Novel phenyl and thiophene dispiro indenoquinoxaline pyrrolidine quinolones induced apoptosisviaG1/S and G2/M phase cell cycle arrest in MCF-7 cells

Article abstract of DOI:10.1039/d0nj02588g

New phenyl and thiophene dispiro indeno quinoxaline pyrrolidine quinolone analogues were synthesized by a one-pot four-component [3+2] cycloaddition reaction between (E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones,o-phenylenediamine, ninhydrin, and benzylamine/thiophenemethylamine. The structure and regio- and stereochemistry of the compounds have been confirmed by various spectroscopic and X-ray diffraction analyses. The observed regio- and stereoselectivity of the compounds were further enlightened by DFT calculations. Further, thein vitrocytotoxic effects of the compounds were evaluated by MTT assay. The compounds exhibited significant inhibition activity towards the tested cancer cells HeLa and MCF-7. Among the synthesized compounds compound9eshowed the highest inhibitory potency against MCF-7 cells. Mechanistic cell death studies revealed that cell death was induced by9eagainst MCF-7 cells by ROS mediated G1/S and G2/M phase cell cycle arrest apoptosis. The apoptotic cell death was quantified by the annexin V-FITC/PI staining method. The binding mode of compound9ewas further examined byin silicomolecular docking studies.

Full text of DOI:10.1039/d0nj02588g

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A Novel Phenyl and Thiophene Dispiro Indenoquinoxaline Pyrrolidine Quinolones Induced
Apoptosis via G1/S and G2/M Phase Cell Cycle Arrest in MCF-7 Cells
Selvaraj Shyamsivappana, Arjunan Saravananb, Raju Vivekc, Thangaraj Suresha, Ramasamy
Shankard Kodiveri Muthukaliannan Gothandame Palathurai Subramaniam Mohana,*
aSchool of Chemical Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India.
bDRDO-BU CLS, Bharathiar University campus, Coimbatore, Tamil Nadu, India.
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cCancer Research Program (CRP), Bio-Nano Therapeutics Research Laboratory, School of Life Sciences,
Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India.
dDepartment of Physics, Bharathiar University, Coimbatore, Tamil Nadu, India.
eSchool of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India
Abstract
A progressive approach towards new phenyl and thiophene dispiro indeno quinoxaline
pyrrolidine quinolone analogues were synthesized by one-pot four-component [3+2]
cycloaddition
reaction
between
(E)-3-arylidene-2,3-dihydro-8-nitro-4-quinolones,
o-
phenylenediamine, ninhydrin, and benzylamine/ thiophenemethylamine. The structure, regio-
and stereochemistry of the compounds have been confirmed by various spectroscopic and X-ray
diffraction analysis. The observed regio- and stereoselectivity of the compounds were further
enlightened by the DFT calculations. Further, the in vitro cytotoxic effects of the compounds
were evaluated by MTT assay. The compounds exhibited significant inhibition activity towards
the tested cancer cells HeLa and MCF-7. Among the synthesized compounds the compound 9e
showed the highest inhibitory potency against MCF-7 cells. The mechanistic cell death studies
revealed that cell death was induced by 9e against MCF-7 cells by ROS mediated G1/S and
G2/M phase cell cycle arrest apoptosis. The apoptotic cell death was quantified by annexin V-
FITC / PI staining method. The binding mode of the compound 9e was further examined by in
silico molecular docking studies.
Key words
one-pot synthesis; cycloaddition reaction; quinolone analogues; in vitro cytotoxic effects; G1/S
and G2/M phase cell cycle arrest; ROS; caspase-3;
Introduction
Cancer is one of the top listed reasons for death worldwide.1 Cervical and breast cancers
are the most common and leading causes of cancer death among women.2 Currently, surgery,
radiation therapy and chemotherapy are important treatment for cancer. In chemotherapy, many
anticancer agents have been approved for cancer treatment. However, during chemotherapy,
most of the drugs are having side effects and they are not specific. Hence, the resistance of the
currently available anticancer drugs making a necessary for the development of new high
1

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efficient anticancer agents with low side effects. Among the different targeted therapy, the
synthesis of apoptosis modulators and gene expression modulators plays an important role in
cancer treatment.3
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8
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In this therapeutic perspective, quinoxaline derivatives occurred in many natural products
and showed a variety of biological activities. In the early reports the quinoxaline moieties were
showed excellent anticancer activities with various mechanism of action like types I1/2 and II Eph
tyrosine kinase inhibitor,4 topoisomerase I and tubulin polymerization dual inhibitor,5
Bromodomain and extra terminal (BET) inhibitor,6 Pim inhibitor,7 Wnt2/β-catenin pathway
inhibitor,8 ERK inhibitors,9 etc. The five-membered heterocycles like pyrrolidine and thiophene
are building blocks of many drugs. In particular, multifunctional polycyclic spiro pyrrolidine
heterocycles exhibits versatile anticancer activities.10 The structure analogs of pyrrolo
quinoxaline showed good proliferation against breast cancer and cervical cancer cells. The
sulphur-containing thiophene heterocycle has demonstrated to be an attractive isostere, ensuing
in enhanced efficacy of drugs.
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On the other hand, nitrogen-containing quinoline heterocyclic derivatives were found in
numerous alkaloids and exhibits interesting anticancer activities through verity of molecular
mechanisms such as topoisomerase IIα (TOP2A) inhibitors for metastatic colorectal cancer,
topoisomerase 1 (Top1) inhibitors,11 c-KIT kinase inhibitor for gastrointestinal stromal tumors,12
14
mutant isocitrate dehydrogenase 1 (mIDH1) inhibitors,13 AXL kinase inhibitors,
aldehyde
dehydrogenase 1A1 (ALDH1A1) inhibitors,15 ataxia telangiectasia mutated (ATM) kinase
inhibitors,16 histone deacetylase 6 inhibitors,17 HSP90 inhibitors,18 Rho-associated protein kinase
(ROCK) inhibitors,19 dihydroorotate dehydrogenase (hDHODH) inhibitors,20 etc. Owing to these
interesting therapeutic properties of quinoline analogues, we have recently reported one class of 8-
nitro-2,3-dihydroquinoline-4(1H)-one hybrids, resulting in better anticancer activities.21
2

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N
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N
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N
MeO
N
N
HN
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Cl
N
O
O
H
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N
O
N
H
Top1 inhibitor
mIDH1 inhibitor
O
F
F
F
O
N
N
O
O
N
N
O
Cl
N
N
O
H
N
O
ATM kinase inhibitor
c-KIT kinase inhibitor
F
H
N
F
S
F
O
O
O
O
N
O
N
AXL kinase inhibitor
Figure 1. Some of recently reported anticancer quinolone analogs
Provoked by the early synthetic and anticancer properties of the quinoxaline, pyrrolidine,
thiophene and quinoline analoges, herein we described a one-pot multicomponent synthesis of
phenyl and thiophene dispiro indenoquinoxaline pyrrolidine quinolone analogues 8a-f & 9a-f via
1,3 dipolar cycloaddition reaction of (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones with
azomethine ylides. It processes mild reaction condition with high yield and substrate extension of
reaction. The stereoselectivity of the compounds was further confirmed by theoretical DFT
calculation methods. The synthesized compounds were examined for their in vitro anticancer
activities against cervical cancer HeLa and breast cancer MCF-7 cells. Among the synthesized
compounds the thiophene containing compound 9e showed better anticancer activity against the
tested cell lines. The apoptotic cell death was further evaluated by fluorescent staining method
and flow cytometry method. Further, we investigated the binding properties of the compounds
with MCF-7 estrogen receptor alpha protein by molecular docking studies.
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Results and Discussion
Chemistry
A new series of phenyl/thiophene dispiro indenoquinoxaline pyrrolidine quinolone
derivatives were obtained by 1,3 dipolar cycloaddition reaction between azomethine ylide and
dipolarophiles. Initially, the dipolarophiles 3a-f were prepared by Knoevengal condensation
reaction between 8-nitro-2,3-dihydroquinoline-4(1H)-one 1 with benzaldehyde derivatives 2a-f.
The pyrrolidine base was used as catalyst and reaction mixture was stirred in ethanol at room
temperature to afford (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones 3a-f in good yield
(Scheme 1).
