H2O2 as positive control f) Cell cycle analysis of compound 3a treated MCF-7
Cells g) Quantification of apoptotic cells by flow cytometric analysis by treating MCF-7 cells for
24 h with compounds 3a, 3f, and 3b. Quadrants; Upper left (necrotic cells), Lower left (live
cells), Lower right (early apoptotic cells) and Upper right (late apoptotic cells.
10 | P a g e

Quantification of Apoptotic Cells by Annexin V-FITC and propidium iodide PI staining
To determine the apoptotic cells quantitatively, the double stain Annexin V-FITC and
propidium iodide PI staining method by flow cytometer was applied. The selected potent
compounds 3a, 3f, and 3b were treated with MCF-7 cells for 24 hours. After treatment, the cells
were stained with Annexin V-FITC and propidium iodide PI and incubated for 30 minutes while
the untreated cells were used as a control to quantify the apoptotic cells. After incubation, the
apoptotic cells were analyzed by flow cytometer. The flow cytometer results of the compound 3a
treated cells showed 98 percentage of apoptotic cells with a favorable mode of action, the
compounds 3f and 3b treated cells were showed 97 and 95 percentage of apoptotic cells (Figure
4g). The control cells have no apoptotic or necrotic activities with 100% gated cells. The results
demonstrated that the compounds have more potential action to kill breast cancer cells.
Detection of Apoptotic Protein Expression and Nuclear DNA Damage
Further, we have investigated whether the compound 3a, 3f, and 3b induced apoptosis on
MCF-7 cells through activation of caspase-3 apoptotic biomarker caspase-3 by gene expression
analysis. Caspases are cysteine-aspartic acid proteases enzymes that play a crucial role in the
activation and implementation of apoptotic markers.[39] Bcl-2 family proteins are served as the
anti-apoptotic activity, the up-regulation of the Bcl-2 protects the apoptosis of cancer cells.[40]
While the down-regulation of the Bcl-2 discharge the cyt C into cytoplasm which results in the
activation of caspase-3.[41] The MCF-7 cells were treated with the selected compounds 3a, 3f,
and 3b for 24 hours. The treated MCF-7 cells were examined by RT-PCR gene expression
analysis. The results specified the up-regulation of apoptotic protein caspase-3 enzyme when
compared with the internal standard control gene β-actin (Figure 5a), and significant down-
regulation of anti-apoptotic Bcl-2 protein was observed in the treated MCF-7 cells. These
activation phenomenons of caspase-3 and down-regulated Bcl-2 proteins have confirmed the
activation of apoptosis in breast cancer cells MCF-7 cells through the intrinsic pathway signaling
of mitochondrial-programmed cell death.
The activation of caspase-3 vitally induces the cellular DNA fragmentation and causes
nuclear damage. DNA-agarose gel electrophoresis method is used to determine the DNA ladder
pattern of fragmentation. The DNA was extracted from the compounds 3a, 3f, and 3b treated
MCF-7 cells. From the agarose-gel results, it was very clearly noted that the compound was
effectively induced DNA laddering pattern in treated MCF-7 cells (Figure 5b). The DNA
laddering pattern indicates the fragmentation of DNA occurs in the MCF-7 cells and causes
apoptosis.
11 | P a g e

Figure 5. a) 3a, 3f, and 3b induced caspase-3 activation by RT-PCR studies b) DNA
fragmentation of compounds 3a, 3f, and 3b treated MCF-7 cells by DNA-agarose gel
electrophoresis method
Scheme 3. Schematic diagram of cell death induced by the compounds
Molecular Binding Affinity of Compounds with ERα
Estrogen Receptor alpha (ERα) is the hormone that presents in most of the breast cancer
cells and promotes the growth of breast cancer cells.[42, 43] Inhibition of the ERα is one of the
important targeted therapy in breast cancer treatment.[44, 45] The computational molecular
docking studies were carried out to calculate the binding mode of the synthesized compounds
with breast cancer ERα protein. Since the breast cancer responsible Estrogen Receptor alpha
(ERα) structure was downloaded from the PDB with PDB id of 3ERT.[46, 47] The PDB
structure is prepared with the help of protein preparation wizard in the Schrodinger Maestro[48]
module, to add the necessary hydrogen atoms and to correct the improper bond orders for
carrying out molecular docking studies. The ligands were prepared with the help of the LigPrep
module of Schrodinger.[49] The grid box was generated on the center of the macromolecule
12 | P a g e

