Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3′-methoxy-4′-(2-amino-2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumour agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.025

A series of new (Z)-3-(3′-methoxy-4′-(2-amino-2-oxoethoxy)benzylidene)indolin-2-one derivatives has been synthesized and evaluated for their cytotoxic activity against selected human cancer cell lines of prostate (PC-3 and DU-145), breast (BT-549 and MDA-MB-231) and non-tumorigenic prostate epithelial cells (RWPE-1). Among the tested, one of the compounds 4p exhibited potent cytotoxicity selectively on prostate cancer cell lines (PC-3 and DU-145; IC₅₀: 1.89 ± 0.6 and 1.94 ± 0.2 μM, respectively). Further experiments were conducted with 4p on PC-3 cancer cells to study the mechanisms of growth inhibition and apoptosis inducing effect. Treatment of PC-3 cells with test compound 4p resulted in inhibition of cell migration through disorganization of F-actin protein. The flow-cytometry analysis results showed that the compound arrested PC-3 cancer cells in the G2/M phase of cell cycle in a dose dependent manner. Hoechst staining and annexin-V binding assay revealed that the compound 4p inhibited tumor cell proliferation through induction of apoptosis. Western blot studies demonstrated that the compound 4p treatment led to activation of caspase-3, increased expression of pro-apoptotic Bax and significantly decreased expression of anti-apoptotic Bcl-2 in human prostate cancer PC-3 cells. In addition, the mitochondrial membrane potential (ΔΦm) was also affected and the levels of intracellular Ca²⁺ were raised.

Full text of DOI:10.1016/j.ejmech.2016.04.025

Accepted Manuscript
Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3'-methoxy-4'-(2-
amino-2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumor agents
Kishna Ram Senwar, T. Srinivasa Reddy, Dinesh Thummuri, Pankaj Sharma, V.G.M.
Naidu, Gannoju Srinivasulu, Nagula Shankaraiah
PII:
S0223-5234(16)30310-5
10.1016/j.ejmech.2016.04.025
EJMECH 8540
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2015
Revised Date: 3 March 2016
Accepted Date: 8 April 2016
Please cite this article as: K.R. Senwar, T.S. Reddy, D. Thummuri, P. Sharma, V.G.M. Naidu, G.
Srinivasulu, N. Shankaraiah, Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3'-
methoxy-4'-(2-amino-2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumor agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.025.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3'-methoxy-4'-(2-amino-
2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumor agents
Kishna Ram Senwar,a T. Srinivasa Reddy,a Dinesh Thummuri,b Pankaj Sharma,a V. G. M.
Naidu,b Gannoju Srinivasulu,a Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER) Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India

ACCEPTED MANUSCRIPT
Design, synthesis and apoptosis inducing effect of novel (Z)-3-(3'-methoxy-4'-(2-amino-
2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumor agents
Kishna Ram Senwar,a T. Srinivasa Reddy,a Dinesh Thummuri,b Pankaj Sharma,a V. G. M.
Naidu,b Gannoju Srinivasulu,a Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER) Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India
*Corresponding author
Dr. Nagula Shankaraiah
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research
Hyderabad, 500037, India
Fax: (+91) 040 23073751
E-mail: shankar.niperhyd@gov.in; shankarnbs@gmail.com
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Abstract
A series of new (Z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)benzylidene)indolin-2-one
derivatives has been synthesized and evaluated for their cytotoxic activity against selected
human cancer cell lines of prostate (PC-3 and DU-145), breast (BT-549 and MDA-MB-231)
and non-tumorigenic prostate epithelial cells (RWPE-1). Among the tested, one of the
compounds 4p exhibited potent cytotoxicity selectively on prostate cancer cell lines (PC-3
and DU-145; IC50: 1.89±0.6 and 1.94±0.2 ꢀM, respectively). Further experiments were
conducted with 4p on PC-3 cancer cells to study the mechanisms of growth inhibition and
apoptosis inducing effect. Treatment of PC-3 cells with test compound 4p resulted in
inhibition of cell migration through disorganization of F-actin protein. The flow-cytometry
analysis results showed that the compound arrested PC-3 cancer cells in the G2/M phase of
cell cycle in a dose dependent manner. Hoechst staining and annexin-V binding assay
revealed that the compound 4p inhibited tumor cell proliferation through induction of
apoptosis. Western blot studies demonstrated that the compound 4p treatment led to
activation of caspase-3, increased expression of pro-apoptotic Bax and significantly
decreased expression of anti-apoptotic Bcl-2 in human prostate cancer PC-3 cells. In addition,
the mitochondrial membrane potential (ꢁΨm) was also affected and the levels of intracellular
Ca2+ were raised.
Key words:
Oxindole, Knoevenagel condensation, Benzylideneindolin-2-one, Heterocycles, Anticancer,
Apoptosis
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1.0. Introduction:
Cancer is one of the leading global health burden and most serious clinical problems in the
world with increasing incidences every year. Despite avoiding behavioural risk factors such
as tobacco, overweight and obesity, and preventive managements like dietary, medication and
vaccination, the disease still affects millions of patients worldwide [1]. Most of the current
anticancer drugs generally act on metabolically active or rapidly proliferating cells, and suffer
from poor selectivity between normal and cancerous cells. The high toxicity and poor
tolerance of the current anticancer drugs, highlighting the need to identify new molecules
with potent antitumor activity, low toxicity and minimum side effects. Therefore, the design
and synthesis of new chemical entities for the effective and safe cure of cancer are an active
area of research in medicinal chemistry.
The indolin-2-one (oxindole) is a privileged scaffold, which represents an important class of
heterocyclic compounds endowed of interesting pharmacological [2–5] and biological
activities such as inhibitors of the MDM2-p53 interaction [6], antimicrobial [7],
cholinesterases [8], histone deacetylases [9], and anticancer properties [10, 11]. Besides,
sunitinib and nintedanib [Fig. 1] are the representative drugs emerged from this class and are
in clinical use for targeted anticancer therapies [12]. The FDA approval of sunitinib and
nintedanib paved the way to design and synthesis of various indolin-2-one based molecules
with diverse activities against cancer [13, 14]. Particularly, the structural modifications at the
C3- and C5-positions of the indolin-2-one ring have led many derivatives possessing
increased antitumor activity. In this context, many synthetic indolin-2-one derivatives with
anticancer activity were developed which inhibit diverse tyrosine and serine/threonine
kinases, for instance, c-Met kinase [15], Pim kinase [16], RET kinase [17], FLT3 kinase [18],
Aurora B kinase [19], glycogen synthase kinase-3 [20], cyclin-dependent kinases (CDKs)
[21], and Polo-like kinase 4 (PLK4) [22]. Hence, we have previously reported the synthesis,
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structure-activity relationship (SAR), and importance of 3-substituted-indolin-2-ones as well
as spiro-fused-indolin-2-one based hybrids as potent cytotoxic agents [23–27].
On the other hand, incorporation of medicinally active moieties into a core bioactive natural
product provides the means for accessing wider range of pharmacological profiles, especially
in the area of anticancer therapeutics. For instance, various heterocyclic ring systems such as
morpholine, pyrrolidine, piperidine, dimethylmorpholine, indoline etc. have been found as
the fundamental scaffold components of several drugs in the market today [28, 29]. The
significance of these moieties are well understood by medicinal chemists, since they play an
important role in molecular properties or whole molecule properties such as three
dimensionality, scaffold rigidity, lipophilicity or polarity, and can determine molecular
reactivity, metabolic stability, cellular activity, and toxicity.
<insert Figure 1 here>
In our quest to develop more potent anticancer molecules, we integrated the structural
features of representative examples of 3-alkenyl-indolin-2-ones by aiming at the
identification of novel small molecules with potent antiproliferative effects on tumor cells.
We hypothesized that the newly designed molecules comprising of 3-(3'-
methoxybenzylidene)indolin-2-ones and saturated heterocyclic secondary amines connected
through oxoethoxy-linker within a single molecule could enhance the cytotoxic activity that
might work through induction of tumor cell apoptosis and cell cycle arrest. In this context, we
herein reported the synthesis and in vitro anticancer evaluation of novel (Z)-3-(3'-methoxy-4'-
(2-amino-2-oxoethoxy)-benzylidene)indolin-2-one derivatives with a view to produce the
promising anticancer agents. Besides, we also explored the effects toward the cell cycle and
possible mechanism of inducing apoptosis on human PC-3 cancer cells.
