Synthesis of imine-pyrazolopyrimidinones and their mechanistic interventions on anticancer activity

Article abstract of DOI:10.1016/j.bmc.2013.07.016

Design, synthesis and anticancer activity of a series of imine-pyrazolopyrimidinones is reported for the first time. Compounds 9d, 9n and 9o in the series show encouraging in vitro anticancer activity with low micromolar IC₅₀ values against prostate (PC3) and breast (MCF7) cancer cell lines. Some notions about structure-activity relationships and plausible mechanism of biological activity are presented.

Full text of DOI:10.1016/j.bmc.2013.07.016

Bioorganic & Medicinal Chemistry 21 (2013) 5782–5793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of imine-pyrazolopyrimidinones and their mechanistic
interventions on anticancer activity
Ashish T. Baviskar b, Uttam C. Banerjee b, Mukesh Gupta c, Rajveer Singh c, Sunil Kumar c,
Manish K. Gupta c, Sanjeev Kumar d, Satish K. Raut f, Madhu Khullar f, Sandeep Singh e, Raj Kumar a,c,
⇑
,1
a Laboratory for Drug Design and Synthesis, Centre for Chemical and Pharmaceutical Sciences, Central University of Punjab, 151 001 Bathinda, India
b Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER), Mohali, S.A.S. Nagar, Sec 67, 160 062 Punjab, India
c Molecular Modeling and Pharmacoinformatics Lab, Indo-Soviet Friendship College of Pharmacy, Moga, 141 001 Punjab, India
d Centre for Biosciences, Central University of Punjab, 151 001 Bathinda, India
e Centre for Genetic Diseases and Molecular Medicine, Central University of Punjab, 151 001 Bathinda, India
f Department of Experimental Medicine and Biotechnology, PGIMER, 160 012 Chandigarh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and anticancer activity of a series of imine-pyrazolopyrimidinones is reported for the
first time. Compounds 9d, 9n and 9o in the series show encouraging in vitro anticancer activity with
low micromolar IC50 values against prostate (PC3) and breast (MCF7) cancer cell lines. Some notions
about structure–activity relationships and plausible mechanism of biological activity are presented.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 8 May 2013
Revised 28 June 2013
Accepted 9 July 2013
Available online 17 July 2013
Keywords:
Synthesis
Imine-pyrazolopyrimidinones
Anticancer agents
Effects on G2/M phase of the cell cycle
Catalytic topoisomerase inhibitors
1. Introduction
alterations/modifications12 such as acetylation,13 methylation14
of resveratrol or hybridization of stilbene-coumarin.15 The pub-
Cancer is a complex, multifactorial disease characterized by
uncontrolled growth of abnormal cells in the body.1 It is comprised
of more than hundred diseases having common features of exces-
sive proliferation, angiogenesis and may also harbour metastatic
properties. It is a leading cause of deaths worldwide after cardio-
vascular diseases.2 As per WHO, 70% of all cancer deaths occurred
in low and medium income countries in 2008, are predicted to con-
tinue rising with an estimate of 13.1 million deaths in the year
2030.3 Although, several anticancer drugs are available, the failure
to achieve the desired therapeutic efficacy with existing agents due
to multi-drug resistance, toxicity or poor bioavailability warrants
evaluation of newer compounds.4–6
lished success stories on a single molecule with multiple phar-
amcophores16–18 having beneficial and reduced or unwanted side
effects encouraged us to rationally design a hybrid pharmacophore
(Fig. 1). The hybrid pharamcophore is designed as a template for
anticancer agent with substitution of one of the aromatic rings of
stilbenes/imines (A) with pyrazolo[3,4-d]pyrimidinone (B).
Pyrazolo[3,4-d]pyrimidines/pyrimidinones have been already
reported as anticancer agents,19 through a variety of investigated
mechanisms such as inhibition of EGFR,20 IGF-1R21 or dual Src/
Abl.22
Naturally occurring stilbenes7,8 especially trans-stilbenes
including resveratrol9 and piceatannol10 are being explored as
anticancer agents. However, the limited bioavailability of the these
stilbenes due to glucuronidation and sulfation regardless of their
greaterabsorption11 has prompted the researchers to rely on their
R1
R1
O
X
X
N
A
O
N
N
R2
R1
N
N
NH
N
X may be N or CH
Target compounds
(5 and 9)
R2
N
X = N
⇑
B
Corresponding author. Fax: +91 163 623 6564.
E-mail address: raj.khunger@gmail.com (R. Kumar).
1
CUPB Library Communication Number P-19.
Figure 1. Design of the target compounds 5 and 9.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.016

A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
5783
10a or 2-chlorobenzaldehyde 10b resulted into condensation fol-
lowed by ring cyclisation to afford 9n and 9o, respectively, via for-
mation of intermediate 11 (Scheme 2). Reduction of imine of 9a
using NaBH4 in methanol afforded 9p (Scheme 3). The products
were purified either by crystallization or column chromatography
using EtOAc/hexane as elution solvents. All new compounds were
fully characterized by 1H and 13C NMR, mass spectral, and elemen-
tal micro-analytical data.
2. Results and discussion
2.1. Synthesis
Preparation of the target compounds 5a–f and 9a–p was accom-
plished by means of the synthetic procedures shown in Scheme 1.
Briefly, nucleophilic substitution of ester of 1 was carried out with
hydrazine hydrate to afford hydrazide 2 which was condensed
with substituted or unsubstituted benzaldehyde (3) to give hydra-
zone derivatives 4.
2.2. Antiproliferative activity
Heating 4 with CH(OEt)3 afforded ring cyclized target com-
pounds 5. For the synthesis of desired compounds 9a–p, 1 was
treated with benzyl chloride to afford a mixture of two regio-iso-
mers (6a and 6b). The major isomer 6a was isolated by crystalliza-
tion from ethyl acetate and treated with hydrazine hydrate to yield
7. Condensation of 7 with various benzaldehyde derivatives 3 re-
sulted into 8 which was further ring cyclized to afford 9a–k using
CH(OEt)3 under acid catalyzed heating conditions. 7 when treated
with CH(OEt)3, 9l was obtained whereas a different product 9m
was isolated when 7 was heated with CH(OEt)3 under acidic condi-
tions. The position of benzyl group in case of 6a and 6b was con-
firmed using UV spectroscopy (6a; kmax (MeOH) = 266 nm and
6b; kmax (MeOH) = 254 nm). Treatment of 7 with anisaldehyde
In order to determine the antiproliferative potential of the tar-
get compounds 5a–f, 9a–k, and 9n–p were screened in vitro
against six cancer cell lines, including THP-1 (leukemia), IGROV-1
(ovary), HeLa (cervix), PC3 (prostate), MCF7 (breast fibroadenoma),
H460 (liver). The IC50 values for tested compounds are summarized
in Supplementary Table 1 (see Supplementary data).23 The com-
pounds that showed the highest in vitro anticancer activity in pros-
tate, breast and liver cancer lines are collected in Table 1. Etoposide
(Etop)24 was used as the positive control. These compounds when
further tested on normal human cells, little or no significant cyto-
toxicity towards human buccal cavity cells (less than 20% apopto-
sis) at highest concentration of 100 lM in comparison to etoposide
O
O
O
OEt
(i)
OEt
N
N
OEt
N
+
N
N
NH2
NH2
H
N
NH2
1
6a
6b
(ii)
(v)
O
O
O
N
NHNH2
N
NHNH2
NH2
N
OEt
NH2
(viii)
N
N
N
N
N
NH2
H
N
2
7
9l
R1
R1
R4
O
R2
(ix)
R2
R3
N
(iii)
(vi)
N
H
H
R3
N
N
O
R4
3
O
R1
9m
3
R1
R4
R2
R3
R2
R3
O
O
N
N
H
N
N
N
H
R4
N
N
NH2
N
H
NH2
4
8
(vii)
(iv)
O
R1
R1
R2
R3
R2
R3
O
O
N
N
N
N
N
O
N
N
N
R4
N
N
R4
N
N
5
N
N
N
9k
H
9
5a; R1, R2, R3 = OMe; R4 = H
5b; R1 = H; R2, R3, R4 = OMe
9a; R1, R2, R3, R4 = H
9b; R1, R3, R4 = H; R2 = Cl
5c; R1, R2 = OMe; R3, R4 = H
5d; R1 = OMe; R2 = OH; R3, R4 = H
5e; R1= OH; R2 = OMe; R3, R4 = H
5f; R1, R2 = -CH2-O-CH2-; R3, R4 = H
9c; R1, R3, R4 = H; R2 = CN
9d; R1 = OMe; R2 = OH; R3, R4 = H
9e; R1 = OH; R2 = OMe; R3, R4 = H
9f; R1, R2 = -CH2-O-CH2-; R3, R4 = H
9g; R1, R2, R3 = OMe; R4 = H
9h; R1 = H; R2, R3 and R4 = OMe
9i; R1, R2 = OMe; R3, R4 = H
9j; R1, R2, R4 = OMe; R3 = H
Scheme 1. Reagents and conditions: (i) BnCl, NaH, MeCN, rt, 12 h; (ii) NH2NH2ꢀH2O, EtOH, reflux, 12 h; (iii) MeOH, reflux, 4 h; (iv) CH(OEt)3, MeCN, reflux, 8 h; (v)
NH2NH2ꢀH2O, EtOH, reflux, 10 h; (vi) EtOH, reflux, 4 h; (vii) CH(OEt)3, MeCN, reflux, 4 h; (viii) CH(OEt)3, reflux, 12 h; (ix) CH(OEt)3, MeCN, H2SO4 (1 drop), reflux, 7 h.

