3-Formylchromone based topoisomerase IIα inhibitors: Discovery of potent leads

Article abstract of DOI:10.1039/c3md00125c

Substituted 3-formylchromones were synthesized and evaluated as inhibitors of the human DNA topoisomerase IIα (hTopo-IIα) enzyme. The results of the decatenation, relaxation and DNA intercalation assays revealed that the compounds (11b, 12a, 12b, 12d, 12e, 13a and 13b) exhibited potent inhibitory activity against the hTopo-IIα enzyme, and are nonintercalating agents. These compounds also possess significant in vitro cytotoxicity (LC₅₀ ranges from 0.5-8.6 μM) against prostate (PC-3) cancerous cell line as seen in comparison to the standard drug etoposide. To further probe the plausible mode of action of 3-formylchromone derivatives, molecular docking studies have also been carried out, which showed that the compounds under investigation fitted well in the ATP binding pocket of hTopo-IIα enzyme with good docking scores and form nonbonding interactions with the crucial residues of the catalytic site. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3md00125c

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3ꢀFormylchromone Based Topoisomerase IIα Inhibitors: Discovery of
Potent Leads
Satyajit Singh,a,b Ashish Triambak Baviskar,c Vaibhav Jain,c Nidhi Mishra,c Uttam Chand
Banerjee,c Prasad V. Bharatam,c Kulbhushan Tikooc and Mohan Paul Singh Ishara*
a b Bioorganic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak
Dev University, Amritsar 143 005, India
bBioorganic Chemistry Laboratory, Department of Pharmaceutical Chemistry, Khalsa College of
Pharmacy,G.T.Road,Amritsar,143002,Punjab,India.
cNational Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar,
Punjab 160 062, India
*Corresponding author:
Mohan Paul Singh Ishar, Ph.D.
Professor
Department of Pharmaceutical Sciences,
Guru Nanak Dev University, Amritsar 143005, India
Ph:
0183ꢀ2258802
Fax: 0183ꢀ2258820
Email: mpsishar@yahoo.com (M.P.S. Ishar)
1

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ABSTRACT
Substituted 3ꢀformylchromones were synthesized and evaluated as inhibitors of human DNA
topoisomerase IIα (hTopoꢀIIα) enzyme. The results of the decatenation, relaxation and DNA
intercalation assays revealed that the compounds (11b, 12a, 12b, 12d, 12e, 13a and 13b)
exhibited potent inhibitory activity against hTopoꢀIIα enzyme, and are nonintercalating
agents. These compounds also possess significant in vitro cytotoxcity (LC50 ranges from 0.5ꢀ
8.6ꢀM) against Prostate (PCꢀ3) cancerous cell line as seen in comparison to the standard drug
etoposide. To further probe the plausible mode of action of 3ꢀformylchromone derivatives,
molecular docking studies have also been carried out, which showed that the compounds
under investigation fitted well in the ATP binding pocket of hTopoꢀIIα enzyme with good
docking score and makes nonbonding interactions with the crucial residues of the catalytic
site.
Key Words: 3ꢀFormylchromone; Topoisomerase IIα inhibitors; DNA nonꢀintercalator;
Cytotoxcity; Molecular Docking.
1. Introduction
The structure of DNA is prone to topological challenges arising from intertwining of
its complementary strands and can generate super helical strain which impairs the vital
processes (transcription, replication, chromatin assembly and chromosome segregation). The
cells rely on “topoisomerases”, essential enzymes, which have the ability to resolve the
topological problems associated with the double helix structure of DNA without changing the
underlying chemical structure of DNA. 1 Two major forms of topoisomerase enzymes (Topoꢀ
I and TopoꢀII) are present in all the cells and are differentiated on the basis of mechanistic
and physical properties. TopoꢀI causes the single strand break in superhelical DNA resulting
2

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in relaxation, and finally relegates the nick. Single strand cut allows the controlled rotation of
broken DNA around the other intact strand and thereby reducing the stress. TopoꢀII requires
ATP for its catalytic activity and it has the ability to create a transient double strand break in
the DNA, allowing a passage of one intact DNA helix through cleaved end and the break is
resealed by enzyme.1,2 Human TopoꢀII exists in two isoforms, α (alpha) and β (beta). Topoꢀ
IIα is required to regulate both cell cycle and growth cycle, and its level increases throughout
the S phase, and peaks at G2/M boundary. Moreover, it is present exclusively in rapidly
proliferating tissues, which suggests that TopoꢀIIα bears the major responsibility toward
events associated with DNA replication and chromosome segregation. In contrast, level of
TopoꢀII β isoform is found to be independent of both cell cycle and growth cycles due to its
presence in cell regardless of their proliferation status. 3
DNA topoisomerases have fascinated both clinical scientists and medicinal chemists
for several decades; these have been recognized as important molecular targets for a variety
of pharmaceutical agents .4 Most of the chemotherapeutic anticancer treatments rely on the
5,6
use of at least one drug that inhibits topoisomerases.