(protein in our case) for carrying out molecular docking as a blind docking approach. The
prepared protein and ligand structures were subjected to molecular docking with the help of the
Glide module.[50]
The docking results of the synthesized compounds revealed that all the synthesized
compounds have a good binding affinity with the selected breast cancer cell protein Estrogen
Receptor alpha (ERα) (Table 3). The selected potent compounds 3a, 3b, and 3f were showed
good binding energy with a docking glide score of -7.675, -6.879, and -7.339 with breast cancer
ERα protein whereas, the standard drug doxorubicin showed glide score of -6.213 with ERα
protein. Particularly, the unsubstituted parent thiosemicarbazone 3a binds very well with breast
cancer protein which shows two crucial hydrogen bonding interaction with LEU 346 and
GLY521 through two NH group of quinoline and NH2 group of thiosemicarbazone, and the
compound 3b and 3f showed one bonding with LEU346 through NH substituent respectively
(Figure 7).
Table 3. Molecular docking studies of compounds 3a-l with estrogen receptor (PDB ID: 3ERT)
Compounds Glide
gscore
Glide
evdw
Glide
ecoul
Glide
energy
-38.556
-38.59
-39.194
-32.337
-38.235
-31.669
-34.244
-30.411
-37.636
-32.99
Interacting Residues
3a
3b
3c
3d
3e
3f
-7.675 -35.701
-6.879 -38.359
-6.659 -39.197
-5.773 -28.454
-7.104 -39.639
-2.854
-0.231
0.003
-3.882
1.404
-4.138
-1.204
-2.236
-0.917
-3.901
-2.446
-2.953
LEU 346, GLY521
LEU 346
LEU 346
THR 347
LEU 387
THR 347
-7.339
-27.53
3g
3h
3i
-5.261 -33.041
-6.481 -28.176
-7.151 -36.719
-7.469 -29.089
H2O, THR 347
THR 347
-
3j
THR 347
3k
3l
-6.113
-6.453 -39.966
-6.213 -21.34
-33.96
-36.406
H2O, THR 347
-42.92 CYS 530, H2O, THR 347
Dox
-16.847 -38.186 ASP351, CYS530, H2O,
THR 347
13 | P a g e

Figure 6. 3-dimensional representation of the molecular interaction between the compounds 3a-l
with breast cancer estrogen receptor alpha (ERα).
14 | P a g e

Figure 7. 2-dimensional representation of the molecular interaction between the compounds 3a-l
with breast cancer estrogen receptor alpha (ERα). The red arrows indicate the hydrogen bonding
interaction with protein the residues.
15 | P a g e

Figure 8. 3-dimensional and 3-dimensional representation of the molecular interaction between
the doxorubicin with breast cancer estrogen receptor alpha (ERα). The red arrows indicate the
hydrogen bonding interaction with protein the residues.
Pharmacokinetic studies of the compounds
In the view of excellent biomedical anticancer properties of compounds, we further
examined the in silico pharmacokinetic properties of the compounds. To determine the in silico
pharmacokinetic drug-like properties such as adsorption, distribution, metabolism, and excretion
(ADME) of the synthesized compounds, the 2D structure of the compounds were subjected to
the computational program by using Qikprop module of Schrodinger software.[51] Further, the
Lipinski’s rule of five and Jorgensen’s rule of three was also predicted for the synthesized
compounds.
The Lipinski’s rules are the molecular weight of drug < 500, octanol/water partition
coefficient QPlogPo/w < 5, donor hydrogen bond of drug ≤ 5, acceptor hydrogen bond of drug ≤
10. The obtained In silico pharmacokinetic study results indicated that the molecular weight of
the selected potent compounds 3a, 3f, and 3b ranges from 265.289 to 293.343 a.m.u., the number
of hydrogen bonding donor of compound 3a was 4 and for 3b and 3f was 3 whereas, the
hydrogen bonding acceptor of the compounds was 5.5. The pharmacokinetic property predictions
revealed that the selected potent compounds 3a, 3b, and 3f satisfied the Lipinski’s rules which
indicate that the synthesized compounds having drug-like properties (Table 4).
Jorgensen’s rules are aqueous solubility of the drug QPlogS > -5.7, Caco-2 cell
permeability QPP Caco > 22nm/s, primary metabolic reactions #metab < 7. The selected
compounds satisfy Jorgensen’s rules with QPlogS between -3.214 to -4.385, QPP Caco 152.497
to 380.844, #metab 3 to 4 which indicates that the selected compounds more likely to be orally
available (Table 5). Furthermore, the compounds showed acceptable values to satisfy the other
pharmacokinetic properties like IC50 value of HERG K+ channels, brain/blood partition
coefficient value QPlogBB, MDCK cell permeability QPP MDCK, skin permeability QPlogKp,
binding ability to blood serum albumin QPlogKhsa, human oral absorption percentage, and Van
16 | P a g e

der Wall’s surface area of polar nitrogen and oxygen atoms PSA (Table 6). Hence, from the
molecular docking and pharmacokinetic properties results clearly showed that the synthesized
compounds had a strong binding site and drug-like properties.
Table 4. Pharmacokinetic Properties of compounds 3a-l according to the Lipinski’s rule of five
Compounds
mol W
<500
Donor HB Acceptor HB QP log Po/w
<5
4
3
3
3
4
3
3
3
4
3
3
3
5
<10
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
14.8
<5
3a
3b
3c
3d
3e
3f
3g
3h
3i
265.289
279.316
293.343
341.387
279.316
293.343
307.37
355.414
299.734
313.761
327.788
375.832
543.526
0.815
1.632
2.002
2.976
1.079
1.91
2.287
3.267
1.274
2.103
2.477
3.455
-0.293
3j
3k
3l
Dox
Table 5. Pharmacokinetic Jorgensen’s rule of three of compounds 3a-l
Compounds QPlogS
> -5.7
QPP Caco
> 22 nm/s
152.497
380.34
#metab
< 7
4
3
3
4
5
4
4
5
4
3
3
4
3a
3b
3c
3d
3e
3f
3g
3h
3i
-3.214
-3.888
-4.207
-5.219
-3.682
-4.385
-4.724
-5.748
-3.872
-4.58
439.136
462.596
152.767
380.844
438.567
463.069
153.511
380.253
442.904
459.342
3.322
3j
3k
3l
Dox
-4.9
-5.937
-2.583
9
17 | P a g e