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A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
O
R1
O
R1
OHC
R2
NHNH2
R2
N
N
H
N
CH
+
(i)
N
N
N
NH2
N
C
R2
7
H
10
R1
11
10a; R1 = H; R2 = OMe
10b; R1 = Cl; R2 = H
R2
O
N
N
N
R1
R2
N
N
H
R1
9n; R1 = H; R2 = OMe
9o; R1 = Cl; R2 = H
Scheme 2. Reagents and conditions: (i) EtOH, reflux, 4 h.
O
O
N
cell cycle analysis confirmed that these compounds lead to in-
creased cell numbers in G2/M phase (13.8% vs 48.1% and 52.2%).
Figure 3A shows that these compounds induce apoptosis through
G2/M arrest.
Many of the drugs also induce free radicals as well as alter var-
ious other signaling cascades inside the cancer cells. We evaluated
our compounds for any oxidant potential using spectrophotomet-
ric and fluorescence microscopy analysis of H2DCFDA. The results
indicate that these compounds increased ROS levels at low concen-
H
N
N
N
N
N
NaBH4
rt, 4 h
N
N
N
9a
N
9p
Scheme 3. Synthesis of 9p.
Table 1
trations while at 5 lM there was more than 1.5 fold increase in free
Anticancer activity of 9d, 9n and 9o
radicals (Fig. 3B and C). Further evaluation for DNA damage was
indicative that these compounds also cause DNA damage at low
concentrations. Comet analysis revealed that there was almost
9% decrease in comet head indicating potential increase in DNA
damage (Fig. 3D and E). Taken together biological analyses of these
compounds indicate their selective cytotoxic potential to cancer
cells and induce free radical stress as well as DNA damage in these
cancer cells. These findings are suggestive of multiple pathways
are being affected by these compounds.
Cd
PC3
Prostate
MCF7
Breast
H460
Liver
IC50
9d
9n
9o
(l
M)a
10.1
17.6
18.1
18.2
29.7
18.1
20.1
20.9
34.3
35.7
39.8
<30
Etop
a
Values are derived from averaging three independent experiments and each
experiment was done in triplicate.
2.4.2. Compounds were selective TopoII
a inhibitors
(30% apoptosis) was observed. This overall suggests that the com-
pounds are cytotoxic to cancer cells only (Fig. 2).
It has been formerly reported that cell cycle arrest at G2/M
phase is also correlated with higher topoisomerase II
a (TopoIIa)
expression in rapidly proliferating cells25 whereas inhibition of
topoisomerase activity is also associated with increased DNA dam-
age.26 It was planned to screen 9d, 9n and 9o against topoisomer-
2.3. Structure–activity relationships
Some general notions about structure–activity relationships
emerged from these studies on the tested compounds (see Supple-
mentary Table 1 in Supplementary data):23 (a) removal of benzyl
group from 9g, 9j, 9d, 9e and 9f resulted in decrease of anticancer
activity as exemplified by 5a, 5c, 5d, 5e and 5f, respectively, (b)
reduction or removal of iminic bond diminishes the activity
(9a > 9p and 9l > 9m), (c) addition of phenyl ring on iminic carbon
enhances the cytotoxic activity (9l < 9a) and (d) on phenyl ring, in
general the activating groups such as methoxy and hydroxy poten-
tiated the activity (9a < 9d, 9e and 9g) whereas the deactivating
groups such as cyano and chloro resulted in dilution of the activity
(9a > 9b and 9c).
ase enzymes (TopoII
human topoisomerase II
kDNA decatenation assay, Kinetoplast DNA (kDNA) was used as a
substrate whereas etoposide (TopoII inhibitor), was used as stan-
dard (Fig. 4A and B). Incubation of kDNA with human topoisomer-
a
and TopoI). The effect of the compounds on
was examined using ATP-dependent
a
ase IIa showed two decatenated products: nicked kDNA (Nck) and
supercoiled (SC)/relaxed (Rel) circular DNA. In agarose gel, cate-
nated kDNA appears at the top as it cannot enter into the gel be-
cause of its overall size while other decatenated products move
into.
The samples containing etoposide showed partial or moderate
decatenation, same as reported in the literature because of its
known reversible inhibition of topoisomerase IIa 27,28
.
Compounds
9d, 9n and 9o were found to be most active against human topoi-
somerase II . Almost no decatenation activity was observed in the
2.4. Mechanistic interventions on anticancer activity
a
2.4.1. Cell cycle analysis and DNA damage studies
presence of these compounds. In comparison to etoposide, com-
In order to execute preliminary mechanistic studies on antican-
cer activity, we focused on exploring the stage(s) of the cell cycle
being affected by selected compounds 9d and 9o. To this end, we
pounds 9d, 9n and 9o had shown higher inhibition of topoisomer-
ase IIa activity. Compounds under investigation were also
subjected to topoisomerase II dependent relaxation (Fig. 4C and
D) in which a negatively supercoiled DNA (pRYG) was measured
using agarose gel electrophoresis. Results obtained were in tune
performed flow cytometry on PC3 cells treated with 10
lM of 9d
and 9o for 24 h, and the results are collected in Figure 3A. Initial

A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
5785
Figure 2. Cytotoxicity (MTT) assay for normal and cancerous cell lines. Compounds 9d (A), 9n (B), 9o (C) were tested in normal buccal cavity (blue bars), prostate cancer PC3
(red bars), breast cancer MCF7 (black bars) and liver cancer H460 (violet bars) cell lines. (D) Etoposide was used as positive control to compare the IC50 values in all four cell
lines. All the experiments were done in triplicate as three independent experiments (total 9 replicates per treatment) and values were averaged to plot the graphs.
with as observed in kDNA decatenation assay. It was detected that
supercoiled form of substrate pRYG plasmid DNA was reduced by
topoisomerase II and appeared as a set of variably relaxed topoi-
somers that migrate more slowly as compared to supercoiled form.
It was noticed that compounds 9d, 9n, and 9o were found to be
2.4.4. Molecular modeling
As 9d emerged to be a potent catalytic inhibitor of TopoIIa, we
were interested to identify its binding interactions with ATPase do-
main of hTopoIIa
29 in the active site by molecular docking simula-
tion using the GOLD software.30 Overall binding mode of 9d with
binding site residue of ATPase domain of hTopoIIa29 was found
similar to that observed with co-crystalized AMPPNP.27,29 The ma-
more potent topoisomerase II
side (IC50 = 78.4 M) as indicated by the calculated IC50 value
46.5
M (Fig. 4E) of the 9d (see Supplementary data).23 Further,
a inhibitors in comparison to etopo-
l
l
jor interactions of 9d with topoisomerases II
lar and van der Waals interactions. The highly conserved Walker A
motif at ATP binding site of topoisomerases II is composed of
a include H-bonds, po-
the tested compounds (9d, 9n, and 9o) were tuned up to be weak
inhibitors of TopoI as compared to camptothecin (C; a standard
hTopoI inhibitor) in topoisomerase I mediated relaxation assay
a
Arg162, Asn163, Gly164, Tyr165, Gly166 and Ala 167. The studies
revealed that the Walker A motif is crucial for binding of catalytic
inhibitors acting on ATPase domain. The ring A of 9d gets posi-
tioned in the vicinity of Walker A motif and involved in two H-
bonds and van der Waals interactions (Fig. 5). In this, the oxygen
atom of OCH3 (ring A) shows a H-bond interaction with backbone
NH of Arg162.The other H-bond is observed between phenolic oxy-
gen and NH ofGly166. The other residues Glu87, Gly160, Asn163,
Gly164, Tyr165 and Gln376 are involved in polar and/or van der
Waals interactions with ring A. The ring-A also showed an effective
indicating their selectivity towards TopoII
a
(Fig. 4).
