Design, synthesis, and anticancer properties of novel benzophenone- conjugated coumarin analogs

Article abstract of DOI:10.1002/ardp.201300298

In the current scenario, development of anticancer drugs with specific targets is of prime importance in modern chemical biology. Observing the importance of benzophenone and coumarin nucleus, it would be worthwhile to design and synthesize novel benzophenone derivatives (8a-o) bearing the coumarin nucleus. Further, they were screened for prospective anticancer activities in vitro against the Michigan Cancer Foundation-7 (MCF-7) and Ehrlich's ascites tumor (EAT) cell lines and their biomarkers, followed by in silico studies regarding phosphoinositide 3-kinase (PI3K) and caspase by molecular docking. Benzophenones have been reported as potential drugs targeting tumor angiogenesis; thus, the formation of neovessels in an in vivo model system like CAM, which is angiogenesis dependent, was observed in the presence of compounds 8a-o. The above findings would help in understanding their putative potential as therapeutic agents for cancer patients. Coumarin-integrated benzophenone conjugates (8a-o) were designed, synthesized, and screened for prospective anticancer activities in vitro against the MCF-7 and EAT cell lines. Molecular docking studies with regard to phosphoinositide 3-kinase and caspase were performed. In addition, neovessel formation was observed in an in vivo model system.

Full text of DOI:10.1002/ardp.201300298

Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
901
Full Paper
Design, Synthesis, and Anticancer Properties of Novel
Benzophenone-Conjugated Coumarin Analogs
V. Lakshmi Ranganatha1, Farhan Zameer2, S. Meghashri3, N. D. Rekha4, V. Girish1,
H. D. Gurupadaswamy1, and Shaukath Ara Khanum1
1
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore, Karnataka, India
Mahajana Life Science Research Laboratory, Department of Biotechnology, Microbiology and Biochemistry,
2
Mahajana Research Foundation, Pooja Bhagavat Memorial Mahajana Post-Graduate Centre, Affiliated to
University of Mysore, Metagalli, Mysore, Karnataka, India
Pronet Informatics Pvt. Ltd, Bangalore, Karnataka, India
Department of Studies in Biotechnology, JSS College of Arts, Commerce and Science, Mysore, Karnataka,
3
4
India
In the current scenario, development of anticancer drugs with specific targets is of prime importance
in modern chemical biology. Observing the importance of benzophenone and coumarin nucleus, it
would be worthwhile to design and synthesize novel benzophenone derivatives (8a–o) bearing the
coumarin nucleus. Further, they were screened for prospective anticancer activities in vitro against
the Michigan Cancer Foundation-7 (MCF-7) and Ehrlich’s ascites tumor (EAT) cell lines and their
biomarkers, followed by in silico studies regarding phosphoinositide 3-kinase (PI3K) and caspase by
molecular docking. Benzophenones have been reported as potential drugs targeting tumor
angiogenesis; thus, the formation of neovessels in an in vivo model system like CAM, which is
angiogenesis dependent, was observed in the presence of compounds 8a–o. The above findings would
help in understanding their putative potential as therapeutic agents for cancer patients.
Keywords: Anticancer agents / Benzophenone / CAM / Caspase / Coumarin / PI3K / Structure–activity relation (SAR)
Received: August 10, 2013; Revised: September 1, 2013; Accepted: September 5, 2013
DOI 10.1002/ardp.201300298
Introduction
In continuation of our previous research toward the
development of antiangiogenic as well as anticancer drugs
[3–6], more specifically benzophenone derivatives, [2-(4-
methoxy-benzoyl)-4-methylphenoxy]-N-(4-chloro-phenyl)-acet-
amide (BP-1, IC50: 42.50 mM) was known to inhibit angio-
genesis. It prevents the angiogenesis-dependent disorders
like mammary carcinoma and rheumatoid arthritis both
in vivo and in vitro, where it down-regulated the vascular
endothelial growth factor (VEGF) gene expression, which is
responsible for angiogenesis. Herein, we report the synthesis
of potential antiangiogenic molecules by combining two
bioactive molecules like benzophenone and coumarin
collectively through amide linkage to achieve pharmacologi-
cally efficacious single molecules. In order to develop novelty
in cancer therapy, recently our group developed new
benzophenone-integrated oxadiazole analogs as potent anti-
cancer drugs [6]. Developing angiogenesis inhibitors is a
desirable anticancer target, which minimizes the side effects
caused by the chemotherapy. Specificity in selecting tumor
Cancer, the second cause of mortality in the world, is
continuing to be a major health hazard in developing as well
as in undeveloped countries [1]. Design and development of
anticancer drugs with fewer or no side effects are important
for the treatment of cancer. The search for such potential
anticancer drugs has led to the discovery of synthetic
molecules with anticarcinogenic activity. Therefore cancer
has become a major challenge to mankind [2], and it has
opened up myriad new avenues for advance drug design and
discoveries.
Correspondence: Dr. Shaukath Ara Khanum, Department of Chemistry,
Yuvaraja’s College, University of Mysore, Mysore 570 005, Karnataka,
India.
E-mail: shaukathara@yahoo.co.in
Fax: þ91 821 2419239
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
endothelial cells and neovessel formation as a target by drugs
is their unique property, which could be developed into
potential antiangiogenic drug for cancer therapeutics [7, 8].
Moreover, benzophenones are a class of compounds
obtained from natural source [9] or by synthetic methods [10].
. They are of great importance fundamentally due to their
diverse biological and chemical properties. Benzophenones
exhibit significant antitumor activity both in vitro and
in vivo [11]. In addition, synthetic benzophenones, such as
2-aminobenzophenone [12] and dihydroxy-4-methoxy benzo-
phenone [13], have proven to be antimitotic and anticancer
agents, respectively. Recently, para-methoxy substituted
benzophenones were evaluated as p38a inhibitors with
high efficacy and selectivity (IC50: 14 nM) [14]. Amino
and methoxy substituted benzophenones have been
reported to be potent cytotoxic agents against a panel of
human cancer cell lines including multi-drug-resistant
(carbonyl) stretching band for ester group and in proton NMR,
the disappearance of broad singlet for OH proton of
substituted phenols 1a–c indicate the formation of com-
pounds 3a–o. Fries rearrangement of compounds 3a–o gave
hydroxy benzophenones 4a–o, which were confirmed by the
disappearance of O–C stretching band and appearance of O–H
stretching band in IR spectra and by the appearance of broad
singlet for OH proton and decrease in one aromatic proton in
proton NMR. Compounds 4a–o on etherification with chloro
ethylacetate gave substituted ethyl esters 5a–o, which was
confirmed by disappearance of O–H stretching band and
–
appearance of O C (carbonyl) stretching band for ester group
–
in IR absorption spectra. The proton NMR observations reveal
the disappearance of broad singlet for OH proton and
appearance of triplet and quartet for CH3 and CH2 protons,
respectively. The substituted ethyl esters 5a–o on treatment
with 99% hydrazine hydrate yield (4-benzoyl phenoxy)-aceto
hydrazides 6a–o, which was clearly evident with the
appearance of N–H and NH2 stretching bands in IR spectra.
