Umbelliferone-oxindole hybrids as novel apoptosis inducing agents

Article abstract of DOI:10.1039/c7nj02578e

In furtherance of our endeavour towards the synthesis of novel bioactive agents, a panel of (E)-3-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene)indolin-2-one derivatives were synthesized using diverse 5-substituted oxindoles and 7-hydroxy-4-methyl-

Full text of DOI:10.1039/c7nj02578e

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Guntuku, S. K. prajapti, S. D. Guggilapu, R. Sonar, G. Vegi and N. B. Bathini, New J. Chem., 2017, DOI:
10.1039/C7NJ02578E.
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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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PAPER
Umbelliferone-oxindole hybrids as novel apoptosis inducing
agents
Atulya Nagarsenkara, Lalita Guntukub, Santosh Kumar Prajaptia, Sravanthi Devi Guggilapua,
Rajkiran Sonar a, Ganga Modi Naidu Vegib* and Bathini Nagendra Babua,c*
†
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In furtherance of our endeavour towards the synthesis of novel bioactive agents, a panel of (E)-3-((7-hydroxy-4-methyl-2-
oxo-2H-chromen-8-yl)methylene)indolin-2-one derivatives were synthesized using diverse 5-substituted oxindoles and 7-
hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (3a). These synthesized analogues were further evaluated for their
in vitro cytotoxic activity against MDA-MB-231 (breast), MCF-7 (breast), DU145 (prostate), PC-3 (prostate) and A549 (lung)
cancer cell lines by employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Among all the synthesized compounds, compounds 7q (IC50 = 9.52 and 9.9 µM) and 7r (IC50 = 15.3 and 11.7 µM) showed
the most significant cytotoxicity towards DU145 and PC-3 cancer cell lines respectively. The compounds 7q and 7r were
found to be comparatively safe towards human normal prostate epithelial (RWPE-1) cells. The apoptosis inducing effect of
compounds 7q and 7r on PC-3 and DU145 cancer cells was further investigated using annexin V-FITC/propidium iodide
staining assay, which confirmed the increase in the percentage of early apoptotic cells. Moreover, the cell cycle analysis
revealed cell cycle arrest particularly at G0/G1 phase in PC-3 and DU145 cells. In addition to that, the treatment with
compounds 7q and 7r led to collapse of the mitochondrial membrane potential (DΨm) and increased levels of reactive
oxygen species (ROS) in PC-3 and DU145 cells. Western blotting was performed to examine the appearance of active forms
of cytochrome c and cleaved PARP (Poly ADP ribose polymerase), indicator proteins of apoptosis in PC-3 cells; the study
confirmed the triggering of mitochondrial mediated apoptotic pathway upon exposure of compounds 7q and 7r.
www.rsc.org/
sunitinib and indirubin are recognized anticancer agents containing
3-alkenyl-oxindole as basic scaffold.
Umbelliferone, also known as 7-hydroxycoumarin, hydrangine,
Introduction
Cancer is alone turning into one of the most devastating health
problems worldwide as the number of deaths due to it is more than
those caused by AIDS, malaria, and tuberculosis combined.1
According to survey if new treatments are not found by 2030, there
will be 27 million people with cancer and 17 million deaths
annually.2 Although there have been untired efforts and screening
of myriads of compounds for anti-cancer activities, the ambiguity
about the cause of origin of cancer, limitations in its detection at
early stage, its direct relation with process of cell division,
skimmetine, and β-umbelliferone, is a common natural product of
the coumarin family (Figure 1).5 Umbelliferone primarily occurs in
many familiar plants from the Apiaceae (Umbelliferae) family such
as carrot, coriander and garden angelica. According to recent
studies, umbelliferone was found to exhibit significant anticancer
effects via the induction of apoptosis, cell cycle arrest and DNA
fragmentation in HepG2 cancer cells.6 Scopoletin,
a naturally
occurring umbelliferone derivative, can induce cell cycle arrest and
metastatic nature of cancer cells and lack of drug penetration to increase apoptosis in human prostate tumor cells and human
leukemia cell line via activation of caspase-3.7
cancer tissue are some of the characteristics of this disease which
prove as stumbling block in the successful treatment of cancer.3
Most of the present anticancer drugs generally act on metabolically
The 3-substituted oxindole forms possess various biological
activities and continue to be a main structural unit of many natural
and pharmacologically active compounds (Figure 1).4 For example,
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 generates the need to
identify new molecules with potent antitumor activity, low toxicity
and minimum side effects.8
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research, Balanagar, Hyderabad, 500037, India. *E-mail: bathini@
niperhyd.ac.in; Tel.: +91-40-23073740; fax: +91-40-23073751
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research, Balanagar, Hyderabad, 500037, India.
