Some new indole-coumarin hybrids; Synthesis, anticancer and Bcl-2 docking studies

Article abstract of DOI:10.1016/j.bioorg.2015.10.001

Hybrid molecules have attracted attention for their improved biological activity, selectivity and lesser side effects profile, distinct from their individual components. In the quest for novel anticancer drug entities, three series of indole-coumarin hybrids - 3-(1-benzyl-1H-indol-2-yl)-2H-chromen-2-ones, 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehydes and 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acids were synthesized. All the synthesized compounds were characterized by spectral techniques like IR, ¹H NMR, ¹³C NMR, mass spectrometry and elemental analysis. In silico docking studies of synthesized molecules with apoptosis related gene Bcl-2 that is recognized to play an important role in tumerogenesis were carried out. Dose-dependent cytotoxic effect of the compounds in human breast adenocarcinoma (MCF-7) and normal cell lines were assessed using MTT assay and compared with that of the standard marketed drug, Vincristine. Compound 4c had a highly lipophilic bromine substituent capable of forming halogen bond and was identified as a potent molecule both in docking as well as cytotoxicity studies. Flow cytometric cell cycle analysis of 4c exhibited apoptotic mode of cell death due to cell cycle arrest in G2/M phase. Structure activity relationship of these hybrid molecules was also studied to determine the effect of steric and electronic properties of the substituents on cell viability.

Full text of DOI:10.1016/j.bioorg.2015.10.001

Accepted Manuscript
Some new indole-coumarin hybrids; Synthesis, anticancer and Bcl-2 docking
studies
Pooja R Kamath, Dhanya Sunil, Abdul Ajees, K.S.R. Pai, Shubhankar Das
PII:
S0045-2068(15)30025-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.10.001
YBIOO 1848
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
24 June 2015
1 October 2015
5 October 2015
Please cite this article as: P.R. Kamath, D. Sunil, A. Ajees, K.S.R. Pai, S. Das, Some new indole-coumarin hybrids;
Synthesis, anticancer and Bcl-2 docking studies, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bioorg.2015.10.001
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Some new indole-coumarin hybrids; Synthesis, anticancer and Bcl-2 docking studies
Pooja R Kamatha, Dhanya Sunila*, Abdul Ajeesb, KSR Paic and Shubhankar Dasd
*aDepartment of Chemistry, Manipal Institute of Technology, Manipal University, 576 104, India.
dhanyadss3@gmail.com
bDepartment of Atomic and Molecular Physics, Manipal University, 576 104, India.
cDepartment of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, 576 104,
India.
dDepartment of Radiation Biology & Toxicology, School of Life Sciences, Manipal University, 576 104, India.
Abstract
Hybrid molecules have attracted attention for their improved biological activity, selectivity and lesser side
effects profile, distinct from their individual components. In the quest for novel anticancer drug entities, three
series of indole-coumarin hybrids - 3-(1-benzyl-1H-indol-2-yl)-2H-chromen-2-ones, 2-(2-oxo-2H-chromen-3-
yl)-1H-indole-3-carbaldehydes and 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acids were synthesized.
1
All the synthesized compounds were characterized by spectral techniques like IR, HNMR, 13CNMR, mass
spectrometry and elemental analysis. In silico docking studies of synthesized molecules with apoptosis related
gene Bcl-2 that is recognized to play an important role in tumerogenesis were carried out. Dose-dependent
cytotoxic effect of the compounds in human breast adenocarcinoma (MCF-7) and normal cell lines were
assessed using MTT assay and compared with that of the standard marketed drug, Vincristine. Compound 4c
had a highly lipophilic bromine substituent capable of forming halogen bond and was identified as a potent
molecule both in docking as well as cytotoxicity studies. Flow cytometric cell cycle analysis of 4c exhibited
apoptotic mode of cell death due to cell cycle arrest in G2/M phase. Structure activity relationship of these
hybrid molecules was also studied to determine the effect of steric and electronic properties of the substituents
on cell viability.
Keywords: Apoptosis. Cytotoxicity. Indole-coumarin. Bcl-2
* Author for correspondence
Dhanya Sunil
Department of Chemistry,
Manipal Institute of Technology,
Manipal-576104, India.
Tel. +91 820 2924412; Email: dhanyadss3@gmail.com

1. Introduction
Breast cancer is the most common cause of cancer deaths in females. The tumour cells will no longer be
sensitive to the signals within the tissue which regulate cellular differentiation, survival, proliferation and cell
death. This will result in accumulation of the cells within the tissue, causing local damage and inflammation [1].
Amongst many therapeutic strategies, chemotherapy produces significant clinical responses. However, these
chemotherapeutic agents have a narrow therapeutic index, exhibit non-specificity and high-systemic toxicity.
Target-selective chemotherapeutics that could reduce off-target binding thereby ensuing side effects remains as
a major challenge.
Heterocyclic compounds exhibit diverse biological activity owing to their unique ability to mimic the
structure of peptides and to bind reversibly to proteins. Amidst them, heterocycles with polycyclic ring system
possess distinct rigid geometry displaying high functional specialization by orienting its substituents in three
dimensional spaces, there by attracting great interest [2]. Indole is a bicyclic heterocyclic scaffold that find
applications in medical therapy due to a variety of valuable biologic activities such as antiviral [3, 4], anti-
inflammatory [5, 6], anti-hyperlipidemic, antihypoglycemic, anti-hypertensive, anti-asthmatic, anti HIV [7],
antidepressant [8] and notably anticancer [9-15] activity. Few marketed anticancer indole derivatives are
vincristine, vinblastine, vinorelbine, vindesine, mitraphylline, cediranib and apaziquone [2]. Coumarin is a
bicyclic heterocycle belonging to benzopyran-2-one family which is constantly drawing interest due to its
versatile pharmacological activities such as anti-HIV [16], anti-alzheimer’s [17], antimalarial [18] and
antioxidant [19]. The cytotoxic potential of coumarin derivatives are also extensively studied [20-23].
A new approach of developing hybrid molecules by combination of different pharmacophores may end up
with compounds having interesting biological profile. Hybrid molecules can be designed to target
simultaneously two different isoenzymes or a receptor and an enzyme to produce potent synergestic effects or as
dual acting drugs. Hence a single molecule having more than one pharmacophore which results in a hybrid
multifunctional entity, where each individual pharmacophore exhibits different modes of action could be more
useful in the treatment of cancer. As indole and coumarin scaffolds have exhibited favorable response as
anticancer agents, these pharmacophores were subjected to structural modifications, to synthesize hybrid
molecules and to investigate their anticancer potency which has been a core interesting strategy for the
researchers.
Vincristine, an antitumor drug bearing indole moiety hinders microtubule polymerization, causes G2/M
arrest and induces Bcl-2 hyperphosphorylation and apoptosis in MCF-7 cells [24, 25]. In accord with our
program to discover potent and selective antagonists that target Bcl-2, indole-coumarin hybrid based small-
molecules were designed and synthesized; molecular modeling and docking studies were carried out and
subsequently the promising molecules were biochemically tested for their anticancer potencies.
2. Materials and methods