In proton NMR, the appearance of NH and NH2 protons and
disappearance of triplet and quartet peaks for CH3 and CH2
protons confirm the formation of product. Finally all the
substituted aceto hydrazides, on treatment with coumarin-3-
carboxylic acid (7) in the presence of TBTU and lutidine as a
coupling reagent, deliver the expected final products 8a–o in
a good yield. These final products are more assertively
confirmed by NMR, IR, mass, and CHN analyses by concurring
the expected data with experimentally determined data
indistinguishably.
cell lines [15]. Analogs of benzophenone exhibit
a
selective toxicity for proliferating endothelial cells by
induction of apoptosis [16], and polyprenylated benzo-
phenone derivatives are also able to induce caspase-
mediated apoptosis [17].
In addition, coumarin derivatives have been found to
exhibit a wide range of biological and controlled therapeutic
activities in view of their extensive occurrence and also due to
low toxicity [18]. Furthermore, they are present in natural and
synthetic compounds possessing biological activity, effective
and specific at different stages of cancer formation. Some of
them have cytostatic properties and the others have cytotoxic
activity [19]. A few naturally occurring coumarins have been
found to exhibit cytotoxicity against a panel of mammalian
cancer cell lines [20].
Pharmacology
Preliminary screening of compounds 8a–o
In continuation of our present and previous work, further
motivated by the above-mentioned biological properties, an
effort was made to achieve new coumarin-conjugated
benzophenone derivatives. Herein, we report the synthesis,
angiogenesis, and anticancer activity of new coumarin
incorporated benzophenones on Michigan Cancer Founda-
tion-7 (MCF-7) and Ehrlich’s ascites tumor (EAT) cancer cell
lines and also caspase and phosphoinositide 3-kinase (PI3K)
enzyme regulation pathways. Further, this study was also
supported by in silico molecular docking. Hence, the results of
this study will hold promising pavement for developing a
safer alternative to the existing anticancer agents.
In the present study, the cytotoxic effect of compounds 8a–o
was investigated on MCF-7 breast cancer cell lines following
48-h exposure using Trypan blue and (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT). This method is
opted as a first target screening method to eliminate those
analogs that do not show any cytotoxic effects. In this
screening, tamoxifen was used as a standard and dimethyl
sulfoxide (DMSO) as negative control, and the results are
reported in Table 1. In the Trypan blue assay, the analogs 8d,
8l, and 8o reveal excellent cell growth inhibition when
compared to the standard with IC50 of 7.7, 9.4, 10.4, and
11.4 mM, respectively. Furthermore, the effect of compounds
8a–o on cell proliferation was tested using MTT assay. The
results observed in MTT assay were concurrent with Trypan
blue (Table 1). The compounds 8d, 8l, and 8o showed
maximum cytotoxicity with an IC50 value of 7.6, 9.3, and 10.2,
respectively, followed by standard tamoxifen ꢀ11.4 mM. It is
obvious from the data that compound 8d with bromo group
at ortho position of phenyl ring exhibited highest activity
against MCF-7 cells in Trypan blue and MTT assay with the IC50
Results and discussion
Chemistry
Synthesis of the title compounds 8a–o was accomplished by a
synthetic procedure as shown in Scheme 1. All the synthesized
compounds were confirmed by IR and proton NMR data. In IR
–
spectra of benzoylated products 3a–o, the appearance of O C
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
Benzophenone-Integrated Coumarin Analogs as Novel Anticancer Agents
903
R4
R3
OH
OH
R1
R
COCl
R2
R1
R
R2
10% NaOH
AlCl3
_
R2
O
O
R
+
°
0 C, Stirring, 3 h
R3
150 170°C
R3
R4
R1
O
R4
_
_
_
_
1a c
2a l
3a o
4a o
g: R2=R3=H, R4=OCH3
h: R2=R3=H, R4=CH3
i: R3=R4=H, R2=CH3
j: R2=R4=H, R3=CH3
k:R2=R3=H, R4=I
a: R2=R3=R4=H
a: R1=H, R=CH3
b: R1=Cl, R=F
c: R=R1=CH3
e
on
cet
Reflux
8 10 h
A
ry
D
K C
O3
b: R3=R4=H, R2=Cl
c: R2=R4=H, R3=Cl
d: R3=R4=H, R2=Br
e: R2=R3=H, R4=Br
f: R2=R3=H, R4=F
2
_
ClCH2COOC2H5
l: R3=H, R2=R4=Cl
O
O
O
H
N
O
H2N
O
R1
R
O
R1
R
NH2-NH2.H2O
OH
Alcohol/Stirring
R2
_
R2
at rt, 2 3 h
+
O
O
R3
R3
O
O
Coumarin carboxylic acid
7
_
_
5a o
6a o
R4
R4
Stirring
12 h at rt
TBTU/LUTIN
DRY DCM
a: R1=R2=R3=R4=H, R=CH3
b: R1=R3=R4=H, R=CH3, R2=Cl
c: R1=R2=R4=H, R=CH3, R3=Cl
d: R1=R3=R4=H, R=CH3, R2=Br
e: R1=R2=R3=H, R=CH3, R4=Br
f: R1=R2=R3=H, R=CH3, R4=F
R4
R3
R2
g: R1=R2=R3=H, R=CH3,R4=OCH3
h: R1=R2=R3=H, R=CH3, R4=CH3
i: R1=R3=R4=H, R=CH3, R2=CH3
j: R1=R2=R4=H, R=CH3, R3=CH3
k: R2=R3=H, R=Cl, R1=F, R4=F
l: R2=R3=H, R=Cl, R1=F, R4=CH3
m: R2=R3=H, R=Cl, R1=F, R4=I
n: R2=R3=R4=H, R=R1=CH3
R1
O
O
O
H
N
N
H
O
R
O
O
o: R1=R3=H, R=CH3, R2=R4=Cl
_
8a o
Scheme 1. Synthesis of benzophenone-conjugated coumarin analogs.
values of 7.7 and 7.6 mM when compared with the standard
drug with an IC50 of 11.4 mM, followed by moderate activity of
9.4 and 9.3 mM, 10.4 and 10.2 mM by compounds 8l with
chloro and fluoro groups at ortho position of phenoxy ring and
methyl group at para position of benzoyl ring and 8o with two
chloro groups at ortho position of phenoxy ring, respectively.