cCentre for Semiochemicals, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad, 500007
Molecular hybridization is an effective tool to design more active
and novel chemical entities by covalently combining two or more
drug pharmacophores into a single molecule.9 Recently, it has been
observed that chromone-pyrimidine, chromone-indolinone,
chromonepyrazole, indole-pyrimidine, indole-indolinone, pyrazine–
† Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/x0xx00000x
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coumarins and indole-pyrazole conjugates demonstrated profound These 5-substituted oxindoles (5a-b) and (6a-g) were then
growth inhibitory activity against different cancer cells.10,11
subjected to Knoevenagel condensation with aldehyde (3a) to give
In this context, we desire to exploit 7-hydroxy-4-methyl-2-oxo-2H- corresponding final compounds (7j-r).
chromene-8-carbaldehyde as the diversity surrogate to condense It is to be noted that all the synthesized target compounds were
with various 5-substituted oxindoles. These umbelliferone-oxindole obtained specifically in thermodynamically stable E configuration
derivatives may have the advantages of tunable anticancer activity, form. The configuration can be easily identified by 1H NMR analysis.
low toxicity and structural diversity. Furthermore, the chemical The deshielding of C4-proton of oxindole could be due to the
processes and reactions implicated are simple and concise, which presence of nearby umbelliferone ring in the E isomer form.
make it practicable for the bulk production of important oxindoles
derivatives.
iii
i
ii
HO
O
O
HO
OH
BnO
O
3b
O
HO
O
O
O
2
1
3a
O
O
O
O
NH
O
N
F
N
N
RCOCl
H
H
R
5a R = Me
HN
O
iv
5b
R = Ph
O
5a-b
N
H
O
O
N
H
N
H
N
N
H
H
R1
R1OH
(b) Semaxanib
(c) Indirubin
(a)
4
Sunitinib
O
F
v
N
H
6a-g
O
O
Ph
6c R1
=
=
=
Ph
6a R1 = Benzhydryl, R1 = H, R2 = H
6b R1 = PMB, R1 = H, R2 = H
6f R1
=
Ph
O
O
O
O
HO
O
Umbelliferone
Figure 1:
O
HO
(e)
O
Scopoletin
O
O
O
Cl
(d)
Herniarin
Scoparone
(g)
(f)
6d R1
Cl
Ph
Ph
Structure of some representative bioactive oxindole (a, b and c) and
OMe
umbelliferone (d, e, f and g) derivatives.
6g R1
=
Ph
6e R1
Results and discussion
O
O
Chemistry
R2O
R
vi
R
The synthesized 3-umbelliferone substituted oxindoles compounds
of the present study comprise two core structural elements: (i) a 5-
substituted oxindole and (ii) a 4-methyl umbelliferone moiety.
The 4-methyl umbelliferone scaffold required for the syntheses of
target compounds was synthesized using resorcinol as the starting
material (Scheme 1). Resorcinol (1) upon treatment with ethyl
acetoacetate and sulphuric acid gave 4-methyl umbelliferone (2).12
Next, the Duff reaction was performed on 4-methyl umbelliferone
using urotropine to give the required 7-hydroxy-4-methyl-2-oxo-2H-
chromene-8-carbaldehyde (3a).13
O
R2O
O
O
O
N
N
R1
O
R1
7a-r
3a or 3b
F
7n R =
R1 = H, R2 = H
Ph
7a R = H, R1 = H, R2 = H
7b R = H, R1 = H, R2 = Bn
7c R = Br, R1 = H, R2 = H
7d R = I, R1 = H, R2 = H
7e R = F, R1 = H, R2 = H
7f R = Me, R1 = H, R2 = H
7g R = NO2, R1 = H, R2 = H
7h R = Br, R1 = Me, R2 = H
7i R = Cl, R1 = Me, R2 = H
7j R = Ac, R1 = H, R2 = H
7k R = Bz, R1 = H, R2 = H
7l R = Benzhydryl, R1 = H, R2 = H
7m R = PMB, R1 = H, R2 = H
Ph
7q R =
R1 = H, R2 = H
Ph
R1 = H, R2 = H
OMe
7o R =
Cl
Ph
Cl
R1 = H, R2 = H
7r R =
7p R =
R1 = H, R2 = H
Ph
Ph
Knoevenagel condensation was employed using oxindole (4) and
aldehyde (3) to give target compound 7a in good yield. Compound
7a was formed as a single isomer with E configuration, confirmed by
1H NMR studies.14 Next, the reaction was tried using O-benzyl
protected aldehyde (3b) and the compound 7b was also formed as
a single isomer. Encouraged by such interesting observations,
different 5-halo substituted oxindole were subjected to
Knoevenagel condensation with aldehyde (3a) to give
corresponding target compounds (7c-e) in good to excellent yields.
