Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4) inhibitors by catalyst-free, green approach

Article abstract of DOI:10.1016/j.ejmech.2015.11.046

Aiming to develop a new target for the anticancer treatment, a series of 5′H-spiro[indoline-3,4′-pyrrolo [1,2-A]quinoxalin]-2-ones has been synthesized by simple, highly efficient and environmentally friendly method in excellent yields under catalyst-free

Full text of DOI:10.1016/j.ejmech.2015.11.046

Accepted Manuscript
Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4
(CDK4) inhibitors by Catalyst-free, green approach
Ahmed Kamal, Rasala Mahesh, V. Lakshma Nayak, Korrapati Suresh Babu, G.
Bharath Kumar, Anver Basha Shaik, Jeevak Sopanrao Kapure
PII:
S0223-5234(15)30382-2
10.1016/j.ejmech.2015.11.046
EJMECH 8236
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 July 2015
Revised Date: 5 November 2015
Accepted Date: 25 November 2015
Please cite this article as: A. Kamal, R. Mahesh, V.L. Nayak, K.S. Babu, G.B. Kumar, A.B. Shaik, J.S.
Kapure, Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4)
inhibitors by Catalyst-free, green approach, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2015.11.046.
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ACCEPTED MANUSCRIPT
Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4)
inhibitors by Catalyst-free, green approach
Ahmed Kamal a,b,c,*, Rasala Mahesh a V. Lakshma Nayak a Korrapati Suresh Babu a G Bharath
,
,
,
Kumar a Anver Basha Shaik a Jeevak Sopanrao Kapure b
,
,
A new class of anticancer agents, pyrrolospirooxindole derivatives have been synthesized by simple,
efficient and environmentally benign method has been developed for the synthesis of in excellent yields
under catalyst-free condition with ethanol. The synthesized compounds evaluated for their
antiproliferative activity, among them, compound 3n showed significant cytotoxicity against the human
prostate cancer (DU-145). Further mechanistic studies such as cell cycle analysis, mitochondrial potential
assay, annexin V-FITC assay and Western blot analysis indicated that 3n induces cell death by apoptosis.

ACCEPTED MANUSCRIPT
Discovery of pyrrolospirooxindole derivatives as novel cyclin
dependent kinase 4 (CDK4) inhibitors by Catalyst-free, green
approach
Ahmed Kamal a,b,c,*, Rasala Mahesh a V. Lakshma Nayak a Korrapati Suresh Babu a G Bharath
,
,
,
Kumar a Anver Basha Shaik a Jeevak Sopanrao Kapure b
,
,
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
c Catalytic Chemistry Research Chair, Chemistry Department, College of Science, King Saud
University, Riyadh 11451, Saudi Arabia
Abstract: Aiming to develop a new target for the anticancer treatment, a series of 5'H-
spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-ones has been synthesized by simple, highly
efficient and environmentally friendly method in excellent yields under catalyst-free conditions
using ethanol as a green solvent. A simple filtration of the reaction mixture and subsequent
drying affords analytically pure products. The synthesized derivatives were evaluated for their
antiproliferative activity against five different human cancer cell lines, among the congeners
compound 3n showed significant cytotoxicity against the human prostate cancer (DU-145). Flow
cytometric analysis revealed that this compound induces cell cycle arrest in the G0/G1 phase and
Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell
death. This was further confirmed by mitochondrial membrane potential ((∆Ψm), Annexin V-
FITC assay and docking experiments. Furthermore, it was observed that there is an increase in
expression levels of cyclin dependent kinase inhibitors like Cip1/p21 and Kip1/p27.
Keywords: Isatin, spirooxindole, ethanol, cytotoxicity, apoptosis.
* Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189;
E-mail addresses: ahmedkamal@iict.res.in

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1. Introduction
Spirocyclic compounds are notable for their molecular architecture as well as the
prominent biological activities [1]. Especially, spirocyclic oxindoles are attractive synthetic
targets because of their widespread occurrence in various natural products and biologically active
molecules [2,3]. The structural rigidity imparted by the spiro-carbon causes conformational
restrictions and this may influence the binding of such compounds to the biological targets
favourably. A selected assortment of natural products incorporating the spirooxindole unit is
depicted in Figure 1. Spirotryprostatin A and B are two natural alkaloids isolated from the
fermentation broth of Aspergillus fumigates, that have been identified as promising inhibitors of
microtubule assembly [4]. Most of these spirooxindole alkaloids were isolated from the
Apocynaceae and Rubiacae species, which play important roles in medicine [5], some other
important spirooxindole natural products include pteropodine (positive modulators of muscarinic
acid M) [6] coerulescine, horsfiline and elacomine (Figure 1). Spirooxindole systems also
constitute the core scaffold of many synthetic pharmaceuticals with a wide range of biological
applications such as antimicrobial [7], antitumor [8], antibiotic [9] and inhibitors of the human
NK-1 receptor [10].
N
OCH3
O
O
Me
NH
N
N
O
H
N
H
H
O
H3CO
O
O
O
N
H
N
HO
N
H
O
OCH3
N
R
H
H
Horsifiline
Elacomine
Cl
Pteropodine
R = OCH3 Spirotryprostatin A
R = H Spirotryprostatin B
Cl
Br
O
N
NH
O
H
N
O
O
N
O
N
N
O
N
HN
HN
NH
COOH
O
O
N
N
N
N
O
O
1PU501
Br
A
3n
B
Figure 1: Chemical structures of biologically active spirooxindole derivatives and CDK4
inhibitors

