Synthesis and bio-evaluation of indole-chalcone based benzopyrans as promising antiligase and antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.015

DNA replication and repair are complex processes accomplished by the concerted action of a network of enzymes and proteins. DNA ligases play a crucial role in these processes by catalyzing the nick joining between DNA strands. As compared to normal cells,

Full text of DOI:10.1016/j.ejmech.2017.11.015

Accepted Manuscript
Synthesis and bio-evaluation of indole-chalcone based benzopyrans as a promising
antiligase and antiproliferative agent
Sampa Gupta, Pooja Maurya, Akanksha Upadhyay, Pragati Kushwaha, Shagun
Krishna, Mohammad Imran Siddiqi, Koneni V. Sashidhara, Dibyendu Banerjee
PII:
S0223-5234(17)30908-X
10.1016/j.ejmech.2017.11.015
EJMECH 9890
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2017
Revised Date: 16 October 2017
Accepted Date: 4 November 2017
Please cite this article as: S. Gupta, P. Maurya, A. Upadhyay, P. Kushwaha, S. Krishna, M.I. Siddiqi,
K.V. Sashidhara, D. Banerjee, Synthesis and bio-evaluation of indole-chalcone based benzopyrans as
a promising antiligase and antiproliferative agent, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.11.015.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and bio-evaluation of indole-chalcone based benzopyrans as a
promising antiligase and antiproliferative agent
1
†
2†
1
1
2
Sampa Gupta , Pooja Maurya , Akanksha Upadhyay , Pragati Kushwaha , Shagun Krishna ,
2#
1#
2#
Mohammad Imran Siddiqi , Koneni V. Sashidhara* , Dibyendu Banerjee*
1
2
Medicinal and Process Chemistry Division, Molecular and structural biology division,
#
†
Academy of Scientific and Innovative Research (AcSIR), India
Both authors contributed equally to this work.
CSIR-Central Drug Research Institute, (CSIR-CDRI), BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow,
2
*
26031, India.
Correspondence and requests for materials should be addressed to D.B (d.banerjee@cdri.res.in, dibyenducdri@gmail.com) and
K.V.S (e-mail: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in)

ACCEPTED MANUSCRIPT
Synthesis and bio-evaluation of indole-chalcone based benzopyrans as
promising antiligase and antiproliferative agents
1
†
2†
1
1
Sampa Gupta , Pooja Maurya , Akanksha Upadhyay , Pragati Kushwaha , Shagun
2
2#
1#
2#
Krishna , Mohammad Imran Siddiqi , Koneni V. Sashidhara* , Dibyendu Banerjee*
1
2
Medicinal and Process Chemistry Division, Molecular and structural biology division,
#
†
Academy of Scientific and Innovative Research (AcSIR), India
Both authors contributed equally to this work.
CSIR-Central Drug Research Institute, (CSIR-CDRI), BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow,
2
*
26031, India.
Correspondence and requests for materials should be addressed to D.B (d.banerjee@cdri.res.in, dibyenducdri@gmail.com)
and K.V.S (e-mail: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in)
ABSTRACT
DNA replication and repair are complex processes accomplished by the concerted action of a
network of enzymes and proteins. DNA ligases play a crucial role in these processes by
catalyzing the nick joining between DNA strands. As compared to normal cells, elevated
levels of human DNA ligase I (hLigI) is reported in some cancers. We studied the inhibition
of hLigI mediated DNA nick sealing activity followed by the antiproliferative activity of
novel indole-chalcone based benzopyran compounds on cancer cells. One molecule called
compound 27 showed a notable preference for inhibition of hLigI as compared to other
ligases and showed enhanced cytotoxicity against colon cancer (DLD-1) cells as compared to
normal cells. Mechanistic studies showed that compound 27 directly interacts with hLigI in a
competitive manner and did not interact with the DNA substrate during ligation. This novel
and potent hLigI inhibitor showed significant inhibition of both monolayer culture as well as
3
D culture of DLD-1 cells that mimic solid tumor. It also affected the migration of DLD-1
cells indicating the potential anti-metastatic activity. This novel hLigI inhibitor could
therefore serve as a promising lead for anticancer drug development.
1

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Keywords: Indole-chalcone based benzopyrans, DNA ligases, 3D culture, antiligase activity,
antiproliferative activity
Abbreviations Used
BER (base excision repair), NER (nucleotide excision repair), HR (homologous
recombination), NHEJ (non-homologous end joining), MMR (mismatch repair), TLS
6
(
translesion synthesis), FA (Fanconi anemia), MGMT (O -methylguanine DNA
methyltransferase), hLigI (human DNA ligase I), hLigIIIβ (human DNA ligase III), hLigIV
human DNA ligase IV), EMSA (Electrophoretic mobility shift assay) FDA (Fluorescein
diacetate), PI Propidium iodide).
(
(
2

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1
. Introduction
The prime objective of each living organism is to transfer its hereditary material, intact and
unaltered, to the next generation [1]. DNA replication and repair are essential molecular
mechanisms required for error-free transfer of genetic material to the new generation, using a
network of enzymes and proteins [2, 3, 4]. Errors in the replication, repair and recombination
of DNA are reasons for genomic instability that can lead to unregulated cellular
multiplication and cancer [5, 6, 7, 8]. Several DNA repair pathways are present inside the
cells to cope with different genetic lesions [9, 10]. Cancer cells often have a slightly different
set of DNA repair pathways from normal cells, to help them propagate despite errors in their
DNA [11]. We therefore hypothesize that it might be a good strategy to target DNA repair
proteins for cancer therapy; specially those proteins that are differentially expressed in cancer
cells. Incidentally, the expression of hLigI has been reported to be elevated in several cancers
like breast, lung and ovarian cancer cells, as compared to normal cells [12, 13]. DNA ligases
have a crucial role in DNA replication and repair. They catalyze the joining of nicks by the
formation of phosphodiester bonds between two adjacent DNA strands [14]. There are three
types of ligases in mammalian cells, viz, ligase I (hLigI), ligase III (hLigIIIβ) and ligase IV
(hLigIV) [15]. Human DNA ligase I is responsible for joining of Okazaki fragments during
the lagging strand synthesis and involved in several DNA repair pathways such as NER (by
hLigI and hLigIII) [16], BER (by hLigI and hLigIII) [17, 18] and microhomology-mediated
end joining (by hLigI and hLigIII) [19, 20] whereas NHEJ is mediated by hLigIV [21]. In this
manner, DNA ligation is a crucial requirement for all cells during replication and repair [22,
2
3, 24].
Inhibiting DNA ligases in cancer cell is shown to impede cell growth due to the accumulation
of DNA strand breaks and lead to apoptotic cell death [13, 25]. In the past, several
compounds were identified that can impede DNA nick sealing by inhibiting DNA ligases [4,
3