In order to elucidate the mode of action of cell death, the best
three synthesized analogs 8d, 8l, and 8o were evaluated for
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
Table 1. IC50 values of compounds 8a–o calculated based on
Trypan blue and MTT assay at 48 h in MCF-7 cells.
their effects on homogeneous caspase and PI3-kinase in MCF-7
cell line. Hence, the compounds 8d, 8l, and 8o were selected
as lead compounds and further investigated.
Trypan blue assay
MTT assay
Compounds
IC50 value (mM)
IC50 value (mM)
Compounds 8d, 8l, and 8o considered as potent
molecules
Angiogenesis in CAM model
Control
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
–
–
98 ꢁ 0.11
29 ꢁ 0.19
69 ꢁ 0.13
7.7 ꢁ 0.09
>100
46 ꢁ 0.15
75 ꢁ 0.18
>100
57 ꢁ 0.14
72 ꢁ 0.17
20 ꢁ 0.10
9.4 ꢁ 0.02
>100
80 ꢁ 0.20
10.4 ꢁ 0.08
11.4 ꢁ 0.06
>100
26 ꢁ 0.18
71 ꢁ 0.14
7.6 ꢁ 0.10
>100
48 ꢁ 0.17
73 ꢁ 0.13
>100
56 ꢁ 0.13
72 ꢁ 0.15
21 ꢁ 0.13
9.3 ꢁ 0.04
>100
84 ꢁ 0.21
10.2 ꢁ 0.12
11.4 ꢁ 0.09
To investigate the antiangiogenic activity of compounds 8a–o
in in vivo models, all the compounds 8a–o were subjected to
CAM angiogenesis assay. In this model, the compounds 8a–o
induced moderate-to-good avasculture zone formation in
the developing embryos. Among all the tested compounds,
compounds 8d, 8l, and 8o more clearly showed regression of
newly formed microvessels, which were found around the
area of disk implanted, compared to the other compounds in
the series (Fig. 1A). This regression of microvessels density in
the CAM model is highly evident for the tumor inhibition and
it correlates to our in vitro results more accurately.
8m
8n
8o
Std
Giemsa and ethidium bromide/acridine orange staining
To study the morphological features of the cells, we subjected
the cells to Giemsa and ethidium bromide/acridine orange
The IC50 values of compounds 8d, 8l, and 8o (values in bold)
were very significant; thus, 8d, 8l, and 8o were chosen as lead
compounds.
Figure 1. Angioprevention effect of compounds 8d, 8l, and 8o. (A) CAM photos illustrate the formation of blood vessels in normal cells
followed by regression in cells treated with compounds 8d, 8l, and 8o. Inhibition of angiogenesis is evident. (B) Giemsa staining of normal cells
and cells treated with compounds 8d, 8l, and 8o highlights the apoptotic morphology of the cells. (C) Representative photographs clearly
indicating apoptotic morphology of cells treated with compounds 8d, 8l, and 8o and stained with ethidium bromide/acridine orange stain.
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Benzophenone-Integrated Coumarin Analogs as Novel Anticancer Agents
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staining. In this, experimental batches of both test and
control smears were stained with Giemsa’s stain and acridine
orange/ethidium bromide stain that highlights the apoptotic
morphology of the cells, when observed under florescent
microscope (Fig. 1B and C). The apoptotic morphology clearly
indicated the potential of the compounds 8d, 8l, and 8o,
respectively, and the same were photographed.
DNA fragmentation assay
DNA fragmentation was performed for elucidating the mode
of action of the investigated compounds, especially with
respect to induction of oligonucleosomal DNA fragmentation
(DNA ladder), which is a characteristic feature of the
programmed cell death or apoptosis. Apoptotic degradation
of DNA was analyzed by agarose gel electrophoresis. The
fragmented DNA observed under UV light clearly demon-
strated the in vivo apoptotic effect of compounds 8d, 8l, and
8o. In the clearly documented Bio-RAD image (Fig. 2), lane 1
indicates control, lane 2 indicates the marker, and lanes 3–5
indicate the apoptotic effect of compounds 8d, 8l, and 8o,
respectively. These results have greater resemblance to our
other in vitro and in vivo results. Further by observing these
facts, we can conclude that the compounds 8d, 8l, and 8i had
more proapoptotic properties when compared to all other
analogs in the series.
Figure 3. Title compounds activate homogeneous caspase in
MCF-7 cells: 1 ꢂ 104 cells/well with 70–80% confluence were
grown for 24 h, treated with 5 mM of each compounds. Flurometric
determination of general caspase was done as described in
Materials and Methods section with an excitation filter of 465 nm
and emission filter of 520 nm (ꢃp < 0.05 vs. negative control).
coumarin analogs, which induced apoptosis via caspase
activation. These results were further confirmed by in silico
analysis for looking into structure–activity relationship for
which the three major caspases, namely 1, 3, and 8, were
considered and molecular docking was carried out.
In the mean time, all the conjugates were tested for PI3K
enzyme inhibition in MCF-7 cells treated at a concentration of
5 mM. This also exhibited the promising results, indicating
the potency of the inhibitors used. As mentioned earlier,
PI3-kinases have a pivotal role in signaling pathways involved
in oncogenic transformation and intracellular protein
trafficking. The benzophenone-integrated coumarin deriva-
tives induced apoptosis via PI3K triggering. Mainly the
compounds 8d, 8l, and 8o showed significant inhibition
against PI3K (Fig. 4). An increased activity in MCF-7 cells by all
the three compounds 8d, 8l, and 8o is outstanding.
