Synthesis of substituted biphenyl methylene indolinones as apoptosis inducers and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.01.063

A new series of biphenyl methylene indolinones has been designed, synthesized and evaluated for their in vitro antiproliferative activity against various cancer cell lines like DU-145 (prostate cancer cell line), 4T1 (mouse breast cancer cell line), MDA-MB-231 (human breast cancer cell line), BT-549 (human breast cancer cell line), T24 (human urinary bladder carcinoma cell line), and HeLa (cervical cancer cell line). Among the series, compound 10e showed potent in vitro cytotoxic activity against HeLa and DU-145 cancer cell lines with IC₅₀ value of 1.74 ± 0.69 μM and 1.68 ± 1.06 μM respectively. To understand the underlying mechanism of most potent cytotoxic compound 10e, various mechanistic studies were carried out on DU-145 cell lines. Cell cycle analysis results revealed that these conjugates affect both G0/G1 and G2/M phase of the cycle, tubulin binding assay resulted that compound 10e interrupting microtubule network formation by inhibiting tubulin polymerization with IC₅₀ value of 4.96 ± 0.05 μM. Moreover, molecular docking of 10e on colchicine binding site of the tubulin explains the interaction of 10e with tubulin. Clonogenic assay indicated inhibition of colony formation by compound 10e in a dose dependent manner. In addition, morphological changes were clearly observed by AO/EB and DAPI staining studies. Moreover, ROS detection using DCFDA, JC-1, and annexin V-FITC assays demonstrated the significant apoptosis induction by 10e.

Full text of DOI:10.1016/j.bioorg.2019.01.063

BioorganicChemistry86(2019)210–223
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of substituted biphenyl methylene indolinones as apoptosis
inducers and tubulin polymerization inhibitors
Kavitha Donthiboinaa, Pratibha Anchib, P.V. Sri Ramyaa, Shailaja Karrib, Gannoju Srinivasulua,
⁎
⁎
Chandraiah Godugub, Nagula Shankaraiaha, , Ahmed Kamala,c,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
b Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
c School of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi 110062, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of biphenyl methylene indolinones has been designed, synthesized and evaluated for their in vitro
antiproliferative activity against various cancer cell lines like DU-145 (prostate cancer cell line), 4T1 (mouse
breast cancer cell line), MDA-MB-231 (human breast cancer cell line), BT-549 (human breast cancer cell line),
T24 (human urinary bladder carcinoma cell line), and HeLa (cervical cancer cell line). Among the series,
compound 10e showed potent in vitro cytotoxic activity against HeLa and DU-145 cancer cell lines with IC50
Methylene indolinones
Cytotoxicity
Tubulin polymerization
Cell cycle
Annexin binding
Molecular modeling
value of 1.74
0.69 µM and 1.68
1.06 µM respectively. To understand the underlying mechanism of most
potent cytotoxic compound 10e, various mechanistic studies were carried out on DU-145 cell lines. Cell cycle
analysis results revealed that these conjugates affect both G0/G1 and G2/M phase of the cycle, tubulin binding
assay resulted that compound 10e interrupting microtubule network formation by inhibiting tubulin poly-
merization with IC50 value of 4.96
0.05 μM. Moreover, molecular docking of 10e on colchicine binding site of
the tubulin explains the interaction of 10e with tubulin. Clonogenic assay indicated inhibition of colony for-
mation by compound 10e in a dose dependent manner. In addition, morphological changes were clearly ob-
served by AO/EB and DAPI staining studies. Moreover, ROS detection using DCFDA, JC-1, and annexin V-FITC
assays demonstrated the significant apoptosis induction by 10e.
1. Introduction
potential biological activities like anticancer, antibacterial, antifungal,
antiviral and anti-angiogenic properties [6,7]. Sunitinib (5 SU11248,
Microtubule dynamics is an attractive biological target for various
synthetic and semi-synthetic chemotherapeutic agents [1,2] as micro-
tubule system is ubiquitous element, serves as primary substrate for
various cellular processes such as cell division, formation, maintenance
of cell shape, regulation of motility, cell signaling, secretion and in-
tracellular transport [3]. Anti-microtubule agents can interrupt highly
dynamic microtubule net work and they are categorized as microtubule
stabilizing agents, vinca site binding agents and colchicine site binding
agents [4]. Most of the studies revealed that biphenyl configuration is
an essential dimension to mimic colchicine binding site, the presence of
planar group is one of the pharmacophoric features required to bind
colchicine site of the tubulin [5a], which is present in several cytotoxic
natural products such as steganacine (1), steganone (2), eupomatilone
(3), and buflavine (4, Fig. 1) [5b].
Fig. 1) having 3-alkenyl oxindole part is significant as orally active
receptor tyrosine kinase (RTK) inhibitor, currently prescribed for the
treatment of renal cell carcinoma and gastrointestinal stromal tumours
[8,9]. Semaxanib (6, SU5416) is a related anticancer, anti-angiogenic
scaffold which is in clinical trials for colorectal cancer [10]. A-432411
is also an another oxindole containing anti-microtubule agent, which
activate spindle check points to induce apoptosis [11]. Based on these
investigations by exemplifying the significance of biaryl compounds
and oxindole derivatives with potent cytotoxic property, herein we
have developed a new series of biaryl moieties which are in conjugation
with 3-alkenyl oxindole (Fig. 2). In recent years, molecular hybridiza-
tion approach is an essential tool in medicinal chemistry which involves
combining of two bioactive scaffolds [12]. Previously, our research
group reported hybridization of bioactive moieties like imidazo-thia-
zoles, 2-anilinopyrydyls and pyrazoles with oxindole which were
On the other hand, oxindole is a privileged scaffold exhibiting
⁎ Corresponding authors at: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
(A. Kamal).
E-mail addresses: shankar@niperhyd.ac.in (N. Shankaraiah), ahmedkamal@iict.res.in (A. Kamal).
https://doi.org/10.1016/j.bioorg.2019.01.063
Received 19 September 2018; Received in revised form 17 January 2019; Accepted 27 January 2019
Availableonline29January2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

K. Donthiboina et al.
BioorganicChemistry86(2019)210–223
Fig. 1. Chemical structures of bioactive steganacine (1), steganone (2), eupomatilone (3), buflavin (4), sunitinib (5), semaxinib (6), and newly synthesized biphenyl
methylene indolinones 10a–r.
evaluated and reported as anti-cancer agents [13–15]. In continuation
of our efforts towards the synthesis of bioactive scaffolds, herein we
attempted to link 3-alkenyl oxindoles with biphenyl moiety which re-
sult in biphenyl methylene indolinones 10a–r and further evaluated for
their in vitro anti-cancer activity.