Some of the clinically important
anticancer drugs targeting topoisomerase type II include doxorubicin, daunorubicin,
etoposide, teniposide, mitoxanthrone, amonafide, and amsacrine 7,8, whereas camptothecin
9,10
derivatives target the topoisomerase type I.
anticancer drugs are also being investigated. 1
Dual topoisomerase I and II inhibitors as
Molecules targeting TopoꢀII are classified into two types: TopoꢀII catalytic inhibitors
and poisons. 11 Molecules/therapeutic agents belonging to catalytic inhibitor class interfere at
one of the stages involving binding of the enzyme to DNA, ATP to the DNAꢀenzyme
complex or ATP hydrolysis. The second class TopoꢀII poisons act by stabilizing the cleavable
complex of TopoꢀII with DNA by the formation of a covalent ternary complex of drug/agentꢀ
3

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cleaved G segment DNA and enzyme. Previous studies indicated that TopoꢀII poisons may
12
induce chromosomal translocation, which lead to specific leukaemias. Consequently, in
recent years efforts have been directed towards the development of TopoꢀII catalytic
inhibitors to avoid cytotoxic effects of TopoꢀII poisons; some of the agents are also capable
of overcoming the multidrug resistance (MDR).13, 14
Chromone moiety is a part of a large number of molecules of medicinal significance
and is capable of interacting with a variety of proteins, enzymes and biological receptors, and
many of chromone derivatives display significant anticancer activity.15 Earlier we had
reported 3ꢀformylchromone derivatives as potential topoisomerase inhibitors, which exhibit
high anticancer activity against Ehrlich Ascites cancer cells in vitro as well as in EAC
implanted mice.16 Some recently reported synthetic and naturally occurring chromone
derivatives as TopoꢀII inhibitors (Fig. 1) include benzoxanthone (1) derivatives, which are
DNA crossꢀlinker 17, and synthetic flavonoid MHY336 (2); the latter arrests the cell cycle in
G2/M or S phase via TopoꢀII dependent mechanism 18. Dietary flavonoid fisetin (3), has been
found to act as a dual inhibitor of TopoꢀI and TopoꢀII in cells.19 Hecht and coworkers have
demonstrated that topopyrones (4ꢀ7) are also strong TopoꢀII poisons.20, 21 In the case of
22
psorospermin (8), a natural antitumor antibiotic , it has been shown that it intercalates with
DNA and its alkylating potential is significantly increased in the presence of TopoꢀII. 23
Prompted by reported anticancer activity of molecules possessing chromone nucleus,
present investigations were aimed at design, synthesis, bioevaluation and molecular modeling
studies of chromone based anticancer agents, targeting human DNA Topo IIα (hTopoꢀIIα)
enzyme. Based on our earlier design considerations 16, it was envisaged that differently
substituted 3ꢀformylchromones and its derivatives could act as potential inhibitors of this key
enzyme, regulating the topological changes of DNA. Herein we report the synthesis of 3ꢀ
4

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formylchromones derivatives, followed by their in vitro evaluation using decatenation,
relaxation and DNA intercalation assays to identify their hTopoꢀIIα inhibitory activity. In
vitro cytotoxic activity of the potent compounds was also tested against PCꢀ3 cancerous cell
line by MTT assay. Finally, molecular docking studies were performed on investigational
compounds to identify their plausible mode of interaction with hTopoꢀIIα enzyme.
S
O
O
OH
O
O
OH
O
OH
O
O
O
O
HO
S
OH
3
2
1
OH
O
O
O
OH
O
O
OH
Cl
O
O
HO
OH
HO
OH
O
4
5
O
O
OCH3
O
O
OH
O
OH
O
OH
O
OH
Cl
OH
O
OH
O
OH
O
O
H
7
6
CH3
8
O
Fig. 1. Chromone based compounds as topoisomerase II inhibitors
Results and discussion
Chemistry
The synthetic approaches adopted for the preparation of the desired compounds are illustrated
in scheme 1. A variety of 3ꢀformylchromones and its derivatives (11aꢀe, 12aꢀe and 13aꢀe)
16, 24
were synthesized by utilizing the protocol established in our previous study
which
involve synthesis of 3ꢀformylchromone and further derivatisation by reaction with Nꢀ
phenylhydroxylamine yielding nitrones (X), which on thermal rearrangement in the presence
5

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of a few drops of acetic acid, leads to the formation of 2ꢀanilinoꢀ3ꢀformylchromones (12aꢀe).
Nꢀ methylation of 12aꢀe was carried out with methyl iodide in the presence of anhydrous
potassium carbonate by refluxing in dry acetone leading to the formation of 2ꢀ(Nꢀ
methylanilino)ꢀ3ꢀformylchromones (13aꢀe, Scheme 1).