Reactions using 5-methyl and 5-NO2 substituted oxindoles furnished
compounds (7f-g) in good yields. N-methyl protected 5-halo
substituted oxindole substrates upon Knoevenagel condensation
with aldehyde (3a) gave higher yields (7h-i).
Scheme 1: Synthetic scheme for the synthesis of umbelliferone linked oxindole derivatives: (i) ethyl
°
°
acetoacetate, H2SO4, 10 C, 4 h; (ii) urotropine, sulfuric acid, acetic acid, 120 C, 4 h; (iii) benzyl
bromide, potassium carbonate, acetonitrile, reflux, 6 h; (iv) AlCl3, DMF, reflux, 6 h; (v) pTSA,
nitromethane, 6 h; (vi) piperidine, ethanol, reflux, 4 h.
Biological evaluation
In vitro anticancer activity. All the newly synthesized compounds
were evaluated for their in vitro cytotoxic activity against MDA-MB-
231, MCF-7, DU145, PC-3 and A549 cancer cell lines by employing
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.16 Sunitinib was used as the reference for this
study. The concentration response course analysis was performed
to determine drug concentrations required to inhibit the growth of
cancer cells by 50% (IC50) after incubation for 48 h. The results from
the in vitro anticancer studies exposed that the synthesized target
compounds exhibited different levels of anticancer properties
(Table 1). From the close examination of the IC50 values, it was
observed that the Knoevenagel product (7a) of oxindole and
aldehyde (3) was active only against DU145 cell lines, while the O-
benzylated product (7b) was found to be inactive. Compound
bearing 5-bromo (7c) substitution showed no activity against all the
tested cell lines, whereas compound with 5-iodo (7d) substitution
To extend the substrate range, diversified 5-substituted oxindoles
were synthesized from oxindole. Oxindole (4) upon treatment with
acid chlorides such as acetyl chloride and benzoyl chloride gave 5-
acylated oxindoles (5a-b).15 Reactions with oxindole and bulky
alcohols in the presence of Lewis acid gave 5-alkylated oxindoles
(6a-g).
2 | J. Name., 2012, 00, 1-3
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displayed remarkable activity against A549 cells along with substituted oxindole rather than the individual units of the target
moderate activity towards DU145 and PC-3 cell lines. Target compounds.
compound with 5-fluro substitution (7e) was specifically active Analysis of the MTT assay results suggests PC-3 and DU145 cells are
against A549, whereas compound with 5-methyl (7f) substitution more sensitive towards the synthesized target compounds. The
was inactive towards all the tested cell lines. The compound with 5- impact of modification of the “R” group at 5th position of oxindole
nitro (7g) substitution was also moderately active against A549 derivatives is interesting in the light of the results of the SAR study,
cells. Compound bearing 5-bromo N-methyl (7h) substitutions which suggests that bulky diphenyl propargyl moiety at 5th position
showed good activity selectively against PC-3 cells, on the other of oxindole is optimal for anticancer activity. Previous literature
side compound with 5-chloro N-methyl (7i) substitutions displayed reports demonstrated that strong electron withdrawing
moderate activity against DU145, PC-3 and A549 cell lines. substitutions such as chloro and fluoro on the aromatic ring
Compound containing 5-acetyl (7j) substitution showed moderate increases the lipophilicity of molecules and are responsible for
activity selectively against PC-3 cells, on the other hand compounds enhanced cytotoxicity.17,18 Interestingly, electron withdrawing
with 5-benzoyl moiety (7k) and 5-benzhydryl group (7l) were found substitutions on diphenyl propargyl moiety at 5th position of
active only against A549 cells; however the compound bearing 5-p- oxindole showed better results than electron donating
methoxy benzyl (PMB) group (7m) displayed good activity against substitutions. Conversely, simple substitutions at this position
PC-3 and A549 cells and was also moderately active against DU145.