1.1. Cytotoxicity assay
The synthesized compounds 10a–r were evaluated for their in vitro
cytotoxicity by dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay [16] against a panel of selected cancer cell lines, viz., DU-
145 (prostate cancer cell line), 4T1 (mouse breast cancer cell line),
MDA-MB-231 (human breast cancer cell line), BT-549 (human breast
cancer cell line), T24 (human urinary bladder carcinoma cell line),
HeLa (human cervical cancer) and compared with reference compound
vincristine sulfate (Table 1). Among the synthesized compounds, 10e
showed significant cytotoxic activity on all the tested cancer cell lines,
and a potent effect on DU-145, and HeLa cell lines with an IC50 value of
The syntheses of target biphenyl methylene indolinones 10a–r were
depicted in Scheme 1, thus includes bromination of substituted ben-
zaldehydes 7a,b using Br2 which resulted 8a,b followed by Suzuki
cross-coupling with Pd(PPh3)4 afforded the key biphenyl 2-carbalde-
hyde intermediates 9a–d in overall good yields. Finally, the target
compounds 10a–r were obtained by the Knoevenagel condensation
between different oxindoles 11a-e and biphenyl 2-carbaldehyde 9a–d
in the presence of piperidine. These compounds were obtained as
mixture of E/Z isomers in the ratio of 9:1. Major isomer E was isolated
by conventional column chromatography and confirmed by using NMR
spectroscopy. Moreover, NOE results confirmed E as major product by
assigning the conformation of representative compound 10e. To know
all proton resonances, 1H NMR of compound 10e was carried out using
gDQFCOSY and ROESY experiments in DMSO‑d6 at 30 °C. NOE was
observed between H1-H2, H4-H6, H4-OMe7 and a very weak interac-
tion was observed between H3-OMe7 in ROESY experiment explains
the geometry of double bond and the configuration of 10e was char-
acterized as E-isomer as depicted in Fig. 3.
1.68
1.06 and 1.74
0.69 μM, respectively. Compounds 10b–d
showed effective cytotoxicity against DU145 cell line; however com-
pounds 10a, 10g, 10i, 10k, 10l, 10m, 10o and 10p showed moderate
cytotoxicity without the selectivity on different cancer cell lines.
Compounds 10f, 10j, 10n, 10q and 10r were found to be inactive at the
tested conc. of 50 μM (IC50 > 50 μM, not included in Table 1) against
all the tested cell lines.
Structure activity relationships (SARs) can be exemplified by using
various substitutions on ring A, B and C (Fig. 4). From the preliminary
screening results, it was observed that most of the compounds are
showing potent to moderate cytotoxicity on tested cancer cell lines.
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condensation can be observed [17]. This happened because of effi-
ciency of DAPI stain diffusion into cell membrane based on the in-
tegrity, i.e. stain penetrates with less efficiency into the intact cell
membrane when compared to the disintegrated cell membrane it dif-
ferentiate the normal and apoptotic cells by forming a fluorescent
complex with the chromatin. To evaluate the compound 10e induced
nuclear changes in the DU-145 cells DAPI staining was used. This study
revealed that, the nuclear structure of control cells was intact with
round shape, while compound 10e treated DU-145 cells showed con-
densed and micronuclei formation along with some horse-shoe shaped
nuclei in a dose depended manner (Fig. 6) indicating the extent of
apoptosis.
1.4. Acrdine orange/ethidium bromide staining
Acridine orange (AO)/Ethidium bromide (EB) is also a fluorescent
dye that can differentiate between live, apoptotic and necrotic cells.
The underlying mechanism is AO has an ability to permeate through the
intact cell membrane and stains nuclei green in colour, while EB can
penetrate through cells which have lost membrane integrity, and stain
the nuclei red in colour. After performing this assay, it was notified that
control cells displayed normal homogenous morphology and appeared
cells green in colour. However upon treatment with compound 10e, it
was clearly visualized that compound induced morphological changes
like cell membrane blebbing, and apoptotic body formations (Fig. 7) in
a dose dependent manner.
Fig. 2. Design of biphenyl-oxindole conjugates via molecular hybridization
approach.
1.5. Measurement of reactive oxygen species (ROS)
Based on the results in Table 1, it is noted that compounds 10b–e
showed significant activity compared to other compounds in this series,
it was showed that OCH3 substitution at 3rd position of ring A, and
presence of F at the 4th position of ring B responsible for the activity.
Compound 10e which is having Cl atom on 6th position of ring C is
most potent on all the tested cancer cell lines, other compounds 10b-d
having substitution at 5th position of ring C were more selective to-
wards prostate (DU-145) and cervical cancer (HeLa) cell lines, whereas
compound 10a not having any substitution on ring C is almost less
active. The compounds 10g and 10h having F atom at ring B was
shifted from 4th position to 3rd position, potency of compounds was
lowered and shifted more likely to cervical cancer cell lines. It was
notified that when OMe group of ring A was replaced with H, cytotoxic
property of compounds 10k–m was decreased. When modifications
were carried out on both A and B rings in the case 10o and 10p activity
was observed only on DU-145 cell line and inactive against remaining
tested cell lines. Most of the compounds were more likely potent to-
wards prostate cancer (DU-145) cell lines compared to other tested cell
lines, hence with most potent compound 10e further mechanistic stu-
dies were performed against DU-145 cell line.
Most of the cytotoxic compounds possess a property of ROS gen-
eration [18], where it initiates oxidative damage to the mitochondrial
membrane potential and permeability. This property can aggravate the
process of apoptosis. We evaluated the effect of compound 10e on ROS
generation by DCFDA (2′-7′-dichlorofluorescin diacetate) assay. DCFDA
is a fluorogenic dye measures hydroxyl, peroxyl, and other reactive
oxygen species (ROS) within cell. The activity initiates after diffusion of
dye into the cell where it gets deacetylated by cellular esterases to a
non-fluorescent compound, later oxidized by ROS into highly fluor-
escent 2′, 7′ - dichlorofluorescein (DCF). The corresponding fluores-
cence intensity displays the extent of generation of ROS. In line with
previous experiments, compound 10e has shown dose dependence in
ROS generation also (Fig. 8). Approximately 2 fold increase in fluor-
escence was emitted in the highest dose treated prostate cancer DU-145
cells.
1.6. Determination of mitochondrial membrane potential
One of the distinctive features of the early stages of programmed
cell death is the disruption of active mitochondria. This mitochondrial
disruption includes changes in the membrane potential and alterations
in the oxidation–reduction potential. To evaluate the effect of com-
pound 10e on mitochondrial membrane-permeable, JC-1 dye based
assay was performed. With this study we have observed a dose de-
pendent increase of J-monomers (disrupted mitochondria cells) formed
from normal polarised J aggregate (Fig. 9). This analysis was re-
presented as P1 (normal cells) and P2 populations (polarised cells).
1.2. Determination of cell morphology
To evaluate the effect of compound 10e on morphology and viabi-
lity of cells, microscopic examination was carried out using phase
contrast microscope, by the treatment of compound 10e on DU-145
cells for 48 h, at concentrations of 1, 2 and 4 µM. Phase contrast ima-
ging resulted that as the concentration of the compound increased there
was a clear cut decrease in cell viability with significant morphological
changes such as cell shrinkage (Fig. 5). These results indicated that
compound 10e induced distinctive morphological changes and affected
viability of the DU-145 cells.
