Synthesis and X-ray study of dispiro 8-nitroquinolone analogues and their cytotoxic properties against human cervical cancer HeLa cells

Article abstract of DOI:10.1039/c8md00482j

A series of unique dispiro analogues containing an oxindole pyrrolidine 8-nitroquinolone hybrid has been obtained through a one-pot three-component 1,3-dipolar cycloaddition of azomethine ylides generated in situ from the condensation of isatins and benzylamine with (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones. The structures of the newly synthesized compounds were characterized by using different spectroscopic techniques and by X-ray diffraction studies of their regio- and stereochemistry. All the synthesized compounds were screened for in vitro cytotoxic activity against the human cervical cancer cell line HeLa. The compounds have exhibited potent inhibition against human cervical cancer cells and insignificant toxicity to normal cells. The compounds 6d, 6a, 6h, 6b, and 6e induced apoptosis of HeLa cells, through ROS influx. The expression levels of proteins involved in the mitochondrion-related pathways were detected, and Western blot analysis showed that apoptosis occurred via activation of caspase-3.

Full text of DOI:10.1039/c8md00482j

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Med. Chem. Commun., 2019, DOI: 10.1039/C8MD00482J.
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DOI: 10.1039/C8MD00482J
Synthesis and X-ray Study of Dispiro 8-Nitroquinolone Analogues and Their Cytotoxic
Properties Against Human Cervical Cancer Cells HeLa
Selvaraj Shyamsivappana, Raju Vivekb, Arjunan Saravananc, Thangaraj Arasakumara, Gopalan
Subashinid, Thangaraj Suresha, Ramasamy Shankare, Palathurai Subramaniam Mohana,*
aSchool of Chemical Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India.
bChemical Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
cDRDO-BU CLS, Bharathiar University campus, Coimbatore, Tamil Nadu, India.
dDepartment of Chemistry, P.S.G.R. Krishnammal College For Women, Coimbatore, Tamilnadu,
India
e Department of Physics, Bharathiar University, Coimbatore, Tamil Nadu, India
Corresponding author: psmohan59@gmail.com and ps_mohan_in@yahoo.com
Abstract
A series of unique dispiro analogues containing oxindole pyrrolidine 8-nitroquinolone
hybrid has been accomplished through a one-pot three-component 1,3-dipolar cycloaddition of
azomethine ylides generated in situ from the condensation of isatins and benzylamine with (E)-
3-arylidene-2,3-dihydro-8-nitro-4-quinolones. The structures of newly synthesized compounds
were characterized by using different spectroscopic techniques and by X-ray diffraction studies
on their regio- and stereochemistry. All the synthesized compounds were screened for In vitro
cytotoxic activity against human cervical cancer cell line HeLa. The compounds have exhibited
potent inhibition against human cervical cancer cells and insignificant toxicity to normal cells.
The compounds 6d, 6a, 6h, 6b, and 6e induced apoptosis of HeLa cells, through ROS influx.
The expression levels of proteins involved in the mitochondrion-related pathways were detected,
Western blot analysis showed that apoptosis occurred via activation of caspase-3.
Keywords
dispiro-oxindole; 1,3-dipolar cycloaddition; cytotoxic; apoptosis
Introduction
Since genesis, cancer remains one of the major health alarming issues in worldwide.1, 2
According to the census, cancer is the second most cause of human mortality after cardiovascular
disease.3-5 Especially cervical cancer is one of the rapidly proliferating tumor prevailing around
the globe. Apart from current standards of treatment for cervical cancer that include surgery
and irradiation techniques, chemotherapy remains a reliable alternative approach for advanced
metastatic cervical cancer therapy.6 However, due to a few confinements and disadvantages,
such as difficulties in isolation, undesirable side effects, intricacy in synthesis, and multidrug
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protection, there is no assured treatment for some of the advanced cancer tumors. To overcome
these limitations, a synthetic investigation of new chemotherapeutics with specified mechanistic
action with upgraded ampleness is very much warranted to take it to clinical studies.