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In the course of our interest to develop nitrogen heterocyclic compounds [11] as
anticancer agents, prompted us to investigate the biological activity of spirooxindole scaffold in
detail. This called for a convenient and practical method for the synthesis of the spirooxindole
unit. Literature survey reveals that 1,3-dipolar cycloaddition reactions have been used
extensively to prepare spirooxindole derivatives [12]. The preparation of such compounds via the
reaction of isatin and N-(2-aminophenyl)pyrrole at room temperature in the presence of different
catalysts [13] such as molecular iodine, ZnCl2, CuCl2, FeCl3, p-TSA, Sulfamic acid and HCl are
also reported. In many cases the desired products were obtained in very low to moderate yields.
Our efforts led to the development of a catalyst-free synthesis of pyrrole fused spirooxindoles in
ethanol (Scheme 1).
Scheme 1: Green synthesis of cytotoxic spirooxindole derivatives
The screening of these new spiro-heterocycles revealed that they possess
significant cyctotoxicity and exhibit cyclin dependent kinase inhibition activities.
Recently, spirooxindole derivatives (B) have been reported as kinase-privileged scaffold
which have emerged as an attractive synthetic target molecules [14], 1PU501 [15]
(protein database code) and oxindole derivative [16] (A) depicted in Figure 1, are the
closest structural analogues of the title compounds that display CDK4 inhibitory activity,
are presently in preclinical research for the treatment of human cancers. The results of
our preliminary studies are presented in the following sections.
2. Results and discussion
2.1. Chemistry
Our studies began with exploring various non-catalytic routes for the cyclocondensation
reaction between 2-(1H-pyrrol-1-yl)aniline (1) and isatin (2) (Scheme 2). Some of the
representative reaction conditions that were investigated are depicted in Table 1. It was observed
that this reaction did not proceed at all at room temperature when run neat or in water,

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acetonitrile, methanol or ethanol (entries 1-5). Interestingly, the product 3a was obtained in 68%
by heating the neat reaction at 100 °C for 12 h (entry 6). Subsequently it was revealed that the
cyclocondensation proceeds efficiently in all the solvents tested under reflux temperatures
(entries 7-10). Reaction in water afforded high yields of products (entry 7), however,
recrystallization from ethanol was required for obtaining analytically pure samples. The reaction
in refluxing ethanol, on the other hand gave a slightly higher yield (entry 10) and pure product
crystallized on cooling the reaction mixture to room temperature. Considering the operational
simplicity and lower temperature ethanol was preferred over water as the solvent.
Table 1: Optimization of reaction conditions for the formation of 3a
Entry Solvent
Temperature (°C)
Time (h) Yielda (%)
1
2
Neat
Water
RT
RT
RT
RT
RT
100
80
12
12
12
12
12
12
12
6
--
--
3
CH3CN
Methanol
Ethanol
Neat
-
4
--
5
--
6
68
72
98
98
97
7
CH3CN
Methanol
Ethanol
Water
8
70
9
80
6
10
100
8
a isolated yields
The scope and generality of the present protocol were then examined by employing
various substituted isatins (Scheme 2) and the results are summarized in Table 2. The reaction
tolerates both electron releasing as well as withdrawing substituents on the isatin component
without any significant deviation in yields (entries 3b-i). N-Protected isatins also afforded the

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corresponding products in excellent yields (entries 3j-n). Importantly, analytically pure products
are obtained by cooling, filtering and drying.
Scheme 2: Synthesis of 5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one derivatives
Table 2: Synthesis of 5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one derivatives
Entry
3a
3b
3c
X
H
R
Time (h)
Yielda (%)
H
8
8.5
8.5
9
98
96
97
98
96
96
97
94
96
97
96
95
96
97
5-F
H
H
7-F
5-Cl
5-Br
5-I
H
3d
3e
H
9
H
10
12
12
8
3f
5-Me
5-OMe
5-NO2
H
H
3g
3h
3i
H
H
benzyl
benzyl
benzyl
pipernyl
pipernyl
10
10
11
10
10
3j
5-Cl
5-Br
H
3k
3l
3m
5-Cl
3n
a - isolated yields
A plausible mechanism of a representative cyclocondensation to furnish 3a may be
advanced as shown in Scheme 3. Schiff base formation from the electron rich 2-pyrrolyl aniline
1 and the highly electrophilic isatin 2a is followed by an intramolecular Friedel-Crafts type

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cyclization at the 2-position of the pyrrole. It may be recalled here that the intermediate 4a has
been prepared and independently converted to the product 3a previously [13a].
Scheme 3: Plausible Mechanism.
2.2 Biological evaluation
2.2.1 Cytotoxic activity
All the newly synthesized compounds (3a-n) were evaluated for their cytotoxic activity
in a panel of five human cancer cell lines, namely Hela (cervical), A549 (lung), DU-145
(prostate), HT-29 (colon) and MDA MB-231 (breast) by employing MTT assay [17], and
doxorubicin was used as the reference drug. The results are summarized in Table 3 and
expressed as IC50 values. The results revealed that some of these compounds exhibit promising
anticancer activity against the tested cell lines. Among them, compound, 3n showed significant
cytotoxicity against human prostate cancer cell line, DU-145 and consequently, it was chosen for
subsequent detailed studies.
Table 3: IC50 valuesa (in µM) for compounds in selected human cancer cell lines
Compound
Helab
4.57
A549c
7.10
DU-145d
6.03
HT-29e
4.11
MDA MB-231f
8.84
3a
3b
3c
3d
3e
3f
15.13
12.40
4.88
23.43
11.32
4.81
12.70
12.66
4.79
15.84
12.69
10.96
16.98
19.03
14.45
14.67
15.62
31.44
15.87
26.30
16.40
16.70
11.15
14.78
12.89
4.90
3.98
29.70
22.38
13.48
13.78
16.13
15.67
12.45
11.00
22.65
27.09
22.09
3g
3h
3i
15.70
17.4