Caspase and PI3-kinase activation assay
In order to elucidate the mode of action of cell death, the best
three synthesized analogs 8d, 8l, and 8o were evaluated
for their effects on homogeneous caspase and PI3-kinase in
MCF-7 cells. The result indicates that the homogeneous
caspase activity was increased in MCF-7 cells by these three
compounds. The caspase activity upon treatment of respective
compounds was significantly high compared to untreated
control, indicating that all these compounds activated
general caspases (Fig. 3) indicating benzophenone-integrated
In silico modeling of compounds 8d, 8l, and 8o with
caspase and PI3-kinase enzymes
Molecular docking of homogeneous caspase results revealed
the atomic contact energy (ACE) and amino acid binding
residues, which are depicted in Table 2. This analysis
Figure 2. DNA fragmentation assay. The fragmented DNA
observed under UV light clearly demonstrated the in vivo apoptotic
effect of the compounds. Lanes 1 and 2 are control and marker,
lanes 3–5 are compounds 8d, 8l, and 8o, respectively.
Figure 4. Enzyme inhibition of PI3K in MCF-7 cells treated at a
concentration of 5 mM.
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Table 2. Molecular docking results exhibiting atomic contact energy (ACE) values and amino acid residues binding pockets for analyzed
caspases against the synthesized ligand molecules.
Caspase-1 (PDB: 2HBZ)
Amino acid residues
Caspase-3 (PDB: 4DCP)
Amino acid residues
Caspase-8 (PDB: 1QDU)
Amino acid residues
Compound
8d
ACE
ACE
ACE
ꢄ163.29
Glu 250
Ser 339
Cys 285
Arg 341
Gln 142
Ile 152
ꢄ212.44
Arg 207
Glu 123
Cys 163
ꢄ83.01
Lys 253
Asp 319
Cys 360
Asp 363
Asp 455
Gln 423
Lys 457
Asp 319
Cys 360
Asp 363
Asp 319
Cys 360
Ser 411
8l
ꢄ131.23
ꢄ117.91
ꢄ235.58
ꢄ195.39
ꢄ212.27
ꢄ242.14
Glu 123
Cys 163
ꢄ77.37
ꢄ138.76
ꢄ217.26
8o
Std
Lys 158
Gln 142
Lys 82
Asp 228
Lys 224
Cys 163
Tyr 204
Arg 207
Ile 176
Cys 285
Asp 288
along with previous studies on caspases proves that they
are cysteine-dependent aspartate-directed proteases that
mediate programmed cell death (apoptosis) and involve both
the extrinsic and intrinsic pathways, among which caspase 1
is an interleukin-1 beta converting enzyme (ICE) homolog.
Caspase 8 is an initiator caspase, which forms part of the
death-inducing signaling complex (DISC) once a death signal
is received by the cell. The formation of this complex allows
for the autocatalytic cleavage of procaspases into active
caspases. Activated caspases allow for the activation of
the effector caspases, including caspase 3. The SAR also
supports the above data, which is evident by the docking
results and the docked amino acid pockets (Fig. 5) mainly
indicating the presence of Cys-Asp-Glu-Gln residues. Taken
Figure 5. Molecular docking of human cas-
pase 1, 3, and 8 receptor with ligand 8d (A–C),
with 8l (D–F), with 8o (G–I), and with standard
tamoxifen (J–L) indicating site of dock of the
ligand to amino acid residues.
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Benzophenone-Integrated Coumarin Analogs as Novel Anticancer Agents
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Table 3. Molecular docking results exhibiting ACE values, amino acid residues binding pockets for analyzed human PI3K against the
synthesized ligand molecules.
ACE
Compound
values
Amino acid residues
Domain
Function
PI3K þ 8d
ꢄ434.28
Val 540, His 541, Arg 542
PI3K_C2 superfamily
Chemotaxis and interference in PI3K metabolism
and ATP binding activity in cytosol and membrane
bound region
PI3K þ 8l
PI3K þ 8o
ꢄ428.35
ꢄ417.53
Val 540
Trp 539, Arg 542, Phen 545
PI3K_C2 superfamily
PI3K_C2 superfamily
ATP binding activity
Interference in small molecule metabolic process a
nd PI3K signaling pathways
PI3K þ Std
ꢄ408.22
Val 540, His 541
PI3K_C2 superfamily
ATP binding activity and chemotaxis
Our docking region: 506–629, indicating PI3K_C2 super family.
together, the above results demonstrate that the synthesized
benzophenone-integrated coumarin analogs have potential
anticancer properties and are mainly directed by caspase-
induced apoptosis mechanism.
at respective positions of the main skeleton of compounds 8a–
o may enhance the biological activity.
Conclusion
In the meantime, the in silico model of compounds 8d, 8l,
and 8o was studied and the results of molecular docking with
ACE values with PI3K inhibition are tabulated in Table 3. The
docking studies and compound microscopic images further
strengthen the evidence to the in vitro results. Here the
docking results of compounds 8d, 8l, and 8o are extremely
similar to that of standard tamoxifen with amino acid packets
(Fig. 6). The above result shows that the different substitutions
In order to develop novel anticancer molecules, we have
designed and synthesized new bioactive benzophenone-
integrated coumarin analogs (8a–o) and evaluated them for
anticancer properties. The anticancer activity of these
compounds was tested against MCF-7 cells by different
methods. Compounds 8d, 8l, and 8o exhibited potent
anticancer properties among the series. In silico results are
also concordant with in vitro and in vivo results more
accurately. Further in vivo investigations are in progress in
our laboratory. By observing the present results, the
compounds 8d, 8l, and 8o can be considered as potent
anticancer drugs for future chemotherapy.
Experimental
All solvents and reagents were purchased from Sigma–Aldrich
Chemicals Pvt Ltd. Melting points were determined on an
electrically heated VMP-III melting point apparatus. The elemen-
tal analysis of the compounds was performed on a Perkin Elmer
2400 elemental analyzer. The FT-IR spectra were recorded using
KBr disks and Nujol on FT-IR Jasco 4100 infrared spectrophotom-
eter. The 1H NMR spectra were recorded using Bruker DRX 400
spectrometer at 400 MHz with TMS as the internal standard.
Mass spectra were recorded on LC–MS/MS (API-4000) mass
spectrometer.