Table 6. Pharmacokinetic Properties of compounds 3a-l
QP log
Compound HERG
QP
log
BB
QPP
MDCK
QP
QP
Percent
Human oral
PSA
7.0 to
200.0
logKp logKhsa
s
< -5
<25 poor, -8.0 to -1.5 to 1.5 Absorption
-3.0 to
1.2
>500
great
-1.0
<25% is low
>80% is high
3a
3b
3c
3d
3e
3f
-4.616 -1.197
-4.908 -0.897
-5.096 -0.944
-6.346 -0.983
179.535
417.285
462.085
492.062
179.89
-4.31
-3.539
-3.322
-2.57
-0.408
-0.207
-0.121
0.161
-0.293
-0.077
0.014
0.302
-0.325
-0.112
-0.023
0.265
-0.59
70.791
82.681
85.965
92.075
72.353
84.322
87.622
93.787
73.533
85.437
88.81
111.694
98.439
97.299
96.307
111.664
98.416
97.289
96.317
111.689
98.351
97.319
96.322
214.575
-4.549
-1.25
-4.506
-3.736
-3.52
-4.818 -0.936
-5.008 -0.984
417.61
3g
3h
3i
461.424
491.841
445.368
-6.243
-4.545
-1.02
-1.06
-2.767
-4.472
3j
-4.834 -0.755 1027.241 -3.706
-5.008 -0.795 1146.514 -3.482
-6.258 -0.843 1201.218 -2.744
3k
3l
94.82
Dox
-6.183 -2.857
1.146
-7.601
1
Conclusion
In conclusion, we successfully synthesized
a
novel 8-nitroquinoline based
thiosemicarbazone analogues which exhibit more potent biomedical antitumor properties on
tested breast cancer MCF-7 cells. All the synthesized compounds were showed effective
inhibition ability of breast cancer cell proliferation especially, the parent unsubstituted compound
3a, and methyl group substituted compounds 3b and 3f showed excellent antitumor activity. The
molecular mechanism of cell death by effective compounds 3a, 3f, and 3b were further examined
by various fluorescent staining, flow cytometry, and gene expression analysis. The tested
compounds induced mitochondrial dysfunction, along with increased the production of ROS
levels and induced the DNA fragmentation. Further, the compound 3a induced G1/S and G2/M
cell cycle arrest and the apoptosis induced by activation of apoptotic biomarker caspase-3 and
down-regulation of anti-apoptotic protein Bcl-2. Additionally, the theoretical molecular docking
and pharmacokinetic studies indicated that the compounds had binds strongly with ER-α receptor
and exerted drug-like properties. Therefore, the newly synthesized 8-nitroquinoline based
thiosemicarbazone analogues may progress as promising biomedical antitumor drugs against
breast cancer.
18 | P a g e