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2.0. Results and Discussion
2.1. Chemistry
The (Z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)-benzylidene)indolin-2-one derivatives 4a–v
were synthesized by employing the Knoevenagel condensation between indolin-2-ones 7a–e
and the corresponding 3-methoxy-4-(2-amino-2-oxoethoxy)-benzaldehydes 11a–e as shown
in Scheme 1. Oxindoles 7a–e were synthesized from the corresponding isatins through
Wolff-Kishner reduction reaction with hydrazine-hydrate in the presence of potassium
hydroxide. According to Sandmeyer’s method, isatins 6a–e were synthesized by converting
the appropriate anilines 5a–e into isonitrosoacetanilides via reaction with chloral hydrate and
hydroxylamine hydrochloride followed by step-wise cyclization in acidic media. The
respective key intermediates 11a–e were synthesized from chloro-acetyl precursors 9a–e by
reacting with vanillin using potassium carbonate as a base. Alkylation of the vanillin with a
variety of heterocyclic amines bearing oxoethoxy-linker introduced the basic functionalities
and scope of diversity. Intermediates 9a–e were synthesized from saturated heterocyclic
secondary amines 8a–e. Further, the (Z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)-
benzylidene)indolin-2-ones bearing different heterocyclic secondary amines viz. morpholino,
piperidin-1-yl, pyrrolidin-1-yl, indolin-1-yl, and 2,6-dimethylmorpholino were synthesized as
title compounds 4a–v. Finally, the compounds 4a–v were unambiguously characterized by
HRMS (ESI), 1H and 13C NMR spectroscopy.
<insert Scheme 1 here>
2.1.1. Stereo-chemical characterization
The 3-alkenyl-indolin-2-ones were reported to exist as either Z or E isomer depending on the
characteristics of the substituents at C3-position of indolin-2-one. Therefore, to establish the
E/Z stereochemistry of the compounds 4a–v, we considered the fact that E and Z isomers of
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benzylidene-indolin-2-ones might have time dependent isomerization lability in polar solvent
(i.e., DMSO-d6). The previous literature reports [30, 31] suggest that the ortho protons (C2'-
or C6'-protons) of the benzylidene ring in Z isomer are deshielded relative to those in E
isomer. The deshielding of C2'- or C6'-protons could be due to the presence of nearby C2-
position carbonyl of indolin-2-one ring in the Z isomer form. As a result, the two isomer
forms could be easily distinguished by 1H and 13C NMR analysis.
The stereochemistry was assigned for one of the representative compounds 4v by detailed
1
13
NOE studies. The H and C NMR assignments for compounds 4v were carried out using
gDQFCOSY, ROESY, HSQC and HMBC experiments in DMSO–d6 at 25 oC on Bruker 500
1
13
MHz ( for H NMR) and 125 MHz (for C NMR), respectively [see ESI†]. The presence of
NOE between H4-H10, H10-H12 and H10-H16 in ROESY experiment conforms the
geometry at double bond was characterized as Z-isomer as depicted in Figure 2.
<insert Figure 2 here>
2.2. Biological Evaluation
2.2.1. In vitro cytotoxic activity
All the synthesized compounds 4a–v were evaluated for their in vitro antiproliferative
activity against selected human cancer cell lines of prostate (PC-3 and DU-145), breast (BT-
549 and MDA-MB-231) and normal human prostate epithelial cells (RWPE-1) by using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Sunitinib was used
as reference standard [32–35] and the in vitro screening results are summarized in Table 1.
The results indicated that the compounds 4a, 4f, 4h, 4i, 4p, 4q, and 4t exhibited significant
cytotoxicity on both prostate cancer cells (PC-3 and DU-145) with IC50 values in the range of
1.89±0.6–6.97±0.6 µM. Gratifyingly, the compound 4p was the most active compound in this
series which exhibited potent cytotoxicity on PC-3 cancer cell line with IC50 of 1.89±0.6 µM,
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whereas the reference standard sunitinib displayed IC50 of 19.6±3.0 µM. The compounds
showing significant cytotoxicity on prostate cancer cell lines (PC-3 and DU-145) were further
tested for their in vitro cytotoxicity on non-tumorigenic prostate epithelial cells (RWPE-1), to
determine the safety of these compounds against non cancer cells. It was interesting to
observe that the tested compounds 4a, 4f, 4h, 4i, 4p, 4q, and 4t were moderately selective
toward cancer cells when compared to non cancer RWPE-1 cells.
From the analysis of IC50 values of these compounds, it is observed that most of the
compounds were less potent against breast cancer cells, BT-549 and MDA-MB-231,
respectively, except compound 4h which is fairly selective towards BT-549 cells. In general,
the cytotoxic activity decreases with heterocyclic ring substituents in the order of indolin-1-yl
> piperidine-1-yl > 2,6-dimethylmorpholino > morpholino > pyrrolidin-1-yl. Indeed, the
presence of indoline ring makes more contribution to the antitumor activity characterized
with most active compounds 4p and 4q; while the derivatives with piperidine group (4f–j)
show a reduced antiproliferative activity. Changing the piperidine ring with other
heterocyclic ring substituents (i.e., 2,6-dimethylmorpholine, morpholine and pyrrolidine)
further reduces the in vitro cytotoxic activity. In addition, the introduction of electron-
donating 3'-methoxy group on to benzylidene ring resulted in modest improvement in the
potency and might be a potential location for further chemical modification. On the other
hand, the activity profile of 4a, 4f and 4p corroborates toward the preference of H-substituent
on C5-position of indolin-2-one in comparison to C5-methyl as well as C5-halogen
counterparts. Among halogenated analogues, the compounds with 5-chloro substitution (4h
and 4t) at C5-position of indolin-2-one are generally more active than the corresponding 5-
fluoro (4b, 4g, 4l and 4s) and 5-bromo analogs (4d, 4n and 4u except 4i, which is equipotent
to 4t). Overall, the applicability of active compounds could not be questioned due to the
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known toxicity of halogens, since the most promising compound 4p bearing no halogen
substituent.
From the structure activity relationships (SARs), it could be inferred in general that the
modifications on the benzylidene ring as well as C5-position of indolin-2-one significantly
influence the cytotoxic activity. The above SARs also suggest that prospective antitumor
agents with better cytotoxic activity may be obtained from a good combination of the
substituents at C5-position of the indolin-2-one ring and the side chain at 4'-position of the
benzylidene ring. Based on the promising cytotoxic activity, most active compound 4p from
this series was taken-up for further mechanism of cell growth inhibition and cell-cycle arrest
studies.
<insert Table 1 here>
2.2.2. Morphology
To examine whether the treatment with the compound could lead to loss of cell viability and
induction of apoptosis, PC-3 cells were treated with different concentrations of the most
potent compound 4p for 48 h. Cells were observed and photographs were taken under a phase
contrast microscope (Nikon). It can be inferred from the Figure 3 that the treatment with
compound 4p resulted in markedly decreased number of viable cells of prostate PC-3 cancer
cells in comparison to the vehicle treated control cells as observed by the distinctive
morphological features of cells including detachment and cell shrinkage. Moreover, the
percentages of viable cells were determined after treatment with increasing concentrations (1,
2 and 4 µM) of compound 4p by using trypan blue assay. It was observed that at
concentrations of 2 or 4 µM, compound 4p displayed significant cytotoxicity (53% and 45%
viable cells, respectively) in PC-3 cells as shown in Figure 3.