2.1. Chemistry
The chemicals used for synthesis and anticancer assays were commercially procured from various
chemical units viz. E. Merck India Ltd, Sigma Aldrich, Himedia and Spectrochem Chemicals Pvt. Ltd. The
progress of reactions and the purity of the synthesized compounds were monitored by thin layer chromatography
using pre coated aluminium sheets with Aluchrosep silica Gel 60/ UV254 and spots were visualized in UV
chamber. Melting points were determined by open capillary method and are uncorrected. IR spectra in KBr
pellets were recorded in Schimadzu FTIR 8400S spectrophotometer. The 1H NMR and 13C NMR were recorded
in a Bruker 300 MHz instrument in DMSO/ CDCl3 using tetramethylsilane as internal standard. Mass spectra
were recorded using an Agilent 6510 series mass spectrometer. The elemental analysis was done in Flash
thermo 1112 series CHN analyser.
Substituted 3-acetyl-coumarin-2-ones (1a-d) were synthesised by Knoevenagel reaction between
different substituted salicylaldehydes and ethyl acetoacetate in presence of catalytic amount of piperidine [26].
Fischer indole synthesis of 1a-d in presence of Eaton’s reagent gave substituted 3-(1H-Indol-2-yl)-chromen-2-
ones (2a-d) [27]. Compounds 2a-d were allowed to undergo benzylation and Vilsmeyer-Haack formylation to
yield substituted 3-(1-benzyl-1H-indol-2-yl)-2H-chromen-2-ones (3a-d) [28] and 2-(2-oxo-2H-chromen-3-yl)-
1H-indole-3-carbaldehydes (4a-d) [29]. The oxidation of 4a-d in presence of potassium permanganate afforded
to give 2-(2-Oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acids (5a-d) [30]. The synthetic scheme for the
preparation of indole-coumarin hybrids is presented in scheme-1.
Scheme-1
2.2. In-silico molecular modelling and docking studies
Vincristine acts as a wedge at the interface of two tubulin molecules and thereby inhibits microtubule
polymerisation. As a consequence, formation of the mitotic spindle of dividing cells cannot occur, which leads
to activation of c-Jun NH2-terminal Kinase (JNK), phosphorylation of Bcl-2, followed by G2/M arrest and
finally apoptosis [31]. In order to understand binding and inhibition of the synthesized hybrid molecules with
Bcl-2 and thereby sensitize cancer cells to apoptosis, in silico docking studies were performed.
The Bcl-2 human sequence (GI: 231632) was obtained from NCBI (www.ncbi.nlm.nih.gov) and the
target structure was selected using protein sequence similarity search (www.ebi.ack.u/pdbe). Crystal structure of
Bcl-2 in complex with a BAX BH3 peptide (PDB ID: 2XA0) was selected due to 100 % sequence identity with
minimum E-value of 2.8e-98 as a target. The Bcl-2 protein is monomeric in it stoichiometry and is present as a
dimer with two chains A and B. Each monomeric unit has a ligand peptide BAX bound to the protein. The
chain A of structure of Bcl-2 PDB ID 2XA0 was selected for docking and the bound peptide was removed from
active site of the receptor. The active site was identified using residue information from PDBsum
(http://www.ebi.ac.uk/pdbsum/).

Structures of the compounds were sketched using MarvinSketch (www.chemaxon.com) and ligand
molecules were converted from 2D to 3D using Open Babel software [32]. Docking study was performed using
AutoDock Vina [33] and input files necessary for AutoDock program were prepared using AutoDock Tools
[34]. The size of the grid box in AutoDock Vina was kept as 25 x 25 x 20 for X, Y, Z and the default setting was
kept for energy range. The automated program yielded nine possible conformations with distinguished binding
energy for each ligand output. The final model was selected based on the binding affinity as well as molecular
contacts. The molecular contacts were calculated using the program CONTACT available in CCP4 suite [35].
Docked complexes were analysed and figures were rendered using PyMOL (www.pymol.org).
2.3. Anticancer studies
The studies to evaluate the anticancer potential of synthesized compounds were carried out in human
breast adenocarcinoma (MCF-7) cells purchased from National Cancer Centre for Cell Science, Pune, India. The
cells were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10 % fetal bovine
serum (FBS) at 37 °C in 5 % CO2 atmosphere.
2.3.1. MTT assay
MTT - based colorimetric cell viability assay is a sensitive in vitro assay for measuring cell
proliferation or cytotoxicity. Exponentially growing cells were harvested from T-25 tissue culture flask and a
stock cell suspension of 1×105 cells / mL was prepared. A sterile 96-well flat bottom tissue culture plate was
seeded with 5×103 cells in 0.1 mL of MEM medium containing 10 % FBS and allowed to attach for 24 h. The
cells were treated with different concentration of test compounds (50-400 µM) in triplicates and incubated for
48 h. The control group cells were treated with medium containing 0.1 % DMSO. MTT reagent (30 µL, 4
mg/mL) was added in to the wells and incubated for 4 h at 37 °C. Medium containing MTT was removed and
the formazan crystals formed in each well were dissolved in 100 µL of DMSO. The absorbance was measured
by an ELISA plate reader at 540 nm [36, 37].
2.3.2. Flow cytometry
Propidium iodide (PI) flow cytometric assay can be used to identify apoptotic cells which are characterized
by DNA fragmentation and consequently the loss of nuclear DNA content. MCF-7 cells (2×106 cells/mL) were
seeded in 6-well plates, treated with 0.5 μM Vincristine and 5 μM test compound. Cells were harvested after 48
h, washed twice with ice-cold PBS and were fixed with 70 % ice-cold ethanol. After centrifugation, 100 mg/L
RNase and 5 g/L PI were added to the cells, and were stained in the dark for 30 min. Detection of cell cycle
distribution was then performed on a BD Accuri™ C6 flow cytometer and related software was used to analyse
the data [38].
3. Results and discussion