Scheme 1 Synthesis of various 3ꢀformylchromone derivatives
Pharmacology
The bioevaluation of the investigational compounds (11aꢀe, 12aꢀe and 13aꢀe) against hTopoꢀ
IIα enzyme was carried out to study decatenation, relaxation processes and mode of
interaction (intercalating or nonintercalating). A screening kit purchased from TopoGEN, Inc.
(Columbus, OH) was utilized for this purpose. To investigate the cytotoxic activity of potent
hTopoꢀIIα inhibitors, they were tested against Prostate (PCꢀ3) cancerous cell line by MTT
assay; cell survival was measured after the treatment of cells with the investigational
compounds for 48 h. Etoposide, a known Topo II inhibiting anticancer drug, was used as a
standard in all the in vitro experimental assays.
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hTopoꢀIIα based decatenation assay
The ATPꢀdependent kDNA decatenation assay was performed to examine the effect of the
investigational compounds on hTopoꢀIIα enzyme and results are presented in Fig. 2A. This
assay was performed using kinetoplast DNA (kDNA) as a substrate and etoposide (known
topoIIꢀinhibiting anticancer drug) as a standard.14, 25 Incubation of kDNA with hTopoꢀIIα
leads to the formation of decatenated products: nicked (Nck) and supercoil (SC)/relaxed (Rel)
circular DNA, which move easily in agarose gel as compared to catenated kDNA because of
latter’s overall size.26 The standard drug etoposide causes the partial or moderate
decatenation. In case of the investigational compounds (11aꢀ13e) almost no decatenation
activity was observed in the presence of compounds 11b, 12a, 12b, 12d, 12e, 13a and 13b,
indicating their good inhibitory activity against hTopoꢀIIα enzyme (Fig. 2A). The
decatenation products (Nck, Rel, and SC) were quantified by analyzing the densitometric data
obtained using Quantity One (BioRad) and the results were compared with standard drug
etoposide (Fig. 2B). In comparison to etoposide, compounds 11b, 12a, 12b, 12d, 12e, 13a
and 13b have shown higher degree of inhibition of hTopoꢀIIα activity.
A
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B.
Fig. 2 (A) Effect of test compounds on Topo IIα mediated kDNA decatenation assay. kDNA was treated with
hTopoIIα in the presence of either 100 ꢁM etoposide or test compounds. Reaction mixture was incubated at 37
°C for 30 min and 1% agarose gel was run in TAE buffer (B) Graphical representation of decatenated products
formed in kDNA decatenation assay.
hTopoꢀIIα based relaxation assay
Further, in order to confirm the hTopoꢀIIα inhibitory activity of investigational molecules,
compounds 11b, 12a, 12b, 12d, 12e, 13a and 13b were selected on the basis of results of
decatenation assay and were subjected to relaxation assay.14, 25 In this assay, negatively
supercoiled DNA (pRYG) was used as a substrate and etoposide as positive control. When
the supercoiled form of substrate plasmid DNA (pRYG) was treated with hTopoꢀIIα, a set of
variably relaxed topoisomers was formed, which migrate more slowly as compared to
supercoiled form in agarose gel. The results of relaxation assay revealed similar trends as
observed in kDNA decatenation assay (Fig. 3A). Quantification of relaxed topoisomers was
done by analyzing the densitometry data using Quantity One (BioRad) and the results were
compared with etoposide (Fig. 3B). It was observed that compounds (11b, 12a, 12b, 12d,
12e, 13a and 13b) were found to be more potent than the standard etoposide as the degree of
relaxation of the substrate pRYG plasmid DNA was less in the presence of compounds under
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investigation (Fig. 3B). The obtained results indicate that investigational compounds are
more potent hTopoꢀIIα inhibitors as compared to the etoposide and the results obtained in
relaxation assay were found to be consistent with kDNA decatenation assay.
A.
B.
Fig. 3 (A) Effect of test compounds on Topo IIα mediated supercoiled pRYG plasmid DNA relaxation assay.
Negatively supercoiled pRYG plasmid DNA was treated with hTopoIIα in the presence of either 100 ꢂM
etoposide or test compounds. Electrophoresis was carried out in 1% agarose gel in TAE buffer without ethidium
bromide. (B) Graphical representation of relative relaxation in pRYG DNA by investigational compounds 11b,
12a, 12b, 12d, 12e, 13a and 13b
DNA intercalation assay
To gain deeper insight, whether active investigational compounds are DNA intercalator or
nonintercalators, DNA intercalation assay was performed with active compounds 11b, 12a,
12b, 12d,12e, 13a and 13b. In this assay negatively supercoiled small circular plasmid DNA,
isolated from E. coli was used as a substrate.27, 28 From the results obtained, it was found that
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in the presence of ethidium bromide (DNA intercalating agent), there was retardation in the
migration of the DNA, whereas, in the case of etoposide (DNA nonintercalator) and our
tested compounds, there was no such retardation, which indicates that the investigational
compounds are DNA nonintercalators (Fig. 4).
