Anticancer activity of dihydropyrazolo[1,5-c]quinazolines against rat C6 glioma cells via inhibition of topoisomerase II

Article abstract of DOI:10.1002/ardp.201800023

The design and synthesis of dihydropyrazolo[1,5-c]quinazolines (1a–h) as human topoisomerase II (TopoII) catalytic inhibitors are reported. The compounds were investigated for their antiproliferative activity against the C6 rat glial cell line. Two compou

Full text of DOI:10.1002/ardp.201800023

Received: 2 February 2018
Revised: 8 April 2018
Accepted: 11 April 2018
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DOI: 10.1002/ardp.201800023
FULL PAPER
Anticancer activity of dihydropyrazolo[1,5-c]quinazolines
against rat C6 glioma cells via inhibition of topoisomerase II
Gagandeep Kaur1
Ravi P. Cholia2
Gaurav Joshi1
Uttam C. Banerjee3
Suyog M. Amrutkar3
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Sourav Kalra4
Anil K. Mantha2
Raj Kumar1
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1 Laboratory for Drug Design and Synthesis,
Department of Pharmaceutical Sciences and
Natural Products, Central University of
Punjab, Bathinda, India
Abstract
The design and synthesis of dihydropyrazolo[1,5-c]quinazolines (1a–h) as human
topoisomerase II (TopoII) catalytic inhibitors are reported. The compounds were
investigated for their antiproliferative activity against the C6 rat glial cell line. Two
compounds, 1b and 1h, were found to be potent cytotoxic agents against glioma cells
and exerted selective TopoII inhibitory activity. Furthermore, the compounds induced
alterations in reactive oxygen species levels as measured by DCFDA assay and were
found to induce cell cycle arrest at the G1 phase at lower concentrations and profound
apoptosis at higher concentrations. The interaction of selected investigational
molecules with TopoII was further corroborated by molecular modeling.
2 Department of Animal Sciences, Central
University of Punjab, Bathinda, India
3 Department of Pharmaceutical Technology
(Biotechnology), National Institute of
Pharmaceutical Education and Research
(NIPER), S. A. S. Nagar, Mohali, India
4 Department of Human Genetics and
Molecular Medicine, Central University of
Punjab, Bathinda, India
Correspondence
Dr. Raj Kumar, Laboratory for Drug Design
and Synthesis, Department of Pharmaceutical
Sciences and Natural Products, Central
University of Punjab, 151 001 Bathinda, India.
Email: raj.khunger@gmail.com,
K E Y W O R D S
cancer, cell cycle, dihydropyrazolo[1,5-c]quinazoline, rat C6 glioma cells, topoisomerases
rajcps@cup.ac.in
Funding information
Bristol-Myer Squibb, USA, Grant number:
34003085
subsequently leading to DNA damage.[7] Numerous studies in the past
have correlated the overexpression of hTopoII as a prognostic marker in
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1
INTRODUCTION
Topoisomerases (Topo) are designated as the magicians in the world of
DNA.[1,2] They are associated with resolving all the topological problems
of DNA. Their overexpression is found in various diseases including
cancer.[3] Human DNA topoisomerases are classified into topoisomerase I
(TopoI) and topoisomerase II (TopoII) and are considered to be the
authenticated targets for numerous classes of potent anti-cancer drugs
including epipodophyllotoxin, etoposide, and the anthracycline derivative
doxorubicin, camptothecin, etc.[2,4,5] The TopoI is involved in breakage of
the DNA single strand whereas TopoII is known to cleave double strand of
phosphate backbone of the DNA and needs ATP for its function
irrespective of hTopoI.[6] The clinically active hTopoII inhibitors induce
cancer cell death by trapping TopoII-DNA complex (covalent complex),
the glioblastomas (GBMs).[8] GBM is considered as the severe and the
most aggressive form of malignant brain tumor.[9] The TopoII is a well-
validated target for GBM chemotherapy.[10,11] Several topoisomerase
inhibitors (poison and catalytic) from natural and synthetic origin act at
different phases of the catalytic cycle.[12–15] Additionally, TopoII
inhibitors are reported to exhibit lack of isoform specificity and
multidrug resistance in tumor cells. Submicromolar inhibitory values
of available TopoII inhibitory drugs have rendered many opportunities
to explore this research area.[16,17] Through our previous experiences in
the field of anticancer drug discovery and development,[18–24] we
thought of designing dihydropyrazolo[1,5-c]quinazolines (1) as TopoII
catalytic inhibitors after considering the interaction models of
phosphoraminophosphonic acid–adenylate ester (ANP), an ATP analog,
aswell asN-acetylpyrazoline (2),[19] previouslyreported TopoIIinhibitor
Gagandeep Kaur and Ravi P. Cholia have contributed equally to this paper.
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Arch Pharm Chem Life Sci. 2018;1800023.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800023

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FIGURE 1 Design of target compounds (1) as topoisomerase II inhibitors
(Figure 1) at the catalytic domain of hTopoII enzyme (PDB entry:
1ZXM).[25] The rationale behind the design of 1 includes that (a) two aryl
groups be flanked by a central heteroaromatic ring and one of the rings
shouldbeabletocoordinatewithMg2+ centerofTopoII;(b)N-COCH3 of
2 may be isosterically replaced with cyclized N-C-N-Ar rigid framework
in order to increase stability and avoid acid mediated cleavage that can
occur with acetamido group of 2 in tumor microenvironment; and (c)
extraphenylringmay not onlyimpart necessarylipophilicityto1tocross
the blood brain barrier in GBM but may also get into the hydrophobic
cavity ofATPdomaininhTopoII. Withthisbackground, wehereinreport
the synthesis and biological evaluation of 1 (Figure 1).
in Scheme 1. Briefly a mixture of 2-nitrobenzaldehyde (A) and
appropriate aryl ketone (B) in glacial acetic acid was stirred using conc.
