Scaffold-hopping of bioactive flavonoids: Discovery of aryl-pyridopyrimidinones as potent anticancer agents that inhibit catalytic role of topoisomerase IIα

Article abstract of DOI:10.1016/j.ejmech.2016.06.024

A strategy of scaffold-hopping of bioactive natural products, flavones and isoflavones, leading to target-based discovery of potent anticancer agents has been reported for the first time. Scaffold-hopped flavones, 2-aryl-4H-pyrido[1,2-a]pyrimidin-4-ones and the scaffold-hopped isoflavones, 3-aryl-pyrido[1,2-a]pyrimidin-4-ones were synthesized via Pd-catalyzed activation–arylation methods. Most of the compounds were found to exhibit pronounced human topoisomerase IIα (hTopoIIα) inhibitory activities and several compounds were found to be more potent than etoposide (a hTopoIIα-inhibiting anticancer drug). These classes of compounds were found to be hTopoIIα-selective catalytic inhibitors while not interfering with topoisomerase I and interacted with DNA plausibly in groove domain. Cytotoxicities against various cancer cells, low toxicity in normal cells, and apoptotic effects were observed. Interestingly, compared to parent flavones/isoflavones, their scaffold-hopped analogs bearing alike functionalities showed significant/enhanced hTopoIIα-inhibitory and cytotoxic properties, indicating the importance of a natural product-based scaffold-hopping strategy in the drug discovery.

Full text of DOI:10.1016/j.ejmech.2016.06.024

Accepted Manuscript
Scaffold-hopping of bioactive flavonoids: Discovery of aryl-pyridopyrimidinones as
potent anticancer agents that inhibit catalytic role of topoisomerase IIα
Garima Priyadarshani, Suyog Amrutkar, Anmada Nayak, Uttam C. Banerjee,
Chanakya N. Kundu, Sankar K. Guchhait
PII:
S0223-5234(16)30502-5
10.1016/j.ejmech.2016.06.024
EJMECH 8685
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2016
Revised Date: 13 June 2016
Accepted Date: 14 June 2016
Please cite this article as: G. Priyadarshani, S. Amrutkar, A. Nayak, U.C. Banerjee, C.N. Kundu,
S.K. Guchhait, Scaffold-hopping of bioactive flavonoids: Discovery of aryl-pyridopyrimidinones as
potent anticancer agents that inhibit catalytic role of topoisomerase IIα, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.024.
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ACCEPTED MANUSCRIPT
Scaffold-hopping of bioactive flavonoids: Discovery of aryl-pyridopyrimidinones as potent
anticancer agents that inhibit catalytic role of topoisomerase-IIα

ACCEPTED MANUSCRIPT
Scaffold-hopping of bioactive flavonoids: Discovery of aryl-
pyridopyrimidinones as potent anticancer agents that inhibit catalytic role
of topoisomerase IIα
a,1
b,1
c
b
Garima Priyadarshani , Suyog Amrutkar , Anmada Nayak , Uttam C. Banerjee ,
c
,a
Chanakya N. Kundu , Sankar K. Guchhait*
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, SAS Nagar, Mohali, Punjab 160062, India
b
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education
and Research (NIPER), Sector 67, SAS Nagar, Mohali, Punjab 160062, India
c
School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India
1
Those authors contributed equally.
*
S.K. Guchhait: Tel.: +91 (0)172 2214683; fax: +91 (0)172 2214692; E-mail addresses: skguchhait@niper.ac.in
KEYWORDS: Flavonoids, Pyrido[1,2-a]pyrimidin-4-ones, Scaffold-hopping, Human
topoisomerase IIα, Anticancer Agent, Drug Discovery.
ABSTRACT: A strategy of scaffold-hopping of bioactive natural products, flavones and
isoflavones, leading to target-based discovery of potent anticancer agents has been reported
for the first time. Scaffold-hopped flavones, 2-aryl-4H-pyrido[1,2-a]pyrimidin-4-ones and the
scaffold-hopped isoflavones, 3-aryl-pyrido[1,2-a]pyrimidin-4-ones were synthesized via Pd-
catalyzed activation–arylation methods. Most of the compounds were found to exhibit
pronounced human topoisomerase IIα (hTopoIIα) inhibitory activities and several compounds
were found to be more potent than etoposide (a hTopoIIα-inhibiting anticancer drug). These
classes of compounds were found to be hTopoIIα-selective catalytic inhibitors while not
interfering with topoisomerase I and interacted with DNA plausibly in groove domain.
Cytotoxicities against various cancer cells, low toxicity in normal cells, and apoptotic effects
were observed. Interestingly, compared to parent flavones/isoflavones, their scaffold-hopped
analogs bearing alike functionalities showed significant/enhanced hTopoIIα-inhibitory and
cytotoxic properties, indicating the importance of a natural product-based scaffold-hopping
strategy in the drug discovery.
1
. Introduction
Scaffold-hopping [1,2] of bioactive template/drug for drug-design to develop novel
molecules with desired properties is an important strategy. Moreover, the new molecule with
changed core becomes patentable. Several examples of drug development by scaffold-
hopping are known, development of tramadol from morphine being one of the earliest

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[
2].The discovery of vardenafil from sildenafil [3], and celecoxib and rofecoxib from Dup
6
97 [4] are certainly amazing. Imigliptin [5], a DPP IV inhibitor, developed by such strategy
is currently under clinical trials of type-2 diabetes mellitus.
Natural products (NPs) have played a valuable role in the drug discovery and
development [6–8]. Newman and Cragg [9] reported that in the case of cancer around 79% of
FDA-approved drugs during a period of 1981-2010 are either natural products or their
based/mimicked-compounds. NPs are chosen through evolutionary process via lead
optimization to interact with various enzymes/proteins and thus represent biologically
relevant regions of the vast chemical space [10]. Despite the immense importance, to the best
of our knowledge, there is no precedence for application of scaffold-hopping in the anti-
cancer drug discovery utilizing natural product as a lead.
In our anticancer research program for drug discovery [11–13] we considered flavones
and isoflavones as valuable templates. They display anticancer activities by various
mechanisms [14]. Flavopiridol, a semisynthetic flavones analog, acts as CDK9 inhibitor, is
FDA-approved orphan drug for acute myeloid leukemia. Aminoflavone as a prodrug is in
phase I clinical trials for solid tumours. Flavones/isoflavones that inhibit topoisomerase II
among other mode of anticancer activities are quercetin, fisetin, genistein, orobol,
kaempferol, myricetin and morin [15,16]. To our surprise, literature survey revealed that there
is no report of scaffold–hopping/modulation of flavones and isoflavones in the discovery of
anticancer agents.
Figure 1: Design of new compounds
We were interested on scaffold-hopping of flavones and isoflavones with consideration of
incorporating the structural features of imidazopyridines that we have previously explored as
potent human DNA topoisomerase IIα (hTopoIIα)-catalytically inhibiting anticancer agents

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[
11]. In addition, the imidazopyridine motif is present in the marketed drugs, such as
zolpidem and zolimidine [17]. We speculated 2-aryl- and 3-aryl-4H-pyrido[1,2-a]pyrimidin-
-ones as potential hTopoIIα–inhibiting anticancer agents (Figure 1).
4
2
. Results and Discussion
2
.1. Chemistry
In transition-metal-catalyzed cross couplings, the exploration of drug/privileged
scaffolds as feasible substrates [18] and the catalyzed arylations via C-O bond cleavage using
phenol derivatives [19,20] as electrophiles alternatives to aryl halides have gained
importance. Recently, we have developed a new approach for the synthesis of 2-aryl-4H-
pyrido[1,2-a]pyrimidin-4-ones from pyridopyrimidin-2,4-diones via Pd-catalyzed enolic
C−O bond activation‒arylation [21] (Figure 2a). Keeping in view, the structural features of
Topo II-inhibiting flavonoids, a series of investigational compounds with relevantly
substituted aryl moieties at C2-position of 4H-pyrido[1,2-a]pyrimidin-4-ones were
synthesized.
In the direction of direct arylation via C-H bond activation of biologically-important
scaffold, recently, we have developed a regioselective Pd-catalyzed Ag(I)-assisted direct C3-
H activation-arylation of pyrido[1,2-a]pyrimidin-4-ones with bromo/iodo-(hetero)arenes to
prepare relevantly substituted 3-aryl-pyrido[1,2-a]pyrimidin-4-ones (Figure 2b) [22].
Following this method, relevant C3-aryl-substituted 4H-pyrido[1,2-a]pyrimidin-4-ones were
synthesized.
To have closer structural resemblance of investigational compounds to naturally-
occurring topo II-inhibiting flavones and isoflavones, (poly)hydroxylated 2- or 3-aryl
substituted 4H-pyrido[1,2-a]pyrimidin-4-ones were prepared via HBr (48% aqueous
solution)-mediated ether cleavage [23] of corresponding methoxy derivatives (Figure 2c).
To evaluate the effectiveness of scaffold-hopping concept on the topoisomerase II
inhibitory activities of flavonoids, several flavones and isoflavones were prepared. These
compounds with alike substitutions completely mimic the structures of investigated potent
topo II-inhibiting 2-arylpyridopyrimidiones and 3-arylpyridopyrimidinones, such that a
comparison in their activities can be made. Three flavones (C1-3) matching with 2-
arylpyridopyrimidinones (3c-e), and four isoflavones (C1-7) matching with 3-
arylpyridopyrimidinones (6d, 6e, 6n, 6o), were prepared following literature reported [24,25]
synthetic routes (Figure 2d).