demonstrated decrease in the activity. Among all compounds
Target compound holding bulky diphenyl propargyl substitution at synthesized, compounds 7q and 7r showed the most significant
5th position of oxindole (7n) showed good activity against MCF-7 cytotoxicity towards all the tested cancer cell lines. These primary
cells and moderate activity against PC-3, DU145 and A549 cell lines. results persuade further investigation on the synthesized
The compound having p-methyl substitution on diphenyl propargyl compounds aiming to the development of novel potential
moiety (7o) was found to be moderately active against all the anticancer agents.
tested cell lines. With p-methoxy substitution on diphenyl propargyl
Acridine orange-Ethidium bromide (AO-EB) staining. Acridine
orange/ethidium bromide (AO/EB) fluorescent staining assay was
performed to distinguish the live, apoptotic and necrotic cells.19 AO
permeates the intact cell membrane and stains the nuclei green,
while EB can stain the cells that have lost their membrane integrity
and tinge the nucleus red. From figures 2A and 2B, it can be seen
that the control cells display normal healthy morphology with intact
nuclear architecture and appeared green in colour.
moiety (7p), the compound was active selectively against prostate
cancer cell lines PC-3 and DU145. Interestingly, when diphenyl
propargyl moiety was substituted with halogens such as fluoro (7q)
and chloro (7r), the anticancer activity of the compounds was
remarkably increased against all the tested cancer cell lines.
Table 1. In vitro anticancer activity of umbelliferone-oxindole derivatives
(IC50 µM)
MDA-MB-
231
>50
>50
>50
C
MCF-7
DU145
PC-3
A549
RWPE-1
7a
7b
7c
7d
7e
7f
>50
>50
>50
19.3±0.6
>50
>50
19.8±0.3
>50
>50
>50
>50
25.9±0.2
ND
>50
>50
ND
9.0±1.4
9.7±2.5
>50
ND
ND
ND
ND
ND
>50
ND
>50
>50
>50
>50
>50
>50
ND
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
1
>50
>50
>50
>50
>50
>50
>50
>50
ND
>50
>50
>50
>50
ND
>50
11.6±1.4
26.0±2.1
>50
>50
>50
35.4±0.1
>50
>50
ND
38.3±0.5
>50
28.0±0.8
>50
49.9±2.0
15.4±0.0
14.5±1.5
31.3±0.3
17.4±0.2
>50
ND
ND
ND
ND
ND
ND
ND
ND
13.6±0.8
22.0±1.6
35.0±2.1
>50
>50
ND
Figure 2A: Morphological changes observed by Acridine orange/Ethidium bromide
staining in PC-3 cells. Apoptotic features such as membrane blebbing, chromatin
condensation and apoptotic body formations were clearly observed in cells treated
with 5 and 10 µM of compounds 7q and 7r.
31.6±0.2
32.6±0.4
24.1±3.2
31.7±0.7
9.52±0.3
15.3±0.2
>50
19.3±2.3
25.5±2.2
17.5±0.5
16.5±0.3
9.9±0.89
11.3±0.3
>50
34.2±0.3
>50
ND
ND
23.5±0.39
15.2±0.4
12.9±0.5
>50
12.4±1.1
7.4±0.28
>50
16.4±0.1
15.3±0.2 25.3±0.58
>50
>50
ND
ND
25.2±1.4
3a
S
>50
>50
>50
>50
7.4±0.5
23.8±0.4
16.3±0.5
12.6±1.0
14.4±0.3
C: Compound;; ND: Not determined; S: Sunitinib
The most active compounds 7q and 7r were also tested on RWPE-1
cells and were found to be safe compared to the prostate cancer
cells.
Additionally, the building blocks of target compounds i.e. oxindole
(1), 4-methyl umbelliferone aldehyde (3), were screened for their
anticancer potential and were found to be inactive against all tested
cell lines. These results suggest that the enhancement of anticancer
activity occurs when the umbelliferone moiety is linked to 5-
Figure 2B: Morphological changes observed by Acridine orange/Ethidium bromide
staining in DU145 cells. Apoptotic features such as membrane blebbing, chromatin
condensation and apoptotic body formations were clearly observed in cells treated
with 5 and 10 µM of compounds 7q and 7r.