Fig. 4. DNA intercalation assay. Negatively supercoiled plasmid was incubated with 1 ꢂg/mL ethidium bromide
or 100 ꢂM etoposide or test compounds. Electrophoresis was carried out in 1% agarose gel in TAE buffer
without ethidium bromide.
Anticancer activity of investigational compounds: Cell survival assay
The cytotoxic activity of the potent investigational compounds (11b, 12a, 12b, 12d, 12e, 13a
and 13b) was investigated in Prostate (PCꢀ3) cells by MTT assay and the results are shown in
Fig. 5. Cell survival was measured after treating cells with the investigational compounds for
48 h. It was observed that with an increasing concentration of the compounds, the cell
viability of PCꢀ3 cells decreased. Among the compounds tested for cytotoxic studies,
compound, 13a was found to be most active as it killed 50% of PCꢀ3 cell at concentration of
0.5 ꢂM, which was less than the standard drug etoposide (LC50ꢀ 7.5 ꢂM). The compounds
13b, 12e and 11b were also found to possess significant cytotoxic potential as they have
lower range of LC50 i.e. 2.1, 2.4 and 5.7 ꢂM, respectively, as compared to the positive
control, etoposide. While the other three investigational compounds 12a, 12b and 12d were
found to possess cytotoxic activity (LC50: 8.2, 8.3 and 8.6 ꢂM) comparable to that of
etoposide.
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Fig. 5 MTT cell survival assay (LC50)
Further the IC50 value for the most active investigational compound (13a) (selected on the
basis of results of biological assays) was determined by performing kDNA decatenation assay
using concentration range from 20 ꢁM to 100 ꢁM and IC50 value for compound 13a was
found to be 67.86 ꢁM. (fig.6 A and B)
Figure.6: (A) Effect of test compound 13a on Topo IIα mediated kDNA decatenation assay. kDNA was treated
with hTopoIIα in the presence of various concentrations of compounds 13a (20 ꢁM to 100 ꢁM). Electrophoresis
was carried out in 1% agarose gel in TAE buffer (B) Graphical plot of decatenated products formed in kDNA
decatenation
assay.
Molecular modeling studies
Nowadays, molecular modeling studies are routinely used to understand the atomistic level
details of proteinꢀligand interactions, and to identify potential inhibitors against disease target
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of interest. But accurate and reliable results can only be obtained if the ligands are considered
in their most stable conformation. In present study, the basic nucleus 3ꢀformylchromone of
the synthesized compounds can exist in two forms due to the rotation of 3ꢀformyl group
assigned as 0o and 180o. Thus, in order to find the most stable conformation of 3ꢀ
formylchromone derivatives (11aꢀe, 12aꢀe and 13aꢀe), the rotamers of the representative
structures (11a, 12a and 13a) were considered and subjected to quantum chemical
calculations. After full optimization of these species in gas phase using density functional
theory (DFT) method at B3LYP/6ꢀ31+G(d,p) level, the conformations with about 180o
rotation (CꢀCꢀCꢀO) in 3ꢀformyl group were named as 11α, 12α and 13α and those with about
0o rotation as 11β, 12β and 13β (Fig. 7). The relative energy differences between each
respective rotamer are shown in Table 1. From the table it is clear that the conformations 11α
ꢀ13α are more stable than their respective rotamers 11ꢀ13 β. The higher stability of all the
alpha conformations is attributed to the fact that intramolecular hydrogen bonding can be
formed between 3ꢀformylꢀH and 4ꢀcarbonyl groups, while this type of interaction is absent in
beta conformations. Moreover in beta conformations, the electrostatic repulsion between the
lone pairs of oxygens in 3ꢀformyl and 4ꢀcarbonyl groups can also makes them less stable as
compared to alpha conformations. The information obtained from the quantum chemical
analysis was further utilized to build the representative 3D stable structure of the compounds
for molecular docking study.
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Fig. 7 Fully optimized rotamers of 3ꢀformylchromone derivatives in gas phase at B3LYP/6ꢀ
31+G(d,p) level. Color representation of atoms is as follows: carbonꢀmagenta, oxygenꢀred,
nitrogenꢀblue, hydrogenꢀwhite.
Table 1 Relative energy difference between 3ꢀformylchromones rotamers.