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<insert Figure 3 here>
2.2.3. In vitro cell migration assay
Wound healing assay was carried out to investigate the effect of compound 4p on migration
potential of PC-3 cells [36]. Wounds were created in the monolayer cultures of PC-3 cells
with a 200 µL pipette tip and cells were allowed to migrate into the wound area for 0, 24, and
48 h. The migration of prostate cancer cells to fill-up the ‘wound’ was recorded by
microscopic observations. As shown in Figure 4, after 24 h the untreated control cells had
filled 43.4% in the scratched area, whereas compound 4p treatment resulted in inhibition of
cell migration with 37.3%, 29.4% and 7.3% cells migrated in the wounded area with the
doses of 1, 2, and 4 µM, respectively. The effect was more prominent with 1 and 2 µM doses
after 48 h. It can be inferred from the results that the compound 4p suppresses the migration
potential of the prostate cancer cells in dose dependent manner and also causes a significant
reduction in cell number, which can be induced by cytostatic, or even cytotoxic activity at the
concentrations used.
<Insert Figure 4 here>
2.2.4. Effect on Structures of F-actin
Previous reports suggested that a migration of cells depends on the formation of membrane
protrusions in response to chemotactic stimuli [37]. The driving force for the formation of
membrane protrusion is localized actin filaments polymerization which leads to stress fibre
assembly and plays a crucial role in cell motility. As 4p inhibits the migration of prostate
cancer cells, it was of interest to investigate the effect on stress fibre formation. The
formation of the actin cytoskeleton in PC-3 cells was established by rhodamine-phalloidin
staining which is red fluorescent dye that specifically binds to F-actin and images were
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captured with confocal microscope. As shown in Figure 5, control cells exhibited a large
number of F-actin extensions and stress fibre formation at the periphery of the cells, whereas
compound treated cells displayed disrupted stress fibres around the nucleus and the numbers
were decreased. Collectively, these results demonstrated that the compound 4p inhibit the
migration of prostate cancer cells through disruption of actin formation.
<insert Figure 5 here>
2.2.5. Cell cycle analysis
In order to further investigate the mechanism of cell growth inhibition of compound 4p, cell
cycle analysis on PC-3 cells was carried out using propidium iodide staining [38]. PC-3 cells
were treated with different concentrations (1, 2, and 4 µM) of compound 4p for 24 h and cell
cycle distribution was observed by flow-cytometric analysis after staining with propidium
iodide. As shown in Figure 6, the treatment with compound 4p resulted in significant
increase in ratio of PC-3 cells in G2/M phase from 19.2% to 55.7% when compared to the
control cells with a concomitant decrease in the number of cells at G0/G1 phase in a dose
dependent manner. The G2/M phase content was more prominent at 4 µM concentration
which indicates that the compound 4p arrested the PC-3 cells in G2/M phase.
<insert Figure 6 here>
2.2.6. Apoptosis detection studies
The ability of tumor cells to evade apoptosis, or programmed cell death, is a hallmark of
human cancers [39]. In addition to cancer cell survival, defects in apoptotic pathways may
also contribute to tumor progression and chemo-resistance. Thus, inducing apoptosis in
cancer cells has emerged as an attractive strategy in cancer therapy. Therefore, it was
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considered of interest to investigate the apoptosis inducing effect of compound 4p on PC-3
cells.
2.2.6.1. Hoechst staining
The morphological changes induced by the compound 4p in prostate cancer cells were
investigated using Hoechst nuclear staining [40]. PC-3 cells were treated with the various
concentrations of the compound 4p (1, 2 and 4 µM) for 24 h and stained with Hoechst 33242.
The results from the Figure 7 demonstrates that the compound 4p treated cells displayed
typical apoptotic characteristics such as chromatin condensation (brightly stained cells as
indicated by arrows) and nuclear fragmentation in a concentration dependent manner,
whereas vehicle treated (DMSO) control cells showed an even distribution of staining of
homogeneous nuclei. These results indicated that the compound 4p could induce apoptosis in
PC-3 cells.
<insert Figure 7 here>
2.2.6.2. Effect of compound 4p on mitochondrial membrane potential (DΨm)
Studies showed that the mitochondria also play a critical role in the intrinsic pathway of
apoptosis as they are the key targets of cellular oxidative stress, which hampers the electron
transport chain, leading to reactive oxygen species (ROS) generation and depolarization of
DΨm [41]. Therefore, to assess the influence of compound 4p on mitochondria, the DΨm
was measured. Treatment of prostate cancer PC-3 cells with compound 4p caused 19–42%
decrease in DΨm compared to control cells (Fig. 8). The compound 4p dose-dependently
induced dissipation of DΨm. At 4 µM, the compound 4p was found to be more potent in
dissipating DΨm which induces 42% loss compared to the control which is indicative of the
lethal effects on mitochondria, thereby promoting the ensuing events of apoptosis.
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<insert Figure 8 here>
2.2.6.3. Effect of compound 4p on intracellular Ca2+ levels
Previous reports suggested that elevated levels of intracellular Ca2+ is one of the major
contributing factors towards the changes in mitochondrial membrane potential DΨm [42].
Therefore, the effect of compound 4p on intracellular Ca2+ was investigated to further
confirm its role in inducing DΨm depolarization. The results from the Figure 9 indicated that
the compound treatment caused increase in the intracellular calcium levels in a dose
dependent manner compared to control in PC-3 cells. At 2 µM, there was a significant
increase in Ca2+ levels compared to control and at 4 µM it was found to be more potent in
increasing Ca2+.
<insert Figure 9 here>
2.2.6.4. AnnexinV-FITC/Propidium iodide dual staining assay
To quantify the percentage of apoptosis induced by compound 4p, the Annexin V-
FITC/Propidium iodide dual staining assay [43] was carried out using prostate PC-3 cancer
cells. The Annexin V-FITC/PI dual staining assay facilitates the detection of live cells (Q1-
LL; AV-/PI-), early apoptotic cells (Q1-LR; AV+/PI-), late apoptotic cells (Q1-UR-
AV+/PI+) and necrotic cells (Q1-UL; AV-/PI+). As shown in Figure 10, the percentage of
total apoptotic cells (early and late apoptotic cells) was increased to 38.2% after treatment
with 4 ꢀM concentration of compound 4p for 24 h, in comparison to the control (9.4%) cells.
The percentage of early and late apoptotic cells were significantly increased with increase in
concentrations of the compound 4p, which indicates that the compound 4p induces apoptosis
in PC-3 cells in a dose dependent manner.
<insert Figure 10 here>
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2.2.7. Western blotting
Caspases play an important role in the apoptosis process. Particularly, the caspase-3 is a
frequently activated death protease which catalyzes the specific cleavage of many key
cellular proteins [44]. We examined the effect of compound 4p on the activation of caspase-3
by western blotting. As shown in Figure 11, compound 4p dose dependently increased the
activation of caspase-3. Subsequently, we demonstrated the effect of compound 4p on Bcl2
family proteins. Apoptosis is associated with changes in the expression of anti-apoptotic
protein Bcl-2 and pro-apoptotic protein Bax [45]. We observed that compound 4p dose
dependently decreased the Bcl2 expression and increased the Bax expression. These results
show that compound 4p induces apoptosis through activation of caspase-3 and up-regulation
of pro-apoptotic Bax and down regulation of anti-apoptotic Bcl-2 proteins.
<insert Figure 11 here>
3.0. Conclusion
In conclusion, we have designed and synthesized a series of (Z)-3-(3'-methoxy-4'-(2-amino-2-
oxoethoxy)-benzylidene)indolin-2-one derivatives and evaluated for their antiproliferative
activity against selected human cancer cell lines. Among them, one of the compounds 4p
displayed potent growth inhibition on PC-3 cells. The exposure of prostate PC-3 cancer cells
to the compound 4p resulted in a remarkable inhibition of in vitro cell migration through
disorganization and disruption of F-actin structures. Flow cytometry analysis indicated that
the compound 4p arrested the cells at G2/M phase of the cell cycle. The apoptosis inducing
effect of the compound was analyzed by Hoechst nuclear staining. Moreover, the compound
4p induces apoptosis in PC-3 cells through collapse of mitochondrial membrane potential and
elevation of intracellular Ca2+ levels. Western blotting analysis demonstrated that compound
4p induces apoptosis through activation of caspase-3 and up-regulation of pro-apoptotic Bax
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and down regulation of anti-apoptotic Bcl-2 proteins. Overall, the simple synthetic
preparation and their biological properties make these derivatives as promising new chemical
entities for the development of cancer therapeutics.