Experimental Section
General methods
All the chemicals and reagents were purchased from Sigma Aldrich and Loba chemicals
and used as such. IR spectra were recorded on JASCO FT IR 4100 spectrometer using KBr
pellets and the absorption frequencies quoted in reciprocal centimeters. Nuclear magnetic
resonance (NMR) spectra were recorded on Bruker advance (400 MHz for 1H and 100 MHz for
13C) using tetramethylsilane (TMS) and DMSO-d6 as the solvent. Chemical shifts were reported
in δ values (ppm) downfield from tetramethylsilane and coupling constants are reported in Hertz
(Hz). The following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q) and
multiplet (m). The reaction courses were monitored by TLC on silica gel precoated F254 Merck
plates. Elemental analyses were performed on a Perkin Elmer 2400 Series II Elemental CHNS
analyzer.
Synthesis of quinoline based thiosemicarbazones 3a-l
The 2,3-dihydro-8-nitro-4-quinolone 2a-c (1 mmol), was dissolved in anhydrous
methanol (10 mL). The solution was heated at reflux for 15 min, and then a catalytic amount of
acetic acid added and a thiosemicarbazide derivatives 1a-d (1.5mmol) added. The reaction
mixture was refluxed for 3-5 h after completion of the reaction (monitored by TLC), the reaction
mixture was evaporated to dryness then the product was isolated by recrystallisation in methanol.
The pure thiosemicarbazone product 3a-l obtained as dark red crystals (85 % yield).
(E)-2-(8-nitro-2, 3-dihydroquinolin-4(1H)-ylidene)hydrazinecarbothioamide 3a
mp 176-182°C. IR (KBr, cm-1): 3239, 2976, 1601, 1507; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.33 (1H, s, N-H), 8.57 (1H, d, J = 6.8 Hz, Ar-H), 8.38 (1H, s, Ar-H), 8.28 (1H, s, NH), 8.09
(2H, dd, J = 1.6, 1.6 Hz NH2), 6.68 (1H, t, J = 8.0 Hz Ar-H), 3.48-3.44 (2H, m, CH2), 2.82 (2H,
t, J = 6.4 Hz, CH2).13C NMR (DMSO-d6) δ ppm 178.79, 143.84, 143.32, 132.91, 132.12, 127.60,
121.54, 114.85, 38.50, 23.45. Anal. Calcd for: C10H11N5O2S C, 45.27; H, 4.18; N, 26.40; O,
12.06; S, 12.09. Found; C, 47.32; H, 4.22; N, 26.36; O, 12.13; S, 12.11.
(E)-N-methyl-2-(8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)hydrazinecarbothioamide 3b
mp 178-183°C. IR (KBr, cm-1): 3262, 2931, 1632, 1562; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.30 (1H, s, N-H), 8.56 (2H, dd, J = 1.2, 1.2 Hz, Ar-H), 8.35 (1H, s, NH), 8.11 (1H, dd, J = 1.6,
1.6 Hz, NH), 6.72 (1H, t, J = 8 Hz, Ar-H), 3.49-3.45 (2H, m, CH2), 3.07 (3H, d, J = 4.6 Hz,
CH3), 2.83 (2H, t, J = 6.8 Hz, CH2).13C NMR (DMSO-d6) δ ppm 178.94, 144.30, 143.61, 133.21,
132.60, 128.09, 122.05, 115.30, 38.98, 31.53, 23.91. Anal. Calcd for: C11H13N5O2S C, 47.30; H,
4.69; N, 25.07; O, 11.46; S, 11.48. Found; C, 47.35; H, 4.71; N, 25.66; O, 11.46; S, 11.52.
(E)-N-ethyl-2-(8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)hydrazinecarbothioamide 3c
mp 178-185°C. IR (KBr, cm-1): 3370, 2926, 1625, 1542; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.27 (1H, s, NH), 8.62 (1H, t, J = 6.4 Hz, NH), 8.54 (1H, d, J = 6.8 Hz, Ar-H), 8.39 (1H. s,
NH), 8.10 (1H, dd, J = 1.6, 1.6 Hz, Ar-H), 6.72 (1H, t, J = 8.0 Hz, Ar-H), 3.66-3.59 (2H, m,
CH2), 3.48-3.44 (2H, m, CH2), 2.81 (2H, t, J = 6.4 Hz, CH2), 1.16 (3H, t, J = 7.2 Hz, CH3). 13C
NMR (DMSO-d6) δ ppm 177.46, 143.81, 143.21, 132.71, 132.14, 127.58, 121.55, 114.80, 38.51,
19 | P a g e