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20.04
4.86
21.01
4.53
2.45
1.78
16.98
4.786
16.59
7.59
13.21
4.71
11.15
3.98
1.16
1.82
17.37
18.97
18.09
15.12
1.63
26.20
26.60
23.68
30.01
2.34
3j
3k
3l
3m
2.11
3n
1.06
1.16
1.67
Doxorubicin
a 50% Inhibitory concentration after 48 h of drug treatment and the values are average of three individual
experiments. b Human cervical cancer. c Human lung cancer. d Human prostate cancer. e human colon cancer. f
Human breast cancer.
2.2.2 Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combined
effect of both cycle block and apoptosis [18-19]. The in vitro screening results revealed that
compound 3n showed significant antiproliferative activity against human prostate cancer cell
line, DU-145. Therefore, it was considered of interest to understand whether this inhibition of
cell growth was on account of cell cycle arrest. In this study DU-145 cells were treated with this
compound at concentrations of 1 and 2 µM for 48 h. The data obtained clearly indicated that this
compound arrested the cell cycle at G0/G1 phase at 1 µM concentration. Interestingly, when the
concentration was increased from 1 µM to 2 µM it was observed that the percentage of cells in
G0/G1 phase decreased and accumulation of cells in subG1 phase increased, which indicates the
onset of apoptosis [20] (Figure 2).

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Figure 2: Cell cycle analysis of compound 3n on DU-145 cells. A: Control cells (DU-145), B: 3n (1 µM) and C: 3n
(2 µM).
Sample
Sub G1 %
8.20
G0/G1 %
81.67
S %
1.59
1.21
2.94
G2/M %
5.96
A: Control (DU-145)
B: 3n (1 µM)
C: 3n (2 µM)
6.98
85.16
4.20
29.12
58.84
4.46
2.2.3 Measurement of mitochondrial membrane potential (∆Ψm)
The maintenance of mitochondrial membrane potential (∆Ψm) is significant for
mitochondrial integrity and bioenergetic function [21]. Mitochondrial changes, including loss of
mitochondrial membrane potential (∆Ψm), are the key events that take place during drug-
induced apoptosis. Mitochondrial injury by compound 3n was evaluated by detecting drops in
mitochondrial membrane potential (∆Ψm). In this study we have investigated the involvement of
mitochondria in the induction of apoptosis by 3n. After 48 h of drug treatment with this
compound at 1 and 2 µM concentrations, it was observed that there is reduced mitochondrial
membrane potential (∆Ψm) of DU-145 cells as assessed by JC-1 staining (Figure 3).

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Figure 3: Drops in membrane potential (∆Ψm) was assessed by JC-1 staining of DU-145 cells treated with
compound 3n and samples were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson). A:
Control cells (DU-145), B: 3n (1 µM) and C: 3n (2 µM).
2.2.4 Annexin V-FITC assay for apoptosis
The apoptotic effect of 3n was further evaluated by Annexin V FITC/PI (AV/PI) dual
staining assay [22] to examine the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis. In this study DU-
145 cells were treated with compound 3n for 48 h at 1 and 2 µM concentrations to examine the
apoptotic effect. It was observed that compound 3n showed significant apoptosis against DU-145
cells as shown in Figure 4.

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Figure 4: Annexin V-FITC staining. A: Control cells (DU-145), B: 3n (1 µM) and C: 3n (2 µM).
Sample
UL %
0.67
UR %
1.63
LL%
95.57
60.64
54.99
LR %
2.13
A: Control
B: 3n (1 µM)
C: 3n (2 µM)
2.00
15.42
16.03
21.95
27.36
1.63
2.2.5 Western blot analysis of CDK4, p21 and p27
One of the characteristics of cancer is uncontrolled cell growth and proliferation. Cyclin
dependent kinases (CDKs) are key regulators of the cell cycle [23] and the proper regulation of
CDK activity is crucial for the ordered execution of the phases of the cycle. Cyclin-dependent
kinase inhibitors (CDKI) are tumor suppressor proteins that bind to active CDK-cyclin
complexes and inhibit their kinase activities [24]. Cip1/p21 is a universal Cyclin-dependent
kinase inhibitor and plays a critical role in the cellular response for cell cycle arrest [25].
Kip1/p27 is another important member of Cyclin-dependent kinase inhibitor which connects the
anti- proliferative signals for cell cycle arrest [26]. Lower level of Kip1/p27 protein is known as
a prognostic marker for many cancers [27]. In this connection, DU-145 cells were treated with
3n at 1 and 2 µM concentrations for 48 h and Western blot analysis was carried out. Results