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O
O
OHC
Pyrrolidine
Rt, EtOH
N
H
R
R
N
H
1
NO2
NO2
3a-f
2a-f
R = H, Br, Cl, F, CH3, OCH3
Scheme 1. Synthesis of (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones 3a-f
To reduce the reaction time and to extend the yield of the product, we optimize the
reaction conditions for the synthesis of dispiro indenoquinoxaline pyrrolidine quinolone
derivatives. The one-pot multicomponent reaction was performed between the (E)-3-
benzylidene-8-nitro-2,3-dihydroquinolin-4(1H)-one 3a, ninhydrin 4, o-phenylenediamine 5 and
benzylamine 6 as a model reaction (Table 1). The reaction was examined with various solvents
like toluene, acetonitrile, 1,4 dioxane, ethanol, methanol, and tetrahydrofuran under reflux
conditions. From the optimized results, the best outcome results were obtained in methanol
solvent to furnish dispiro indenoquinoxaline pyrrolo quinolone derivatives in higher yield (97%)
with minimum reaction time (Table 1, entry 4). We extend the scope of the reaction with different
p-benzaldehyde derivatives for dipolarophile (Br, Cl, F, OCH3, and CH3) and thiophene
methylamine for azomethine ylide and which has no significant change on the yield of the product.
Having optimized reaction conditions of desired product, we extend all the subsequent
cycloaddition reactions of (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones 3a-f (1 mmol),
ninhydrin 4(1mmol), o-phenylenediamine 5 (1 mmol) and benzylamine 6/ Thiophene
methylamine 7 (1 mmol) in MeOH at reflux condition for 2-3 hours to obtain desired compound
dispiro indenoquinoxaline pyrrolidine quinolone derivatives 8a-f & 9a-f (Scheme 2).
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Table 1. Optimization of the reaction condition for the synthesis of compound 7a
O
H2N
N
OH
OH
O
H2N
H2N
4
N
5
O
MeOH
Reflux
NH
H
O
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N
H
H
NO2
N
H
7a
3a
NO2
6
S.No.
Solvents
Temperature Time (h) Yield a (%)
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2
3
4
5
6
Acetonitrile
1,4 Dioxane
Ethanol
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
8
8
58
64
3
86
97
Methanol
Tolune
2
5
66
Tetrahydrofuran
10
No reaction
aIsolated yield, reaction condition: (E)-3-benzylidene-8-nitro-2,3-dihydroquinolin-4(1H)-one 3a
(1mmol), ninhydrin 4 (1 mmol), o-phenylenediamine 5 (1 mmol) and benzylamine 6 (1 mmol).
N
H2N
O
O
O
N
H2N
H2N
NH
OH
OH
6
H
N
H
H
5
4
NO2
O
N
8a-f
O
R
MeOH
N
N
N
H2N
Reflux
2-3 hours
O
S
7
NH
H
N
H
H
N
R
H
S
NO2
NO2
3a-f
R = H, Br, Cl, F, CH3, OCH3
9a-f
R
Scheme 2. Synthesis of phenyl/thiophene dispiro indenoquinoxaline pyrrolidine quinolone
derivatives 8a-f & 9a-f
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1
The formation of the cycloadduct 8a was characterized by IR, H & 13C NMR
spectroscopy, single crystal X-ray analysis, and elemental analysis. The IR spectrum of
compound 8a showed an intense absorption band at 1683 cm-1 for the carbonyl group and 1504
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9
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cm-1 for the nitro group of quinolone. The H NMR spectrum of the compound 8a showed a
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broad singlet at δ 7.77 ppm for quinolone N-H and the doublet at δ 2.32 ppm for pyrrolidine N-
H. The pyrrolidinylmethine protons have appeared at δ 4.90 (J = 6.0 Hz) and δ 4.06 (J = 4.0 Hz)
as a doublet that representing the regioselectivity of the cycloaddition reaction. The signals at δ
3.80 ppm as a doublet of doublet attributed to aliphatic CH2 protons for the quinoline ring. The
13
other protons appeared and captured for aromatic regions. In C NMR spectrum the peaks at δ
64.09 and 71.46 ppm were assignable to spirocarbons, δ 46.66, 54.45, and 58.88 ppm for
aliphatic and 191.74 ppm, for 4-quinolone carbonyl carbon respectively. The other carbons peak
appeared in aromatic regions.
X-ray crystallography analysis
The structure of the compound 9f was further confirmed by single crystal X-ray
diffraction analysis.
Figure 2. ORTEP diagram of compound 9f (CCDC No. 1980179)
Molecular geometry and crystal packing
The molecule was crystallized in the centrosymmetric monoclinic space group with four
molecules in the unit cell. The molecular structure contains two five-membered heterocyclic
rings. Among them, sulphur containing ring adopts planar conformation. However, the nitrogen
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contained five-membered ring adopts envelope conformation with C25 atom in the terminal
position. Similarly, another six-membered ring also adopts envelop conformation with C16 as a
terminal atom. According to Cremer & Pople puckering analysis, the puckering coordinations for
the five-membered ring is q2 = 0.359(2) Å and 2 = 286.6(3); and for six-membered ring is q2 =
0.369(2) Å, q2 = -0.229(2) Å and 2 = 302.7(3). The packing of molecules on monoclinic lattice
viewed down a-axis is shown in Figure 3.
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Table 2. Hydrogen bonding geometry (Å, °)
D-H...A
d(D-H) d(H...A) d(D...A) <(DHA)
C(17)-H(17A)...N(3)#1
C(37)-H(37)...O(2)#2
N(2)-H(39)...O(2)
0.97
0.93
2.52
2.6
3.067(2) 115.3
3.260(3) 128.3
0.78(2) 2.08(2) 2.664(2) 131(2)
Symmetry transformations used to generate equivalent atoms: #1 x,-y+1/2,z-1/2 #2 x+1,y,z
Figure 3. Packing of the molecules viewed down along the b-axis of the unit cell. H-bonds are
shown as dashed lines.
The fused quinoxaline ring and five-membered sulphur containing thiophene rings are
oriented with the dihedral angle of 22.4(1). The five-membered sulphur containing the ring and
the phenyl plane of the methoxyphenyl group are oriented with the dihedral angle of 68.9(1).
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The molecular structure features on intramolecular N-H...O interaction. The crystal packing is
achieved through weak C-H...N and C-H...O intermolecular interactions.
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The possible mechanism for the formation of dispiroheterocyclic derivative 8a was
portrayed in Scheme 2. The reaction of ninhydrin 4 and o-phenylenediamine 5 affords an
indenoquinoxaline intermediate 4b, which was further reacted with Benzylamine 6 to generate
the azomethine ylide dipole 10 in situ. The subsequent 1,3 cycloaddition reaction of 10 over (E)-
3-benzylidene-8-nitro-2,3-dihydroquinolin-4(1H)-one 3a leads the formation of the desired
product 8a as single regioisomer (Scheme 3).
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O
H2N
OH
OH
H2N
H
MeOH
O
O
5
4
N
H2N
-H2O
N
N
N
N
10
6
N
H
4
8
N
10
N
27
O
N
N
NH
H
O
N
H
H
22
NO2
N
H
3a
8a
NO2
H
N
O
H
N
NH
N
N
O
H
H
N
NO2
8'a
N
N
H
3a
NO2
Scheme 3. Schematic representation of the plausible mechanism of dispiro indenoquinoxaline
pyrrolidine quinolone derivative 8a
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Quantum Chemical Calculations
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The regio- and stereoselectivity of the compound 9f was further examined by quantum
chemical DFT calculations. To understand the regioselectivity of the compound 9f, we carried
out the Frontier molecular orbital (FMO) and Fukui function calculation. The energy values of
FMO analysis indicated that the energy gap between HOMOdipole 10 - LUMOdipolarophile 3e (2.061
eV) is lower than that of LUMOdipole 10 - HOMOdipolarophile 3e (2.816 eV) (Figure 4). The results
revealed that the LUMO of the dipolarophile 3f and the HOMO of the dipole 10 interactions was
more possible and it controls the cycloaddition reaction than the LUMO of dipole 10 and HOMO
dipolarophile 3f because of lower energy difference, which is concord with the experimental
results.
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Figure 4. HOMO and LUMO energies of dipolarophile 3f and dipole 10 calculated at the
B3LYP/LANL2DZ level of theory.