Synthesis and characterization
The reaction sequence for different title compounds (7a–o) is
outlined in Scheme 1. The starting materials substituted phenyl
benzoates (3a–o) were synthesized according to a reported
procedure [21] through the reaction of substituted phenols
(1a–c) with substituted acid chlorides (2a–l) in the presence of 10%
sodium hydroxide. The phenyl benzoates (3a–o) on subjecting to
Fries rearrangement afforded hydroxy benzophenones (4a–o).
Ethyl (2-aroyl-4 methylphenoxy) acetates (5a–o) were achieved
in excellent yield by reacting compounds 4a–o with ethyl
Figure 6. (A–D) Molecular surface of docking in the order Std,
compounds 8d, 8l, and 8o. (E–H) Amino acid pockets and (I–M) are
the compound microscopic images, which indicates the effect of
5 mM concentration of potent inhibitors starting from untreated
cells, Std, compounds 8d, 8l, and 8o, respectively.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
ꢄ1
–
chloroacetate in the presence of anhydrous potassium carbonate
and dry acetone. Compounds 5a–o treated with 99% hydrazine
hydrate gave different hydrazides (6a–o). Finally, compounds
6a–o on coupling with coumarin 3-carboxylic acid (7) in the
presence of O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyl uronium
tetrafluoroborate (TBTU) and 2,6-dimethyl pyridine (lutidine) as a
coupling agent furnished 8a–o in excellent yield.
5a: Yield 90%. m.p. 49–52°C; IR (Nujol): 1664 (C O), 1760 cm
–
1
–
(ester, C O); H NMR (CDCl ): d 1.2 (t, 3H, CH of ester), 2.3 (s, 3H,
–
3
3
CH3), 4.1 (q, 2H, CH2 of ester), 4.5 (s, 2H, OCH2), 7.1–7.7 (m, 8H,
Ar–H). Anal. calcd. for C18H18O4 (298): C, 72.48; H, 6.04. Found:
C, 72.46; H, 6.02%.
General procedure for the preparation of (4-benzoyl
phenoxy)aceto hydrazides (6a–o)
General procedure for the preparation of phenyl benzoates
(3a–o)
To compounds 5a–o (0.01 mol) in ethanol (10 mL), 99% hydrazine
hydrate (0.01 mol) was added dropwise and continuously stirred
for 2 h at room temperature to achieve white solid compounds
6a–o. Solid was recrystallized with methanol to get pure products
(6a–o). Compound 6a is taken as a representative example to
explain characterization data [24].
Substituted benzoates (3a–o) were synthesized by benzoylation of
substituted phenols (1a–c) with corresponding benzoyl chlorides
(2a–l, 1:1) using 10% sodium hydroxide solution. The reaction
mass was stirred for 2–3 h at 0°C. Reaction was monitored by thin
layer chromatography (TLC) using 4:1 n-hexane/ethyl acetate
solvent mixture. After completion of the reaction the oily
product was extracted with ether layer. Ether layer was washed
with 10% sodium hydroxide solution (3 ꢂ 50 mL) followed by
water (3 ꢂ 30 mL) and then dried over anhydrous sodium
sulfate and the solvent was evaporated under pressure to afford
compounds 3a–n. Compound 3a is taken as a representative
example to explain characterization data [21].
–
6a: Yield 80%. m.p. 125–128°C; IR (Nujol): 1630 (C O), 1670
–
ꢄ1
1
–
(amide, C O), 3120–3220 cm (NH NH2); H NMR (CDCl3): d 2.2
–
(s, 3H, Ar–CH3), 4.35 (bs, 2H, NH2), 4.63 (s, 2H, CH2), 6.96–7.68
(m, 8H, Ar–H), 9.30 (bs, 1H, CONH). Anal. calcd. for C16H16N2O3
(285): C, 67.59; H, 5.67; N, 9.85. Found: C, 67.65; H, 5.74; N, 9.91%.
General procedure for the preparation of 2-oxo-2H-
chromene-3-carboxylic acid N0-[2-(4-benzoyl-2-methyl-
phenoxy)-acetyl]-hydrazide derivatives (8a–o)
3a: Yield 90%. Pale yellow liquid; IR (Nujol): 1715 cmꢄ1 (C O);
–
–
1H NMR (CDCl3): d 2.45 (s, 3H, Ar–CH3), 7.0–8.2 (m, 9H, Ar–H). Anal.
calcd. for C14H12O2 (212): C, 79.22; H, 5.70. Found: C, 79.18;
H, 5.76%.
To the mixture of compounds 6a–o (0.0037 mol) in dry dichloro-
methane (15 mL), lutidine (1.2 vol.) was added at 25–30°C
followed by coumarin 3-carboxylic acid (7, 0.0037 mol) and
stirred at 25–30°C for 30 min. The reaction mass was cooled to
0–5°C and TBTU (0.0037 mol) was added in lots over a period of
30 min by maintaining temperature below 5°C. The reaction
mass was stirred overnight and monitored by TLC using
chloroform/methanol (9:1). The reaction mass was diluted with
20 mL of dichloromethane followed by 1.5 N HCL solution (20 mL).
The organic layer was washed with water (25 mL), dried over
anhydrous sodium sulfate, concentrated to syrupy liquid, and
recrystallized twice by diethyl ether to afford compounds 8a–o.
General procedure for the preparation of (4-hydroxy
phenyl)phenyl-methanones (4a–o)
Substituted (4-hydroxy phenyl)phenyl-methanones commonly
known as hydroxy benzophenones (4a–o) were synthesized by
Fries rearrangement. Compounds 3a–o (0.001 mol) were treated
with anhydrous aluminum chloride (0.002 mol) as a catalyst at
150–170°C without solvent for about 2–3 h. Then the reaction
mixture was cooled to room temperature and quenched with
6 N HCl in the presence of ice water. The reaction mixture
was stirred for about 2–3 h; solid was filtered and recrystallized
with methanol to obtain compounds 4a–o. Compound 4a is
taken as a representative example to explain characterization
data [22].