1.7. Annexin V-FITC/propidium iodide staining assay
To quantify the apoptotic effect of compound 10e, Annexin V-FITC/
Propidium Iodide staining assay was carried out [19]. This study fa-
cilitates the detection of live cells (Q1-LL; AV−/PI−), early apoptotic
cells (Q2-LR; AV+/PI−), late apoptotic cells (Q3-UR; AV+/PI+) and
necrotic cells (Q4-UL; AV−/PI+). This assay reveals that compound
10e has shown apoptosis in a dose dependent manner with an incre-
ment in late apoptotic cells [control (0%), at 2.5 μM (7.3%), at 5 μM
1.3. DAPI staining study
DAPI is a fluorescent stain binds to A-T rich sequence of nucleic acid
in rigid manner by which nuclear damage through chromatin
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BioorganicChemistry86(2019)210–223
Scheme 1. Synthetic strategy for biphenyl methylene indolinones 10a–r.
(19.6%) and at 10 μM (20.9%)] (Fig. 10).
manner, in comparison with control (13.3%) G2/M population at
0.5 μM (13.4%), 1 μM (15.8%) increased, along with this G0/G1 po-
pulation was also increased control (26%), 0.5 μM (28%), and 1 μM
(37%).
1.8. Clonogenic assay
As cancer cells proliferate from a single cell to solid tumours by
forming colonies, inhibition of colony formation was considered as an
important property of anti-cancer molecule, compound 10e was eval-
uated by clonogenic assay to asses its effect on the colony formation.
This study proved that 10e was effective in inhibiting the colony for-
mation even at a low concentration and maximum colonies disappeared
at high dose i.e 2.5 µM (Fig. 11).
1.10. Tubulin polymerization assay
Cytotoxic assay results proved that biphenyl methylene indolinones
have anti-proliferative activity against majority of the tested cancer cell
lines. It was observed from the literature that most of the alkenyl oxi-
ndoles, as well as biphenyl compounds exhibit their cytotoxic activity
by inhibiting tubulin polymerization [23] as tubulin is the most critical
element for mitosis. To investigate the correlation between cytotoxicity
and antimitotic activity, potent compound 10e was evaluated for tu-
bulin polymerization inhibition in a cell free in vitro assay (Fig. 4) using
podophyllotoxin as standard. The IC50 of compound 10e was found to
1.9. Flow cytometric analysis
In vitro screening results proved that compound 10e has shown
eloquent cytotoxic effect therefore the effect of compound 10e on cell
cycle progression was examined by flow cytometric analysis [20–22].
DU-145 cell were treated with 0.5 and 1 μM concentrations of com-
pound 10e. From Fig. 12, the results clearly explained that 10e was
showing effect on both G0/G1 as well as G2/M in a dose dependent
be 4.96
0.057 μM by comparing with control. These results indicate
that molecular target of these biphenyl methylene indolinones in-
hibiting the tubulin.
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BioorganicChemistry86(2019)210–223
Fig. 4. SAR analysis of biphenyl methylene indolinones.
hydrogen bond acceptor and involved in the hydrogen bond interaction
with the side chain NH2 of Asn101 (d = 1.72 Å). In addition, the oxi-
ndole N-H established a hydrogen bond interaction with the back bone
carbonyl oxygen of Thr353 (d = 2.11 Å). Moreover, various hydro-
phobic interactions were noted between 10e and the active site re-
sidues, Ala180, Val181, Tyr224, Leu248, Ala250, Leu255, Met259,
Ala316, Ala354, and Val355 that stabilizes the binding of 10e in the
active site cavity of α/β-tubulin interface.
To attain further insight of our findings, we manifested the super-
imposition of colchicine (the co-crystallized ligand) and the best docked
pose of 10e in the colchicine binding domain at the α/β-tubulin in-
terface. The superimposition poses from Fig. 14 demonstrate that 10e
accommodates in the active site cavity in a similar mode as that of
colchicine and evidently, the 3,4,5-trimethoxy phenyl moiety of 10e
superimposes with the trimethoxy phenyl motif of colchicine (see
Fig. 15). Altogether, these molecular docking simulation studies furn-
ished a rationalization for the good tubulin polymerization inhibitory
activity of 10e.
Fig. 3. Schematic representation of characteristic NOEs of compound 10e.
1.11. Molecular docking simulation study
Molecular docking simulation studies were carried out with the
promising compound 10e in order to understand the mode of binding
and type of interactions with the active site of the tubulin (PDB ID:
3E22) [24], by using the GLIDE docking module of Schrödinger suite
2014-3 [25]. Docking study revealed that the top ranked conformation
of compound 10e binds effectively in the colchicine binding site of the
tubulin. As can be noticed from the Fig. 13, compound 10e displayed
two hydrogen-bonding interactions with the colchicine-binding domain
at the α/β-tubulin interface that appear to play a critical role in
binding. The oxygen atom of 4-OMe group on the phenyl ring acts as
2. Conclusion
In conclusion, a series of new biphenyl methylene indolinones
10a–r was synthesized and evaluated for their in vitro cytotoxic po-
tential using a panel of cell lines i.e. DU-145 (prostate cancer cell line),
4T1 (mouse breast cancer cell line), MDA-MB-231 (human breast
cancer cell line), BT-549 (human breast cancer cell line), T24 (human
urinary bladder carcinoma cell line), HeLa (cervical cancer cell line).
Compound 10e showed potent cytotoxic activity against prostate
Table 1
In vitro antiproliferative activity (IC50 value in μM)a of compounds 10a–r.
Compounds
DU-145b
4T1c
MDA -MB-231d
BT549e
T24f
HeLag
10a
27.71
2.27
2.16
8.78
1.67
39.95
> 50
27.77
> 50
2.71
1.70
2.16
> 50
> 50
> 50
> 50
10.02
> 50
> 50
31.77
26.91
> 50
> 50
> 50
> 50
127.47
15.88
3.14
4.55
1.98
> 50
> 50
> 50
0.81
10b
0.98
18.40
35.56
> 50
13.75
8.32
3.31
10c
1.29
1.45
1.67
1.06
1.04
3.61
> 50
> 50
2.08
10d
1.05
0.52
0.69
1.08
10e
1.68
1.42
2.07
1.93
1.75
1.74
10g
> 50
> 50
> 50
17.48
> 50
> 50
7.42
0.88
28.9
> 50
> 50
> 50
6.64
3.02
10h
> 50
21.09
> 50
> 50
> 50
> 50
> 50
ND
> 50
37.40
13.60
> 50
> 50
> 50
> 50
107.40
16.40
> 50
8.94
2.53
10i
4.04
1.47
3.77
6.50
1.98
10k
2.04
1.77
4.77
1.96
10l
6.64
> 50
> 50
> 50
> 50
ND
10m
35.56
> 50
> 50
ND
1.56
10o
0.63
5.68
2.48
10p
33.24
120.22
Vincristine sulfate (nM)
12.07
0.1
a
Concentration required for 50% inhibition and mean
Prostate cancer cell line.
SD of three individual experiments performed in triplicate.
b
c
d
e
f
Mouse breast cancer cell line.
Human breast cancer cell line.
Human breast cancer cell line.
Human urinary bladder carcinoma cell line.
Cervical cancer cell line. ND: Not determined.
g
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Fig. 5. Phase contrast images of DU-145 cells treated with different concentration of (1, 2, 4 µM) compound 10e and observed for the compound induced mor-
phological changes at 200× magnification.
Fig. 6. DAPI stain for nuclear morphology in DU-145 prostate cancer cell lines after 48 h post treatment. The images were taken with fluorescence microscope with a
DAPI filter at 200× magnification.
cancer cell line with IC50 1.68
1.06 µM, to evaluate the underlying
that compound 10e was able to arrest G2/M phase of the cell cycle.