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1
4, 20, 26, 27]; however, none of the inhibitors were potent enough to reach clinical trials on
their own. Hence, there is a need for identification and characterization of more potent
molecules. In order to find suitable DNA ligase inhibitors we searched the literature which
suggested that indoles and their derivatives are highly versatile and common nitrogen-based
heterocycles that are frequently used in the synthesis of important FDA approved anticancer
drugs [28]. Indoles have recently attracted interest in anticancer research due to activity
against several cancer cells. They are reported as potent inducers of apoptosis through a novel
cell-based HTS caspase assay [29]. Singh et al have also reported indoles as highly promising
anti-cancerous agents. They found that indole derivatives show appreciable inhibition of
tumor growth in different cancer cell lines [30]. On the other hand, chalcones have attracted
attention due to their wide range of potential biological activities [31, 32, 33, 34]. Rawat et al
showed chalcone-resveratrol as a promising anticancer agent with selectivity towards certain
ovarian, lung and breast cancer cell lines [35]. Our group has previously worked on chalcones
and reported coumarin-chalcone hybrids as anticancer agents against four human cancer cell
lines: KB (oral squamous cell carcinoma), C33A (cervical carcinoma), MCF-7 (breast
adenocarcinoma) and A549 (lung cancer) [36]. Another pharmacophore with anticancer
property is benzopyran. Recently, Sui et al showed benzopyrans as novel anticancer agents
having an EC50 value of 2 nM [37]. William Kemnitzer et al demonstrated benzopyrans as
potent caspase cascade activators and inducers of apoptosis in solid tumors [38]. Based on
our long experience of working on molecular hybridization techniques [39], we designed a
novel series of hybrid compounds in order to identify molecules that can inhibit DNA ligation
and cell proliferation, and may serve as drug-like molecules. The design for this series is
schematically represented in scheme A (figure 1). The molecules were synthesized and tested
for their antiligase and antiproliferative activities in cancer cells. Finally, a detailed study of
4

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the most active compound was carried out in order to verify the mode of action and
cytotoxicity in in vitro and 3D tumor models.
2
. Results and Discussion
2
.1 Chemistry
The synthetic strategy of indole-chalcone based benzopyran molecules is depicted in scheme
1
. In the first step, the Duff formylation on ortho substituted phenol (1-4) was carried out in
o
the presence of HMTA with TFA as solvent at 120 C for 4h followed by acid hydrolysis with
1
0% H SO which gave aromatic dicarbaldehydes (5-8) [40]. The regioselective chalcones
2 4
(9-16) were prepared from these dicarbaldehydes by reacting them with para-substituted
acetophenones in the presence of conc. HCl as catalyst in dioxane [36]. Several research
groups have worked upon multicomponent reactions taking salicylaldehyde, indole and
different neucleophiles as reactive components in different reaction conditions to give
benzopyran hybrids [41, 42, 43]. In a similar strategy, we carried out multicomponent
reactions with chalcone intermediates, malononitrile (as neucleophile) and substituted 2-
methyl indole in the presence of L-proline as catalyst in acetonitrile to give our target
compounds (17-29) in quantitative yields. The reaction conditions were optimized for the
choice of catalyst and the solvent. The structures of all the synthesized compounds were
1
13
characterized with the help of H and C NMR spectral analysis coupled with mass
spectrometry, which were in good agreement with the proposed structures (Supporting
Information).
2
.2. Inhibition of human DNA ligase I activity and cytotoxicity of synthesized compounds
The nick sealing activity of thirteen indole-chalcone based benzopyrans were determined for
purified hLigI protein by gel-based, fluorescence-labelled DNA-ligation assays at 10 µM
concentration as shown in figure 2. Among these thirteen compounds, four compounds ( 20,
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2
3, 26 and 27) showed >50% inhibition, two compounds (22 and 28) showed >45%
inhibition, and other seven compounds showed <20% inhibition of nick sealing activity. All
thirteen compounds were tested for their cytotoxic effect on various cancer cell lines viz.
MDA-MB-231 (breast cancer), DLD-1 (colon cancer), HeLa (cervical cancer), HepG2 (liver
cancer) and 4T1 (mouse breast cancer cell line) at 10 µM concentrations. The % inhibition of
cancer cell proliferation by indole-chalcone based benzopyrans is represented in figure 3.
Among them, five compounds (24, 25, 27, 28 and 29) showed robust antiproliferative activity
against tested cancer cell lines.
We found one compound which contains both thiazole and tert-butyl group in it (compound
2
7) which possesses both antiligase (69.42 ± 3.05 % inhibition of nick sealing activity) and
antiproliferative activity in various cancerous cell lines as depicted in figure 2 and 3. The IC50
values of compound 27 were calculated in various cancerous cell lines like MDA-MB-231,
DLD-1, HeLa, HepG2 and 4T1 (IC <10 µM) from three independent experiments and
5
0
plotted with the help of GraphPad Prism software version 5 (San Diego California USA).
Compound 27 showed maximum antiproliferative activity against DLD-1 cells (IC = 4.6
5
0
µM). When further checked for cytotoxicity against the normal cell line HEK-293, we
observed a three-fold lower cytotoxicity (IC = 12.4 µM) as shown in figure 4. Therefore, we
5
0
selected compound 27 for detailed studies of mechanism of action.
.3 Structure activity relationship (SAR) studies
2
From the available biological activity results and the diverse chemical structures of
synthesized compounds, a preliminary structure activity relationship (SAR) was established.
We found that the compounds showing > 50% inhibition in DNA ligation assay mainly
contained tert-butyl and NH group at C-8 and C-2 positions of benzopyran rings,
2
respectively. In addition, the imperative role of these substituents was supported in docking
studies. Further, substitution in the indole ring did not bring significant effect in inhibition of
6

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hLigI activity. Moreover, when some of the structures were modified by replacing aryl group
from chalcone with a thiazole moiety, they showed marked cytotoxic activity towards various
cancer cell lines. Preliminary SAR analysis revealed that tert-butyl, amino and thiazole
moieties are essential for inhibition of DNA nick sealing activity of hLigI and for cytotoxic
activity against cancer cell lines. This was most evident in compound 27, which emerged as
the potential lead. The SAR is summarized in a pictorial diagram in figure 5.
2
.4 Specific activity of compound 27 against various ATP-dependent DNA ligases
For determination of specific activity against various ATP-dependent DNA ligases, we
performed a DNA nick-sealing assay in a concentration dependent manner against three
different human DNA ligases viz., hLigI, hLigIIIβ, and hLigIV/XRCC4 and the non-human
T4 DNA ligase. We found that compound 27 inhibited hLigI in a concentration dependent
manner, with some overlapping activity against hligIV/XRCC4; however, the compound was
completely inactive against hligIIIβ and T4 DNA ligase as shown in figure 6. Hence, this
compound specifically inhibits the activity of hligI with some overlapping activity against
hligIV/XRCC4 as depicted in figure 6.
2
.5 Interaction study of compound 27 with hLigI and DNA
DNA ligation activity in the presence of compound 27 can be inhibited in two ways, either by
direct interaction with human DNA ligase I or by interaction with the DNA to indirectly
obstruct protein binding. To study the interaction of compound 27 with both protein and
DNA, we performed in silico molecular docking followed by gel electrophoretic mobility
shift assay (EMSA) and DNase I cleavage protection assay. In silico docking of compound
2
7 into the DNA binding site of hLigI (PDB ID-1X9N) showed that the compound binds to
hLigI with a predicted binding energy of -14.66 kJ/mole (Figure 7). As seen in figure 7, one
7

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hydrogen atom of amino group attached with a chromene ring forms a hydrogen bond with
Lys770, while a second hydrogen atom interacts with Gly773. Further, a hydrogen bond was
observed with Arg451. Two of these residues (Lys770 and Arg451) are involved in direct
hydrogen bond formation with DNA in the crystal structure and are therefore, anticipated to
have a significant effect on the binding of hLigI with DNA [26, 44 and 45]. Therefore, these
interactions of compound 27 with hLigI play an important role in the stability of the docked
complex; further, the interaction of these amino acid residues with compound 27 can explain
the potent hLigI inhibition observed in the in vitro assays.
In the EMSA study, we observed a significantly reduced interaction between hLigI and DNA
in the presence of increasing concentrations of compound 27 (31.25, 62.5, 125, 250 and 500
µM), indicating that compound 27 interacts with hLigI by replacing the interacting DNA
substrate, as shown in figure 8. Thus, hLigI was inhibited by a competitive mode of
inhibition.
DNase I cleavage protection assay showed that unlike DNA interacting molecules such as
doxorubicin (DNA intercalator), DAPI (DNA minor grove binder) and methyl green (DNA
major groove binder); there was a complete cleavage of DNA in the presence of ampicillin
(negative control) and compound 27 (at 250, 500 and 700µM concentrations), even at very
high concentrations. This suggested that compound 27 does interact with DNA either through
intercalation or through DNA minor/ major groove binding. Therefore, DNA incubated with
compound 27 was left free for cleavage by DNase I (figure 9).
2
.6 Compound 27 induces morphological changes in DLD-1 cells
We studied the morphological changes in DLD-1 cells induced by compound 27. The bright
field images of DLD-1 cells showed significant morphological changes after 24 and 48 h of
treatment with compound 27 at 5 and 10 µM concentrations, as compared to the control cells
8