2-Oxo-2H-chromene-3-carboxylic acid N0-[2-(4-benzoyl-2-
methyl-phenoxy)acetyl]-hydrazide
–
–
8a: Yield 85%. m.p. 190–193°C; IR (Nujol): 1630 (C O), 1670
ꢄ1
(amide C O), 3120–3220 cm (NH–NH); 1H NMR (CDCl3): d 2.2
–
4a: Yield 72%. m.p. 110–112°C; IR: (Nujol) 1640 (C O), 3510–
–
–
–
3600 cmꢄ1 (OH); 1H NMR (CDCl3): d 2.35 (s, 3H, CH3), 6.71–7.70
(m, 8H, Ar–H), 12.0 (bs, 1H, –OH). Anal. calcd. data for C14H12O2
(212.08): C, 79.22; H, 5.70. Found: C, 72.23; H, 5.69%.
(s, 3H, Ar–CH3), 4.8 (s, 2H, CH2), 7.0–8.0 (m, 12H, Ar–H), 8.9 (s, 1H,
coumarin Ar–H), 10.5–10.9 (d, 2H, –NHNH); MS: m/z 476. Anal.
calcd. for C26H20N2O6: C, 68.42; H, 4.42; N, 6.14. Found: C, 68.45;
H, 4.44; N, 6.19%.
General procedure for the preparation of ethyl(4-benzoyl
phenoxy)aceto esters (5a–o)
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(2-chloro-
Compounds 5a–o were obtained by refluxing a mixture of
compounds 4a–n (0.013 mol) and ethyl chloroacetate (0.026 mol)
in dry acetone (50 mL) and anhydrous potassium carbonate
(0.019 mol) for 8–9 h. The reaction mixture was cooled and solvent
was removed by distillation. The residual mass was triturated
with cold water to remove potassium carbonate and extracted
with ether (3 ꢂ 50 mL). The ether layer was washed with 10%
sodium hydroxide solution (3 ꢂ 50 mL) followed by water
(3 ꢂ 30 mL) and then dried over anhydrous sodium sulfate
and evaporated to dryness to obtain crude solid, which, on
recrystallization with ethanol, afforded compounds 5a–n.
Compound 5a is taken as a representative example to explain
characterization data [23].
benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8b: Yield 88%. m.p. 200–202°C; IR (Nujol): 1620 (C O), 1675
–
ꢄ1
(amide C O), 3140–3250 cm (NH–NH); 1H NMR (CDCl3): d 2.4
–
–
(s, 3H, Ar–CH3), 4.6 (s, 2H, CH2), 7.1–8.0 (m, 11H, Ar–H), 8.8 (s, 1H,
coumarin Ar–H), 10.6–10.9 (d, 2H, –NHNH); MS: m/z 511. Anal.
calcd. for C26H19ClN2O6: C, 63.61; H, 3.90; N, 5.71. Found: C, 63.68;
H, 3.99; N, 5.80%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(3-chloro-
benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8c: Yield 80%. m.p. 180–185°C; IR (Nujol): 1620 (C O), 1675 (amide
–
–
C O), 3140–3250 cm (NH–NH); 1H NMR (CDCl3): d 2.4 (s, 3H,
ꢄ1
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
Benzophenone-Integrated Coumarin Analogs as Novel Anticancer Agents
909
Ar–CH3), 4.6 (s, 2H, CH2), 7.1–8.0 (m, 11H, Ar–H), 8.7 (s, 1H,
coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 491. Anal.
calcd. for C26H19ClN2O6: C, 63.61; H, 3.90; N, 5.71. Found: C, 63.68;
H, 3.97; N, 5.80%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-methyl-4-
(3-methyl-benzoyl)-phenoxy]-acetyl}-hydrazide
–
8j: Yield 80%. m.p. 200–204°C; IR (Nujol): 1625 (C O), 1675 (amide
–
–
C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.2 (s, 3H,
ꢄ1
–
Ar–CH3), 2.3 (S, 3H, CH3), 4.1 (s, 2H, CH2), 7.3–8.3 (m, 11H, Ar–H),
8.7 (s, 1H, coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 471.
Anal. calcd. for C27H22N2O6: C, 68.93; H, 4.71; N, 5.95. Found: C,
68.99; H, 4.77; N, 5.96%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(2-bromo-
benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8d: Yield 85%. m.p. 202–205°C; IR (Nujol): 1625 (C O), 1675
–
ꢄ1
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.5
–
–
(s, 3H, Ar–CH3), 4.3 (s, 2H, CH2), 7.2–8.1 (m, 11H, Ar–H), 8.9 (s, 1H,
coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 536. Anal.
calcd. for C26H19BrN2O6: C, 58.33; H, 3.58; N, 5.23. Found: C, 58.40;
H, 3.65; N, 5.29%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-chloro-6-
fluoro-4-(4-fluoro-benzoyl)-phenoxy]-acetyl}-hydrazide
–
8k: Yield 78%. m.p. 207–209°C; IR (Nujol): 1625 (C O), 1675 (amide
–
–
C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 4.4 (s, 2H,
ꢄ1
–
CH2), 7.2–8.1 (m, 10H, Ar–H), 8.7 (s, 1H, coumarin Ar–H), 10.4–10.9
(d, 2H, –NHNH); MS: m/z 514. Anal. cald. for C25H15ClF2N2O6: C,
58.55; H, 2.95; N, 5.46. Found: C, 58.59; H, 2.99; N, 5.48%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(4-bromo-
benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8e: Yield 80%. m.p. 210–212°C; IR (Nujol): 1625 (C O), 1675
–
ꢄ1
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.5
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-chloro-6-
–
–
(s, 3H, Ar–CH3), 4.3 (s, 2H, CH2), 7.1–8.0 (m, 11H, Ar–H), 8.6 (s, 1H,
coumarin Ar–H), 10.6–10.9 (d, 2H, –NHNH); MS: m/z 536. Anal.
calcd. for C26H19BrN2O6: C, 58.33; H, 3.58; N, 5.23. Found: C, 58.40;
H, 3.65; N, 5.29%.
fluoro-4-(4-methyl-benzoyl)-phenoxy]-acetyl}-hydrazide
–
8l: Yield 88%. m.p. 175–177°C; IR (Nujol): 1625 (C O), 1675 (amide
–
ꢄ1
C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.5 (s, 3H,
–
–
Ar–CH3), 4.4 (s, 2H, CH2), 7.1–8.2 (m, 10H, Ar–H), 8.7 (s, 1H,
coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 510. Anal.