Name
B3LYP/6ꢀ31+G(d,p)
Relative energy (kcal/mol)
0.00
11α
11β
5.06
0.00
5.17
12α
12β
0.00
7.39
13α
13β
From the decatenation, relaxation and DNA intercalation assays performed on
synthesized 3ꢀformylchromone derivatives, it was established that compounds 11b, 12a, 12b,
12d, 12e, 13a and 13b are nonintercalating agents and possess potent hTopoꢀIIα inhibitory
activity. In addition, these compounds also possess good cytotoxcity (LC50 ranges from 0.5ꢀ
8.6 ꢀM) activity against PCꢀ3 cancerous cell line. Further the previous computational study
established that several designed molecules bind to the ATPase domain of hTopoꢀIIα and the
possible interactions between ligands and enzyme are already made available.14 Thus, to
obtain insight into the molecular mechanism of action of 3ꢀformylchromone derivatives,
molecular docking of the synthesized compounds was carried out in the ATPase domain of
hTopoꢀIIα enzyme (PDB ID: 1ZXM). 29
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Molecular docking studies were performed using FlexX program30, which uses
incremental construction algorithm. The docking protocol was already established as a
reliable one in previous report14, which revealed the importance of interaction with Mg2+ ion
in the active site cavity. The detailed intermolecular nonꢀbonding interactions (hydrogen
bonding, hydrophobic and metal ion interactions) and FlexX docking score of substrate ATP
and active compounds with the ATPase domain of hTopoꢀIIα is provided in Table 2.
Table 2. FlexX docking results for some of the synthesized compounds and substrate ATP in
presence of two conserved water molecules in hTopoꢀIIα ATP binding site.
Compound aLC50
FlexX
Score
Hydrogen bonding
Interactions
Hydrophobic
interactions
Interaction
with Mg
cation
(ꢁM)
Backbone
Side chain
ꢀ84.06
Ser149, Arg162,
Asn163, Gly164,
Tyr165, Gly166,
Ala167
Asn91, Asn120,
Ser148, Ser149,
Asn150, Lys168,
Gln376, Lys378
Ile125, Ile141,
Phe142, Ile217
Ionic
interaction
ATP
(Substrate)
5.7
8.2
8.6
2.5
ꢀ29.10
ꢀ27.78
ꢀ25.38
ꢀ23.42
Gly164, Tyr165,
Gly166, Ala167
Ser148, Asn150,
Gln376
Ile141
π interaction
π interaction
π interaction
11b
12a
12d
12e
Ser149
Ser148 (2),
Asn150
Ile141, Phe142
ꢀ
Ser149, Gly164,
Tyr165, Gly166
Ser148, Asn150,
Gln376, Lys378
Ser149
Asn91, Ser148 (2), Ile141, Phe142 π interaction
Asn150 (2)
0.5
8.3
ꢀ23.09
ꢀ22.00
Ser149
Ser149
Ser148, Asn150
Ile141, Phe142, π interaction
Ala167
13a
12b
Ser148 (2),
Asn150 (3)
Ile141, Phe142
π interaction
2.1
ꢀ18.77
Ser149
Ser148 (2),
Asn150 (3)
Ile141, Phe142, π interaction
Ala167
13b
aThe values were calculated by noting down the concentration of the compound at which 50% of the
PCꢀ3 cancerous cell gets killed (see cell survival assay).
Binding pose analysis of the active 3ꢀformylchromone derivatives 11b, 12a, 12b, 12d,
12e, 13a and 13b showed that they can fit well in the active site of ATPase domain and
exhibit good docking score (Table 2). Interestingly, it was found that the best docked
conformation (highest FlexX score among 30 conformations) of each of the active 3ꢀ
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formylchromones can exist in the binding pocket in a less stable structural representation
(11β, 12β and 13β). This shows that during the process of molecular recognition, the
compounds exhibit induced fit effect to maximize the interactions with the active site
residues of protein and as a result, the energy difference between the stable (α) and less stable
(β) structures can be easily overcome.
All the compounds showed key interactions with the amino acid residues of ATP
binding pocket of hTopoꢀIIα enzyme (Table 2) as observed in the docked pose of ATP. Some
of the compounds (11b and 12d) formed one or more hydrogen bonding interactions with the
Pꢀloop, the so called Walker A consensus sequence (Gly161, Arg162, Asn163, Gly164,
31
Tyr165, Gly166, and Ala167) of ATPase domain. Siteꢀdirected mutagenesis studies have
established the importance of these residues in the binding of substrate and catalytic
inhibitors.32ꢀ34 Binding model of the compounds suggested that chromone ring of these
ligands can effectively make π interaction with the Mg2+ ion, which is a requisite feature to
be an hTopoꢀIIα ATPase inhibitor.13,25 Moreover, recent review article by Mahadevi and
Sastry also showed that cationꢀπ interactions play significant role in the process of molecular
recognition.35
The most active compound 13a (LC50 = 0.5 ꢀM) displayed a FlexX docking score of
ꢀ23.09 (Table 2) and it showed several hydrogen bonding interactions with the active site
residues of ATP binding pocket. Binding model of compound 13a is depicted in Fig. 8A. The
3ꢀformyl group of chromone ring showed hydrogen bonding with the main chain ꢀNH of
Ser149, and 4ꢀoxo with the side chains of Ser148 and Asn150. The 2ꢀ[methyl(phenyl)amino]ꢀ
group of compound 13a was further stabilized by hydrophobic interactions with the side
chains of Ile141, Phe142 and Ala167. In addition, the chromone nucleus of compound 13a
could also form NHꢃ ꢃ ꢃπ interactions with the side chain of Lys168 and Gln376.