2.4.3. Compounds were catalytic TopoII
a
inhibitors
Since our compounds expressed topoisomerase II inhibitory
activity and also indicated genotoxicity through generation of
ROS, so we next planned to answer the question whether these
compounds are catalytic inhibitors of Topo II or the poisons. We
performed the DNA relaxation assay by setting up competition
between ATP and our compound 9d.23 Nuclear lysate from HeLa
cells was used as source of topoisomerase and incubated with
varying concentration of 9d with constant amount of ATP. Our
results signposted that compound 9d was able to inhibit the
relaxation induced by topoisomerase in increasing concentration,
thus indicating that our compounds are catalytic inhibitors of
topoisomerase activity while also induce free radicals and DNA
damage through secondary mechanisms (see Supplementary
data).23
cation-
p
interaction with the Mg2+. The Mg2+ ion is an important
The
for catalytic activity at the ATPase domain of hTopoIIa 25,29
.
nitrogen (at intermediate chain) is involved in polar interactions
with carbonyl function of Ile141 and Thr147. The carbonyl function
at ring B oriented towards Ser148 and Ser149 and shows many po-
lar interactions. The ring B also shows arene-hydrogen interaction
with the side chain of Ile141. The guanidine group of Arg98 func-
tions as a backbone acceptor to the methylene group present in

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A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
Figure 3. (A) FACS analysis for cell cycle profiling of control and drug treated PC3 cells. The first graph shows untreated control cells, the middle showing PC3 cells treated
with 5 M of 9d and the right side graph shows PC3 cells treated with 10 M of compound 9o. (B) Spectrophotometric analysis of DCFDA intensity in PC3 cells treated with
l
l
compound 9d (white bars) and 9o (grey bars) to evaluate the levels of free radicals in cells. (C) PC3 cells treated with 9d and 9o compounds were stained with DCFDA and
captured under fluorescent microscope. The green color represents DCFDA staining. (D) Comet assay pictures showing formation of comet in treated PC3 cells. (E) Comets
were analyzed for percentage of DNA in head and tail. Total 30 cells were scored using Cometscore15 software Version 1.0.1.0 and all the values were averaged and standard
errors were calculated.
between ring C and D. The ring D lies parallel to the side chain car-
elevated ROS levels, thus causing DNA damage. The compounds
were also found to be potent and selective catalytic inhibitors of
hTopoIIa inhibitors indicated by ATP-dependent decatenation
and relaxation assays which was further supported by molecular
modeling studies. We propose that anticancer activity expressed
by the compounds is a result of hijacking/interfering of multiple
pathways through regulation of free radicals, DNA damage and cat-
bonyl group of Asn95 and showed a unique lone pair-
p interac-
tion.31 The other residues surrounding to the ring D include
Asn120 and Lys123. Thus the present study indicates that 9d is a
catalytic inhibitor acting via occupying the ATP binding pocket of
ATPase domain of hTopoIIa and making favorable interactions
with its key residues.
alytic-TopoIIa activity concurrently in cancer cells. Further de-
tailed investigations of SARs as well as explorations of antitumor
activity are presently in progress.
3. Conclusions
In conclusion, we have designed and synthesized some com-
pounds that show encouraging anticancer activity in vitro with
low micromolar IC50 values compared to etoposide against pros-
tate and breast cancer cell lines tested and with low toxicity to
the normal cells. Our preliminary mechanistic studies of biological
activity of 9d, 9n, and 9o suggest their effect on the G2/M phase of
the cell cycle. The experiments suggested that these compounds
provoke several stress pathways in the cancer cells including
4. Experimental
4.1. Chemistry
The reagents for the synthesis of compounds were purchased
from Sigma–Aldrich, Loba and CDH, India and used without further
purification. All yields refer to isolated products after purification.

A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
5787
Figure 4. (A). Effect of test compounds on TopoII
a
mediated kDNA decatenation assay. (B) Graphical representation of decatenated products formed in TopoII
a
mediated
kDNA decatenation assay. (C) Effect of test compounds on TopoII
products formed in TopoII mediated supercoiled plasmid pRYG DNA relaxation assay. (E) Inhibition of topoisomerase II
ranging from 10 to 100 M in kDNA decatenation assay. (F) Effect of test compounds on TopoI mediated supercoiled plasmid DNA relaxation assay.
a
mediated supercoiled plasmid pRYG DNA relaxation assay. (D) Graphical representation of relaxed
a
a
activity by compound 9d at various concentrations
l
Products were characterized by by spectroscopic data (IR, 1H NMR,
13C NMR and MS spectra). NMR experiments were measured in
CDCl3/DMSO-d6 relative to TMS (0.00 ppm). IR (KBr pallets) spectra
were recorded on a Fourier transform infrared (FT-IR) Thermo
spectrophotometer. Melting points were determined in open capil-
laries and were uncorrected. The purity of the all the tested com-
pounds was assessed by reverse phase analytical HPLC. All the
target compounds were unreported and their physical data is pre-
sented as below:
was stirred in a 250 mL round bottom flask for 1 h under ice cold
conditions. Benzylchloride (6 mmol, 1.5 equiv) was added to the
reaction mixture and further stirred at room temperature for
12 h. The mixture was extracted with ethyl acetate (3 ꢁ 10 mL).
The organics were washed, dried on anhydrous Na2SO4, evaporated
on a rotary evaporator to afford the crude product. The crude prod-
uct was recrystallized (MeOH) to get pure compound 6a. Yield:
60%, white solid; mp: 161–162 °C; IR (KBr): 3465, 3301, 1693,
1605 cmꢂ1
;
1H NMR (400 MHz, DMSO-d6): d = 1.24 (t, 3H,
J = 6.96 Hz, CH3), 4.17 (q, 2H, J = 6.92 Hz, OCH2), 5.35 (s, 2H, CH2),
7.35–7.24 (m, 5H), 8.08 (s, 1H, CH); 13C NMR (100 MHz, DMSO-
d6): d = 55.09, 59.52, 98.09, 127.61, 127.78, 128.17, 128.25,
128.72, 128.93, 133.63, 137.28, 157.07, 164.14.
4.1.1. Ethyl 5-amino-1-benzyl-1H-pyrazole-4-carboxylate (6a)
A solution of 3-amino-4-carbethoxy pyrazole (1, 4 g, 1 mmol,
1 equiv) in acetonitrile (60 mL) and sodium hyride (1.5 g, 2 equiv)

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A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
was then dried to remove methanol under vacuum to give 4.
Then 2 mL of triethylorthoformate was added and the reaction
mixture was refluxed for 4 h (TLC). The crude compound obtained
was purified by column chromatography to give 5b using ethyl
acetate:hexane (1:3) as eluents. Yield: 75%, white solid; mp:
212–215 °C; IR (KBr): 3192, 1709, 1592 cmꢂ1
;
1H NMR
(400 MHz, DMSO-d6): d = 3.79 (s, 3H, –OCH3), 3.89 (m, 6H, –
OCH3) , 7.01 (d, 1H, J = 8.5 Hz, CH), 7.78 (d, 1H, J = 8.5 Hz, CH),
8.36 (s, 1H, CH), 8.44 (s, 1H, N–CH), 9.14 (s, 1H, N@CH); 13C
NMR (100 MHz, DMSO-d6): d = 56.61, 61.01, 62.64, 105.94,
109.19, 118.85, 122.39, 141.93, 148.32, 154.60, 157.70, 162.43.
Anal. Calcd for C15H15N5O4: C, 54.71; H, 4.59; N, 21.27. Found:
C, 54.79; H, 4.65; N, 21.24.
4.1.6. 5-(3,4-Dimethoxybenzylideneamino)-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5c)
A solution of 2 (50 mg, 3 mmol, 1 equiv) in methanol (5 mL) and
3,4-dimethoxy benzaldehyde (76 mg, 1.3 equiv) was refluxed for
4 h in a 10 mL round bottom flask. After 4 h, methanol was distilled
off to give 4. Triethylorthoformate (2 mL) and acetonitrile (1 mL)
were added to crude residue. The reaction mixture was refluxed
for 5 h (TLC). The excess of triethylorthoformate and acetonitrile
were distilled off and the crude compound obtained was purified
by column chromatography to give 5c using ethyl acetate:hexane
(1:1.5) as eluents. Yield: 70%, white solid; mp: 252–256 °C; IR
Figure 5. Docked conformation of 9d at the ATPase domain of hTopoIIa.