3.1. 2-(6-Chloro-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4b) and 2-(6-Bromo-2-oxo-2H-
chromen-3-yl)-1H-indole-3-carbaldehyde (4c) as potential Bcl-2 antagonists
In parallel with biochemical studies, in silico docking was performed to examine the structure of
Bcl-2 and its interaction with all the synthesized compounds. In the analysis, all four 2-(2-oxo-2H-chromen-3-
yl)-1H-indole-3-carbaldehydes molecules are well fit into the active site of Bcl-2 as depicted in Fig 1. The Bcl-2
residues that are proposed to interact with the four ligand molecules are represented in Fig 2. The corresponding
affinity scores and the molecular contacts are summarized in Table 1.
Table 1 Ligand molecules and Bcl-2 docking affinities and residues predicted in silico docking to be with in
4.0Å of each other.
Molecular Contacts**
Affinity*
Compound
(kcal/mol)
Ligand atoms BCL-2 protein atoms Distance ( in 4.0Å)
O12
O12
O12
O13
O22
O22
Gln118 O
Met115 O
Leu119 N
Tyr108 OH
Gln118 NE2
Tyr108 OH
3.95
3.22
3.82
2.63
2.59
2.30
-7.7
4a
O12
O12
Cl
Gln118 NE2
Tyr108 OH
Gln118 O
2.52
2.42
3.79
2.74
2.50
3.46
-6.7
4b
Cl
Arg129 NH1
His120 N
Cl
Cl
His120 O
O12
O12
Br
Gln118 NE2
Tyr108 OH
Gln118 O
2.67
2.36
3.70
2.62
2.58
3.60
-6.4
4c
Br
Arg129 NH1
His120 HN
His120 O
Br
Br
O12
O12
O13
O22
O22
Gln118 NE2
Tyr108 OH
Gln118 NE2
Tyr108 OH
Gln118 NE2
2.34
1.94
3.11
2.37
3.24
-6.3
4d

O23
Gln118 O
3.31
*Results obtained from Autodock vina.
**Results obtained from CONTACT program of CCP4 suite.
Over all, four Bcl-2 amino acids Tyr108, Met115, Gln118 and Leu119 are predicated to be within
4.0 Å of molecule 4a. Similarly compound 4b and 4c are observed to interact with Tyr108, Gln118, His120 and
Arg129 and compound 4d with Tyr108 and Gln118. The Bcl-2 amino acids Tyr108 and Gln118 are the
common residues that interacted with all four compounds and it is interesting to notice that Gln118 makes
strong interactions with the peptide BAX [39]. Halogen atoms are always attractive and play a major role in
biological molecules [40, 41, 7]. In our case, the molecules 4b and 4c contains Cl and Br atoms and are well fit
with in the cavity of Bcl-2 protein as compared to 4a and 4d (Fig2). Moreover, the molecules 4b and 4c interact
with two positively charged amino acids His120 and Arg129 of Bcl-2. This type of interaction is not observed
with either molecule 4a or 4d. As both halogen (chlorine and bromine) atoms have strong interactions with Bcl-
2 protein, these molecules may act as good candidates for further biochemical studies.
Fig.1
Fig.2
3.2. 2-(6-Bromo-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4c) induces cytotoxicity and shows
selectivity in MCF-7 cells
The cytotoxicity induced by the new indole-coumarin hybrids was determined using MTT assay by
analysing dose-response inhibition curves (Graph Pad prism, 4). Cell proliferation was estimated by MTT
reduction by live cells when treated with compounds (2a-d), (3a-d), (4a-d) and (5a-d) on MCF-7 and normal
kidney (VERO) cells. The marketed anticancer drug vincristine was used as the standard drug. The results of
MTT assay in terms of their IC50 values are summarized in Table 2. Neither the substituted 3-(1H-Indol-2-yl)-
chromen-2-ones nor 3-(1-benzyl-1H-indol-2-yl)-chromen-2-ones were active against the two cell lines.
Compounds 4c, 5b and 5c were found to be potent against MCF-7 cell lines. The selectivity index of 4c, 5b and
5c for MCF-7 cells was very high which indicated the high selectivity for these molecules towards MCF-7 cells
than to normal cells.
Table 2 In vitro cytotoxicity data (IC50, µM) for compounds (2a-d), (3a-d), (4a-d) and (5a-d)
Compound IC50 (µM) IC50 (µM) Compound IC50 (µM) IC50 (µM)
MCF-7
0.3
Vero
0.9
MCF-7
73.2
Vero
>100
9.9
4a
4b
4c
Vincristine
>100
>100
>100
>100
>100
7.4
2a
2b
>100