Furthermore, compound 10e induced morphological changes and
apoptosis in DU-145 cell lines displayed by acridine orange/ethidium
bromide, DAPI staining studies, annexin V-FITC/propidium iodide
staining assay, analysis of mitochondrial membrane potential and ROS
assays. Moreover clonogenic assay proved that compound 10e interrupt
mechanism with respect to cytotoxicity, various experiments were
performed. Tubulin polymerization assay resulted that compound 10e
was able to interfere in mitosis by inhibiting tubulin polymerization;
molecular docking also suggested that compound 10e bind to the col-
chicine binding site of the tubulin. Flow cytometric analysis revealed
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BioorganicChemistry86(2019)210–223
Fig. 7. Prostate cancer DU-145 cells treated with increased concentrations of compound 10e and stained with AO/EB. Decreased number of viable cells and apoptotic
characteristics (indicated by blue arrows) were observed at 200× magnification.
colony formation of cancerous cells. We believe that 10e could be po-
tential lead for the development of chemical library which would serves
as a potential drug to treat prostate cancer.
formation was detected by visualizing spots in UV light. All melting
points were taken and are uncorrected. 1H spectra were recorded on
300 and 500 MHz, 13C spectra were recorded on 75, 100 and 125 MHz
spectrometers using tetramethylsilane as internal standard. Chemical
shifts are reported in parts per million (ppm) downfield from tetra-
3. Experimental section
methylsilane. Spin multiplicities are described as
s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet).
Coupling constants are reported in hertz. HRMS analyses were acquired
on single quadruple and carried out using ESI technique 70 eV. Column
All the starting materials, reagents and solvents used were com-
mercially available and used directly without any further purification.
TLC was performed on 0.25 mm silica gel 60-F254 plates. Product
Fig. 8. DCFDA imaging depicting effect of compound 10e on generation of reactive Oxygen Species (ROS) in a.u at concentration 1, 2 and 4 µM in DU-145 prostate
cancer cell lines. Graph represents the intensity of fluorescence emitted at excitation and emission wavelengths of 480 and 535 nm at various doses in comparison to
control. *** < 0.001, **0.05 vs control. Data was analysed by one way ANOVA followed by Tukey’s multiple comparision.
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Fig. 9. Effect of compound 10e on mitochondrial membrane potential (ΔΨm) in DU-145 prostate cancer cells upon treatment at 1, 2 and 4 µM. Cells were incubated
with JC-1 dye and analysed by flow cytometer (BD FACSVerse™, USA). Values in the figures represents the J aggregates and J monomers as P1 and P2 monomers
populations respectively.
chromatography was performed by using silicagel of 60–120 mm with
hexane and ethyl acetate as eluents. Melting points were determined
with an Electro thermal melting point apparatus and were uncorrected.
3.3. General procedure for the synthesis of biphenyl methylene indolinones
(10a-r)
Oxindole (9 mmol) was added to a solution of biphenyl 2-carbal-
dehyde (10 mmol) in ethanol (5 mL). To that mixture catalytic amount
of piperdine (1 mL) was added and the reaction mixture was allowed to
stir at 80 °C for 4 h. Completion of reaction was confirmed by TLC. The
solvent was removed by rotary evaporation and the product obtained
was washed with methanol for 4–5 times. After complete air drying, the
final compounds biphenyl methylene indolinones 10a–r were obtained
as pure solids with yield of 80%.
3.1. General procedure for the preparation of substituted 2-bromo
benzaldehydes (8a-b) [26]
500 mL round bottom flask was charged with corresponding ben-
zaldehydes 7a,b (25.5 mmol), methylene chloride (100 mL) and acetic
acid (200 mL). The RBF was cooled to 0 °C in an ice-bath and bromine
(25.5 mmol) in methylene chloride (10 mL) was added drop-wise via
addition funnel over 15 min. After stirring at 0 °C for half an hour,
aqueous sodium thiosulfate was added and the mixture was extracted
three times with CH2Cl2. The combined organic layers were washed
with saturated aqueous sodium bicarbonate, brine and dried over so-
dium sulfate. Removal of the solvent under reduced pressure gave a
solid, which was recrystallized (EtOAc/hexane) to give 8a,b (91%) as
colorless needles.
3.3.1. (E)-3-((4′-Fluoro-4,5,6-trimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10a)
Yellow solid; M.p: 199–200 °C; Rf = 0.4 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.52 (s, 1H), 7.60 (d,
J = 7.6 Hz, 1H), 7.26–7.21 (m, 2H), 7.16 (t, J = 8.9 Hz, 2H), 7.05 (s,
1H), 7.04–6.99 (m, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.86 (d, J = 7.7 Hz,
1H), 3.88 (s, 3H), 3.82 (s, 3H), 3.59 (s, 3H), 2.26 (s, 3H). 13C NMR
(125 MHz, DMSO‑d6): δ 168.4, 160.06 (d, J = 243.5 Hz, 1C), 152.1,
151.1, 143.0, 142.8, 134.8, 133.3 (d, J = 5.2 Hz, 1C), 131.4 (d,
J = 3.6 Hz, 1C), 129.9, 129.7 (d, J = 8.1 Hz, 1C), 128.6, 127.3, 123.6
(d, J = 17.4 Hz, 1C), 122.8, 121.1, 121.0, 114.5, 114.3, 110.1, 108.3,
60.7, 60.6, 56.2, 14.1 ppm; HRMS calculated for [M+H]+
C25H23FNO4: 420.16056 found: 420.16036.
3.2. General procedure for the preparation of substituted biphenyl 2-
carbaldehydes 9a–d [27]
To a solution of 2-bromo-substituted benzaldehyde 8a,b (1 mmol) in
3 mL ethanol, appropriate aryl boronic acid (1.1 mmol), Pd(PPh3)4
(1 mol%) sodium carbonate (1.2 mmol, dissolve in a minimum amount
of water) was added. The reaction mixture was heated at 80 °C for 6 h.
The solvent was removed by rotary evaporation and the product was
extracted with ethyl acetate (3 × 10 mL), the organics were washed
with water (2 × 10 mL), and dried over anhydrous sodium sulfate. The
solvent was evaporated, and the residue was purified by column
chromatography on silica gel, eluting with hexane/EtOAc mixture.
3.3.2. (E)-5-Chloro-3-((4′-fluoro-4,5,6-trimethoxy-3′-methylbiphenyl-2-
yl)methylene)indolin-2-one (10b)
Yellow solid; M.p: 234–235 °C; Rf = 0.5 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.67 (s, 1H), 7.59 (s, 1H),
7.23–7.23 (m, 1H), 7.20–7.13 (m, 1H), 7.12 (s, 1H), 7.05 (s, 1H), 6.88
(d, J = 7.8 Hz, 1H) 3.89 (s, 3H), 3.84 (s, 3H), 3.59 (s, 3H), 2.25 (s, 3H)
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Fig. 10. Annexin V-FITC/propidium iodide dual staining assay. DU-145 cells were treated with compound 10e and labelled with Annexin V-FITC/PI and examined
for apoptosis using flow-cytometer. Cells in the lower left quadrant (Q1-LL: AV-/PI-): live cells; lower right quadrant (Q2-LR: AV-/PI-): early apoptotic cells; upper
right quadrant (Q3-UR: AV+/PI+): late apoptotic cells and upper left quadrant (Q4-UL: AV−/PI+): necrotic cells.