H2SO4 as catalyst to afford appropriate 1,3-diaryl propenone (3a,
b)[19,26,27] which on reaction with hydrazine hydrate in methanol
yielded appropriate pyrazoline (4a,b).[19,26,27] The pyrazolines so
obtained were further oxidized in the presence of I2/DMSO to yield
pyrazole derivatives (5a,b). The aromatic nitro group of the appropriate
pyrazoles was reduced using SnCl2-HCl in methanol to procure the
intermediates aryl-1H-pyrazol-5-yl anilines (6a,b).
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2.1.1 Synthesis of dihydropyrazolo[1,5-c]quinazoline
derivatives as target molecules (1a–h)
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RESULTS AND DISCUSSION
The intermediates aryl-1H-pyrazol-5-yl anilines (6a,b) so formed were
cyclo-condensed with glyoxylic acid as well as with aryl aldehydes to
afford the target dihydropyrazolo[1,5-c]quinazoline compounds
(1a–h; Figure 2). All the final compounds were characterized by 1H,
13C NMR, IR, mass, and elemental analyses.
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2.1 Chemistry
Compounds 6a,b as precursors for the synthesis of the target
compounds 1a–h were prepared by using synthetic procedures shown
SCHEME 1 Synthesis of the target molecules 1a–h. Reagents and conditions (a) CH3COOH, H2SO4 (1 drop), rt, 24 h; (b) NH2NH.H2O,
MeOH, rt, 0.5 h; (c) I2, DMSO, reflux, 4 h; (d) SnCl2-HCl, MeOH, reflux, 6 h; (e) aryl aldehydes/glyoxylic acid, MeOH/MeCN, rt or reflux

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FIGURE 2 Chemical structures of the target molecules synthesized
Interestingly reaction of 6a with glyoxylic acid performed in
presence ofmethanolledtotheformationof1b, amethylesterof1aasa
major product (1b; 90%), along with 8 (minor product; 10%) (Figure 3).
The formation of 1b could be due to in situ esterification of initially
formed 1a by MeOH (used as solvent) under non-acidic conditions.
Whereas 8 was formed as a result of decarboxylation of 1a followed by
oxidation. Further, attempt to solely prepare 1a was made successful by
the reaction of 6a with glyoxylic acid (NMR) in MeCN in 5 min at 0°C.
hPBMCs (Figure 4) was observed, even at the highest concentration
(25 μM) of tested compounds, after treatment for 48 h.
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2.2.2 Intracellular reactive oxygen species (ROS)
measurement by DCFDA assay
The anticancer molecules tend to alter the intracellular ROS upon
treatment.[18–20,29] In order to know exact pattern of alteration in ROS
levels, rat C-6 glial cells were treated with the representative
compounds 1a, 1b, 1d, and 1h for 48 h and measurements were done
using DCFDA fluorescence assay. It was observed that at lower
concentration (1 μM) all the test compounds showed increase in ROS
production compared to normal control. The trend was reversed at
higher concentration of 5 and 25 μM of the test compounds. Overall it
may be concluded that all the compounds showed significant alteration
in ROS levels (Figure 5).
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2.2 Biological studies
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2.2.1 Antiproliferative activity against rat C6 glioma
cells
To assess the antiproliferative potential of the synthesized compounds
rat C6 glioma cells were utilized. The tumors derived from rat C6 cell
implants exhibit similar morphology and vascularization grade compa-
rable to human glioblastoma.[28] It was observed that all the
investigational molecules (1a–h) exhibited profound antiproliferative
activity (Table 1; IC50 < 10 µM) in rat C6 glioma cells at 48 h treatment.
Twocompounds1band1hportrayedmostpotentcytotoxicitywithIC50
values 3.98 and 3.86 μM, respectively, comparable to positive control
etoposide (IC50 = 4.32 μM). In order to ascertain preferential cytotoxic-
ity of 1b and 1h, normal cells, i.e., human peripheral blood mononuclear
cells (hPBMCs), were treated with these compounds at 1, 5, and 25 μM
concentrations. To our satisfaction no significant cytotoxicity toward
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2.2.3 Redefining selectivity of investigational
molecules (1b and 1h) as hTopoIIα inhibitors
hTopoII mediated DNA decatenation assay
Etoposide is a well-established anticancer compound that works by
inhibiting TopoII enzyme. In the present study, the most potent
compounds 1b and 1h were analyzed for inhibition of TopoII
mediated decatenation in comparison to etoposide as a reference
FIGURE 3 Synthesis of 1a, 1b, and 8

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TABLE 1 Antiproliferative activity of investigational compounds
1a–h analyzed via MTT assay in rat-derived C6 glioma cells
Entry
Rat C6 glioma cells SDa (IC50; µM)
1a
6.45 0.94
1b
3.98 1.35
1c
7.78 1.24
1d
4.72 0.97
1e
8.11 1.22
1f
9.32 1.76
1g
7.83 1.24
1h
3.86 0.98
Etoposide
4.32 0.63
FIGURE 5 Percent alteration in intracellular ROS levels in rat C6
glial cells in response to treatment with synthesized compounds at
concentrations of 1, 5, and 25 μM at 48 h. Measured via DCFDA
assay
aValues are derived from averaging three independent experiments and
each experiment was done in triplicate.
standard.[18,19,29] In this assay, kDNA (kinetoplast DNA), the substrate
for decatenation is unable to migrate during agarose gel electrophoresis
and hence remains in the agarose well itself. hTopoIIα enzyme
decatenates kDNA into relatively smaller and circular form and unlike
kDNA they migrate with ease inside the agarose gel during electropho-
resis. Due to etoposide mediated hTopoIIα inhibition, the two forms
have not been observed in the gel image (Figure 6A). Also in the
presence of compounds 1 and 2, these forms were absent and further
densitometric analysis revealed significant inhibition of hTopoIIα by the
investigational compounds in comparison to etoposide. Degree of
inhibition among them is in the order: etoposide < 1b < 1h (Figure 6B).