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Figure 2: Synthetic route for the 2/3- arylpyridopyrimidinones and parent compounds (flavones and
isoflavones)
2
.2. Biological Studies
Synthesized compounds were investigated for their ability to inhibit hTopoIIα and for
the mode of action. A screening kit purchased from TopoGEN, Inc. (Columbus, OH) was
used. The screening protocols as provided by the supplier and reported in literature [11–
1
3,26,27] with changed concentration as per our requirement were followed.
2
.2.1. Inhibition of hTopoIIα-mediated kDNA decatenation
The compounds (series 3 and series 6) as well as their parent compounds (C1-7) were
screened for the inhibitory activities of hTopoIIα-mediated decatenation of kinetoplast DNA
kDNA). The decatenation of kDNA by hTopoIIα results in the formation of small
(
minicircles. The extent of hTopoIIα inhibition was measured by comparing the amount of
decatenated products formed in the presence and absence of inhibitor (Figure 3). Many of the

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synthesized compounds 3a, 3c-e, 3m, 3o, 3s, 6c-f, 6n, 6o, 6q, 6t, 6u, 6w and C1-7 were
found to be relatively more potent than Etoposide (a hTopoIIα-inhibiting anticancer drug)
against hTopoIIα.
Figure 3: a) Inhibition of hTopoIIα-mediated kDNA decatenation: kDNA was treated with hTopoIIα in the
absence or presence of 100 ꢀM etoposide (E) or investigated compounds; b) Quantification of decatenation
products.
2
.2.2. Cytotoxic potential of selected compounds in cancer cells
The cytotoxic potential of selected compounds (3a, 3c-e, 3m, 3o, 3s, 6c-f, 6n, 6o, 6q,
t, 6u, 6w) as well as the parent compounds (C1-7) were investigated by a MTT assay using
6
kidney cancer cells (HEK-293) and normal monkey kidney epithelial cells (Vero). Etoposide
was used as positive control. The cells were treated with compounds in increasing
concentrations for 48 h and then MTT assay was performed. All the compounds showed a
characteristic cytotoxic profile in both the cells (Table 1). A dose dependent decrease of cell
viability was noted in cells and more growth inhibition was noted in HEK-293 compared to
Vero cells at the same concentration (Figure 4).

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Table 1: IC50 values in HEK-293 and Vero cells
Compounds
LC50 (µM) HEK- LC (µM) Vero Compounds
LC50
(µM) LC50
(µM)
50
2
93
HEK-293
Vero
3
3
3
3
3
3
3
6
6
6
6
6
a
c
17 ±2.4
15.5±2.3
9 ± 1.8
50±1.5
6o
7 ± 2.6
60 ± 2.7
52±1.4
6q
6t
3 ± 2.1
9 ± 1.6
2 ± 1.3
6 ± 2.4
8 ± 1.6
12 ± 1.6
8.5 ± 1.6
10 ± 1.3
12 ± 1.2
8 ± 1.2
12 ± 1.6
60 ± 2.5
38 ± 2.0
50 ± 1.9
52 ± 2.6
48 ± 1.3
52 ± 1.3
45 ± 1.3
50 ± 1.3
52 ± 1.5
48 ± 1.3
42 ± 1.6
d
e
46±2.5
8 ± 1.2
60±2.1
6u
6w
C1
C2
C3
C4
C5
C6
C7
m
o
s
3 ± 2.0
48 ±1.8
47.5±2.1
32±1.4
2.4 ± 1.5
13 ± 1.6
15 ±2.3
3 ± 0.3
c
48 ± 2.3
40 ± 2.1
38 ± 1.4
46 ± 1.3
44 ± 1.9
55 ± 1.5
d
e
19 ± 2.5
9 ± 2.1
f
n
3.5 ± 1.2
22 ± 1.5
Etoposide
Next, the cytotoxicity of selective potent compounds (3m, 3o, 6d, 6u, 6n and 6q)
were tested in other cancer cells, lung (A-549), oral (H-357), colon (HCT-116), cervical
(HeLa), and breast (MCF-7) (Table 2a).
Figure 4: Viability of cells in HEK-293 and Vero cells. Compounds 3m, 3o, 6d, 6u, 6n and 6q were found to
be relatively highly cytotoxic to HEK-293 cells compared to Vero cells.
In these cells, significant toxicity with IC (concentration at which 50% cell death in
5
0
culture was observed) ranging from 1.5 to 4 ꢀM were observed. To determine the potential of

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the compounds in long term cytotoxic effect even after end of treatment, a clonogenic cell
survival assay using HEK-293 cells was performed (Table 2b). A dose dependent decrease in
colony formation was observed in investigated compounds. The LC values (concentration at
5
0
which fifty percent cell death in culture was observed) indicated that they were more potent
than etoposide with 5-10 fold higher activities.
Table 2:
a) IC50 (µM ) of compounds in MTT assay
b) LC50 (µM ) in
clonogenic assay using
HEK-293
Compound
MCF-7
HeLa
A-549
HCT-116
H-357
3
m
2.5 ± 0.2
1.8 ± 0.2
3.0 ± 0.3
2 ± 0.2
3 ± 0.2
2 ± 0.3
2.4 ± 0.3
2.1 ± 0.2
2.1 ± 0.2
2.8 ± 0.2
2.5 ± 0.3
2 ± 0.2
3.5 ± 0.2
2.5 ± 0.2
3.2 ± 0.2
3 ± 0.2
2.5 ± 0.2
2.3 ± 0.2
3 ± 0.2
2.5 ± 0.1
2 ± 0.9
3
o
d
u
n
q
6
2.8 ± 0.2
1.5 ± 0.2
3 ± 0.3
1.8 ± 0.1
2.4 ± 0.3
2.5 ± 0.1
3.8 ± 0.7
20 ± 0.1
6
6
6
3 ± 0.3
2.8 ± 0.3
3 ± 0.2
4 ± 0.2
2.5 ± 0.3
3 ± 0.2
2 ± 0.2
1.5± 0.2
Etoposide
For further studies of properties and mode of action, the compounds 3o and 6d were
then selected as representative examples, one from each series, based on their overall superior
effects of hTopoIIα-inhibitory activities, cytotoxicities and anti-clonogenic potential.
2
.2.3. Inhibition of hTopoI-mediated DNA relaxation
The compounds 3o and 6d were screened for inhibition of hTopoI-mediated
relaxation of negatively supercoiled DNA. In the presence of compounds almost no inhibition
of DNA-relaxation was observed as compared to camptothecin (Figure 5a). Hence,
investigated compounds of 100 µM concentration did not act as hTopoI inhibitors.
Next, mode of action for hTopoIIα inhibition was studied.
Figure 5: a) hTopoI-mediated DNA relaxation assay (Negatively supercoiled DNA was treated with hTopoI in
the absence/presence of 100 µM camptothecin (C) or investigated compounds). b) hTopoIIα DNA cleavage
complex assay (Negatively supercoiled DNA (pUC19) was treated with hTopoIIα and immediately incubated
with 100 µM etoposide or investigated compounds). c) DNA intercalation assay using SC DNA (pUC19)
2
.2.4. hTopoIIα-DNA cleavage complex formation
A hTopoIIα poisons e.g. etoposide stabilizes a TopoII-cleaved DNA complex
(cleavage complex) which generally appears as linear band (Lin)[11]. In the presence of

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selected active compounds C1-4, 3m, 3o, 6d, 6n and 6u (100 µM) no such linear band was
observed (Figure 5b). It indicates that the investigated compounds with upto 100 µM did not
act as hTopoIIα poisons.
2
.2.5. DNA-binding
The binding of ligand with DNA takes place either by DNA intercalation or by
groove binding[28].
DNA intercalation
DNA intercalators like ethidium bromide (EtBr) retards the movement of SC DNA in
the gel electrophoresis. In the presence of etoposide, compound 3o or compound 6d, no such
retardation of DNA movement was observed (Figure 5c), suggesting the non-intercalating
nature of the investigated compounds.
Mode of DNA-binding
In order to check the specificity of the compounds (3m, 3o and 6d) towards the
DNA, an in vitro DNA-ligand binding assay was carried out (Figure 6a-e). In principle, pure
DNA provides maximum absorbance at 260 nm, however, when a ligand binds to the DNA,
the absorbance decreases. In our experiment, it was observed that pure DNA, which gave an
exact spectrum with λmax 260 nm, on treatment with investigated compounds of increasing
concentrations showed hypochromism in the absorption. This indicates possible binding of
the compounds to the DNA. The results were validated by taking EtBr (a known DNA
intercalator) as a positive control [29].
Figure 6: a) DNA-binding of compound 3m; b) compound 3o; c) compound 6d; d) EtBr; e) CD based ligand-
DNA binding of compounds EtBr, 3o and 6d.