Fig. 11. Effect of compound 10e on clonogenic assay. Control cells are having multiple colonies, while compound 10e at concentrations of 0.25, 0.5, 1 and 2.5 µM
showing inhibition of number of colonies respectively when compared to control. Images were taken at 40× magnification. Graph represents the number of average
colonies represented as mean
Tukey’s multiple comparision.
s.e.m of three independent experiments. *** < 0.001, ** < 0.05 vs control. Data was analysed by one way ANOVA followed by
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Fig. 12. Cell cycle analysis of DU-145 cells upon treatment with compound 10e by flow cytometry. DNA histogram represents different stages of cell cycle i.e.,
SubG1, G0/G1, S, G2/M. Each phase of treated cells cell cycle results were compared with DMSO control cells, cell cycle analysis. 10,000 events were analysed for the
results.
ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.6, 160.7 (d, J = 243.7 Hz,
1C), 152.7, 151.7, 143.9, 142.1, 137.0, 133.9 (d, J = 5.1 Hz, 1C), 131.7
(d, J = 3.5 Hz, 1C), 130.3 (d, J = 8.0 Hz, 1C), 129.9, 129.6, 128.6,
126.8, 125.3, 124.2 (d, J = 17.4 Hz, 1C), 123.1, 122.9, 114.9 (d,
J = 22.2 Hz, 1C), 111.9, 108.9, 61.3, 61.2, 56.1, 14.5 ppm; HRMS
calculated for [M+H]+C25H21ClFNO4: 454.1215 found: 454.1223.
157.67 (d, J = 235.2 Hz, 1C), 152.8, 151.7, 143.7, 139.7, 137.0, 133.9,
131.7, 130.2, 129.3, 128.7, 127.4, 124.2 (d, J = 17.4, Hz, 1C), 122.57
(d, J = 8.9 Hz, 1C), 116.7 (d, J = 23.6 Hz, 1C), 115.0 (d, J = 22.2 Hz,
1C), 113.0 (d, J = 8.3 Hz, 1C), 111.3 (d, J = 8.3 Hz, 1C), 110.2 (d,
J = 25.9 Hz, 1C), 108.7, 61.3, 61.1, 56.6, 14.6 ppm; HRMS calculated
for [M+H]+C25H21F2NO4: 438.1517 found: 438.1513.
3.3.3. (E)-5-Bromo-3-((4′-fluoro-4,5,6-trimethoxy-3′-methylbiphenyl-2-
yl)methylene)indolin-2-one (10c)
3.3.5. (E)-6-chloro-3-((4′-fluoro-4,5,6-trimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10e)
Yellow solid; M.p: 239–240 °C; Rf = 0.4 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.66 (s, 1H), 7.7 (s, 1H),
4.42 (d, J = 7.8 Hz 1H), 7.28 (s, 1H), 7.21–7.12 (m, 2H), 7.11 (s, 1H),
7.05 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.59 (s,
3H), 2.2 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.4, 161.5 (d,
J = 243.7 Hz, 1C), 152.7, 151.7, 143.9, 142.4, 137.0, 133.9, 132.6,
131.7, 130.4, 130.3, 128.5, 126.6, 125.7, 124.3 (d, J = 17.5 Hz, 1C),
123.7, 114.9 (d, J = 22.1 Hz, 1C), 112.9, 112.5, 108.8, 61.3, 61.2, 56.5,
14.2 ppm; HRMS calculated for [M]+C25H21BrFNO4: 497.0638 found:
497.0635.
Yellow solid; M.p: 240–241 °C; Rf = 0.4 (30% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.69 (s, 1H), 7.60 (d,
J = 8.3 Hz, 1H), 7.21 (s, 1H), 7.19–7.11 (m, 2H), 7.07 (s, 1H), 7.05 –
6.97 (m, 2H), 6.88 (d, J = 1.7 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H), 3.58
(s, 3H), 2.25 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.3,
161.0 (d, J = 243.6 Hz, 1C), 152.3, 151.1, 144.1, 143.3, 135.6, 133.9,
133.4, 131.3, 129.7 (d, J = 8.2 Hz 1C), 128.7, 128.3, 126.1, 124.1,
123.7, 123.6, 120.9, 119.9, 114.4 (d, J = 22.2 Hz), 110.0, 108.2, 60.7,
60.6, 56.0, 14.1 ppm; HRMS calculated for [M+H]+ C25H21ClFNO4:
454.1215 found: 454.1223.
3.3.4. (E)-5-Fluoro-3-((4′-fluoro-4,5,6-trimethoxy-3′-methylbiphenyl-2-
yl)methylene)indolin-2-one (10d)
3.3.6. (E)-3-((3′-Fluoro-4,5,6-trimethoxybiphenyl-2-yl)methylene)indolin-
2-one (10f)
Yellow solid; M.p: 233–234 °C; Rf = 0.4 (50% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.55 (s, 1H), 7.30 (dd,
J = 9.2, 2.2 Hz, 1H), 7.23 (s, 1H), 7.16 (t, J = 9.0 Hz, 2H), 7.12 (s, 1H),
7.09 (dd, J = 9.0, 2.4 Hz, 1H), 7.05–7.00 (m, 1H), 6.86 (dd, J = 8.5,
4.6 Hz, 1H), 3.89 (m, 3H), 3.83 (s, 3H), 3.60 (s, 3H), 2.25 (s, 3H) ppm;
13C NMR (125 MHz, DMSO‑d6): δ 168.8, 160.6 (d, J = 243.6 Hz, 1C),
Yellow solid; M.p: 193–194 °C; Rf = 0.4 (40% ethyl acetate/n-
hexane); 1H NMR (300 MHz, CDCl3): δ 8.62 (s, 1H), 7.67 (d, J = 7.5 Hz,
1H), 7.36–7.20 (m, 4H), 7.08–6.99 (m, 3H), 6.94–6.88 (m, 2H), 4.01 (s,
3H), 3.86 (s, 3H), 3.68 (s, 3H) ppm; 13C NMR (125 MHz, CDCl3): δ
169.9, 163.2 (d, J = 246.1 Hz, 1C), 152.6, 151.5, 143.6, 141.8, 137.4,
137.0, 129.8, 129.4, 129.1, 128.9, 127.4, 126.6, 123.0, 121.7, 121.5,
Fig. 13. Effect of compound 10e on tubulin polymerization inhibition.
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Fig. 14. Docking pose of the most potent compound 10e (green colour ball and stick) in the colchicine binding site of α/β-tubulin interface and the interacting amino
acids (PDB ID: 3E22). The red dashed lines represent hydrogen bonds.
117.6 (d, J = 21.8 Hz, 1C), 114.7 (d, J = 20.9 Hz, 1C), 110.4, 108.3,
61.1, 61.0, 56.1 ppm; HRMS calculated for [M+H]+C24H20FNO4:
406.1455 found: 406.1449.