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(figure 10). The observed morphological changes were shrinkage of cells and detachment of
cells from the culture surface that are the characteristic of cells undergoing apoptosis. We
observed that in the presence of compound 27, the cells undergo apoptosis in a concentration
and time dependent manner (figure 10).
2
.7 Compound 27 inhibits colony formation in DLD-1 cell line
The colony-forming assay determines the ability of a cell to proliferate indefinitely, thereby
retaining its reproductive ability to form a colony or a clone. Colony formation by DLD-1
cells was observed by staining with crystal violet. DLD-1 cells were treated with compound
2
7 (at 1.25, 2.5 and 5 µM concentrations) for 24 h and 48 h and cell colonies were grown for
1
0 days before staining. Figure 11 confirms inhibition of colony formation in the presence of
compound 27 in a time and concentration dependent manner.
2
.8 Compound 27 inhibits migration of colon cancer cells
Cell migration is a highly coordinated and multi-step process that plays a critical role in
progression of cancer. Cancer cells often have a highly metastatic nature. Wound-healing
assay is a simple and inexpensive method to study the directional migration of cells in vitro
[
46]. Thus, we studied the effects of compound 27 on the migration of colon cancer cells by
wound-healing assay in DLD-1 cells, after treatment with different concentrations of the
compound (2.5 and 5 µM) for 12, 24 and 48 h. Percentage open area in untreated and treated
cells were calculated at different time points with the help of Image-J software. The bright
field images and graphs represented in figure 12 showed a significant reduction in % open
area of control cells as compared to treated cells. Decrease in % open area indicates increased
migration. Therefore, we determined that compound 27 could significantly inhibit cellular
migration at 2.5 and 5 µM concentrations suggesting that it might have anti-metastatic
activity against colon cancer cells.
9

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2
.9 Compound 27 induces apoptosis in DLD-1 cells
In order to check the effect of compound 27 on cellular apoptosis, DLD-1 cells were exposed
to different concentrations of compound 27 for 48 h, and stained with Annexin V-FITC and
PI. Flow cytometry analysis of stained cells showed the induction of apoptosis in DLD-1
cells in a dose dependent manner (2.5, 5 and 10µM). At 5 µM concentration 17.38 ± 2.23 %
cells were in early apoptosis and 14.85 ± 2.18 % cells in late apoptosis whereas at 10 µM
concentration percentage of early and late apoptotic cells increased upto 26.53 ± 1.51% and
5
0.96 ± 6.98% respectively, as shown in figure 13. Hence, the induction of apoptosis was
responsible for death of DLD-1 cells after treatment with compound 27.
2
.10 Effect of compound 27 on DLD-1 3D spheroids imitating solid tumors
3
D spheroids are good mimics of in vivo tumor system in in vitro study and provide
physiologically more relevant information than 2D cultures [47]. In previous experiments, we
checked the effectiveness of compound 27 against DLD-1 cells in 2D culture. Thereafter, in
order to investigate the potential effect of compound 27 on solid tumors, we developed 3D-
spheroids of DLD-1 cells and treated them with compound 27 for 72 h, followed by live dead
staining with FDA and PI. The dye FDA produces green fluorescence when it enters the
viable cells whereas PI, the nuclear staining dye cannot pass through the cell membrane of
viable cells but can enter dead cells via the disordered membranes and stains the nuclear
DNA to produce red fluorescence. In control spheroids, there was significantly less number
of dead cells as compared to cells treated with increasing concentrations of compound 27
(6.25, 12.5 and 25µM). In contrast, the spheroids treated with compound 27 showed
increased cell death with a distinct necrotic center after 72 h of treatment, highlighted by the
increased red fluorescence visible in figure 14. More dead cells appear at the center because
1
0

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of lower levels of nutrients and oxygen supply and increased level of debris, CO and
2
,
acidosis, as compared to cells present at the periphery [48].
3
. Conclusions
In this study, we identified a novel hLigI inhibitor (compound 27) that induced
antiproliferative activity against cancer cell lines (DLD-1, MDA-MB-231, HeLa, HepG2 and
4
T1), but most prominently targeted the colon cancer cell line DLD-1 (Figure 3). The
compound specifically inhibited hLigI mediated nick sealing activity in a competitive manner
by interacting with the DNA binding residues of the protein (Figure 7 & 8). Compound 27
inhibited the migration of DLD-1 cells suggesting a potential anti-metastatic activity (Figure
1
2). Compound 27 induced apoptosis in monolayer cultures and more interestingly, was
effective against 3D-spheroids of DLD-1 cells that resembled solid tumors. Therefore, this
novel class of hLigI inhibitor is a promising lead for drug development for colorectal cancer.
The impact of ligase inhibitors in combination with radiation and DNA damaging
chemotherapeutic agents is a subject of further research in our laboratory.
4
4
. Experimental Section
.1. General Procedure: All chemical reagents were procured commercially and used
without further purification. Column chromatography was carried out on silica gel (60-120
and 100-200 mesh). All reactions were monitored by TLC (silica gel plates with fluorescence
1
13
F254). Melting points were uncorrected. The H and C NMR spectra were recorded on 400,
00 MHz and 100, 125 MHz, respectively, using DMSO- d as solvents and TMS as internal
5
6
standard. All chemical shifts values were given in ppm and their multiplicities expressed as:
s = singlet, brs = broad singlet, d = doublet, brd = broad doublet, dd = double doublet, t =
-1
triplet, q = quartet, m = multiplet. IR spectra were recorded in the range of 500 ~ 4000 cm .
High performance liquid chromatography analyses for checking purity of targeted
1
1

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compounds were performed on Waters 2998, and detection was done by UV detector. All
final compounds were found to be >95% pure by HPLC (Supporting Information).
4
.2. General synthetic procedure for the preparation of 4-Hydroxy-5-alkyl
isophthalaldehydes (5-8).
-Alkyl phenol (1.0 equiv.) and hexamethylenetetramine (HMTA) (1.2 equiv.) were
2
dissolved in TFA (25 mL) and the solution was heated at 120 ºC for 3 h. After cooling to
room temperature, 10 % aq.H SO (25 mL) was added and the temperature maintained at 90-
2
4
1
00 ºC for two more hours. The solution was basified with NaHCO to pH=8 and extracted
3
with CHCl (50 mL x 3). The combined organic layers were dried over anhy. Na SO ,
3
2
4
filtered, and concentrated to dryness under reduced pressure. The crude product thus obtained
was purified on a silica gel column (100-200 mesh) using ethylacetate-hexane (12:88, v/v) as
eluent to afford compounds 5-8 in good yields [39].
4
.3. General synthetic procedure for preparation of compounds (9-16)
A solution of 4-hydroxy-5-alkyl isophthalaldehyde 5-8 (1.0 equiv.) and 4-substituted
acetophenone (1.0 equiv.) in dioxane (15 mL) was treated with conc. HCl (2 mL). The
resultant solution was refluxed for 8-10 h. After completion of the reaction as checked by
TLC, the reaction mixture was cooled and the excess dioxane was removed under reduced
pressure. The residue thus obtained was suspended in water (50 mL) and extracted 3-fold
with CHCl (50 mL). The combined organic layers were dried on anhydrous Na SO , filtered,
3
2
4
and concentrated to dryness under reduced pressure. The residue was purified by silica gel
230-400 mesh) column chromatography using ethyl acetate-hexane (10:90, v/v) as eluent to
afford regioselective parachalcones 9-16 in moderate yields [49].
(
4
.4. General procedure for the synthesis of compounds (17-29)
1
2