>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4d
5a
5b
5c
5d
48.1
>100
5.5
>100
87.5
15.4
>100
22.0
2c
2d
3a
3b
3c
3d
13.5
>100
The halogen substituted aldehyde and acid showed better activity compared to unsubstituted and
hydroxy substituted compounds. The standard drug vincristine exhibited an IC50 value of 0.3 µM and selectivity
index of 3. Compound 4c was selected for studying the mechanism of cytotoxicity and cell death because of its
inhibitory effect with low IC50 value in MCF-7 cells and its high selectivity index when compared to vincristine.
3.3. 2-(6-Bromo-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4c) affects the cell cycle in G2/M phase
As 4c could effectively inhibit cancer cell growth in vitro, this inhibitory activity may be attributable to
its ability to interfere with the cell cycle. The cell cycle distribution on treatment with 4c was measured by
fluorescence activated flow cytometric cell sorting analysis by PI-labelled cells. The histograms obtained in cell
cycle analysis studies are presented in Fig 3. The standard drug vincristine is found to induce cell cycle arrest at
the G2/M phase by promoting microtubule depolymerization in MCF-7 cell lines. The histogram of vincristine
treated cells was observed on similar lines during our cell cycle analysis studies confirming the previously
published data. Interestingly, a change in the normal cell cycle pattern leading to a cell cycle arrest at the G2/M
phase was observed in MCF-7 cells upon incubation with 4c. Fig 3 shows that 64 % of the cell population was
in the G2/M phase when treated with vincristine, and 46 % on incubation with 4c. Accumulation of apoptotic
cells (21 %) was observed in subG1 phase of the histogram. In contrast, the histogram of control DMSO treated
cells exibited a standard cell cycle pattern with G1 and G2/M peaks seperated by S peak and a cell population of
only 15 % in G2/M phase. The Sub G1 peak that constitutes mostly dead cells was not prominent. Thus, these
results suggest that the inhibitory effect of 4c on MCF-7 cells are due to a G2/M arrest of the cell cycle which
can lead to apoptosis.
Fig. 3
3.4. Structure activity relationship studies
The broad variety of the synthesized compounds was designed in order to gain insight into the
influence of halogen and hydroxyl groups on the coumarin nucleus and also benzyl, aldehyde and acid groups
on the indole moiety of the hybrid molecules on their anticancer activity. In the present study, N- benzylation of
the indole nucleus is found to have no effect on anticancer activity. Benzylation might have deactivated the
weakly basic amino group of the indole ring. The presence of an aldehydic or acid group was found to increase
the cytotoxic potential as well as the selectivity index of the compounds towards MCF-7 cells. The carboxylic
group is often highly ionized in physiological pH and cannot cross the biological membranes or is subjected to

rapid clearance from the body due to its high hydrophilicity. To compensate the loss in lipophilicity of an active
compound, it is usually necessary to attach a halogen atom at an appropriate place. Aldehyde 4c and acids 5b
and 5c with halogen substituents in the coumarin ring exhibited better anticancer potential than unsubstituted or
hydroxyl substituted aldehydes and acids, where there is a proper balance between hydrophilic and lipophilic
nature of the molecule. The introduction of a hydroxyl group in a molecule changes the partition coefficient
toward more hydrophilicity.
The presence of non-covalent halogen bonds has a potential significance because of their distinct
interaction with ligand recognition, binding site, molecular folding and in assembly of proteins and nucleic
acids. Halogens are believed to polarize with covalently attached atom to generate electropositive centres, thus
acting as a lewis acid to pair with oxygen and nitrogen that is similar to hydrogen bonds, both in strength and
directionality and hence they are termed as charge transfer bonds. As a result, the bromine interaction in 4c,
depending on the geometry of the halogen bond is estimated to be stronger than the analogous hydrogen bond in
this environment. Drug candidates that are substituted with bromine atoms (induces an important lipophilic
contribution even larger than that of a methyl group) are interestingly found to increase the lipophilicity of the
molecule which in turn helps in easy penetration of drug through the lipid membranes and tissues thus
enhancing the intercellular drug delivery. It also increases protein-ligand stability there by contributing to the
binding affinity and selectivity of the molecule [42]. It is also found that halogen bond containing molecules are
involved in the inhibition of several classes of protein kinases like MEK, CDK1 and CDK2 [41].
The better activity of molecule 4c was also analysed using steric and electronic effects caused by
bromine atom. The obstruction of the hybrid molecule by means of bromine substitution can also impose certain
conformations for certain functions. The bulky bromine atoms prevent the free rotation and maintain the planes
of the aromatic rings. The steric effects of halogens are ascribed to their inductive electron-attracting properties
which are maximal for chlorine and bromine. The mesomeric donor effect of bromine is usually not considered
in biological media [43]. The better anticancer activity of compound 4c as opposed to 4b could be attributed to
higher lipophilic nature of bromine when compared to chlorine.
4. Conclusion
In the present study, three new series of indole-coumarin hybrid derivatives having different halogen and
hydroxyl substitution on coumarin ring and benzyl, aldehyde and acid substituents on indole nucleus have been
synthesised and their biologic activities are described. Bromine substituted aldehyde and acid displayed good in
vitro cytotoxic activity against MCF-7 cells. 2-(6-Bromo-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde
(4c) was found to display dose dependent cytotoxicity and high selectivity towards MCF-7 cells when compared
to standard microtubule-interfering chemotherapeutic agent, vincristine. Cell cycle analysis of MCF-7 cells on
treatment with 4c revealed cell cycle arrest in the G2/M phase confirming its action via the apoptotic pathway.
From the current investigation, the structure activity relationships of the indole-coumarin hybrid aldehyde series