The most favorable attack site for the formation of the cycloadducts was also calculated
by theoretical DFT calculations. The local chemical Fukui function parameter for electrophilic
-
+
(fk ) and nucleophilic (fk ) were determined through NBO atomic charges. The determined local
chemical parameters were shown in Table 3. The results indicated that the C27 site of
+
azomethine ylide 10 had fk value of -0.107 a.u. which was higher nucleophilic value than the C4
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+
-
site of 10 (fk value of -0.145 a.u.). The electrophilic (fk ) values of dipolarophile 3f showed that
-
+
the C22 site had a higher fk value of -0.008 a.u. than C8 site (fk value of -0.145 a.u.). It reveals
that the higher nucleophilic C27 site of azomethine ylide 10 attacks first on the higher
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9
+
electrophilic site C22 of dipolarophile 3f. Further, the C8 site of dipolarophile 3f (fk value of
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0.023 a.u.) undergoes to nucleophilic attack on the C4 site of dipole 10. These theoretical results
were matched well with experimental results. The transition state (TS) structures of reactant 10,
3f and the product 9f were optimized at the B3LYP/LANL2DZ level of theory (Figure 5).
Table 3. Local chemical reactivity Fukui function parameters
+
-
Reactant
Site nucleophilic (fk ) (a.u) electrophilic (fk ) (a.u)
Dipolarophile 3f
Dipolarophile 3f
C28
C22
0.023
-0.099
-0.145
-0.107
-0.057
-0.008
-0.125
-0.072
Azomethine ylide 10 C4
Azomethine ylide 10 C27
Figure 5. The transition state (TS) structures for 1,3 dipolar cycloaddition reaction of reactant 10
& 3f and product 9f optimized at B3LYP/LANL2DZ level of theory
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The stability of the cycloadduct 9f and the reactant was calculated by Hartrees at the
above level theory. It was found to be 9f (-2010.5162) was more stable than the reactant. The
results indicated that the formation of the product was exothermic. The low activation and
reaction energy indicated the formation of the compound 9f at the MO6-2X/6-31G⁄ levels.
Further, the correlation between practically obtained single-crystal XRD values of 9f and
theoretically optimized geometrical values of 9f was shown in Figure 6. The results indicate that
the observed experimental bond angle and bond length values of 9f were correlated with
theoretically optimized values of 9f. The correlation coefficient was found to be 0.930 and 0.888
at M062x/6-31g** level of theory.
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Figure 6. Correlation coefficients between experimental single crystal XRD values and
theoretically optimized values. a) bond angle b) bond length
Biology
In Vitro Cytotoxic Activity Dispiro Indenoquinoxaline Pyrrolidine Quinolone Analogues
Investigation of the early literature revealed that the nitro attached quinolines had good
inhibition properties against cancer cells. Recently our group also reported 8-nitroquinoline
based dispiro pyrrolo oxindole and pyrano quinoline derivatives, which showed good inhibition
properties against breast cancer MCF-7 and cervical cancer HeLa cells.22, 23 Further, pyrrolidine,
quinoxaline, and thiophene moieties also showed promising anticancer activity against MCF-7
and HeLa cells.24-26 These observed reports suggest that 8-nitroquinoline incorporated spiro
pyrrolo quinoxaline compounds may inhibit the growth of MCF-7 and HeLa cells. Hence, to
examine the in vitro anticancer activities of the synthesized compounds 8a-f & 9a-f, we have
selected specified cell lines of MCF-7 and HeLa. The non-tumorigenic epithelial breast cells
MCF10A was used to determine the toxicity of the compounds.
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Initially, all the synthesized compounds were treated with normal epithelial breast cells
MCF10A at different concentrations up to 100 µM. After treatment, it was observed that up to
100 µM the compounds did not show significant toxicity towards normal cell lines. Hence, to
investigate the anticancer effects of compounds, the selected HeLa and MCF-7 cells were treated
with compounds at below 100 µM concentration. To compare the anticancer potency of the
compounds, the anticancer drug Doxorubicin (Dox) was used as a positive control. The results
were summarized by IC50 values which indicates the concentrations required to inhibit the 50%
viability of the tested cancer cells HeLa and MCF-7 (Table 4).
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Table 4. MTT cytotoxicity assay of 8a-f & 9a-f against MCF-7 and HeLa cells
IC50 of MCF-7 IC50 of HeLa
S. No
R
Compounds
Cells (µM)
28±0.8
29±1.7
33±1.4
35±1.3
22±1.2
24±0.8
23±1.5
27±1.4
28±1.3
28±1.7
17±1.3
21±0.8
16±1.4
Cells (µM)
29±0.7
29±1.6
33±1.2
37±0.4
25±1.1
26±1.7
25±1.2
28±1.3
30±1.1
30±1.5
19±1.2
23±1.3
18±1.4
1
H
F
8a
8b
8c
2
3
Cl
4
Br
8d
8e
5
CH3
OCH3
H
6
8f
7
9a
8
9
F
9b
9c
Cl
10
Br
9d
9e
11
CH3
OCH3
-
12
9f
Control
DOX
The in vitro cytotoxicity effects of the synthesized compounds were investigated MTT
cytotoxic assay. From the obtained MTT results it was observed that all the synthesized
compounds inhibit the growth of the tested cancer cells Hela (IC50< 37±0.4 μM) and MCF-7
(IC50 < 35±1.3μM). Among the synthesized compounds the thiophene substituted 4-methyl
benzaldehyde derivative 9e showed highest inhibition activity with an IC50 value of 17±1.3 μM
against MCF-7 cells and 19±1.2 μM against HeLa cells. The sulfur-containing five-membered
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thiophene ring attached compounds 9a-f showed better activity against both cell lines than the
phenyl ring attached compounds 8a-f. Further, we observed that the electron-donating methoxy
and methyl group substituted compounds 8e, 8f, 9e, and 9f showed better activity than the
electron-withdrawing F, Cl, Br substituted compounds 8b, 8c, 9d, 9b, 9c, and 9d. Among both
tested cell lines, the compounds showed better inhibition activity towards the MCF-7 cells than
the HeLa cells. However, the compounds exposed no significant toxicity against the normal
MCF10A cells at below 100 µM concentrations. From the results, we observed that the compounds
showed good inhibition properties against tested cell lines, and nontoxic towards normal cells.
Structure-Activity Relationship (SAR)
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Based on the MTT results, the impact of the substitutions on the synthesized compounds
towards the cytotoxicity was investigated. From the MTT results, it was observed that the
thiophene derivatives 9a-f are more active than the phenyl derivatives 8a-f (Table 4). Among the
compounds, the thiophene attached 4-methyl benzaldehyde derivative 9e showed the highest
activity with an IC50 value of 17±1.3 μM against MCF-7 cells and 19±1.2 μM against HeLa cells
which are very close value for the reference compound Dox. Among the phenyl ring attached
compounds 8a-f the 4-methyl benzaldehyde derivative 8e showed the highest activity with an
IC50 value of 24±0.8 μM & 25±1.1μM against MCF-7 & HeLa cells than other derivatives. From
these results, it reveals that the electron-donating methyl-substituted derivatives had better
activity than the other R substituted compounds.
On the investigation of R substituted compounds, the electron-donating methoxy, and
methyl-substituted thiophene and phenyl derivatives 8e, 8f, 9e, and 9f showed better activity
with an IC50 value of 22±1.2, 24±0.8, 17±1.3, and 21±0.8 μM against MCF-7 and 25±1.1,
26±1.7, 19±1.2, and 23±1.3 μM against HeLa cell lines. Among both electron-donating groups,
the methyl group derivatives showed better activity than the methoxy substituted derivatives.
The parent benzaldehyde derivatives 8a and 9a exhibit good activity than electron-withdrawing
group substituted compounds. When compared only electron-withdrawing halogen-substituted
compounds the fluorine substituted compounds 8b and 9b showed higher activity than 8c, 8d,
9c, and 9d. From the above observations we conclude that potential activity of substitution
orders are thiophene compounds 9a-f >phenyl 8a-f, at R substitution CH3> OCH3> H > F> Cl>
Br. Based on the MTT results, the potent compound 9e was chosen for further mechanistic
studies toward MCF-7 cells.