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Para substituted chalcones (1 equiv.) (9-16) and malononitrile (1 equiv.) were stirred in
acetonitrile in the presence of L-proline catalyst at room temperature for half an hour. To this
o
reaction mixture different 2-methyl indole (0.8 equiv.) were added and was heated to 90 C
on an oil bath. Reaction mixture was stirred till the completion of reaction as monitored by
TLC. Afterwards, the reaction mixture was evaporated to dryness under vacumm and was
partitioned between ethyl acetate and water. The combined organic layer was dried and
purified by silica gel column chromatography to get pure products 17-29 [50].
(
E)-2-amino-8-(tert-butyl)-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)-
H-chromene-3-carbonitrile (17).
Yellow solid, yield: 75%; mp: 193 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.51 (s, 9H), 2.39
4
1
6
(
s, 3H), 2.51 (s, 3H), 5.04 (s, 1H), 6.76 (t, J = 7.9 Hz, 1H), 6.87 (s, 2H, NH ), 6.92 (t, J = 7.9
2
Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.31 (s, 1H), 7.34 (d, J = 8.1 Hz,
1
3
2
1
3
H), 7.56-7.60 (m, 3H), 7.96 (d, J = 8.2 Hz, 2H), 10.86 (s, 1H, NH); C NMR (DMSO-d ,
6
t
00 MHz) δ: 11.8 (CH of indole), 21.6 (CH of Ph), 30.4 (CH of Bu), 31.9 (CH of pyran),
3
3
3
5.2 (C(CH ) ), 56.7 (C-CN), 111.2, 115.1, 117.7, 118.8, 120.5, 121.1 (Ar), 121.7, 125.1
3
3
(CN), 125.7, 126.7, 128.9, 129.1, 129.7, 130.5, 132.4, 135.5, 135.9, 137.8 (Ar), 143.8, 143.9,
+
1
49.6 (Ar), 159.4 (C-NH ), 189.1(C=O); ESI-MS (m/z): 502.1 (M+H) ; HRMS m/z calcd for
2
+
C H N O (M+H) 502.2495, found 502.2491.
33
31
3
2
(E)-2-amino-8-(tert-butyl)-6-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl)-4-(2-methyl-1H-
indol-3-yl)-4H-chromene-3-carbonitrile(18).
1
Yellow solid, yield: 78%; mp: 190 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.49 (s, 9H), 2.50
6
(
s, 3H), 3.80 (s, 3H), 5.03 (s, 1H), 6.76 (t, J = 7.4 Hz, 1H), 6.89 (s, 2H, NH ), 6.91 (t, J = 7.5
2
Hz, 1H), 7.01(d, J = 8.2 Hz, 3H), 7.21 (d, J = 8.0 Hz, 1H), 7.28 (brs, 1H), 7.50 (d, J = 15.6
Hz, 1H), 7.54 (s, 1H), 7.60 (d, J = 15.6 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 10.84 (s, 1H, NH);
1
3
t
C NMR (DMSO-d , 100 MHz) δ:11.8 (CH ), 30.1 (CH of Bu), 31.6 (CH of pyran), 35.0
6
3
3
1
3

ACCEPTED MANUSCRIPT
(
C(CH ) ), 55.5 (OCH ), 60.8 (C-CN), 110.6 , 113.8, 114.2, 117.9, 119.4, 119.8, 121.2 (Ar),
3
3
3
1
21.4, 124.0, 125.5, 126.8, 128.1, 130.8, 131.0, 131.1, 131.9, 135.5, 137.6 (Ar) 143.5, 149.2
+
(
Ar-O), 158.1 (C-NH ), 163.4 (Ar-OMe), 188.9 (C=O); ESI-MS (m/z): 518.2 (M+H) ;
2
+
HRMS m/z calcd for C H N O (M+H) 518.2444, found 518.2442.
3
3
31
3
3
(E)-2-amino-8-(tert-butyl)-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(3, 4, 5-trimethoxyphenyl)
prop-1-en-1-yl)-4H-chromene-3-carbonitrile(19).
1
Yellow solid, yield: 75%; mp: 147 ºC; H NMR (DMSO-d , 500 MHz) δ: 1.50 (s, 9H), 2.51
6
(s, 3H), 3.75 (s, 3H), 3.86 (s, 3H), 5.05 (s, 1H), 6.76 (t, J = 7.6 Hz, 1H), 6.88 (s, 2H, NH2),
6
.92 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.31 (s, 2H), 7.34
1
3
(
s, 1H), 7.56-7.59 (m, 3H), 10.86 (s, 1H, NH); C NMR (DMSO-d , 100 MHz) δ: 11.7
6
t
(
CH ), 30.3 (CH of Bu), 31.8 (CH of pyran), 35.1 (C(CH ) ), 56.6 (OCH ), 60.6 (C-CN),
3
3
3 3
3
1
06.6, 111.2, 115.0, 117.7, 118.8, 120.5, 121.1 (Ar), 121.8, 125.2 (CN), 126.1, 126.7, 128.6,
30.5, 132.4, 133.5, 135.8, 137.8 142.3, 143.9 (Ar), 149.6 (Ar-O), 153.3(Ar-OMe), 159.5(C-
1
+
+
NH ), 188.5(C=O); ESI-MS (m/z): 578.1 (M+H) ; HRMS m/z calcd for C H N O (M+H)
2
35 35
3
5
5
78.2655, found 578.2649.
(E)-2-amino-8-(tert-butyl)-6-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)-4-(2-methyl-1H-
indol-3-yl)-4H-chromene-3-carbonitrile (20).
1
Yellow solid, yield: 77%; mp: 157 ºC H NMR (DMSO-d , 400 MHz) δ: 1.51 (s, 9H), 2.51
6
(
s, 3H), 5.04 (s, 1H), 6.76 (t, J = 7.5 Hz, 1H), 6.87 (s, 2H, NH ), 6.92 (t, J = 7.4 Hz, 1H), 7.01
2
(d, J = 7.9 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.32 (s, 1H), 7.59-7.61 (m, 5H), 8.07 (d, J = 8.6
1
3
Hz, 2H), 10.86 (s, 1H, NH); C NMR (DMSO-d , 100 MHz) δ: 11.8 (CH ), 30.4 (CH of
6
3
3
t
Bu), 31.9 (CH of pyran), 35.2 (C(CH ) ), 56.7 (C-CN), 111.2, 115.0, 117.7, 118.8, 120.5,
3
3
1
1
21.0, 121.4, 125.1, 125.9, 126.7, 129.1, 129.3, 130.4, 130.9, 132.4, 135.9, 136.7, 137.8,
+
38.3, 144.7 (Ar), 149.8 (Ar-O), 159.4 (C-NH ), 188.7 (C=O); ESI-MS (m/z): 522.0 (M+H) ;
2
+
HRMS m/z calcd for C H ClN O (M+H) 522.1948, found 522.1948.
3
2
28
3
2
1
4