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The absorption spectrum of individual compound was found to be different from EtBr-DNA
absorption spectra, indicating the occurrence of hypochromism due to interaction of
compound with the DNA, not due to property of the compound itself. The binding constant
6
-1
-6
-1
-6 -1
(
Kd) were were found to be 2.43 x 10- M , 3.4 x 10 M and 2.5 x 10 M for compounds
3
m, 3o and 6d respectively (Figure 7).
Figure 7: a) Graph showing the binding constant (K ) of compound 3m. b) Graph showing the binding constant
d
of compound 3o. c) Graph showing the binding constant of compound 6d.
In addition, circular dichroism (CD) spectral analysis was performed to measure the
conformational changes in the DNA duplex structure due to binding with compounds 3o and
6
d (Figure 4e). The CD spectra showed that bands at 275 nm (due to base stacking) and at
2
45 nm (due to the B-conformation of DNA) did not change significantly in the presence of
the tested compounds (3o and 6d), while EtBr (a well-known DNA intercalator) used as a
positive control showed changes in the absorption spectra (shape and absorbance) [29]. The
DNA-ligand interaction studies all together signify that the investigated compounds may
interact with DNA through groove binding and not by DNA intercalation.
2
.2.6. Apoptosis measured by DAPI-nuclear staining
To study the cytotoxicity of investigated compounds caused due to possible
apoptosis, a fluorescence assay was performed using DAPI which binds only to the nucleus
of the cells and not to the cytoplasm. HEK-293 cells were treated with compound 3o of
various concentrations and then stained with DAPI prior to observe under microscope. A
dose dependent increase of deformation, shrinkage and bubble shaped nuclei in comparison
with untreated cells was noted. More than 5 fold increased apoptotic nuclei were noted at 5
µM of compound 3o (Figure 8a and 8b).

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Figure 8: DAPI-stained nuclei of HEK-293 cells after treatment with compound 3o: a) Images were taken under
a fluorescence microscope at 40X magnification (scale bar 10 µM). Images 1, 2, 3, and 4 represent cells treated
with compound 3o of 0, 1, 2.4 and 5 ꢀM, respectively; b) Bar diagram showing the number of apoptotic and
non-apoptotic nuclei.
2
.2.7. Apoptosis measured by annexin V-FITC/PI dual staining in HEK 293 cells
To further confirm the apoptosis and different phases of apoptosis a more sensitive
and reliable assay was carried out by sorting the cells after annexinV-FITC/PI dual stain.
Annexin-V/FITC binds to living cells, whereas PI stains apoptotic cells. The distribution of
cells in Q1, Q2, Q3 and Q4 represent the normal, early apoptotic, apoptotic and late apoptotic
cells, respectively (Figure 9). In treating the cells with compound 3o, a dose dependent
increase in total apoptotic cells (early apoptotic + apoptotic + late apoptotic cells) was noted.
More than 22 percent total apoptotic cells were noted at 5ꢀM of compound 3o, which was
significantly higher compared to untreated cells (1%).
Figure 9: Analysis of Annexin V –FITC/PI stained cells after treatment with compound 3o in HEK-293 cells
2
.2.8. Inhibition of invasiveness of cancer cells
Cancer cells invade easily into the matrix of basement membrane. The effect of
compound 3o in the invasive property was measured by matrigel cell invasion assay. It was
noted that with increase in concentration of compound 3o the percentage of cell invaded into

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the matrix decreased in comparison with untreated cells. A 5 fold decrease in the
invasiveness was observed in 5 µM of compound 3o with respect to untreated cells (Figure
1
0).
Figure 10: Assay for matrigel invasion in HEK- 293 cell for compound 3o: Histogram showing the number of
cell invaded into the matrix.
The comparison of topoisomerase II inhibitory and antiproliferative activities of investigated
potent scaffold-hopped analogs with the parent flavones and isoflavones led to the interesting
findings. To our glad, the scaffold-hopped analogs of isoflavones (6d-e, 6n, 6o) were found
to be superior compared to parent isoflavones (C4-7) in both of topo II inhibitory and
antiproliferative activities, and more potent than etoposide. Although less active than their
parent compounds (C1-3), the scaffold-hopped analogs of flavones (3c-e) were found to be
significantly potent in topo II inhibitory and antiproliferative activities and higher efficacious
compared to etoposide. In addition, these scaffold-hopped analogs belong to the new
molecular motifs (2/3-arylpyrido[1,2-a]pyrimidinone) that are outside the patent issues.
3
. Conclusion
The present study represents for the first time a scaffold-hopping strategy of bioactive
natural product in the anticancer drug discovery. Based on scaffold-hopping of flavones and
isoflavones with consideration of the structure of marketed relevant drugs and the anticancer
agents previously explored by us, 2-aryl-4H-pyrido[1,2-a]pyrimidin-4-ones and 3-aryl-4H-
pyrido[1,2-a]pyrimidin-4-ones were considered as potential hTopoIIα inhibitors. They were
synthesized by Pd-catalyzed C-O activation–arylation and arene C–H bond arylation
approaches, respectively. These compounds found to exhibit significant topoisomerase II
inhibitory and anticancer activities. Compounds 3a, 3c-e, 3m, 3o, 3s, 6c-f, 6n, 6o, 6q, 6t, 6u,
6
w showed higher topo II inhibition and cytotoxicities than etoposide (a topo II–inhibiting

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anticancer drug), while they are relatively less toxic to normal cells. These compounds
inhibited the topo II-activity by blocking the catalytic role of hTopoIIα in DNA-process while
not interfering with topoisomerase I and interacted with DNA plausibly in groove domain.
Compounds (3m, 3o, 6d, 6u, 6n and 6q) were found to be cytotoxic in other cancer cells,
lung, oral, colon, cervical, and breast cancer cells as well. These compounds inhibited the
invasiveness as well as clonogenic properties of cancer cells. They showed their
cytotoxicities by apoptosis.
An appealing attribute of this scaffold-hopping strategy is that the analogs compared
to the parent flavones and isoflavones, bearing alike functionalities showed
significant/superior hTopoIIα-inhibitory and cytotoxic properties. Taken all together, these
suggest the importance of scaffold-hopping approach in this class of compounds. The present
study will certainly encourage for the medicinal chemistry programme of such NPs-based
scaffold-hopping strategy in accomplishing rapid drug discovery.
4
. Experimental
4
.1. Chemistry
4
.1.1. General considerations
All reactants and reagents were obtained from commercial source and used without
further purification. Bruker Avance III-400 (400 MHz) spectrometer was used for recording
1
13
H NMR and C NMR spectra were measured on a Bruker Avance III-400 (100 MHz)
spectrometer with complete proton decoupling. Data were reported as follows: chemical
shifts in ppm from tetramethylsilane as an internal standard in CDCl /CD OD/DMSO-d
6
3
3
integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, td =
triplet of doublet, dt = doublet of triplet, ddd = doublet of doublet of doublet, br = broad), and
coupling constants (Hz). The Infrared (IR) spectra were recorded on a Perkin Elmer FTIR
with ATR & IR Microscope spectrometer. High-resolution mass spectra (HRMS) were
performed on Bruker maxis Q-TOF. Elemental analyses were done on Elementar Vario EL
spectrometer. Melting points determined are uncorrected. Analytical HPLC was used to
determine the purity of the compounds and the purity was found to be greater than 95%.
4
.2.1.Typical experimental procedure for the synthesis of 2-aryl-4H-pyrido[1,2-
a]pyrimidin-4-ones (3a-r):[21]
A mixture of the 4H-pyrido[1,2-a]pyrimidin-4-one (1 mmol), and K CO (2.5 eq) in
2
3
1
,4-dioxane (2 mL) and H O (1 mL) taken in a round-bottomed flask and p-TsCl (1.3 eq) was
2
added to it. The mixture was stirred under open air for 1h at rt (25-28 °C). The boronic acid