4.1.2. 5-Amino-1-benzyl-1H-pyrazole-4-carbohydrazide (7)
A solution of 6a (200 mg, 1 mmol, 1 equiv) in ethanol (10 mL)
and hydrazine hydrate 98% (2 mL, 5 equiv) was refluxed in 25 mL
round bottom flask for 10 h. White crystals of 7 obtained on cool-
ing were filtered, washed with diethyl ether and dried. Yield: 80%,
White crystalline; IR (KBr): 3465, 3301, 1693, 1605 cmꢂ1; 1H NMR
(400 MHz, DMSO-d6): d = 4.20 (s, 2H, D2O exchangeable NH2), 5.04
(s, 2H,D2O exchangeable NH2), 5.35 (s, 2H, CH2), 7.35–7.26 (m, 3H),
7.20 (d, J = 7.64 Hz, 2H), 7.92 (s, 1H, CH), 9.01 (s, 1H, D2O exchange-
able NH); 13C NMR (100 MHz, DMSO-d6): d = 55.17, 99.64, 128.11,
128.94, 130.09, 137.56, 156.75, 164.91.
(KBr): 3300, 1693, 1580 cmꢂ1 1H NMR (400 MHz, DMSO-d6):
;
d = 3.84 (s, 3H, –OCH3), 3.97 (s, 3H, –OCH3), 7.11 (d, 1H,
J = 8.1 Hz, CH), 7.41 (d, 1H, J = 8.1 Hz, CH), 7.51 (s, 1H, CH), 8.25
(s, 1H, CH), 8.43 (s, 1H, N–CH), 8.94 (s, 1H, N@CH);13C NMR
(100 MHz, DMSO-d6): d = 56.04, 56.21, 105.90, 109.71, 111.97,
125.03, 125.46, 148.07, 149.59, 153.15; MS (APCI): m/z = 300.33
[M+1]+, m/z = 164.13 [M+1]+ a fragment peak. Anal. Calcd for
C14H13N5O3: C, 56.18; H, 4.38; N, 23.40. Found: C, C, 56.21; H,
4.41; N, 23.49.
4.1.3. Synthesis of 5a–5f
4.1.7. 5-(3-Methoxy-4-hydroxybenzylideneamino)-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5d)
A
solution of 3-amino-4-carbethoxy pyrazole (1, 200 mg,
1 mmol, 1 equiv) in ethanol (2 mL) and hydrazine hydrate 98%
(2 mL, 4 equiv) was refluxed for 12 h in 25 mL round bottom
flask. White crystals were filtered off, washed with diethyl ether
and dried to afford the 2 which was used for the next step as
follows:
A solution of 2 (30 mg, 2 mmol, 1 equiv) in methanol (5 mL)
and 3-methoxy-4-hydroxy benzaldehyde (42 mg, 1.3 equiv) was
refluxed for 4 h in 10 mL round bottom flask. The reaction mix-
ture was then dried to remove methanol under vacuum to give
4. Then 2 mL of triethylorthoformate was added and the reaction
mixture was refluxed for 4 h (TLC). The crude compound obtained
was purified to give 5d by column chromatography using ethyl
acetate:hexane (1:3) as eluents. Yield: 78%, brown solid; mp:
4.1.4. 5-(3,4,5-Trimethoxybenzylideneamino)-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5a)
239–241 °C; IR (KBr): 3113, 1708, 1601 cmꢂ1
;
1H NMR
A solution of 2 (30 mg, 2 mmol, 1 equiv) in methanol (5 mL) and
3,4,5-trimethoxy benzaldehyde (54 mg, 1.3 equiv) was refluxed for
4 h in 10 mL round bottom flask. The reaction mixture was then
dried to remove methanol under vacuum at rotary evaporator to
give 4. Then 2 mL of triethylorthoformate was added and the reac-
tion mixture was refluxed for 8 h. White crystals obtained on cool-
ing were filtered off, washed with diethyl ether and dried to afford
5a. Yield: 59%, Yellowish white solid; mp: 169–171 °C. IR (KBr):
(400 MHz, DMSO-d6): d = 3.84 (s, 3H, –OCH3), 6.93 (d, 1H,
J = 7.9 Hz, CH), 7.31 (d, 1H, J = 7.9 Hz, CH), 7.50 (s, 1H, CH), 8.27
(s, 1H, CH), 8.43 (s, 1H, N–CH), 8.84 (s, 1H, N@CH); 13C NMR
(100 MHz, DMSO-d6): d = 56.49, 60.70, 105.85, 106.44, 107.15,
128.20, 141.51, 148.16, 153.65, 166.68. Anal. Calcd for
C13H11N5O3: C, 54.74; H, 3.89; N, 24.55. Found: C, 54.77; H,
3.93; N, 24.62.
3177, 3088, 1701, 1611 cmꢂ1 1H NMR (400 MHz, DMSO-d6):
;
d = 3.76 (m, 3H, –OCH3), 3.86 (m, 6H, –OCH3), 7.26 (m, 2H, CH),
8.28 (s, 1H, CH), 8.48 (s, 1H, N–CH), 9.03 (s, 1H, N@CH); 13C NMR
(100 MHz, DMSO-d6): d = 56.49, 60.70, 105.85, 106.44, 107.15,
128.20, 141.51, 148.16, 153.65, 166.68. Anal. Calcd for
4.1.8. 5-(3-Hydroxy-4-methoxybenzylideneamino)-1H-
pyrazolo[5,4-d]pyrimidin-4(5H)-one (5e)
A solution of 2 (30 mg, 2 mmol, 1 equiv) in methanol (5 mL) and
3-hydroxy-4-methoxy benzaldehyde (42 mg, 1.3 equiv) was re-
fluxed for 4 h in 10 mL round bottom flask. The reaction mixture
was then dried to remove all methanol under vacuum to give 4.
Then 2 mL of triethylorthoformate was added and the reaction
mixture was refluxed for 4 h (TLC). The crude compound obtained
was purified by column chromatography to give 5e using ethyl
acetate:hexane (1:3) as eluents. Yield: 63%, light brown solid;
mp: 242–244 °C; 1H NMR (400 MHz, DMSO-d6): d = 3.85 (s, 3H, –
OCH3), 7.08 (d, 1H, J = 7.6 Hz, CH), 7.28 (d, 1H, J = 7.6 Hz, CH),
C15H15N5O4: C, 54.71; H, 4.59; N, 21.27. Found: C, 54.68; H, 4.61;
N, 21.32.
4.1.5. 5-(2,3,4-Trimethoxybenzylideneamino)-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5b)
A solution of 2 (30 mg, 2 mmol, 1 equiv) in methanol (5 mL)
and 2,3,4-trimethoxy benzaldehyde (54 mg, 1.3 equiv) was re-
fluxed for 4 h in 10 mL round bottom flask. The reaction mixture

A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
5789
7.40 (s, 1H, CH), 8.26 (s, 1H, CH), 8.43 (s, 1H, N–CH), 8.85 (s, 1H,
N@CH). Anal. Calcd for C13H11N5O3: C, 54.74; H, 3.89; N, 24.55.
Found: C, 54.80; H, 3.92; N, 24.58.
triethylorthoformate was added and the reaction mixture was
refluxed for 4 h (TLC). The crude compound obtained was purified
by column chromatography to give 9c using ethyl acetate:hexane
(1:2) as eluents. Yield: 58%, Creamish white solid; mp: 226–
4.1.9. 5-((Benzo[d][1,3]dioxol-6-yl)methyleneamino)-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5f)
230 °C; IR (KBr): 3442, 3114, 2224, 1694, 1585 cmꢂ1 1H NMR
;
(400 MHz, DMSO-d6): d = 5.52 (s, 2H, CH2), 7.37 (m, 5H, CH), 8.02
(d, 2H, J = 7.8 Hz, CH), 8.08 (d, 2H, J = 7.8 Hz, CH), 8.46 (s, 1H, CH),
8.87 (s, 1H, N–CH), 9.39 (s, 1H, N@CH); 13C NMR (100 MHz,
DMSO-d6): 56.76, 107.07, 128.52, 128.61, 129.14, 129.44, 131.02,
133.40, 136.52, 137.51, 148.36, 155.81, 162.08; MS (APCI): m/
z = 355.27 [M+1]+, m/z = 227.53 [M+1]+ a fragment peak. Anal.
Calcd for C20H14N6O: C, 67.79; H, 3.98; N, 23.72. Found: C, 67.87;
H, 3.93; N, 23.77.