4.0. Materials & Methods
4.1. Chemistry
All the starting materials and other reagents were commercially available of the best grade
and were used without further purification; substituted anilines, vanillin, piperidine,
morpholine, pyrrolidine, piperidine, dimethylmorpholine, indoline and chloro-acetyl chloride
(Spectrochem); chloral hydrate, NH2OH.HCl and NH2NH2.H2O (Aldrich); and ethylene
glycol, DMF and acetonitrile (Finar Chemicals). The progress of the reactions were
monitored by thin layer chromatography (TLC), performed on MERCK pre-coated silica gel
60-F254 aluminum plates. Spots were visualized by UV light. All melting points were
1
13
recorded on Stuart® SMP30 melting point apparatus and are uncorrected. H and C NMR
spectra were recorded on Bruker Avance 500 MHz spectrometer and referenced to residual
solvents. Chemical shifts are reported in parts per million (ppm) and referenced to residual
1
DMSO˗d6 (δ 2.50) (for H spectra) or DMSO˗d6 (δ 39.52) (for 13C spectra). Spin
multiplicities are described as s (singlet), brs (broad singlet), d (doublet), dd (double doublet),
t (triplet), q (quartet), or m (multiplet). Coupling constants are reported in hertz (Hz). HRMS
analyses were acquired on Agilent Q-TOF-Mass Spectrometer 6540-UHD and carried out in
the ESI techniques at 70 eV.
4.1.1. General procedure for synthesis of oxindoles (7a–e)
To a mixture of isatin (6a–e, 1.0 g) and hydrazine-hydrate (5 mL, ~30 mmol) in ethylene
glycol, potassium hydroxide (10 equiv.) was added and the resulting reaction mixture was
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o
stirred at 110–130 C for 2–3 h (reaction monitored by TLC). The reaction mixture was
cooled to room temperature, poured into ice-cold water (50 mL), acidified to pH 2 with 6N
hydrochloric acid and extracted with ethyl acetate (3 x 30 mL). The organic extracts were
combined, washed with brine (15 mL), dried over sodium sulfate and concentrated in vacuo.
The obtained residue was purified by column chromatography using ethyl acetate–hexane as
eluents to furnish 7a–e in moderate yields.
4.1.2. General procedure for the synthesis of (Z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)-
benzylidene)indolin-2-one derivatives (4a–v)
To a solution of appropriate oxindole (7a–e, 0.3 mmol) in ethanol (3 mL) was added
corresponding aldehydes (11a–e, 0.32 mmol) and a catalytic amount of piperidine. The
reaction mixture was stirred at reflux for 6–12 h (reaction monitored by TLC). After cooling,
the precipitate was filtered, washed with cold ethanol, and dried in air to furnish pure (Z)-3-
(3'-methoxy-4'-(2-amino-2-oxoethoxy)-benzylidene)indolin-2-ones title compounds of
formula (4a–v) as yellow/brown/orange solids in moderate to excellent yields.
4.1.2.1.
(Z)-3-(3'-Methoxy-4'-(2-morpholino-2-oxoethoxy)benzylidene)indolin-2-one
(4a).Yellow solid, yield 85%; Mp: 234–236 oC; 1H NMR (500 MHz, DMSO˗d6): δ 3.43–3.51
(m, 4H), 3.55–3.65 (m, 4H), 3.86 (s, 3H), 4.94 (s, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.98 (t, J =
8.7 Hz, 2H), 7.18(t, J = 7.7 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.81 (dd, J = 1.6,
8.6 Hz, 1H), 8.64 (d, J = 1.6 Hz, 1H), 10.56 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ 41.6,
44.8, 55.5, 66.0, 66.1, 66.2, 109.2, 112.5, 115.4, 119.2, 120.9, 124.1, 125.4, 126.9, 127.6,
128.2, 137.2, 140.2, 148.0, 149.2, 165.6, 167.4; HRMS (ESI): m/z calcd. for C22H23N2O5,
395.1607, found 395.1596 [M+H]+.
4.1.2.2. (Z)-5-Fluoro-3-(3'-methoxy-4'-(2-morpholino-2-oxoethoxy)benzylidene)indolin-2-one
o
1
(4b). Yellow solid, yield 83%; Mp: 259–261 C; H NMR (500 MHz, DMSO˗d6): δ 3.44–
15

ACCEPTED MANUSCRIPT
3.50 (m, 4H), 3.55–3.64 (m, 4H), 3.86 (s, 3H), 4.95 (s, 2H), 6.77–6.83 (m, 1H), 6.96–7.03
(m, 2H), 7.60 (dd, J = 2.4, 9.0 Hz, 1H), 7.78–7.86 (m, 2H), 8.66 (d, J = 1.6 Hz, 1H), 10.57 (s,
13
1H); C NMR (125 MHz, DMSO˗d6): δ 41.6, 44.7, 55.5, 65.9, 66.0, 66.1, 106.6 (d, JCF
=
25.2 Hz), 109.9, (d, JCF = 8.1 Hz),112.5, 114.3 (d, JCF = 23.7 Hz), 115.5, 123.7 (d, JCF = 2.5
Hz), 126.9 (d, JCF = 8.8 Hz), 127.3, 127.4, 136.4, 139.0, 148.0, 150.1, 157.9 (d, JCF = 233.2
Hz), 165.6, 167.4; HRMS (ESI): m/z calcd. for C22H22FN2O5, 413.1513, found 413.1514
[M+H]+.
4.1.2.3. (Z)-5-Chloro-3-(3'-methoxy-4'-(2-morpholino-2-oxoethoxy)benzylidene)indolin-2-one
o
1
(4c). Yellow solid, yield 87%; Mp: 253–254 C; H NMR (500 MHz, DMSO˗d6): δ 3.43–
3.50 (m, 4H), 3.55–3.65 (m, 4H), 3.85 (s, 3H), 4.95 (s, 2H), 6.83 (d, J = 8.2 Hz, 1H), 6.98 (d,
J = 8.5 Hz, 1H), 7.20 (dd, J = 2.1, 8.2 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.84 (dd, J = 1.8, 8.6
Hz, 1H), 7.88 (s, 1H), 8.65 (d, J = 1.9 Hz, 1H), 10.68 (s, 1H); 13C NMR (125 MHz,
DMSO˗d6): δ 41.6, 44.7, 55.6, 65.9, 66.0, 66.1, 110.5, 112.5, 115.5, 119.2, 122.9, 125.3,
127.3, 127.4 (2C), 127.5, 138.8, 139.3, 148.0, 150.2, 165.5, 167.2; HRMS (ESI): m/z calcd.
for C22H22ClN2O5, 429.1217, found 429.1211 [M+H]+.
4.1.2.4. (Z)-5-Bromo-3-(3'-methoxy-4'-(2-morpholino-2-oxoethoxy)benzylidene)indolin-2-one
o
(4d). Yellow solid, yield 82%; Mp: 246–248 C; 1H NMR (500 MHz, DMSO˗d6): δ 3.43–
3.51 (m, 4H), 3.55–3.65 (m, 4H), 3.85 (s, 3H), 4.95 (s, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.99 (d,
J = 8.5 Hz, 1H), 7.20 (dd, J = 1.9, 8.2 Hz, 1H), 7.85 (dd, J = 1.8, 8.6 Hz, 1H), 7.88 (s, 1H),
13
7.93 (d, J = 1.9 Hz, 1H), 8.65 (d, J = 1.6 Hz, 1H), 10.69 (s, 1H); C NMR (125 MHz,
DMSO˗d6): δ 41.6, 44.7, 55.5, 65.9, 66.0, 66.1, 111.0, 112.5, 113.0, 115.5, 121.9, 122.8,
127.4 (2C), 127.8, 130.3, 139.1, 139.3, 148.0, 150.2, 165.5, 167.0; HRMS (ESI): m/z calcd.
for C22H22BrN2O5, 473.0712, found 473.0673 [M+H]+.