calcd. for C26H18ClFN2O6: C, 61.37; H, 3.57; N, 5.50. Found: C,
61.42; H, 3.61; N, 5.53%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(4-fluoro-
benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8f: Yield 82%. m.p. 225–228°C; IR (Nujol): 1625 (C O), 1675 (amide
–
–
C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.5 (s, 3H,
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-chloro-6-
ꢄ1
–
Ar–CH3), 4.3 (s, 2H, CH2), 7.1–8.0 (m, 11H, Ar–H), 8.9 (s, 1H,
coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 475. Anal.
calcd. for C26H19FN2O6: C, 65.82; H, 4.04; N, 5.90. Found: C, 65.88;
H, 4.10; N, 5.97%.
fluoro-4-(4-iodo-benzoyl)-phenoxy]-acetyl}-hydrazide
–
8m: Yield 85%. m.p. 235–238°C; IR (Nujol): 1625 (C O), 1675
–
ꢄ1
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 4.2
–
–
(s, 2H, CH2), 7.3–8.4 (m, 10H, Ar–H), 8.5 (s, 1H, coumarin Ar–H), 10.4–
10.9 (d, 2H, –NHNH); MS: m/z 622. Anal. calcd. for C25H15ClFIN2O6: C,
48.37; H, 2.44; N, 4.51. Found: C, 48.42; H, 2.48; N, 4.53%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(4-
methoxy-benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
–
8g: Yield 90%. m.p. 197–199°C; IR (Nujol): 1625 (C O), 1675
2-Oxo-2H-chromene-3-carboxylic acid N0-[2-(4-benzoyl-
–
ꢄ1
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.2
–
–
2,6-dimethyl-phenoxy)-acetyl]-hydrazide
(s, 3H, Ar–CH3), 3.7 (S, 3H, OCH3), 4.3 (s, 2H, CH2), 6.7–7.9 (m, 11H,
Ar–H), 8.6 (s, 1H, coumarin Ar–H), 10.5–10.9 (d, 2H, –NHNH); MS:
m/z 487. Anal. calcd. for C27H22N2O7: C, 66.66; H, 4.56; N, 5.76.
Found: C, 66.70; H, 4.59; N, 5.80%.
–
8n: Yield 82%. m.p. 229–232°C; IR (Nujol): 1625 (C O), 1675
–
ꢄ1
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.2
–
–
(s, 3H, Ar–CH3), 2.3 (S, 3H, CH3), 4.6 (s, 2H, CH2), 7.1–8.2 (m, 11H,
Ar–H), 8.8 (s, 1H, coumarin Ar–H), 10.2–10.8 (d, 2H, –NHNH);
MS: m/z 471. Anal. calcd. for C27H22N2O6: C, 68.93; H, 4.71; N, 5.95.
Found: C, 68.99; H, 4.77; N, 5.96%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-methyl-4-
(4-methyl-benzoyl)-phenoxy]-acetyl}-hydrazide
8h: Yield 85%. m.p. 188–191°C; IR (Nujol): 1625 (C O), 1675
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[4-(2,6-
–
–
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.2
ꢄ1
–
–
dichloro-benzoyl)-2-methyl-phenoxy]-acetyl}-hydrazide
(s, 3H, Ar–CH3), 2.5 (S, 3H, CH3), 4.3 (s, 2H, CH2), 7.3–8.2 (m, 11H,
Ar–H), 8.9 (s, 1H, coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH);
MS: m/z 471. Anal. calcd. for C27H22N2O6: C, 68.93; H, 4.71; N, 5.95.
Found: C, 68.99; H, 4.77; N, 5.96%.
–
8o: Yield 70%. m.p. 215–219°C; IR (Nujol): 1625 (C O), 1675
–
(amide C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.3
ꢄ1
–
–
(s, 3H, Ar–CH3), 4.4 (s, 2H, CH2), 7.4–8.3 (m, 10H, Ar–H), 8.6 (s, 1H,
coumarin Ar–H), 10.2–10.7 (d, 2H, –NHNH); MS: m/z 526. Anal.
calcd. for C26H18Cl2N2O6: C, 59.44; H, 3.45; N, 5.33. Found: C,
59.49; H, 3.50; N, 5.38%.
2-Oxo-2H-chromene-3-carboxylic acid N0-{2-[2-methyl-4-
(2-methyl-benzoyl)-phenoxy]-acetyl}-hydrazide
–
8i: Yield 78%. m.p. 178–180°C; IR (Nujol): 1625 (C O), 1675 (amide
–
–
Biology
C O), 3130–3240 cm (NH–NH); 1H NMR (CDCl3): d 2.2 (s, 3H,
ꢄ1
–
Ar–CH3), 2.4 (S, 3H, CH3), 4.4 (s, 2H, CH2), 7.2–8.2 (m, 11H, Ar–H),
8.8 (s, 1H, coumarin Ar–H), 10.4–10.9 (d, 2H, –NHNH); MS: m/z 471.
Anal. calcd. for C27H22N2O6: C, 68.93; H, 4.71; N, 5.95. Found:
C, 68.99; H, 4.77; N, 5.96%.
Cell culture and in vitro compounds treatment
MCF-7 breast cancer cells were procured from National
Centre for Cell Sciences (NCCS), Pune, India. MCF-7 cells were
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910
V. Lakshmi Ranganatha et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
routinely maintained in DMEM (Sigma, St. Louis, MO, USA)
supplemented with 10% heat-inactivated FCS (Himdeia,
Mumbai). The EAT cells were collected from JSS College of
Pharmacy, Mysore.
and left for l h at 4°C. The supernatant was subjected to
phenol/chloroform/isoamyl alcohol (25:24:1) extraction and
chloroform extraction. DNA was precipitated by adding
1:2 vol. of ice-cold ethanol. The precipitated DNA was
dissolved in 50 mL of TE buffer (pH 8.0). The DNA was digested
with 20 mL/mL of RNase at 37°C for 1 h. The DNA was
quantified and equal concentration of DNA (50 mg) was
resolved on 1.5% agarose gel, viewed under UV light, and
documented using Bio-RAD.
Trypan blue dye exclusion assay
The effect of compounds 8a–o on the viability of MCF-7 breast
cancer cells was determined by Trypan blue dye exclusion
assay [3]. MCF-7 breast cancer cells treated with or without
compounds were harvested and resuspended in 0.4% Trypan
blue and the viable cells were counted using hemocytometer.
IC50 values were estimated after 48 h of treatment.
PI3 kinase assay
The cells were washed with PBS and lysed in lysis buffer.