In the therapeutic context, quinoline analogues have wide occurrence among natural
products and exhibit diversified biological activities depending on the structure type.7
Particularly a large variety of quinolines are reported to exhibit substantial anti-cancer activity
through a variety of mechanisms, such as inhibition of dehydrogenase 1A1 (ALDH1A1),8 cell
cycle arrest in the G2 phase,9 Inhibition of Ataxia Telangiectasia Mutated (ATM) kinase,10
inhibition of topoisomerase,11 inhibition of tubulin polymerization,12 inhibition of Heme
Oxygenase-1,13 and inhibition of tyrosine kinases14 etc. Among the numerous varieties of natural
products, we have found that 2,3 dihydro-4-quinolone ring system is found to exhibit interesting
pharmacological properties.15-17 Especially nitro group attached quinolines have shown
considerable influence on inhibiting cancer cell growth.18-20 Recently, we have reported such
type of 8-nitroquinolone derivatives which have specific biological activities, like cell
reinforcement antibacterial and anticancer activities.21
Further, the pronounced anticancer activity of structurally diverse spiro pyrrolidine
oxindoles was described in a number of recent publications.22-24 The spirooxindole-pyrrolidine
core exhibit various biological functions such as targeting tubulin polymerization,25 inhibition of
PLK4,26 inhibition mammalian cell cycle at G2/M phase,27 inhibition of p53-MDM2,28 29 etc.
Based on these observations our strategy was oriented towards the synthesize of novel quinoline
encapsulated spiro oxindole system.
Despite the presence of various synthetic methodologies, the multicomponent 1,3-dipolar
cycloaddition of electron-deficient alkenes with azomethine ylides seems very attractive to
access spiro pyrrolidine containing compounds due to the simplicity, mild reaction conditions,
and atom economy.30 In particular, azomethine ylides generated in situ from the decarboxylative
condensation of α-amino acids and isatins are most common. Herein, we report the
multicomponent 1,3-dipolar cycloaddition of (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones
with azomethine ylides generated in situ from isatins and benzylamine to afford a new series of
dispiro 8-nitroquinolone analogues. All the synthesized compounds were examined for their
regioselectivity, high yielding MCR conversion, and cytotoxic impacts on human cervical cancer
HeLa cell line by MTT assay. The toxicity of selected potent compounds was evaluated by their
cell morphology and cell death that were assessed by staining methods that clearly to indicate
cell death that was actuated by apoptosis. We have also evaluated the potential cytotoxic
mechanism of selective potent dispiro 8-nitroquinolone analogue 6d that involved caspase-3
dependent apoptosis.
2. Results and Discussion
2.1 Chemistry
The synthesis of dispiro 8-nitroquinolone analogues 6a-l were achieved through a 1,3-
dipolar cycloaddition reaction protocol. Initially, the dipolarophiles 3a-d were synthesized by
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Knoevenagel condensation of 8-nitro-2,3-dihydro-1H-quinolin-4-one 1 with aromatic aldehydes
2a-d in the presence of pyrrolidine base in ethanol affording (E)-3-arylidene-2,3-dihydro-8-nitro-
4-quinolones 3a-d in quantitative yields.31 In the next step one pot three-component 1,3-dipolar
cycloaddition reaction carried out between (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones 3a-
d, and the dipole generated from isatin derivatives 5a-c and benzylamine 4 (Scheme 1).
O
O
R1
NH
O
O
MeOH
Reflux
Pyrrolidine
Rt, EtOH
N
H
N H
N
H
R
1
H
3a-d
R1
NO2
NO2
O
H
N
H
NH2
OHC
NO2
O
N
R
H
6a-l
4
5a-c
R
2a-d
R = Cl, Br, F, OCH3 R1 = Cl, Br, F
Scheme 1. Synthesis of dispiro 8-nitroquinolone analogues 6a-l
In order to optimize the reaction conditions, a one-pot three-component reaction of (E)-3-
(4-bromobenzylidene)-8-nitro-2,3-dihydroquinolin-4(1H)-one 3b(1mmol), benzylamine 4 (1.2
mmol) and 5-chloroisatin 5a were chosen as a model reaction, kept under reflux condition in
various solvents (Table 1). From optimization study, we have observed that the best result was
obtained when the methanol solvent was used under reflux condition for 2 h, which provides
dispiro oxindole pyrrolidine 8-nitroquinolone 6b with an excellent yield (96%) (Table 1, entry
5). We have extended further, by replacing the substituent in the aryl group (OCH3, Cl, Br, F,)
and isatin (Cl, Br, F) but found no impact on the yield of the products (Scheme 1, Table1).
