N-Benzylation of 6-aminoflavone by reductive amination and efficient access to some novel anticancer agents via topoisomerase II inhibition

Article abstract of DOI:10.1007/s11030-020-10079-1

Series of novel N-benzyl derivatives of 6-aminoflavone (9a–n) were synthesized and evaluated for anticancer and topoisomerase II enzyme inhibition activity. All the synthesized compounds were screened for in vitro anticancer activity against human breast cancer cell line (MCF-7) and human lung cancer cell line (A-549). Among the synthesized compounds, 9f and 9g were found to be the most potent anticancer agents against human breast cancer cell line (MCF-7) with IC₅₀ values of 9.35?μM and 9.58?μM, respectively. Compounds 9b, 9c and 9n exhibited promising anticancer activity against human lung cancer cell line (A-549) with 43.71%, 46.48% and 44.26% inhibition at the highest concentration of 10?μM, respectively. Compounds 9c, 9f and 9g have ability to inhibit the topoisomerase II enzyme. Compound 9f showed most potent topoisomerase II enzyme inhibition activity with IC₅₀ value of 12.11?μM. Further, these compounds have a high potential to be developed as a promising topoisomerase II inhibitors.

Full text of DOI:10.1007/s11030-020-10079-1

Molecular Diversity
https://doi.org/10.1007/s11030-020-10079-1
ORIGINAL ARTICLE
N‑Benzylation of 6‑aminofavone by reductive amination and efcient
access to some novel anticancer agents via topoisomerase II inhibition
Nitin M. Thorat1,2 · Aniket P. Sarkate3 · Deepak K. Lokwani4 · Shailee V. Tiwari5 · Rajaram Azad6 ·
Shankar R. Thopate1,7
Received: 28 November 2019 / Accepted: 23 March 2020
© Springer Nature Switzerland AG 2020
Abstract
Series of novel N-benzyl derivatives of 6-aminofavone (9a–n) were synthesized and evaluated for anticancer and topoi-
somerase II enzyme inhibition activity. All the synthesized compounds were screened for in vitro anticancer activity against
human breast cancer cell line (MCF-7) and human lung cancer cell line (A-549). Among the synthesized compounds, 9f and
9g were found to be the most potent anticancer agents against human breast cancer cell line (MCF-7) with IC50 values of
9.35 µM and 9.58 µM, respectively. Compounds 9b, 9c and 9n exhibited promising anticancer activity against human lung
cancer cell line (A-549) with 43.71%, 46.48% and 44.26% inhibition at the highest concentration of 10 µM, respectively.
Compounds 9c, 9f and 9g have ability to inhibit the topoisomerase II enzyme. Compound 9f showed most potent topoi-
somerase II enzyme inhibition activity with IC50 value of 12.11 µM. Further, these compounds have a high potential to be
developed as a promising topoisomerase II inhibitors.
Keywords Aminofavones · Buchwald coupling · Reductive amination · Anticancer agent · Topoisomerase II enzyme
inhibitor · Malic acid
Introduction
[1]. Scientifc fndings have shown that the favones inhibit
mitosis [2, 3], angiogenesis [4] and enzymes such as tyros-
ine kinase [5], topoisomerase [6–8], aromatase [9], epoxide
hydrase [10], and tubulin polymerization [11–14]. Most of
these compounds are either natural products or derivatives
(Fig. 1). Aminofavones are a class of compounds having
nitrogen directly attached to aromatic sp2 carbon, exhib-
its diverse biological activity. Substituted aminofavones
(Fig. 1) show anti-tubercular, cytotoxic and kinase inhibitory
activity with anti-proliferative efects [15, 16]. Compound
Processes of cell growth viz. proliferation, migration, and
angiogenesis are regulated by the various growth factors and
mitogen-stimulated signaling networks. Any dysregulation
of these factors can lead to cancer. Flavones are known to
have anticancer activity through modulation of these factors
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10079-1) contains
supplementary material, which is available to authorized users.
4
* Shankar R. Thopate
Department of Pharmaceutical Chemistry, R.C. Patel
srthopate@gmail.com
Institute of Pharmaceutical Education and Research, Shirpur,
Maharashtra 425405, India
1
Department of Chemistry, Prof. John Barnabas Post
5
Department of Pharmaceutical Chemistry, Durgamata
Graduate School for Biological Studies, Ahmednagar
College, Ahmednagar, Station Road, Ahmednagar,
Maharashtra 414001, India
Institute of Pharmacy, Dharmapuri, Parbhani,
Maharashtra 431401, India
6
Department of Animal Biology, University of Hyderabad,
2
Department of Chemistry, Maharaja Jivajirao Shinde
Hyderabad 500046, India
Arts, Science, Commerce College, Shrigonda,
Maharashtra 413701, India
7
Department of Chemistry, Shri Sadguru Gangageer Maharaj
Science, Gautam Arts and Sanjivani Commerce College,
Kopargaon, Dist. Ahmednagar, Maharashtra 423 601, India
3
Department of Chemical Technology, Dr. Babasaheb
Ambedkar Marathwada University, Aurangabad,
Maharashtra 431004, India
Vol.:(0123456789)
1 3

Molecular Diversity
B
O
O
O
O
HO
A
C
OH
Flavonoid
Naphthaflavone
Chrysin
Epoxide hydrase activity
Aromatase inhibition
OH
OH
NH2
H
N
N
F
NH2
HO
O
OH
O
O
O
O
F
HO
F
OH
O
NH2
OH
O
Luteolin
Tyrosine kinase
inhibition
AFP464
Phase I clinical trial:
Cancer
Rohitukine
Cyclin-dependent
kinases (CDKs) inhibitors
OH
O
OCH3
O
OH
O
O
O
HO
O
OH
H
OH
O
OH
CH3
O
O
Fisetin
Psorospermin
MHY336
Fig. 1 Biologically active favonoid structures
AFP646 is in phase-I clinical trial for cancer treatment [17].
A synthetic favonoid MHY336 has ability to arrest the cell
cycle in G2/M or S phase via Topo-II-dependent mecha-
nism [18]. Dietary favonoid fsetin acts as a dual inhibi-
tor of Topo-I and Topo-II in cells [19]. Psorospermin, a
natural antitumor antibiotic [20], intercalates DNA, and its
alkylating potential is signifcantly increased in the pres-
ence of Topo-II [21, 22]. Unfortunate negligence of nature
to aminofavonoids as natural products might be attributed
to intricacy in the synthesis of aminofavones [15, 16], par-
ticularly those in which amino group is attached to A ring.