A solution of 2 (30 mg, 2 mmol, 1 equiv) in methanol (5 mL) and
piperonal (38 mg, 1.2 equiv) was refluxed for 4 h in 10 mL round
bottom flask. The reaction mixture was then dried to remove
methanol under vacuum to give 4. Then 2 mL of triethylorthofor-
mate was added and the reaction mixture was refluxed for 6 h
(TLC). The crude compound obtained was purified by column chro-
matography to give 5f using ethyl acetate:hexane (1:3) as eluents.
Yield: 71%, white solid; mp: 251–252 °C; IR (KBr): 3435, 3190,
1718, 1576 cmꢂ1
;
1H NMR (400 MHz, DMSO-d6): d = 6.14 (s, 2H,
4.1.13. 5-(3-Methoxy-4-hydroxybenzylideneamino)-1-benzyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9d)
O–CH2–O), 7.10 (d, 1H, J = 7.45, CH), 7.40 (d, 1H, J = 7.45, CH),
7.49 (s, 1H, CH), 8.28 (s, 1H, CH), 8.43 (s, 1H, N–CH), 8.92 (s, 1H,
N@CH); 13C NMR (100 MHz, DMSO-d6): d = 101.92, 105.27,
105.89, 108.58, 126.43, 126.56, 147.40, 148.04, 151.07, 166.25;
MS (APCI): m/z = 284.13 [M+1]+, m/z = 164.33 [M+1]+ a fragment
peak. Anal. Calcd for C13H9N5O3: C, 55.13; H, 3.20; N, 24.73. Found:
C, 55.18; H, 3.25; N, 24.79.
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
3-methoxy-4-hydroxy benzaldehyde (26 mg, 1.3 equiv) was re-
fluxed for 4 h in 10 mL round bottom flask. The reaction mixture
was then dried to remove all ethanol under vacuum to give 8. Then
2 mL of triethylorthoformate was added and the reaction mixture
was refluxed for 4 h (TLC). The crude compound obtained was
purified by column chromatography to give 9d using ethyl
acetate:hexane (1:2) as eluents. Yield: 60%, Yellowish white solid;
4.1.10. 1-Benzyl-5-(benzylideneamino)-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (9a)
mp: 199–201 °C; IR (KBr): 3263, 1686, 1595 cmꢂ1
;
1H NMR
A solution of 7 (200 mg, 8 mmol, 1 equiv) in ethanol (5 mL)
and benzaldehyde (136 mg, 1.5 equiv) was refluxed for 4 h in
50 mL round bottom flask. The reaction mixture was then dried
to remove all ethanol under vacuum to give 8. Then 2 mL of tri-
ethylorthoformate was added and the reaction mixture was re-
fluxed for 4 h. White crystals obtained on cooling were filtered
off, washed with diethyl ether and dried to afford 9a. Yield:
79%, white solid; mp: 207–209 °C; IR (KBr): 3425, 3106, 1683,
(400 MHz, CDCl3): d = 3.96 (s, 3H, –OCH3), 5.45 (s, 2H, CH2), 6.91
(d, 1H, J = 8.16 Hz, CH), 7.21 (m, 1H, CH), 7.38 (m, 5H, CH), 7.50
(s, 1H, CH), 8.05 (s, 1H, CH), 8.26 (s, 1H, N–CH), 9.14 (s, 1H, N@CH);
13C NMR (100 MHz, CDCl3): 56.10, 57.75, 107.90, 108.25, 114.48,
125.29, 125.35, 128.58, 128.63, 128.93, 129.15, 134.27, 147.12,
148.65, 149.80, 156.49, 157.40, 163.66; MS (APCI): m/z = 376.13
[M+1]+, m/z = 227.40 [M+1]+ a fragment peak. Anal. Calcd for
C20H17N5O3: C, 63.99; H, 4.56; N, 18.66. Found: C, 63.83; H, 4.66;
1591 cmꢂ1
;
1H NMR (400 MHz, CDCl3): d = 5.45 (s, 2H, CH2),
N, 18.69.
7.82 (m, 2H), 7.52 (m, 3H), 7.38 (m, 5H), 8.05 (s, 1H, CH), 8.29
(s, 1H, N–CH), 9.47 (s, 1H, N@CH); 13C NMR (100 MHz, CDCl3):
d = 57.75, 107.91, 128.50, 128.56, 128.76, 128.87, 128.92,
129.15, 132.02, 133.19, 134.29, 149.21, 156.77, 157.28, 162.36;
MS (APCI): m/z = 330.20 [M+1]+, m/z = 227.47 [M+1]+ a fragment
peak. Anal. Calcd for C19H15N5O: Calcd C, 69.29; H, 4.59; N,
21.26. Found: C, 69.35; H, 4.63; N, 21.24.
4.1.14. 5-(3-Hydroxy-4-methoxybenzylideneamino)-1-benzyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9e)
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
3-hydroxy-4-methoxy benzaldehyde (26 mg, 1.3 equiv) was re-
fluxed for 4 h in 10 mL round bottom flask. The reaction mixture
was then dried to remove all ethanol under vacuum to give 8.
Then 2 mL of triethylorthoformate was added and the reaction
mixture was refluxed for 4 h (TLC). The crude compound obtained
was purified by column chromatography to give 9e using ethyl
acetate:hexane (1:2) as eluents. Yield: 65%, brown solid; mp:
4.1.11. 5-(4-Chlorobenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9b)
A mixture of 7 (200 mg) in ethanol (5 mL) and 4-chloro benzal-
dehyde (157 mg, 1.3 equiv) was refluxed for 4 h in 10 mL. round
bottom flask. The reaction mixture was dried to remove all ethanol
under vacuum to give 8. Crude product was dissolved in 2 mL ace-
tonitrile and 2 mL of triethylorthoformate and the reaction mixture
was refluxed for 4 h (TLC). White crystals obtained on cooling were
filtered, washed with diethyl ether and dried to afford 9b. Yield:
78%; brownish solid; Molecular formula: C19H14ClN5O; Molecular
weight: 363; mp: 240–241 °C; IR (KBr, cmꢂ1): 1720, 1635, 1610,
740; 1H NMR (300 MHz, CDCl3): d = 5.46 (s, 2H, CH2), 7.39 (m,
5H, CH), 7.44 (d, 2H, J = 7.8, CH), 7.77 (d, 2H, J = 7.5, CH), 8.05 (s,
1H, CH), 8.29 (s, 1H, N–CH), 9.53 (s, 1H, N@CH); MS (APCI): m/
z = 364 [M+1]+, m/z = 227.47 [M+1]+ a fragment peak. Anal. Calcd
for C19H14ClN5O: Calcd C, 62.73; H, 3.88; N, 19.25. Found C,
62.80; H, 3.86; N, 19.28.
208–210 °C; IR (KBr): 3453, 1685, 1596 cmꢂ1
;
1H NMR
(400 MHz, CDCl3): d = 3.96 (s, 3H, –OCH3), 5.45 (s, 2H, CH2),
5.81 (s, 1H, D2O exchangeable OH), 6.91 (d, 1H, J = 8.2 Hz, CH),
7.28 (d, 1H, J = 8.2 Hz, CH), 7.38–7.36 (m, 5H, CH), 7.50 (s, 1H,
CH), 8.04 (s, 1H, CH), 8.26 (s, 1H, N–CH), 9.24 (s, 1H, N@CH);
13C NMR (100 MHz, CDCl3): 57.06, 57.75, 107.95, 110.33, 113.04,
122.86, 126.58, 128.57, 128.63, 128.92, 129.15, 134.29, 146.06,
149.03, 150.01, 156.67, 157.37, 162.66; MS (APCI): m/z = 376.20
[M+1]+, m/z = 227.47 [M+1]+ a fragment peak. Anal. Calcd forC20-
H17N5O3: C, 63.99; H, 4.56; N, 18.66. Found: C, 63.85; H, 4.60;
N, 18.61.