1hS) were docked onto the ATPase domain of hTopoII (PDB entry:
1ZXM).[25,32] AMP-PNP in ATPase domain presented some important
key amino acid interactions that include the H-bond interactions
between carbonyl of ASN120 with the NH2 of adenine moiety and
between side chain residue SER149 and SER148 with ─OH of ribose
unit. The backbone residue interactions (GLY166, ALA167, ASN163,
TYR165) and side chains residues interactions (SER148, LYS168,
GLU87, ASN91, and GLN376) were observed with phosphate groups.
The Mg++ ion exists in an octahedral geometric state having ionic
interaction with the oxygen attached to the three phosphate groups.
The conserved region (Walker A motif) comprising ARG162, ASN163,
GLY164, TYR165, GLY166, and ALA167 helps binding of inhibitors at
ATPase domain.
DNA intercalation assay
A distinctive characteristic of intercalative drugs is the unwinding of
DNA, e.g., ethidium bromide, AMSA, chloroquine. In gel electrophore-
sis, ethidium bromide (EtBr) impedes the migration of supercoiled DNA
(scDNA) due to intercalative action.[30,31] 1b and 1h did not show any
such retardation of scDNA (as observed in Figure 6C) which proves
DNA non-intercalative nature of investigated compounds.[18,19,29]
The binding mode of the top scoring compound, 1hR (Figure 7A),
revealed that the ─OH group forms an H-bond interaction with ─NH2
of SER148 (3.2 Å) and THR147 (Figure 7C); and the oxygen atom of OH
group interacts with ammonium ion of backbone residue LYS168
(2.8 Å). The hydroxy benzene ring showed π–cation interactions with
Mg++ 903 (3.2 Å). The π–cation interaction was also observed between
ARG98 and aromatic ring attached quinazoline ring. The NH of the
quinazoline ring formed a hydrogen bond with the SER149. The
compound 1h (R), however, has shown hydrophobic interactions with
ILE217 and polar interactions with THR215 and ASN91 (Figure 7). The
overall binding pattern of compounds 1hR and 1bR were found to be
similar as of AMP-PNP (Figure 7B).
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2.3 Molecular docking and prediction of
physiochemical parameters
In order to theoretically investigate the binding patterns of 1b and 1h
at TopoIIα active site, the stereoisomers of 1b (1bR, 1bS) and 1h (1hR,
The compounds 1b and 1h were predicted for their pharmaceuti-
cally relevant properties such as blood brain barrier, partition
coefficient, bioavailability, pharmacokinetics, etc. using QikProp
module in MAESTRO 11.4.[33] The indispensable lead generation
and optimization property of the module suggested that 1b and 1h
have blood brain barrier coefficient (QPlogBB) of −1.68 and −2.09,
respectively (range: −3.0 to 1.2), and can cross blood brain barrier.
Moreover, the partition coefficient (QPlogPo/w) predicted was in the
normal range i.e., −2.0 to 6.5. QPPCaco represents the gut cell
absorption of the compounds ranges from 25 for poor gut absorbed
FIGURE 4 Effect of cytotoxity by compounds 1b and 1h on
hPBMC. No cytotoxicity was found up to 25 µM at 48 h

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FIGURE 6 (A) Agarose gel image for decatenation assay: kDNA and hTopoIIα were incubated in presence and absence of 100 μM
etoposide (E) and investigational compound 1b and 1h (100 μM). (B) Relative decatenation of kDNA by investigational compounds 1b and 1h
in comparison to etoposide after quantification. (C) Agarose gel image for DNA intercalation assay. Supercoiled DNA (pUC 19 plasmid) was
incubated in the presence and absence of ethidium bromide (final concentration 1 μg/mL) and investigational compounds 1b and 1h
FIGURE 7 (A) 3D Binding mode of 1hR at the ATPase domain of topoisomerase II. (B) Docked compounds 1hR (pink) and 1bR (orange) at
ATPase domain of topoisomerase II. (C) 2D interaction diagram of 1hR in hTopoIIα

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TABLE 2 Important physiochemical properties of the compounds that help in the drug penetrance to brain, gut and oral absorption
Entry
Compound number
QPlogPo/w
3.845
QPPCaco
2061.737
1663.569
QPlogBB
−0.209
Human oral absorption
1
2
1b
1h
3
1
5.268
−0.144
compounds while >500 for the compounds with good gut absorption.
The human oral absorption depends on the number of rotatable bonds,
number of metabolites, logP, cell permeability, and solubility. The
number 1 depicts low oral absorption, 2 represents medium oral
absorption, and 3 represents high oral absorption. Compound 1h was
predicted to have low human oral absorption as compared to 1b
(Table 2).
having mitotic catastrophy[20] at the higher drug concentration
(Figure 8).