Table 1 Optimization of the reaction condition for the synthesis of compound 6b
S. No
Solvent
Toluene
1,4- Dioxane
Acetonitrile
Ethanol
Temperature Time (h) Yield a (%)
1
2
3
4
5
6
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
10
12
12
4
2
10
65
63
54
83
96
-
Methanol
Tetrahydrofuran
a Isolated yield after recrystallization.
With the optimized results in hand, all subsequent cycloaddition reactions were explored
by refluxing a mixture of (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones 3a-d (1mmol),
benzylamine 4 (1.2 mmol) and isatin 5a-c in MeOH for 2-3 h.
The structure of the synthesized cycloadduct 6a-l was characterized by IR, 1H NMR, 13C
NMR, HR mass, X-ray diffraction, and elemental analysis. The IR spectrum of compound 6b
showed two intense absorption bands at 1700 and 1614 cm-1 characteristic for 8-nitro-4-
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1
quinolone and oxindole ring carbonyl functionalities respectively. The H NMR spectrum of
product 6b showed a sharp singlet at δ 10.52 and the doublet, resonating at δ 8.37 which were
accounted for isatin and quinoline NH. Multiplet at δ 4.06-3.99 has accounted for quinoline N-
CH2 and the pyrrolidinylmethine proton that appeared as a doublet at δ 4.57 (J = 10.0 Hz)
demonstrating the regiochemistry of the cycloaddition reaction. In case of other possible
regioisomer 6′l, the pyrrolidinylmethine proton would have appeared as a singlet in the 1H NMR
spectrum. In 13C NMR spectrum, the peak at δ 45.90 assignable to quinolone N-CH2 carbon, at δ
54.07, 58.42 for pyrrolo C-H carbons, at δ 63.05, 69.78 for spirocarbons, at 179.56, and 191.04
for amide and 4-quinolone carbonyl carbons respectively. In the HR mass spectrum, the presence
of a molecular ion peak at m/z = 631.0583 confirmed the formation of 6b. The regio- and
stereochemistry of the cycloaddition product 6l were further confirmed by single crystal X-ray
analysis (Figure 2).
10.52, s
Cl
69.78
179.56
2.52, d, 1H, J = 2.0 Hz)
NH
O
N H
191.04
4.06-3.99, m
45.90
O
H
5.43 J = 5.2 Hz
63.05
H
N
H
NO2
8.37, d, J = 5.2Hz
Br
4.57, d, J = 10.0 Hz
6b
Figure 1. 1H and 13C NMR chemical shift values for products 6b
A plausible mechanism for the regio- and stereoselective formation of the hybrid
heterocycles 6a is portrayed in Scheme 3. The azomethine ylide 8 generated in situ from the
reaction of isatin 4 with benzylamine 5. The subsequent reaction of 8 with the electron deficient
β-carbon of the dipolarophile 3a-d furnished the cycloadducts 6a-l as a single regioisomer.
Despite the presence of four stereocenters in the cycloadducts 6a-l, it leads to the selective
formation of only one of the possible diastereomers, since the electron-rich carbon of the dipole
adds to the electron-deficient β-carbon of the α,β-unsaturated system via path A, which is not
possible in path B.