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indicated that there was significant reduction of CDK4 expression level. Furthermore, the levels
of p21 and p27 cyclin dependent kinase inhibitors were significantly increased as seen in Figure
5.
Figure 5: Effect of compound (3n) on CDK4, p21 and p27 level. DU-145 cells were treated with this compound at
1 and 2 µM concentrations for 48 h. The cell lysates were collected and expression level of CDK4, p21 and p27
were determined by Western blot analysis. β-Actin was used as a loading control.
2.3 Molecular docking studies
To investigate the possible binding mode in the ATP binding domain of the CDK4 mimic
model, we performed docking studies on the most active conjugate 3n of the series to show the
binding interactions that may explain CDK4 selectivity. Coordinates of protein structure were
obtained from Protein Data Bank (PDB ID 1GIH) [15]. Docking was accomplished by using
AutoDock 4.2 software and Fig.6A shows comparison of binding of potent CDK4 inhibitor
(PDB code: 1PU501) within the crystal structure of CDK4 that mimics CDK2 (PDB code: 1GIH,
resolution 2.80 Å) with the most active conjugate 3n into the binding pocket of the same protein.
Whereas Fig. 6B and 6C show the similarity in the binding pattern of the central urea linker of
co-crystallized 1PU501 and docked 3n conjugate. In fig 6C peptidic NH of Leu83 shows
important interaction 2.9 Å from oxygen of benzo[d][1,3]dioxol-5-ylmethyl ring. The binding
model places a carbonyl oxygen of oxindole core at 4.2 Å from the backbone of His84 and 3.9 Å
from NH of Gln85. Additionally NH of pyrrolo[1,2-a]quinoxalin ring is found within 4.5-5.8 Å
from the backbone carbonyl oxygen of Leu83. The benzo[d][1,3]dioxol-5-ylmethyl ring of
conjugate 13 fitted at close proximity within residues Phe82, Leu83 and His84. This interaction
is closely similar to pyridine ring of the co-crystallized 1PU501 suggesting the occurrence of

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similar interactions. Fig 6D shows the superimposition of co-crystallized 1PU501 and conjugate
3n, where in pyridine ring of co-crystallized 1PU501 overlaps closely with the
benzo[d][1,3]dioxol-5-ylmethyl ring of the conjugate 3n with different orientation. In summary
docking studies indentifies key interactions with backbone groups of hinge region and suggests
that conjugate 3n directly binds to ATP binding site of CDK4.
Figure 6: Molecular docking of 3n within the ATP binding pocket of CDK4. (6A) View of the X-ray
crystallographic structure of the binding pocket of CDK4 mimic of CDK2 kinase domain (PDB code: 1GIH,
resolution 2.8 Å) showing amino acids involved in binding. (6B) X-ray crystallographic structure of co-crystallized
ligand (PDB code: 1PU501) within the binding pocket of CDK4. (6C) Docked structure of 3n within CDK4, (6D)
superimposition of the co-crystallized ligand (red) 3n docked structure (green).

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3. Conclusion
In conclusion, a simple, mild, efficient and environmentally benign method for the
synthesis of 5'H-spiro[indoline-3,4'-pyrrolo(1,2-a)quinoxalin]-2-ones has been developed
without using a catalysts in ethanol. The advantages of this method include its simplicity of
operation, cleaner reactions, absence of side products and higher yields. Further, the purification
of the product is simple, involving a simple filtration and washing. The synthesized compounds
are tested for their cytotoxocity and they exhibit IC50 values in the range 31.44 - 1.16 µm.
Compound 3n with piperonyl substitution on 5-chloroisatin nitrogen displayed the highest
cyctotoxicity in the series. Its IC50 values are comparable to that of the positive control,
doxorubicin. Further mechanistic studies such as cell cycle analysis, mitochondrial potential
assay, annexin V-FITC assay and Western blot analysis indicated that 3n induces cell death by
apoptosis.
4. Experimental section
4.1. Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich), St. Louis, MO,
USA), Lancaster (Alfa Aeser, Johnson Matthey Company, Ward Hill, MA, USA), or
Spectrochem Pvt. Ltd. (Mumbai, India) and were used without further purification. Reactions
were performer by TLC performed on silicagel glass plate containing 60 GF-254, and
1
visualization was achieved by UV light or iodine indicator. H and 13C NMR spectra were
determined in DMSO-d6 by using Varian and Avance instruments. Chemical shifts are expressed
in parts per million (
δ in ppm) downfield from internal TMS and coupling constants are
expressed in Hz. 1H NMR spectroscopic data are reported in the following order: multiplicity (s,
singlet; brs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet), coupling
constants in Hz, number of protons. ESI mass spectra were recorded on a Micro mass Quattro
LC using ESI+ software with capillary voltage 3.98 kV and an ESI mode positive ion trap
detector. Melting points were determined with an Electro thermal melting point apparatus, and
are uncorrected.
4.2. General procedure for the synthesis of N-benzyl isatins
Isatin (1mmol), corresponding benzyl bromide (1mmol) and K2CO3 (3nmol) were heated
in acetonitrile for 1-2 hours. After completion of the reaction, acetonitrile was removed on
rotavopar and the product was extracted using ethyl acetate. The organic layer was dried over