demonstrate that bromine substituent in the coumarin pharmacophore showed increased antitumor activity.
Presence of bromine might have contributed to the lipophilic nature of molecule and hence favour the passage of
biomembranes. Bromine, when usually incorporated into a molecule generates a reactive alkylating intermediate
more easily when compared to fluorine or chlorine and benefits in long term treatment. The results of the
biochemical assays carried out were in complete agreement with the in silico docking studies done in order to
predict the possible anticancer activity of the three series of indole-coumarin hybrids. The docking studies have
provided excellent information about the binding affinity of molecules and have helped to understand the weak
force of interactions. Due to the high incidence of undesirable side effects induced by the majority of current
anticancer drugs and by considering the high selectivity index value when compared to vincristine, compound
4c appears as promising and interesting lead for the design of improved selective therapeutic agents to fight
cancer.
5. Experimental
5.1. General procedure for the synthesis of substituted 3-(1H-Indol-2-yl)-chromen-2-ones (2a-d)
Phenyl hydrazine (0.001 mol, 0.098 g) and substituted 3-acetyl-chromen-2-ones (1a-d) (0.001 mol) in ethanol
with catalytic quantity of glacial acetic acid were refluxed for 6 h to yield respective phenyl hydrazones.
Further, substituted phenyl hydrazones (0.004 mol) was heated for 30 - 60 min at 90-95 °C with a mixture of
methane sulfonic acid (0.015 mol, 0.97 mL) and phosphorus pentoxide (0.003 mol, 0.85 g) resulting in the
formation of target compound which was recrystallized using dimethylformamide
5.2. General procedure for the synthesis of substituted 3-(1-Benzyl-1H-indol-2-yl)-chromen-2-one (3a-d)
Powdered potassium carbonate (0.011 mol, 1.52 g) was added to a solution of 2a-d (0.002 mol) in DMF and
was stirred at r.t. for 30 min. Benzyl chloride (0.002 mol, 0.23 mL) was added, mixture stirred vigorously and
refluxed for 6 h. The reaction mixture was cooled and poured in to ethanol water mixture (1:10). The
precipitated N-benzylated indole-coumarin derivative was filtered, washed with water and recrystallized using
ethanol-water mixture.
5.2.1. 3-(1-Benzyl-1H-indol-2-yl)-chromen-2-one (3a)
Brown solid (44 %); m.p. 138-140 °C; Rf. 0.68; IR (KBr) [cm-1]: 3030 (Ar. C-H str.), 2915 (methyl C-H asym.
str.), 2845 (methyl C-H sym. str.), 1735 (lactone C=O str.), 1510 (Ar. C=C str.), 1203 (Ar. C-O-C str.), 1361
1
(C-N str.); H NMR (400 MHz, DMSO-d6) [ppm]: δ 4.58 (s, 2H, CH2), δ 6.88 (s, 1H, indole 3-H), δ 6.94 (m,
1H, phenyl 4-H), δ 7.00-7.15 (d, 2H, phenyl 2-H and 6-H), δ 7.25 (d, 1H, coumarin 8-H), δ 7.42-7.45 (t, 2H,
phenyl 3-H and 5-H), δ 7.50 (d, 1H, indole 7-H), δ 7.55 (d, 1H, indole 4-H), δ 7.71-7.73 (m, 2H, indole 5-H and
6-H), δ 7.84-7.90 (m, 3H, coumarin 5-H, 6-H and 7-H), δ 8.45 (s, 1H, coumarin 4-H); 13C NMR (100 MHz,
DMSO-d6) δ = 55.82, 105.23, 112.11, 118.72, 120.03, 120.14, 120.25, 121.26, 121.68, 121.76, 123.48, 128.34,
129.36, 129.87, 130.41, 131.25, 131.62, 134.11, 134.18, 134.92, 137.71, 138.14, 151.63, 158.52; MS (m/z): 351
(M+); Anal. Calcd. for C24H17NO2: C, 82.03; H, 4.88; N, 3.99; found: C, 82.19; H, 4.90; N, 4.00.
5.2.2. 3-(1-Benzyl-1H-indol-2-yl)-6-chloro-chromen-2-one (3b)