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The FlexX docking score for the best docked compound 11b (LC50 = 5.7 ꢀM) was
found to be ꢀ29.10. Its binding model (Fig. 8B) revealed that 3ꢀformyl group can form
intermolecular hydrogen bonding interactions with the main chain ꢀNH of Walker A
consensus sequence viz. Gly164, Tyr165, Gly166 and Ala167. Moreover, the 3ꢀformyl group
also showed hydrogen bonding with the side chain –NH2 of Gln376, which is one of the
amino acid residues of the QTKꢀloop (Gln376ꢀThr377ꢀLys378), a transducer domain. The 4ꢀ
oxo group of chromone ring showed additional hydrogen bonding interactions with the side
chains of Ser148 and Asn150. The chromone nucleus could also be involved in hydrophobic
interaction with the Ile141 and (NHꢃ ꢃ ꢃπ) interaction with side chain of Lys168.
Thus, molecular docking studies performed on some of the 3ꢀformylchromone
derivatives suggested that they could act as catalytic inhibitors possibly via occupying the
ATPase domain of hTopoꢀIIα enzyme.
A.
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B
Fig. 8 Binding mode of 3ꢀformylchromones derivatives into the active site of hTopoꢀIIα ATPase domain. (A)
Most active compound 13a and (B) best docked compound 11b are displayed in magenta color with stick
representation (carbon atomꢀmagenta, oxygen atomꢀred, chlorine atomꢀgreen and hydrogen atomꢀwhite),
magnesium ion in green sphere, two water molecules in stick representation and hydrogen bonding interactions
in yellow dashed lines. Enzyme is represented in cartoon structure with side chains displayed as lines.
Conclusion
The differently substituted 3ꢀformylchromones were synthesized and evaluated for their
inhibitory action on hTopoꢀIIα enzyme. The results of the decatenation and relaxation assays
showed that the compounds under investigations possess significant hTopoꢀIIα inhibitory
activity. The DNA intercalation assay established that these compounds are DNA nonꢀ
intercalators, thus more specific for binding to the hTopoꢀIIα. Screening of the compounds
for their cytotoxic nature revealed that some of the compounds being more cytotoxic than the
positive control etoposide (LC50ꢀ 7.5 ꢂM) and the most active compound 13a exhibits LC50
value of 0.5 ꢁM against PCꢀ3 cancerous cell line. Molecular docking studies have revealed
that these compounds act as catalytic inhibitor of hTopoꢀIIα and block the ATPase domain of
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the enzyme. From this comprehensive study, it can be concluded that these compounds shall
serves as useful ‘Leads’ for further design and development of potent topoisomerase II
catalytic inhibitors, useful for combating deadly disease cancer.
Experiments
Synthesis ꢀ General
General procedure for the synthesis of 3ꢀformylchromones (11aꢀe)
Variously substituted phenols were treated with acetylcholride (1.1 molar equivalents); the
contents were heated for one hour. The reaction mixture was further subjected to thermal
Fries rearrangement using anhydrous AlCl3 (2 eq). The contents were heated for 6ꢀ7 h, and
subsequently were fused with dilute HCl; reaction mixture was partitioned between ethyl
acetate and water. Extracted ethyl acetate was dried over anhydrous Na2SO4 and filtered. The
solvent was removed under reduced pressure to obtain substituted oꢀhydroxyacetophenones.
To a cooled stirred solution of various substituted oꢀhydroxyacetophenones and N, Nꢀ
dimethylꢀformamide was added phosphorus oxychloride dropꢀwise with constant stirring and
the reactants were allowed to stand overnight, and then treated with ice cold water to obtain
yellow crystalline solid, which was filtered and washed with diethyl ether. The crude solid
was recrystallized from acetone to get variously substituted 3ꢀformylchromones.
General procedure for the conversion of chromones to 2ꢀanilinoꢀ3ꢀformylchromones (12aꢀ
e)
To a clear solution of chromones (10 g) in dry benzene (150 mL) was added Nꢀ
phenylhydroxylamine (1 molar equivalent) dissolved in dry benzene (20 mL), leading to the
formation of nitrone. The obtained nitrone was further subjected to refluxing under
anhydrous conditions in presence of few drops of acetic acid for 5ꢀ6 h. After the conversion,
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solvent was removed under reduced pressure and 2ꢀanilinoꢀ3ꢀformylchrome was further
recrystallized from chloroform:hexane (1:2)
General procedure for the conversion of 2ꢀanilinoꢀ3ꢀformylchromes to 2ꢀ(Nꢀ
methylanilino)ꢀ3ꢀformylchromones (13aꢀe)
2ꢀAnilinoꢀ3ꢀformylchromes (12aꢀe, 5.0 g) were dissolved in dry acetone (150 mL) and to the
solution was added K2CO3 (excess) and methyl iodide (2.0 molar equivalents). The contents
were refluxed with stirring under anhydrous condition and the progress of reaction was
monitored by TLC. After the completion of reaction (6ꢀ7 h), contents were filtered hot and
residue was washed with acetone, and, solvent was removed under reduced pressure, 2ꢀ(Nꢀ
methylanilino)ꢀ3ꢀformylchromone were recrystallized from chloroform:hexane (1:2).