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anhydrous Na2SO4 and concentrated. The product obtained was used for next step without
further purification.
4.2.1 General procedure for the synthesis of 5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-
one derivatives
A mixture of 2-(1H-pyrrol-1-yl) aniline and (1 mmol) isatin (1 mmol) in ethanol was
refluxed at 80 °C for 6 h. The progress of reaction was monitored by TLC. After completion of
the reaction, cooled to room temperature, the precipitated product was filtered, washed with
ethanol and dried under reduced pressure to afford the pure products.
4.3. 1-benzylindoline-2,3-dione
0
1
Light red solid (Yield : 99%); 126-128 C; H NMR (300 MHz, CDCl3): δ 4.93 (s, 2H),
6.78 (d, = 8.07 Hz, 1H), 7.09 (t, = 7.47 & 7.62 Hz, 1H), 7.28-7.36 (m, 5H), 7.48 (dd, = 1.37
& 7.93 Hz, 1H), 7.61 (d,
= 7.47 Hz, 1H); ESI-MS: m/z = 238 (M+H)+.
J
J
J
J
4.3.1. 1-benzyl-5-chloroindoline-2,3-dione
Light red solid (Yield: 98%); 142-144 C; H NMR (300 MHz, CDCl3): δ 4.93 (s, 2H),
6.72 (d, = 8.39 Hz, 1H), 7.29-7.37 (m, 5H), 7.43 (dd, = 2.13 & 8.39 Hz, 1H), 7.57 (d, = 8.39
0
1
J
J
J
Hz, 1H); ESI-MS: m/z = 272 (M+H)+.
4.3.2. 1-benzyl-5-bromoindoline-2,3-dione
0
1
Red solid (Yield : 98%); 106-108 C; H NMR (300 MHz, CDCl3): δ 4.92 (s, 2H), 6.67
= 1.67 & 8.39 Hz, 1H), 7.30-7.37 (m, 5H), 7.59 (dd, = 2.13 & 8.39 Hz, 1H), 7.70 (d,
2.89 Hz, 1H); ESI-MS: m/z = 316 (M)+.
(dd,
J
J
J =
4.3.3. 1-(benzo[d][1,3]dioxol-5-ylmethyl)indoline-2,3-dione
Red solid (Yield : 97%); 101-1030C; 1H NMR (300 MHz, CDCl3): δ 4.83 (s, 2H), 5.94 (s,
2H), 6.77 (d, = 7.78 Hz, 1H), 6.80-6.83 (m, 3H), 7.09 (dd, = 0.61 & 7.62 Hz, 1H), 7.50 (dd,
= 1.22 & 7.78 Hz, 1H), 7.61 (d,
= 7.47 Hz, 1H); ESI-MS: m/z = 282 (M+H)+.
4.3.4. 1-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloroindoline-2,3-dione
J
J
J
J
0
1
Red solid (Yield : 97%); 116-118 C; H NMR (300 MHz, CDCl3): δ 4.82 (s, 2H), 5.95
(s, 2H), 6.76-6.80 (m, 4H), 7.46 (dd,
MS: m/z = 316 (M+H)+.
J = 1.98 & 8.39 Hz, 1H), 7.56 (d, J = 1.83 Hz, 1H); ESI-
4.3.5. 5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3a)

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0
0
1
White solid; 173-175 C (Yield : 98%); 142-144 C; H NMR (300 MHz, DMSO-d6): δ
5.55-5.57 (m, 2H), 6.21 (t, J = 2.83 & 6.23 Hz, 1H), 6.77-6.82 (m, 2H), 6.89-6.96 (m, 2H), 7.02
(t, J = 7.55 & 15.10 Hz, 1H), 7.25-7.34 (m, 4H), 9.71 (brs, 1H); 13C NMR (75 MHz, DMSO-d6):
δ 60.38, 104.84, 109.55, 109.72, 113.32, 114.75, 114.80, 117.71, 121.82, 124.05, 124.13, 125.03,
125.54, 129.23, 130.59, 134.92, 142.03, 177.00; ESI-MS: m/z = 288 (M+H)+; HRMS (ESI) m/z
for C18H14ON3 calculated m/z: 288.11314, found m/z: 288.11190 (M+H)+.
4.3.6. 5-fluoro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3b)
1
Light red solid (Yield : 96%); 142-144 0C; H NMR (300 MHz, DMSO-d6): δ 5.58 (dd,
J
=
1.32 & 3.39 Hz, 1H), 5.75 (brs, 1H), 6.22 (t, J = 3.21 & 6.42 Hz, 1H), 6.77-6.86 (m, 3H),
6.91-7.01 (m, 2H), 7.06 (dd, J = 2.64 & 7.74 Hz, 1H), 7.27 (q, J = 1.51 & 2.83 Hz, 1H), 7.34 (d,
J = 7.93 Hz, 1H), 9.82 (brs, 1H); 13C NMR (75 MHz, DMSO-d6): δ 61.29, 105.57, 110.14,
110.83, 113.35, 113.92, 115.16, 115.48, 116.31, 119.00, 124.60, 124.90, 132.05, 134.15, 137.74,
157.20, 160.39, 177.52; ESI-MS: m/z = 328 (M+Na)+; HRMS (ESI) m/z for C18H12ON3FNa
calculated m/z: 328.08566, found m/z: 328.08457 (M+H)+.
4.3.7. 7-fluoro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3c)
White solid (Yield : 97%); 136-138 0C; 1H NMR (300 MHz, DMSO-d6): δ 5.54 (dd, J =
1.32 & 3.39 Hz, 1H), 6.18-6.21 (m, 2H), 6.76-6.84 (m, 2H), 6.91-7.14 (m, 4H), 7.28 (d, J = 1.13
13
Hz, 1H), 7.35 (d, J = 7.74 Hz, 1H), 10.39 (brs, 1H); C NMR (75 MHz, DMSO-d6): δ 60.60,
105.01, 109.84, 113.42, 114.91, 116.32, 117.96, 120.81, 122.52, 123.97, 124.25, 124.93, 129.37,
133.47, 134.59, 176.66; ESI-MS: m/z = 306 (M+H)+; HRMS (ESI) m/z for C18H13ON3F
calculated m/z: 306.10372, found m/z: 306.10348 (M+H)+.
4.3.8. 5-chloro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3d)
Light red solid (Yield : 98%); 173-175 0C; 1H NMR (300 MHz, DMSO-d6): δ 5.58 (dd,
J
=
1.51 & 3.58 Hz, 1H), 5.85 (brs, 1H), 6.22 (t, J = 3.21 & 6.42 Hz, 1H), 6.77-6.86 (m, 3H), 6.94
(dt, J = 1.13 & 7.93 Hz, 1H), 7.22-7.34 (m, 4H), 9.94 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ
60.56, 109.87, 110.83, 113.44, 114.83, 114.99, 117.82, 123.84, 124.27, 124.84, 125.17, 126.46,
129.09, 132.67, 134.74, 140.85, 176.75; ESI-MS: m/z = 322 (M+H)+; HRMS (ESI) m/z for
C18H13ON3Cl calculated m/z: 322.07417, found m/z: 322.07392 (M+H)+.
4.3.9. 5-bromo-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3e)