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(
1.2 eq) and Pd(PPh ) (3 mol %) were subsequently added and the mixture was heated at 100
3
4
°
C under open air. Upon completion of reaction as indicated by TLC (1-1.5 h), the resultant
mixture was cooled to rt and extracted with EtOAc (2x25 mL). The combined organic
solution was washed with water (2x5 mL) and brine (1x5 mL), dried with anhyd. Na SO ,
2
4
and concentrated under reduced pressure. The column chromatographic purification of crude
mass was performed on neutral alumina using EtOAc-hexane (30-40%) as eluting solvent
afford the arylated products (3a-r).
For spectral data and scanned spectra of compounds 3c-e, 3g-r, see: Guchhait, S. K.;
Priyadarshani, G. Synthesis of 2-arylpyridopyrimidinones, 6-aryluracils, and tri-and
tetrasubstituted conjugated alkenes via pd-catalyzed enolic C–O bond activation–arylation. J.
Org. Chem. 2015, 80, 6342−6349.
Characterization data for 2-aryl-4H-pyrido[1,2-a]pyrimidin-4-ones (3a, 3b, 3f, 3i):
2
-(3,4-Dimethoxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3a):[30] White solid, 259
1
mg, 92%, m.p. 168-170 °C; H NMR (400 MHz, CDCl ):
δ
9.05 (ddd, J = 7.0 Hz, J = 0.9 Hz,
3
J = 0.9 Hz, 1H), 7.75-7.67 (m, 4H), 7.12-7.09 (m, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H),
1
3
4
.01 (s, 3H), 3.95 (s, 3H) ppm; C NMR (100 MHz, CDCl ):
δ
161.5, 158.6, 151.6, 151.3,
3
1
50.9, 149.1, 136.1, 129.8, 127.3, 126.6, 120.6, 114.9, 111.0, 110.1, 98.9, 56.0, 55.9 ppm; IR:
-1
νmax 3072, 3004, 1678, 1638, 1486, 1269, 1134, 1022 cm ; HRMS (ESI) m/z: calcd. for
+
C H N O [M+H] 283.1082, found: 283.1074.
16
15
2
3
2
9
1
6
1
-(3,5-Dimethoxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3b): White solid, 253 mg,
1
0%, m.p. 160-162 °C; H NMR (400 MHz, CDCl ):
δ 9.07 (ddd, J = 7.2 Hz, J = 1.2 Hz, J =
3
.2 Hz, 1H), 7.76-7.74 (m, 2H), 7.24 (d, J = 2.3 Hz, 2H), 7.16-7.13 (m, 1H), 6.88 (s, 1H),
1
3
.60 (dd, J = 2.3 Hz, J = 2.3 Hz, 1H), 3.88 (s, 6H) ppm; C NMR (100 MHz, CDCl ):
δ
3
61.9, 161.1, 158.6, 150.9, 139.4, 136.1, 127.3,126.8, 115.2, 105.4, 103.0, 100.4, 55.5 ppm;
-1
IR: νmax 3110, 2939, 1670, 1632, 1457, 1439, 1152, 1063 cm ; HRMS (ESI) m/z: calcd. for
+
C H N O [M+H] 283.1082, found: 283.1073.
16
15
2
3
2
-(3-Methoxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3f): White solid, 232 mg, 92%,
1
m.p. 176-178 °C; H NMR (400 MHz, CDCl ):
δ 9.06 (ddd, J = 7.1 Hz, J = 1.0 Hz, J = 1.0
3
Hz, 1H), 7.74-7.73 (m, 2H), 7.66-7.63 (m, 2H), 7.40 (dd, J = 8 Hz, J = 7.8 Hz, 1H), 7.14-
.10 (m, 1H), 7.03 (ddd, J = 8.2 Hz, J = 2.5 Hz, J = 0.9 Hz, 1H), 6.90 (s, 1H), 3.90 (s, 3H)
7
13
ppm; C NMR (100 MHz, CDCl ):
δ
161.9, 160.0, 158.6, 150.9, 138.8, 136.1, 129.8, 127.3,
3
1
26.8, 119.8, 116.6, 115.2, 112.6, 100.3, 55.4 ppm; IR: νmax 3082, 2995, 1670, 1639, 1442,

ACCEPTED MANUSCRIPT
-1
+
1
253, 1037 cm ; HRMS (ESI) m/z: calcd. for C H N O [M+H] 253.0977, found:
15 13 2 2
2
53.0974.
2
-(4-Hydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3i):[30] Off-white solid, 174 mg,
1
7
3%, m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ
10.06 (s, 1OH), 8.92 (ddd, J = 7.1
6
Hz, J = 0.7 Hz, J = 0.7 Hz, 1H), 8.07 (d, J = 8.8 Hz, 2H), 7.93 (ddd, J = 8.8 Hz, J = 6.7 Hz, J
1.6 Hz, 1H), 7.69 (dd, J = 8.7 Hz, J = 0.8 Hz, 1H), 7.29 (ddd, J = 8.2 Hz, J = 6.9 Hz, J =
=
1
3
1
1
.3 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 6.85 (s, 1H) ppm; C NMR (100 MHz, DMSO-d ): δ
6
60.8, 160.5, 158.1, 151.0, 137.9, 129.5, 127.7, 127.3, 126.5, 116.2, 115.9, 97.2 ppm; IR:
-1
νmax 3277, 2832, 1658, 1630, 1450, 1239, 1015 cm ; HRMS (ESI) m/z: calcd. for
+
C H N O [M+H] 239.0821, found: 239.0816.
14
11
2
2
4
.2.2. Typical experimental procedure for the synthesis of 3-aryl-4H-pyrido[1,2-
a]pyrimidin-4-ones (6a-s):[22]
Pyridopyrimidinone (1 mmol), K CO (3 mmol, 414 mg, 3 eq), Ag CO (1 mmol, 275 mg, 1
2
3
2
3
eq), PCy (0.2 mmol, 56 mg, 0.2 eq), Pd(OAc) (0.1 mmol, 22 mg, 0.1 eq) were weighed in
3
2
air to a sealed tube equipped with magnetic stirring bar. PivOH (1 mmol, 0.12 mL, 1 eq) and
bromoarene (2 mmol, 2 eq) were added to the tube. The tube was back filled with argon and
toluene (3 mL) and water (1 mL) were added. The mixture was heated at 110 °C for 18-24 h
until complete consumption of the substrate as indicated by TLC. The resultant mixture was
cooled to rt and concenterated under reduced pressure. The mixture was extracted with
EtOAc (2x50 mL) and washed with 10% aqueous ammonia solution (3x20 mL). The
combined organic solution was washed with water (2x5 mL) and brine (1x5 mL), dried with
anhyd. Na SO , and concentrated under reduced pressure. The column chromatographic
2
4
purification of crude mass was performed on silica gel partially de-acidified by passing
triethylamine (1-2 mL) using EtOAc-hexane (25-40%) as eluting solvent to afford the
arylated products (6a-s).
For spectral data and scanned spectra of compounds 6a-s, see: Guchhait, S. K.;
Priyadarshani, G. Pd-Catalyzed Ag (I)-promoted C3-arylation of pyrido [1,2-a] pyrimidin-4-
ones with bromo/iodo-arenes. J. Org. Chem. 2015, 80, 8482–8488.

ACCEPTED MANUSCRIPT
4
.2.3.Typical experimental procedure for the synthesis of (poly)hydroxyaryl substituted-
pyridopyrimidinones (3s-t, 6t-w):[23]
The methoxy compound (1.5 mmol) was taken into 48% HBr (7 mL) and refluxed at 100 °C.
The reaction was continued until complete consumption of the starting material as monitored
by TLC (3-5 h). The mixture was cooled to room temperature and neutralized with 0.1 N
NaOH. Solids were collected and purified by neutral alumina column chromatography using
5
% methanol in dichloromethane.
Characterization data for compounds 3s, 3t, 6t-w:
2
1
8
=
-(3,4-Dihydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3s):[30] Light yellow solid,
1
29 mg, 34%, m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ 9.04 (d, J = 6.4 Hz, 1H),
6
.23 (dd, J = 7.4 Hz, J = 6.4 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.56-7.41 (m, 3H), 6.92 (d, J
13
8.1 Hz, 1H), 6.77 (s, 1H) ppm; C NMR (100 MHz, DMSO-d ):
δ
157.2, 156.7, 149.8,
6
1
3
2
49.3, 146.1, 141.8, 128.6, 124.6, 122.5, 120.4, 118.4, 116.4, 115.1, 97.9 ppm; IR: νmax 3544,
-1
+
407, 1659, 1615, 1277, 1134 cm ; HRMS (ESI) m/z: calcd. for C H N O Na [M+Na]
1
4
10
2
3
77.0589, found: 277.0580.
2
-(3,5-Dihydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (3t): Light brown solid, 110
1
mg, 29%, m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ 8.94 (d, J = 6.5 Hz, 1H), 7.96
6
(ddd, J = 8.6 Hz, J = 6.7 Hz, J = 1.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), ), 7.33 (ddd, J = 8.1
Hz, J = 6.9 Hz, J = 1.3 Hz, 1H), 7.01 (d, J = 2.2 Hz, 2H), 6.72 (s, 1H), 6.40 (dd, J = 2.2 Hz, J
1
3
=
2.1 Hz, 1H) ppm; C NMR (100 MHz, DMSO-d ):
δ
161.4, 159.6, 158.1, 150.9, 138.8,
6
1
38.1, 127.4, 126.6, 116.6, 105.7, 105.6, 98.8 ppm; IR: νmax 3286, 3113, 1668, 1634, 1164
-1
+
cm ; HRMS (ESI) m/z: calcd. for C H N O [M+H] 255.0770, found: 255.0763.
1
4
11
2
3
3
-(3,4-Dihydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (6t): Yellow solid, 87 mg,
1
2
3%, m.p. 160-162 °C; H NMR (400 MHz, DMSO-d ):
δ
9.08 (ddd, J = 7.2 Hz, J = 1.4 Hz,
6
J = 0.8 Hz, 1H), 9.06 (s, 1OH), 9.00 (s, 1OH), 8.50 (s, 1H), 7.93 (ddd, J = 8.4 Hz, J = 6.7
Hz, J = 1.6 Hz, 1H), 7.71 (dd, J = 8.7 Hz, J = 1.1 Hz, 1H), ), 7.39 (ddd, J = 8.2 Hz, J = 7.0
Hz, J = 1.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.10 (dd, J = 8.2 Hz, J = 2.2 Hz, 1H), 6.80 (d,
1
3
J = 8.2 Hz, 1H) ppm; C NMR (100 MHz, DMSO-d ):
δ
156.4, 152.0, 150.1, 145.6, 145.3,
6
1
1
2
36.8, 127.6, 126.5, 125.8, 119.9, 117.0, 116.3, 115.9 (2C) ppm; IR: νmax 3219, 2941, 1682,
-1
+
486, 1282, 1016 cm ; HRMS (ESI) m/z: calcd. for C H N O [M+H] 255.0770, found:
14
11
2
3
55.0762.