4.1.15. 5-((Benzo[d][1,3]dioxol-6-yl)methyleneamino)-1-
benzyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9f)
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
piperonal (23 mg, 1.2 equiv) was refluxed for 4 h in 10 mL round
bottom flask. The reaction mixture was then dried to remove all
ethanol under vacuum to give 8. Then 2 mL of triethylorthoformate
was added and the reaction mixture was refluxed for 8 h. White
crystals obtained on cooling were filtered off, washed with diethyl
ether and dried to afford 9f. Yield: 68%, white solid; mp: 241–
4.1.12. 5-(4-Cyanobenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9c)
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
4-cyano benzaldehyde (21 mg, 1.2 equiv) was refluxed for 4 h in
10 mL round bottom flask. The reaction mixture was then dried
to remove all ethanol under vacuum to give 8. Then 2 mL of

5790
A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
243 °C; IR (KBr): 3053, 1687, 1596 cmꢂ1
;
1H NMR (400 MHz,
5H, CH), 7.52 (s, 1H, CH), 8.04 (s, 1H, CH), 8.27 (s, 1H, N–CH),
DMSO-d6): d = 5.51 (s, 2H, CH2), 6.15 (s, 2H, –O–CH2–O), 7.08 (d,
1H, J = 7.84 Hz, CH), 7.38–7.36 (m, 6H, CH), 7.46 (s, 1H, CH), 8.37
(s, 1H, CH), 8.82 (s, 1H, N–CH), 8.98 (s, 1H, N@CH); 13C NMR
(100 MHz, DMSO-d6): 56.72, 102.43, 106.26, 107.18, 109.08,
126.77, 127.27, 128.50, 128.59, 129.13, 130.61, 136.58, 148.00,
148.59, 151.43, 155.55, 157.64, 165.51; MS (APCI): m/z = 374.27
[M+1]+, m/z = 227.47 [M+1]+ a fragment peak. Anal. Calcd for
9.21 (s, 1H, N@CH); 13C NMR (100 MHz, CDCl3): d = 107.92,
108.50, 110.61, 124.68, 125. 82, 128.56, 128.90, 129.12, 134.29,
148.74, 149.51, 152.72, 156.62, 157.40, 163.21; MS (APCI): m/
z = 390.27 [M+1]+, m/z = 227.47 [M+1]+ a fragment peak. Anal.
Calcd for C21H19N5O3: C, 64.77; H, 4.92; N, 17.98. Found: C,
64.72; H, 4.98; N, 18.10.
C
20H15N5O3: C, 64.34; H, 4.05; N, 18.76. Found: C, 64.31; H, 4.09;
4.1.19. 5-(2,4,5-Trimethoxybenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9j)
N, 18.81.
For the synthesis of compound-5, mixture of 2 (200 mg) in eth-
anol (5 mL) and 2,4,5-trimethoxy benzaldehyde (168 mg,
1.3 equiv) was refluxed for 4 h in 10 mL. round bottom flask. The
reaction mixture was dried to remove all ethanol under vacuum
to give 8. Crude product was dissolved in 2 mL acetonitrile and
2 mL of triethylorthoformate and the reaction mixture was re-
fluxed for 4 h (TLC). White crystals obtained on cooling were fil-
tered, washed with diethyl ether and dried to afford 9j. Yield:
70%; color: white solid; mp: 228–230 °C; IR (KBr, cmꢂ1): 1710,
1635, and 1620; 1H NMR (300 MHz, CDCl3): d = 3.86 (s, 3H, –
OCH3), 3.92 (s, 3H, –OCH3), 3.96 (s, 3H, –OCH3), 5.45 (s, 2H, CH2),
6.49 (s, 1H, CH), 6.91 (s, 1H, CH), 7.37 (7.36–7.38, m, 5H, CH),
8.23 (s, 1H, CH), 8.45 (s, 1H, N–CH), 9.35 (s, 1H, N@CH); MS (APCI):
m/z = 420 [M+1]+ m/z = 227.47 [M+1]+ a fragment peak. Anal. Calcd
forC22H21N5O4: C, 63.00; H, 5.05; N, 16.70. Found: C, 63.09; H, 5.03;
N, 16.68.
4.1.16. 5-(3,4,5-Trimethoxybenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9g)
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
3,4,5-trimethoxy benzaldehyde (32 mg, 1.3 equiv) was refluxed
for 4 h in 10 mL round bottom flask. The reaction mixture was then
dried to remove all ethanol under vacuumto give 8. Then 2 mL of
triethylorthoformate was added and the reaction mixture was re-
fluxed for 4 h (TLC). The crude compound obtained was purified
by column chromatography to give 9g using ethyl acetate:hexane
(1:2) as eluents. Yield: 75%, white solid; mp: 191–194 °C; IR
(KBr): 3434, 1693, 1609, 1591 cmꢂ1 1H NMR (400 MHz, CDCl3):
;
d = 3.92 (s, 9H, –OCH3), 5.45 (s, 2H, CH2), 7.08 (s, 2H, CH), 7.38–
7.36 (m, 5H, CH), 8.05 (s, 1H, CH), 8.29 (s, 1H, N–CH), 9.32 (s, 1H,
N@CH); 13C NMR (100 MHz, CDCl3): 56.26, 57.77, 61.02, 105.59,
107.86, 128.29, 128.58, 128.75, 128.95, 129.16, 134.26, 141.50,
149.00, 153.53, 156.69, 157.27, 162.46;MS (APCI): m/z = 420.13
[M+1]+, m/z = 227.40 [M+1]+ a fragment peak. Anal. Calcd for
4.1.20. 5-((Furan-2-yl)methyleneamino)-1-benzyl-1H-
C22H21N5O4: C, 63.00; H, 5.05; N, 16.70. Found: C, 63.08; H, 5.10;
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9k)
N, 16.72.
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
2-furaldehyde (15 mg, 1.2 equiv) was refluxed for 4 h in 10 mL
round bottom flask. The reaction mixture was then dried to remove
all ethanol under vacuum to give 8. Then 2 mL of triethylorthofor-
mate was added and the reaction mixture was refluxed for 4 h
(TLC). The crude compound obtained was purified by column chro-
matography to give 9k using ethyl acetate:hexane (1:2) as eluents.
Yield: 55%, brown solid; mp: 158–161 °C; IR (KBr): 3427, 3092,
4.1.17. 5-(2,3,4-Trimethoxybenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9h)
A solution of 7 (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
2,3,4-trimethoxy benzaldehyde (32 mg, 1.3 equiv) was refluxed
for 4 h in 10 mL round bottom flask. The reaction mixture was
then dried to remove all ethanol under vacuumto give 8. Then
2 mL of triethylorthoformate was added and the reaction mixture
was refluxed for 4 h (TLC). The crude compound obtained was
purified by column chromatography to give 9h using ethyl ace-
tate:hexane (1:2) as eluents. Yield: 73%, Yellowish white solid;
1692, 1607 cmꢂ1 1H NMR (400 MHz, CDCl3): d = 5.45 (s, 2H,
;
CH2), 6.34 (m, 1H, CH), 7.07 (m, 1H, CH), 7.38–7.36 (m, 5H, CH),
7.69 (m, 1H, CH), 8.21 (s, 1H, CH), 8.40 (s, 1H, N–CH), 9.42 (s, 1H,
N@CH); 13C NMR (100 MHz, CDCl3): 112.41, 117.94, 128.58,
128.76, 128.96, 129.17, 146.33, 149.31, 150.24; MS (APCI): m/
z = 320.20 [M+1]+, m/z = 227.40 [M+1]+ a fragment peak. Anal.
Calcd for C17H13N5O2: C, 63.94; H, 4.10; N, 21.93. Found: C,
63.97; H, 4.13; N, 21.97.
mp: 155–157 °C; IR (KBr): 3433, 3069, 1694, 1592 cmꢂ1
;
1H
NMR (400 MHz, CDCl3): d = 3.88 (s, 3H, –OCH3), 3.93 (s, 3H, –
OCH3), 3.96 (s, 3H, –OCH3), 5.45 (s, 2H, CH2), 6.78 (d, 1H,
J = 8.9 Hz, CH), 7.37–7.34 (m, 5H, CH), 7.84 (d, 1H, J = 8.9 Hz,
CH), 8.05 (s, 1H, CH), 8.24 (s, 1H, N–CH), 9.37 (s, 1H, N@CH);
13C NMR (100 MHz, CDCl3): 56.16, 57.72, 60.97, 62.14, 107.89,
107.92, 119.27, 122.11, 128.58, 128.89, 129.13, 134.30, 141.86,
148.44, 154.67, 156.29, 157.35, 157.51, 160.51; MS (APCI): m/z =
420.20 [M+1]+, m/z = 227.53 [M+1]+ a fragment peak. Anal. Calcd
for C22H21N5O4: C, 63.00; H, 5.05; N, 16.70. Found; C, 63.04; H,
5.11; N, 16.76.