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Detection of Morphological Changes of Compound 9e Treated Cancer Cells
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The selected breast cancer MCF-7 cells were treated with potent compound 9e in two
different concentrations 25 & 50 μM and incubated for 24 hours. After incubation, the
morphological changes of the compound treated MCF-7 cells were detected by phase-contrast
microscopy. From the phase-contrast microscopy images, it was observed that the compound 9e
treated MCF-7 cells were appeared to be rounded, shrunken, and detached from neighbor cells,
whereas the untreated control cells were showed normal morphology (Figure 7). The
morphological changes indicated that the selected potent compound 9e was significantly induced
cell death in the tested MCF-7 cells.
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Figure 7. Morphological analysis of untreated control and compound 9e treated MCF-7 cells
Detection of Apoptotic Cells by AO/EB Dual Staining
The apoptotic cell death induced by the selected compound 9e was detected by acridine
orange/ ethidium bromide (AO/EB) staining method. Acridine orange (AO) is a green
fluorescent dye that stains a nuclear DNA of live cells, whereas ethidium bromide (EB) is an
orange-red dye that stains only damaged DNA of late apoptotic and dead cells.27 The MCF-7
cells were treated with compound 9e with two different concentrations of 25 & 50 μM and
incubated for 24 hours. After incubation, the control untreated and compound 9e treated cells
were stained with AO/EB mixture. After staining, the cells were observed under fluorescence
microscopy. It was observed that the compound 9e treated cells were showed chromatin
condensed migrated nuclear morphology with densely red and orange fluorescence which
indicates the necrotic and apoptotic cells, whereas the untreated control cells appeared as healthy
normal morphology with green fluorescence (Figure 8). The results indicated that the compound
9e was significantly inducing apoptosis in treated MCF-7 cells with highly fragmented and
condensed chromatin.
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Figure 8. Detection of apoptosis on the compound 9e treated MCF-7 cells by AO/EB staining
method using fluorescence microscopy.
Detection of Compound 9e Induced Nuclear Damage Fluorescent DAPI Staining
The apoptotic nuclear damage induced by the compound 9e was further detected by
DAPI staining method. DAPI (4’,6-diamidino-2-phenylindole) is the stain, which binds only
with nuclear adenine-thymine of double-stranded DNA and emits blue fluorescence. This
method is used to distinguish the condensed chromatin or damaged nuclear DNA by using
fluorescence microscopy.28 The DAPI stains effectively with the nuclear damaged apoptotic cells
and produces stronger blue fluorescence. The compound 9e was treated with MCF-7 cancer cells
with 25 & 50 μM concentrations and incubated for 24 hours. After incubation, the treated MCF-7
cells were stained DAPI, and the morphological changes were detected by using fluorescence
microscopy. The DAPI stained compound 9e treated MCF-7 cells exhibited bright blue
fluorescence apoptotic morphology with condensed chromatins and nuclear fragmentations,
whereas the untreated control cells were showed normally stained morphology (Figure 9). The
observed DAPI results indicated that the compound 9e significantly induced apoptotic nuclear
damage in tested MCF-7 cells.
Figure 9. Apoptosis detection of the compound 9e treated MCF-7 cells by fluorescence
microscopy. After treatment, the cells were stained with DAPI.
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Detection of Intracellular ROS-Generation by DCFH-DA staining
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8
In recent literature, it is found that the quinoline analoges induced apoptosis in cancer
cells through generation of reactive species.29, 30 Ghattass and his co-workers have reported the
quinoxaline compounds which effectively induced ROS associated DNA damage in MCF-7
cells.31 A series of quinoxaline-2-carbonitrile-1,4-dioxide derivatives increase the intracellular
ROS production and exhibited better cytotoxicity against MCF-7 cell lines.32 And many spiro
pyrrolidine compounds also exhibited better anticancer activity through ROS mechanisms;
recently our group also reported one set of dispiro quinolone pyrrolidine compounds that exhibit
better anticancer activity through ROS mediated mechanism.22 The above reports suggest that
the dispiro indenoquinoxaline pyrrolo thiophene quinolone compound 9e may increase the
intracellular ROS generation.
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Figure 10. a) Detection of increased intracellular ROS production of compound 9e treated MCF-
7 cells by fluorescence microscopy b) Detection of fluorescence intensity of compound treated
MCF-7 cells compared with H2O2 treated cells as a positive control
Hence, after the treatment of compound 9e, the MCF-7 cells were further investigated for
the production of intracellular ROS by DCFH-DA assay. The DCFH-DA (2’,7’-
dichlorodihydrofluorescein diacetate) is a nonfluorescent compound, but in the presence of
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intracellular ROS, the DCFH-DA rapidly oxidized into green fluorescence DCF
(dichlorofluorescein). The compound 9e treated MCF-7cells were incubated for 24 hours. After
incubation, the cells were further treated with DCFH-DA for 30 minutes and cells were observed
under fluorescence microscopy. Interestingly, the compound 9e treated MCF-7 cells were
showed an increased level of green fluorescence, which indicates the increased level of ROS
(Figure 10a). The increased level of intracellular ROS was quantified by the percentage of cells
with enhanced green fluorescence intensity (Figure 10b). The results showed that the compound
9e induced apoptosis mainly through the action of ROS-mediated mechanisms.
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The Compound 9e Promotes G1/S and G2/M Cell Cycle Arrest in MCF-7 Cells
The mechanism of cell death induced by compound 9e on MCF-7 cells was further
evaluated by flow cytometry cell cycle analysis. The cell cycle results of control untreated cells
showed 77.03% of G0-G1Phase, 13.68% of G1/S phase and 9.39% of G2/M phase. In the
compound 9e, treated cells showed 51.57% of G0-G1Phase, 23.66% of G1/S phase and 22.99%
of G2/M phase (Figure 11). The cell cycle results indicated that after treatment of the compound
9e the cells were significantly increased in the G1/S phase and G2/M phase cells whereas the G0-
G1 phase cells were decreased when compared to the control cells. The increased cells of the
G1/S phase and G2/M phase indicated that the compounds 9e induced G1/S and G2/M phase cell
cycle arrest in treated MCF-7 cells.
Figure 11. Cell cycle analysis of compound 9e treated MCF-7 cells by flow cytometry
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Quantification of Apoptotic Cells by Annexin V-FITC and Propidium Iodide PI staining
The apoptotic cells were further quantified by annexin V-FITC and propidium iodide PI
double staining method using a flow cytometer. The annexin V-FITC is a green color stain that
binds only with the plasma membrane of early and late apoptotic cells, whereas PI is the red stain
which only binds with membrane lost late apoptotic and dead cells.33 The MCF-7 cells were
treated with compound 9e for 24 hours and then stained with annexin V-FITC and PI. The
untreated cells were used as a control to quantify the apoptotic cells. After staining the apoptotic
cells were quantified by flow cytometer. Interestingly, the flow cytometer results of the 50 μM
compound treated cells showed 47 percentages of apoptotic cells with a favorable mode of action
(Figure 12). But the control cells have no apoptotic or necrotic cells with 97.5 % gated cells. The
results indicated that the compound 9e has more potential for action to kill the cancer cells.
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Figure 12. Quantification of apoptotic cells by flow cytometric analysis by treating MCF-7 cells
for 24 h with compounds 9e. Quadrants: Upper left (necrotic cells), Upper right (late apoptotic
cells), lower right (early apoptotic cells), lower left (live cells).
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Compounds Induced Caspase-3 Activation via Mitochondrial-Programmed Cell Death
Further, we have investigated whether the compound 9e induced apoptosis on MCF-7
cells through apoptotic biomarker of caspase-3. After 24 hours treatment of compound 9e, the
MCF-7 cells were analyzed for protein expression by western blot analysis. The results indicated
that the compound increased the production of caspase-3 in treated MCF-7 cells when compared
with the internal standard control gene β-actin (Figure 13). This up-regulation of the caspase-3
proteins has induced apoptosis in compound treated MCF-7 cells.
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Figure 13. Western blot protein expression analysis of compound 9e treated MCF-7 cells
3. Insilco Molecular docking
The binding mode of the compound 9e with breast cancer Estrogen Receptor alpha (ERα)
was further determined by molecular docking studies. The breast cancer estrogen receptor alpha
(ERα) structure was downloaded from the PDB with PDB id of 3ERT.34, 35 The docking results
showed that the potent compound 9e was binding well with breast cancer estrogen receptor alpha
with two hydrogen bond interactions with ASP351 and CYS530 through the NH group of
pyrrolidine ring and the carbonyl group of quinolone ring (Figure 14). The docking results
indicated that the active compound 9e binds well with breast cancer estrogen receptor ERα.