ACCEPTED MANUSCRIPT
3
1
8
6
-(3,5-Dihydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (6u): Yellow solid, 72 mg,
1
9%, m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ 9.13 (d, J = 6.9 Hz, 1H), 8.53 (s, 1H),
6
.11 (dd, J = 7.6 Hz, J = 7.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), ), 7.52 (dd, J = 7.0 Hz, J =
1
3
.7 Hz, 1H), 6.68 (d, J = 1.9 Hz, 2H), 6.25 (dd, J = 1.9 Hz, J = 1.9 Hz, 1H) ppm; C NMR
158.7 (3C), 155.9, 149.6, 139.4, 135.6, 128.5, 124.4, 118.1, 115.8,
(100 MHz, DMSO-d6):
δ
-1
1
07.0, 102.6 ppm; IR: νmax 3310, 3098, 1675, 1038 cm ; HRMS (ESI) m/z: calcd. for
+
C H N O Na [M+Na] 277.0589, found: 277.0584.
14
10
2
3
3
-(4-Hydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (6v): Yellow solid, 189 mg, 53%,
1
m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ 9.61 (s, 1OH), 9.08 (d, J = 6.6 Hz, 1H),
6
8
.53 (s, 1H), 7.93 (dd, J = 7.5 Hz, J = 7.2 Hz, 1H), 7.72-7.66 (m, 3H), 7.39 (dd, J = 6.4 Hz, J
1
3
=
6.3 Hz, 1H), 6.84 (d, J = 7.9 Hz, 2H) ppm; C NMR (100 MHz, DMSO-d ): δ 157.4,
6
1
3
2
56.5, 152.0, 150.2, 136.8, 129.9, 127.6, 126.5, 125.4, 117.0, 115.9, 115.6 ppm; IR: νmax
-1
+
300, 3068, 1654, 1628, 1444, 1217 cm ; HRMS (ESI) m/z: calcd. for C H N O [M+H]
1
4
11
2
2
39.0821, found: 239.0805.
3
-(3-Hydroxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one (6w): Yellow solid, 160 mg, 45%,
1
m.p. >200 °C; H NMR (400 MHz, DMSO-d ):
δ 9.50 (s, 1OH), 9.10 (dd, J = 7.2 Hz, J = 0.6
6
Hz, 1H), 8.56 (s, 1H), 7.97 (ddd, J = 8.7 Hz, J = 6.7 Hz, J = 1.5 Hz, 1H), 7.73 (d, J = 8.8 Hz,
1
H), 7.42 (ddd, J = 8.2 Hz, J = 7.0 Hz, J = 1.4 Hz, 1H), ), 7.31-7.30 (m, 1H), 7.26-7.20 (m,
13
2
H), 6.75 (ddd, J = 7.4 Hz, J = 2.0 Hz, J = 0.6 Hz, 1H) ppm; C NMR (100 MHz, DMSO-
157.6, 156.3, 152.9, 150.7, 137.5, 136.1, 129.7, 127.9, 126.5, 119.4, 117.3, 115.7,
d6):
δ
-1
1
15.5, 114.9 ppm; IR: νmax 3300, 3105, 1678, 1632, 1483, 1255 cm ; HRMS (ESI) m/z:
+
calcd. for C H N O Na [M+Na] 261.0640, found: 261.0634.
1
4
10
2
2
4
.2.4. Synthesis and characterization data of flavones (C1-C3):
The control compounds were synthesized following the literature reported method [21].
2
-(Benzo[d][1,3]dioxol-5-yl)-4H-chromen-4-one (C1):[31] White solid, 143 mg, 54%, m.p.
1
1
7
98-200 °C; H NMR (400 MHz, CDCl +DMSO-d ):
δ
8.18 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H),
3
6
.70 (ddd, J = 8.6 Hz, J = 7.2 Hz, J = 1.7 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.51 (dd, J = 8.3
Hz, J = 1.8 Hz, 1H), 7.42 (ddd, J = 8.0 Hz, J = 0.9 Hz, J = 0.9 Hz, 1H), 7.37 (d, J = 1.8 Hz,
1
3
1
H), 6.94 (d, J = 8.2 Hz, 1H), 6.70 (s, 1H), 6.08 (s, 2H) ppm; C NMR (100 MHz,
CDCl +DMSO-d ): 178.2, 163.0, 156.0, 150.6, 148.4, 133.7, 125.5, 125.4, 125.1, 123.7,
δ
3
6
-1
1
21.4, 117.9, 108.7, 106.3, 106.2, 101.9 ppm; IR: νmax 2992, 1642, 1446, 1244, 1024 cm ;

ACCEPTED MANUSCRIPT
+
MS (ESI) m/z 289 (M + Na ); Anal. Calcd. for C H O : C%, 72.18; H%, 3.79; Found: C%,
1
6
10
4
7
2.42; H%, 3.68.
2
-(3,4,5-Trimethoxyphenyl)-4H-chromen-4-one (C2):[32] White solid, 140 mg, 45%, m.p.
1
1
59-161 °C; H NMR (400 MHz, CDCl ):
δ
8.23 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 7.71 (ddd,
3
J = 8.6 Hz, J = 1.6 Hz, J = 1.3 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 7.9 Hz, J = 7.2
1
3
Hz, 1H), 7.14 (s, 2H), 6.77 (s, 1H), 3.96 (s, 6H), 3.94 (s, 3H) ppm; C NMR (100 MHz,
CDCl3): 178.3, 163.3, 156.2, 153.6, 141.2, 133.7, 127.0, 125.7, 125.3, 123.9, 118.1, 107.4,
03.8, 61.0, 56.4 ppm; IR: νmax 2936, 1636, 1606, 1243, 1123 cm ; MS (ESI) m/z 335 (M +
δ
-1
1
+
Na ); Anal. Calcd. for C H O : C%, 69.22; H%, 5.16; Found: C%, 69.31; H%, 5.37.
1
8
16
5
2
-(4-Methoxyphenyl)-4H-chromen-4-one (C3):[33] White solid, 169 mg, 67%, m.p. 158-
1
1
60 °C; H NMR (400 MHz, CDCl ):
δ
8.22 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 7.88 (d, J = 8.9
3
Hz, 2H), 7.68 (ddd, J = 8.6 Hz, J = 7.1 Hz, J = 1.7 Hz, 1H), 7.54 (dd, J = 8.4 Hz, J = 0.6 Hz,
1
3
1
1
4
H), 7.40 (ddd, J = 8.0 Hz, J = 1.0 Hz, J = 1.0 Hz, 1H), 7.02 (d, J = 8.9 Hz, 2H), 6.74 (s, 1H),
1
3
.88 (s, 3H) ppm; C NMR (100 MHz, CDCl ):
δ
178.4, 163.4, 162.4, 156.2, 133.6, 128.0,
3
25.7, 125.1, 124.0, 123.9, 117.9, 114.5, 106.2, 55.5 ppm; IR: νmax 3050, 2992, 1645, 1265,
-1
+
122, 1024 cm ; MS (ESI) m/z 275 (M + Na ); Anal. Calcd. for C H O : C%, 76.18; H%,
1
6
12
3
.79; Found: C%, 76.46; H%, 4.71.
4
.2.5. Synthesis and characterization data of isoflavones (C4-C7):
The control compounds were synthesized following the literature reported method[30]. 3-
Iodo-4H-1-benzopyran-4-one was synthesised following the literature report. 3-Iodo-4H-1-
benzopyran-4-one (1 mmol, 272 mg), K CO (3 mmol, 414 mg, 3 eq), boronic acid (1.2 eq)
2
3
and Pd(PPh ) (0.1 mmol, 115 mg, 10 mol %) were taken in a round-bottom flask and 1,4-
3
4
dioxane (3 mL) and H O (0.5 mL) were added to the mixture. The mixture was heated at 110
2
°
C for 18-24 h until complete consumption of the substrate as indicated by TLC. It was
cooled to rt and concenterated under reduced pressure. The mixture was extracted with
EtOAc (2x50 mL) and washed with water (2x5 mL) and brine (1x5 mL), dried with anhyd.
Na SO , and concentrated under reduced pressure. The column chromatographic purification
2
4
of crude mass was performed on silica gel using EtOAc-hexane (25-40%) as eluting solvent
to afford the isoflavones (C4-C7).
3
-(3,4,5-Trimethoxyphenyl)-4H-chromen-4-one (C4): Off-white solid, 193 mg, 62%, m.p.
1
1
17-119 °C; H NMR (400 MHz, CDCl ):
δ
8.32 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 8.05 (s,
3