38.40, 23.43, 14.51. Anal. Calcd for: C12H15N5O2S C, 49.13; H, 5.15; N, 23.87; O, 10.91; S,
10.93. Found; C, 49.15; H, 5.16; N, 23.91; O 10.88; S, 10.89.
(E)-2-(8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)-N-phenylhydrazinecarbothioamide 3d
mp 175-180°C. IR (KBr, cm-1): 3389, 2926, 1628, 1467; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.65 (1H, s, NH), 10.11 (1H, s, NH), 8.68 (1H, d, J = 7.2 Hz, Ar-H), 8.40 (1H, s, NH), 8.13
(1H, dd, J = 1.4, 1.4 Hz, Ar H), 7.59 (2H, d, J = 7.2 Hz, Ar-H), 7.39 (2H, t, J = 8.0 Hz, Ar-H),
7.23 (1H, t, J = 7.6 Hz, Ar-H), 6.73 (1H, t, J = 8.0 Hz, Ar-H), 3.53-3.49 (2H, m, CH2), 2.91 (2H,
t, J = 6.8 Hz, CH2). 13C NMR (DMSO-d6) δ ppm 177.40, 145.07, 144.44, 139.62, 133.87,
132.59, 128.56, 128.43, 126.70, 125.98, 121.73, 115.34, 39.00, 24.21. Anal. Calcd for:
C16H15N5O2S C, 56.29; H, 4.43; N, 20.51; O, 9.37; S, 9.39. Found; C, 56.34; H, 4.42; N, 20.54;
O, 9.42; S, 9.41.
(E)-2-(6-methyl-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)hydrazinecarbothioamide 3e
mp 176-183°C. IR (KBr, cm-1): 3287, 2979, 1582, 1513 1H NMR (400 MHz, DMSO-d6): δ ppm
10.29 (1H, s, NH), 8.43 (1H, s, Ar-H), 8.32 (1H, s, NH), 8.24 (1H, s, NH), 8.12 (1H, s, Ar-H),
7.89 (1H, s, Ar-H), 3.44-3.40 (2H, m, CH2), 2.80 (2H, t, J = 6.8 Hz, CH2) 2.25 (3H, s, CH3). 13C
NMR (DMSO-d6) δ ppm 178.71, 143.30, 142.25, 133.86, 131.77, 126.95, 124.21, 121.36, 38.52,
23.61, 19.45. Anal. Calcd for: C11H13N5O2S C, 47.30; H, 4.49; N, 25.07; O, 11.46; S, 11.48.
Found; C, 47.32; H, 4.51; N, 25.11; O, 11.48; S, 11.46.
(E)-N-methyl-2-(6-methyl-8-nitro-2,3-dihydroquinolin-4(1H)-
ylidene)hydrazinecarbothioamide 3f
mp 178-183°C. IR (KBr, cm-1): 3295, 2919, 1628, 1545; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.37 (1H, s, NH), 8.56 (1H, d, J = 4.4 Hz, Ar-H), 8.36 (1H, s, NH), 8.28 (1H, s, NH), 7.89 (1H,
s, Ar-H), 3.06 (3H, d, J = 4.4 Hz, CH3), 2.78 (2H, t, J = 6.8 Hz, CH2), 2.50 (2H, t, J = 2.0 Hz
13
CH2), 2.27 ( 3H, s, CH3). C NMR (DMSO-d6) δ ppm 178.92, 143.86, 142.74, 134.14, 132.25,
127.50, 124.59, 121.89, 39.03, 31.59, 24.11, 20.13. Anal. Calcd for: C12H15N5O2S C, 49.13; H,
5.15; N, 23.87; O,10.91; S, 10.93. Found; C, 49.15; H, 5.16; N, 23.89; O, 10.88; S, 10.94.
(E)-N-ethyl-2-(6-methyl-8-nitro-2,3-dihydroquinolin-4(1H)-
ylidene)hydrazinecarbothioamide 3g
mp 180-184°C. IR (KBr, cm-1): 3316, 2914, 1600, 1506; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.24 (1H, s, NH), 8.59 (1H, t, J = 5.6 Hz, NH), 8.34 (1H, s, Ar-H), 8.27 (s, 1H, NH), 7.92 (1H,
s, Ar-H), 3.68-3.61 (2H, m, CH2), 3.45-3.41 (2H, m, CH2), 2.79 (2H, t, J = 6.8 Hz, CH2), 2.29
(3H, s, CH3), 1.18 (3H, t, J = 7.2 1Hz, CH3). 13C NMR (DMSO-d6) δ ppm 177.42, 143.59,
142.28, 133.69, 131.81, 127.09, 124.07, 121.42, 38.56, 38.41, 23.67, 19.72, 14.60. Anal. Calcd
for: C13H17N5O2S C, 50.80; H, 5.57; N, 22.78; O, 10.41; S, 10.43. Found; C, 50.84; H, 5.59; N,
22.77; O, 10.42; S, 10.42.
(E)-2-(6-methyl-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)-N-
phenylhydrazinecarbothioamide 3h
mp 179-185°C. IR (KBr, cm-1): 3370, 2926, 1625, 1542; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.69 (1H, s, NH), 10.12 (1H, s, NH), 8.50 (1H, s, Ar-H), 8.33 (1H, s, NH), 7.94 (1H, s, Ar-H),
7.53 (2H, d, J = 7.6 Hz, Ar-H), 7.39 (2H, t, J = 7.6 Hz, Ar-H), 7.24 (1H, t, J = 7.2 Hz, Ar-H),
20 | P a g e