16

ACCEPTED MANUSCRIPT
4.1.2.5. (Z)-3-(3'-Methoxy-4'-(2-morpholino-2-oxoethoxy)benzylidene)-5-methylindolin-2-one
o
1
(4e). Orange solid, yield 91%; Mp: 214–216 C; H NMR (500 MHz, DMSO˗d6): δ 2.30 (s,
3H), 3.43–3.50 (m, 4H), 3.55–3.64 (m, 4H), 3.85 (s, 3H), 4.93 (s, 2H), 6.71 (d, J = 7.8 Hz,
1H), 6.96 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 7.50 (s, 1H), 7.70 (s, 1H), 7.81 (dd, J
= 1.7, 8.5 Hz, 1H), 8.65 (d, J = 1.7 Hz, 1H), 10.44 (s, 1H); 13C NMR (125 MHz, DMSO˗d6):
δ 20.8, 41.6, 44.8, 55.5, 66.0, 66.1, 66.2, 108.9, 112.5, 115.4, 119.7, 124.3, 125.4, 126.8,
127.7, 128.6, 129.6, 137.9, 138.0, 148.0, 149.6, 165.6, 167.5; HRMS (ESI): m/z calcd. for
C23H25N2O5, 409.1763, found 409.1800 [M+H]+.
4.1.2.6. (Z)-3-(3'-Methoxy-4'-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)indolin-2-one (4f).
Yellow solid, yield 84%; Mp: 212–214 oC; 1H NMR (500 MHz, DMSO˗d6): δ 1.36–1.65 (m,
6H), 3.39–3.47 (m, 4H), 3.85 (s, 3H), 4.90 (s, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 8.5
Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.7 Hz,1H), 7.67 (d, J = 7.4 Hz, 1H), 7.74 (s,
13
1H), 7.80 (dd, J = 1.8, 8.5 Hz, 1H), 8.65 (d, J = 1.8 Hz, 1H), 10.56 (s, 1H); C NMR (125
MHz, DMSO˗d6): δ 23.9, 25.2, 25.9, 42.2, 45.2, 55.5, 66.4, 109.2, 112.3, 115.4, 119.2, 120.9,
124.1, 125.4, 126.9, 127.5, 128.2, 137.2, 140.2, 148.0, 149.8, 165.0, 167.4; HRMS (ESI): m/z
calcd. for C23H25N2O4, 393.1814, found 393.1838 [M+H]+.
4.1.2.7. (Z)-5-Fluoro-3-(3'-methoxy-4'-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)indolin-
o
2-one (4g). Yellow solid, yield 84%; Mp: 221–223 C; 1H NMR (500 MHz, DMSO˗d6): δ
1.40–1.63 (m, 6H), 3.38–3.46 (m, 4H), 3.85 (s, 3H), 4.91 (s, 2H), 6.78–6.82 (m, 1H), 6.96 (d,
J = 8.5 Hz, 1H), 7.00 (dt, J = 2.6, 9.5 Hz, 1H), 7.60 (dd, J = 2.5, 9.1 Hz, 1H), 7.80 (dd, J =
1.8, 8.6 Hz, 1H), 7.82 (s, 1H), 8.67 (d, J = 1.9 Hz, 1H), 10.57 (s, 1H); 13C NMR (125 MHz,
DMSO˗d6): δ 23.9, 25.3, 25.9, 42.2, 45.2, 55.5, 66.4, 106.6 (d, JCF = 25.3 Hz), 109.9 (d, JCF
=
8.3 Hz), 112.4, 114.3 (d, JCF = 23.7 Hz), 115.5, 123.7 (d, JCF = 2.4 Hz), 127.0 (d, JCF = 8.7
Hz), 127.3, 127.4, 136.4, 139.0, 148.0, 150.3, 157.9 (d, JCF = 233.3 Hz), 165.0, 167.5; HRMS
(ESI): m/z calcd. for C23H24FN2O4, 411.1720, found 411.1690 [M+H]+.
17

ACCEPTED MANUSCRIPT
4.1.2.8. (Z)-5-Chloro-3-(3'-methoxy-4'-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)indolin-
o
1
2-one (4h). Yellow solid, yield 87%; Mp: 230–232 C; H NMR (500 MHz, DMSO˗d6): δ
1.41–1.63 (m, 6H), 3.38–3.46 (m, 4H), 3.85 (s, 3H), 4.91 (s, 2H), 6.83 (d, J = 8.2 Hz, 1H),
6.96 (d, J = 8.5 Hz, 1H), 7.21 (dd, J = 2.0, 8.2 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.83 (dd, J =
1.8, 8.6 Hz, 1H), 7.88 (s, 1H), 8.66 (d, J = 1.8 Hz, 1H), 10.68 (s, 1H); 13C NMR (125 MHz,
DMSO˗d6): δ 23.9, 25.3, 25.9, 42.2, 45.2, 55.5, 66.3, 110.6, 112.4, 115.5, 119.2, 122.9,
125.3, 127.3, 127.4, 127.5 (2C), 138.8, 139.3, 148.0, 150.3, 165.0, 167.2; HRMS (ESI): m/z
calcd. for C23H24ClN2O4, 427.1425, found 427.1461 [M+H]+.
4.1.2.9. (Z)-5-Bromo-3-(3'-methoxy-4'-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)indolin-2-
one (4i). Orange solid, yield 83%; Mp: 224–225 oC; 1H NMR (500 MHz, DMSO˗d6): δ 1.40–
1.62 (m, 6H), 3.39–3.46 (m, 4H), 3.85 (s, 3H), 4.91 (s, 2H), 6.78 (d, J = 8.2 Hz, 1H), 6.96 (d,
J = 8.5 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.84 (dd, J = 1.8, 8.5 Hz, 1H), 7.88 (s, 1H), 7.93 (s,
13
1H), 8.65 (d, J = 1.7 Hz, 1H), 10.69 (s, 1H); C NMR (125 MHz, DMSO˗d6): δ 23.9, 25.3,
25.9, 42.2, 45.2, 55.5, 66.3, 111.1, 112.4, 113.0, 115.5, 122.0, 122.7, 127.3, 127.5, 127.8,
130.3, 139.2, 139.3, 148.0, 150.3, 165.0, 167.1; HRMS (ESI): m/z calcd. for C23H24BrN2O4,
473.0899, found 473.0922 [M+H]+.
4.1.2.10. (Z)-3-(3'-Methoxy-4'-(2-oxo-2-(piperidin-1-yl)ethoxy)benzylidene)-5-methylindolin-
o
1
2-one (4j). Orange crystalline solid, yield 89%; Mp: 213–214 C; H NMR (500 MHz,
DMSO˗d6): δ 1.40–1.61 (m, 6H), 2.30 (s, 3H), 3.39–3.45 (m, 4H), 3.85 (s, 3H), 4.89 (s, 2H),
6.72 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 7.50 (s, 1H), 7.70
(s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 8.66 (s, 1H), 10.45 (s, 1H); 13C NMR (125 MHz,
DMSO˗d6): δ 20.8, 23.9, 25.3, 25.9, 42.2, 45.2, 55.5, 66.4, 108.9, 112.3, 115.3, 119.7, 124.2,
125.4, 126.9, 127.6, 128.6, 129.6, 136.9, 138.0, 148.0, 149.8, 165.0, 167.5; HRMS (ESI): m/z
calcd. for C24H27N2O4, 407.1971, found 407.1968 [M+H]+.
18

ACCEPTED MANUSCRIPT
4.1.2.11.
(Z)-3-(3'-Methoxy-4'-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)benzylidene)indolin-2-one
o
1
(4k). Yellow solid, yield 86%; Mp: 210–212 C; H NMR (500 MHz, DMSO˗d6): δ 1.74–
1.82 (m, 2H), 1.86–1.94 (m, 2H), 3.30–3.36 (m, 2H), 3.48 (t, J = 6.8 Hz, 2H), 3.85 (s, 3H),
4.82 (s, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 7.18
(t, J = 7.7 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.81 (dd, J = 1.7, 8.6 Hz, 1H), 8.64
(d, J = 1.8 Hz, 1H), 10.56 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ 23.5, 25.7, 44.7, 45.6,
55.5, 66.5, 109.2, 112.4, 115.3 119.2, 120.9, 124.0, 125.4, 126.9, 127.5, 128.2, 137.3, 140.2,
148.0, 149.9,165.2, 167.4; HRMS (ESI): m/z calcd. for C22H23N2O4, 379.1658, found
379.1631 [M+H]+.