ACCEPTED MANUSCRIPT
(E)-2-amino-8-(sec-butyl)-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(3, 4, 5-trimethoxyphenyl)
prop-1-en-1-yl)-4H-chromene-3-carbonitrile (21).
1
Yellow solid, yield: 70%; mp: 220 ºC; H NMR (CDCl , 400 MHz) δ: 0.93-0.97 (m, 3H),
3
1
3
7
.34 (dd, J = 4.6Hz, J = 2.3Hz, 3H), 1.69-1.74 (m, 2H), 2.49 (s, 3H), 3.17-3.25 (m, 1H),
1 2
.89 (s, 6H), 3.94 (s, 3H), 4.66 (s, 2H), 5.14 (s, 1H), 6.90-6.95 (m, 1H), 7.05-7.19 (m, 6H),
1
3
.25-7.28 (m, 1H), 7.33 (s, 1H), 7.56 (t, J = 15.7Hz, 1H), 8.09 (s, 1H, NH); C NMR
(
CDCl , 100 MHz) δ:11.8 (CH ), 12.1, 20.6, 30.3, 31.6, 33.7, 56.4, 60.7, 60.9 (Aliphatic),
3 3
1
1
5
06.2, 110.6, 113.9, 117.9, 119.4, 121.1, 121.5, 123.4, 125.8, 126.9, 127.5, 131.3, 132.1,
33.5, 135.3, 135.5, 142.4, 144.2, 148.1, 153.1, 158.6 (C-NH ), 189.7 (C=O); ESI-MS (m/z):
2
+
+
78.1 (M+H) ; HRMS m/z calcd for C H N O (M+H) 578.2655, found 578.2660.
3
5
35
3
5
(E)-2-amino-8-(tert-butyl)-4-(5-methoxy-2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(p-tolyl)prop-1-
en-1-yl)-4H-chromene-3-carbonitrile(22).
1
Yellow solid, yield: 72%; mp: 163 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.51 (s, 9H), 2.39
6
(
s, 3H), 2.51 (s, 3H), 3.60 (s, 3H), 5.00 (s, 1H), 6.53 (s, 1H), 6.58 (dd, J = 6.2Hz, J = 2.4Hz,
1 1
1
7
2
1
H), 6.89 (s, 2H, NH ), 7.11 (d, J = 8.7Hz, 1H), 7.34 (d, J = 8.1Hz, 3H), 7.58-7.61 (m, 3H),
2
1
3
.97 (d, J = 8.2Hz, 2H), 10.69 (s, 1H,NH); C NMR (DMSO-d , 100 MHz) δ:11.9 (CH ),
6
3
1.6, 30.4, 31.9, 35.2, 55.5, 56.5 (Aliphatic), 100.2, 109.9, 111.7, 115.0, 121.1, 121.7, 125.1,
25.7, 127.0, 128.9, 129.1, 130.6, 130.9, 133.1, 135.5, 137.9, 143.9, 149.5, 153.2, 159.6 (C-
+
+
NH ), 189.2 (C=O); ESI-MS (m/z): 532.1 (M+H) ; HRMS m/z calcd for C H N O (M+H)
2
34 33
3
3
5
32.2600, found 532.2602.
(E)-2-amino-8-(tert-butyl)-4-(5-chloro-2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(p-tolyl)prop-1-
en-1-yl)-4H-chromene-3-carbonitrile (23).
1
Yellow solid, yield: 75%; mp: 175 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.52 (s, 9H), 2.38
6
(s, 3H), 2.53 (s, 3H), 5.03 (s, 1H), 6.91-6.96 (m, 4H), 7.23 (d, J = 8.4 Hz, 1H), 7.34 (d, J =
8
.0 Hz, 3H), 7.56 (d, J = 15.6Hz, 1H), 7.60 (s, 1H), 7.64 (d, J = 15.7Hz, 1H), 7.97 (d, J = 8.1
1
5

ACCEPTED MANUSCRIPT
1
3
Hz, 2H), 11.09 (s, 1H, NH); C NMR (DMSO-d , 100 MHz) δ:11.8 (CH ), 21.6, 30.4, 31.8,
6
3
3
1
1
5
5.2, 56.4, 79.6 (Aliphatic), 112.6, 115.3, 116.9, 120.3, 120.9, 121.8, 123.5, 124.7, 125.9,
27.7, 128.9, 129.1, 129.7, 130.7, 134.3, 135.5, 137.9, 143.7, 143.8, 149.6, 159.4 (C-NH2),
+
+
89.1 (C=O); ESI-MS (m/z): 536.2 (M+H) ; HRMS m/z calcd for C H ClN O (M+H)
3
3
30
3
2
36.2105, found 536.2109.
E)-2-amino-8-isopropyl-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-
yl)-4H-chromene-3-carbonitrile (24).
(
1
Yellow solid, yield: 70%; mp: 183 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.32-1.35 (m, 6H),
6
2
.52 (s, 3H), 3.46-3.53 (m, 1H), 5.05 (s, 1H), 6.77 (t, J = 7.3 Hz, 1H), 6.84 (s, 2H, NH ), 6.93
2
(t, J = 7.3Hz, 1H), 6.99 (d, J = 7.9Hz, 1H), 7.23 (d, J = 7.9Hz, 2H), 7.28 (t, J = 4.7Hz, 1H),
7
8
2
1
1
.54 (d, J = 15.6 Hz, 1H), 7.64 (d, J = 15.6Hz, 1H), 7.72 (s, 1H), 8.02 (d, J = 4.3Hz, 1H),
13
.26 (d, J = 3.3 Hz, 1H), 10.86 (s, 1H,NH); C NMR (DMSO-d , 100 MHz) δ:11.8 (CH ),
6
3
2.8, 23.4, 26.9, 31.8, 56.8 (Aliphatic), 111.2, 114.7, 117.7, 118.8, 120.5, 121.1, 121.3,
24.4, 125.1, 126.8, 129.0, 129.2, 130.8, 132.5, 134.1, 135.8, 135.9, 136.5, 143.2, 145.9,
+
48.1, 159.9 (C-NH ), 181.9 (C=O).; ESI-MS (m/z): 480.1 (M+H) ; HRMS m/z calcd for
2
+
C H N O S (M+H) 480.1746, found 480.1748.
29
25
3
2
(E)-2-amino-8-isopropyl-4-(5-methoxy-2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(thiophen-2-
yl)prop-1-en-1-yl)-4H-chromene-3-carbonitrile (25).
1
Light yellow solid, yield: 72%;mp: 156 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.32-1.36 (m,
6
6
H), 2.50 (s, 3H), 3.44-3.51 (m, 1H), 3.60 (s, 3H), 5.01 (s, 1H), 6.49 (d, J = 2.3Hz, 1H), 6.58
(
dd, J = 6.2Hz, J =2.4Hz, 1H), 6.86 (s, 2H), 7.11 (d, J = 8.7Hz, 1H), 7.25 (s, 1H), 7.28-7.31
1
2
(
m, 1H), 7.54-7.68 (m, 2H), 7.72 (s, 2H), 8.03 (dd, J = 3.9Hz, J =1.0Hz, 1H), 8.27 (dd, J =
1 2 1
1
3
2
2
1
.8Hz, J =1.0Hz, 1H), 10.69 (s, 1H, NH); C NMR (DMSO-d , 125 MHz) δ:11.9 (CH ),
2 6 3
2.8, 23.4, 27.2, 31.9, 55.4, 56.7 (Aliphatic), 100.5, 109.7, 111.7, 114.7, 121.1, 121.4, 124.4,
25.2, 127.1, 129.0, 129.2, 130.8, 130.9, 133.2, 134.0, 135.8, 136.4, 143.2, 145.9, 148.2,
1
6