Brown solid (29 %); m.p. 258-260 °C; Rf. 0.85; IR (KBr) [cm-1]: 3067 (Ar. C-H str.), 2928 (methyl C-H asym.
str.), 2850 (methyl C-H sym. str.), 1704 (lactone C=O str.), 1515 (Ar. C=C str.), 1245 (Ar. C-O-C str.), 1150
(C-N str.), 798 (C-Cl str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 4.59 (s, 2H, CH2), δ 6.87 (s, 1H, indole 3-
H), δ 6.95 (m, 1H, phenyl 4-H), δ 7.01-7.15 (d, 2H, phenyl 2-H and 6-H), δ 7.26 (d, 1H, coumarin 8-H), δ 7.43-
7.48 (t, 2H, phenyl 3-H and 5-H), δ 7.52 (d, 1H, indole 7-H), δ 7.59 (d, 1H, indole 4-H), δ 7.71-7.74 (m, 2H,
indole 5-H and 6-H), δ 7.81 (d, 1H, coumarin 7-H), δ 7.89 (s, 1H, coumarin 5-H), δ 8.45 (s, 1H, coumarin 4-H);
13C NMR (100 MHz, DMSO-d6) δ = 55.85, 105.21, 112.15, 118.70, 120.00, 120.13, 120.20, 121.22, 121.65,
121.75, 123.46, 128.34, 129.31, 129.85, 130.47, 131.19, 131.58, 134.00, 134.15, 134.97, 137.75, 138.13,
151.62, 158.58; MS (m/z): 385 (M+); Anal. Calcd. for C24H16ClNO2: C, 74.71; H, 4.18; N, 3.63; found: C,
74.88; H, 4.19; N, 3.64.
5.2.3. 3-(1-Benzyl-1H-indol-2-yl)-6-bromo-chromen-2-one (3c)
Brown solid (40 %); m.p. 220-224 °C; Rf. 0.58; IR (KBr) [cm-1]: 3031 (Ar. C-H str.), 2923 (methyl C-H asym.
str.), 2854 (methyl C-H sym. str.), 1709 (lactone C=O str.), 1519 (Ar. C=C str.), 1243 (Ar. C-O-C str.), 1151
(C-N str.), 806 (C-Br str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 4.60 (s, 2H, CH2), δ 6.90 (s, 1H, indole 3-
H), δ 6.98 (m, 1H, phenyl 4-H), δ 7.03-7.17 (d, 2H, phenyl 2-H and 6-H), δ 7.31 (d, 1H, coumarin 8-H), δ 7.43-
7.48 (t, 2H, phenyl 3-H and 5-H), δ 7.54 (d, 1H, indole 7-H), δ 7.59 (d, 1H, indole 4-H), δ 7.72-7.75 (m, 2H,
indole 5-H and 6-H), δ 7.85 (d, 1H, coumarin 7-H), δ 7.95 (s, 1H, coumarin 5-H), δ 8.51 (s, 1H, coumarin 4-H);
13C NMR (100 MHz, DMSO-d6) δ = 55.86, 105.23, 112.14, 118.71, 120.01, 120.15, 120.23, 121.24, 121.69,
121.77, 123.47, 128.35, 129.34, 129.86, 130.48, 131.22, 131.60, 134.10, 134.19, 134.99, 137.76, 138.15,
151.66, 158.58; MS (m/z): 430 (M+); Anal. Calcd. for C24H16BrNO2: C, 66.99; H, 3.75; N, 3.26; found: C,
67.20; H, 3.77; N, 3.27.
5.2.4. 3-(1-Benzyl-1H-indol-2-yl)-7-hydroxy-chromen-2-one (3d)
Dark brown solid (19 %); m.p. 140-142 °C; Rf. 0.85; IR (KBr) [cm-1]: 3375 (OH str.), 3058 (Ar. C-H str.), 2920
(methyl C-H asym. str.), 2851 (methyl C-H sym. str.), 1720 (lactone C=O str.), 1523 (Ar. C=C str.), 1254 (Ar.
C-O-C str.), 1211 (C-N str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 4.76 (s, 2H, CH2), δ 5.12 (s, 1H, OH), δ
6.14 (s, 1H, indole 3-H), δ 6.70 (s, 1H, coumarin 8-H), δ 6.87 (d, 1H, coumarin 6-H), δ 6.92 (d, 2H, phenyl 2-H
and 6-H), δ 7.01 (m, 1H, phenyl 4-H), δ 7.08-7.02 (t, 2H, phenyl 3H and 5H), δ 7.10 (d, 1H, indole 7-H), δ 7.18-
7.23 (m, 2H, indole 5-H and 6-H), δ 7.43 (d, 1H, indole 4-H), δ 7.69 (d, 1H, coumarin 5-H), δ 8.51 (s, 1H,
coumarin 4-H); 13C NMR (100 MHz, DMSO-d6) δ = 55.80, 105.21, 112.11, 114.48, 118.70, 120.01, 120.09,
120.12, 120.15, 121.18, 121.63, 121.71, 128.31, 129.75, 129.57, 131.31, 131.62, 134.21, 134.15, 134.92,
137.71, 138.24, 156.63, 158.53; MS (m/z): 367 (M+); Anal. Calcd. for C24H17NO3: C, 78.46; H, 4.66; N, 3.81;
found: C, 78.65; H, 4.67; N, 3.82.
5.3 General procedure for the synthesis of substituted 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde
(4a-d)
Phosphorous oxychloride (0.038 mol, 3.5 mL) was added drop wise to dimethyl formamide (0.051 mol, 4.1 mL)
maintained at -10 – 0 °C and stirred for 30 min. 2a-d (0.0001 mol) dissolved in minimum quantity of DMF was
added part by part to the reaction mixture maintained at 0-10 °C, stirred for 30 min, warmed at 60-70 °C for 5 h