ACCEPTED MANUSCRIPT
1
H), 7.70 (ddd, J = 8.6 Hz, J = 7.1 Hz, J = 1.7 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.45 (ddd, J
1
3
=
8.0 Hz, J = 1.0 Hz, J = 1.0 Hz, 1H), 6.80 (s, 2H), 3.90 (s, 6H), 3.88 (s, 3H) ppm; C NMR
176.3, 156.1, 153.3, 153.0, 138.3, 133.7, 127.3, 126.4, 125.3, 124.5,
(100 MHz, CDCl3):
δ
-1
1
18.1, 106.3, 60.8, 56.2 ppm; IR: νmax 3069, 2976, 1644, 1118 cm ; MS (ESI) m/z 335 (M +
+
Na ); Anal. Calcd. for C H O : C%, 69.22; H%, 5.16; Found: C%, 69.49; H%, 5.17.
1
8
16
5
3
1
8
-(4-Methoxyphenyl)-4H-chromen-4-one (C5):[25] White solid, 118 mg, 47%, m.p. 140-
1
42 °C; H NMR (400 MHz, CDCl ):
δ 8.31 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.67 (dd, J =
3
.2 Hz, J = 7.3 Hz, 1H), 7.51 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.42 (dd, J = 7.8
1
3
Hz, J = 7.3 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 3.84 (s, 3H) ppm; C NMR (100 MHz,
CDCl3): 176.4, 159.7, 156.2, 152.5, 133.5, 130.1, 126.4, 125.1, 125.0, 124.5, 124.1, 118.0,
14.0, 55.3 ppm; IR: νmax 3007, 2954, 1626, 1245, 1181 cm ; MS (ESI) m/z 275 (M + Na );
Anal. Calcd. for C H O : C%, 76.18; H%, 4.79; Found: C%, 76.47; H%, 4.76.
δ
-1
+
1
1
6
12
3
3
-(4-Methylphenyl)-4H-chromen-4-one (C6):[34] White solid, 118 mg, 50%, m.p. 156-158
1
°
C; H NMR (400 MHz, CDCl ):
δ
8.31 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 8.00 (s, 1H), 7.67
3
(ddd, J = 8.6 Hz, J = 7.1 Hz, J = 1.7 Hz, 1H), 7.48-7.45 (m, 3H), 7.41 (ddd, J = 8.0 Hz, J =
1
3
1
.0 Hz, J = 1.0 Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 2.39 (s, 3H) ppm; C NMR (100 MHz,
CDCl3):
18.0, 21.3 ppm; IR: νmax 3008, 1633, 1276, 1112 cm ; MS (ESI) m/z 259 (M + Na ); Anal.
Calcd. for C H O : C%, 81.34; H%, 5.12; Found: C%, 81.24; H%, 5.10.
δ
176.3, 156.2, 152.8, 138.1, 133.5, 129.2, 128.9, 128.8, 126.4, 125.4, 125.2, 124.6,
-1
+
1
1
6
12
2
3
-(Naphthalen-1-yl)-4H-chromen-4-one (C7):[35] White solid, 114 mg, 42%, m.p. 122-124
1
°
C; H NMR (400 MHz, CDCl ):
δ
8.36 (dd, J = 7.9 Hz, J = 1.4 Hz, 1H), 8.03 (s, 1H), 7.94-
3
1
3
7
1
1
.89 (m, 2H), 7.76-7.72 (m, 2H), 7.57-7.43 (m, 6H) ppm; C NMR (100 MHz, CDCl ): δ
3
76.5, 156.5, 154.2, 133.8, 133.7, 132.5, 129.8, 129.2, 128.4, 128.3, 126.6, 126.4, 126.0,
-1
25.7, 125.5, 125.40, 125.37, 124.5, 118.2 ppm; IR: νmax 3043, 1637, 1464, 1116 cm ; MS
+
(
ESI) m/z 295 (M + Na ); Anal. Calcd. for C H O : C%, 83.81; H%, 4.44; Found: C%,
1
9
12
2
8
3.54; H%, 4.41.
4
.3. Biology
Synthesized compounds were investigated for their ability to inhibit human DNA
topoisomerase IIα (hTopoIIα) and for the investigation of mode of action. A screening kit
purchased from TopoGEN, Inc. (Columbus, OH) was used. The screening protocols as

ACCEPTED MANUSCRIPT
provided supplier and reported in literature[11,13,36] were followed. The concentrations of
the reagents were varied according to need of our experiments.
4
.3.1. hTopoIIα-mediated DNA decatenation assay
In hTopoIIα-mediated DNA decatenation assay, kinetoplast DNA (kDNA) was used as a
substrate. All the reactions were carried out in micro centrifuge tubes on ice. Reaction
mixture contains 4 units of purified hTopoIIα, 150 ng of catenated kDNA, and 100 ꢀM
investigated compound or standard drug (dissolved in DMSO) were combined in freshly
prepared 5x complete reaction buffer. The final volume of reaction mixture was brought to 20
ꢀ
L with distilled H O. In the standard assay method, the order of addition for assay
2
components was H O, 5x complete buffer (buffer A: 0.5 M Tris-HCl (pH 8), 1.5 M sodium
2
chloride, 100 mM magnesium chloride, 5 mM dithiothreitol, 300 ꢀg of bovine serum
albumin/mL and buffer B: 20 mM ATP in water), kDNA, followed by either test compound
or standard drug and finally hTopoIIα. The reaction mixture was incubated at 37 ºC for 30
min. Further the reaction was stopped with 10% SDS, followed by digestion with proteinase
K then incubated at 37 ºC for 15 min. Topological forms of kDNA were determined by
running 1% agarose gel electrophoresis in Tris-acetate-EDTA (TAE) buffer containing 0.5
ꢀ
g/mL ethidium bromide and further de-stained with water for 20 min. The bands of kDNA
and decatenated products were visualized by Gel Doc EZ imager (BioRad, U. S. A.) and the
products were quantified by using ImageLab (BioRad).
4
.3.2. hTopoIIα-mediated DNA relaxation assay
In relaxation assay, a hot spot sequence for hTopoIIα containing supercoiled plasmid (pRYG)
was used as a substrate. In the reaction mixture following components were added, freshly
prepared 5x complete buffer (A: 0.5M Tris- HCl,pH 8), 1.50 M sodium chloride, 100 mM
magnesium chloride, 5 mM dithiothreitol, 300 ꢀg of bovine serum albumin/mL and buffer B:
2
0 mM ATP in water), 250 ng of supercoiled pHOT, followed by either test compound or
etoposide (100 ꢀM) and finally topoisomerase II (4 units). The final volume of reaction
mixture was brought to 20 ꢀL with distilled H O. Relaxation was carried out at 37 ºC for 30
2
min. The reaction was stopped with addition of 10% SDS followed by digestion with
proteinase K and further incubated at 37 ºC for 15 min. Electrophoresis was performed in a
1
% agarose gel in TAE buffer without ethidium bromide. Further the gel was stained with
ethidium bromide (0.5 ꢀg/mL) for 15 min, de-stained with water for 20 min, and
photographed using Gel Doc EZ imager (BioRad, U. S. A.) of the quantification of products
was carried out using ImageLab (BioRad).

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4
.3.3. Cleavage complex assay
Cleavage complex assay was performed using negatively small circular supercoiled plasmid
pHOT) as a substrate, following above mentioned protocol of the relaxation assay with
(
modification that 8 units of hTopoIIα was added 3-5 min before the addition of the
compound. Cleavage complex formation was started by incubating reaction mixture at 37 ºC
for 20 min and stopped with 10% SDS, followed by digestion with proteinase K with
subsequent incubation at 45 ºC for 30 min. After addition of the loading buffer, the reaction
was heated at 70 ºC for 2 min. Electrophoresis was performed in a 1% agarose gel in TAE
buffer containing ethidium bromide (0.5 ꢀg/mL). Further the gel was de-stained with water
for 20 min and photographed using Gel Doc EZ imager (BioRad, U. S. A.).Quantification of
the products was carried out using ImageLab (BioRad).
4
.3.4. DNA-intercalation assay
In this assay, 250 ng plasmid (pUC19) was incubated at 37 ºC for 20 min with 1 ꢀg/ mL
ethidium bromide, 100 ꢀM standard drug (etoposide) or investigated compounds.
Electrophoresis was carried out in a 1% agarose gel in TAE buffer without ethidium bromide.
Further the gel was stained with ethidium bromide (0.5 ꢀg/mL) for 15 min, de-stained with
water for 20 min, and photographed using Gel Doc EZ imager (BioRad, U. S. A.).
4
.3.5. Topo I-mediated relaxation assay
Negatively supercoiled DNA (pRYG) was used as a substrate in TopoI mediated DNA
relaxation assay. Reaction mixture contained freshly prepared 5x complete buffer (100 mM
Tris–HCl pH 7.9, 10 mM EDTA, 1.5 M NaCl, 1% Bovine Serum Albumin, 1 mM
Spermidine and 50% glycerol), 250 ng of pRYG, followed by either test compound or
standard drug camptothecin (100 µM) and finally topoisomerase I (4 units). The final volume
of reaction mixture was brought to 20 ꢀL with distilled H2O. Reaction mixture was incubated
at 37 °C for 30 min and was quenched with the addition of 10% SDS followed by digestion
with proteinase K at 37 °C for 15 min. Electrophoresis of the each sample was carried out in
1
% agarose gel in TAE buffer without ethidium bromide. Gel was stained with ethidium
bromide (0.5 µg/mL) for 15 min. Further, the gel was de-stained with water for 20 min and
photographed using Gel Doc EZ imager (BioRad, U. S. A.).
4
.3.6. CD based drug-DNA binding study:
Reaction mixture for CD spectroscopic study was containing 50 mM Tris-HCl buffer of pH
7
2
.5, compound solution (100 ꢀM) and CT-DNA solution (500 µg/mL), incubated at 25 °C for
h. The CD spectra of the CT-DNA solution (500 µg/mL) recorded in the presence and