ACCEPTED MANUSCRIPT
+
1
53.2, 159.9 (C-NH ), 181.9 (C=O).; ESI-MS (m/z): 510.0 (M+H) ; HRMS m/z calcd for
2
+
C H N O S (M+H) 510.1851, found 510.1847.
30
27
3
3
(E)-2-amino-8-(tert-butyl)-4-(5-chloro-2-methyl-1H-indol-3-yl)-6-(3-(4-methoxyphenyl)-3-
oxoprop-1-en-1-yl)-4H-chromene-3-carbonitrile (26).
1
Light yellow solid, yield: 72%;mp: 202 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.51 (s, 9H),
6
2
.52 (s, 3H), 3.84 (s, 3H), 5.02 (s, 1H), 6.92-6.93 (m, 4H), 7.04 (d, J = 8.5Hz, 2H), 7.22 (d, J
8.4Hz, 1H), 7.33 (s, 1H), 7.53 (d, J = 15.6Hz, 1H), 7.58 (s, 1H), 7.64 (d, J = 15.6Hz, 1H),
=
1
3
8
3
1
1
.06 (d, J = 8.4Hz, 2H), 11.09 (s, 1H); C NMR (DMSO-d , 100 MHz) δ:11.8 (CH ), 30.4,
6 3
1.8, 35.2, 55.9, 56.4 (Aliphatic), 112.7, 114.4, 115.3, 116.9, 120.4, 121.0, 121.8, 123.5,
24.7, 125.9, 127.7, 128.8, 130.8, 130.9, 131.4, 134.4, 137.9, 143.3, 149.5, 159.5 (C-NH2),
+
63.6 (Ar-OMe), 187.9 (C=O).; ESI-MS (m/z): 536.1 (M+H) ; HRMS m/z calcd for
+
C H ClN O (M+H) 552.0626, found 552.2047.
33
30
3
2
(
E)-2-amino-8-(tert-butyl)-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(thiophen-2-yl)prop-1-en-
-yl)-4H-chromene-3-carbonitrile (27).
Light yellow solid, yield: 70%; mp: 208 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.52 (s, 9H),
1
1
6
2
.52 (s, 3H), 5.05 (s, 1H), 6.76 (t, J = 7.2Hz, 1H), 6.88 (s, 2H), 6.92 (t, J = 7.1Hz, 1H), 7.02
(
(
d, J = 8.0Hz, 1H), 7.23 (d, J = 8.0Hz, 1H), 7.28 (t, J = 4.6Hz, 1H), 7.32 (s, 1H), 7.58-7.60
13
m, 3H), 8.01 (d, J = 4.3Hz, 1H), 8.24 (d, J = 3.4Hz, 1H), 10.86 (s, 1H); C NMR (DMSO-
d , 100 MHz) δ:11.8 (CH ), 30.4, 31.9, 35.2, 56.7, 79.6 (Aliphatic), 111.2, 115.0, 117.7,
6
3
1
1
18.8, 120.5, 121.1, 121.3, 125.1, 125.9, 126.7, 129.0, 129.3, 130.3, 132.4, 134.1, 135.8,
+
37.9, 143.4, 145.8, 149.7, 159.4 (C-NH ), 181.9 (C=O).; ESI-MS (m/z): 494.2 (M+H) ;
2
+
HRMS m/z calcd for C H N O S (M+H) 494.1902, found 494.1897.
3
0
27
3
2
(E)-2-amino-8-methyl-4-(2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)-
4
H-chromene-3-carbonitrile (28).
1
7

ACCEPTED MANUSCRIPT
1
Light yellow solid, yield: 72%;mp: 202 ºC; H NMR (DMSO-d , 500 MHz) δ: 2.39 (s, 3H),
6
2
.51 (s, 3H), 5.05 (s, 1H), 6.78 (t, J = 7.2Hz, 1H), 6.83 (s, 2H, NH ), 6.93 (t, J = 7.8Hz, 1H),
2
6
.97 (d, J = 8.1Hz, 1H), 7.17 (s, 1H), 7.23 (d, J = 8.0Hz, 1H), 7.28 (t, J = 4.0Hz, 1H), 7.50 (d,
J = 15.5Hz, 1H), 7.62 (d, J = 15.5Hz, 1H), 7.73 (s, 1H), 8.02 (d, J = 4.9Hz, 1H), 8.22 (d, J =
1
3
3
5
1
.7Hz, 1H), 10.87 (s, 1H, NH); C NMR (DMSO-d , 100 MHz) δ:11.8 (CH ), 15.9, 31.6,
6 3
6.9 (Aliphatic), 111.2, 114.4, 117.7, 118.8, 120.5, 121.1, 121.4, 124.1, 126.1, 126.8, 128.9,
29.3, 130.5, 132.7, 133.9, 135.8, 135.9, 142.9, 145.9, 149.1, 159.8 (C-NH ), 181.9 (C=O).;
2
+
+
ESI-MS (m/z): 452.0 (M+H) ; HRMS m/z calcd for C H N O S (M+H) 452.1433, found
2
7
21
3
2
4
52.1432.
(E)-2-amino-8-(tert-butyl)-4-(5-methoxy-2-methyl-1H-indol-3-yl)-6-(3-oxo-3-(thiophen-2-
yl)prop-1-en-1-yl)-4H-chromene-3-carbonitrile (29).
1
Light yellow solid, yield: 70%; mp: 209 ºC; H NMR (DMSO-d , 400 MHz) δ: 1.51 (s, 9H),
6
2
6
8
.52 (s, 3H), 3.60 (s, 3H), 5.00 (s, 1H), 6.52 (s, 1H), 6.58 (dd, J = 6.5Hz, J = 2.1Hz, 1H),
1 2
.89 (s, 2H, NH ), 7.11 (d, J = 8.6 Hz, 1H), 7.28 (t, J = 4.0Hz, 1H), 7.34 (s, 1H), 7.59 (s, 2H),
2
1
3
.03 (d, J = 4.4Hz, 1H), 8.25 (d, J = 3.1Hz, 1H), 8.32 (s, 1H), 10.69 (s, 1H, NH); C NMR
(
DMSO-d , 100 MHz) δ:11.9, 30.4, 31.9, 35.2, 55.5, 56.5, 79.6 (Aliphatic), 100.2, 109.9,
6
1
1
11.7, 115.0, 121.1, 121.4, 125.2, 125.9, 127.0, 129.2, 130.3, 130.9, 133.1, 134.1, 135.8,
37.9, 143.4, 145.8, 149.6, 153.2, 159.6 (C-NH ), 181.9 (C=O).; ESI-MS (m/z): 524.3
2
+
+
(
M+H) ; HRMS m/z calcd for C H N O S (M+H) 524.2008, found 524.2004.
31 29 3 3
4
.5. Biological Methods
4
.5.1. In vitro DNA nick sealing (ligation) assay
DNA ligation assays were performed as previously described [26]. For the generation of
linear duplex DNA ligase substrate, we used three different single strand DNA oligomers, 52-
mer (5′-GTACGTCGATCGATTGGTAGATCAGTGTCTATGTATGTCAGTGAGATAGT
1
8