Figure 14. The molecular interaction of the compound 9e with breast cancer estrogen receptor
alpha (ERα)
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Conclusion
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7
In conclusion, we have prepared a new series of phenyl/thiophene dispiro
indenoquinoxaline pyrrolidine quinolone analogues via one-pot four-component 1,3
8
9
cycloaddition
reaction
between
ninhydrin,
o-phenylenediamine,
benzylamine/
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thiophenemethylamine, and (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones. The reaction
conditions were optimized and obtained the products with good yield in less reaction time with
high regio and stereoselectivity. The structural assignments and regio and stereoselectivity of the
compounds were confirmed by various spectroscopic and single crystal XRD techniques. The
regio and stereoselectivity of the compounds were further confirmed by the theoretical DFT
method. The in vitro cytotoxic effects of these compounds were assessed against cervical cancer
HeLa and MCF-7 cells. Among the tested compounds the thiophene electron-donating methyl-
substituted compound 9e exhibited better inhibition against the tested cancer cells MCF-7 and
HeLa. The selected potent compound 9e exhibited remarkable morphological and apoptotic
changes towards the tested cell line MCF-7. The cell cycle analysis indicated that the compound
9e induced G1/S and G2/M phase cell cycle arrest apoptosis in tested cancer cells. The
mechanistic studies indicated that the compound induced apoptosis through intracellular ROS
mediated caspase-3 activation. The molecular docking studies showed the compound having a
good binding property with breast cancer estrogen receptor alpha.
Experimental Section
Chemistry
General methods
All the chemicals and solvents were purchased from Sigma Aldrich and Loba Chemie in
analytical grade. The IR spectra were recorded by JASCO FT-IR 4100 spectrometer using KBr
palate and the frequency values were quoted in reciprocal of centimeters. The 1H and 13C NMR
were recorded by Bruker advance 400 MHz in the DMSO-d6 solvent. The chemical shift δ values
were reported in ppm downfield from TMS (tetramethylsilane) and the coupling constant J
values were reported in Hertz (Hz). The single-crystal structure was analyzed by Bruker AXS
KAPPA APEX-2 diffractometer equipped with a graphite monochromator. The elemental
analyses were recorded by a Perkin Elmer 2400 series II elemental CHNS analyzer. The
abbreviations used in the manuscript are singlet(s), doublet (d), triplet (t), multiplet (m),
centimeter (cm), hours (h) and minutes (min).
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General procedure for synthesis of (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones
(3a-f)
An equimolar ratio of 8-nitro-2,3-dihydroquinoline-4(1H)-one 1 (1 mmol) and aromatic
aldehydes 2a-f (1 mmol) was dissolved in ethanol, few drops of pyrrolidine base was added as a
catalyst. The mixture was stirred at room temperature for 1 hour; the obtained solid product was
filtered and dried. The product was recrystallized by ethanol to obtain pure product 3a-f in good
yield (90-95%).
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General procedure for the synthesis of indenoquinoxaline dispiropyrrolo quinolone derivatives
8a-f & 9a-f
An equimolar mixture of ninhydrin 4 (1mmol) and o-phenylenediamine 5 (1mmol) in
10ml methanol and stirred for 10 min at room temperature. To the mixture the benzylamine 6/
thiophenemethylamine 7 (1 mmol) and (E)-3arylidine-8-nitro-2,3-dihydroquinolin-4(1H)-ones
3a-f (1mmol) were added, and the reaction mixture was refluxed in a water bath. The progress of
the reaction was monitored by TLC, after completion of the reaction the mixture was cooled to
room temperature and obtained solid product was filtered and dried which afforded a single
product 8a-f & 9a-f. The purest form of the product will be obtained through recrystallization
using chloroform/methanol in a 1:1 ratio.
Crystallographic data collection and refinement
The slow evaporation solution growth technique is used and block-shaped good quality
single crystals of the title compound 9f were obtained. Good transparent crystal from them was
taken for the X-ray intensity data collection with the X-ray of the wavelength of 0.71073 Å at
room temperature. The Bruker AXS KAPPA APEX-2 diffractometer equipped with a graphite
monochromator was used for the data collection. The details of parameters regarding the data
collection and structure solution were given in Table S1.
The structure was solved by direct methods and refined by full-matrix least-squares
calculations using SHELXL-2014. All the H atoms were placed geometrically calculated bond
distances with -CH = 0.93 Å and constrained to ride on the parent atom with Uiso(H) = 1.2
Ueq(parent carbon atom). The H atom attached to nitrogen was located from an electron density
map and refined isotropically. The ORTEP view of the molecule drawn at 50% probability
thermal displacement ellipsoids with the atom numbering scheme is shown in Figure 1. During
the refinement, it was identified that the methyl group of the methoxyl phenyl is disordered over
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position with the site occupancies of 0.5 each. The disorder was resolved with SADI and DELU
constraints. It was shown in Figure.1 through atomic labels C32 and C32'.
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4-(phenyl)-pyrrolidine (1H)-spiro [2.3ʹ] indeno[1,2-b]quinoxaline-spiro [3.3ʹʹ]-8ʹʹ-
nitro-1ʹʹH-quinolin-4(2ʹʹH)-one 8a
1
Yellow solid; Yield (96%); mp: 204-212°C; IR (KBr): 1683, 1504 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.43 (d, 1H J = 8.0 Hz, Ar-H), 8.16 (d, 1H, J = 7.6 Hz, Ar-H), 7.95-7.85 (m, 4H,
Ar-H), 7.77 (brs, 1H, Q-NH), 7.70-7.68 (m, 2H, Ar-H), 7.61-7.59 (m, 3H, Ar-H), 7.42-7.21 (m, 9H,
Ar-H), 6.55 (t, 1H, J = 8.0 Hz, Ar-H), 5.99 (dd, 1H, J = 6.0 Hz ), 4.90 (d, 1H, J = 10.0 Hz), 4.06 (d,
1H J = 4.0 Hz), 3.81 (dd, 1H, J = 9.6 Hz), 2.32 (d, 1H J = 14.4 Hz ); 13C NMR (100 MHz, DMSO-d-
6): δ 46.66, 54.45, 58.88, 64.09, 71.46, 114.77. 120.60, 122.46, 127.11, 127.24, 127.41, 128.07,
128.46, 128.84, 128.87, 129.14, 129.42, 129.69, 129.94, 130.42, 131.40, 131.84, 136.18, 136.43,
136.88, 140.30, 141.67, 142.83, 144.56, 147.58, 153.70, 165.51, 191.74; Anal. Calcd for
C38H27N5O3; C, 75.86; H, 4.52; N, 11.64; O, 7.98; Found C, 75.82; H, 4.49; N, 11.62; O, 7.93.