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0
1
Light yellow solid (Yield : 96%); 111-113 C; H NMR (300 MHz, DMSO-d6): δ 5.52-
5.61 (m, 1H), 6.03 (brs, 1H), 6.22 (t, J = 3.21 & 6.42 Hz, 1H), 6.76-6.83 (m, 3H), 6.93 (t, J =
7.55 & 15.10 Hz, 1H), 7.27-7.41 (m, 4H), 9.99 (s, 1H); 13C NMR (75 MHz, DMSO-d6): δ 60.58,
105.04, 109.75, 111.26, 113.43, 114.05, 114.80, 114.97, 118.20, 123.97, 124.22, 124.63, 128.09,
132.01, 132.60, 137.17, 141.00, 176.63; ESI-MS: m/z = 366 (M)+; HRMS (ESI) m/z for
C18H13ON3Br calculated m/z: 366.02365, found m/z: 366.02362 (M)+.
4.3.10. 5-iodo-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3f)
Light blue solid (Yield : 96%); 107-109 0C; 1H NMR (300 MHz, DMSO-d6): δ 5.58 (dd,
J = 1.32 & 3.39 Hz, 1H), 5.94 (brs, 1H), 6.22 (t, J = 3.02 & 6.23 Hz, 1H), 6.71-6.83 (m, 3H),
6.94 (t, J = 7.36 & 15.86 Hz, 1H), 7.24-7.34 (m, 2H), 7.54-7.60 (m, 2H), 9.98 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): δ 60.23, 83.81, 104.80, 109.83, 111.89, 113.51, 114.67, 115.14, 117.58,
123.71, 124.20, 124.83, 133.38, 133.47, 134.84, 137.80, 142.07, 176.25; ESI-MS: m/z = 414
(M)+; HRMS (ESI) m/z for C18H13ON3I calculated m/z: 414.00978, found m/z: 414.00897 (M)+.
4.3.11. 5-methyl-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3g)
0
1
Light red solid (Yield : 97%); 216-218 C; H NMR (300 MHz, DMSO-d6): δ 2.28 (s,
3H), 4.26 (brs, 1H), 5.58 (dd, J = 1.32 & 3.39 Hz, 1H), 6.23 (t, J = 3.21 & 6.42 Hz, 1H), 6.61 (d,
J = 7.93 Hz, 1H), 6.67 (dd, J = 1.32 & 7.74 Hz, 1H), 6.86 (dt, J = 1.32 & 7.55 Hz, 1H), 6.94 (dt,
J = 1.32 & 7.55 Hz, 1H), 7.04 (d, J = 7.17 Hz, 1H), 7.18 (s, 1H), 7.25-7.28 (m, 1H), 7.36 (d, J =
13
7.55 Hz, 1H), 8.09 (brs, 1H); C NMR (75 MHz, DMSO-d6): δ 20.54, 60.48, 104.85, 109.37,
109.80, 113.42, 114.77, 117.52, 123.97, 124.20, 125.69, 125.77, 129.60, 130.87, 131.00, 135.26,
139.67, 177.12; ESI-MS: m/z = 302 (M+H)+; HRMS (ESI) m/z for C19H16ON3 calculated m/z:
302.12879, found m/z: 302.12832 (M+H)+.
4.3.12. 5-methoxy-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3h)
0
1
Light red solid (Yield : 94%); 261.263 C; H NMR (300 MHz, DMSO-d6): δ 3.74 (s,
3H), 5.57 (dd, J = 1.51 & 3.58 Hz, 1H), 5.74 (brs, 1H), 6.21 (t, J = 3.21 & 6.42 Hz, 1H), 6.76-
6.96 (m, 5H), 7.27 (t, J = 1.51 & 2.83 Hz, 1H), 7.33 (d, J = 7.93 Hz, 1H), 7.54 (s, 1H), 9.62 (s,
13
1H); C NMR (75 MHz, DMSO-d6): δ 55.28, 60.74, 104.72, 109.84, 110.06, 111.56, 113.73,
114.56, 114.66, 115.38, 117.38, 123.91, 124.24, 125.64, 132.04, 135.39, 135.50, 155.08, 176.95;
ESI-MS: m/z = 318 (M+H)+; HRMS (ESI) m/z for C19H16O2N3 calculated m/z: 318.12370,
found m/z: 318.12220 (M+H)+.