This indeed limited structure activity relationship (SAR)
studies of aminofavones and related derivatives. Simon and
co-workers have synthesized diferently substituted 6-amino-
favones and shown that structural modifcations particularly
in B-ring would not have much impact on anticancer activity
[23]. To improve inhibitory potency of 6-aminofavone and
to build SAR, a series of N-benzyl derivatives of 6-amino-
favones was designed as a novel anticancer agents. Herein,
the present work reports the synthesis and anticancer activity
of these novel favones.
Results and discussion
Chemistry
Multistep synthetic protocol with synthesis of 1,3-diketone
5 from 4-bromophenol was initiated (1). 5-Bromo-2-hy-
droxyacetophenone (3) from 1 by methylation followed by
Friedel–Craft acylation and in situ demethylation was pre-
pared. To execute the Bekar–Venkataraman rearrangement,
compound 3 was benzoylated by using benzoyl chloride
and pyridine to get 5-bromo-2-benzoyloxy-acetophenone
(4) which on further base catalyzed Bekar–Venkataraman
rearrangement resulting in 1,3-diketone (5). Diketone 5 is
then cyclized to 6-bromofavone (6) employing malic acid
under solvent free reaction condition [24]. The Buchwald
coupling reaction of 6-bromofavone (6) with benzophenone
imine, Pd (II) catalyst and xantphos as ligand, Cs2CO3 as
a base in dioxane at 90 °C delivered imine 7 in 72% yield.
Hydrolysis of imine 7 with TFA in THF at room tempera-
ture gave 6-aminofavone 8 in excellent yield. Kónya et al.
1 3

Molecular Diversity
have reported direct amination of 6-bromofavone; however,
emphasized steric hindrance in the reacting amine is a lim-
iting factor in this reaction [15]. α-Branching in reacting
amine, hampers yield of the coupling reaction. However,
to overcome this limitation, reductive amination protocol
is used to synthesize novel 6-(1-arylmethanamino)-2-phe-
nyl-4H-chromen-4-ones (9a–n). It improved the yields and
efcacy of parallel synthetic protocol to intended target mol-
ecules. Various aldehydes were employed for synthesizing
imines which were then reduced to corresponding arylmeth-
anamino derivatives (9a–n) using NaBH(OAc)3 in one-pot
reaction protocol to get excellent yield (Table 1).
From SAR (Table 2), it is confrmed that 2-phenyl-4H-
chromen-4-one is essential for the anticancer activity. Vari-
ous 6-(substituted benzyl amino)-2-phenyl-4H-chromen-
4-one derivatives were synthesized to study the effect
of substituent on benzyl amino ring. Compound 9f i.e.,
6-(4-Chlorobenzylamino)-2-phenyl-4H-chromen-4-one with
4-chloro substitution on the benzylamino group was found
to be potent anticancer agent among all synthesized deriva-
tives against human breast cancer cell line (MCF-7). Com-
pound 9g with 4-bromo substitution on the benzylamino
group showed good anticancer activity against MCF-7 cell
line. Substituents like m-NO2 (9a), p-ethoxy (9k), 3, 4-di-
methoxy (9m) and 3,4-dichloro (9c) on benzylamino group
resulted in decrease in the activity.
Biology
Compound 9c with 2,3-dichloro substituent on ben-
zylamino group showed good (46.48%) inhibition against
the human lung cancer cell line (A-549), while replacing
2,3-dichloro substituent either by nitrile (9b, 43.71%) or
p-methoxy (9j, 41.96%) resulted in decrease in activity.
Other substituents like 4-bromo (9g, 36.51), 5-Bromo-2-hy-
droxy (9d, 35.69%) and 3-NO2 (9a, 21.08) exhibited lesser
inhibition against human lung cancer cell line (A-549).
Interestingly, when benzyl group was replaced by quino-
lin-2-ylmethyl group, compound 9n showed good in vitro
anticancer activity against both the selected cell lines. This
means that the replacement of benzyl group by some other
heterocyclic ring may result in good anticancer activity. In
general, electron withdrawing groups such as chloro- and
bromo substituents on benzyl group exhibit good anticancer
activity against both cell lines. The benzyl substitution with
dichloro group (9c) was found to be more potent than that
of with p-chloro group (9b) against A-549 cell lines. The
para-furo (9e) substituted derivatives were not found to be
as good anticancer agents as that of para-chloro (9b) and
para-bromo (9g) substituted derivatives.
Initially in vitro anti-proliferative efcacy of the synthe-
sized N-benzyl derivatives of 6-aminofavone (9a–n) was
evaluated against human breast cancer cell line (MCF-7)
and human lung cancer cell line (A-549) using MTT assay.
Doxorubicin (standard antitumor drug) was used as a posi-
tive control. Cancer cells were treated with synthesized
derivatives at various concentrations such as 0.1 uM, 1 uM
and 10 uM to determine the % cancer cell inhibition and %
cancer cell viability so as to predict the in vitro anticancer
activity.
Table 2 shows that most of the synthesized compounds
have shown good anticancer activity against human breast
(MCF-7) and lung (A-549) cancer cell lines. 9f was found
to be the most potent anticancer compound among the syn-
thesized derivatives against human breast cancer cell line
(MCF-7) with 52.90% cancer cell line inhibition. 9g was
found to be the second most potent anticancer compound
among the synthesized derivatives against human breast can-
cer cell line (MCF-7) with 52.17% cancer cell line inhibi-
tion. Other compounds such as 9a, 9k and 9n showed good
anticancer activity against human breast cancer cell line
(MCF-7) with 42.03%, 30.67% and 44.76% cancer cell line
inhibition, respectively.
Measurement of topoisomerase II activity
New derivatives 9(a–n) also displayed good anti-prolif-
erative activity against human lung cancer cell line (A-549).
Thus, compound 9c exhibited 46.48% inhibition against
human lung cancer cell line (A-549), while compounds 9n,
9b and 9j showed 44.26%, 43.71% and 41.96% of human
lung cancer cell line inhibition, respectively, at 10 uM con-
centration. Compounds 9f, 9g and 9n were found to be more
potent anticancer agent than that of standard drug Doxo-
rubicin against MCF-7 cells. Compound 9a was found to
be equipotent to that of Doxorubicin against MCF-7 cells.
Compounds 9b, 9c, and 9n were found to be more potent
anticancer agents than that of Doxorubicin against A-549
cells. Compound 9j with p-methoxy group was found to be
equipotent to that of Doxorubicin against A-549 cells.