4.1.21. Synthesis of 9l
A solution of 7 (400 mg, 1 mmol, 1 equiv) in triethylorthofor-
mate (2 mL) was refluxed for 12 h (TLC). The crude compound ob-
tained was purified by column chromatography to give 9l using
ethyl acetate:hexane (1:5) as eluents. Yield: 57%, Creamish white
solid; mp: 201–203 °C; IR (KBr): 1703, 1617, 1584 cmꢂ1 1H NMR
;
(400 MHz, DMSO-d6): d = 1.34 (t, J = 7.04 Hz, 3H, CH3), 4.32 (q,
J = 6.99 Hz, 2H, OCH2), 5.49 (s, 2H, CH2), 7.36–7.32 (m, 5H), 8.15
(s, 1H, CH), 8.50 (s, 1H, N–CH), 8.78 (s, 1H, N@CH); 13C NMR
(100 MHz, DMSO-d6): d = 14.40, 56.67, 64.64, 107.16, 128.43,
128.52, 129.10, 130.10, 136.17, 148.08, 155.55, 157.93, 168.74;
MS (APCI): m/z = 298.27 [M+1]+, m/z = 227.47 [M+1]+ a fragment
peak. Anal. Calcd for C15H15N5O2: C, 60.60; H, 5.09; N, 23.56.
Found: C, 60.68; H, 5.23; N, 23.53.
4.1.18. 5-(3,4-Dimethoxybenzylideneamino)-1-benzyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (9i)
A solution of 7 (40 mg, 1 mmol, 1 equiv) in ethanol (5 mL) and
3,4-dimethoxy benzaldehyde (43 mg, 1.5 equiv) was refluxed for
4 h in 10 mL round bottom flask. The reaction mixture was then
dried to remove all ethanol under vacuum to give 8. Then 2 ml of
triethylorthoformate was added and the reaction mixture was re-
fluxed for 4 h (TLC). The crude compound obtained was purified
by column chromatography to give 9i using ethyl acetate:hexane
(1:2) as eluents. Yield: 76%, white solid; mp: 209–211 °C; IR
4.1.22. Synthesis of 5-amino-1,5-dihydro-1-benzyl-
pyrazolo[3,4-d]pyrimidin-4-one (9m)
A solution of 7 (400 mg, 1 mmol, 1 equiv) in acetonitrile (2 mL)
and triethylorthoformate (1 mL) was refluxed for 7 h. One drop of
H2SO4 was also added. White crystals obtained on cooling were
(KBr): 3425, 3106, 1683, 1591 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
d = 3.95 (s, 3H, –OCH3), 3.96 (s, 3H, –OCH3), 5.45 (s, 2H, CH2),
6.91 (d, 1H, J = 8.4 Hz, CH), 7.27 (d, 1H, J = 8.4 Hz, CH), 7.38 (m,

A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
5791
filtered off, washed with diethyl ether and dried to afford 9m.
Yield: 54%, white solid; mp: 184–186 °C; IR (KBr): 3328, 1697,
the mouth was rinsed atleast 2 h before with mouthwash. Cells
were harvested in PBS and washed three times with antibiotic con-
taining PBS to remove the contaminants. Cells were cultured in
1586 cmꢂ1 1H NMR (400 MHz, DMSO-d6): d = 5.47 (s, 2H, D2O
;
exchangeable NH2), 5.60 (s, 2H, CH2), 7.30–7.35 (m, 6H), 8.17 (s,
1H, CH), 8.72 (s, 1H, N–CH); 13C NMR (100 MHz, DMSO-d6):
d = 56.65, 106.67, 128.42, 128.51, 129.09, 129.56, 136.67, 150.91,
158.31, 158.59. Anal. Calcd for C12H11N5O: C, 59.74; H, 4.60; N,
29.03. Found: C, 59.58; H, 4.30; N, 29.21.
DMEM media with 10% FBS, 50 U/mL penicillin G, 50
lg/mL strep-
tomycin sulfate and 1.25 g/mL amophotericin B (fungizone). The
l
cells were incubated at 37 °C with 5% CO2 and 95% humidity con-
ditions. For experiments, cells were seeded in equal numbers after
trypan blue cell counting (5000 cells per well of 96-well plate and
100,000 cells per well of 6 well plate). Afterwards cells were
washed once with sterile 1ꢁ PBS and cultured with serum free
media for 8 h for synchronization. The compounds as well as eto-
poside (positive control) were dissolved in cell culture grade
DMSOupto concentration of 100 mM and further dilutions were
done in serum free DMEM media. The total amount of media per
4.1.23. Synthesis of 5-(4-methoxybenzylideneamino)-1-benzyl-
6,7-dihydro-6-(4-methoxyphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (9n)
A mixture of 7 (200 mg) in ethanol (5 mL) and anisaldehyde
(1.6 mL, 1.3 equiv) was refluxed for 4 h in 10 mL round bottom
flask. White crystals obtained on cooling were filtered, washed
with diethyl ether and dried to afford 9n. Yield: 70%; color: white
solid; mp: 161–163 °C; IR (KBr, cmꢂ1): 3200, 1730, and 1635; 1H
NMR (300 MHz, CDCl3): d = 3.84 (s, 3H, –OCH3), 3.92 (s, 3H, –
OCH3), 5.27 (s, 2H, CH2), 6.92 (d, 2H, J = 8.1 Hz, CH), 7.05 (d, 2H,
J = 7.8, CH), 7.26 (s, 1H, CH), 7.37 (m, 5H,CH), 7.74 (d, 2H,
J = 8.1 Hz, CH), 7.90 (s, 2H, J = 7.8 Hz, CH), 8.11 (s, 1H, CH), 9.06
(s, 1H, N@CH), 11.53 (s, D2O Exchangeable proton NH); MS (APCI):
m/z = 468 [M+1]+.Anal. Calcd for C27H25N5O3: C, 69.36; H, 5.39; N,
14.98. Found; C, 69.41; H, 5.63; N, 14.84.
well (200 lL per well of 96 well plate and 2 mL per well for six well
plates) was kept constant and all the treatment volumes were
accommodated within these ranges only.
4.2.2. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay
MTT assay was carried out in 96-well plates where total volume
of media was 200
l
l/well. Briefly cells after the treatments were
l/mL well of
washed with 1ꢁ PBS and were mixed with 100
l
MTT (5 mg in 10 mL of 1ꢁ PBS) and incubated at room temperature
in dark for 4 h to allow formation of formazan crystals. Each well
4.1.24. Synthesis of 5-(2-chlorobenzylideneamino)-1-benzyl-
6,7-dihydro-6-(2-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (9o)
was then mixed with 100 ll of DMSO to dissolve the crystals fol-
lowed by ELISA readings at 570 nm. The results were then plotted
in graphs to calculate IC50 and dose dependent response to the
treatment.
A mixture of 7 (200 mg) in ethanol (5 mL) and 2-chlorobenzal-
dehyde (1.57 mL, 1.3 equiv) was refluxed for 4 h in 10 mL round
bottom flask. White crystals obtained on cooling were filtered,
washed with diethyl ether and dried to afford 9o. Yield: 76%; color:
white solid; mp: 231–233 °C; IR (KBr, cmꢂ1): 3100, 1720, and
1630; 1H NMR (300 MHz, DMSO-d6): d = 5.18 (s, 2H, CH2), 7.38
(m, 12H, CH), 7.45 (m, 2H, CH), 8.97 (s, 1H, CH), 9.36 (1H, N@CH),
11.63 (s, 1H, D2O exchangeable proton NH); MS (APCI): m/z = 477
[M+1]+. Anal. Calcd forC27H25N5O3: C, 69.36; H, 5.39; N, 14.98.
Found: C, 69.26; H, 5.32; N, 14.79.
4.2.3. Analysis of total reactive oxygen species
Free radicals were measured using DCFDA dye (Sigma) in two
different ways; using fluorescence microscopy and spectropho-
tometry. For this assay cells were treated for short periods (2 h).