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NH2
R1
O
MeOH
N
MeOH
R1
H
O
N
N
-H2O
O
O
H
5
4
NH
NH
R1
R1
8
7
R1
H
NH
O
N H
12
HN
O
N
63
path A
O
H
H
O
N
H
NO2
28
27
N
R
H
R
6a-l
3a-d
NO2
O
NH
63
R1
12
N
O
H
NH
H
pathB
R1
O
N
NH
H
H
O
NO2
28
27
N
R
H
NO2
R
6'a-l
3a-d
Scheme 2. The Plausible mechanism of formation of compound 6a-l
X-ray Crystallography
The structure of compound 6l was further confirmed by single crystal X-ray analysis (Figure 2)
Figure 2. ORTEP diagram of compound 6l
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Molecular geometry and crystal packing
The triclinic centrosymmetric lattice of compound 6l contains two molecules in the unit
cell. The convergent of the structure solution is confirmed from the R factor of 4.8 %.
According to Cremer & Pople conformational analysis,32 the central five-membered ring is
observed to be in envelope conformation on nitrogen atom (N3) with the puckering values of q2
= 0.363(2) Å and 2 = 176.0(3). The phenyl ring is oriented with the dihedral angles of 30.2(1),
62.0(1) and 32.6(1) with fluorophenyl, methoxyphenyl and nitrophenyl groups, respectively.
Table 2. Hydrogen bonding geometry in 6l crystal
D-H...A
N(2)-H(2)...O(2)#1
N(4)-H(4)...O(1)#2
C(16)-H(16)...O(4)
C(9)-H(9)...O(1)
C(32)-H(32A)...O(1)
N(2)-H(2)...O(2)
d(D-H)
0.86
0.86
0.98
0.98
0.97
0.86
d(H...A)
2.32
d(D...A)
2.982(2)
2.872(2)
2.828(2)
3.147(2)
3.132(2)
2.618(2)
<(DHA)
134
2.03
168
2.30
113
2.58
117
2.50
123
2.03
125
Symmetry transformations used to generate equivalent atoms: #1 -x+2,-y+1,-z+1 #2 -x+1,-y+1,-z+1
Figure 3. Packing of the molecules 6l in triclinic unit cell is viewed down along a-axis. H-bonds
are shown as dashed lines.
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The hydrogen bonding dimensions are listed in Table 2 and the packing arrangement of
molecules is depicted in Figure 3. Intermolecular interactions, especially classical and non-
classical hydrogen bonds, are playing a crucial role in the formation of crystalline solids and
their physiochemical properties. These hydrogen bonding interactions can be classified and
notated with graph-set nomenclature which is useful in comparing the stability molecular
conformations and crystalline lattices between the similar molecules.33 The molecular structure
features intramolecular classical N-H...O and non-classical C-H...O interactions. The classical
intramolecular hydrogen bond leads to self-associated S(6) motif. Furthermore, the crystal
packing is stabilized through C-H...O and - interactions. The substituted Fluorine is expected
to participate in the intermolecular interactions. But, it is observed to be a hydrophobic region of
the crystalline lattice. The different hydrogen bond motifs with the necessary graph-set notations
2
are depicted in Figure 4. Through the two intermolecular N-H...O hydrogen bonds, ring R2 (8)
1
and R2 (4) motifs are formed. A bifurcated or three centered N-H...O hydrogen bond is observed
around N2 donor atom.
2
1
Figure 4. Self associated ring S(6), ring R2 (8) and R2 (4) motifs are shown. H-bonds are shown
as dashed lines.
Quantum Chemical Analysis
The relative stereochemistry of cycloadduct 6l, was studied by the quantum chemical
density functional theory (DFT) method. The Frontier molecular orbital (FMO) and Fukui
calculations were carried out to understand the regioselectivity of cycloadduct 6l. The FMO
calculation revealed that the energy gap between HOMO 8 –LUMO 3c (6.029 eV) is higher than
that of LUMO8-HOMO 3c (4.373 eV) (Figure 5). This suggests that the HOMO orbital of
dipole 8 (azomethine ylide) and LUMO of dipolarophile 3c interaction (normal electron demand
condition) is more feasible than the HOMO of dipolarophile 3c and LUMO of a dipole 8 (inverse
electron demand condition) due to the lower energy gap, which is in good agreement with the
experimental observation.