4.1.2.12.
(Z)-5-Fluoro-3-(3'-methoxy-4'-(2-oxo-2-(pyrrolidin-1-
o
1
yl)ethoxy)benzylidene)indolin-2-one (4l). Orange solid, yield 85%; Mp: 244–246 C; H
NMR (500 MHz, DMSO˗d6): δ 1.75–1.81 (m, 2H), 1.87–1.94 (m, 2H), 3.29–3.35 (m, 2H),
3.48 (t, J = 6.8 Hz, 2H), 3.85 (s, 3H), 4.83 (s, 2H), 6.76–6.82 (m, 1H), 6.96 (d, J = 8.5 Hz,
1H), 7.00 (dt, J = 2.6, 8.6 Hz, 1H), 7.60 (dd, J = 2.5, 9.1 Hz, 1H), 7.80 (dd, J = 1.8, 8.6 Hz,
1H), 7.82 (s, 1H), 8.66 (d, J = 1.9 Hz, 1H), 10.57 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ
23.5, 25.7, 44.7, 45.6, 55.5, 66.4, 106.6 (d, JCF = 25.2 Hz), 109.9 (d, JCF = 8.3 Hz), 112.4,
114.3 (d, JCF = 23.8 Hz), 115.4, 123.7 (d, JCF = 2.4 Hz), 127.0 (d, JCF = 8.9 Hz), 127.3, 127.4,
136.4, 139.1, 148.0, 150.3, 157.9 (d, JCF = 233.3 Hz), 165.2, 167.5; HRMS (ESI): m/z calcd.
for C22H22FN2O4, 397.1564, found 379.1580 [M+H]+.
4.1.2.13.
(Z)-5-Chloro-3-(3'-methoxy-4'-(2-oxo-2-(pyrrolidin-1-
o
1
yl)ethoxy)benzylidene)indolin-2-one (4m). Yellow solid, yield 88%; Mp: 246–247 C; H
NMR (500 MHz, DMSO˗d6): δ 1.74–1.80 (m, 2H), 1.86–1.92 (m, 2H), 3.27–3.34 (m, 2H),
3.47 (t, J = 6.8 Hz, 2H), 3.85 (s, 3H), 4.82 (s, 2H), 6.82 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 8.5
Hz, 1H), 7.19 (dd, J = 2.1, 8.2 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 1.8, 8.6 Hz,
1H), 7.86 (s, 1H), 8.64 (d, J = 1.9 Hz, 1H), 10.67 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ
19

ACCEPTED MANUSCRIPT
23.5, 25.6, 44.7, 45.6, 55.4, 66.4, 110.5, 112.4, 115.4, 119.2, 122.8, 125.3, 127.3, 127.4,
127.5 (2C), 138.8, 139.2, 147.9, 150.3, 165.1, 167.2; HRMS (ESI): m/z calcd. for
C22H22ClN2O4, 413.1268, found 413.1234 [M+H]+.
4.1.2.14.
(Z)-5-Bromo-3-(3'-methoxy-4'-(2-oxo-2-(pyrrolidin-1-
o
1
yl)ethoxy)benzylidene)indolin-2-one (4n). Brown solid, yield 85%; Mp: 221–223 C; H
NMR (500 MHz, DMSO˗d6): δ 1.74–1.80 (m, 2H), 1.86–1.92 (m, 2H), 3.30–3.34 (m, 2H),
3.47 (t, J = 6.8 Hz, 2H), 3.84 (s, 3H), 4.82 (s, 2H), 6.77 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.5
Hz, 1H), 7.32 (dd, J = 1.9, 8.2 Hz, 1H), 7.83 (dd, J = 1.8, 8.6 Hz, 1H), 7.87 (s, 1H), 7.91 (d, J
= 1.8 Hz, 1H), 8.64 (d, J = 1.8 Hz, 1H), 10.68 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ
23.5, 25.6, 44.7, 45.6, 55.4, 66.4, 111.0, 112.4, 112.9, 115.5, 121.9, 122.7, 127.3, 127.5,
127.8, 130.2, 139.1, 139.3, 147.9, 150.3,165.1, 167.0; HRMS (ESI): m/z calcd. for
C22H22BrN2O4, 459.0742, found 459.0741 [M+H]+.
4.1.2.15.
(Z)-3-(3'-Methoxy-4'-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)benzylidene)-5-
o
methylindolin-2-one (4o). Orange solid, yield 88%; Mp: 226–228 C; 1H NMR (500 MHz,
DMSO˗d6): δ 1.74–1.80 (m, 2H), 1.86–1.92 (m, 2H), 2.29 (s, 3H), 3.29–3.34 (m, 2H), 3.47 (t,
J = 6.8 Hz, 2H), 3.84 (s, 3H), 4.80 (s, 2H), 6.70 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H),
6.98 (d, J = 7.8 Hz, 1H), 7.49 (s, 1H), 7.68 (s, 1H), 7.80 (dd, J = 1.7, 8.5 Hz, 1H), 8.64 (d, J =
1.6 Hz, 1H), 10.43 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ 20.8, 23.5, 25.6, 44.7, 45.6,
55.4, 66.4, 108.9, 112.4, 115.3, 119.7, 124.2, 125.4, 126.9, 127.6, 128.6, 129.6, 136.9, 138.0,
147.9, 149.8, 165.2, 167.5; HRMS (ESI): m/z calcd. for C23H25N2O4, 393.1814, found
393.1812 [M+H]+.
4.1.2.16. (Z)-3-(4'-(2-(Indolin-1-yl)-2-oxoethoxy)-3'-methoxybenzylidene)indolin-2-one (4p).
Yellow solid, yield 89%; Mp: 239–241 oC; 1H NMR (500 MHz, DMSO˗d6): δ 3.20 (t, J = 8.3
Hz, 2H), 3.88 (s, 3H), 4.18 (t, J = 8.4 Hz, 2H), 5.04 (s, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.95–
20

ACCEPTED MANUSCRIPT
7.07 (m, 3H), 7.13–7.20 (m, 2H), 7.27 (d, J = 7.3 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.75 (s,
1H), 7.81 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 8.68 (d, J = 1.9 Hz, 1H), 10.57 (s,
1H); 13C NMR (125 MHz, DMSO˗d6): δ 27.7, 45.8, 55.5, 66.3, 109.2, 112.5, 115.4, 115.8,
119.2, 120.9, 123.6, 124.1, 124.9, 125.4, 126.9, 127.1, 127.6, 128.2, 131.5, 137.3, 140.2,
142.7, 147.9, 149.8, 165.4, 167.4; HRMS (ESI): m/z calcd. for C26H23N2O4, 427.1658, found
427.1651 [M+H]+.
4.1.2.17. (Z)-3-(4'-(2-(Indolin-1-yl)-2-oxoethoxy)-3'-methoxybenzylidene)-5-methylindolin-2-
one (4q). Orange solid, yield 91%; Mp: 244–246 oC; 1H NMR (500 MHz, DMSO˗d6): δ 2.30
(s, 3H), 3.20 (t, J = 8.0 Hz, 2H), 3.88 (s, 3H), 4.18 (t, J = 8.2 Hz, 2H), 5.04 (s, 2H), 6.72 (d, J
= 7.7 Hz, 1H), 6.94–7.10 (m, 3H), 7.16 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.1 Hz, 1H), 7.50 (s,
1H), 7.71 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 8.69 (s, 1H), 10.44 (s,
13
1H); C NMR (125 MHz, DMSO˗d6): δ 20.8, 27.7, 45.8, 55.5, 66.3, 108.9, 112.5, 115.4,
115.8, 119.7, 123.6, 124.3, 124.9, 125.5, 126.9, 127.0, 127.7, 128.6, 129.6, 131.5, 136.9,
138.0, 142.7, 147.9, 149.7, 165.4, 167.5; HRMS (ESI): m/z calcd. for C27H25N2O4, 441.1814,
found 441.1816 [M+H]+.
4.1.2.18.