Biological assayꢀ General
All the reagents required for the testing of new chemical entities were purchased from
TopoGEN, Inc. (Columbus, OH). The testing of the compounds was performed using a
commercially available TopoꢀII drug screening kit. All the synthesized compounds and
etoposide were dissolved in DMSO at a concentration of 1 mM as a stocked solution and
stored at ꢀ20 °C. Topo I and II inhibition assay were performed as described in the supplier
manual with minor modifications as per the requirement.
hTopo IIα mediated DNA decatenation assay
Topo IIα mediated DNA inhibition activity for the synthesized compounds were performed
as follows. Reaction mixture containing freshly prepared 5x complete reaction assay buffer
(buffer A: 0.5 M TrisꢀHCl (pHꢀ8), 1.50 M sodium chloride, 100 mM magnesium chloride, 5
mM dithiothreitol, 300 ꢁg of bovine serum albumin/mL; buffer B: 20 mM ATP in water),
150 ng catenated kDNA (substrate), 100 ꢁM drug or test compound dissolved in DMSO
followed by 2ꢀ4 units of purified hTopoꢀIIα were incubated at 37 °C for 30 min. The reaction
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was then terminated with addition of 10% SDS, followed by digestion with proteinase K.
Further the reaction mixture was again incubated at 37 °C for 15 min. 20 ꢀL of each sample
were then subjected to 1% agarose gel electrophoresis in Trisꢀacetateꢀ EDTA (TAE) buffer
containing 0.5 ꢁg/mL ethidium bromide and further destained with water for 20 min. The
bands were analyzed under UV transꢀilluminator and the decatenated kDNA products were
quantified with QuantityOne (BioRad).
hTopo IIα mediated DNA relaxation assay
Supercoiled plasmid DNA (pRYG) was used as substrate in Topo II mediated DNA
relaxation assay. Reaction mixture contained freshly prepared 5x complete buffer (A:
0.5MTrisꢀ HCl (pHꢀ8), 1.50 M sodium chloride, 100 mM magnesium chloride, 5 mM
dithiothreitol, 300 ꢁg of bovine serum albumin/mL; buffer B: 20 mM ATP in water), 250 ng
of supercoiled plasmid DNA (pRYG), followed by either test compound or standard drug
(100 ꢁM) and finally hTopoꢀIIα (2ꢀ4 units) in a total of 20 ꢁL. Reaction mixture was then
incubated at 37 °C for 30 min and stopped with addition of 10% SDS followed by digestion
with proteinase K. Further the reaction mixture was again incubated at 37 °C for 15 min.
Electrophoresis of the each sample was carried out in a 1% agarose gel in TAE buffer without
ethidium bromide. Gel was stained with ethidium bromide (0.5 ꢁg/mL) for 15 min and
destained with water for 20 min. The bands were analyzed under UV transꢀilluminator and
quantification of the products was carried out as mentioned in decatenation assay.
DNA Intercalation Assay
In this assay, negatively supercoiled plasmid isolated from E. coli was used as a substrate.
250 ng plasmid was incubated with 1 ꢁg/mL ethidium bromide, 100 ꢁM standard drug
(etoposide), or with the investigational compound at 37 °C for 20 min. Samples were then
loaded in 1% agarose gel and electrophoresis was carried out in TAE buffer. Further the gel
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was stained with ethidium bromide (0.5 ꢁg/mL) for 15 min and destained with water for 20
min and photographed using BioRad.
MTT cell survival assay
Cell lines and cell culture
PCꢀ3 (human prostate adenocarcinoma cells) was obtained from National Centre for Cell
Science (NCCS), Pune, India. The cells were cultured in DMEM, containing 10% FBS and
1% penicillinꢀstreptomycin. DMEM, FBS and Etoposide were obtained from Sigma
Chemicals and Invitrogen. Cell cultures were maintained in flasks under standard conditions:
o
incubation at 37 C and 5% CO2. All the subcultures were used prior to passage 15. Cells
were routinely subꢀcultured using 0.25% trypsin. For treatment, cells were cultured in the
presence of increasing concentrations (1ꢀM, 5 ꢀM, 10 ꢀM, 25 ꢀM, 50 ꢀM, 100 ꢀM) of
compounds for 48 hours.