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Fluorescence microscopic images of 7q and 7r treated PC-3 and PC-3 and DU145 cells were treated with designated concentrations
DU145 cells have clearly demonstrated morphological changes of compounds 7q and 7r independently for 24 h. They were stained
which are the characteristic features of apoptotic cells such as cell with propidium iodide and samples were further analysed by flow
shrinkage, membrane blebbing, chromatin condensation and cytometry. Treatment with the compounds (7q and 7r) at 5 and 10
apoptotic body formation. This confirms that the compounds 7q µM concentrations in PC-3 cells displayed rise in G0/G1 population
and 7r induced cell death in PC-3 and DU145 cancer cells.
from 44.96% (control) to 61.18% and 60.01% respectively in a dose
dependent manner. Similarly, 7q and 7r treated DU145 cells
exhibited increase in G0/G1 population of cells from 64.23%
(control) to 70.04% and 72.43% respectively in a dose dependent
manner. Notably, reference compound Sunitinib (S) has
demonstrated increase in G0/G1 population from 44.96% (control)
to 62.14% and 64.23% (control) to 77.1% in PC-3 and DU145 cell
lines at 5 µM respectively. Significant increase in Sub-G1 population
in DU145 cells also indicates exertion of apoptosis by the indicated
compounds. Overall, the results suggest clear induction of G0/G1
cell cycle arrest by the test compounds.
DAPI nucleic acid staining. DAPI (4',6-diamidino-2-phenylindole) is a
fluorescent dye that stains nucleus by binding strongly to DNA and
thus identifies the nuclear damage or chromatin condensation. The
DAPI stains nucleus of the apoptotic cells as bright colored due to
the condensed nucleus which is
a distinguishing apoptotic
characteristic. DAPI staining technique was carried out according to
earlier reported method.20 It can be inferred from the figures 3A
and 3B that the nuclear structure of untreated cells was intact,
whereas compounds 7q and 7r treated cells exhibited horse
shaped, fragmented and pyknotic (shrunken) nuclei.
Figure 3A: Assessment of Nuclear morphological changes by DAPI staining in PC-3 cells
after 48 h. 7q and 7r treated PC-3 cells have displayed nuclear apoptotic characteristics
such as nuclear fragmentation and shrunken nuclei.
Figure 4B: Effect of compounds on cell cycle distribution as analyzed by flow cytometry
using propidium iodide staining method in DU145 cells. DU145 cells were treated with
designated conc. of 7q, 7r and Sunitinib (S) for 24 h. The population of cells in each cell
cycle phase was numerically depicted. Data shown here is representative of three
independent experiments.
Figure 3B: Assessment of Nuclear morphological changes by DAPI staining in DU145
cells after 48 h. 7q and 7r treated DU145 cells have displayed nuclear apoptotic
characteristics such as nuclear fragmentation and shrunken nuclei.
Cell cycle analysis. Many of the cytotoxic compounds exert their
growth inhibitory effect by arresting the cell cycle at a specific
phase of a cell cycle. In vitro screening results revealed that the
compounds 7q and 7r showed significant activity against PC-3 and
DU145 cells. Thus, we herein examined the effect of the
compounds 7q and 7r on cell cycle using propidium iodide staining
method (Figure 4A and 4B).21,22
Annexin V binding assay. The apoptosis inducing effect of
compounds 7q and 7r on PC-3 and DU145 cancer cells was further
investigated using annexin V-FITC/propidium iodide staining
assay.23 PC-3 and DU145 cells were treated with 5 and 10 µM of
compounds 7q and 7r independently for 24 h and stained with
Annexin V-FITC and propidium iodide, and samples were analysed
by flow cytometry.
Figure 4A: Effect of compounds on cell cycle distribution as analyzed by flow cytometry
using propidium iodide staining method in PC-3 cells. PC-3 cells were treated with
designated conc. of 7q, 7r and Sunitinib (S) for 24 h. The population of cells in each cell
cycle phase was numerically depicted. Data shown here is representative of three
independent experiments.
Figure 5A: Annexin V- FITC /PI binding assay in PC-3 cells. PC-3 cells were treated with
designated conc. of 7q, 7r and Sunitinib (S) for 24 h.
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As depicted in figure 5A, 7q and 7r treated PC-3 cells demonstrated
rise in the total percentage of apoptotic (early and late apoptotic
cells- Annexin V +ve cells) and dead cells from 4.08% (untreated) to
53.48% and 43.7% respectively in a dose dependent manner.