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2.4.2 Prediction of cell death mode
To examine the mode of cell death, we performed annexin-V versus
propidium iodide staining utilizing the investigational compounds 1b
and 1h, at 5 and 25 μM concentration. The assay is based on the
principle that annexin-V binds to the apoptotic populations (detects
the release of phosphatidylserine) under normal conditions whereas
propidium iodide does not stain live or early/late apoptotic cells. In late
apoptotic and necrotic cells, the integrity of the plasma and nuclear
membranes declines allowing propidium iodide to pass through the
membranes which further intercalate with nucleic acids and display red
fluorescence.[18–20,29] Compounds 1b and 1h induced early apoptosis
at 5 μM and the mode of cell death was late apoptosis at 25 μM
(Figure 9).
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2.4 Cell cycle analysis and prediction of mode of
cell death
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2.4.1 Cell cycle analysis
The inhibition of specific target by the anticancer compounds tend to
cause cell death and halt the cell cycle progression[24] by arresting cell
cycle.[18–20,29] To investigate the specific phase of cell cycle arrested
by our investigational compounds (1b, 1h), rat C6 glioma cells were
utilized and treatment was given at two concentrations, 5 and 25 μM
for 24 h. The results showed significant changes in G1 pattern (33.59%
in control, 87.06% in 1b, 82.21% in 1h) treated at 5 μM concentration.
The compounds at 25 μM induced increase in sub-G1 population
several folds (82.21–87.06%) indicative of cell death. This peculiar
feature may be attributed to the fact that compounds may be toxic to
the glioma cells independent of the status of cell cycle or cells might be
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CONCLUSION
In summary, we have designed, synthesized, and evaluated the
dihydropyrazolo[1,5-c]quinazolines (1a–h) as inhibitors of rat C6-
glioma cells. The most potent inhibitors 1b and 1h were found to be
FIGURE 8 Cell cycle analysis using propidium iodide. Rat C6 glioma cells were treated with 1b and 1h at 5 and 25 μM concentration. The
bar graph represents the percent cell count (DNA) at various stages of cell cycle induced by investigational compounds

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FIGURE 9 Mode of cell death prediction by PI versus annexin V assay. The investigational compounds 1b and 1h exhibited rat C6 glioma
cell death via apoptosis
non-cytotoxic toward normal cells (HPBMCs) at highest concen-
tration. The compounds were able to alter the intracellular ROS
levels and inhibited TopoII which was further corroborated through
in silico studies. Compounds delayed cell cycle, caused G1 phase
arrest at low concentration and induced apoptosis at higher
concentration. All the above annotations indicated multiple modes
of mechanisms involved in anticancer effects of compounds.
Further results on their detailed antitumor potential and effect of
receptor inhibition of down targets, formulation and SAR will be
published in due course of time. The experiments were conducted
in accordance to protocol no. CUPB/cc/14/IEC/4483 approved by
Institutional Ethics Committee of Central University of Punjab,
Bathinda, as per guidelines issued by Indian Council of Medical
Research (ICMR), Govt. of India.
Stuart melting point apparatus (SMP-30) with open glass capillary
tubes and were uncorrected. Infrared (IR) spectra of compounds were
recorded with KBr on a Bruker FT-IR spectrophotometer. 1H and
13C nuclear magnetic resonance (NMR) spectra were recorded at
Panjab University, Chandigarh in CDCl3/d6-DMSO on a Bruker Avance
II (400 MHz) NMR spectrometer using TMS (δ = 0) as an internal
standard. Mass spectra were recorded on TOF-MS/ESI at Panjab
University, Chandigarh and MS/EI at Central University of Punjab,
Bathinda.
The NMR spectra of compounds 1a,b and the InChI codes of the
investigated compounds together with some biological activity data
are provided as Supporting Information.
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4.1.2 General procedure for the synthesis of
chalcones 3a,b
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4
EXPERIMENTAL
A mixture of 2-nitrobenzaldehyde (1 mmol) and appropriate aryl
ketone (1 mmol) in glacial acetic acid was stirred in 100 mL round
bottomed flask (RBF). Conc. sulfuric acid (4–5 drops) was added to the
reaction mixture in ice cold condition and further stirred at room
temperature for 24 h. Completion of the reaction was determined by
thin layer chromatography (TLC). After the completion, the reaction
mixture was poured on ice-cold water and filtered. The solid was
washed with water and dried to obtain the crude product. The
crude product was recrystallized from methanol to get pure
compound.[19,26,27]
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4.1 Chemistry
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4.1.1 General
All the chemical and biological reagents were purchased from Sigma–
Aldrich, Loba-Chemie Pt. Ltd., S.D. Fine Chemicals, Sisco Research
Laboratory, and Avra Synthesis Ltd. and were used without further
purification. Sartorius analytical balance (BSA224S-CW) and Mettler
Toledo were used for the weighing purposes. JSGW heating mantle,
Tarson spinot digital, ILMVAC digital rotary evaporater, digital hop top,
and NSW oven/vacuum oven were used during reaction. TLC
monitored the progress of the reaction, using n-hexane/ethyl acetate
and chloroform/methanol as the mobile phase on pre-coated Merck
TLC (TLC silica gel 60, F254) plates in JSGW UV/fluorescent analysis
cabinet and/or iodine chamber. Melting points were recorded on
Physical data of the representative compound 3a
Yield: 85%, 142.37 mg, creamy white solid, mp: 89–91°C; IR (KBr,
cm−1): 1666 (C O), 1610 (C C), 1510 (N O), 1342 (N O), 1014
(C─N). 1H NMR (CDCl3, 400 MHz): 8.12 (1H, d, J = 15.9 Hz), 8.02 (3H,
bs), 7.69–7.74 (2H, m), 7.50–7.57 (4H, m), 7.34 (1H, d,
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J = 15.9 Hz).13C NMR (CDCl3, 75 MHz): 190.22, 148.34, 140.03,
137.20, 133.56, 133.09, 131.08, 130.31, 129.13, 128.62, 126.97,
and 124.86.