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.
Figure 5. HOMO and LUMO energies of dipole 8 and dipolarophile 3c calculated at the
B3LYP/6-31G* level of theory.
+
The DFT based local chemical reactivity fukui function parameters, for nucleophilic (fk )
-
and electrophilic attack (fk ), were calculated through the NBO atomic charges. From the
calculated local chemical reactivity parameters (Table 3) the reaction mechanism was at C63 site
-
of azomethineylide 8 which shows fk value -0.0058 a.u. and attack the C28 position of
+
+
dipolarophile 3c (fk value 0.0067 a.u.). Further at the C27 site (fk value 0.0092a.u.) of
dipolarophile 3c, and it underwent nucleophilic attack on C12 site (0.0101 a.u.) of
azomethineylide 8. These results were well matched with the experimental observation.
Table 3. Local chemical reactivity fukui function parameters
+
-
Reactant
Site fk (a.u ) fk (a.u)
Dipolarophile 3c
Dipolarophile 3c
C27 0.0092
C28 0.0067
0.0028
-0.1273
Azomethineylide 8 C12 -0.1817 0.0101
Azomethineylide 8 C63 -0.1656 -0.0058
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Figure 6. The transition state structures of reactant 8 and 3c and product 6l optimized by
B3LYP/6-31G* level of theory.
The stability of the product 6l was found to be more stable compared to the reactant 3c
and the corresponding total energies are found to be -1928.5826 and -1928.6386 by Hartrees at
above level of theory. Moreover, low activation energy and reaction energy indicate the
formation of compound 6l would quite favor’s at MO6-2X/6-31G⁄ levels. The results indicate the
formation of compound 6l was found to be exothermic in nature. All the computational
calculations were performed using the GAUSSIAN 09W program.34
The selected optimized geometrical values of theoretical (DFT) and single crystal XRD
values of the product 6l are presented in the supplementary table 2 and the corresponding
correlation coefficient graphs are shown in Figure 7. In addition to that, the selected
experimentally observed geometrical values of bond length, bond angle and torsional angle
values are correlated well with theoretically predicted values and the corresponding correlation
coefficient are found to be 0.997, 0.925 and 0.999 at M062x/6-31g** level of theory.
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Figure 7. Correlation coefficient graphs of a) Dihedral angle b) bond length and c) bond angle
values with XRD values
2.2 Biology
2.2.1 Invitro Cytotoxicity of synthesized compounds 6a-l against the human cancer cell line
The recent reports revealed that the nitroquinolines have a significant ability to inhibit
cancer cell growth. In particular, nitroquinolines were found to be most active against cervical
cancer cell line.19,20 Further the oxindole and pyrrole containing heterocycles have shown
promising anticancer activities against human cervical cancer cell line Hela.35,36 These reports
suggest that the quinoline, pyrrole, and oxindole containing compounds might possibly inhibit
the growth of cervical cancer cells. Hence, we used specified cervical cancer cell line HeLa to
evaluate the In vitro cytotoxic effects of the newly synthesized dispiro pyrrolo oxindole 8-
nitroquinolones analogues 6a-l.
The well-known anticancer drug Doxorubicin (Dox) was selected as the reference drug to
compare the in vitro anticancer potency of the synthesized compounds by MTT [3-(4,5-
dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] assay. The IC50 values of synthesized
compounds and Dox against tested cervical cancer cell line are summarized in Table 4. The
MTT experimental results have well demonstrated that all the compounds show the ability to
inhibit cell proliferation in a dose-dependent concentration manner. Generally, the synthesized
compounds had broad-spectrum of anticancer activity, showing favorable inhibition against the
tested cancer cell line HeLa (IC50< 27±1.5μM). Specifically, 6d exhibited excellent inhibition
against human cancer cell line HeLa with the IC50 values of 16±0.2 μM and the compounds 6a,
6h, 6b, and 6e also exhibit better activity, and the rest showed moderate activity.