In order to shed light on core antitumor mechanism, the
most active compounds (9b, 9c, 9f, 9g and 9n) were further
evaluated to predict their ability to inhibit topoisomerase
II enzyme. The catalytic activity of Topo II was evaluated
according to the procedure reported by Patra et al. [18]. Dox-
orubicin was used as a standard drug in this assay. Doxoru-
bicin inhibits the progression of topoisomerase II, enzyme
which relaxes supercoils in DNA for transcription. Results
of measurement of topoisomerase II activity are given in
Table 3.
The synthesized compounds 9b, 9c, 9f, 9g and 9n have
an ability to inhibit topoisomerase II enzyme. Compounds
9f and 9g were found to be potent topoisomerase II enzyme
inhibitor among tested synthesized compounds. Compounds
9f and 9g have shown IC50 value of 12.11 µM and 12.79 µM,
1 3

Molecular Diversity
Table 1 Synthesis of novel N-benzyl derivatives of 6-aminofavones
O
O
Br
Br
c
b
a
d
Br
OH
Br
O
HO
BzO
1
2
3
4
OH
O
O
f
e
Br
N
Br
O
O
O
O
5
6
7
O
O
O
g
h
H
N
H
N
H
R1
O
9a-n
8
NC
O
O
O
O
O
H
N
H
N
H
N
Cl
O2N
Cl
O
9a, 83%
9c, 83.4%
9b, 85%
Br
F
Cl
O
O
O
O
H
H
N
H
N
N
OH
O
O
9d, 82.8%
9e, 86.7%
9f, 85.8%
Br
O
O
Br
Br
O
H
N
O
O
H
N
H
N
O
O
9g, 88.2%
O
9i,81.2%
9h, 82.7%
O
O
O
O
O
O
H
N
H
N
O
O
H
N
O
O
9j, 86.6%
9k, 84.2%
9l, 82.8%
O
O
O
O
O
H
N
H
N
N
O
9m, 86.2%
9n, 80.2%
1 3

Molecular Diversity
Table 1 (continued)
Reagents and Reaction Conditions: (a) K2CO3, Methyl iodide, Acetonitrile, 50 °C, 6 h; (b) acetyl chloride, anhy. AlCl3, 6 h, r.t; (c) benzoyl
chloride, C5H5N, 20 min, 3% HCl; (d) KOH, C5H5N, 15 min, 10% AcOH; (e) malic acid, oil bath preheated at 140 °C; (f) Pd2(dba)3, CS2CO3,
benzophenone imine, and xantphos, dioxane, 16 h, 90 °C; (g) TFA, THF, r.t. 2 h; (h) aldehyde, sodium triacetoxy borohydride,TFA, DCM, 0 °C
to r.t. 45 min
Table 2 Anticancer activity of
MCF-7
A-549
novel N-benzyl derivatives of
6-aminofavones (at 10uM)
% Viability
% Inhibition
SD
% Viability
% Inhibition
SD
Control
100
0
1.08
4.27
1.66
1.13
1.09
0.21
1.09
1.02
3.88
0.88
3.57
0.60
0.91
2.15
0.03
0.98
100
0
2.00
3.26
0.67
0.03
2.72
0.70
0.49
0.45
4.65
1.23
1.02
3.14
1.55
4.45
3.32
1.34
9a
57.96
92.77
78.93
92.47
91.72
47.09
47.82
102.95
102.754
103.91
69.32
99.49
75.87
55.23
57.78
42.03
7.22
21.06
7.52
8.27
52.90
52.17
0
78.91
56.28
53.51
64.30
83.83
90.25
63.48
90.88
94.37
58.03
83.38
92.96
85.33
55.73
56.33
21.08
43.71
46.48
35.69
16.16
9.749
36.51
9.11
9b
9c
9d
9e
9f
9g
9h
9i
0
0
5.62
9j
41.96
16.61
7.03
14.66
44.26
43.67
9k
30.67
0.50
24.12
44.76
42.22
9l
9m
9n
Doxorubicin
SD standard deviation; human breast cancer cell line: MCF-7 and human lung cancer cell line: A-549
Table 3 Topoisomerase II enzyme inhibition, docking score and MM
pairs of DNA and forms H bonds with amino acid residue
Asp479 and DNA guanine fragment (DG13). Certainly, the
molecular docking study showed that all docked compounds
had been placed in between DNA base pair and had good
contact with Topo II residues and DNA fragments. Analy-
sis of interaction of docked compounds with Topo II-DNA
complex revealed existence of H-bonding interaction with
amino acid residue Gln778, and pi–pi stacking interactions
with DG13, DC8, and DG12 DNA fragments. It was also
revealed that in most of the compounds, carbonyl group of
favone moiety stabilities ligand–protein complex by form-
ing H-bond with amino acid residue Gln778. Similarly
N-benzyl group support the complex by establishing pi–pi
stacking interaction with various fragments of DNA. These
results indicate that most of the compounds showed similar
binding pattern with binding site amino acid residues as that
of Doxorubicin (Figs. 2, 3).
GBSA binding free energy of selected compounds
Compound
IC50 µMa
Docking score MM GBSA
Binding free energy
9b
35.46 0.05
28.18 0.21
12.11 0.15
12.79 0.28
30.92 0.16
−5.69
−6.69
−7.14
−6.63
−8.90
−23.25
9c
−33.11
−28.25
−30.66
−56.61
−42.33
9f
9g
9n
Doxorubicin
0.94 0.05 −11.18
a
Three independent experiments were performed for each concentra-
tion
respectively, as compared to the Doxorubicin (0.94 µM). The
synthesized compounds are not as strong topoisomerase II
enzyme inhibitor as that of standard drug Doxorubicin.
Docking study
Materials and methods
The activity of N-benzyl derivatives of 6-aminofavone was
justifed by its predicted binding mode at the DNA cleavage
site of topoisomerase II. Analysis of human Topo II co-crys-
tal structure complexed with DNA and etoposide (PDB code
3QX3) showed that etoposide is incorporated between base
Chemistry
All reactions were performed in oven-dried glassware.
All reagents and solvents were obtained from commercial
1 3

Molecular Diversity
Fig. 2 Binding pose of N-benzyl derivative of 6-aminofavone at DNA cleavage site of topoisomerase II
suppliers and used as received. Analytical thin-layer chro-
matography (TLC) was performed on precoated Merck silica
gel plates (60F-254), visualized with a UV254 lamp and
stained with KMnO4. Melting points are uncorrected and
were determined in open capillary tubes using parafn oil
bath. 1H and 13C NMR spectra were obtained as solutions
in deuterated solvents. Standard 1H NMR (400 MHz) and
13C NMR (100 MHz) were recorded on a Varian Mercury
spectrometer in DMSO-d6 and CDCl3 solution and with
tetramethylsilane as an internal standard. IR spectra were
recorded on Perkin Elmer Model 1600 series FTIR instru-
ment. Mass spectra were recorded on Agilent 1200SL-6100
LC/MS (ES-API) instrument. High-resolution mass spectra
(HRMS) were performed with a QTOF Micromass Mass
Spectrometer in electro spray ionization mode.