2.86 (2H, t, J = 6.8 Hz, CH2), 2.51-2.50 (2H, m, CH2), 2.26 (3H, s, CH3). 13C NMR (DMSO-d6)
δ ppm 177.49, 145.29, 142.89, 139.67, 134.69, 132.28, 128.61, 127.86, 127.05, 126.13, 124.69,
121.61, 39.05, 24.41, 20.09. Anal. Calcd for: C17H17N5O2S C, 57.45; H, 4.82; N, 19.70; O, 9.00;
S, 9.02. Found; C, 56.48; H, 4.85; N, 19.66; O, 9.06; S, 9.05.
(E)-2-(6-chloro-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)hydrazinecarbothioamide 3i
mp 180-186°C. IR (KBr, cm-1): 3327, 2984, 1603, 1561; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.28 (1H, s, NH), 8.62 (1H, d, J = 2.4 Hz, Ar-H), 8.43 (1H, s, NH), 8.31 (2H, d, J = 12.4 Hz,
NH2), 8.05 (1H, d, J = 2.4 Hz, Ar-H), 3.48-3.45 (2H, m, CH2), 2.83 (2H, t, J = 6.8 Hz, CH2). 13C
NMR (DMSO-d6) δ ppm 179.26, 143.15, 142.61, 132.59, 132.34, 126.54, 123.86, 119.49, 38.85,
23.75. Anal. Calcd for: C10H10N5O2SCl C, 40.07; H, 3.36; N, 23.37; O, 10.68; S, 10.70; Cl,
11.83. Found; C, 40.09; H, 3.38; N, 23.40; O, 10.71; S, 10.68; Cl, 11.85.
(E)-2-(6-chloro-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)-N-
methylhydrazinecarbothioamide 3j
mp 183-188°C. IR (KBr, cm-1): 3287, 2979, 1682, 1513; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.42 (1H, s, NH), 8.74 (1H, d, J = 4.4 Hz, Ar-H), 8.57 (1H, d, J = 2.4 Hz, NH), 8.49 (1H, s,
NH), 8.06 (1H, d, J = 2.4 Hz, Ar-H), 3.05 (3H, d, J = 4.4 Hz, CH3), 2.80 (2H, t, J = 6.8 Hz,
CH2), 2.51-2.50 (2H, m, CH2). 13C NMR (DMSO-d6) δ ppm 178.91, 143.13, 142.47, 132.67,
132.05, 126.54, 123.93, 119.35, 38.86, 31.62, 23.74. Anal. Calcd for: C11H12N5O2SCl C, 42.11;
H, 3.85; N, 22.32; O, 10.20; S, 10.22; Cl, 11.30. Found; C, 42.12; H, 3.86; N, 22.36; O, 10.21; S,
10.23, Cl, 11.29.
(E)-2-(6-chloro-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)-N-
ethylhydrazinecarbothioamide 3k
1
mp 181−186°C. IR (KBr, cm-1): 3313, 2925, 1565, 1529; H NMR (400 MHz, DMSO-d6): δ
ppm 10.23 (1H, s, NH), 8.76 (1H, t, J = 6.0, NH), 8.53 (1H, d, J = 2.8 Hz, Ar-H), 8.44 (1H, s,
NH), 8.06 (1H, d, J = 2.8 Hz, Ar-H), 3.69-3.62 (2H, m, CH2), 3.49-3.45 (2H, m, CH2), 2.82 (2H,
t, J = 6.8 Hz, CH2), 1.18 (3H, t, J = 7.2 Hz, CH3). 13C NMR (DMSO-d6) δ ppm 177.94, 143.12,
142.59, 132.69, 132.03, 126.54, 123.94, 119.31, 38.92, 38.89, 23.76, 15.00. Anal. Calcd for:
C12H14ClN5O2S C, 43.97; H, 4.30; Cl, 10.82 N, 21.37; O, 9.76; S, 9.78. Found; C, 44.03; H,
4.35; Cl, 10.76 N, 21.35; O, 9.66; S, 9.81.
(E)-2-(6-chloro-8-nitro-2,3-dihydroquinolin-4(1H)-ylidene)-N-
phenylhydrazinecarbothioamide 3l
mp 182-186°C. IR (KBr, cm-1): 3331, 2928, 1626, 1519; 1H NMR (400 MHz, DMSO-d6): δ ppm
10.72 (1H, s, NH), 10.29 (1H, s, NH), 8.73 (1H, s, Ar-H), 8.72 (1H, s, Ar-H), 8.52 (1H, s, NH),
8.08 (1H, d, J = 2.4 Hz, Ar-H), 7.51(2H, d, J = 2.4 Hz, Ar-H), 7.41 (2H, t, J = 8.0 Hz, Ar-H),
7.26 (2H, t, J = 7.6 Hz, Ar-H), 3.52-3.48 (2H, m, CH2), 2.90 (2H, t, J = 6.8 Hz, CH2). 13C NMR
(DMSO-d6) δ ppm 177.27, 143.30, 142.77, 139.26, 132.23, 132.13, 128.09, 126.84, 126.33,
125.72, 123.18, 118.88, 38.43, 23.55. Anal. Calcd for: C16H14N5O2SCl C, 51.13; H, 3.75; N,
18.63; O, 8.15; S, 8.53; Cl, 9.43. Found; C, 51.15; H, 3.76; N, 18.61; O,8.17; S, 8.55; Cl, 9.41.
21 | P a g e