4-(4ʹʹʹ-flurophenyl)-pyrrolidine (1H)-spiro [2.3ʹ] indeno[1,2-b]quinoxaline-spiro
[3.3ʹʹ]-8ʹʹ-nitro-1ʹʹ H-quinolin-4(2ʹʹH)-one 8b
1
Yellow solid; Yield (98%); mp: 207-215°C; IR (KBr): 1681, 1508 cm-1; H NMR (400
MHz, DMSO-d6) : δ 8.45 (dd, 1H, J = 7.6 Hz, Ar-H ), 8.16 (dd, 1H, J = 7.6 Hz, Ar-H ), 7.95-
7.85 (m, 4H, Ar-H ), 7.77 (brs, 1H, Q-NH), 7.71-7.58 ( m, 5H, Ar-H ), 7.34-7.31 (m, 3H, ArH )
7.26-7.22 (m, 5H, Ar-H ), 6.57 (t, 1H, J = 8.0 Hz, Ar-H ), 5.91 (dd, 1H, J = 5.6 Hz), 4.87 (d, 1H,
J= 10.0 Hz, -CH ), 4.07 (d, 1H, J = 4.0 Hz, -CH), 3.76 (dd, 1H, J = 8.8 Hz, -CH), 2.34 (d, 1H, J
13
= 14. Hz, -CH) ; C NMR (100MHz, DMSO-d6): δ 46.61, 53.81, 58.62, 64.56, 71.53, 114.81,
115.13, 115.34, 120.60, 122.49, 127.24, 127.34, 128.12, 128.84, 128.88, 129.16, 129.46, 129.70,
130.39, 131.44, 131.86, 132.99, 133.02, 136.20, 136.44, 140.28, 141.69, 142.73, 144.57, 147.42,
153.70, 160.06, 162.48, 165.35, 191.73; Anal. Calcd for C38H26FN5O3; C, 73.66; H, 4.23; F,
3.07; N, 11.30; O, 7.75; Found C, 73.64; H, 4.24; F, 3.05; N, 11.26; O, 7.73;
4-(4‴-chlorophenyl)-pyrrolidine (1H)-spiro [2.3′] indeno[1,2-b]quinoxaline-spiro
[3.3″]-8″-nitro-1″H-quinolin-4(2″H)-one 8c
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9
Yellow solid; Yield (92%); mp: 210-218˚C; IR (KBr): 1684, 1507 cm-1; H NMR (500
MHz, DMSO-d6): δ 8.44 (d, 1H J = 8.0 Hz, Ar-H), 8.14 (d, 1H, J = 8.0 Hz, Ar-H), 7.93-7.84 (m,
5H, Ar-H), 7.71 (brs, 1H, NH), 7.69-7.67 (m, 1H, Ar-H), 7.62-7.56 (m, 3H, Ar-H), 7.46-7.44 (d,
2H, J = 7.0 Hz Ar-H), 7.32-7.29 (m, 3H, Ar-H), 7.24-7.20 (m, 3H, Ar-H), 6.55 (t, 1H, J = 8.0 Hz,
Ar-H), 5.89 (dd, 1H, J = 6.0 Hz ), 4.84 (d, 1H, J = 10.0 Hz, -CH), 4.07 (d, 1H, J = 4.0 Hz,), 3.72
(dd, 1H, J = 9.5 Hz), 2.33 (d, 1H, J = 14.5 Hz ) ; 13C NMR(100 MHz, DMSO-d6): δ 47.03, 54.43,
59.16, 64.89, 72.03, 115.35, 121.10, 122.93, 127.72, 127.82, 127.86, 128.65, 128.95, 129.29,
129.43, 129.70, 130.01, 130.22, 130.90, 131.98, 132.28, 132.32, 136.38, 136.72, 136.89, 140.75,
142.13, 143.10, 145.06, 147.81, 154.15, 165.74, 192.15; Anal. Calcd for C38H26ClN5O3; C, 71.75;
H, 4.12; Cl, 5.57; N, 11.01; O, 7.55; Found C, 71.71; H, 4.11; Cl, 5.51; N, 11.05; O, 7.46;
1
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4-(4‴-bromophenyl)-pyrrolidine (1H)-spiro [2.3′] indeno[1,2-b]quinoxaline-spiro
[3.3″]-8″-nitro-1″H-quinolin-4(2″H)-one 8d
1
Yellow solid; Yield (93%); mp: 208-216˚C; IR (KBr): 1692, 1503 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.46 (dd, 1H J = 7.6 Hz, Ar-H), 8.16 (dd, 1H, J = 8.0 Hz, Ar-H), 7.95-7.85
(m, 4H, Ar-H), 7.71-7.69 (m, 2H, Ar-H), 7.64-7.58 (m, 6H, Ar-H), 7.35-7.30 (m, 3H, Ar-H),
7.26-7.20 (m, 3H, Ar-H), 6.57 (t, 1H, J = 8.0 Hz, Ar-H), 5.91 (dd, 1H, J = 5.6 Hz ), 4.84 (d, 1H,
J = 10.0 Hz, -CH), 4.09 (d, 1H, J = 4.0 Hz), 3.73 (dd, 1H J = 9.2 Hz), 2.36 (d, 1H J = 14.4 Hz) ;
13C NMR(100 MHz, DMSO-d6): δ 46.56, 54.02, 58.64, 64.38, 71.58, 114.84, 120.37, 120.60,
122.47, 127.24, 127.32, 128.16, 128.83, 128.89, 129.19, 129.48, 129.74, 130.37, 131.37, 131.47,
131.86, 132.26, 136.22, 136.37, 136.45, 140.28, 141.69, 142.65, 144.58, 147.31, 153.68, 165.23,
191.64; Anal. Calcd for C38H26BrN5O3; C, 67.06; H, 3.85; Br, 11.74; N, 10.29; O, 7.05; Found
C, 67.02; H, 3.87; Br, 11.73; N, 10.24; O, 7.01;
4-(4‴-methylphenyl)-pyrrolidine (1H)-spiro [2.3′] indeno[1,2-b]quinoxaline-spiro
[3.3″]-8″-nitro-1″H-quinolin-4(2″H)-one 8e
1
Yellow solid; Yield (97%); mp: 209-215˚C; IR (KBr): 1684, 1506 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.38 (dd, 1H, J = 6.4 Hz, Ar-H), 8.12 (dd, 1H, J = 5.6 Hz, Ar-H), 7.92-7.83
(m, 4H, Ar-H), 7.68-7.50 (m, 6H, Ar-H), 7.32-7.27 (m, 3H, Ar-H), 7.24-7.16 (m, 5H, Ar-H),
6.53 (t, 1H, J = 8.0 Hz, Ar-H), 5.92 (dd, 1H, J = 5.6 Hz ), 4.83 (d, 1H, J = 10.0 Hz, -CH), 3.96
(d, 1H, J = 4.0 Hz), 3.78 (dd, 1H, J = 9.2 Hz), 2.32 (d, 1H, J = 25.6 Hz) 2.25 (s, 3H, CH3) ; 13C
NMR(100 MHz, DMSO-d6): δ 20.58, 46.61, 54.05, 58.78, 64.05, 71.41, 114.72, 120.55, 122.45,
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127.14, 127.20, 127.37, 128.01, 128.79, 128.83, 129.04, 129.08, 129.38, 129.63, 129.77, 130.37,
131.35, 131.79, 133.70, 136.08, 136.13, 136.38, 140.25, 141.62, 142.89, 144.52, 147.60, 153.66,
165.49, 191.76; Anal. Calcd for C39H29N5O3; C, 76.08; H, 4.75; N, 11.37; O, 7.80; Found C,
76.06; H, 4.76; N, 11.34; O, 7.78;
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4-(4‴-methoxyphenyl)-pyrrolidine (1H)-spiro [2.3′] indeno[1,2-b]quinoxaline-spiro [3.3″]-
8″-nitro-1″H-quinolin-4(2″H)-one 8f
Yellow solid; Yield (94%); mp: 207-215˚C; IR (KBr): 1685, 1571 cm-1; 1H NMR (500
MHz, DMSO-d6): δ 8.40 (dd, 1H, J = 6.5 Hz, Ar-H), 8.16-8.08 (m, 1H, Ar-H), 7.93-7.83 (m, 5H,
Ar-H), 7.72-7.65 (m, 2H, ArH), 7.58-7.55 (m, 4H, Ar-H), 7.31-7.28 (m, 3H, Ar-H), 7.22-7.18 (m,
3H, Ar-H), 6.94 (d, 2H, J = 7.0 Hz, ArH) 6.53 (t, 1H, J = 8.0 Hz, Ar-H), 5.87 (dd, 1H, J = 6.0 Hz,
ArH ), 4.79 (d, 1H, J = 10.0 Hz, -CH), 4.00 (d, 1H, J = 4.0 Hz), 3.82 (dd, 1H, J = 5.0 Hz ) 3.71 (s,
3H, OCH3), 2.31 (d, 1H J = 14.5 Hz); 13C NMR (100 MHz, DMSO-d6): δ 47.12, 54.24, 55.41, 59.20,
64.78, 71.90, 114.33, 115.22, 121.07, 122.97, 124.71, 127.69, 127.88, 128.55, 128.97, 129.27,
129.39, 129.61, 129.81, 129.94, 130.18, 130.86, 130.91, 131.42, 131.88, 132.27, 136.65, 136.83,
140.75, 142.09, 143.42, 145.03, 148.10, 154.16, 158.6, 166.01, 192.36; Anal. Calcd for C39H29N5O4;
C, 74.15; H, 4.63; N, 11.09; O, 10.13; Found C, 74.11; H, 4.61; N, 11.05; O, 10.06;
4'-(phenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno[1,2-b]
quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9a
1
Yellow solid; Yield (96%); mp: 205-215˚C; IR (KBr): 1686, 1574 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.38 (dd, 1H J = 6.4 Hz, Ar-H), 8.13 (dd, 1H, J = 6.4 Hz, Ar-H), 7.93-7.82
(m, 4H, Ar-H), 7.67 (d, 3H, J = 6.4 Hz, Ar-H), 7.55 (d, 1H, J = 4.8 Hz, Ar-H), 7.42-7.35 (m, 3H,
Ar-H), 7.29-7.20 (m, 4H, Ar-H), 6.96 (d, 1H, J = 3.2 Hz, Ar-H), 6.89 (m, 1H, Ar-H), 6.52 (t, 1H,