5‑Bromo‑2‑hydroxyacetophenone (3) To a stirred solution
of 4-bromo anisole (2, 1.0 g, 5.37 mmol) in 10 mL dichlo-
romethane, acetyl chloride (0.42 g, 5.37 mmol) was added
at 0 °C. After 5 min of stirring anhydrous AlCl3 (0.78 g,
5.91 mmol) was added portion wise, then reaction mixture
was stirred for 6 h. After completion of the reaction (TLC
check), the mixture was poured on crushed ice, product
precipitates out. The precipitate thus obtained was fltered
of, washed with diethyl ether and dried to get white solid.
(0.9 g, 79%); m.p. 58–60 °C; IR (KBr): 3363, 1663, 845,
671 cm−1; 1H NMR (CDCl3, 400 MHz) δ 12.70 (s, 1H), 7.84
(s, 1H), 7.55 (t, J=8 & 4 Hz, 1H), 6.90 (d, J=8 Hz, 1H),
2.64 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 203.5, 161.3,
139.1, 132.9, 120.9, 120.5, 110.4, 26.7; LCMS (ES-API)
m/z: 214.87 (M+H)+.
1‑Bromo‑4‑methoxybenzene (2) Methyl iodide (1.31 g,
9.24 mmol) was added at RT to a stirred solution of
4-bromophenol (1, 1.0 g, 5.78 mmol) and K2CO3 (1.59 g,
11.56 mmol) in 10 mL acetonitrile. Reaction mixture was
refuxed for 6 h at 50 °C. After completion of the reac-
tion (TLC check), the mixture was cooled to room tem-
perature, fltered through Celite bed and washed with ethyl
acetate (3×20 mL). Organic layer was washed with water
(3 × 40 mL), dried over anhydrous Na2SO4 concentrated
on vaccuo to get colorless oil (0.91 g, 84%); b.p. 223 °C;
5‑Bromo‑2‑benzoyloxy‑acetophenone (4) To a stirred
solution of 5-bromo-2-hydroxyacetophenone (3, 1.0 g,
4.67 mmol) in 10 mL pyridine benzoyl chloride (0.78 g,
5.60 mmol) was added at 0 °C. After 20 min of stirring,
reaction mixture was poured into ice cooled solution of
3% hydrochloric acid, product precipitates out. The crude
product was fltered of washed with water and recrystal-
lized from methanol to give yellow solid; m.p. 82–85 °C; IR
(KBr): 3363, 1755, 1663, 845, 671 cm−1; 1H NMR (CDCl3,
400 MHz) δ 8.20–8.18 (m, 2H), 7.96 (d, J = 2.4 Hz, 1H),
7.69–7.64 (m, 2H), 7.55–7.51 (m, 2H), 7.13 (d, J=8.8 Hz,
1H), 2.53 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 196.0,
164.8, 148.3, 136.1, 134.1, 132.9, 130.5, 130.3, 128.9,
125.8, 114.3, 29.7; LCMS (ES-API) m/z: 318.0 (M+H)+.
1
IR (KBr): 1657, 1339, 840, 670 cm−1; H NMR (CDCl3,
400 MHz) δ 7.40 (d, J=8 Hz, 2H), 6.81 (d, J=8 Hz, 2H),
3.81 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 158.1, 132.2,
115.7, 112.8, 55.4; LCMS (ES-API) m/z: 186.96 (M+H)+.
1 3

Molecular Diversity
Fig. 3 Binding pose of compounds at DNA cleavage site of topoi-
somerase II. a Doxorubicin, b compound 9f, c compound 9g and d
compound 9n. Black colored wire representation of atoms indicates
amino acid residues, cyan colored wire representation of atoms are
fragments of DNA, yellow colored dotted line indicates H-bonding
and cyan colored dotted line indicates pi–pi stacking
1‑(5‑Bromo‑2‑hydroxyphenyl)‑3‑phenylpropane‑1,3‑dione
(5) To a solution of 5-bromo-2-benzoyloxy-acetophenone
(4, 1.0 g., 3.14 mmol) in 7.5 mL of pyridine, potassium
hydroxide (0.26 g., 4.71 mmol) was added and reaction
mixture was stirred at 50 °C for 15 min. After completion
of the reaction (TLC check), the mixture was poured in ice
cooled solution of 10% aq. acetic acid (50 mL) product
precipitates out. The solid thus obtained was fltered of,
washed with 10% aq. acetic acid and dried to get yellow
solid (0.89 g, 80%), m.p. 108–109 °C; IR (KBr): 3363, 1663,
1H), 7.60–7.49 (m, 4H), 6.91 (d, J =8.8 Hz, 1H), 6.76 (s,
1H); 13C NMR (DMSO-d6, 100 MHz) δ 186.5, 183.4, 172.7,
159.1, 138.4, 137.1, 135.1, 133.1, 131.7, 129.2, 128.9,
128.2, 127.5, 122.5, 121.0, 120.3, 110.8, 96.0; LCMS (ES-
API) m/z: 318.0 (M+H)+.
6‑Bromo‑2‑phenyl‑4H‑chromen‑4‑one (6) The mixture of
1-(2-hydroxyphenyl)-3-aryl-1,3-propanedione (5, 1.0 g.,
3.14 mmol) and malic acid (0.42 g., 3.14 mmol) was heated
in a preheated oil bath at 140 °C for 10 min. After comple-
tion of the reaction (TLC check), the mixture was allowed
to cool at room temperature, water (10 mL) and ethyl acetate
1
845, 671 cm−1; H NMR (CDCl3, 400 MHz) δ 15.4 (bs,
1H), 12.05 (s, 1H), 7.97–7.95 (m, 2H), 7.86 (d, J= 2 Hz,
1 3

Molecular Diversity
(20 mL) were added. Organic layer was washed with aq.