Table 4. In vitro cytotoxic activity by MTT assay
IC50 of
(µM) HeLa
R1
S. No
R
Compounds
1
2
3
Cl
Br
F
Cl
Cl
Cl
6a
6b
6c
18±1.1
21±0.1
27±1.5
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4
OCH3
Cl
Cl
Br
Br
Br
Br
F
6d
6e
16±0.2
23±0.6
25±1.3
26±0.4
19±0.4
25±0.5
26±1.6
20±0.8
24±1.6
14±0.3
5
6
Br
6f
7
F
6g
8
9
OCH3
Cl
6h
6i
10
Br
F
6j
11
F
F
6k
12
OCH3
F
6l
Control
DOX
2.2.2 Structure-activity relationship (SAR)
Based on the result obtained from the MTT assay, we have tried to understand the role of
different substitutions on cytotoxic activity:
All the synthesized compounds 6a-l, have exhibited good cytotoxic activity. This might
be due to the presence of nitro and oxo group on quinoline ring and the substitution attached to
the pyrrolidine group of the synthesized spiro heterocycle. As it is clear from Table 4 that the
compounds 6a, 6b, 6d, 6e, and 6h are most active amongst reported compounds. Compound 6d
has an IC50 of 16±0.2μM against cervical cancer cell HeLa, which is close to the reference
compound doxorubicin (14±0.3 μM) and similarly compound 6h exhibit IC50 value 19±0.4 μM
against HeLa cell line respectively. From these results, we conclude that compound substituted
with electron donating methoxy group on aryl ring showed good activity, which revealed that
substitution on aromatic ring plays important role in the activity. From the comparison of
compound 6d and 6h, it is clear that chloro substitution on oxinode ring possesses more active
than bromo substitution as shown in Table 4. In the case of the 4-chlorophenyl group-bearing
compounds 6a and 6e the IC50 values show that the compound 6a is more active, which also
revealed that chloro substitution on oxinode ring induces more active than bromo substitution.
From the results, the other fluoro substituted compounds show moderate activity. From the SAR
studies the order of cytotoxicity effects of compounds is 6d> 6a> 6h> 6b> 6e.
2.2.3. Detection of cell death by morphological analysis
The potential cytotoxic effect of compounds on HeLa cells were investigated by
morphological changes and were observed under phase contrast microscopy. HeLa cells were
treated with compounds 6d, 6a, 6h, 6b, and 6e at IC50 concentration for 24 h. Figure 8 shows
that after treatment of HeLa cells, they were appeared to be swollen and detached from neighbor
cells with the completely disintegrated membrane, rounded, shrunken which indicate that the
synthesized compounds probably induce cell death at IC50 concentrations whereas, untreated
HeLa cells showed healthy and normal morphology.
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Figure 8. Morphological analysis of compounds 6d, 6a, 6h, 6b, and 6e treated HeLa cancer cells
compared with untreated HeLa as the control. The morphological changes like shrinkage and
rounding of apoptotic cells were observed under a phase-contrast microscope.
2.2.4. Hemolytic activity
To evaluate hemocompatibility and non-toxicity of compounds on normal cells the
hemolytic activity was performed. Human blood sample RBCs was used for experiment and the
human blood samples were provided by Mouthee hospital, Coimbatore, Tamil Nadu. PBS and
H2O were used as positive and negative control to compare the activity. It was observed that the
control solution wherein the erythrocyte suspension treated with distilled H2O in 1:1 ratio
appeared dark red due to the release of hemoglobin (Hb) from RBC lysis. However, the
erythrocyte suspension treated with compounds at IC50 concentrations did not appear in red
which indicates that no noticeable hemolytic effect on RBC. Our present hemolytic findings
validate the active compounds exhibited the low hemolytic activity of less than 7% when used at
IC50 concentration, whereas the water as negative control showed 100% hemolysis. The
experimental results are suggesting that the active compounds 6d, 6a, 6h, 6b, and 6e have
exhibited minimum or low toxicity to human blood cells.