ACCEPTED MANUSCRIPT
absence of investigated compounds and ethidium bromide (100 µM). EtBr (100 ꢀM) was
used as a positive control (known DNA intercalating agent CD spectroscopic measurements
were carried out at 25 °C with cuvette having path length 1 mm using CD spectrometer
(Jasco J-815) with the sensitivity of 100 millidegree and in continuous scanning mode with a
speed of 100 nm/min; band-width was 1 nm and the number of accumulations was three.
Scans were taken from 220 to 340 nm[37,38].
4
.3.7. Measurement of Drug -DNA interaction using UV-Vis spectrophotometer
To measure the drug –DNA binding interaction a UV-Vis absorption spectrophotometric
study was carried out with 1 cm path length quartz cuvette using UV-Vis spectrophotometer;
(UV-2450 Shimadzu). The spectra were scanned in the wavelength range 220-340 nm. Stock
solutions of the compounds (3m, 30 and 6d) were prepared in DMSO. This stock of each
compound was diluted to various concentrations in 50 mM Tris-HCl/NaCl buffer (pH 7.5).
For this assay a fixed concentration of DNA was used i.e. 50ng/mL. The drug DNA solutions
were incubated for 1 h in 37ºC incubator to allow the formation of drug-DNA complex. Then
the absorption spectra for different concentrations were taken within the wave length range
2
20-340 nm[29].
4
.3.8. Calculation of binding constant (Kd)
The binding isotherm (θ) for 3m, 3o and 6d was calculated by following formula:
θ = (A - A )/ (A - A ),
0
i
0
α
Here, A is the absorbance of DNA in the absence of compound at 260 nm, A is the
0
i
absorbance of DNA in the presence of any concentration, and A is the absorbance of DNA at
α
a concentration for which maximum binding took place. K was calculated that is correspond
M
to the half saturation point and the reciprocal of K was given by K (binding constant).
M
d
4
.3.9. MTT assay
The anchorage dependant cell viability of the investigational compounds, etoposide and
parent compounds were measured using MTT cell viability assay. Briefly, exponentially
growing cells (8000-10,000cells/well) were seeded in 96 well flat bottom tissue culture
plates. Then cells were treated with increasing concentrations of the compounds for 48 h
prior to harvest. Then media was aspirated and washed once with 1XPBS. Then 0.05% MTT
solution was added to each well and incubated in 37°C for 5 h to allow formation of
formazan crystals. The formazan crystals were dissolved by adding 100 µL of 0.2% NP-40
detergent solution and incubated in dark for 1 h. The colour intensity was measured
spectrophotometrically at 570 nm by using microplate reader (Mithras LB 940, Berthold,

ACCEPTED MANUSCRIPT
Germany). The data obtained were presented graphically as percent viability against
concentrations. The fifty percent viability (IC ) was obtained from the graph. Each data
5
0
point was calculated in triplicate and all the assays were performed at least thrice[11].
.3.10. Clonogenic assay
4
The colony forming ability of cells after exposure to the compounds were measured using a
well-defined assay known as colony forming assay or clonogenic assay. Briefly, HEK 293
cells (500/well) in 80-90% confluent were seeded and grown for 24 h. Then cells were treated
varied concentrations of the compounds for 48 h. Then media was aspirated and replaced
with the fresh media and allowed to grow for 6 days. Then plates were washed twice with 1X
PBS and followed by stainied with 0.2% crystal violet for 1 h. After washing with water and
air drying the plate, the colonies were counted. The data were calculated and graphically
represented as percent survival against concentrations. Each data point was calculated in
triplicate and all the assays were performed at least three times[39].
4
.3.11. Detection of apoptotic nuclei by DAPI stain
Apoptotic nuclei after treatment with compound 3o was determined using DAPI saining
6
method. Briefly ~1 × 10 cells were seeded in 40-mm dishes. After reaching 70–80%
confluency, cells were treated with increasing concentration of compound 3o (0–10 ꢀM) for
4
8 h. Cells were washed with ice-cold PBS and fixed with ice-chilled acetone: methanol (1:1)
for 10 min at -20°C. Fixed cells were washed with ice cold PBS followed by incubation with
the DAPI and 1h in dark. Excess DAPI was removed by repeated washing with chilled 1×
PBS, and the samples were observed under fluorescence microscope (Nikon, Tokyo, Japan)
at 40× magnification. The data provided here are from one of three independent
experiments[39].
4
.3.12. Matrigel invasion assay:
Matrigel invasion assay was carried out to study the ability of cells invade through the matrix.
This assay was carried out with little modification in the protocol described by Mousseau et
al. 2014. For this assay a matrix was prepared in a 6 well cell culture plate using egg albumin
after carefully taking the albumin inside laminar hood and allowed to settle down by heating
it in a hotpot at 65ºc temperature. The cells were seeded above the matrix and allowed to
grow for next 24 hour. Cells were treated with increasing concentration of compound for 48
hours. After the required time period media were discarded followed by washing with 1X
PBS. Cells were stained with 0.2% crystal violet after fixing with 4% para formaldehyde.

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Excess stains were washed with 1X PBS and invaded colonies were counted using colony
counter. Data were the replica of three independent experiments[40].
4
.3.13. Annexin-V/FITC/PI dual staining:
Annexin-V/FITC/PI dual staining is used to detect the distribution of cells in early apoptotic,
late apoptotic and necrosis phases. For this assay, cells were seeded in a 6 well cell culture
5
plate at a density of 1 x10 cells per well. 70–80 percent confluent cells were treated with
varying concentrations of compound 3o for 48 h. Then, the cells were harvested, washed and
stained with Annexin-V/FITC and PI according to manufacturer’s protocol (Sigma) and
finally sorted by FACS and data were analysed by FCS express 5.0 software. Data given here
is representative of one of three independent experiments[39].
4
.3.14. Statistical analysis:
Statistical significance was determined by paired t test (*p<0.05), (**p<0.005),
***p<0.001).
•
(

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Abbreviations
hTopoIIα, Human topoisomerase IIα; kDNA, Kinetoplast DNA; Nck, Nicked; Rel, Relaxed;
Lin, Linear; SC, Supercoiled; TAE, Tris-acetate-EDTA; MTT, 3-(4,5-Dimethylthiazol-2-yl)-
2
,5-diphenylte-trazolium bromide.
Acknowledgment
We gratefully acknowledge financial support from DBT, DST, and CSIR, Government of
India, New Delhi, for this investigation. GP and SMA are thankful to NIPER for their
fellowships. AN is thankful to ICMR for providing fellowship.
Supporting Information
Details of the analytical data of compounds are provided in the supporting information.
References
[
[
[
[
[
1] N. Brown, E. Jacoby, On Scaffolds and Hopping in Medicinal Chemistry, Mini-
Reviews Med. Chem. 6 (2006) 1217–1229. doi:10.2174/138955706778742768.
2] H. Sun, G. Tawa, A. Wallqvist, Classification of scaffold-hopping approaches, Drug
Discov. Today. 17 (2012) 310–324. doi:10.1016/j.drudis.2011.10.024.
3] N.T. Southall, Ajay, Kinase Patent Space Visualization Using Chemical Replacements,
J. Med. Chem. 49 (2006) 2103–2109. doi:10.1021/jm051201m.
4] P. Singh, A. Mittal, Current Status of COX-2 Inhibitors, Mini-Reviews Med. Chem. 8
(2008) 73–90. doi:10.2174/138955708783331577.
5] C. Shu, H. Ge, M. Song, J.H. Chen, H. Zhou, Q. Qi, et al., Discovery of Imigliptin, a
Novel Selective DPP-4 Inhibitor for the Treatment of Type 2 Diabetes, ACS Med
Chem Lett. 5 (2014) 921–926. doi:10.1021/ml5001905.
[
[
[
6] F.E. Koehn, G.T. Carter, The evolving role of natural products in drug discovery, Nat.
Rev. Drug Discov. 4 (2005) 206–220. doi:10.1038/nrd1657.
7] M.S. Butler, The Role of Natural Product Chemistry in Drug Discovery, J. Nat. Prod.
6
7 (2004) 2141–2153. doi:10.1021/np040106y.
8] G.M. Cragg, P.G. Grothaus, D.J. Newman, Impact of natural products on developing
new anti-cancer agents, Chem. Rev. 109 (2009) 3012–3043. doi:10.1021/cr900019j.

ACCEPTED MANUSCRIPT
[
[
9] D.J. Newman, G.M. Cragg, Natural Products As Sources of New Drugs over the 30
Years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311–335. doi:10.1021/np200906s.
10] J. Rosén, J. Gottfries, S. Muresan, A. Backlund, T.I. Oprea, Novel Chemical Space
Exploration via Natural Products, J. Med. Chem. 52 (2009) 1953–1962.
doi:10.1021/jm801514w.
[
11] A.T. Baviskar, C. Madaan, R. Preet, P. Mohapatra, V. Jain, A. Agarwal, et al., N-
Fused Imidazoles As Novel Anticancer Agents That Inhibit Catalytic Activity of
Topoisomerase IIα and Induce Apoptosis in G1/S Phase, J. Med. Chem. 54 (2011)
5
013–5030. doi:10.1021/jm200235u.
[
[
12] M. Kashyap, S. Kandekar, A.T. Baviskar, D. Das, R. Preet, P. Mohapatra, et al.,
Indenoindolone derivatives as topoisomerase II-inhibiting anticancer agents,
Bioorganic Med. Chem. Lett. 23 (2013) 934–938. doi:10.1016/j.bmcl.2012.12.063.
13] A.T. Baviskar, S.M. Amrutkar, N. Trivedi, V. Chaudhary, A. Nayak, S.K. Guchhait, et
al., Switch in Site of Inhibition: A Strategy for Structure-Based Discovery of Human
Topoisomerase IIα Catalytic Inhibitors, ACS Med. Chem. Lett. 6 (2015) 481–485.
doi:10.1021/acsmedchemlett.5b00040.
[
[
[
[
14] Y. Li, H. Fang, Wenfang Xu, Y. Li, H. Fang, W. Xu, Recent Advance in the Research
of Flavonoids as Anticancer Agents, Mini-Reviews Med. Chem. 7 (2007) 663–678.
doi:10.2174/138955707781024463.
15] Y. Yoshinori, K. Sho-Zou, N. Hirofumi, Induction of mammalian topoisomerase ii
dependent dna cleavage by nonintercalative flavonoids, genistein and orobol,
Biochem. Pharmacol. 39 (1990) 737–744. doi:10.1016/0006-2952(90)90153-C.
16] A. Constantinou, R. Mehta, C. Runyan, K. Rao, A. Vaughan, R. Moon, Flavonoids as
DNA Topoisomerase Antagonists and Poisons: Structure-Activity Relationships, J.
Nat. Prod. 58 (1995) 217–225. doi:10.1021/np50116a009.
17] N. Chernyak, V. Gevorgyan, General and efficient copper-catalyzed three-component
coupling reaction towards imidazoheterocycles: one-pot synthesis of alpidem and
zolpidem.,
Angew.
Chem.
Int.
Ed.
Engl.
49
(2010)
2743–6.
doi:10.1002/anie.200907291.