NaHCO3 (3 × 40 mL) and dried over anhydrous Na2SO4
then concentrated in vaccuo to get crude product. The crude
product was purifed by column chromatography on silica
gel using hexane-ethylacetate (85:15) solvent system to give
white solid (0.82 g, 87%); m.p. 194–196 °C; IR (KBr): 1645,
1604, 1568, 1130, 756 cm−1; 1H NMR (CDCl3, 400 MHz)
δ 8.20–8.10 (m, 3H), 8.01 (dd, J=10.8 & 2.4 Hz, 1H), 7.83
(d, J = 8.8 Hz, 1H), 7.65–7.55 (m, 3H), 7.12 (s, 1H); 13C
NMR (CDCl3, 100 MHz) δ: 176.3, 163.4, 155.1, 137.3,
132.5, 131.3, 129.6, 127.3, 126.9, 125.3, 121.7, 118.3,
107.4; HRMS (ESI) m/z [M+H]+: calcd for C15H10BrO2:
300.9864, found: 300.9867.
yellow solid (1.37 g, 81%); m.p. 200–202 °C; IR (KBr):
1
3416, 3342, 1733, 1609, 1565, 768, 677 cm−1; H NMR
(CDCl3, 400 MHz) δ 8.10–8.01 (m, 2H), 7.55–7.58 (m, 3H),
7.54 (d, J=8.8 Hz, 1H), 7.06–7.11 (m, 2H), 6.87 (s, 1H),
3.30 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 178.4, 163.0,
150.0, 144.1, 132.1, 131.3, 128.9, 126.2, 124.7, 122.2,
119.0, 107.9, 106.7; LCMS (ES-API) m/z: 238.9 (M+H)+;
HRMS (ESI) m/z [M+H]+: calcd for C15H12NO2: 238.0868,
found: 238.0874.
General procedure for the synthesis of 6‑amino‑2‑phe‑
nyl‑4H‑chromen‑4‑ones (9a–9n) To a solution of 6-amino-
2-phenyl-4H-chromen-4-one (8, 1 g., 4.21 mmol) and
substituted aromatic aldehydes (4.21 mmol) in 1,2-dichlo-
roethane (10 mL), sodium triacetoxy borohydride (1.34 g.,
6.34 mmol) was added at 0 °C. Trifuoroacetic acid (0.96 g.,
8.43 mmol) was added to the above solution and reaction
mixture was stirred at 0 °C to RT for 45 min. After comple-
tion of the reaction (TLC check), the mixture was diluted
by adding DCM, organic layer was frst washed with aq.
NH4Cl and then with saturated solution of brine. Organic
layer was dried over anhydrous Na2SO4 and concentrated in
vacuo. The crude product was purifed through column chro-
matography on silica gel using hexane-ethylacetate (85:15)
solvent system to give the corresponding favones deriva-
tives in high yields.
6‑(Benzhydrylidene‑amino)‑2‑phenyl‑chromen‑4‑one
(7) The 6-bromo favone (6, 1.0 g., 3.33 mol), CS2CO3
(1.62 g., 5.65 mmol), benzophenone imine (0.6 g.,
3.33 mmol) was dissolved in dioxane (10 mL). Then it
was degassed by argon gas for 20 min, xantphos (50 mg.,
0.10 mmol) and Pd2(dba)3 (9 mg., 0.01 mmol) was added.
The reaction mixture was stirred for 16 h at 90 °C. After
completion of the reaction (TLC check), the mixture was
cooled to room temperature, diluted with ethyl acetate and
fltered through Celite bed. The fltrate was concentrated in
vacuo. To this residue MeOH was added, the solid obtain
was fltered and washed with diethyl ether. The crude prod-
uct was purifed by column chromatography on silica gel
using hexane-ethylacetate (80:20) solvent system to give
yellow solid (0.96 g, 72%); m.p. 248–249 °C; IR (KBr):
3442, 3063, 1637, 1616, 1455, 1361, 1290, 1025, 908, 775,
696 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.89 (d, J=8 Hz
2H), 7.80–7.77 (m, 2H), 7.60–7.54 (m. 1H), 7.50–7.52
(m, 4H), 7.45 (t, J = 8 & 5 Hz, 2H), 7.40–7.38 (m, 1H),
7.28–7.29 (m, 1H), 7.17 (t, J=8 & 4 Hz, 3H), 7.11 (t, J=8
& 4 Hz, 2H), 6.76 (s, 1H); 13C NMR (CDCl3, 100 MHz)
δ 178.3, 169.9, 163.0, 152.6, 148.8, 139.7, 135.7, 131.9,
131.4, 129.4, 129.0, 128.3, 126.2, 124.1, 118.3, 116.0,
107.1; LCMS (ES-API) m/z: 402.14 (M + H)+: HRMS
(ESI) m/z [M+H]+: calcd for C28H20NO2: 402.1489, found:
402.1492.
6‑(3‑Nitrobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9a) Yellow solid, yield 82.6%, m.p. 157–160 °C; IR (KBr):
1
3280, 3062, 1762, 1222, 1188, 830, 504 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 8.25 (s, 1H), 8.12–8.10 (m, 1H),
8.05–8.03 (m, 2H), 7.87–7.85 (m, 1H), 7.67–7.63 (m, 1H),
7.59–7.57 (m, 4H), 7.23–7.20 (m, 1H), 6.98–6.96 (m, 1H),
6.91–6.90 (m, 1H), 6.88 (s, 1H), 4.54–4.52 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 177.3, 172.7, 159.2, 144.3,
134.0, 132.0, 130.0, 129.0, 128.4, 127.6, 126.2, 124.9,
121.2, 119.9, 118.0, 105.4, 103.1, 46.4; LCMS (ES-API)
m/z: 372.0 (M+H)+ HRMS (ESI) m/z [M+H]+: calcd for
C22H17N2O4: 373.1188, found: 373.1195.
6‑(4‑Cyanobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9b) Yellow solid, yield 85.2%, m.p. 167–168 °C; IR
6‑Amino‑2‑phenyl‑4H‑chromen‑4‑one (8) To a solution of
6-(benzhydrylidene-amino)-2-phenyl-chromen-4-one (7,
1.0 g., 2.49 mmol) in 40 mL of THF, trifuoroacetic acid
(0.28 g., 2.49 mmol) was added at 0 °C. The reaction mix-
ture was stirred for 2 h at room temperature. After com-
pletion of the reaction (TLC check), the mixture was con-
centrated in vacuo. The residue was neutralized by addition
of aq. NaHCO3 solution and then extracted in ethyl acetate
(2 × 10 mL). Combined organic extract was dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude
product was purifed by column chromatography on silica
gel using hexane-ethylacetate (95:5) solvent system to give
(KBr): 3287, 3043, 2222, 1613, 1497, 671, 543 cm−1
;
1HNMR (DMSO-d6, 400 MHz) δ 8.05–8.03 (m, 2H), 7.81
(d, J = 8.4 Hz, 2H), 7.62–7.56 (m, 6H), 7.18 (dd, J = 9.6
& 3.2 Hz, 1H), 6.92 (d, J = 2.8 Hz, 1H), 6.88–6.86 (m, 2
H), 4.48–4.47 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
δ 178.5, 163.1, 149.9, 144.9, 144.4, 132.7, 132.1, 131.5,
129.1, 127.9, 126.3, 124.9, 121.2, 119.4, 118.9, 111.3, 106.8,
104.8, 48.0; LCMS (ES-API) m/z: 353.0 (M+H)+; HRMS
(ESI) m/z [M+H]+: calcd for C23H17N2O2: 353.1290, found:
353.1286.