Physical data of the representative compound 6a
Yield: 95%, 88.24 mg, yellow solid, mp: 165–166°C; IR (KBr, cm−1):
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3360 (N─H), 3285 (N─H), 1613 (C N), 1579 (CC), 1248 (C─N).
1H NMR (CDCl3, 400 MHz): 13.23 (D2O exchangeable NH, 1H, bs),
7.81 (1H, s), 7.79 (1H, s), 7.31–7.54 (4H, m), 7.00 (2H, s), 6.75 (1H, d,
J = 7.91 Hz), 6.61 (1H, t, J = 7.32 Hz), 6.22 (D2O exchangeable NH,
2H, bs).
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4.1.3 General procedure for the synthesis of
pyrazolines 4a,b
To a solution of appropriate chalcone (1 mmol) in methanol (5 mL) was
added hydrazine hydrate (2 mmol). The reaction mixture was stirred for
2 h. After completion of the reaction (TLC), precipitate formed was
filtered and washed with excess methanol to obtain the pure product
4a and 4b.[26,27]
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4.1.6 Synthesis of 2-phenyl-5,6-
dihydropyrazolo[1,5-c]quinazoline-5-carboxylic acid
(1a)
To a solution of 6a (0.1 g, 0.4255 mmol) in acetonitrile was added
glyoxylic acid (31.4 mg, 0.4355 mmol) (50% aqueous soln) and mixture
was stirred at 10–15°C for 5 min. After the completion of reaction
(TLC), reaction mixture was evaporated using rotary evaporator and
solid product was obtained. The compound was further purified using
flash chromatography to obtain 1a.
Yield: 98%, creamy white solid, mp: 190–192°C. 1H NMR (CDCl3,
400 MHz, δ with TMS = 0): 12.4 (1H, s), 7.84 (2H, d, J = 7.29 Hz), 7.51
(1H, d, J = 7.16 Hz), 7.38–7.45 (4H, bs), 7.30 (1H, m), 7.11 (1H, m), 7.04
(1H, s), 6.90 (1H, d, J = 8 Hz), 6.80 (1H, m), 6.01 (1H, d). 13C NMR
(CDCl3, 100 MHz, δ with TMS = 0): 169.75, 151.07, 139.43, 138.34,
132.95, 129.07, 128.09, 127.57, 125.14, 123.65, 118.53, 114.75,
112.84, 96.49, and 68.41. Anal. calcd. for C17H13N3O2: C, 70.09; H,
4.50; N, 14.42. Found: C, 70.15; H, 4.62; N, 14.39.
Physical data of the representative compound 4a
Yield: 97%, 102.3 mg, orange crystalline solid, mp: 140–142°C IR (KBr,
cm−1): 3310 (N─H stretch), 1575 (N─H bend), 1517 (N O), 1350

(N O), 1605 (C N), 1056 (C─N); 1H NMR (CDCl3, 400 MHz): 7.96
(2H, t, J = 8.4 Hz), 7.65 (3H, m), 7.41 (4H, m), 6.05 (D2O exchangeable
NH, 1H, s), 5.42 (1H, t, J = 13.2 Hz), 3.80, (1H, m), 3.01 (1H, m).
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4.1.4 General procedure for the synthesis of
pyrazoles 5a,b
To a solution of appropriate pyrazolines (4a,b) in DMSO was added
catalytic amount of molecular iodine and reaction mixture was
refluxed at 130–140°C for 4–6 h. After the completion of the
reaction (TLC), the reaction mixture was poured in ice cold water;
solid product was extracted using ethyl acetate. Excess iodine was
removed adding aq. sodium thiosulfate. Organic layer was washed
with brine (10 mL × 3), dried over sodium sulfate, and was finally
evaporated under reduced pressure using rotary evaporator to
obtain the product (5a,b).[26,27]
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4.1.7 Synthesis of methyl 2-phenyl-5,6-
dihydropyrazolo[1,5-c]quinazoline-5-carboxylate
To a solution of 6a (0.1 g, 0.4255 mmol) in methanol was added
glyoxylic acid (31.4 mg, 0.4355 mmmol) (50% aqueous solution) and
stirred at 10–15°C for 5 min. After the completion of reaction (TLC),
reaction mixture was evaporated using rotary evaporator and solid
product was obtained. The compound was further purified using flash
chromatography to obtain 1b.
1H NMR (CDCl3, 400 MHz): 7.84 (2H, d, J = 5.8 Hz), 7.51 (1H, d,
J = 6.4 Hz), 7.40 (2H, s), 7.32 (1H, d, J = 6.16 Hz), 7.14 (1H, s), 7.04 (1H,
s), 6.88 (3H, t, J = 10 Hz), 6.11 (1H, s), 3.67 (3H, s). 13C NMR (CDCl3,
100 MHz): 168.40, 151.56, 138.43, 138.19, 132.51, 128.91, 128.05,
127.38, 125.05, 123.98, 123.36, 118.86, 114.70, 96.17, 68.08, and
52.13. Anal. calcd. for C18H15N3O2: C, 70.81; H, 4.95; N, 13.76. Found:
C, 70.97; H, 5.08; N, 14.11.
Physical data of the representative compound 5a
Yield: 92%, 91.31 mg, brownish solid, mp: 194–196°C IR (KBr, cm−1):
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3456 (N─H stretch), 1664 (C C), 1620 (C N), 1524 and 1348
(N O), 1144 (C─N). 1H NMR (CDCl3, 400 MHz): 7.67–7.76 (3H, m),
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7.56–7.63 (3H, m), 7.33–7.51 (5H, m). 13C NMR (CDCl3, 100 MHz):
149.02, 146.37, 146.16, 132.09, 130.97, 129.04, 128.74, 128.04,
126.47, 125.62, 124.56, 123.82, and 102.59.