2.2.5. The Compounds Induced Intracellular ROS production
The favorable potency of compounds 6d, 6a, 6h, 6b, and 6e toward HeLa cancer cells
prompted us to investigate the preferred mechanism of action. Very recently, quinolines have
been reported as anti-cancer agents by inducing oxidative stress-mediated DNA damage.37
Banerjee and co-workers reported a novel bis-arylidene oxindole which generated the excess of
ROS and eventually killed cancer cells via apoptosis.38 The oxindole alkaloids were extracted
from Uncaria tomentosa which induced apoptosis of HT29 cells through ROS-mediated
activation and DNA repair.39 Above studies suggest that both quinoline and oxindole containing
compounds could serve as ROS-generating agents for cancer therapy. From the literature, we
assume that our synthesized molecules incorporating oxindole and quinoline nucleus might
possibly induce cell death through ROS-mediated mechanisms.
Hence, we used the DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) assay to
determine the intracellular ROS levels. After, treatment of HeLa cells with selective active
compounds 6d, 6a, 6h, 6b, and 6e at IC50 concentration, following DCFH-DA treatment for 30
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min. In the presence of ROS, DCFH-DA is rapidly oxidized into fluorescence
dichlorofluorescein (DCF), which increases the green fluorescence. Interestingly, as shown in
Figure 9a, the synthesized compounds enhanced the green fluorescence intensity, the percentage
of cells with green fluorescence, significantly higher than that of the control. The increase of
ROS generation in treated HeLa cells were quantified by the determining of percentage cells
with the significantly increased high level of green fluorescence in a flow cytometer Figure 9b.
This result supported the hypothesis that the selective active compounds 6d, 6a, 6h, 6b, and 6e
induced cell death mainly through ROS-mediated mechanisms.
Figure 9. a) Detection of intracellular ROS of compounds 6d, 6a, 6h, 6b, and 6e treated HeLa
cancer cells compared with untreated HeLa as control by fluorescence microscope b) ROS
fluorescence intensity of HeLa cells after incubation of compounds compared with positive
control H2O2
2.2.7. Detection of apoptotic cells by DAPI staining of nuclear damage
DAPI (4’, 6-diamidino-2-phenylindole) is a prevalent nuclear fluorescent stain which
strongly binds to the adenine-thymine clusters of the minor section of two-fold stranded DNA
and distinguishes the chromatin condensation or nuclear damage. Binding of DAPI to DNA
produces enhanced fluorescence clearly owing to the removal of water molecules from both
DAPI and the minor groove. DAPI stains the apoptotic cells as bright colored due to the
compressed nucleus that indicates a typical apoptotic feature.
Fluorescence microscopy analysis of DAPI stained cells was undertaken to study nuclear
changes and apoptotic body development, both of which are also involved in the apoptosis.40 The
cervical cancer HeLa cells treated with compounds 6d, 6a, 6h, 6b, and 6e exhibited apoptotic
morphology. Morphological changes characterized by cytoplasmic and nuclear shrinkage,
chromatin condensation and apoptotic body formation were observed after treatment, whereas in
the untreated control cells have appeared as rounded and homogeneously stained with DAPI. The
bright patches which indicate condensed chromatin and apoptotic bodies that are hallmarks of
early and late apoptosis (Figure 10). In contrast, the active 6d, 6a, 6h, 6b, and 6e compounds
induced significant cell death as indicated by DAPI staining.
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Figure 10. Detection of apoptosis by fluorescence microscopy in HeLa cells after treatment
compounds 6d, 6a, 6h, 6b, and 6e. The cells were stained with DAPI.
2.2.8. Detection of apoptotic cells by Annexin V- FITC and propidium iodide (PI) staining
And Activation of caspase-3
The apoptotic effect of active compound 6d on HeLa cells was further evaluated by flow
cytometry using dual fluorescence of Annexin V FITC/PI (AV/PI) dual staining assay. The cells
in early apoptosis bind only to Annexin V, whereas dead and late stage apoptotic cells bind to
both Annexin V-FITC and propidium iodide.41 In this study, HeLa cells were treated with the
compound 6d for 48 h at IC50 concentrations to examine the apoptotic effect. It was observed
that the compound 6d showed significant apoptosis against HeLa cells. The results indicated that
control group of untreated cells showed no apoptotic as well as necrotic activities with 100 %
gated cells, whereas the percentage of apoptotic cells was higher in the case of cells treated with
effective compound 6d which showed 95.68 % apoptotic cells (Figure 11a).