1 3

Molecular Diversity
6‑(2,3‑Dichlorobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9c) Yellow solid, yield 83.4%, m.p. 168–171 °C; IR (KBr):
J=2.8 Hz, 1H), 6.89 (s, 1H), 6.80–6.77 (m, 1 H), 4.35–4.33
(m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 178.6, 162.9,
149.8, 145.4, 137.7, 132.2, 131.9, 131.4, 129.3, 129.1,
126.3, 124.9, 121.4, 121.2, 119.2, 106.8, 104.6, 47.9; LCMS
(ES-API) m/z: 408.0 (M+H)+; HRMS (ESI) m/z [M+H]+:
calcd for C22H17BrNO2: 406.0442, found: 406.0446.
1
3299, 3068, 1685, 1288, 1096, 811, 770 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 8.06–8.04 (m, 2H), 7.60–7.55 (m,
5H), 7.36–7.33 (m, 2H), 7.21–7.18 (m, 1H), 6.86–6.90 (m,
3H), 4.47–4.47 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
δ 178.3, 162.6, 149.3, 145.5, 138.6, 134.3, 132.9, 131.9,
131.3, 129.0, 128.9, 127.3, 126.7, 126.0, 124.5, 121.1,
119.0, 106.3, 103.5, 46.1; LCMS (ES-API) m/z: 396,397.9,
400 (M + H)+; HRMS (ESI) m/z [M + H]+: calcd for
C22H16Cl2NO2: 396.0558, found: 396.0552.
6 ‑ ( 5 ‑ B r o m o ‑ 2 ‑ m e t h o x y b e n z y l a m i n o ) ‑ 2 ‑ p h e ‑
nyl‑4H‑chromen‑4‑one (9h) Yellow solid, yield 82.7%, m.p.
173–176 °C; IR (KBr): 3366, 3067, 1684, 1450, 1029, 708,
549 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 8.05–8.03 (m,
2H), 7.58–7.56 (m, 4H), 7.42- 7.32 (m, 2H), 7.18 (dd, J=5.6
& 3.2 Hz, 1H), 7.01 (d, J=8 Hz, 1H), 6.92–6.89 (m, 2H),
6.67–6.65 (m, 1H), 4.30–4.29 (m, 2H), 3.89 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ 178.6, 162.8, 156.5, 149.6,
145.6, 132.1, 131.2, 131.2, 131.0, 129.0, 128.9, 128.8, 126.2,
124.8, 121.2, 119.0, 110.9, 106.6, 104.6, 55.6, 43.2; LCMS
(ES-API) m/z: 437.9 (M+H)+; HRMS (ESI) m/z [M+H]+:
calcd for C23H19BrNO3: 436.0558, found: 436.0548.
6 ‑ ( 5 ‑ B r o m o ‑ 2 ‑ h y d r o x y b e n z y l a m i n o ) ‑ 2 ‑ p h e ‑
nyl‑4H‑chromen‑4‑one (9d) Yellow solid, yield 82.8%,
m.p. 188–191 °C; IR (KBr): 3287, 3105, 1719, 1643, 1413,
808, 548 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 9.99 (s,
1H), 8.10–7.90 (m, 2H), 7.57–7.54 (m, 4H), 7.31–7.10 (m,
3H), 6.99–6.93 (m, 2H), 6.92 s, 1H), 6.61 (d, J = 6.4 Hz,
1H), 4.27–4.27 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
δ 178.4, 162.6, 154.6, 149.4, 146.0, 131.9, 131.2, 131.1,
130.7, 128.9, 127.3, 126.0, 124.5, 121.7, 118.9, 117.2, 111.0,
106.3, 104.0, 43.51; LCMS (ES-API) m/z: 423.7 (M+H)+;
HRMS (ESI) m/z [M + H]+: calcd for C22H17BrNO3:
422.0392, found: 422.0398.
6 ‑ ( 5 ‑ B r o m o ‑ 2 ‑ e t h o x y b e n z y l a m i n o ) ‑ 2 ‑ p h e ‑
nyl‑4H‑chromen‑4‑one (9i) Yellow solid, yield 81.2%,
m.p. 180–184 °C; IR (KBr): 3374, 3038, 1618, 1506, 910,
753 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 7.90 (s, 2H),
7.51–7.27 (m, 8H), 7.07–7.03 (m, 1H), 6.77 (s, 2H), 4.39
(s, 2H), 4.07 (d, J = 5.4 Hz, 2H), 1.45 (m, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 178.6, 162.8, 155.9, 149.7, 145.7,
132.2, 131.3, 131.0, 129.2, 128.4, 128.9, 126.2, 124.9, 122.2,
121.2, 119.0, 112.9, 106.7, 104.7, 63.9, 43.3, 14.8; LCMS
(ES-API) m/z: 450.1 (M+H)+; HRMS (ESI) m/z [M+H]+:
calcd for C24H21BrNO3: 450.0705, found: 450.0707.
6‑(4‑Fluorobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9e) Yellow solid, yield 86.7%, m.p. 132–135 °C; IR (KBr):
3288, 3066, 1613, 1537, 1431, 842 cm−1; 1H NMR (DMSO-
d6, 400 MHz) δ 7.91 (s, 2H), 7.63–7.26 (m, 8H), 7.08–7.02
(m, 3H), 6.78 (s, 1H), 4.38 (s, 2H); 13C NMR (DMSO-d6,
100 MHz) δ 178.6, 162.9, 149.8, 145.4, 137.7, 132.2, 131.9,
131.4, 129.3, 129.1, 126.3, 124.9, 121.4, 121.2, 119.2, 106.8,
104.6, 47.9; LCMS (ES-API) m/z: 346.2 (M+H)+; HRMS
(ESI) m/z [M + H]+: calcd for C22H17FNO2: 345.1165,
found: 345.1172.