J = 8.0 Hz, Ar-H), 6.27 (dd, 1H, J = 6.4 Hz), 4.90 (d, 1H, J = 10.0 Hz), 4.33 (d, 1H, J = 4.0 Hz)
13
3.83 (dd, 1H, J = 9.6 Hz), 2.30 (d, 1H J = 14.4 Hz) ; C NMR(100 MHz, DMSO-d6): δ 46.49,
54.72, 58.81, 60.05, 71.13, 114.76, 120.61, 122.29, 124.17, 124.43, 126.25, 127.13, 127.22,
128.53, 128.79, 128.85, 129.17, 129.41, 129.65, 129.68, 130.47, 131.78, 136.12, 136.49, 144.44,
146.97, 165.28, 191.23. Anal. Calcd for C36H25N5O3S; C, 71.15; H, 4.15; N, 11.52; O, 7.90; S,
5.28; Found C, 71.12; H, 4.16; N, 11.50; O, 7.86; S, 5.24;
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4'-(4-fluorophenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno
[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9b
7
8
9
Yellow solid; Yield (94%); mp: 204-214˚C; IR (KBr): 1681, 1614 cm-1; 1H NMR (400
MHz, DMSO-d6): δ 8.40 (dd, 1H J = 5.2 Hz, Ar-H), 8.14 (dd, 1H, J = 6.4 Hz, Ar-H), 7.93-7.83
(m, 4H, Ar-H), 7.72-7.57 (m, 4H, Ar-H), 7.38 (dd, 1H, J = 4.0 Hz, Ar-H), 7.27-7.20 (m, 5H, Ar-
H), 6.96 (d, 1H, J = 2.4 Hz, Ar-H), 6.89-6.88 (m, 1H, Ar-H), 6.53 (t, 1H, J = 8.4 Hz, Ar-H ),
6.21 (dd, 1H, J = 6.4 Hz), 4.88 (d, 1H, J = 10.0 Hz), 4.34 (d, 1H, J = 4.4 Hz) 3.79 (dd, 1H, J =
8.8 Hz), 2.33 (d, 1H J = 14.4 Hz) ; 13C NMR(100 MHz, DMSO-d6): δ 46.43, 54.04, 58.57, 60.48,
71.19, 114.80, 115.22, 115.43, 120.61, 122.32, 124.15, 124.50, 126.29, 127.12, 128.79, 128.86,
129.19, 129.45, 129.66, 130.44, 131.41, 131.59, 131.79, 132.63, 136.14, 136.37, 140.20, 141.66,
144.45, 146.82, 147.09, 153.58, 160.12, 165.13, 191.20 Anal. Calcd for C36H24FN5O3S; C,
69.11; H, 3.87; F, 3.04; N, 11.19; O, 7.67; S, 5.12; Found C, 69.13; H, 3.89; F, 3.01; N, 11.17;
O, 7.64; S, 5.10;
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4'-(4-chlorophenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno
[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9c
Yellow solid; Yield (96%); mp: 203-211˚C; IR (KBr): 1684, 1589 cm-1; 1H NMR (400
MHz, DMSO-d6): δ 8.40 (dd, 1H, J = 5.2 Hz, Ar-H), 8.14 (dd, 1H, J = 5.2 Hz, Ar-H), 7.94-7.84
(m, 4H, Ar-H), 7.77 (brs, 1H, Q-NH), 7.68-7.66 (m, 1H, Ar-H), 7.59 (d, 1H, J = 5.2 Hz, Ar-H),
7.49 (d, 2H, J = 8.4 Hz, Ar-H) 7.39 (dd, 1H, J = 4.0 Hz, Ar-H), 7.24-7.20 (m, 3H, Ar-H), 6.97
(d, 1H, J = 2.8 Hz, Ar-H), 6.91 (m, 1H, Ar-H ), 6.54 (t, 1H, J = 8.0 Hz, Ar-H ), 6.20 (dd, 1H, J =
6.0 Hz), 4.87 (d, 1H, J = 10.4Hz), 4.36 (d, 1H, J = 4.0 Hz), 3.77 (dd, 1H, J = 9.2 Hz), 2.34 (d,
13
1H J = 14.4 Hz) ; C NMR(100 MHz, DMSO-d6): δ 46.39, 54.18, 58.62, 60.33, 71.22, 114.83,
120.62, 122.30, 124.19, 124.57, 126.33, 127.13, 128.54, 128.80, 128.89, 129.22, 129.48, 129.70,
130.45, 131.46, 131.63, 131.79, 131.91, 135.55, 136.17, 136.38, 140.20, 141.67, 144.46, 146.75,
147.01, 153.58, 165.05, 191.15 Anal. Calcd for C36H24ClN5O3S; C, 67.34; H, 3.77; Cl, 5.52; N,
10.91; O, 7.47; S, 4.99; Found C, 67.32; H, 3.79; Cl, 5.51; N, 10.89; O, 7.45; S, 4.98;
4'-(4-bromophenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno
[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9d
Yellow solid; Yield (94%); mp: 206-216˚C; IR (KBr): 1682, 1591 cm-1; 1H NMR (400
MHz, DMSO-d6): δ 8.41 (dd, 1H, J = 6.4 Hz, Ar-H), 8.14 (dd, 1H, J = 8.0 Hz, Ar-H), 7.94-7.85
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(m, 4H, Ar-H), 7.68-7.57 (m, 6H, Ar-H), 7.39 (dd, 1H, J = 4.0 Hz, Ar-H), 7.23-7.21 (m, 3H, J =
8.4 Hz, Ar-H) 6.97 (d, 1H, J = 3.2 Hz, Ar-H), 6.91-6.89 (m, 1H, Ar-H), 6.54 (t, 1H, J = 8.0 Hz,
Ar-H ), 6.20 (dd, 1H, J = 5.6 Hz), 4.86 (d, 1H, J = 10.0 Hz), 4.36 (d, 1H, J = 4.0 Hz), 3.76 (dd,
7
8
9
13
1H, J = 9.2 Hz), 2.35 (d, 1H J = 14.4 Hz) ; C NMR(100 MHz, DMSO-d6): δ 46.39, 54.24,
10
11
12
13
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58.59, 60.28, 71.23, 114.83, 120.50, 120.62, 122.30, 124.20, 124.58, 126.33, 127.13, 128.79,
128.89, 129.22, 129.48, 129.70, 130.44, 131.46, 131.79, 132.00, 135.97, 136.17, 136.38, 140.20,
141.67, 144.46, 146.74, 146.99, 153.57, 165.03, 191.14. Anal. Calcd for C36H24BrN5O3S; C,
62.98; H, 3.52; Br, 11.64; N, 10.20; O, 6.99; S, 4.67; Found C, 62.96; H, 3.56; Br, 11.61; N,
10.18; O, 6.95; S, 4.64;
4'-(4-methylphenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno
[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9e
Yellow solid; Yield (96%); mp: 202-210˚C; IR (KBr): 1685, 1607 cm-1; 1H NMR (400
MHz, DMSO-d6): δ 8.35 (dd, 1H, J = 6.4 Hz, Ar-H), 8.11 (dd, 1H, J = 5.2 Hz, Ar-H), 7.91-7.79
(m, 4H, Ar-H), 7.66 (dd, 1H, J = 4.8 Hz, Ar-H), 7.55-7.46 (m, 3H, Ar-H), 7.35 (dd, 1H, J = 4.0
Hz, Ar-H), 7.24-7.18 (m, 5H, Ar-H), 6.95 (d, 1H, J = 2.4 Hz, Ar-H), 6.88-6.86 (m, 1H, Ar-H),
6.51 (t, 1H, J = 8.0 Hz, Ar-H), 6.22 (dd, 1H, J = 6.0 Hz), 4.85 (d, 1H, J = 9.6 Hz), 4.24 (d, 1H, J
= 4.4 Hz), 3.82 (dd, 1H, J = 9.2 Hz), 2.32 (d, 1H J = 14.4 Hz), 2.27 (s, 3H, CH3); 13C NMR (100
MHz, DMSO-d6): δ 20.60, 46.47, 54.36, 58.75, 60.07, 71.12, 114.74, 120.60, 122.32, 124.15,
124.37, 126.23, 127.13, 128.79, 128.86, 129.14, 129.41, 129.57, 129.64, 130.47, 131.37, 131.75,
133.35, 136.12, 136.26, 136.35, 140.20, 141.64, 144.44, 147.07, 147.30, 153.59, 165.31, 191.29
Anal. Calcd for C37H27N5O3S; C, 71.48; H, 4.38; N, 11.26; O, 7.72; S, 5.16; Found C, 71.50; H,
4.39; N, 11.24; O, 7.69; S, 5.14;
4'-(4-methoxyphenyl)-8''-nitro-5'-(thiophen-2-yl)-1'',2''-dihydro-4''H-dispiro[indeno
[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-quinolin]-4''-one: 9f
Yellow solid; Yield (93%); mp: 204-215˚C; IR (KBr): 1683, 1602 cm-1; 1H NMR (400
MHz, DMSO-d6): δ 8.39 (dd, 1H J = 5.6 Hz, Ar-H), 8.14 (dd, 1H, J = 6.0 Hz, Ar-H), 7.94-7.83
(m, 4H, Ar-H), 7.68 (dd, 1H, J = 6.0 Hz Ar-H), 7.60 (s, 1H, q-NH), 7.57 (d, 1H, J = 5.2 Hz, Ar-
H), 7.38 (dd, 1H, J = 3.6 Hz, Ar-H), 7.26-7.21(m, 3H, Ar-H), 6.99-6.96 (m, 3H, Ar-H), 6.91-
6.89 (m, 1H, J = 3.6, 3.2 Hz, Ar-H), 6.54 (t, 1H, J = 8.0 Hz, Ar-H), 6.20 (dd, 1H, J = 6.0 Hz),
4.84 (d, 1H, J = 10.4 Hz), 4.30 (d, 1H, J = 4.4 Hz), 3.83 (dd, 1H, J = 8.4 Hz), 3.75 (s, 3H,
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OCH3), 2.34 (d, 1H J = 14.4 Hz) ; 13C NMR (100 MHz, DMSO-d6): δ 46.49, 54.03, 54.93,