ACCEPTED MANUSCRIPT
[
18] S.K. Guchhait, S. Kandekar, M. Kashyap, N. Taxak, P. V Bharatam, C–H Bond
Functionalization Under Metalation–Deprotonation Process: Regioselective Direct
Arylation of 3-Aminoimidazo[1,2-a]pyrazine, J. Org. Chem. 77 (2012) 8321–8328.
doi:10.1021/jo301065s.
[
[
19] J.W. Dankwardt, Nickel-Catalyzed Cross-Coupling of Aryl Grignard Reagents with
Aromatic Alkyl Ethers: An Efficient Synthesis of Unsymmetrical Biaryls, Angew.
Chemie. 116 (2004) 2482–2486. doi:10.1002/ange.200453765.
20] F. Kakiuchi, M. Usui, S. Ueno, N. Chatani, S. Murai, Ruthenium-Catalyzed
Functionalization of Aryl Carbon-Oxygen Bonds in Aromatic Ethers with
Organoboron Compounds, J. Am. Chem. Soc. 126 (2004) 2706–2707.
doi:10.1021/ja0393170.
[
21] S.K. Guchhait, G. Priyadarshani, Synthesis of 2-Arylpyridopyrimidinones, 6-
Aryluracils, and Tri- and Tetrasubstituted Conjugated Alkenes via Pd-Catalyzed
Enolic C–O Bond Activation–Arylation, J. Org. Chem. 80 (2015) 6342–6349.
doi:10.1021/acs.joc.5b00771.
[
[
[
[
22] S.K. Guchhait, G. Priyadarshani, Pd-Catalyzed Ag(I)-Promoted C3-Arylation of
Pyrido[1,2-a]pyrimidin-4-ones with Bromo/Iodo-Arenes, J. Org. Chem. 80 (2015)
8
482–8488. doi:10.1021/acs.joc.5b01573.
23] N.C.W. Johansson, J. O.; Hansen, H. C.; Chiacchia, F. S.; Wong, Pharmaceutical
compositions for the prevention and treatment of complex diseases and their delivery
by insertable medical devices., PCT/US2006/029827, 2007.
24] A.-S. Lin, K. Nakagawa-Goto, F.-R. Chang, D. Yu, S.L. Morris-Natschke, C.-C. Wu,
et al., First Total Synthesis of Protoapigenone and Its Analogues as Potent Cytotoxic
Agents, J. Med. Chem. 50 (2007) 3921–3927. doi:10.1021/jm070363a.
25] D.A. Vasselin, A.D. Westwell, C.S. Matthews, T.D. Bradshaw, M.F.G. Stevens,
Structural Studies on Bioactive Compounds. 40. 1 Synthesis and Biological Properties
of Fluoro-, Methoxyl-, and Amino-Substituted 3-Phenyl-4 H -1-benzopyran-4-ones
and a Comparison of Their Antitumor Activities with the Activities of Related 2-
Phenylbenzo, J. Med. Chem. 49 (2006) 3973–3981. doi:10.1021/jm060359j.

ACCEPTED MANUSCRIPT
[
[
26] D. Darpan, G. Joshi, S.M. Amrutkar, A.T. Baviskar, H. Kler, S. Singh, et al., Synthesis
and biological evaluation of new 2,5-dimethylthiophene/furan based N-acetyl
pyrazolines as selective topoisomerase II inhibitors, RSC Adv. 6 (2016) 14880–14892.
doi:10.1039/C5RA25705K.
27] A. Negi, J.M. Alex, S.M. Amrutkar, A.T. Baviskar, G. Joshi, S. Singh, et al.,
Imine/amide–imidazole conjugates derived from 5-amino-4-cyano-N1-substituted
benzyl imidazole: Microwave-assisted synthesis and anticancer activity via selective
topoisomerase-II-α inhibition, Bioorg. Med. Chem. 23 (2015) 5654–5661.
doi:10.1016/j.bmc.2015.07.020.
[
[
[
28] R. Palchaudhuri, P.J. Hergenrother, DNA as a target for anticancer compounds:
methods to determine the mode of binding and the mechanism of action, Curr. Opin.
Biotechnol. 18 (2007) 497–503. doi:10.1016/j.copbio.2007.09.006.
29] R. Preet, B. Chakraborty, S. Siddharth, P. Mohapatra, D. Das, S.R. Satapathy, et al.,
Synthesis and biological evaluation of andrographolide analogues as anti-cancer
agents, Eur. J. Med. Chem. 85 (2014) 95–106. doi:10.1016/j.ejmech.2014.07.088.
30] C. La Motta, S. Sartini, L. Mugnaini, F. Simorini, S. Taliani, S. Salerno, et al.,
Pyrido[1,2- a ]pyrimidin-4-one Derivatives as a Novel Class of Selective Aldose
Reductase Inhibitors Exhibiting Antioxidant Activity, J. Med. Chem. 50 (2007) 4917–
4
927. doi:10.1021/jm070398a.
[
[
[
[
31] X. Huang, E. Tang, W.-M. Xu, J. Cao, Lewis Acid Catalyzed Solid-Phase Synthesis of
Flavonoids Using Selenium-Bound Resin, J. Comb. Chem. 7 (2005) 802–805.
doi:10.1021/cc0498231.
32] Z. Du, H. Ng, K. Zhang, H. Zeng, J. Wang, Ionic liquid mediated Cu-catalyzed
cascade oxa-Michael-oxidation: efficient synthesis of flavones under mild reaction
conditions, Org. Biomol. Chem. 9 (2011) 6930-6933. doi:10.1039/c1ob06209c.
33] J. Zhao, Y. Zhao, H. Fu, K 2 CO 3 -Catalyzed Synthesis of Chromones and 4-
Quinolones through the Cleavage of Aromatic C–O Bonds, Org. Lett. 14 (2012) 2710–
2
713. doi:10.1021/ol300908g.
34] M. Rao, V. Venkatesh, D. Jadhav, Pd-Catalyzed Efficient Cross-Couplings of 3-

ACCEPTED MANUSCRIPT
Iodochromones with Triaryl-bismuths as Substoichiometric Multicoupling
Organometallic Nucleophiles, Synlett. 2009 (2009) 2597–2600. doi:10.1055/s-0029-
1
217959.
[
[
35] Y. Hoshino, N. Miyaura, A. Suzuki, Novel synthesis of isoflavones by the palladium-
catalyzed cross-coupling reaction of 3-bromochromones with arylboronic acids or its
esters., Bull. Chem. Soc. Jpn. 61 (1988) 3008–3010. doi:10.1246/bcsj.61.3008.
36] S. Jiménez-Alonso, H.C. Orellana, A. Estévez-Braun, A.G. Ravelo, E. Pérez-Sacau, F.
Machín, Design and synthesis of a novel series of pyranonaphthoquinones as
topoisomerase II catalytic inhibitors, J. Med. Chem. 51 (2008) 6761–6772.
doi:10.1021/jm800499x.
[
[
37] R. Preet, B. Chakraborty, S. Siddharth, P. Mohapatra, D. Das, S.R. Satapathy, et al.,
Synthesis and biological evaluation of andrographolide analogues as anti-cancer
agents, Eur. J. Med. Chem. 85 (2014) 95–106. doi:10.1016/j.ejmech.2014.07.088.
38] S.K. Samanta, D. Dutta, S. Roy, K. Bhattacharya, S. Sarkar, A.K. Dasgupta, et al.,
Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular Cytotoxicity with
Involvement of C-7-OH and −NH Functional Groups, J. Med. Chem. 56 (2013) 5709–
5
721. doi:10.1021/jm400290q.
[
[
39] V. Chaudhary, S. Das, A. Nayak, S.K. Guchhait, C.N. Kundu, Scaffold-hopping and
hybridization based design and building block strategic synthesis of pyridine-annulated
purines: discovery of novel apoptotic anticancer agents, RSC Adv. 5 (2015) 26051–
2
6060. doi:10.1039/C5RA00052A.
40] Y. Mousseau, S. Mollard, H. Qiu, L. Richard, R. Cazal, A. Nizou, et al., In vitro 3D
angiogenesis assay in egg white matrix: comparison to Matrigel, compatibility to
various species, and suitability for drug testing, Lab. Investig. 94 (2014) 340–349.
doi:10.1038/labinvest.2013.150.