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4.1.5 General procedure for reduction of
2-nitropyrazoles 5a,b to their aniline derivatives 6a,b
To a mixture of 5a and 5b in methanol was added SnCl2 (3–4
equivalents). The reaction mixture was refluxed for 4 h. After the
completion of the reaction (TLC), methanol was evaporated under
reduced pressure using rotary evaporator. Solid product was than
extracted with ethyl acetate (10 mL × 3). Excess of SnCl2 was
precipitated in water by neutralizing the reaction mixture with 5%
NaOH. Organics were washed with brine (10 mL × 3), dried over
anhydrous Na2SO4, and evaporated under reduced pressure using
rotary evaporator to obtain the final product 6a,b.[26,27]
|
4.1.8 Synthesis of 4-(2-phenyl-5,6-
dihydropyrazolo[1,5-c]quinazolin-5-yl)phenol (1c)
A mixture of 6a (0.1 g, 0.425 mmol) and 4-hydroxybenzaldehyde
(0.70 g, 0.425 mmol) was dissolved in methanol and refluxed for
1 h. After the completion of the reaction (TLC), the reaction
mixture was left to cool. Precipitated solid was obtained by
decanting extra methanol and recrystallized from methanol to
obtain 1c.[26,27]

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4.1.9 Synthesis of 2-methoxy-4-(2-phenyl-5,6-
left to cool. Precipitated solid was obtained by decanting extra
methanol and recrystallized from methanol to obtain 1h.[26,27]
dihydropyrazolo[1,5-c]quinazolin-5-yl)phenol (1d)
A mixture of 6a (100 mg, 0.425 mmol) and 4-hydroxy, 3-methoxy
benzaldehyde (64.6 mg, 0.425 mmol) was dissolved in methanol
and refluxed for 1 h. After the completion of the reaction (TLC), the
reaction mixture was left to cool. Precipitated solid was obtained
by decanting extra methanol and recrystallized from methanol to
obtain 1d.[26,27]
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4.2 Biological assays
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4.2.1 Cell culture and treatment
Rat C6 glioma cells were generously gifted by Prof. Gursharan Kaur,
GNDU, Amritsar, India. They were grown in DMEM media containing
10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin and
were maintained at 37°C in a 5% CO2 humidified incubator. When the
cells became 70–80% confluent, culture medium was removed and
discarded. Then the cells were rinsed with PBS to remove all the traces
of serum that contains trypsin inhibitor. After that, trypsin-EDTA
0.25% (w/v) solution was added to the flask. It was then allowed to
incubate until cells were detached from the surface (trypsinization).
Subsequently, trypsin was inactivated by the addition of media
containing serum (1 mL). Centrifugation was done on 1200 rpm at
37°C for 10 min for harvesting the cells. Further, supernatant was
disposed and resuspension of the cell pellet was done using 2 mL of the
media. The cell number was counted using automated cell counter. The
cells were transferred to fresh media every 3 days. The maintenance of
C6 cultured cell line was done in 25 or 75 cm2 flasks containing DMEM
medium supplemented with 10% FBS, 1× antibiotic solution and
afterward incubated at 37°C in a humidified atmosphere containing
5% CO2 and 95% humidity.[18–20,29]
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4.1.10 Synthesis of 2-methoxy-5-(2-phenyl-5,6-
dihydropyrazolo[1,5-c]quinazolin-5-yl)phenol (1e)
A mixture of 6a (100 mg, 0.425 mmol) and 4-hydroxybenzaldehyde
(64.6 mg, 0.425 mmol) was dissolved in methanol and refluxed for 1 h.
After the completion of the reaction (TLC), the reaction mixture was
left to cool. Precipitated solid was obtained by decanting extra
methanol and recrystallized from methanol, to obtain 1e.[26,27]
|
4.1.11 Synthesis of 4-(2-phenyl-5,6-
dihydropyrazolo[1,5-c]quinazolin-5-yl)benzonitrile (1f)
A mixture of 6a (100 mg, 0.425 mmol) and 4-cyanobenzaldehyde
(55.67 mg, 0.425 mmol) was dissolved in methanol and refluxed for
1 h. After the completion of the reaction (TLC), the reaction mixture
was left to cool. Precipitated solid was obtained by decanting extra
methanol and recrystallized from methanol to obtain 1f.[26,27]
The cells were seeded in 25 cm2 flasks and the treatment
was given when 70–80% confluency was obtained. The treatment
time of different test compounds was as described earlier in the
section 2.
|
4.1.12 Synthesis of methyl 2-(4-chlorophenyl)-
pyrazolo[1,5-c]quinazoline-5-carboxylate (1g)
Similarly, HPBMC culture was done as per previous published
protocol.[18–20] A total of 5 mL of fresh blood was drawn from healthy
individual as per the protocol no. CUPB/cc/14/IEC/4483 approved by
Institutional Ethics Committee of Central University of Punjab,
Bathinda. The protocol used was standard operating procedure,
provided by Institutional Ethics Committee of Central University of
Punjab according to guidelines issued by Indian Council of Medical
Research (ICMR), Govt. of India.
To a solution of 6b (0.1 g, 0.371 mmol) in methanol was added glyoxylic
acid (0.042 mL, 0.371 mmol) (50% aqueous soln) at 10–15°C. The
reaction mixture was stirred for 5 min. After the completion of reaction
(TLC), reaction mixture was evaporated using rotary evaporator and
solid mixture of products was obtained which was further purified by
column chromatography to obtain 1g.