6‑(4‑Methoxybenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9j) Yellow solid, yield 86.6%, m.p. 134–136 °C; IR (KBr):
3296, 3107, 1613, 1484, 1177, 769 cm−1; 1H NMR (DMSO-
d6, 400 MHz) δ 8.06–8.03 (m, 2H), 7.60–7.52 (m, 4H), 7.30
(d, J = 8 Hz, 2H), 7.18 (dd, J = 8.8 & 2.8 Hz, 1H), 6.96
(d, J = 2.8 Hz, 1H), 6.92–6.89 (m, 3H), 6.66–6.64 (m, 1
H), 4.28–4.26 (m, 2H), 3.66 (s, 3H); 13C NMR (DMSO-
d6, 100 MHz) δ 178.1, 162.2, 150.1, 146.1, 132.1, 131.8,
131.1, 130.7, 128.8, 128.6, 125.9, 124.4, 121.4, 118.7,
113.7, 106.1, 102.8, 55.1, 47.2; LCMS (ES-API) m/z: 358.0
(M+H)+; HRMS (ESI) m/z [M+H]+: calcd for C23H20NO3:
358.1443, found: 358.1441.
6‑(4‑Chlorobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9f) Yellow solid, yield 85.8%, m.p. 164–166 °C; IR
(KBr): 3285, 3061, 1791, 1611, 1047, 999, 578 cm−1
;
1H NMR (DMSO-d6, 400 MHz) δ 7.94–7.92 (m, 2H),
7.55–7.54 (m, 4H), 7.53–7.43 (m, 1H), 7.34–7.29 (m,
5H), 7.04 (t, J=8 Hz, 1H), 6.81 (s, 1H), 4.41 (s, 2H); 13C
NMR (DMSO-d6, 100 MHz) δ 178.5, 162.9, 149.7, 145.4,
137.1, 132.1, 131.3, 129.0, 128.9, 128.8, 126.2, 124.8,
121.1, 119.1, 106.9, 104.4, 100.0, 47.7; LCMS (ES-API)
m/z: 362.8(M+H)+; HRMS (ESI) m/z [M+H]+: calcd for
C22H17ClNO2: 362.0869, found: 362.0873.
6‑(4‑Ethoxybenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9k) Yellow solid, yield 84.2%, m.p. 159–162 °C; IR (KBr):
1
3306, 3107, 1683, 1452, 1292, 767, 686 cm−1; H NMR
6‑(4‑Bromobenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9g) White solid, yield 88.2%, m.p. 174–176 °C; IR (KBr):
3287, 3037, 1745, 1611, 1135, 793 cm−1; 1H NMR (DMSO-
d6, 400 MHz) δ 8.05–8.03 (m, 2H), 7.61–7.51 (m, 6H), 7.38
(s, 1H), 7.34 (s, 1H), 7.18 (dd, J=8.8 & 2.8 Hz, 1H), 6.94 (d,
(DMSO-d6, 400 MHz) δ 8.08–8.01 (m, 2H), 7.60–7.51 (m,
4H), 7.28 (d, J = 8.4 Hz, 2H), 7.17 (dd, J = 6.4 & 2.4 Hz,
1H), 6.96 (d, J = 6.8 Hz, 1H), 6.91–6.88 (m, 3H), 6.64
(d, J = 3.6 Hz, 1H), 4.27–4.25 (d, J = 8 Hz, 2H), 3.98 (q,
J = 14 & 6.8 Hz, 2H), 1.30 (t, J = 6.8 Hz, 3H); 13C NMR
1 3

Molecular Diversity
(DMSO-d6, 100 MHz) δ 178.6, 162.7, 158.4, 149.5, 145.7,
132.2, 131.2, 130.4, 128.9, 128.1, 126.2, 124.8, 121.2,
118.9, 114.7, 106.6, 104.1, 63.4, 48.0, 14.8; LCMS (ES-
API) m/z: 372.9 (M+H)+; HRMS (ESI) m/z [M+H]+: calcd
for C24H21NO3: 372.1599, found: 372.1599.
of the human topoisomerase II beta (PDB ID: 3QX3), in
which drugs simultaneously interact with both DNA and
enzymes [25] was taken from RCSB Protein Data Bank. The
protein was prepared by using protein preparation wizard
for docking using ‘protein preparation wizard’ in Maestro
v10.3. It contains two chains, a chain along with DNA and
Mg2+ ion. The ligand within active site and all water mol-
ecules were removed, while magnesium ion was allowed to
remain with the charge of + 2. The active site was defned to
include all atoms within 6.5A° radius of native ligand. The
bond orders and formal charges were added to hetero-groups
and hydrogen’s were added to all atoms in the structure.
Side chains that are not close to the binding cavity and do
not participate in salt bridges were neutralized and termini
were capped by adding ACE and NMA residue. After prep-
aration, structure was refned to optimize hydrogen bond
network using OPLS_2005 force feld. The minimization
was terminated when the energy converged or the RMSD
reached a maximum cut of of 0.30 Å. The extra precision
(XP) docking mode for all compounds was performed on
generated grid of protein structure [26]. Final evaluation of
ligand–protein binding was done with Glide score [27–29].
Docked pose of ligands was further rescored using
Prime MM-GBSA approach which is used to estimate free
binding energy of enzyme–ligands complex. Energies of
ligand–receptor complexes were calculated using Prime
MM-GBSA with fexibility set for residue surrounding 5
Å region of ligand binding site. Binding energy of receptor
and ligand is as calculated by Prime Energy, a Molecular
Mechanics+Implicit Solvent Energy Function (kcals/mol).
The binding free energy ΔGbind is estimated as
6 ‑ ( 2 ,
4 ‑ D i m e t h o x y b e n z y l a m i n o ) ‑ 2 ‑ p h e ‑
nyl‑4H‑chromen‑4‑one (9l) Yellow solid, yield 82.8%,
M.p. 126–129 °C; IR (KBr): 3359, 3038, 1613, 1523, 1274,
1
1120, 719 cm−1; H NMR (DMSO-d6, 400 MHz) δ 8.09–
8.01 (m, 2H), 7.57–7.53 (m, 4H), 7.18–7.13 (m, 2H), 6.93
(d, J=2.8 Hz, 1H), 6.88 (s, 1H), 6.59 (d, J=2.4 Hz, 1H),
6.47–6.45 (m, 2H), 4.21–4.20 (m, 2H), 3.84 (s, 3H), 3.73 (s,
3H); 13C NMR (DMSO-d6, 100 MHz) δ 178.3, 162.4, 160.1,
158.3, 149.4, 146.2, 131.9, 131.1, 129.5, 126.9, 126.0,
124.5, 121.5, 118.9, 118.7, 106.2, 103.9, 103.5, 98.3, 55.3,
55.2, 42.8; LCMS (ES-API) m/z: 388.2 (M +H)+; HRMS
(ESI) m/z [M+H]+: calcd for C24H22NO4: 388.1549, found:
388.1544.