Protein concentrations were determined by bicinchoninic
acid protein assay. Lysates (400 mg of protein) were incubated
with the substrate at 4°C overnight, followed by further
incubation with protein. After the incubation, PI3K assays
were performed as per the manufacturer’s instructions
(PI3K AlphaScreen Assay kit, Echelon Biosciences). The
proximity of the interaction between the donor and acceptor
was read at a luminescent signal of 600 nm using a plate
reader (Table 2).
MTT assay
The effect of compounds 8a–o on cell proliferation of MCF-7
breast cancer cells was determined by MTT assay as described
previously [3]. Cells treated with or without compounds were
incubated for 48 h. MTT reagent (5 mg/mL) was added and
the color change due to proliferating cells was estimated.
Choriallanotoic membrane (CAM) assay
Inhibition of neovessel formation following the treatment
with compounds 8d, 8l, and 8o (10 mM) in fertilized egg CAM
was analyzed [4] and changes in the microvessel density were
photographed by using Nikon D3200 camera.
Caspase activation assay
Activated caspase was estimated using fluorimetric homoge-
neous caspase assay (Roche) as per manufacturer’s protocol.
Briefly, 1 ꢂ 104 cells/well were seeded in 100 mL DMEM
supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37°C in a
CO2 incubator. After treatment, caspase substrate was added
to pre-diluted incubation buffer and incubated for 2 h at
37°C. The developed fluorochrome was proportional to the
concentration of activated caspase, which was determined
fluorimetrically in the form of free R110 at lmax ¼ 521 nm.
Giemsa and acridine orange/ethidium bromide
staining
EAT cells were obtained from the JSS College of Pharmacy,
Mysore. The cells were centrifuged at 3000 rpm for 5 min
and the packed cells were diluted 1:6 times with phosphate
buffer saline. Two milliliters of the diluted cells were
treated with the synthesized compounds 8d, 8l, and 8o and
incubated for 4 h at 37°C. Untreated EAT cells served as
control. At the end of 4 h, the samples were centrifuged,
smears were made from the cell pellet obtained and fixed with
methanol/acetic acid (3:1), and the morphological features
of the cells were observed using different stains. Batches
of both test and control smears were stained with Giemsa’s
stain and acridine orange/ethidium bromide stain that
highlights the apoptotic morphology of the cells when
observed under bright field microscope and fluorescent
microscope, respectively [25].
Molecular docking
The caspases 1, 3, and 8 and PI3K of human have been
associated with tumorigenesis and apoptotic characteristics
in cancer. The crystal structure of caspases 1, 3, and 8 was
studied, which are responsible for the activation of the
corresponding precursor with PDB: 2HBZ, PDB: 4DC, and
PDB: 1QDU, respectively. Further crystal structure of PI3K
(accession: CAA03853.1) was built using CPH models server
3.0. Energy computations were performed on the molecule
using GROMOS96 implementation of Swiss-PDB viewer.
Electrostatic point charges on the molecules were calculated.
The ligand was docked into the active site using the molecular
docking software PATCH Dock with default parameters.
PATCH Dock is an algorithm for calculating the docking
modes of small molecules into protein-binding sites based
on their shape complementarities. In this, we have used
ChemScore, a scoring function that is derived from regression
against receptor–ligand binding free energies.
DNA fragmentation assay
DNA was isolated from both treated and untreated cells after
the incubation of EAT cells with compounds for 4 h. Reactions
were terminated using ice-cold Hank’s balanced salt solution
(HBSS) and the supernatant was discarded after centrifuga-
tion. Cells were lysed with 50 mM Tris–HCl buffer, pH 8.0, and
0.5% SDS, which was incubated for 30 min at 37°C. The cell
lysate was subjected to 8 M potassium acetate precipitation
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Arch. Pharm. Chem. Life Sci. 2013, 346, 901–911
Benzophenone-Integrated Coumarin Analogs as Novel Anticancer Agents
911
Statistical analysis
[10] F. Karrer, H. Meier, A. Pascual, J. Fluor. Chem. 2000, 10, 381–
384.
All experiments were repeated in triplicates in three indepen-
dent sets. Data are expressed as mean value ꢁ standard deviation
(SD). The comparisons between mean values were tested using
Duncan’s new multiple-range test at a level of pꢅ 0.05.
[11] H. P. Hsieh, J. P. Liou, Y. T. Lin, N. Mahindroo, J. Y. Chang,
Y. N. Yang, S. S. Chern, U. K. Tan, C. W. Chang, T. W. Chen,
C. H. Lin, Y. Y. Chang, C. C. Wang, Bioorg. Med. Chem. Lett. 2003,
13, 101–105.
[12] J. P. Liuo, C. W. Chang, J. S. Song, Y. N. Yang, C. F. Yeh,
H. Y. Tseng, Y. K. Lo, Y. L. Chang, C. M. Chang, H. P. Haiesh,
J. Med. Chem. 2002, 45, 2556–2562.
V. Lakshmi Ranganatha gratefully acknowledges the financial support
provided by the Department of Science and Technology, New Delhi,
under INSPIRE-Fellowship scheme (IF110555). And all authors are
thankful to the Principal, Yuvaraja’s College, University of Mysore, the
Director of Mahajana Research Foundation, Mahajana Education
Society, and Head of the Department of Biotechnology, for their support
and encouragement throughout the execution of this work. Finally
we extend our sincere thanks to Prof. Aravind Balaji for language
corrections.
[13] Y. Nakagawa, T. Suzuki, Chem. Biol. Interact. 2002, 139, 115–
128.
[14] L. Revesz, E. Blum, F. E. Di Padova, T. Buhl, R. Feifel, H. Gram,
P. Hiestand, U. Manning, G. Rucklin, Bioorg. Med. Chem. Lett.
2004, 14, 3601–3605.
[15] M. Schlitzer, M. Bohm, I. Sattler, Bioorg. Med. Chem. 2002, 10,
615–620.
[16] S. Iyer, D. J. Chaplin, D. S. Rosenthal, A. H. Boulares, L. Y. Li,
M. E. Smulson, Cancer Res. 1998, 58, 4510–4514.
The authors have declared no conflict of interest.
[17] K. Balasubramanyam, M. Altaf, A. V. Radhika, V. Swaminathan,
R. Aarthi, P. Parag, T. P. Sadhal Kundu, J. Biol. Chem. 2004, 279,
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