Similarly, as shown in figure 5B, DU145 cells treated with indicated
concentrations of 7q and 7r displayed increase in the total
apoptotic dead population from 4.38% (untreated) to 54.63% and
46.41% respectively in
a concentration dependent manner.
Figure 6B: The effect of compounds 7q and 7r on intracellular ROS generation. a)
DU145 cells were treated with various conc. of 7q for 3 h, with or without 3mM NAC
Reference compound Sunitinib (S) has also displayed rise in
apoptotic, dead population from 4.08% (control) to 39.72% and pretreatment for 1 h. b) DU145 cells were treated with various conc. of 7r for 3 h, with
and without 3 mM NAC pretreatment for 1 h. ROS generation was measured using
4.38% (control) to 58.81% in PC-3 and DU145 cells respectively.
DCFDA staining by flow cytometry and geomean DCFDA fluorescence were represented
in histogram. Values are represented as Mean ±SEM (n=3). Statistical analysis was
performed with one way Annova followed by Dunnet’s test. *P<0.05 **P<0.01,
***P<0.001 , when compared to ctrl. NAC treated groups are compared with their
respective compound treated controls. # P<0.05,## P<0.01,### P< 0.001.
Assessment of mitochondrial membrane potential (DΨm):
Increase in intracellular ROS can cause oxidative stress and lead to
the loss of mitochondrial membrane potential. Thus, the effect of
compounds 7q and 7r on mitochondrial membrane potential (DΨm)
was determined by staining with lipophilic cationic JC-1 dye.25
Healthy polarised mitochondria stains red due to potential
dependent formation of J-aggregates, while depolarised
mitochondria in apoptotic cells stains green because of J-
monomers. Dose dependent increase in green to red fluorescent
Figure 5B: Annexin V- FITC /PI binding assay in DU145 cells. DU145 cells were treated
with designated conc. of 7q, 7r and Sunitinib (S) for 24 h.
population ratio from 0.55 to 1.31 and 0.9 was observed with
treatment of compounds 7q and 7r respectively in PC-3 cells.
DU145 cells exposed to the indicated concentrations of compounds
Determination of intracellular ROS generation: The reactive
7q and 7r also showed enhancement in green to red fluorescence
ratio from 0.16 to 5.63 and 0.45 respectively (Figure 7).
oxygen species generation is one of well characterised mechanisms
of many anticancer drugs. Hence, in next array of experiments, we
assessed the generation of intracellular reactive oxygen species by
compounds 7q and 7r in PC-3 and DU145 cells using DCFDA staining
method.24
Figure 7: Detection of mitochondrial membrane potential (DΨm) by JC-1 staining. a)
Quantitative representation of population of cells with green to red fluorescence ratio
in PC-3 cells. b) Quantitative representation of population of cells with green to red
fluorescence ratio in DU145 cells. Cells were treated with designated conc. of 7q, 7r
Figure 6A: The effect of compounds 7q and 7r on intracellular ROS generation. a) PC-3
cells were treated with various conc. of 7q for 3 h, with or without 3mM NAC
and Sunitinib (S) for 24 h and incubated with 2 μM JC-1 probe and further analyzed by
pretreatment for 1 h. b) PC-3 cells were treated with various conc. of 7r for 3 h, with
flow cytometer (FACS verse, Becton Dickinson, US).
and without 3 mM NAC pretreatment for 1 h. ROS generation was measured using
DCFDA staining by flow cytometry and geomean DCFDA fluorescence are represented
in histogram. Values were represented as Mean ±SEM (n=3). Statistical analysis was
The compounds 7q and 7r displayed prominent effect on green to
performed with one way Annova followed by Dunnet’s test. *P<0.05
red fluorescence ratio than the reference compound Sunitinib (S).
**P<0.01,***P<0.001 , when compared to ctrl. NAC treated groups are compared with
their respective compound treated controls. # P<0.05,## P<0.01,### P< 0.001.
Thus, the results clearly indicate loss of mitochondrial membrane
potential by the compounds (7q and 7r) and suggest the
Treatment with compounds 7q and 7r for 3 h resulted in significant
increase in DCFDA fluorescence in a dose dependent manner,
signifying ROS accumulating property of compounds (Figure 6A and
6B). Treatment with N-acetyl cysteine (NAC) prior to compound
treatment has decreased DCFDA fluorescence intensity; this
indicated that compounds induced cytotoxicity by ROS generation.
involvement of mitochondria dependent apoptotic pathway in their
mechanism of action.