(Z)-3-(4'-(2-(2,6-Dimethylmorpholino)-2-oxoethoxy)-3'-
o
methoxybenzylidene)indolin-2-one (4r). Yellow solid, yield 85%; Mp: 233–234 C; 1H NMR
(500 MHz, DMSO˗d6): δ 1.09 (d, J = 6.2 Hz, 3H), 1.09 (d, J = 6.2 Hz, 3H), 2.30 (t, J = 12.5
Hz, 1H), 2.73 (t, J = 10.7 Hz, 1H), 3.38–3.48 (m, 1H), 3.51–3.60 (m, 1H), 3.78 (d, J = 13.0
Hz, 1H), 3.85 (s, 3H), 4.18 (d, J = 12.9 Hz, 1H), 4.87 (d, J = 14.7 Hz, 1H), 5.00 (d, J = 14.7
Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.94–7.02 (m, 2H), 7.18 (dt, J = 1.0, 7.6 Hz, 1H), 7.67 (d, J
= 7.5 Hz, 1H),7.74 (s, 1H), 7.81 (dd, J = 1.9, 8.6 Hz, 1H), 8.65 (d, J = 1.9 Hz, 1H), 10.56 (s,
13
1H); C NMR (125 MHz, DMSO˗d6): δ 18.3, 18.6, 46.6, 49.7, 55.5, 66.2, 71.1, 71.3,109.2,
112.5, 115.3, 119.2, 120.9, 124.1, 125.4, 126.9, 127.6, 128.2, 137.2, 140.2, 148.0, 149.7,
165.3, 167.4; HRMS (ESI): m/z calcd. for C24H27N2O5, 423.1920, found 423.1917 [M+H]+.
21

ACCEPTED MANUSCRIPT
4.1.2.19.
(Z)-3-(4'-(2-(2,6-Dimethylmorpholino)-2-oxoethoxy)-3'-methoxybenzylidene)-5-
o
1
fluoroindolin-2-one (4s). Yellow solid, yield 83%; Mp: 246–248 C; H NMR (500 MHz,
DMSO˗d6): δ 1.10 (d, J = 6.1 Hz, 3H), 1.10 (d, J = 6.1 Hz, 3H), 2.30 (t, J = 11.5 Hz, 1H),
2.73 (t, J = 10.9 Hz, 1H), 3.39–3.46 (m, 1H), 3.51–3.59 (m, 1H), 3.77 (d, J = 12.9 Hz, 1H),
3.85 (s, 3H), 4.18 (d, J = 12.8 Hz, 1H), 4.88 (d, J = 14.7 Hz, 1H), 5.01 (d, J = 14.7 Hz, 1H),
6.77–6.82 (m, 1H), 6.96–7.02 (m, 2H), 7.60 (dd, J = 2.4, 9.0 Hz, 1H), 7.78–7.84 (m, 2H),
8.66 (d, J = 1.7 Hz, 1H), 10.57 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ 18.3, 18.6, 46.6,
49.7, 55.5, 66.2, 71.1, 71.3, 106.6 (d, JCF = 25.2 Hz), 109.9 (d, JCF = 7.9 Hz), 112.5, 114.3 (d,
JCF = 24.1 Hz), 115.4, 123.8, 127.0 (d, JCF = 8.7 Hz), 127.3, 127.4, 136.4, 139.0, 148.0,
150.1, 157.9 (d, JCF = 233.3 Hz), 165.3, 167.5; HRMS (ESI): m/z calcd. for C24H26FN2O5,
441.1826, found 441.1821 [M+H]+.
4.1.2.20.
(Z)-5-Chloro-3-(4'-(2-(2,6-dimethylmorpholino)-2-oxoethoxy)-3'-
o
methoxybenzylidene)indolin-2-one (4t). Yellow solid, yield 89%; Mp: 231–233 C; 1H NMR
(500 MHz, DMSO˗d6): δ 1.10 (d, J = 6.2 Hz, 3H), 1.10 (d, J = 6.2 Hz, 3H), 2.30 (t, J = 11.9
Hz, 1H), 2.73 (t, J = 11.1 Hz, 1H), 3.39–3.47 (m, 1H), 3.51–3.59 (m, 1H), 3.77 (d, J = 12.9
Hz, 1H), 3.85 (s, 3H), 4.18 (d, J = 12.8 Hz, 1H), 4.88 (d, J = 14.7 Hz, 1H), 5.01 (d, J = 14.7
Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 7.20 (dd, J = 1.9, 8.2 Hz, 1H),
7.80 (d, J = 1.8 Hz, 1H), 7.84 (dd, J = 1.5, 8.6 Hz, 1H), 7.88 (s, 1H), 8.65 (d, J = 1.5 Hz, 1H),
13
10.68 (s, 1H); C NMR (125 MHz, DMSO˗d6): δ 18.3, 18.6, 46.6, 49.6, 55.4, 66.2, 71.1,
71.3, 110.5, 112.5, 115.5, 119.2, 122.9, 125.3, 127.3, 127.4 (2C), 127.5, 138.8, 139.3, 148.0,
150.1, 165.2, 167.2; HRMS (ESI): m/z calcd. for C24H26ClN2O5, 457.1530, found 457.1526
[M+H]+.
4.1.2.21.
(Z)-5-Bromo-3-(4'-(2-(2,6-dimethylmorpholino)-2-oxoethoxy)-3'-
methoxybenzylidene)indolin-2-one (4u). Orange solid, yield 83%; Mp: 237–238 oC; 1H NMR
(500 MHz, DMSO˗d6): δ 1.10 (d, J = 6.1 Hz, 3H), 1.10 (d, J = 6.1 Hz, 3H), 2.30 (t, J = 11.8
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Hz, 1H), 2.73 (t, J = 11.1 Hz, 1H), 3.39–3.46 (m, 1H), 3.51–3.59 (m, 1H), 3.77 (d, J = 12.9
Hz, 1H), 3.85 (s, 3H), 4.18 (d, J = 12.9 Hz, 1H), 4.88 (d, J = 14.7 Hz, 1H), 5.01 (d, J = 14.7
Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 7.33 (dd, J = 1.6, 8.2 Hz, 1H),
7.84 (dd, J = 1.5, 8.5 Hz, 1H), 7.88 (s, 1H), 7.92 (d, J = 1.3 Hz, 1H), 8.65 (d, J = 1.4 Hz, 1H),
10.69 (s, 1H); 13C NMR (125 MHz, DMSO˗d6): δ 18.3, 18.6, 46.6, 49.6, 55.5, 66.2, 71.1,
71.3, 111.1, 112.6, 113.0, 115.5, 122.0, 122.8, 127.4 (2C), 127.8, 130.3, 139.2, 139.3, 148.0,
150.1, 165.2, 167.1; HRMS (ESI): m/z calcd. for C24H26BrN2O5, 501.1005, found 501.1006
[M+H]+.
4.1.2.22.
(Z)-3-(4'-(2-(2,6-Dimethylmorpholino)-2-oxoethoxy)-3'-methoxybenzylidene)-5-
o
1
methylindolin-2-one (4v). Orange solid, yield 90%; Mp: 214–215 C; H NMR (500 MHz,
DMSO˗d6): δ 1.09 (d, J = 6.1 Hz, 3H), 1.09 (d, J = 6.1 Hz, 3H), 2.28 (s, 3H), 2.28–2.32 (m,
1H), 2.73 (dd, J = 10.9, 13.0 Hz, 1H), 3.41–3.44 (m, 1H), 3.51–3.57 (m, 1H), 3.77 (d, J =
13.0 Hz, 1H), 3.85 (s, 3H), 4.18 (d, J = 13.0 Hz, 1H), 4.87 (d, J = 14.5 Hz, 1H), 4.99 (d, J =
14.5 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H),
7.49 (s, 1H), 7.69 (s, 1H), 7.81 (dd, J = 1.9, 8.6 Hz, 1H), 8.65 (d, J = 1.9 Hz, 1H), 10.45 (s,
13
1H); C NMR (125 MHz, DMSO˗d6): δ 18.3, 18.6, 20.8, 46.6, 49.7, 55.5, 66.2, 71.1, 71.3,
109.0, 112.5, 115.3, 119.7, 124.3, 125.4, 126.8, 127.7, 128.6, 129.6, 136.9, 138.0, 148.0,
149.6, 165.3, 167.5; HRMS (ESI): m/z calcd. for C25H29N2O5, 437.2076, found 437.2070
[M+H]+.