Yield: 60%, creamish white, mp: 215–217°C. 1H NMR (CDCl3,
400 MHz): 7.80–7.75 (2H, m), 7.52 (1H, dd, J = 7.72 Hz), 7.38 (2H, dd,
J = 6.72 Hz), 7.26 (1H, d, J = 5.52 Hz), 7.00 (1H, s), 6.95 (1H, d,
J = 7.76 Hz), 6.83 (1H, d, J = 7.52 Hz), 6.00 (1H, d, J = 1.92 Hz), 4.98
(D2O exchangeable NH, 1H, s), 3.68 (3H, s). 13C NMR (CDCl3,
100 MHz): 169.18, 151.71, 138.64, 138.04, 135.95, 133.80, 131.59,
129.69, 128.82, 127.15, 120.93, 115.56, 114.09, 97.29, 68.79, and
53.22. Anal. calcd. for C18H14ClN3O2: C, 63.63; H, 4.15; Cl, 10.43; N,
12.37. Found: C, 63.02; H, 4.34; N, 12.56.
|
4.2.2 Evaluation of anti-proliferative activity of the
synthesized compounds (MTT assay)
Rat C6 glial cells were counted on the automated cell counter.
About 8000–10000 cells were seeded in each well of the 96 well
plates. The plate was incubated at 37°C with 5% CO2 for 24 h. At
the end of the 24 h, treatment was given to the cells in
concentrations of 1, 5, and 25 μM. The cells were further incubated
for 48 h. The media was removed from each well and MTT solution
(5 mg/10 mL) was added. This was incubated in the dark for 4 h. At
the end of 4 h, the MTT solution was disposed from each well and
the intracellular precipitate was dissolved in DMSO solution and
the absorbance was read at 570 nm using a microplate reader
(BioTek).[18–20,29]
|
4.1.13 Synthesis of 4-(2-(2-chlorophenyl)-5,6-
dihydropyrazolo[1,5-c]quinazolin-5-yl)phenol (1h)
A mixture of 6b (0.1 mg, 0.371 mmol) and 4-hydroxybenzaldehyde
(0.45 mg, 0.371 mmol) was dissolved in methanol and refluxed for 1 h.
After the completion of the reaction (TLC), the reaction mixture was

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KAUR ET AL.
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4.2.3 Intracellular ROS measurement by DCFDA
assay
was done using protein preparation wizard of maestro 11.4. The
protein was preprocessed for the addition of the polar hydrogens,
cysteine bridges creation, charges, filling missing side chains, etc. This
preprocess step also corrects the ionization and tautomeric states of
amino acid residues and therefore modifies the total Gasteiger charges
on the protein structure. The optimization of the interactions between
the side chains and ligand with side chains was done. The energy
minimization of the X-ray structure was done at the RMSD value
of 0.3 Å. This energy minimization also ensures the placement of
hydrogen atoms in an optimized geometry. Chemical structures of
ligands were drawn in ChemDraw professional and minimized by using
OPLS3 force-field (LigPrep module) where 1000 iteration cycles were
run, to get the steepest energy minimized structure. The Grid was
generated with the aid of Receptor grid generation module, which is a
user defined three-dimensional grid particular selecting the residues of
ATPase domain for docking. In the present study, the location and
dimensions of the grid box was taken from the coordinates of the
centroid of the already embedded ATP molecule, with the help of
Maestro visualizer. Each docking experiment was done by implicating
the Lamarckian Genetic Algorithm (LGA) which was set with the total
1000 conformations, 50 different runs with termination after 250000
energy evaluation. Population size was set out to be 150. Finally, the
conformation of lowest predicted binding free energy of the most
occurring binding modes in the hTopoIIα active pocket was selected.
The treated and control rat C6 glial cells (1 × 105), cultured in 96-well
plates were equilibrated in PBS and incubated in the dark for 30 min
with 100 μM of H2DCF-DA (Invitrogen) [Ex478 nm/Em518 nm]. After
washing twice with PBS, fluorescence was then read at the excitation
wavelength of 478 nm and emission wavelength of 518 nm using
BioTek Microplate Reader as per the protocol described.
|
4.2.4 Topoisomerases assays for investigational
compounds
hTopoIIα mediated activity in the presence of investigational
compounds were probed using commercially available assay kit
procured from TopoGEN, Inc., USA.
hTopoII mediated DNA decatenation assay
For this assay, reaction components such as freshly prepared 5 ×
buffer, 150 ng of kDNA (substrate), 100 μg drug or test compound
(previously dissolved in DMSO solvent), 2 units of hTopoIIα enzyme,
assay buffer and nuclease free water were added sequentially in a
1.5 mL sterile centrifuge tube. Nuclease free water was added to this
reaction mixture to adjust the final reaction volume to 20 μL. Assay
buffer was prepared by mixing buffer A (0.5 M Tris-HCl of pH 8, 1.5 M
sodium chloride, 100 mM magnesium chloride, 5 mM dithiothreitol,
300 mg bovine serum albumin) and buffer B (20 mM ATP in water) in
1:1 ratio. The reaction mixture was incubated in a water bath at
37°C for 30 min. Reaction was stopped by adding (SDS) sodium
dodecyl sulfate (final concentration 0.10% w/v) and proteinase K (final
concentration 0.1 mg/mL). The catenated and decatenated forms of
kDNA were analyzed by agarose gel (1% w/v) electrophoresis in 1×
Tris-acetate-EDTA (TAE) running buffer containing ethidium bromide
(final concentration 0.5 μg/mL) at 3.5 V/cm for 1 h 20 min. Gel was
destained in water till the bands were sufficiently visible for imaging.