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4.3.13. 5-nitro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3i)
Yellow solid (Yield : 96%); 239.241 0C; 1H NMR (300 MHz, DMSO-d6): δ 5.58 (dd, J =
1.32 & 3.39 Hz, 1H), 5.88 (s, 1H), 6.24 (t, J = 3.21 & 6.42 Hz, 1H), 6.84 6.23 (t, J = 7.74 &
14.35 Hz, 2H), 6.95-7.04 (m, 2H), 7.30 6.23 (q, J = 1.32 & 2.83 Hz, 1H), 7.35 6.23 (d, J = 7.55
13
Hz, 1H), 8.21-8.26 (m, 2H), 10.61 (brs, 1H); C NMR (75 MHz, DMSO-d6): δ 60.07, 104.90,
109.28, 109.69, 113.31, 114.72, 115.07, 118.16, 120.74, 123.67, 124.13, 125.98, 131.42, 133.81,
142.35, 148.19, 176.98; ESI-MS: m/z = 355 (M+Na)+; HRMS (ESI) m/z for C18H12O3N4Na
calculated m/z: 355.08016, found m/z: 355.08021 (M+H)+.
4.3.14. 1-benzyl-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3j)
0
1
White solid (Yield : 97%); 106-108 C; H NMR (300 MHz, DMSO-d6): δ 4.74 (d, J =
15.67 Hz, 1H), 4.89 (d, J = 15.67 Hz, 1H), 5.55 (dd, J = 1.51 & 3.58 Hz, 1H), 5.59 (brs, 1H),
6.23 (t, J = 3.21 & 6.42 Hz, 1H), 6.77 (d, J = 7.74 Hz, 1H), 6.84-6.86 (m, 2H), 6.97 (dt, J = 1.13
& 7.93 Hz, 1H), 7.07 (t, J = 7.55 & 15.67 Hz, 1H), 7.24-7.44 (m, 9H); 13C NMR (75 MHz,
DMSO-d6): δ 42.76, 60.15, 104.88, 108.70, 109.85, 113.42, 114.94, 115.02, 117.99, 122.60,
124.10, 124.26, 125.02, 125.29, 126.67, 127.02, 128.17, 129.36, 129.99, 134.76, 135.18, 142.57,
175.24; ESI-MS: m/z = 412 (M+H)+; HRMS (ESI) m/z for C25H20ON3 calculated m/z:
378.16009, found m/z: 378.16028 (M+H)+.
4.3.15. 1-benzyl-5-chloro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3k)
0
1
White solid (Yield : 96%); 119-121 C; H NMR (300 MHz, DMSO-d6): δ 4.72 (d, J =
15.86 Hz, 1H), 4.988 (d, J = 15.67 Hz, 1H), 5.56 (dd, J = 1.32 & 3.39 Hz, 1H), 6.25 (t, J = 3.21
& 6.42 Hz, 1H), 6.33 (brs, 1H), 6.72 (d, J = 8.30 Hz, 1H), 6.79-6.88 (m, 2H), 6.94-7.01 (m, 1H),
7.19-7.49 (m, 9H); 13C NMR (75 MHz, DMSO-d6): δ 42.83, 60.27, 104.92, 109.98, 113.49,
114.95, 115.21, 118.07, 123.84, 124.34, 124.56, 125.18, 126.61, 127.14, 127.50, 128.25, 129.14,
132.05, 134.52, 134.81, 141.21, 174.94; ESI-MS: m/z = 412 (M+H)+; HRMS (ESI) m/z for
C25H19ON3Cl calculated m/z: 412.12112, found m/z: 412.12156 (M+H)+.
4.3.16. 1-benzyl-5-bromo-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin]-2-one (3l)
0
1
White solid (Yield : 95%); 97-99 C; H NMR (300 MHz, DMSO-d6): δ 4.69 (d, J =
15.67 Hz, 1H), 4.90 (d, J = 15.67 Hz, 1H), 5.59 (dd, J = 1.32 & 3.39 Hz, 1H), 5.94 (s, 1H), 6.26
(t, J = 3.02 & 6.42 Hz, 1H), 6.66 (d, J = 8.30 Hz, 1H), 6.82-6.87 (m, 2H), 6.98 (dt, J = 1.13 &
13
7.74 Hz, 1H), 7.24-7.40 (m, 7H), 7.47 (s, 1H), 7.52 (d, J = 1.88 Hz, 1H); C NMR (75 MHz,

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DMSO-d6): δ 43.08, 60.40, 105.20, 110.01, 110.44, 113.70, 115.05, 115.18, 115.42, 118.65,
124.19, 124.46, 126.74, 127.31, 128.29, 128.37, 132.08, 132.25, 134.06, 134.77, 141.68, 174.86;
ESI-MS: m/z = 478 (M+Na)+; HRMS (ESI) m/z for C25H18ON3BrNa calculated m/z: 478.05255,
found m/z: 478.05167 (M+H)+.
4.3.17. 1-(benzo[d][1,3]dioxol-5-ylmethyl)-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]quinoxalin] -2-
one (3m)
White solid (Yield : 96%); 131-133 0C; 1H NMR (300 MHz, DMSO-d6): δ 1H NMR (300
MHz, DMSO-d6): δ 4.67-4.78 (t, 2H), 4.53-4.54 (dd, J = 3.39 & 3.39 Hz, 1H), 5.98 (d, 2H),
6.18-6.21 (t, J = 2.64 & 5.30 Hz, 1H), 6.75-6.83 (m, 4H), 6.93-7.01 (dt, J = 2.18 & 7.93 Hz,
2H), 7.11 (Brs, 3H), 7.34-7.41 (dt, J = 8.30 & 13.59 Hz, 2H), 7.54-7.57 (d, J = 6.79 Hz, 2H);
13C NMR (75 MHz, DMSO-d6): δ 42.71, 60.24, 100.51, 104.92, 107.08, 107.77, 109.89, 109.96,
113.45, 114.93, 115.15, 118.14, 120.07, 123.87, 124.31, 124.47, 125.19, 127.58, 128.39, 129.11,
131.92, 134.35, 141.02, 146.52, 147.46, 174.85; ESI-MS: m/z = 422 (M+H)+; HRMS (ESI) m/z
for C26H20O3N3 calculated m/z: 422.14992, found m/z: 422.14976 (M+H)+.
4.3.18.
quinoxalin]-2-one (3n)
White solid (Yield : 97%); 125-127 0C; 1H NMR (300 MHz, DMSO-d6): δ 4.27 (brs, 1H),
1-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloro-5'H-spiro[indoline-3,4'-pyrrolo[1,2-a]
4.58 (d, J = 15.41 Hz, 1H), 4.84 (d, J = 15.41 Hz, 1H), 5.56 (dd, J = 1.52 & 3.50 Hz, 1H), 5.91
(d, J = 1.22 Hz, 2H), 6.24 (t, J = 3.20 & 6.40 Hz, 1H), 6.72-6.79 (m, 4H), 6.89 (dt, J = 1.22 &
7.78 Hz, 1H), 6.99 (dt, J = 1.22 & 7.78 Hz, 1H), 7.08 (t, J = 7.62 & 15.10 Hz, 1H), 7.27-7.30
(m, 2H), 7.38 (d, J = 8.85 Hz, 1H), 7.43 (d, J = 8.08 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): δ
42.66, 60.17, 100.49, 104.91, 107.19, 107.75, 108.78, 109.82, 113.43, 114.95, 115.03, 118.10,
120.10, 122.63, 124.17, 124.26, 125.02, 125.24, 128.88, 129.36, 129.94, 134.64, 142.47, 146.45,
147.42, 175.21; ESI-MS: m/z = 456 (M+H)+; HRMS (ESI) m/z for C26H19O3N3Cl calculated
m/z: 456.11095, found m/z: 456.11104 (M+H)+.
4.4. Biology
4.4.1. Anti cancer activity