For microscopy cells were seeded in 6-well plate on sterile cover
slips at a density of 100,000 cells per well followed by described
treatments. Afterwards cells were incubated with DCFDA dye at
37 °C for 30 min in dark followed by fixation in 1% paraformalde-
hyde. The cover slips were then mounted upside down on slides
using mounting media and observed under fluorescent microscope
(Olympus).34 For spectrophotometric analysis, cells were taken
into solution using trypsin and washed with 1ꢁ PBS. Staining
was done by incubating the cells with respective dye in 1ꢁ PBS
on ice for 20 min. Cells were than washed three times with 1ꢁ cold
PBS and OD was taken in spectrophotometer. The measurement
parameters are: for DCFDA, 488 nm excitation and 540 nm
emission.34
4.1.25. 1-Benzyl-5-(benzylamino)-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-one (9p)
To a solution of 9a (30 mg, 1 mmol, 1 equiv) in ethanol (5 mL)
was added sodium borohydride (2 equiv). The reaction mixture
was stirred at rt for 4 h (TLC). The crude compound obtained was
purified by column chromatography to give 9p using ethyl ace-
tate:hexane (1:1) as eluents. Yield: 50%, semi-solid; IR (KBr):
3476, 2923, 2853, 1726, 1636 cmꢂ1 1H NMR (300 MHz, CDCl3):
;
d = 4.11 (s, 2H, CH2), 5.46 (s, 2H, CH2), 6.84 (s, 1H, D2O exchange-
able proton NH), 7.29–7.33 (m, 10H, CH), 7.84 (s, 1H, N@CH); 13C
NMR (75 MHz, CDCl3): 54.42, 56.68, 106.99, 127.99, 128.55,
128.84, 129.11, 129.36, 129.60, 129.92, 136.61, 137.51, 151.20,
157.77, 158.28; MS (APCI): m/z = 332.33 [M+1]+. Anal. Calcd for
4.2.4. Cell cycle analysis
Cells were resuspended in 500
glucose) followed by addition of 5 mL of chilled 70% ethanol and
l
l of ice cold 1ꢁ PBS (with 0.1%
incubated for 30 min on ice. Cells were than washed twice with
C
19H17N5O: C, 68.87; H, 5.17; N, 21.13. Found: C, 68.93; H, 5.23;
10 mL of ice cold PBS. Cells were then resuspended in 300
propidium iodide (Sigma, 69 M in 38 mM sodium citrate pH
7.4) followed by 20 l of RNase (10 mg/mL solution in water). Sam-
ll of
N, 21.09.
l
l
4.2. Biology
ples were then incubated at 37 °C for 45 min and observed using
FACS Calibur (BD). The analysis was done using FACS DIVA
software.
4.2.1. Cell culture and treatments
All the cell lines were procured from National cell repository
situated at NCCS, Pune. MCF7 (breast adenocarcinoma), PC3 (pros-
tate), H460 (liver), THP-1 (leukemia), IGR-OV-1 (ovarian) and HeLa
(cervix) cell lines representing different human cancers were
grown in DMEM media supplemented with 10% fetal bovine serum
(FBS) and antibiotic solution (1ꢁ Penstrip, all the reagents from
Invitrogen). Buccal cavity cells were harvested and culture accord-
ing to the earlier described protocol by Weisberg et al.32,33 Briefly
4.2.5. Single cell gel electrophoresis, SCGE or Comet assay
DNA fragmentation caused by the essential oil in the breast can-
cer cells was determined by single cell gel electrophoresis accord-
ing to the modified method of Olive and Banath.35 In brief, 1 mL
suspension of 15 ꢁ 105 cells/mL was mixed with 1 mL of 1% low-
gelling-temperature agarose at 40 °C and coated on the slides pre-
coated with 1% agarose. These were kept in the lysis solution

5792
A. T. Baviskar et al. / Bioorg. Med. Chem. 21 (2013) 5782–5793
(1.2 M NaCl, 100 mM Na2EDTA, 0.1% SDS, 0.26 M NaOHwith pH
>13) overnight at 4 °C in dark. Rinsed with rinsing solution
(0.03 M NaOH, 2 mM Na2EDTA with pH 12.3) 2–3 times and sub-
merged in the same solution in the Comet Assay Electrophoresis
unit (Sci. Plas, UK). Electrophoresis was done in rinse solution for
30 min at a voltage of 15 V and 40 mA current. Cells were analyzed
by examining at 50 comet images from each slide under fluores-
cent microscope (Olympus Magnus). Scoring of the comets ob-
tained was done by using Cometscore15 software Version 1.0.1.0.
further incubated at 37 °C for 15 min. 1% Agarose gel was run until
the dye front was at the bottom of the gel. Gel were then stained
with ethidium bromide and destained with water for 20 min and
photo documented the results using QuantityOne (BioRad).
4.3. Molecular docking
Three dimensional (3D) X-ray structures of human topoisomer-
ase I (PDB entry: 1K4T;38 resolution: 2.10 Å) and the ATPase do-
main of human topoisomerase II
resolution: 1.86 Å) were procured from protein data bank. The
topoisomerase II (1ZXM) is a homodimer consisting of two iden-
-imidodi-
a
(PDB entry: 1ZXM;29
4.2.6. Topoisomerase assays
All the reagents required for the testing of new chemical enti-
ties were purchased from TopoGEN, Inc. (Columbus, OH). The test-
ing of the compounds was performed using a commercially
available topoisomerase II drug screening kit. All the synthesized
compounds and etoposide were dissolved in DMSO at a concentra-
tion of 1 mM as a stocked solution and stored at ꢂ20 °C. TopoI and
II inhibition assay were performed as described in the supplier
manual with minor modifications as per the requirement.
a
tical subunits, each contains AMPPNP (50-adenylyl-b,
c
phosphate) and Mg2+ ion at the ATP binding pocket. The
structure of 9d was drawn in MOE2011.10 and energy minimized
using the MMFF94x force field, to a gradient less than 0.001 kcal/
mol.39 A flexible docking was carried out using GOLD 5.0.130 in
the active site of ATPase domain of hTopoIIa. The GOLD performs
genetic algorithm based ligand docking to optimize the conforma-
tion of ligand at the receptor binding site. It utilizes Gold Score fit-
ness function to evaluate the various conformations of ligand at the
binding site and comprises of four components: protein–ligand
hydrogen bond energy, protein–ligand van der Waals (vdw) en-
ergy, ligand internal vdw energy, and ligand torsional strain en-
ergy. The compound was docked ten times and each pose was
ranked according to the Gold Score. To validate the adopted dock-
ing procedure, the co-crystallized structure of AMPPNP was ex-
tracted from the pdb file (1ZXM)29 and further docked at the
4.2.7. hTopoIIa mediated DNA decatenation assay
hTopoII mediated DNA inhibition activity for the synthesized
a
compounds were performed as follow. Reaction mixture contain-
ing freshly prepared 5ꢁ complete reaction assay buffer (buffer
(A) 0.5 M Tris–HCl (pH 8), 1.50 M sodium chloride, 100 mM mag-
nesium chloride, 5 mM dithiothreitol, 300
albumin/mL; buffer (B) 20 mM ATP in water), 150 ng catenated
kDNA (substrate), 100 M drug or test compound dissolved in
lg of bovine serum
l
DMSO followed by 2–4 units of purified human topoisomerase II
were incubated at 37 °C for 30 min. The reaction was then termi-
nated with addition of 10% SDS, followed by digestion with pro-
teinase K. Further the reaction mixture was again incubated at
ATPase binding site of topoisomerases IIa. The RMSD value of
0.432 Å between co-crystalized and docked conformation of AMP-
PNP validates and ensures the reliability of the adopted docking
procedure.
37 °C for 15 min. 20
agarose gel electrophoresis in Tris–acetate–EDTA (TAE) buffer con-
taining 0.5 g/mL ethidium bromide and further distained with
ll of each sample were then subjected to 1%
Acknowledgments
l
water for 20 min. The bands were analyzed under UV trans-illumi-
nator and the decatenated kDNA products were quantified with
QuantityOne (BioRad).
R.K., S.S., and S.K. thank Vice Chancellor, Central University of
Punjab for providing the financial support and infrastructure to
peruse in vitro screening of the compounds on cell lines. A.T.B.
and U.C.B. gratefully acknowledge the financial aid from DBT Gov-
ernment of India, New Delhi to carry out hTopoI and hTopoII eval-
uation studies. Authors also thank Professor K.L. Dhar for his
feedbacks in the synthetic part of the manuscript.
4.2.8. TopoIIa mediated DNA relaxation assay
Supercoiled plasmid DNA (pRYG)36,37 was used as substrate in
TopoII mediated DNA relaxation assay. Reaction mixture contained
freshly prepared 5x complete buffer ((A) 0.5M Tris–HCl (pH 8),
1.50 M sodium chloride, 100 mM magnesium chloride, 5 mM dithi-
Supplementary data
othreitol, 300
ATP in water), 250 ng of supercoiled plasmid DNA (pRYG), followed
by either test compound or standard drug (100 M) and finally
topoisomerase II (2–4 units) in a total of 20 L. Reaction mixture
lg of bovine serum albumin/mL; buffer (B) 20 mM
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.07.016.
l
l
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. Electro-
phoresis of the each sample was carried out in a 1% agarose gel in
TAE buffer without ethidium bromide. Gel was stained with ethi-
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