and poured in to crushed ice. The pH was adjusted to 7.5-8 by addition of sodium hydroxide solution and
extracted using ethyl acetate. The crude aldehyde so obtained was recrystallized using ethanol: DMF mixture.
5.3.1.
2-(2-Oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4a)
Dark brown solid (87.12 %); decomposes at 200 °C; Rf. 0.28; IR (KBr) [cm-1 ]: 3310 (N-H str.), 3108 (Ar. C-H
str.), 1728 (lactone C=O str.), 1650 (C=O str.), 1499 (Ar. C=C str.), 1357 (Ar. C-O-C str.); 1H NMR (400 MHz,
DMSO-d6) [ppm]: δ 7.02 (m, 1H, indole 6-H ), δ 7.21 (t, 1H, indole 5-H), δ 7.35 (d, 1H, indole 7-H), δ 7.50 (d,
1H, indole 4-H), δ 7.66 (d, 1H, coumarin 8-H), δ 7.93-7.96 (m, 2H, coumarin 6-H and 7-H), δ 8.09 (d, 1H,
coumarin 5-H), δ 8.48 (s, 1H, coumarin 4-H), δ 9.85 (s, 1H, CHO), δ 9.92 (s, 1H, indole N-H); 13C NMR (100
MHz, DMSO-d6) δ = 105.24, 112.17, 120.02, 120.13, 120.25, 121.26, 121.69, 121.78, 123.48, 128.45, 129.59,
129.89, 130.59, 134.94, 138.18, 151.67, 162.05, 190.05; MS (m/z): 289 (M+); Anal. Calcd. for C18H11NO3: C,
74.73; H, 3.83; N, 4.84; found: C, 74.98; H, 3.85; N, 4.85.
5.3.2.
2-(6-Chloro-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4b)
Dark brown solid (78.12 %); decomposes at 218 °C; Rf. 0.82; IR (KBr) [cm-1]: 3325 (N-H str.), 3109 (Ar. C-H
str.), 1700 (lactone C=O str.), 1650 (C=O str.), 1550 (Ar. C=C str.), 1374 (Ar. C-O-C str.), 750 (C-Cl str.); 1H-
NMR (400 MHz, DMSO-d6) [ppm]: δ 7.04 (t, 1H, indole 6-H ), δ 7.21 (t, 1H, indole 5-H), δ 7.35 (d, 1H, indole
7-H), δ 7.50 (d, 1H, indole 4-H), δ 7.68 (d, 1H, coumarin 8-H), δ 7.95 (d, 1H, coumarin 7-H), δ 8.10 (s, 1H,
coumarin 5-H), δ 8.50 (s, 1H, coumarin 4-H), δ 9.86 (s, 1H, CHO), δ 9.94 (s, 1H, indole N-H); 13C NMR (100
MHz, DMSO-d6) δ = 105.20, 112.13, 120.00, 120.11, 120.20, 121.25, 121.68, 121.77, 123.48, 128.34, 129.32,
130.00, 130.47, 134.99, 138.15, 151.62, 162.02, 190.00; MS (m/z): 323 (M+); Anal. Calcd. for C18H10ClNO3; C,
66.78; H, 3.11; N, 4.33; found: C, 66.96; H, 3.13; N, 4.34.
5.3.3.
2-(6-Bromo-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4c)
Dark brown solid (82 %); m.p. 230-232 °C; Rf. 0.1; IR (KBr) [cm-1]: 3305 (N-H str.), 3100 (Ar. C-H str.), 1724
(lactone C=O str.), 1643 (C=O str.), 1548 (Ar. C=C str.), 1388 (Ar. C-O-C str.), 813 (C-Br str.); 1H-NMR (400
MHz, DMSO-d6) [ppm]: δ 7.06 (m, 1H, indole 6-H ), δ 7.22 (t, 1H, indole 5-H), δ 7.35 (d, 1H, indole 7-H), δ
7.50 (d, 1H, indole 4-H), δ 7.70 (d, 1H, coumarin 8-H), δ 7.97 (d, 1H, coumarin 7-H), δ 8.13 (s, 1H, coumarin 5-
H), δ 8.52 (s, 1H, coumarin 4-H), δ 9.86 (s, 1H, CHO), δ 9.94 (s, 1H, indole N-H); 13C NMR (100 MHz,
DMSO-d6) δ = 105.23, 112.15, 120.01, 120.14, 120.23, 121.24, 121.68, 121.77, 123.47, 128.35, 129.33, 129.86,
130.48, 134.98, 138.16, 151.66, 162.01, 190.03; MS (m/z): 368 (M+); Anal. Calcd. for C18H10BrNO3: C, 58.72;
H, 2.74; N, 3.80; found: C, 58.87; H, 2.75; N, 3.81.
5.3.4.
2-(7-Hydroxy-2-oxo-2H-chromen-3-yl)-1H-indole-3-carbaldehyde (4d)
Orange solid (76 %); m.p. 210-212 °C; Rf. 0.5; IR (KBr) [cm-1]: 3600 (O-H str.), 3352 (N-H str.), 3080 (Ar. C-
1
H str.), 1724 (lactone C=O str.), 1623 (C=O str.), 1546 (Ar. C=C str.), 1261 (Ar. C-O-C str.); H-NMR (400
MHz, DMSO-d6) [ppm]: δ 5.33 (s, 1H, O-H ), δ 7.01 (m, 1H, indole 6-H ), δ 7.19 (t, 1H, indole 5-H), δ 7.33 (d,
1H, indole 7-H), δ 7.56 (d, 1H, indole 4-H),δ 6.69 (d, 1H, coumarin 6-H ), δ 6.85 (s, 1H, coumarin 8-H), δ 7.43
(d, 1H, coumarin 5-H ), δ 8.18 (s, 1H, coumarin 4-H), δ 9.89 (s, 1H, CHO), δ 9.96 (s, 1H, indole N-H); 13C
NMR (100 MHz, DMSO-d6) δ = 105.24, 112.14, 120.00, 120.13, 120.22, 121.21, 121.67, 121.76, 123.48,