Gel image was taken using GelDoc EZ imager (BioRadTM) and
quantitated by Image lab software.
|
4.4 Cell cycle analysis
Cellular samples (C6 previously treated with investigational com-
pounds 1b and 1h for 48 h and control) were prepared for staining by
propidium iodide (PI). The standard protocol set up by our laboratory
was utilized for the analysis.[18–20,29] The cells approximately 106 were
trypsinized and transferred to each tube. The cells were further
centrifuged at 1200 rpm for 5 min and washed with 1× PBS. Thereafter
cells were fixed using chilled methanol and incubated for 3 h at −20°C.
Post 3 h, cells were subjected to centrifugation at 2500 rpm and
washed once with 1× PBS. A total of 50 μL of propidium iodide (along
with 50 μL ribonuclease A) was added to each well and incubated for
30 min at room temperature in dark. The DNA content was then
measured using flow cytometer (BD Accuri).
DNA intercalation assay
For this assay, 150 ng of scDNA was incubated with EtBr and
investigational compounds 1b and 1h at 37°C for 20 min. The reactions
were analyzed by 1% w/v agarose gel electrophoresis in 1× TAE
running buffer at 3.5 V/cm for 30 min. After electrophoresis, gel was
first stained with EtBr solution for 30 min and later destained with
water for sufficient time till the bands were sufficiently visible for
imaging. Gel image was taken using Gel Doc EZ imager (BioRad™).
|
4.5 Prediction of mode of cell death
For predicting whether the cell death induced by investigational
compounds (1b and 1h) is apoptosis or necrosis PI versus
Annexin V assay was done. The assay was conducted in accordance to
our previously reported laboratory protocols.[18–20,29] C6 cells previously
treated with investigational compounds (1b and 1h) for 48 h were
trypsinized and were centrifuged at 1200 rpm for 5 min and washed with
1× PBS. The cellular samples (approx. 106 cells in each tube) were taken
and stained with Annexin V and PI dye (50 μL each dye for each sample).
The samples were incubated for 30 min at room temperature in the dark
and mode of cell death was analyzed using flow cytometer (BD Accuri).
|
4.3 Molecular docking methodology
3-Diminesional co-crystal structure of hTopoIIα with ATP was
retrieved from Protein Data Bank (PDB 1ZXM) and was further edited
for protein preparation by removing excess crystallized water
molecules, chains (using PyMol visualizer). The protein preparation

KAUR ET AL.
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4.6 hPBMC culture and MTT assay
[17] B. Dwarakanath, D. Khaitan, R. Mathur, Indian J. Exp. Biol. 2004, 42,
649.
hPBMCs were obtained from whole blood after treatment with RBC
lysis buffer and were then suspended in RPMI media supplemented
with 10% fetal bovine serum (FBS), 1× antibiotic solution and were
incubated at 37°C with 5% CO2 and 95% humidity. The PBMCs were
counted on the automated cell counter (Invitrogen). MTT assay was
done as mentioned under section 4.2.2.
[18] M. Chauhan, G. Joshi, H. Kler, A. Kashyap, S. M. Amrutkar, P. Sharma,
K. D. Bhilare, U. C. Banerjee, S. Singh, R. Kumar, RSC Adv. 2016,
6, 77717.
[19] G. Joshi, S. M. Amrutkar, A. T. Baviskar, H. Kler, S. Singh, U. C.
Banerjee, R. Kumar, RSC Adv. 2016, 6, 14880.
[20] G. Joshi, H. Nayyar, S. Kalra, P. Sharma, A. Munshi, S. Singh, R. Kumar,
Chem. Biol. Drug Des. 2017, 90, 995.
[21] G. Kaur, R. P. Cholia, A. K. Mantha, R. Kumar, J. Med. Chem. 2014, 57,
10241.
[22] G. Joshi, P. K. Singh, A. Negi, A. Rana, S. Singh, R. Kumar, Chem. Biol.
Interact. 2015, 240, 120.
ACKNOWLEDGMENTS
RK, GJ, and SK are thankful to Central University of Punjab and Bristol-
Myer Squibb, USA [Grant No. 34003085] for providing infrastructure
and funds.
[23] A. T. Baviskar, U. C. Banerjee, M. Gupta, R. Singh, S. Kumar, M. K.
Gupta, S. Kumar, S. K. Raut, M. Khullar, S. Singh, Biorg. Med. Chem.
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[24] S. Kalra, G. Joshi, A. Munshi, R. Kumar, Eur. J. Med. Chem. 2017, 142,
424.
[25] H. Wei, A. J. Ruthenburg, S. K. Bechis, G. L. Verdine, J. Biol. Chem.
2005, 280, 37041.
CONFLICT OF INTEREST
[26] D. Kumar, G. Kaur, A. Negi, S. Kumar, S. Singh, R. Kumar, Bioorg. Chem.
2014, 57, 57.
Authors declare no conflict of interest.
[27] D. Kumar, R. Kumar, Tetrahedron Lett. 2014, 55, 2679.
[28] J. M. Gunnersen, V. Spirkoska, P. E. Smith, R. A. Danks, S. S. Tan, Glia
2000, 32, 146.
ORCID
[29] R. P. Cholia, S. Kumar, M. Kaur, R. Kumar, M. Dhiman, A. K. Mantha,
Metab. Brain Dis. 2017, 32, 1705.
Raj Kumar
http://orcid.org/0000-0001-5113-6627
[30] R. Palchaudhuri, P. J. Hergenrother, Curr. Opin. Biotechnol. 2007, 18,
497.
[31] G. Priyadarshani, S. Amrutkar, A. Nayak, U. C. Banerjee, C. N. Kundu,
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https://doi.org/10.1002/ardp.201800023