4.2. Biological Evaluation
4.2.1. MTT assay
The cytotoxic activity of the compounds (4a–v) was determined using MTT assay. For
preliminary screening, 1 × 104 cells per well were seeded in 100 µL DMEM, supplemented
with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at 37 °C in
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a CO2 incubator. Compounds 4a–v, diluted to the desired concentrations (for preliminary
screening; 20 µM concentration) in culture medium, were added to the wells with respective
vehicle control. After 48 h of incubation, 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) (5 mg mL−1) was added to each well and the plates were
o
further incubated at 37 C for 4 h. Then, the supernatant from each well was carefully
o
removed, formazan crystals were dissolved in 200 µL of DMSO and incubated at 37 C for
10 min. The absorbance at 570 nm was measured on a spectrophotometer (SpectraMax,
Molecular devices).
The compounds which exhibited >50% inhibition of cell viability at 20 µM in preliminary
screening; were further selected to generate drug response curve (DRC). The compounds 4a–
4v and standard sunitinib with series of concentrations (6 serial concentrations; 20–0.625
µM); MTT assay was performed as described above. IC50 values were determined from DRC
plot by linear regression method: % cell inhibition (from control OD) versus different
concentrations (µM). All the values were expressed as Mean ± SEM of three independent
experiments in which each treatment was performed in triplicate wells.
4.2.2. Morphology
PC-3 cells were plated in 6 well culture plates with a cell density of 1 × 105 cells/mL and
allowed to adhere for overnight. The cells were incubated with the 1, 2, and 4 µM
concentrations of the compound 4p. After 48 h treatment, cells were observed for the
morphological changes and photographs were taken under a phase contrast microscope
(Nikon). For trypan blue assay, the cells were harvested by trypsinization, mixed with trypan
blue (0.4 %), and loaded on to the haemocytometer to count the dead and viable cells. Each
sample was assayed for triplicates.
4.2.3. In vitro Cell migration Assay/Wound healing assay
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The wound-healing assay is a proven method to study in vitro cell migration. PC-3 cells (5 ×
105 cells/well) were cultured in 6 well plates for 24 h. The confluent monolayers were then
scratched with 200 µL pipette tip. The wounded monolayers were washed twice with PBS to
remove non-adherent cells. Then, media containing the different concentrations (1, 2, and 4
µM) of the compound 4p were added to each well. Cells which migrated across the scratched
wound were photographed under the phase contrast microscope (Nikon) at 0, 24 and 48 h
time interval after treatment. The cells that migrated to the wounded areas were counted
using Nikon NIS elements software and the percentage of migrated cells in each experimental
condition was calculated in comparison to control cells (DMSO exposed cells).
4.2.4. Effect on Structures of F-actin
PC-3 cells (5 × 105 cells/mL) were grown on cover slips in a 6 well plates for 24 h and were
treated with 1, 2, and 4 µM concentrations of the compound 4p. After 12 h treatment, cells
were washed with PBS and stained with rhodamine phalloidin (R-145, Life technologies), a
red fluorescent dye that specifically stains F-actin structures [46]. Hoechst 33242 was used to
stain the nucleus. Images were captured using confocal microscope. (Nikon)
4.2.5. Flow cytometry analysis
To determine the effect of compound 4p on the cell cycle, PC-3 cells were seeded in 6 well
plates at a density of 1 × 106 cells/well. After 24 h of incubation, cells were treated with
compound 4p in three different concentrations (1, 2 and 4 ꢀM). Cells treated with DMSO
were used as control. 24 h after treatment, both floating and trypsinised cells were harvested
o
and washed with PBS. Cells were fixed with 70% ethanol for 30 min at 4 C. Then, cells
were centrifuged (1200 rpm for 5 min), the supernatant was discarded and the pellet was
washed with PBS and stained the cells with propidium iodide staining buffer (PI (200 ꢀg),
0.1% (v/v) Triton X-100, 2 mg DNAse-free RNAse A (Sigma) in 10 mL PBS) for 15 min at
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ambient temperature in absence of light. Later, samples were analyzed for propidium iodide-
DNA fluorescence from 10000 events by flow cytometry using BD-C6 accuri flow
cytometer.
4.2.6. Hoechst staining
Human prostate cancer PC-3 cells were grown on cover slips in a 6 well plates (5 × 104
cells/well) and allowed to adhere for 24 h. The culture medium containing the compound 4p
with 1, 2, and 4 µM concentrations were added to the cells. After 24 h incubation, culture
medium was removed; cells were washed with PBS and fixed with 4% paraformaldehyde
solution at 4 ºC for 10 min. Then, cells were washed twice with PBS and stained with
Hoechst 33242 (5 µg/mL) for 30 min at room temp. The excess dye was removed by washing
twice with PBS and cell suspensions were mounted on slides and were examined for
morphological changes under confocal fluorescence microscope using 350 nm excitation and
460 nm emission (Nikon, magnification 40X).
4.2.7. Measurement of Mitochondrial Membrane Potential (MMP)
PC-3 cells were cultured in 6 well plates at a density of 5 × 105 cells/mL and allowed to grow
over night. The cells were treated with 1, 2, and 4 µM concentrations of the compound 4p.
After 24 h incubation, the adherent cells were collected by trypsinsation, washed with PBS
and resuspended in a solution of PBS containing Rhodamine-123 (10 µg/mL). After
incubation for 30 min at room temperature, cells were washed twice with PBS and re-
suspended in PBS. The samples were analyzed for Rhodamine123 fluorescence using
spectroflourometer.
4.2.8. Measurement of intracellular calcium
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Intracellular Ca2+ concentration was determined in PC-3 cells using a reported method
Thushara et al. [42]. The PC-3 cells (2 × 105 cells/mL) were plated in 24 well plates and
allowed to adhere for overnight. The cells were treated with increasing doses (1, 2 and 4 µM)
of the compound 4p for a period of 24 h. After treatment, cells were incubated with 2 µM of
fura-2/AM, a fluorescence Ca2+ indicator, for 1 h. The cells were then washed twice with
Hanks’ balanced saline solution containing 10 mM Hepes and 10 mM glucose. The images
were captured using excitation/emission at 340 and 380 nm using fluorescence microscope
(Nikon).
4.2.9. AnnexinV-FITC/Propidium iodide dual staining assay
The Annexin V-FITC/propidium iodide dual staining assay was carried out using PC-3 cells,
to quantify the percentage of apoptotic cells [43]. Briefly, PC-3 cells (1 × 106/mL per well)
were plated in six-well plates and allowed to grow for 24. Cells were treated with increasing
concentrations of compound 4p (1, 2 and 4 ꢀM) for 24 h and were collected by trypsinisation.
The collected cells were washed twice with ice-cold PBS, then incubated with 200
ꢀL1×binding buffer containing 5 ꢀL Annexin V-FITC, and then in 300 ꢀL1×binding buffer
containing 5 ꢀL Propidium iodide (PI) for 5 min at room temperature in the dark. After 15
min incubation, cells were analysed for apoptosis using BD-c6 accuri flow-cytometer.
4.2.10. Western blotting
For western blotting, PC-3 cells were treated with compound 4p for 24 h. Whole cell extracts
were prepared using RIPA (Sigma-Aldrich, St. Louis, MO, USA). Western blotting analysis
was performed using anti-Caspase-3 (rabbit, 1:1000, Santa Cruz, CA), anti-Bcl2 (rabbit,
1:1000, Santa Cruz, CA), anti-Bax (rabbit, 1:500, Santa Cruz, CA), anti-β-actin (rabbit,
1:1000, Santa Cruz, CA) and HRP-conjugated secondary antibodies (Santa Cruz, CA). The
antigen-antibody complex was visualized with an ECL detection kit (Amersham Bioscience).
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For subsequent antibody treatment, membranes were stripped in stripping buffer and re-
probed with another antibody [47]. The immune blots were quantified by densitometry
scanning with NIH ImageJ software.
Acknowledgements
K.R.S. and P.S. are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, for
the award of NIPER fellowship. NS gratefully acknowledges SERB, DST, Govt. of India for
research grant (YSS-2015-001709).
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