Western blotting analysis PARP (Poly ADP ribose polymerase) is
116 kda protein that gets cleaved into 89 kda protein fragment
during caspases activation and this cleavage is prominent hallmark
of the apoptosis induction. Intrinsic or mitochondria mediated
apoptotic pathway can be evidenced with loss of mitochondrial
membrane potential usually preceded by the release of cytochrome
c into cytosol. Thus, to investigate the changes in protein expression
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levels of proteins cytochrome (cytosolic) and cleaved PARP in PC-3,
western blotting analysis was performed (Figure 8).26 Treatment
with 7q has clearly exhibited 1.88 fold increase in cytosolic levels of
cytochrome c at 10 µM, while exposure to 7r has shown marked
Acknowledgements
The authors thank the Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers (Govt. of India) for
increase (8.65 fold) in the protein levels of cleaved PARP. These providing funds and Dr. G. Srinivasalu, NIPER Hyderabad for
protein changes were compared with standard drug Sunitinib (S).
Increase in cleaved PARP level with compound 7r (6.54 fold at 5
µM) was more pronounced than with Sunitinib treatment (4.53 fold
at 5 µM).
NMR studies.
References
1. World Health Organization, Fact sheet No. 297, February, 2012.
2. S. Reinberg, Health Day News, World Health Organization, 2008.
3. J. J. Guidry, A. Greisinger, L. A. Aday, R. J. Winn, S. Vernon and T.
A. Throckmorton, Oncol. Nurs. Forum, 1996, 23, 1393.
4. A. Nagarsenkar, S. K. Prajapti, S. D. Guggilapu, S. Birineni, S. S.
Kotapalli, R. Ummanni and B. N. Babu, Med. Chem. Commun.,
2016, 7, 646.
5. L. K. A. M. Leal, A. A. G. Ferreira, G. A. Bezerra, F. J. A. Matos
and G. S. B. Viana, J. Ethnopharmacol., 2000, 70, 151.
6. S. –M. Yu, D. –H. Hu and J. –J. Zhang, Mol. Med. Rep., 2015, 12,
3869.
7. E. -K. Kim, K. -B. Kwon, B. -C. Shin, E. –A. Seo, Y. -R. Lee, J. -S.
Kim, J. -W. Park, B. -H. Park and D. -G. Ryu, Life Sci., 2005, 77,
824.
8. S. K. Prajapti, A. Nagarsenkar, S. D. Guggilapu, K. K. Gupta, L.
Allakonda, M. K. Jeengar, V. G. M. Naidu, and B. N. Babu,
Bioorg. Med. Chem. Lett., 2016, 26, 3024.
9. S. K. Prajapti, S. Shrivastava, U. Bihade, A. K. Gupta, V. G. M.
Naidu, U. C. Banerjee and B. N. Babu, Med. Chem. Commun.,
2015, 6, 839.
Figure 8: Western blotting analysis of apoptotic protein expression. a) PC-3 cells were
treated with 2.5, 5, 10 μM conc. of 7q and standard drug sunitinib 5 μM for 48 h. b) PC-
3 cells were treated with 2.5, 5, 10 μM conc. of 7r and standard drug Sunitinib (S) 5 μM
for 48 h. Changes in expression levels of Cyt C and Cleaved PARP are represented. Data
shown here is representative of one of the three independent experiments.
Representative histograms showing quantitative results for relative levels of Cyt-C of 7q
treated PC-3 cells and cleaved PARP of 7r treated PC-3 cells. Values are the means ±
SEM (n = 3).
10. P. Singh, M. Kaur and W. Holzer, Eur. J. Med. Chem., 2010, 45,
4968.
11. R. Goel, V. Luxami and K. Paul, RSC Adv., 2015, 5, 37887.
12. X. Zhai, Q. Huang, N. Jiang, D. Wu, H. Zhou and P. Gong,
Molecules, 2013, 18, 2904.
13. G. G. Muccioli, N. Fazio, G. K. Scriba, W. Poppitz, F. Cannata, J.
H. Poupaert, J. Wouters, and J. D. Lambert, J. Med. Chem., 2006,
49, 417.
14. B. N. Babu, A. Nagarsenkar, V. G. M. Naidu, G. Lalita, S. D.
Guggilapu, S. K. Prajapti; (E)-3-((7-hydroxy-4-methyl-2-oxo-2H-
chromen-8-yl) methylene) indolin-2-one derivatives as
anticancer agents, Indian Patent (2017) Application no.