6‑(3,4‑dimethoxybenzylamino)‑2‑phenyl‑4H‑chromen‑4‑one
(9m) Yellow solid, yield 86.2%, m.p. 138–141 °C; IR (KBr):
1
3288, 3106, 1613, 1484, 1152, 936, 541 cm−1; H NMR
(DMSO-d6, 400 MHz) δ 8.08–8.01 (m, 2H), 7.57–7.53
(m, 4H), 7.18 (dd, J = 8.8 & 2.8 Hz, 1H), 7.01–6.99 (d,
J = 2.8 Hz, 2H), 6.92–6.89 (m, 3H), 6.68–6.61 (m, 1 H),
4.27–4.25 (m, 2H), 3.74 (s, 3H), 3.71 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz) δ 178.6, 162.6, 160.4, 158.5, 149.4,
146.0, 132.2, 131.2, 129.9, 128.9, 126.1, 124.7, 121.4,
119.0, 118.8, 106.6, 104.5, 103.9, 98.7, 65.8, 55.4, 43.5;
LCMS (ES-API) m/z: 388.2 (M + H)+; HRMS (ESI) m/z
[M+H]+: calcd for C24H22NO4: 388.1548, found: 388.1551.
ΔGbind = G(C) − G(P) − G(L)
6 ‑ ( ( Q u i n o l i n ‑ 2 ‑ y l ) m e t h y l a m i n o ) ‑ 2 ‑ p h e ‑
nyl‑4H‑chromen‑4‑one (9n) Brown solid, yield 80.3%, m.p.
146–149 °C; IR (KBr): 3347, 3037, 1696, 1198, 773 cm−1;
1H NMR (DMSO-d6, 400 MHz) δ 8.42 (d, J=8.6 Hz, 1H),
8.10–7.89 (m, 4H), 7.82–7.69 (m, 1H), 7.70–7.50 (m, 7H),
7.26–7.21 (m, 1H), 7.03–7.01 (m, 1H), 6.84 (s, 1H), 4.68–
4.70 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 178.4,
162.0, 159.6, 153.3, 149.8 145.3, 138.3, 132.2, 132.08,
130.6, 129.1, 126.3, 126.2, 124.9, 122.2, 119.3, 124.9,
122.2,119.3, 118.7, 106.7, 103.7, 48.8; LCMS (ES-API)
m/z: 379.0 (M+H)+; HRMS (ESI) m/z [M+H]+: calcd for
C25H19N2O2: 379.1450, found: 379.1446.
where ΔGBind is total binding free energy, G(C) is binding
energy of protein ligand complex, G(L) is binding energy of
ligand and G(P) is binding energy of protein.
Biological assay
In vitro anticancer screening
All newly synthesized compounds (9a–n) were evaluated for
their in vitro anticancer activity against human breast cancer
cell line (MCF-7) and human lung cancer cell line (A-549)
by MTT assay. Two cell lines MCF-7 and A-549 (NCCS,
Pune, India) were grown in DMEM medium containing 10%
fetal bovine serum [Life technologies (Gibco)] and 0.7%
antibiotics. Cells were seeded into 96 well microtiter plates
in 100 µL of media at plating density of 5000 cells/well.
Seeded cells were incubated at 37 °C, 5% CO2, 95% air and
100% humidity for 24 h. At 24 h, old media was changed
with fresh media followed by treatment with each compound
Docking study
Molecular docking study was performed in Maestro 9.1
using Glide v. 6.8 (Schrodinger LLC). All compounds were
built using Maestro build panel and optimized to lower
energy conformers using Lig prep v3.5 (Schrodinger, Inc.,
New York, NY, USA). The X-ray crystallographic structure
1 3

Molecular Diversity
at 10 µM, 1 µM, and 0.1 µM. After 24 h treatment, cell
viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphe-
nyltetrazolium bromide (MTT), cells were incubated with 20
µL of MTT (5 mg/mL) in PBS for 4 h at 37 °C. The medium
was removed and formazan crystals dissolved in DMSO.
MTT reduction was quantifed by measurement of absorb-
ance at 570 nm using a multimode reader, Synergy Mx of
BioTek [30]. Statistical employed methods were Sigma Plot
10.0 and Microsoft Excel 2010.
placed in between DNA base pair at active site of enzyme.
Consequently, these N-benzyl derivatives of 6-aminofavone
may serve as lead scafold for development of novel anti-
proliferative agents. Our eforts in this regard are underway.
Acknowledgements We thank Dr. D. K. Mhaske, Dr. L. R. Patil
(Maharaja Jivajirao Shinde College, Shrigonda) and Dr. R. J. Barnabas
(Ahmednagar College, Ahmednagar) for their constant support. NMT
acknowledges the fnancial support by BCUD, SPPU, Pune (Grant No:
13SCI 000031).
Measurement of topoisomerase II inhibitory activity
Compliance with ethical standards
Conflict of interest There are no conficts to declare.
The most active anticancer agents from the synthesized
series were further evaluated for prediction of their mode
of action using Topo II drug screening kit (TopoGEN, Inc.,
Columbus) according to procedure reported by Patra et al.
[18]. Doxorubicin was used as standard in this evaluation.
The reaction was started with incubation of a mixture
consisted of human Topo II (2 μl), substrate super coiled
pHot1 DNA (0.25 μg), 50 μg/mL test compound (2 μl), and
assay bufer (4 μl) in 37 °C for 30 min. In order to terminate
reaction, 10% sodium dodecylsulphate (2 μl) and proteinase
K (50 μg/mL) were added at 37 °C for 15 min. followed
by incubation for 15 min at 37 °C. Then, DNA was run on
1% agarose gel in Bio Rad gel electrophoresis system for
1–2 h followed by staining with GelRedTM stain for 2 h and
destained for 15 min with TAE bufer. The gel was imaged
via Bio Rad’s Gel Doc TMEZ system. Both super coiled and
linear strands DNA were incorporated in the gel as markers
for DNA-Topo II intercalators. The results were reported as
IC50 (50% inhibition concentration) values [31].
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