ACCEPTED MANUSCRIPT
AC-3′), 25-mer (5′-CTGATCTACCAATCGATCGACGTAC-3′) and 27-mer (5′-
/
5Cy3/GTACTATCTCACTGACATACATAGACA-3′) and annealed them together to form
a ds nicked DNA substrate for DNA ligase I nick sealing activity, in the presence of ATP.
The reaction mixture (20 µL) contained 1 pmol of labeled DNA substrate and 0.2 pmol of
purified hLigI in a ligation buffer containing Tris-Cl (50 mM, pH 7.5), MgCl2 (10 mM),
BSA (0.25 mg/mL), NaCl (100 mM), and ATP (500 µM). The nicked duplex DNA was
incubated with purified hLigI in the absence or presence of inhibitors at 37 °C for 30 min.
After 30 min, 10 µL of stop buffer (90% formamide and 10% of 50 mM EDTA) was added to
stop the reaction. This reaction mixture was loaded onto a denaturing gel containing 7 M urea
and 12% acrylamide used for the separation of ligated and un-ligated DNA products. Ligated
duplex DNA appeared higher in the gel due to the larger size, whereas un-ligated product
would appear lower due to smaller size. If an inhibitor is added to the reaction mixture, this
would lead to an inhibition of ligation and a corresponding loss of labelled ligated product in
the gel. The percentage inhibition of ligation was estimated by measuring the density (by GE
Image Quant LAS 4010) of ligated product in the lane without inhibitor and comparing it
with the lanes containing different inhibitors or with the same inhibitor at different
concentrations. Experiments were carried out in triplicate and average values are represented
graphically.
4
.5.2. Cell culture and viability assays
To determine whether the synthesized compounds could affect proliferation of cancer cells,
various types of cancerous cell lines were cultured. The human breast cancer (MDA-MB-
2
31) and hepatic cancer (HepG2) cell lines (ATCC, Manassas, VA) were grown in DMEM.
Human cervical cancer cell line (HeLa) obtained from NCCS (National Centre for Cell
Science), Pune, India, human colon cancer cell line DLD-1 (ECACC, Salisbury, UK) and
1
9

ACCEPTED MANUSCRIPT
mouse breast cancer cell line 4T1 (ATCC, Manassas, VA) were cultured in RPMI 1640.
Human embryonic kidney (HEK-293) cell line was obtained from (ATCC, Manassas, VA)
and cultured in EMEM. All media were supplemented with 10% (v/v) fetal bovine serum
(FBS) and 1% antibiotic–antimycotic solution. To study the antiproliferative activity of
compounds, 10 mM stock solutions of compounds were prepared by dissolving them in
DMSO (Sigma St. Louis, MO). MTT assay was done as described by ATCC, Manassas, VA.
Briefly, cells were seeded in 96 well plates in 100 µL media per well and incubated
overnight. After overnight cell adhesion, various concentrations of compounds dissolved in
DMSO were added to cells and DMSO alone was used as vehicle control. Cells were grown
for 48h to test the effect of compounds. For MTT assay 10 µL of MTT (5mg/ml) was added
to each well and incubated for 3 h. The formazan crystals formed by live cells were dissolved
by adding DMSO and shaking until completely dissolved at room temperature. Absorbance
was measured at 590 nm on the plate reader (Epoch Microplate Reader, Biotek, USA). The
measurement of control cells was defined as 100% growth and the percentage growth
inhibition of treated cells was calculated from other measurements. Each experiment was
done in triplicate. Percentage inhibition of cell growth (anti proliferative activity) was
measured by the formula: [(Absorbance of control cells - Absorbance of inhibitor treated
cells)/ Absorbance of control cells] X 100 and reported as average of three independent
biological replicates.
4
.5.3. Electrophoretic mobility shift assay (EMSA)
EMSA was performed to check the interaction of compound 27 with DNA substrate and
hLigI as described previously [14]. Three different oligos were used to generate a double
stranded, non-ligatable nicked DNA substrate. One was a 5’-FAM labelled 27-mer oligo with
a dideoxy modified 3’ end (5’-/56-FAM/GTACTATCTCACTGACATACATAGAC/3ddc/-
2
0

ACCEPTED MANUSCRIPT
3
’), where FAM labelling and dideoxy modification was used for easy detection of DNA
substrate and for creating the non ligatable nick, respectively. The other two oligos were 25-
mer (5’-CTGATCTACCAATCGATCGACGTAC-3’) and 52-mer (5’-GTACGTCGATCG
ATTGGTAGATCAGGGTCTATGTATGTCAGTGAGATAGTAC-3’) which were annealed
to form a non-ligatable nicked DNA substrate for ligase I. In EMSA, the reaction volume of
2
1
0µL contained 10 pmole of hligI with different concentrations of compound (31.25, 62.5,
25, 250, 500 µM) and 2 pmole non-ligatable nicked DNA substrate in a ligation buffer
containing Tris-Cl (50 mM, pH7.5), MgCl (10 mM), BSA (0.25 mg/ml), NaCl (100mM),
2
ATP (500 mM) were incubated for two hours on ice. 10µL of native gel buffer (50mM TrisCl
pH7.5, 20% glycerol, 0.05% bromophenol blue) was added and samples were separated by
electrophoresis in 6.5% native polyacrylamide gel and bands were detected by GE Image
Quant LAS4010. Represented graphically after three independent experiments in figure 8.
4
.5.4. DNase I protection assay
We performed the DNase I protection assay to test the interaction of compound 27 with DNA
as described by Cardew et al [51]. For this experiment 100 ng of linearized plasmid DNA
(pUC18) was incubated with the DNA intercalating agent doxorubicin at 100 µM
concentration and then competed with DAPI, methyl green, ampicillin (all at 500 µM
concentration) and compound 27 at three different concentrations (250, 500, 700µM) for 30
min at 37˚C in an incubation buffer similar to ligation buffer. After incubation, 0.0025U of
DNase1 was added to the reaction mixture for DNA cleavage and incubated for 5 min at
3
7˚C. After 5 min, the stop buffer containing 7 M Urea, 20 mM Tris-Cl (pH. 7.5), 50 mM
EDTA, 20% glycerol and bromophenol blue, was added to stop the reaction. The samples
were then resolved in 1% agarose gel and visualized under UV transilluminator and image
captured with GE Image Quant LAS4010.
2
1