3.3.8. (E)-6-Chloro-3-((3′-fluoro-4,5,6-trimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10h)
Yellow solid; M.p: 219–220 °C; Rf = 0.5 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): δ 8.39 (s, 1H), 7.58 (d, J = 8.2 Hz,
1H), 7.35 (s, 1H), 7.34–7.30 (m, 1H), 7.10 (s, 1H), 7.07–7.02 (m, 1H),
7.01–6.97 (m, 2H), 6.91–6.87 (m, 2H), 4.01 (s, 3H), 3.85 (s, 3H), 3.66
(s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO‑d6): δ 168.5, 163.0 (d,
J = 245.9 Hz, 1C), 152.0, 150.7, 143.4, 142.7, 137.0, 135.4, 134.2,
128.7, 128.2, 128.0, 126.2, 125.9, 122.9, 120.1, 119.4, 116.8 (d,
J = 21.4 Hz, 1C), 113.8 (d, J = 20.9 Hz, 1C), 110.0, 107.4, 60.3, 60.2,
55.4 ppm; HRMS calculated for [M+H]+C24H19ClFNO4: 440.0987
found: 440.1068.
3.3.7. (E)-5-Chloro-3-((3′-fluoro-4,5,6-trimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10g)
Yellow solid; M.p: 231–232 °C; Rf = 0.5 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): δ 8.49 (s, 1H), 7.75 (s, 1H), 7.38
(s, 1H), 7.36–7.32 (m, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H),
7.08–6.99 (m, 3H), 6.82 (d, J = 8.2 Hz, 1H), 4.02 (s, 3H), 3.90 (s, 3H),
3.67 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.5, 161.9 (d,
J = 243.7 Hz, 1C), 153.1, 151.7, 143.9, 142.1, 138.2, 136.8, 130.3,
129.9, 128.9, 128.6, 127.2, 127.1, 125.3, 123.1, 123.0, 117.6 (d,
J = 21.2 Hz, 1C), 114.9 (d, J = 20.7 Hz, 1C), 111.9, 108.9, 61.3, 61.2,
56.5 ppm; HRMS calculated for [M+H]+C24H19ClFNO4: 440.0987
found: 440.1068.
3.3.9. (E)-5-Fluoro-3-((3′-fluoro-4,5,6-trimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10i)
Yellow solid; M.p: 225–226 °C; Rf = 0.4 (50% ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): δ 8.48 (s, 1H), 7.75 (s, 1H),
7.39–7.30 (m, 2H), 7.22–7.15 (m, 2H), 7.08–6.99 (m, 3H), 6.82 (d,
Fig. 15. Superimposition of co-crystallized ligand (magenta) and the best docked pose of compound 10e (green) in the colchicine binding site of α/β-tubulin
interface (PDB ID: 3E22).
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J = 8.2 Hz, 1H), 4.03 (s, 3H), 3.89 (s, 3H), 3.68 (s, 3H) ppm; 13C NMR
(125 MHz, DMSO‑d6): δ 168.8, 161.6 (d, J = 245.9 Hz, 1C), 158.6 (d,
J = 235.9 Hz, 1C), 152.8, 151.7, 143.8, 139.6, 137.0, 133.9, 131.8,
130.2, 129.3, 128.7, 127.5, 124.3, 122.6, 116.8, 111.3, 110.1, 108.7,
61.3, 61.1, 56.6 ppm; HRMS calculated for [M+H]+C24H19F2NO4:
424.1360 found: 424.1364.
468.0605 found: 468.0616.
3.3.15. (E)-3-((3′-Fluoro-4,5-dimethoxybiphenyl-2-yl)methylene)indolin-
2-one (10o)
Yellow solid; M.p: 233–234 °C; Rf = 0.6 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): δ 8.29 (s, 1H), 7.71 (d, J = 7.6 Hz,
1H), 7.56 (s, 1H), 7.38 (s, 1H), 7.35 (q, J = 7.9, 14.0 Hz, 1H), 7.23 (t,
J = 7.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.09–7.03 (m, 2H), 6.98 (s,
1H), 6.93–6.89 (m, 2H), 4.00 (s, 3H), 3.88 (s, 3H) ppm; 13C NMR
(125 MHz, CDCl3): δ 170.1, 162.3 (d, J = 247.0 Hz, 1C), 150.3, 148.1,
141.9, 141.6, 137.5, 135.2, 129.8 (d, J = 8.2 Hz, 1C), 129.6, 126.7,
125.7, 125.0, 122.9, 121.9, 121.5, 116.5 (d, J = 21.8 Hz, 1C), 114.5 (d,
J = 20.8 Hz, 1C), 112.7, 112.2, 110.4, 56.2, 56.0 ppm; HRMS calcu-
lated for [M+H]+C23H19FNO3: 376.1343 found: 376.1351.
3.3.10. (E)-5-Bromo-3-((3′-fluoro-4,5,6-trimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10j)
Yellow solid; M.p: 237–238 °C; Rf = 0.4 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, CDCl3): δ 8.49 (s, 1H), 7.38–7.32 (m, 3H),
7.17 (s, 1H), 7.08–6.99 (m, 3H), 6.78 (d, J = 7.6 Hz, 1H), 4.02 (s, 3H),
3.91 (s, 3H), 3.67 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ 169.6,
162.4 (d, J = 245.7 Hz, 1C), 152.7, 151.7, 1441.2, 140.6, 138.7, 137.3,
132.1, 129.6, 129.4, 129.3, 128.2, 126.7, 125.9, 123.5, 117.6 (d,
J = 22.0, 1C), 114.8 (d, J = 21.3, 1C), 114.1, 111.8, 108.1, 61.8, 61.0,
56.2 ppm; HRMS calculated for [M+H]+C24H19BrFNO4: 484.0560
found: 480.0561.
3.3.16. (E)-5-Chloro-3-((3′-fluoro-4,5-dimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10p)
Yellow solid; M.p: 275–276 °C; Rf = 0.5 (30% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.69 (s, 1H), 7.59 (s, 1H),
7.53–7.44 (m, 1H), 7.42 (s, 1H), 7.34 (s, 1H), 7.29–7.20 (m, 3H),
7.19–7.13 (m, 2H), 6.90 (d, J = 7.1 Hz, 1H), 3.93 (s, 2H), 3.82 (s, 1H)
ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.8, 162.2 (d, J = 242.3 Hz,
1C), 151.0, 148.6, 142.2, 142.0, 137.3, 135.2, 130.7 (d, J = 8.4 Hz,
1C), 129.6, 126.5, 126.2, 125.3, 124.2, 123.4, 122.7, 116.8 (d,
J = 21.8 Hz, 1C), 114.8 (d, J = 20.9 Hz, 1C), 113.9, 112.7, 111.8, 56.3,
56.3 ppm; HRMS calculated for [M+H]+C23H18 ClFNO3: 410.0959
found: 410.1121.