In vitro cytotoxicity measurements
All inꢂvitro experiments for cell proliferation/inhibition were performed in triplicates. For the
PCꢀ3 cell growth inhibition assay, 2.0 X 104 cells/well were plated in 96ꢀwell microtiter
plate. After 24 hours cells were incubated in the presence and absence of increasing
concentration of positive control etoposide or the test compounds (1ꢀM, 5 ꢀM, 10 ꢀM, 25
o
ꢀM, 50 ꢀM, 100 ꢀM) at 37 C and 5% CO2. Cell proliferation was determined by the MTT
(3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) method (MTT obtained from
HiꢀMedia). After 48 hours incubation, cells were treated with MTT solution for 4 hours in a
o
cell culture incubator at 37 C and 5% CO2. MTT which is a tetrazolium salt, is converted
into insoluble formazan by mitochondrial dehydrogenases in live cells. Formazan is dissolved
in DMSO (Merk) and absorbance was measured at dual wavelength of 550nm and 630nm on
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ELISA plate spectrophotometer (flex station).The total number (percentage) of viable cells
relative to viable cells in untreated control is calculated.
Molecular modeling
Ab initio density functional theory (DFT)36 based geometry optimization calculations of the
3ꢀformylchrmone rotamers were performed using GAUSSIAN09 software package.37 The
gas phase geometry of all the species were fully optimized using B3LYP (Becke3, Lee,
Yang, Parr) methods with 6ꢀ31+G(d,p) basis sets without any geometrical constraint.
Frequencies were computed analytically for all the optimized species to characterize
stationary points as minima and to estimate the zero point vibrational energies (ZPE). The
calculated ZPE values (at 298.15K) were scaled by a factor of 0.9806 for the B3LYP levels.
38
The 3D structure of the compounds (considering stable conformation revealed from
quantum chemical analysis) under study were built using SYBYL 7.1 molecular modeling
package39, installed on a Silicon Graphics Fuel Work station running IRIX 6.5. Gasteigerꢀ
Hückel partial charges were applied to the atoms of the compound and Powell method with
Tripos force field was used for minimization. Finally, a database of ligands was created in
SYBYL. For the purpose of docking, crystal structure of the ATPase domain of hTopoꢀIIα
[29] bound to AMPPNP (5`ꢀadenylylꢀβ,γꢀimidodiphosphate, a nonꢀhydrolyzable ATP analog,
PDB ID: 1ZXM, resolution 1.86 Å) was utilized. This PDB structure composed of a
homodimer, each chain having bound ligand AMPPNP and Mg2+ ion forming distorted
octahedral coordination (3ꢀpoint contacts with phosphate groups, one with Asn91 and two
with conserved water molecules 927 and 928) in an active site. The enzyme structure with
two conserved water molecules in the active site was subjected to protein preparation by
implementing the methodology reported earlier.14
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Docking simulation was performed using FlexX (version 1.2)30 programme
14
incorporated in SYBYL 7.1 package. It was established in previous study that score and
poses obtained while keeping the two conserved water molecules in coordination with Mg2+
ion give more accurate results. Thus, in this work also we have employed the same protocol
for molecular docking. For the protocol validation bound ligand AMPPNP and substrate ATP
were docked initially, and then analyzed for their binding pose and hydrogen bonding
interactions with the key residues. Afterwards, a database of ligands (11b, 12a, 12b, 12d,
12e, 13a and 13b) was subjected to docking by using FlexX multiple ligand docking mode.
At the end of docking, 30 poses per ligand were generated which were sorted according to
their FlexX score, reflecting binding affinity with the enzyme. Top most poses were visually
inspected and analyzed for their interactions with Mg2+ ion and active site residues.
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Figures and Schemes
S
O
O
OH
O
O
OH
O
O
OH
O
O
O
HO
S
OH
OH
3
2
1
O
O
O
OH
O
O
OH
Cl
O
O
HO
OH
HO
OH
O
4
5
O
O
OCH3
O
O
OH
O
OH
O
OH
O
OH
Cl
OH
O
OH
O
OH
O
O
H
7
6
CH3
8
O
Fig. 1. Chromone based compounds as topoisomerase II inhibitors

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A
B.
Fig. 2 (A) Effect of test compounds on Topo IIα mediated kDNA decatenation assay. kDNA was treated with
hTopoIIα in the presence of either 100 ꢀM etoposide or test compounds. Reaction mixture was incubated at 37
°C for 30 min and 1% agarose gel was run in TAE buffer (B) Graphical representation of decatenated products
formed in kDNA decatenation assay.

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A.
B.
Fig. 3 (A) Effect of test compounds on Topo IIα mediated supercoiled pRYG plasmid DNA relaxation assay.
Negatively supercoiled pRYG plasmid DNA was treated with hTopoIIα in the presence of either 100 ꢁM
etoposide or test compounds. Electrophoresis was carried out in 1% agarose gel in TAE buffer without ethidium
bromide. (B) Graphical representation of relative relaxation in pRYG DNA by investigational compounds 11b,
12a, 12b, 12d, 12e, 13a and 13b