128.31, 129.39, 129.85, 130.41, 134.99, 138.16, 156.63, 162.09, 190.01; MS (m/z): 305 (M+); Anal. Calcd. for
C18H11NO4: C, 70.82; H, 3.63; N, 4.59; found: C, 71.10; H, 3.65; N, 4.60.
5.4. General procedure for the synthesis of substituted 2-(2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic
acids (5a-d)
Potassium permanganate (0.001 mol, 0.158 g) solution was added to 0.001 mol of 4a-d dissolved in 10 ml
pyridine and stirred at r.t for 3 h. 50 mL 1% sodium hydroxide solution was added to the reaction mixture and
further stirred for 2 h at 50 °C, cooled, filtered and washed with water. The filtrate was acidified with 6N HCl to
pH 4, precipitate was filtered, washed with water and recrystallized using glacial acetic acid
5.4.1.
2-(2-Oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acid (5a)
Straw yellow solid (30 %); decomposes at 180 °C; Rf. 0.3; IR (KBr) [cm-1]: 3390 (COOH str.), 3350 (N-H str.),
1
3018 (Ar. C-H str.), 1714 (lactone C=O str.), 1630 (C=O str.), 1590 (Ar. C=C str.), 1356 (Ar. C-O-C str.); H
NMR (400 MHz, DMSO-d6) [ppm]: δ 7.28-7.30 (m, 2H, coumarin 7-H and 6-H ), δ 7.57-7.54 (m, 2H, indole 5-
H and 6-H), δ 7.87 (d, 1H, indole 4-H), δ 8.10-8.12 (d, 1H, indole 7-H), δ 8.20-8.23 (d, 2H, coumarin 5-H and
8-H ), δ 8.49 (s, 1H, coumarin 4-H), δ 9.98 (s, 1H, indole N-H), δ 12.42 (s, 1H, COOH); 13C NMR (100 MHz,
DMSO-d6) δ = 112.04, 120.05, 120.19, 120.28, 121.26, 121.67, 123.48, 125.09, 128.35, 129.38, 129.87, 130.50,
131.26, 137.75, 138.18, 151.69, 158.56, 170.18; MS (m/z): 305 (M+); Anal. Calcd. for C18H11NO4: C, 70.82; H,
3.63; N, 4.59; found: C, 71.03; H, 3.64; N, 4.60.
5.4.2.
2-(6-Chloro-2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acid (5b)
Straw yellow solid (50 %); decomposes at 163 °C; Rf. 0.9; IR (KBr) [cm-1]: 3400 (COOH str.), 3300 (N-H str.),
3036 (Ar. C-H str.), 1720 (lactone C=O str.), 1632 (C=O str.), 1492 (Ar. C=C str.), 1357 (Ar. C-O-C str.), 785
(C-Cl str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 7.30-7.32 (d, 2H, coumarin 7-H and 8-H ), δ 7.57-7.54 (m,
2H, indole 5-H and 6-H), δ 7.87 (d, 1H, indole 4-H), δ 8.21 (d, 1H, indole 7-H), δ 8.20 (s, 1H, coumarin 5-H), δ
8.45 (s, 1H, coumarin 4-H), δ 9.97 (s, 1H, indole N-H), δ 12.41 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-
d6) δ = 112.14, 120.00, 120.12, 120.21, 121.23, 121.68, 123.49, 125.02, 128.36, 129.34, 129.85, 130.49, 131.21,
137.75, 138.15, 151.63, 158.57, 170.00; MS (m/z): 339 (M+); Anal. Calcd. for C18H10ClNO4: C, 63.64; H, 2.97;
N, 4.12; found: C, 63.89; H, 2.99; N, 4.13.
5.4.3.
2-(6-Bromo-2-oxo-2H-chromen-3-yl)-1H-indole-3-carboxylic acid (5c)
Straw yellow solid (45 %); m.p. 220-222 °C; Rf. 0.4; IR (KBr) [cm-1]: 3402 (COOH str.), 3379 (N-H str.), 3048
(Ar. C-H str.), 1724 (lactone C=O str.), 1643 (C=O str.), 1525 (Ar. C=C str.), 1388 (Ar. C-O-C str.), 810 (C-Br
str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 7.31-7.35 (d, 2H, coumarin 7-H and 8-H ), δ 7.52-7.57 (m, 2H,
indole 5-H and 6-H), δ 7.87 (d, 1H, indole 4-H), δ 8.21 (d, 1H, indole 7-H), δ 8.23 (s, 1H, coumarin 5-H), δ 8.49
(s, 1H, coumarin 4-H), δ 9.98 (s, 1H, indole N-H), δ 12.42 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6) δ =
112.15, 120.01, 120.14, 120.23, 121.24, 121.69, 123.47, 125.03, 128.34, 129.35, 129.86, 130.48, 131.23,
137.76, 138.16, 151.65, 158.58, 170.01; MS (m/z): 384 (M+); Anal. Calcd. for C18H10BrNO4: C, 56.27; H, 2.62;
N, 3.65; found: C, 56.40; H, 2.63; N, 3.66.

5.4.4.
2-(7-Hydroxy-2-oxo-H-chromen-3-yl)-1H-indole-3-carboxylic acid (5d)
Straw yellow solid (23.8 %); m.p. 198-200 °C; Rf. 0.8; IR (KBr) [cm-1]: 3550 (COOH str.), 3440 (O-H str.),
3320 (N-H str.), 3108 (Ar. C-H str.), 1718 (lactone C=O str.), 1640 (C=O str.), 1560 (Ar. C=C str.), 1380 (Ar.
C-O-C str.); 1H NMR (400 MHz, DMSO-d6) [ppm]: δ 5.38 (s, 1H, O-H ), δ 6.70 (s, 1H, coumarin 8-H ), δ 6.87
(d, 1H, coumarin 6-H), δ 7.40-7.10 (d, 1H, coumarin 5-H), δ 7.55-7.51 (m, 2H, indole 5-H and 6-H), δ 7.86 (d,
1H, indole 4-H), δ 8.18 (d, 1H, indole 7-H), δ 8.20 (s, 1H, coumarin 4-H), δ 9.96 (s, 1H, indole N-H), δ 12.40
(s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6) δ = 112.05, 120.04, 120.18, 120.20, 121.23, 121.61, 123.47,
125.00, 128.31, 129.35, 129.84, 130.47, 131.25, 137.73, 138.17, 151.66, 158.57, 170.10; MS (m/z): 321 (M+);
Anal. Calcd. for C18H11NO5: C, 67.29; H, 3.45; N, 4.36; found: C, 67.54; H, 3.47; N, 4.37.
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Scheme 1. Protocol for the synthesis of different substituted indole-coumarin derivatives.
Fig.1. Bcl-2 docking: Docking results from Autodock vina is shown for compounds 4a (A), 4b (B), 4c (C) and
4d (D). Bcl-2 is shown as surface and coloured green. Ligand molecules 4a-d shown as ball and stick model
are coloured cyan (carbon), red (oxygen), blue (nitrogen), white (hydrogen), yellow (chloride) and brown
(bromine). The inset shows the close view of binding pocket of Bcl-2 docked with compound 4c.
Fig. 2. Bcl-2 molecular interactions: Bcl-2 protein amino acids interacting with compounds 4a (A), 4b (B), 4c
(C) and 4d (D) is shown in ball and stick model. Bcl-2 amino acids carbons are coloured green and ligand
carbons are shown in cyan color. For the clarity purpose only polar hydrogens are shown and remaining
residues are coloured as Figure 1. Interactions are shown in dotted line and distances (Å) between amino acids
and corresponding ligand atoms are marked.
Fig. 3. Cell cycle analysis: Normal cell cycle pattern observed in control. G2/M phase cell cycle arrest seen on
treatment with vincristine and compound 4c

Highlights
 Synthesis of novel indole-coumarin hybrids mediated by eaton’s reagent is proposed.
 A new antimitotic agent, 4c is identified with high selectivity to MCF7 cells.
 Presence of bromine atom in coumarin ring is found to enhance anticancer activity in
SAR studies.
 Inhibition of apoptosis related protein Bcl-2 is observed in in silico docking studies.