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The Anti cancer activity of the compounds was determined using MTT assay [28]. 1×104
cells/well were seeded in 100µl DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37°C in a CO2 incubator. After 24 h of incubation
all the synthesized compounds were added to the cells and incubated for 48 h. After 48 h of drug
treatment, 10 µl MTT (3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) (5
mg/mL) was added to each well and the plates were further incubated for 4 h. Then the
supernatant from each well was carefully removed, formazon crystals were dissolved in 100 µl
of DMSO and absorbance at 570 nm wavelength was recorded.
4.4.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
through the cell-cycle phases. DU-145 cells were incubated for 48 h with compound 3n at
concentrations of 1 and 2 µM. Untreated and treated cells were harvested, washed with
phosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol, and stained with propidium
iodide (Sigma–Aldrich). Cell-cycle analysis was performed by flow cytometry (Becton
Dickinson FACS Caliber instrument) [29].
4.4.3. Mitochondrial membrane potential
DU-145 (1×106 cells/well) cells were cultured in six-well plates. After plating, cells were
treated with compound 3n at 1 and 2 µM concentrations for 48 h. After 48 h of treatment, cells
were collected by trypsinization and washed with PBS followed by resuspending in JC-1 (5
0
µg/ml) and incubated at 37 C for 15 min. Cells were rinsed three times with medium and
suspended in pre warmed medium. The cells were then subjected to flow cytometric analysis on
a flow cytometer (Becton Dickinson) in the FL1, FL2 channel to detect mitochondrial potential
[30].
4.4.4. Annexin staining assay for apoptosis
DU-145 (1×106) were seeded in six-well plates and allowed to grow overnight. The
medium was then replaced with complete medium containing compound 3n at 1 and 2 µM
concentrations. After 48 h of drug treatment, cells from the supernatant and adherent monolayer
cells were harvested by trypsinization, washed with PBS at 3000 rpm. Then the cells were
stained with Annexin VFITC and propidium iodide using the Annexin-V-FITC apoptosis
detection kit (Sigma aldrich). Flow cytometry was performed using a FACScan (Becton
Dickinson) equipped with a single 488-nm argon laser as described earlier [31].

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4.4.5. Protein extraction and Western blot analysis
DU-145 cells were treated with compound 3n at 1 and 2 µM concentrations for 48 h. The
cell lysates were obtained by lysing the cells in ice-cold radioimmunoprecipitation assay (RIPA)
buffer (Thermo Fischer Scientifics Ltd.). After centrifugation at 12000 rpm for 10 min, the
protein in the supernatant was quantified by the Bradford method (BIO-RAD) by using a
Multimode Varioskan instrument (Thermo Fischer Scientifics Ltd.). Protein (50 µg per lane) was
applied in 12% SDS polyacrylamide gel. After electrophoresis, the protein was transferred to a
polyvinylidine difluoride (PVDF) membrane (Thermo Fischer Scientifics Ltd.). The membrane
was blocked at room temperature for 2 h in Tris-buffered saline (TBS) with 0.1% Tween 20
(TBST) containing 5% Bovine Serum Albumin (BSA). The membrane was washed with TBST
for 5 min, then primary antibody was added and the membrane was incubated for overnight.
After incubation the membrane was incubated with the corresponding horseradish peroxidase
labeled secondary antibody (Cell Signaling Technology-CST) at room temperature for 1 h.
Membranes were washed with TBST three times for 15 min, and the blots were visualized with
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). Images were captured by using
the chemiluminescence (vilber lourmat) [32].
4.4.5. Molecular docking procedure:
Optimizations of all compounds were performed in Gaussian 09 by using PM3
semiempirical methods. The protein cocrystal structure was downloaded from RSCB-Protein
Data Bank with ligand (PDB ID 1GIH)1. Autodock 4.2 software [33] was used to perform the
docking studies. The visualization and analysis of the interactions were performed by using
PyMOL (ver. 0.99).
Acknowledgements
R.M. and K.S.B acknowledge CSIR, New Delhi for the award of a senior research
fellowship. We also acknowledge CSIR for financial support under the 12th Five Year plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301). We thank Dr Rajeev S. Menon for
reviewing this manuscript.

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Research Highlights
1). A simple, greener and highly efficient method
2). Catalyst-free, ethanol is used as green solvent
3). 14 examples have been tested for their antiproliferative activity, compound 3n showed significant
cytotoxicity with IC50 1.16 µM
4). Compound 3n induces cell cycle arrest in the G0/G1 phase
5). Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell death.