X-ray Crystallography
The crystallographic data collections and their relevant calculations of crystal and molecular
structure elucidation were done with Bruker AXS KAPPA APEX-2 diffractometer equipped with a
graphite monochromator. The crystal and molecular structures were solved by direct methods and
refined by full-matrix least-squares calculations using SHELXL–2014. The completion of the structure
refinement and the convergence of the structure is affirmed by the reliability index (R-factor) for F2 >
2(F2) of 4.33%. The unit cell parameters, details of data collection and structure refinement
parameters are given in Table 1. All the hydrogen atoms were positioned geometrically and refined
using a riding model, with C–H = 0.93 Å (for aromatic -CH) and 0.97 Å (for -CH2) and N-H = 0.86 Å
and Uiso(H) = 1.2 Ueq(parent atom). The structure of the molecules in the asymmetric part with the
atomic scheme is shown in Fig. 1 as a thermal displacement ellipsoid plot. Drawings of the molecular
structure and the packing diagram were obtained using Mercury and PLATON programs.
Crystallographic data of 3a, 3c, and 3i
3a
C10H11N5O2S
265.3
3c
C12H15N5O2S
586.7
3i
C10H10ClN5O2S
599.48
Molecular formula
Formula weight
Temperature
293(2) K
293(2) K
293(2) K
0.71073 Å
Monoclinic
P 21/c
0.71073 Å
Triclinic
P -1
Wavelength
Crystal system
Space group
0.71073 Å
Triclinic
P -1
a = 10.2262(10) Å;
 = 90°.
b = 14.7417(14) Å;
 = 99.997(2) °.
c = 8.2269(8) Å;
a = 5.257(16) Å;
 = 62.49(4)°.
b = 16.30(5) Å;
 = 81.61(3)°.
c = 17.51(6) Å;
Unit cell dimensions
a = 8.2875(8) Å;
 = 74.673(2)°.
b = 12.0212(12) Å;
 = 88.191(2)°.
c = 14.5640(15) Å;
 = 79.993(2)°.
1377.9(2) Å3, 2
1.445 Mg/m3
0.434 mm-1
 = 90°.
 = 84.90(4)°.
1221.4(2) Å3 , 4
1.443 Mg/m3
0.268 mm-1
1316(7) Å3, 2
1.480 Mg/m3
0.256 mm-1
Volume, Z
Density (calculated)
Absorption
coefficient
552
616
F(000)
Crystal size
616
0.24  0.20  0.18 mm3
0.21 × 0.18 × 0.15
mm3
0.22  0.18  0.14
mm3
2.022 to 24.992°.
2.329 to 24.999°.
Theta range for data
collection
1.783 to 24.996°.
Index ranges
-12h12, -17k17, - -6h6, -19k19, -
-9  h  9, -14  k 
14, -17  l  17
13243
9l9
20l20
11296
27840
Reflections collected
2146 [R(int) = 0.0309] 4620 [R(int) = 0.0355]
Independent
reflections
4836 [R(int) =
0.0270]
97.20% 97.00%
Completeness to
97.10%
22 | P a g e

theta = 25.242°
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Refinement method
Full-matrix least-
squares on F2
2146 / 0 / 188
4620 / 0 / 387
Data / restraints /
parameters
4836 / 0 / 343
1.14
1.062
Goodness-of-fit on
F2
1.054
R1 = 0.0498, wR2 =
0.1153
R1 = 0.0312, wR2 =
0.0730
Final R indices
[I>2sigma(I)]
R1 = 0.0433, wR2 =
0.1083
R1 = 0.0627, wR2 =
0.1219
0.281 and -0.173 e.Å-3
R1 = 0.0400, wR2 =
0.0771
0.248 and -0.315 e.Å-3
R indices (all data)
R1 = 0.0589, wR2 =
0.1154
Largest diff. peak
and hole
0.441 and -0.242 e.Å-
3
Materials
MTT (3-(4,5- dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), DAPI (4’,6-
diamidino-2-phenylindole), AO (Acridine orange), EB (Ethidium Bromide), DCFH-DA (2’,7’-
dichlorodihydrofluorescein diacetate), FITC (fluorescent isothiocyanate), PI (propidium iodide)
and other analytical grade chemicals and reagents were obtained from Sigma-Aldrich and Hi-
Media. The human breast cancer (MCF-7) and cervical cancer (HeLa) were purchased from
National Center for Cell Sciences (NCCS), Pune, India and were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 1% (v/v) antibiotic antimycotic solution
and 10% (v/v) fetal bovine serum. The cells were grown and maintained in the incubator at 37° C
in 5% CO2 atmosphere. The compound’s stock solution was prepared in sterile phosphate-
buffered saline (PBS) and the stock solutions were diluted by using DMEM to obtain required
concentrations. The phase-contrast microscope (Nikon ECLIPSE, TS100) was used to identify
the morphological changes of non treated control and compounds treated cells. The animal
experimental studies were performed by using female BALB/c nude mice of about a month and
half old after acquiring earlier endorsement of the Institutional Animal Ethical Committee,
adhering to the guidelines of the committee for control and supervision of experiments on
animals constituted by the Animal Welfare Division of India.
In vitro Cytotoxicity study
Cell survival was estimated by the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. The cells were seeded in 100 mL of growth medium at a
density of 4000 cells/well in 96-well plate and permitted to set up for 24 h. Then the medium in
wells was placed with 100µL of growth medium containing serially diluted compounds. The
cells were then incubated for 72 h at 37° C in humidified 5% CO2, at which time the growth
media was drawn and change with MTT in IMDM growth media and incubated for 4 hours. The
MTT solution was then suctioned from the wells, and then 100 mL of acidified ethanol was
added to each well and allowed to 15 min for lysis step, the viability of cells was estimated
spectrophotometrically by absorbance at 570 nm and foundation revised at 690 nm. The assay
23 | P a g e