3.3.11. (E)-3-((4′-Fluoro-4,5-dimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10k)
Yellow solid; M.p: 228–229 °C; Rf = 0.5 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.51 (s, 1H), 7.64 (d,
J = 7.6 Hz, 1H), 7.38 (s, 1H), 7.32 (d, J = 6.1 Hz, 1H), 7.26 (s, 1H),
7.24–7.20 (m, 1H), 7.18 (d, J = 9.3 Hz, 1H), 7.17–7.11 (m, 1H), 7.08
(s, 1H), 6.95–6.86 (m, 2H), 3.91 (s, 3H), 3.79 (s, 3H), 2.27 (s, 3H) ppm;
13C NMR (75 MHz, CDCl3): δ 168.1, 159.2 (d, J = 244.8 Hz, 1C), 148.8,
146.4, 141.4, 134.9, 134.2, 131.3, 128.2, 127.7, 127.6, 125.3, 123.4,
123.2 (d, J = 18.1 Hz, 1C), 121.3, 120.3, 119.6 (d, J = 11.5 Hz, 1C),
113.5 (d, J = 22.5 Hz, 1C), 111.6, 110.9, 109.1, 54.7, 54.6, 13.1 ppm;
HRMS calculated for [M+H]+C24H20FNO3: 390.1500 found: 390.1509.
3.3.17. (E)-5-Fluoro-3-((3′-fluoro-4,5-dimethoxybiphenyl-2-yl)methylene)
indolin-2-one (10q)
Yellow solid; M.p: 218–219 °C; Rf = 0.4 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.57 (s, 1H), 8.44 (s, 1H),
7.50–7.43 (m, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.27–7.21 (m, 2H),
7.20–7.14 (m, 2H), 7.12–7.06 (m, 1H), 6.89–6.83 (m, 1H), 3.93 (s, 1H),
3.81 (s, 2H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 169.0, 149.9 (d,
J = 227.4 Hz, 1C), 149.7 (d, J = 245.3 Hz, 1C), 142.3, 139.5, 139.90,
137.3, 134.9, 130.7, 127.0, 126.5, 124.3, 123.5, 116.8, 116.6, 114.7,
113.9, 112.6, 111.2, 110.2, 109.9, 56.1 ppm; HRMS calculated for [M
+H]+C23H18 F2NO3: 394.1255 found: 394.1250.
3.3.12. (E)-5-Chloro-3-((4′-fluoro-4,5-dimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10l)
Yellow solid; M.p: 290–291 °C; Rf = 0.6 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.66 (s, 1H), 7.62 (s, 2H),
7.44–7.22 (m, 4H), 7.23–7.06 (m, 3H), 6.90 (s, 1H), 3.92 (s, 3H), 3.81
(s, 3H), 2.28 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.9,
159.8 (d, J = 234.5 Hz, 1C), 151.1, 148.3, 141.9, 137.6, 135.8, 133.1,
129.7, 129.6, 129.5, 125.80, 125.2, 124.7 (d, J = 17.3 Hz, 1C), 124.1,
123.5, 122.7, 115.2 (d, J = 22.6 Hz, 1C), 113.9, 112.7, 111.8, 56.3,
56.2, 14.5 ppm; HRMS calculated for [M+H]+C24H19ClFNO3:
424.1110 found: 424.1118.
3.3.18. (E)-6-Chloro-3-((3′-fluoro-4,5-dimethoxybiphenyl-2-yl)
methylene)indolin-2-one (10r)
Yellow solid; M.p: 278–279 °C; Rf = 0.5 (30% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.70 (s, 1H), 7.60 (d,
J = 8.2 Hz, 1H), 7.47 (dd, J = 14.1, 7.2 Hz, 1H), 7.36 (s, 1H), 7.32 (s,
1H), 7.24 (t, J = 8.7 Hz, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.14 (s, 1H),
6.98 (d, J = 7.4 Hz, 1H), 6.90 (s, 1H), 3.92 (s, 3H), 3.80 (s, 3H) ppm;
13C NMR (125 MHz, DMSO‑d6): δ 169.0, 162.4 (d, J = 244.2 Hz, 1C),
150.8, 148.7, 144.5, 142.3 (d, J = 8.0 Hz, 1C), 136.4, 134.9, 134.2,
130.7, 126.5, 126.1, 124.5 (d, J = 7.3 Hz, 1C), 121.4, 120.7, 116.8,
114.8 (d, J = 20.8 Hz, 1C), 113.8, 112.6, 110.4, 56.3, 56.2 ppm; HRMS
calculated for [M+H]+C23H18 ClFNO3: 410.0959 found: 410.1121.
3.3.13. (E)-6-Chloro-3-((4′-fluoro-4,5-dimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10m)
Yellow solid; M.p: 295–296 °C; Rf = 0.6 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.67 (s, 1H), 7.64 (s, 1H),
7.42 – 7.25 (m, 3H), 7.25–7.11 (m, 2H), 7.09 (s, 1H), 7.02–6.99 (m,
1H), 6.90 (s, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 2.27 (s, 3H) ppm; 13C NMR
(125 MHz, DMSO‑d6): δ 169.1, 160.8 (d, J = 237.5 Hz, 1C), 150.8,
148.3, 144.5, 136.7, 136.0, 135.6, 134.1, 133.2, 129.6, 125.7, 124.8,
124.6, 124.3, 121.3, 120.7, 115.1, 113.7, 112.5, 110.4, 56.3, 56.2,
14.6 ppm; HRMS calculated for [M+H]+C24H19ClFNO3: 424.1110
found: 424.1118.
4. Biology
4.1. Material and methods
3.3.14. (E)-5-Bromo-3-((4′-fluoro-4,5-dimethoxy-3′-methylbiphenyl-2-yl)
methylene)indolin-2-one (10n)
Prostate cancer (DU-145) mouse breast cancer cell line (4T1),
human breast cancer cell line (MDA-MB-231), human urinary bladder
carcinoma cell line (T24), cervical cancer cell lines (HeLa) were ob-
tained from National center for cell Science (NCCS, Pune). Cells were
cultured in respective RPMI (Roswell Park Memorial Institute medium,
Sigma Aldrich, St. Louis, USA) and Dulbecco's Modified Eagle's medium
(DMEM), supplemented with 10% fetal bovine serum (FBS) (Gibco,
USA) and 1% stabilized antibiotic, penicillin-streptomycin mixture
(Gibco, USA). Cells were maintained in a CO2 incubator at 37 °C with
Yellow solid; M.p: 285–286 °C; Rf = 0.5 (40% ethyl acetate/n-
hexane); 1H NMR (500 MHz, DMSO‑d6): δ 10.67 (s, 1H), 7.78 (s, 1H),
7.47–7.38 (m, 2H), 7.34 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 7.23–7.14
(m, 2H), 7.11 (s, 1H), 6.86 (d, J = 8.2 Hz, 1H), 3.92 (s, 3H), 3.82 (s,
3H), 2.28 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6): δ 168.7, 160.40,
150.9, 148.1, 142.23, 137.6, 135.9, 135.8, 133.2, 132.2, 129.7, 125.60,
125.4, 124.8, 124.6, 124.1, 115.2, 113.9, 112.9, 112.6, 112.3, 56.3,
56.2, 14.6 ppm; HRMS calculated for [M+H]+C24H19 BrFNO3:
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5% CO2 and 90% relative humidity. Trypsin-EDTA required for cell
culture was obtained from (Gibco, USA). Fluorescent dyes acridine or-
ange (AO), 4′, 6-diamidino-2-phenylindole (DAPI) were from Sigma
Aldrich, St. Louis, USA, and Ethidium Bromide (EB) from Himedia
limited India, while Dichlorofluorescin diacetate (DCFDA) and JC-1
dyes were procured from Invitrogen, USA.