201741003441.
15. K. Lackey, M. Cory, R. Davis, S. V. Frye, P. A. Harris, R. N. Hunter,
D. K. Jung, O. B. McDonald, R. W. McNutt, M. R. Peel, R. D.
Rutkowske, J. M. Veal and E. R. Wood, Bioorg. Med. Chem. Lett.,
2000, 10, 223.
Conclusion
In the current study, a series of (E)-3-((7-hydroxy-4-methyl-2-oxo-
2H-chromen-8-yl)methylene)indolin-2-one
derivatives
were
synthesized, and were further evaluated for their in vitro anticancer
potentials. An initial screening was performed against MDA-MB-
231, MCF-7, DU145, PC-3 and A549 cancer cell lines. In MTT assay,
the compounds 7q and 7r were found to be the most active against
the PC-3 and DU145 (prostate cancer) cell lines. Most importantly,
the compounds 7q and 7r were found to be comparatively safe
towards normal cell line RWPE-1. The detailed studies like AO/EB
staining, DAPI nuclear staining and Annexin
V binding assay
suggested that compounds 7q and 7r induced apoptosis in PC-3 and
DU145 cells. The cell cycle analysis confirmed that the compounds
7q and 7r target the G0/G1 phase of PC-3 and DU145 cell cycle in a
dose-dependent manner. Moreover, the compounds 7q and 7r
16. V. G. M. Naidu, U. M. Bandari, A. K. Giddam, K. R. D. Babu, J.
Ding, K. S. Babu, B. Ramesh, R. R. Pragada and P.
Gopalakrishnakone, Asian Pac. J. Trop. Med., 2013, 6, 337.
treatment resulted in collapse of mitochondrial membrane 17. R. Gudipati, R. N. R. Anreddy and S. Manda, Saudi Pharm. J.,
2011, 19, 153.
potential and elevated intracellular ROS levels in PC-3 and DU145
cells. Investigation of expression levels of apoptotic proteins
cytochrome c and cleaved PARP in PC-3 cells revealed that
compounds 7q and 7r trigger mitochondrial mediated intrinsic
pathway thereby inducing apoptosis in cancer cells. Overall, the
current studies demonstrated that dual pharmacophores strategy
can be useful in the synthesis of promising chemical entities for the
development of cancer therapeutics.
18. Y. Lu, C. -M. Li, Z. Wang, C. R. Ross II, J. Chen, J. Dalton, W. Li,
and D. D. Miller, J. Med. Chem., 2009, 52, 1701.
19. D. Ribble, N. B. Goldstein, D. A. Norris and Y. G. Shellman, BMC
Biotechnol., 2005, 5:12.
20. S. G. Kennedy, E. S. Kandel, T. K. Cross and N. Hay, Mol. Cell
Biol., 1999, 19, 5800.
21. S. D. Guggilapu, G. Lalita, T. Srinivasa Reddy, S. K. Prajapti, A.
Nagarsenkar, S. Ramu, B. Uma Rani, U. Jaya Lakshmi, V. G. M.
Naidu, S. K. Bhargava and B. N. Babu, Eur. J. Med
Chem., 2017, 128, 1.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
22. A. Nagarsenkar, L. Guntuku, S. D. Guggilapu, K. Danthi Bai, G.
Srinivasulu, V. G. M. Naidu and B. N. Babu, Eur. J. Med. Chem.,
2016, 124, 782.
23. B. Kloesch, T. Becker, E. Dietersdorfer, H. Kiener and G. Steiner,
Int. Immunopharmacol., 2013, 15, 400.
24. E. Eruslanov and S. Kusmartsev, Methods Mol. Biol., 2010, 594,
57.
25. S. D. Guggilapu, G. Lalita, T. Srinivas Reddy, A. Nagarsenkar, D.
K. Sigalapalli, V. G. M. Naidu, S. K. Bhargava and B. N. Babu, Eur.
J. Med. Chem. 2017, 138, 83.
26. S. Shrivastava, P. Kulkarni, D. Thummuri, M. K. Jeengar, V. G. M.
Naidu, M. Alvala, G. B. Reddy and S. Ramakrishna, Apoptosis,
2014, 19, 1148.
This journal is © The Royal Society of Chemistry 20xx
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TABLE OF CONTENTS
TEXT
Umbelliferone-oxindole hybrids as novel apoptosis inducing agents
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