ACCEPTED MANUSCRIPT
4
.5.5. In vitro cell migration assay
The migration of DLD-1 cells was assessed by the wound healing assay as described by
Mishra et al [52]. Cells were seeded in 6 well plates and grown at 70-80 % confluence. The
wound was created with a 200 µL pipette tip in the middle of the well and then cells were
treated with different concentrations of compound 27 for 12, 24 and 36 h. Wound closure was
measured after different time intervals and imaged with phase contrast microscopy under an
inverted microscope (Ziess Primovert using a 10 x phase contrast objective) and quantified
by Image-J software. Average values were represented in graphical form after three
independent experiments.
4
.5.6. Colony formation assay
Colony formation assay was performed as described previously by Chen et al with some
modifications [45], 400 cells of DLD-1 cell line were seeded per well in 12 well plates and
grown overnight in RPMI media containing 10% FBS. Thereafter, different concentrations
(1.25, 2.5 and 5µM) of compound 27 were added and cells were grown for 24 h and 48 h.
After the desired time point, the culture medium was replaced with fresh medium and cells
were grown for another 10 days, exchanging the medium every third day. To observe the
colonies formed, the cells were washed with 1X PBS and stained and fixed with 0.5% crystal
violet and 6% glutaraldehyde for 30 min at room temperature. Finally, the excess stain was
washed with tap water, plates were dried and images were captured.
4
.5.7. Measurement of Apoptosis by Annexin V-FITC assay
To measure compound 27 induced apoptosis in colon cancer DLD-1 cells, we performed
Annexin V-FITC staining by using a commercially available kit (BD Biosciences, San Diego,
CA). Different concentrations of compound 27 (2.5, 5 and 10µM) were added to DLD-1 cells
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for 48 h. After 48 h of treatment, the cells were harvested, washed with cold PBS and stained
with Annexin V-FITC and propidium iodide (PI) in binding buffer at room temperature in the
dark, according to manufacturer's instructions. Analysis of the stained cells was done by
fluorescence-activated cell sorter using a FACS-Calibur instrument (BD Biosciences, San
Diego, CA) equipped with CellQuest 3.3 Software and average values were plotted
graphically from three independent experiments.
4
.5.8. Generation of DLD-1 cell spheroids and study of antiproliferative activity of compound
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DLD-1 spheroids were generated via the ‘hanging drop’ technique as described previously
3
with some modifications [48, 53]. In short, 5 X 10 DLD-1 cells/ 10 µL RPMI was placed on
the lid of a non-adhesive PBS containing petri dish. Spheroids were incubated for 3 days at
3
7˚C, in a 5% CO2 incubator. The spheroids were transferred to 0.7% agarose coated 24 well
plates containing 10 % RPMI medium. The medium was exchanged after every third day.
After 7 days the spheroids were treated with compound 27 at different concentrations (25,
1
2.5 and 6.25) for 72 h. Spheroids were stained with staining solution containing 2µg/ml
FDA (Fluorescein diacetate) and 20µg/ml PI (Propidium iodide) in PBS and incubated for 20
min in the dark, as previously described [54]. The stained spheroids were washed with PBS
and then visualized and imaged under Nikon Eclipse Ti fluorescence microscope.
4
.5.9. Molecular modelling studies
To model the binding of compound 27 with hLigI, we performed the in silico docking of
compound 27 with the crystal structure of hLigI retrieved from the PDB database (PDB
ID:1X9N) [15]. The co-crystallized nicked DNA and Adenosine Monophosphate moieties
were removed before performing the docking experiments. Subsequently, the hydrogen atoms
were added to the protein structure. Finally, the energy minimization was accomplished
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applying the Sybyl7.1 software suite [55]. The details of the structure preparation and
validation of docking protocol are published by us earlier [26]. The structure of compound 27
was drawn using sketch module of Sybyl7.1 software suite. Further, this structure was
minimized using MMFF4 force field by using Sybyl7.1. Docking was performed using the
FlexX module of Sybyl7.1 [56].
4
.5.10. Statistical analysis
All experiments were performed in triplicate and reported as mean ± SEM. Statistical
analysis were performed through the Graph Pad Prism software version 5 (San Diego
California USA). For the measurement of DNA ligation, cytotoxicty, EMSA and apoptosis,
one-way ANOVA followed by Bonferroni post-test were used to assess the statistical
significance between control and treated groups. A value of p<0.05 was considered
statistically significant. ANOVA showed a significant difference from the control: *(P<0.05),
*
*(P < 0.01), ***(P < 0.001), ns (not significant).
Acknowledgment
The authors acknowledge CSIR-CDRI, Govt. of India, for financial and infrastructural
support. Instrumentation facilities from SAIF, CDRI, are gratefully acknowledged. Financial
support is also acknowledged from Department of Biotechnology (DBT), Govt. of India
(
Grant-BT/PR6421/GBD/27/436/2012), the Department of Science and Technology (DST),
Govt. of India (Grant-SB/FT/LS-163/2012) and CSIR Network projects GENESIS
BSC0121) and Biopros (BSC0106). We acknowledge Dr. S. P. Singh (Technical Officer),
(
CSIR-CDRI, for technical assistance. The authors are also thankful to Sophisticated
Analytical Instrument Facility (SAIF) CDRI, Lucknow, India, for providing spectral analysis
data of chemical compounds and for support with cell based flow cytometry experiments. SG
is thankful to CSIR, New Delhi for financial support. PM is thankful to DBT for financial
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support. AU and PK are thankful to UGC, New Delhi for financial support. This manuscript
bears the CDRI manuscript number 116/2017/KVS.
Additional Information-The authors declare no competing financial interests.
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Captions to illustrations
Figure 1: Strategy for designing indole-chalcone based benzopyran molecules.
Figure 2: Effect of 13 indole-chalcone based benzopyrans on the nick sealing activity of
human DNA ligase I. Denaturing gel images showing inhibition of hLigI nick sealing activity
in the presence of test compounds at 10 µM concentration and the graphical representation of
percentage inhibition of nick sealing activity present in purified hLigI.
Figure 3: Percentage antiproliferative activity of indole-chalcone based benzopyrans in
various cancer cell lines (A) MDA-MB-231, (B) DLD-1, (C) HeLa, (D) HepG2 and (E) 4T1.
Doxorubicin was used as a control to compare antiproliferative activity.
Figure 4: IC values of compound 27 in various cancer (MDA-MB-231, DLD-1, HeLa,
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HepG2 and 4T1) and normal (HEK-293) cell lines.
Figure 5: Cartoon representation of SAR studies shows the groups important for inhibition of
DNA nick sealing activity and anti-proliferative activity.
Figure 6: Inhibition of DNA nick sealing (ligation) activity by compound 27 at 3.125, 6.25,
1
2.5, 25 and 50 µM concentrations against various human (hLigI, hLigIII and
hLigIV/XRCC4) and non-human DNA ligases (T4 DNA ligase).
Figure 7: In silico docking of compound 27 at the DNA binding site of hLigI (crystal
structure 1X9N) represented as a cartoon. Compound 27 is shown in purple sticks, the
interacting residues of hLigI are shown as cyan sticks; black dashed lines represent the
hydrogen bonds.
Figure 8: Electrophoretic Mobility Shift Assay (EMSA) for compound 27 with DNA-protein
complex shows a concentration dependent inhibition of complex formation, suggesting a
competitive mode of inhibition.
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Figure 9: DNase I protection assay for DNA incubated with compound 27 and various other
controls. The representative agarose gel shows that unlike Doxoburicin, Methyl green and
DAPI compound 27 was not able to protect DNA from DNase digestion, suggesting that it
does not bind to DNA.
Figure 10: Bright field images of DLD-1 cells after treatment with compound 27 at various
concentrations (2.5, 5 and 10 µM) for 24 and 48 h shows changes in cellular morphology,
indicating cellular stress.
Figure 11: Images of colony formation assay showed inhibition of colony formation by
DLD-1 cells in the presence of compound 27 in a dose and time dependent manner.
Figure 12: Bright field images of inhibition of DLD-1 cell migration in the presence of
compound 27. The graph shows significant decrease in % open area in control versus treated
cells after different time points, indicating reduced cellular migration.
Figure 13: Induction of apoptosis in DLD-1 cells after treatment of compound 27 at different
concentrations for 48 h. The percentage population of early and late apoptotic cells at
different concentrations of the compound is represented graphically.
Figure 14: Evaluation of cell viability in 3D spheroids of DLD-1 cells in the presence of
compound 27 at different concentrations (from left to right: phase-contrast image, FDA-
signal, PI-signal, composite of FDA and PI signal). (Scale bar: 200 µm).
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H C
3
N
Cl
N
H C
3
O
Br
O
N
HN N
H
N
CH3
Cl
O
H C
3
Indole derivatives showing anti-cancer activity
N
O
CN
R
R
HN
H C
3
O
NH2
R4
N
R3
O
R2
X
O
CN
R2
O
R3
OMe
O
NH2
MeO
Br
R1
O
O
R1
A
CN
Chalcone derivatives having anti-
cancer activity
Indole-Chalcone based Benzopyrans
H C
3
N
CH3
O
NH2
(Our prototype)
Benzopyran derivatives with
potential anti cancer activity
Figure 1: Strategy for designing indole-chalcone based benzopyran hybrids.
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