To investigate whether the compounds 6d induced apoptosis of HeLa cells was mediated
via the caspase-3 pathway, the caspase-3 content was evaluated by Western blot analysis.
Caspases are enzymes of cysteine-aspartic acid proteases and perform the well-known role to the
activation of an execution of apoptosis markers.42 Importantly, the activation of the caspase-3
enzyme is to induce cellular DNA fragmentation which leads to apoptotic cell death.43 After
treatment with the selected compounds for 24 h, the western blot results (Figure 11b) indicate
that the up-regulation of apoptotic activator enzyme caspase-3, as compared to untreated control,
in HeLa cells. From these results, the apoptotic pathways activated the caspase-3 leading to
apoptosis in HeLa cells.
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Figure 11. a) Detection of apoptosis by using the Annexin V-FITC and PI method by treating
HeLa cells for 48 h with compound 6d. Quadrants; Upper left (necrotic cells), Lower left (live
cells), Lower right (early apoptotic cells) and Upper right (late apoptotic cells. b) Effect of
compound 6d induced caspase-3 activation in HeLa cells determined by Western blot analysis
3. Insilco Molecular docking
In order to theoretically calculate the binding mode, molecular docking studies have been
carried out for the compound 6d against cervical cancer suppressor gene 4 protein (PDB ID:
5X5O)44 of HeLa cervical cancer cell line using Glide module of Schrodinger. The active
compound 6d bind very well with cervical cancer protein 5X5O with Glide score of -4.2,
kcal/mol, which shows two crucial hydrogen bonding interaction with ASP92 through NH
substituent of oxindole ring, SER89 with C=O of indole ring (Figure 12b). From these results,
we observed that the synthesized compound 6d have a good mode of binding activity with
cervical cancer cell HeLa.
Based on the results of western blot analysis the interaction of compound 6d with
Caspase-3 was theoretically calculated. The compound 6d bind very well with caspase-3 by
hydrophobic interaction Figure 12c. The binding energy was observed to be -6.02 kcal/mol.
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Figure 12. a) Three-dimensional representation of the molecular interaction of compound 6d
with the protein 5X5O. b) Two-dimensional representation of the molecular interaction of
compound 6d with the protein 5X5O. c) Molecular interaction of compound 6d with Caspase-3
4. Conclusion
In summary, we have synthesized structurally novel dispiro 8-nitroquinolone analogues
via one pot three component 1,3-dipolar cycloaddition reactions from the corresponding (E)-3-
arylidene-8-nitro-2,3 dihydro-4-quinolone as a dipolarophile and azomethine ylides generated
from isatin and benzylamine. The standardized procedure adapted on 1,3-dipolar cycloaddition
of compounds had fetched the excellent yield in short reaction time with high regio and
stereoselectivity. The structures of the synthesized compounds were well characterized by
spectroscopic and single crystal XRD analysis. All the synthesized compounds screened for their
invitro cytotoxic activity against the cervical cancer cell line (HeLa). Compared to Dox,
synthesized novel compounds exhibited strong inhibition against the tested cancer cell line HeLa
and was less toxic to human normal cells (Human RBC cells), indicating their good selectivity
toward cancer cells. The selected potent compound 6d induced typical morphological changes
and remarkable apoptosis of HeLa cells. Additionally, the results suggested that treatment of
HeLa cells with 6d analogue increased the intracellular ROS levels and apoptosis induced by
activation of caspase-3 apoptosis. Based upon the Insilco molecular docking results, it is clearly
evident that the active compounds are having good binding activity with the HeLa cell line
receptor.
Conflicts of interest
There are no conflicts to declare.
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