58.66, 60.31, 71.13, 114.72, 120.59, 122.35, 124.09, 124.34, 126.23, 127.12, 128.15, 128.78,
128.84, 129.14, 129.40, 129.65, 130.45, 130.74, 131.35, 131.76, 136.09, 136.34, 140.21, 141.64,
144.44, 147.11, 147.29, 153.59, 158.31, 165.30, 191.35. Anal. Calcd for C37H27N5O4S; C, 69.69;
H, 4.27; N, 10.98; O, 10.04; S, 5.03; Found C, 69.66; H, 4.25; N, 10.95; O, 10.02; S, 5.01;
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Biology
In Vitro Cytotoxicity Study
The cytotoxicity of the synthesized compounds were estimated by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MCF-7 and HeLa cells were
seeded in 100 mL of growth medium in 96 well plates (4000 cells/ well) and allowed for 24
hours. Then serially diluted compounds were added to the wells and incubated for 48 hours at
37° C in humidified 5% CO2, and then the growth media was drawn and changed with MTT in
IMDM and incubated for 4 hours. The MTT solution was suctioned and 100 mL of acidified
ethyl alcohol was added to each well and allowed for lysis step. The cell viability was analyzed
by spectrophotometer absorbance at 570 nm. The assay was repeated 3 times as triplicate. The
IC50 values were calculated by absorbance that reduced 50% when compared to untreated
control wells.
AO/EB (Acridine Orange/ Ethidium Bromide) staining assay
To detect the apoptotic cells AO/EB dual staining assay was performed. The cells were
seeded and grown in a 24-well plate and treated with 25 µM and 50 µM of the compound. The
MCF-7 cells were incubated for 24 hours and then the cells were washed twice with PBS. The
washed fresh cells were then stained with 10 µL of AO/EB and incubated for 1 hour. After
incubation, the cells were washed with PBS and the apoptotic morphological changes were
analyzed by a fluorescence microscope (Nikon ECLIPSE, TS100) at excitation filter of 480/30
nm.
DAPI Staining assay
The nuclear damage of compound treated cells were determined by DAPI (4’,6-
diamidino-2-phenylindole) staining assay. The MCF-7 cells were seeded and grown in a 24-well
plate and treated with 25 µM and 50 µM of the compound. The cells were incubated for 24 hours
and then the cells were washed twice with PBS. The washed fresh cells were then fixed with
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methanol: acetic acid (3:1 v/v) and stained with 5µg/mL of DAPI and incubated for 1 hour. After
incubation, the cells were washed with PBS and the nuclear damaged blue fluorescence cells
were analyzed by fluorescence microscope (Nikon ECLIPSE, TS100) at an excitation
wavelength of 510nm.
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DCFH-DA Assay for Detection of Intracellular ROS Generation
The intracellular ROS production was examined by DCFH-DA (2’,7’-dichloro
dihydrofluorescin diacetae) staining assay. The MCF-7 cells were seeded and grown in a 24-well
plate and treated with 25 µM and 50 µM of the compound. The cells were incubated for 24 hours
and then the cells were washed twice with PBS. The washed fresh cells were then stained with 5
µM of DCFH-DA and incubated for 1 hour. After incubation, the cells were washed with PBS
and the apoptotic morphological changes were analyzed by a fluorescence microscope (Nikon
ECLIPSE, TS100) at the excitation wavelength of 480nm.
Cell cycle analysis by flow cytometry
The cellular DNA content and distribution of cells for cell cycle were analyzed by flow
cytometry. The MCF-7 cells were seeded and grown in a 24-well plate and treated with IC50
concentration of the compound. The untreated MCF-7 cells were used as control. The cells were
incubated for 24 hours, and then the cells fixed with 700 µL of 70 % ice-cold ethyl alcohol and
re-suspended with 20 mg/mL of PBS containing RNase and incubated for 1 hour. After that, the
cells were stained with 50 mg/mL of propidium iodide and incubated for 30 minutes. The stained
cells were analyzed for DNA content and cell cycle phase distribution by flow cytometry (BD
Bioscience, San Jose, CA, USA)
FITC Annexin V-FITC Propidium Iodide staining Assay
The apoptotic cells were quantified by annexin V-FITC and propidium iodide (PI)
staining assay using flow cytometry method. The MCF-7 cells were seeded and grown in a 24-
well plate and treated with IC50 concentration of the compound. The untreated MCF-7 cells were
used as control. The cells were incubated for 24 hours, and then the cells were suspended 200 µL
of binding buffer and stained with 10 µL of annexin V-FITC and 5 µL of PI and incubated for 30
minutes in dark. After incubation, the cells were washed with PBS and the apoptotic cells were
analyzed by Flow cytometer (BD, FACS Calibur, USA).
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Western blot analysis
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The expression levels of caspase protein in the treated cells were analyzed by western
blot analysis. Western blot analysis for expression of the caspase was incubated for 1h in 5%
powdered non-fat milk in PBS (pH 7.4) with antibodies for 30 min in 5% non-fat dry milk with
goat anti-mouse serum (1:2000) and visualized. Subsequently, Western blot analysis was
performed for cell extracts after determining their protein content by Bradford assay. 50 μg
protein samples were resolved by 12% SDS-polyacrylamide gel electrophoresis and then
transferred to nitrocellulose membrane (Pall Corporation, USA). The membranes were immersed
in blocking buffer (5% skim milk in PBS) for 1 h at room temperature and incubated overnight
with primary antibodies. After normalization with the corresponding expression of β-actin, the
protein expression was determined.
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Statistical Analysis
The Statistical Program for Social Science Software (SPSS) was used for statistical
analysis. All the data expressed in ± 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 the
experiments were performed for three times in triplicate.
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