was performed three times in triplicate. The IC50 values were determined as the drug
concentration that reduced the absorbance to 50% of that in untreated control wells and got from
in any event three separate trails.
Acridine Orange / Ethidium Bromide (AO/EB) staining assay
The compounds treated cells were stained with AO/EB dual dye to detect apoptotic cells.
The cells were grown in 24-well plate and treated with synthesized compounds at IC50
concentrations. After incubation, the culture media were removed and washed twice with PBS.
Fresh washed media were added in each well and stained by adding 10 µL of AO/EB mix (10
mg mL-1 of AO and EB in PBS) and then incubated for 10 minutes. After incubation, the cells
were washed with PBS and fluorescent morphological changes were recorded by using a
fluorescence microscope (Nikon ECLIPSE, TS100) with an excitation filter of 480/30 nm.
Detection of intracellular ROS production
The intracellular ROS generation of compounds treated cells was determined by DCFH-
DA (2’,7’-dichlorodihydrofluorescein diacetate) staining assay. Initially, the tested cells were
seeded in 24-well plate and grown for 24 h, and then the cells were treated with the compounds
for 24 h at IC50 concentration. After treatment, the cells were washed twice with PBS. The
washed treated cells were suspended in 1mL of DMEM with 5 µM DCFH-DA and incubated for
10 min at 37°C. After incubation, the cells were investigated under flow cytometer for DCF
fluorescence response at an excitation wavelength of 488 nm emission wavelength of 530 nm.
The fluorescence information was recorded with the Cell Quest program (BD biosciences). The
Win MDI software was used to analyze flow cytometric data and the ROS generation was
determined by the DCF green fluorescence of cells and expressed in the terms of percentage of
cells. The fluorescence images of cells were recorded by using a fluorescence microscope (Nikon
ECLIPSE, TS100).
DAPI Staining
The nuclear damage of compounds treated cells were detected by DAPI (4’,6-diamidino-
2-phenylindole) staining method. Initially, the tested cells were seeded in a 24-well plate and
treated with compounds for 24 h at IC50 concentration. After incubation, the cells were fixed in
methanol: acetic acid (3:1 v/v) and stained with 5 µg/mL of DAPI and incubated for 20 min.
After incubation, the nuclear damaged blue fluorescence cells were analyzed by using a
fluorescence microscope (Nikon ECLIPSE, TS100) excitation filter at 510 to 590 nm.
FITC Annexin V-FITC PI staining
The apoptotic cancer cells were detected by Annexin V-FITC and propidium iodide (PI)
staining method by using Flow cytometry analysis. In this method, the cancer cells were treated
with the synthesized compounds for 24h at IC50 concentration. The treated cancer cells were
suspended in binding buffer (200µL), and the suspension was stained with 10µL of Annexin V-
FITC and 5µL of propidium iodide (PI) for 20-30 minutes in dark at 25°C. After the incubation,
300 µL of binding buffer was gently mixed with cell suspension and the sample was analyzed by
Flow cytometer (BD, FACS Calibur, USA). The early apoptotic cells bind only with Annexin
V/FITC, while the late apoptotic or dead cells bind with both Annexin V-FITC and PI.
24 | P a g e

RT-PCR gene expression analysis
The apoptotic genes expressed in the compounds treated cancer cells were examined by
RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) gene expression analysis. In this
method, the cells were grown in 24-well plate and treated with compounds for 24 h at IC50
concentrations. After treatment, the expression of apoptotic signaling genes, caspase-3 and Bcl-2
were examined by RT-PCR using β-actin as the internal control. Total RNA was isolated from
treated cancer cells in each well by using the conventional triazole method. 3µg of RNA was
used to cDNA conversion by reverse transcription of M-MLV Reverse Transcriptase. Then semi-
quantitative PCR analysis was carried out to quantify the expression of caspase-3 and Bcl-2 by
using the above RT product. The PCR products were resolved on an agarose gel and stained with
ethidium bromide to visualize under UV light. The reactions were performed in triplicate. The
primers used were
β actin
Forward primer: 5´-CTGTCTGGCGGCACCACCAT-3´
Reverse primer: 5´-GCAACTAAGTCATAGTCCGC-3´,
Bcl-2
Forward primer: 5´-AGATGTCCAGCCAGCTGCACCTGAC-3´
Reverse primer: 5´-AGATAGGCACCCAGGGTGATGCAAGCT-3´,
Caspase-3
Forward primer: 5´-TTTGTTTGTGTGCTTCTGAGCC-3´
Reverse primer: 5´-ATTCTGTTGCCACCTTTCGG-3´.
Analysis of DNA double-strand breaks
DNA double-strand breaks of compounds treated cells were analyzed by the DNA-
agarose gel electrophoresis method. In this method, the cells were seeded in a 24-well plate and
treated with the compounds for 24 h at IC50 concentration. After treatment, the cells were
collected and washed with PBS and lysed (Lysis solution: 10 mM of Tris HCL pH- 7.4, EDTA
and 0.5% of Triton X-100). The cell lysates were incubated for 1h with 200 mg/mL of RNase A
followed by 200 mg/mL of proteinase K at 37° C. After incubation the samples were extracted
with chloroform and DNA was precipitated by using 0.3 M of sodium acetate at -20° C. Finally
the DNA samples were kept running on 1.5% agarose gel at 60V and visualized by ethidium
bromide staining under UV light.
Statistical Analysis
Statistical analysis was performed with the Statistical Program for Social Sciences
software (SPSS). All data were expressed as means ± standard deviation, and a statistically
significant difference was considered to be present at *p<0.05 as significant or **p<0.01 as
highly significant. All assays were carried out in triplicates with three independent experiments.
25 | P a g e

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