4.1.6. Measurement of mitochondrial membrane potential by JC-1 staining
DU-145 cells were cultured in 6 well plates at a density of 5 × 105
cells/mL and allowed to grow over night. After adhering, cells were
treated with 1, 2, and 4 µM concentrations of the compound 10e. After
48 h of incubation, cells were incubated with JC-1 stain [29] (2.5 mg/
mL) later adhered cells were collected by trypsinsation, washed with
PBS and resuspended in a solution of PBS. The samples were then
analysed for JC-1 fluorescence using flow-cytometer (BD FACS-
VerseTM, USA).
4.1.1. Cytotoxic activity
Cytotoxic activity of compounds of the series 10a-r was carried out
by MTT assay. Cells were plated at a density of 5 × 103 cells per well in
a 96 well plate supplemented with 10% FBS and after 24 h of incuba-
tion at 37 °C in 5% CO2 they were treated with respective concentration
of the compounds dissolved in the culture media with vehicle controls
and known standards. The test compounds were initially dissolved in
DMSO and made up the required concentration with media. Then after
48 h of treatment, 100 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide) reagent (0.5 mg/mL) dissolved in serum
free media was added to each well and incubated for 4 h. Then the
media was aspirated and the formazan crystals were dissolved in 200 µL
of DMSO and absorbance was taken at 570 nm in multimode plate
reader (Spectra Max M4, Molecular devices, US).
4.1.7. Clonogenic assay
Prostate cancer DU-145 cells were plated at a density of 250 cells/
well and left to adhere for overnight. After adhering, cells were exposed
to compound 10e at doses of 0.25, 0.5, 1 and 2.5 µM for 24 hr. Post to
incubation, treatment was washed with media, cells were maintained in
complete media and allowed to grow them as colonies for 10 days. Cells
were observed for colonies during this period. After 10 days colonies
which were formed were stained with 1% crystal violet stain for 4 h.
The colonies with cells greater than 25 were considered and counted
under phase contrast microscope and representative images were cap-
tured. (Colony formations were calculated as percentage to control
cells).
4.1.2. Cell morphology by phase contrast imaging
4.1.8. Annexin V binding assay
DU-145 cells were plated with density of 1 × 105 cells/mL in 6 well
culture plates and allowed to adhere for overnight by incubating at
37 °C in an atmosphere of 5% CO2 for 48 h. Cells were incubated with
In order to determine the apoptosis induced cell death annexin V
FITC and propidium iodide staining was performed by using the
Annexin V FITC apoptosis detection kit according to manufacturer’s
protocol (Sigma Aldrich). In brief, cells were seeded at a density of
1 × 106 cells per well in a 6 well plate and allowed to adhere for
overnight. The cells were treated with different concentrations of
2.5 µM, 5 µM and 10 µM compounds along with the control. After 24 h
of incubation, the cells were harvested, washed with PBS and stained
with Annexin V FITC and propidium iodide. Early and late apoptotic
cells were quantified using Accuri C6 flow cytometer based software
(BD Biosciences).
various concentrations of compound 10e (1,
2
and 4 µM).
Morphological changes and viability were studied after 48 h of in-
cubation with compound 10e, and respective images were captured by
using phase contrast microscope (Nikon, Inc. Japan).
4.1.3. DAPI staining
Compound 10e induced morphological changes in the nucleus were
studied using DAPI staining [28]. Here cells were seeded at density of
2 × 105 cells/well and allowed to adhere for overnight. Next day cells
were incubated with compound 10e for 48 h. After incubation cells
were washed with PBS and solubilized with 0.1% Tween 20 for 5 min
after fixing with 4% para formaldehyde followed by staining with 1 mM
DAPI. Control and treated cells were observed with fluorescence mi-
croscope with wavelengths of excitation at 359 nm and emission at
461 nm using DAPI filter in fluorescent microscope at 200 magnifica-
tions and representative images were captured.
4.1.9. Flow cytometry based for cell cycle analysis
DU-145 cells were seeded with the density of 1 × 106/well in six
well plate for 24 h. After 24 h, cells were subjected to starvation (serum
free) for 6 h followed by treatment with 0.5 and 1 µM concentrations of
compound 10e in 5% complete medium for 24 h, later adhered cells
were collected by trypsinisation, washed twice with PBS (pH 7.4) and
fixed with 70% ice cold ethanol for overnight at −20 °C. Following
fixation cells were rewashed twice with PBS and stained with (450 µL)
propidium Iodide (PI) staining buffer for 15 mins in dark at room
temperature (5). The stained samples were then analysed for PI fluor-
escence by flow cytometry (BD FACSVerseTM, USA). 10,000 events
were considered for analysing results.
4.1.4. Acridine orange (AO) and ethyl bromide (EB) staining
DU-145 cells were plated with density of 2 × 105 cells/mL. Upon
adhering for overnight, cells were incubated after treatment with dif-
ferent concentrations of compound 10e (1, 2 and 4 µM) and maintained
at 37 °C in an atmosphere of 5% CO2 for 48 h. For AO/EB staining, both
the dyes of 1 mg/mL concentration were taken and added into wells
reach concentration of 10 µg/mL. After 10 min of incubation, the cells
were visualized under fluorescence microscope (Nikon, Inc. Japan)
with wavelengths of excitation 488 nm and emission 550 nm at 200
magnifications and representative images were captured.
4.1.10. Tubulin polymerization assay
To evaluate the effect of 10e on tubulin polymerization, fluores-
cence based in vitro tubulin polymerization assay was performed ac-
cording to the manufacturer’s protocol (BK011, Cytoskeleton, Inc.). The
reaction mixture for the experiment is having Porcine brain tissue
(2 mg/mL) in 80 mM PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA,
1.0 mM GTP and glycerol in the presence or absence of test compound
10e (final concentration of 10, 5, 2.5, 1.25 µM). Podophyllotoxin was
used as standard in this assay at 5 µM final concentration.
Polymerization was monitored by increase in the fluorescence at 37 °C.
Tubulin polymerization was followed by a time dependent increase in
fluorescence due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission at
440 nm (excitation wavelength is 360 nm) was measured by using a
spectramax M4 multi mode micro plate detection System. The IC50
value was calculated from the compound concentration required to
inhibit 50% of tubulin assembly in comparison to control.
4.1.5. Measurement of reactive oxygen species (ROS) generation by DCFDA
staining
DU-145 cells with a density of 2 × 105 cells/mL were plated in 6
well plates and allowed to adhere for overnight. Later the cells were
treated with concentration 1, 2 and 4 µM of compound 10e for 48 h.
The media was replaced with culture medium containing DCFDA dye
(10 mM) and incubated in dark for 30 min. The intensity of this fluor-
escence from cells was determined by florescence spectroscopy
(SpectraMax, Molecular devices) at an excitation and emission wave-
length of 480 and 535 nm respectively and the corresponding images
were captured by fluorescent microscope.
222

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Acknowledgements
The authors are thankful to DoP, Ministry of Chemicals & Fertilizers,
Govt. of India, for the award of NIPER fellowship. Dr. N. Shankaraiah
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