Synthesis and in vitro cytotoxic activity of novel pyrazolo[3,4-d] pyrimidines and related pyrazole hydrazones toward breast adenocarcinoma MCF-7 cell line

Article abstract of DOI:10.1016/j.bmc.2011.09.036

New series of pyrazolo[3,4-d]pyrimidines (7a-e and 13a-d) and pyrazole hydrazones 17a-d were synthesized and evaluated for their antiproliferative activity against human breast adenocarcinoma MCF-7 cell line. Most of the tested compounds exploited potent

Full text of DOI:10.1016/j.bmc.2011.09.036

Bioorganic & Medicinal Chemistry 19 (2011) 6808–6817
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro cytotoxic activity of novel pyrazolo[3,4-d]pyrimidines and
related pyrazole hydrazones toward breast adenocarcinoma MCF-7 cell line
a
c
Ghaneya Sayed Hassan a, Hanan Hassan Kadry b, , Sahar Mahmoud Abou-Seri , Mamdouh Moawad Ali ,
⇑
Abeer Essam El-Din Mahmoud c
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, PO Box 11562, Egypt
b Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Biochemistry Department, Division of Genetic Engineering and Biotechnology, National Research Centre, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2011
Revised 12 September 2011
Accepted 20 September 2011
Available online 24 September 2011
New series of pyrazolo[3,4-d]pyrimidines (7a–e and 13a–d) and pyrazole hydrazones 17a–d were syn-
thesized and evaluated for their antiproliferative activity against human breast adenocarcinoma MCF-7
cell line. Most of the tested compounds exploited potent to moderate growth inhibitory activity, in par-
ticular compound 7e exhibited superior potency to the reference drug cisplatin (IC50 = 7.60 and 13.29 lM,
respectively). The antitumor activity of the new compounds was accompanied by significant increase in
the activity of superoxide dismutase with concomitant decrease in the activities of catalase and glutathi-
one peroxidase and reduced glutathione level. Accordingly, the overproduction of hydrogen peroxide,
nitric oxide and other free radicals allowed reactive oxygen species (ROS)-mediated tumor cells death,
as monitored by reduction in the synthesis of protein and nucleic acids.
Keywords:
Pyrazolo[3,4-d]pyrimidine
Pyrazole
Cytotoxic activity
Oxidative stress
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
over, a number of guanylhydrazone derivatives were able to induce
apoptosis and to cause oxidative stress in HT-29 colon cancer and
Although there have been great advances in the detection and
treatment of cancer, it remains one of the greatest medical chal-
lenges, with the incidence of some malignancies continuing to
increase.1 For many tumor types, established treatments such
as cytotoxic chemotherapy and radiotherapy provide only tran-
sient therapeutic benefits despite severe side effects.2 Therefore,
the need for better treatments has stimulated research to devel-
op new efficient chemotherapeutic agents for management of
cancer.
In the past few years, several studies have been devoted to the
antiproliferative activity of aroylhydrazone derivatives. Where, a
plethora of hydrazone compounds has been reported to have
inhibitory effect on the growth of a number of tumor cells includ-
ing leukemia, colon, ovarian, renal, lung and breast cancer cell
lines.3–5 For example, a series of 2-arylamino-6-trifluoromethyl-
3-(hydrazonocarbonyl)pyridines 1 was found to have excellent
growth inhibitory activity against the full panel of human tumor
cell lines.6 In addition, aroylhydrazones of 2-phenylindole-3-car-
baldehydes were able to cause cell cycle arrest accompanied by
apoptosis in MCF-7 human breast adenocarcinoma cell line.7 More-
HL-60 leukemia cell lines.8,9
On the other hand, extensive studies have been conducted on
pyrazolo[3,4-d]pyrimidines as anticancer agents. Several members
of this family were found to induce apoptosis and/or reduce cell
proliferation in different solid tumor and leukemia cell lines.10–15
Different mechanisms account for the cytotoxic activity of this
class of compounds, where they have been reported to act as
epidermal growth factor (EGFR) inhibitors,11,16 mammalian target
of rapamycin (mTOR) inhibitors,10 Src or dual Src/Abl inhibi-
tors,17–19 cyclin dependent kinase (CDK) inhibitors,20 glycogen
synthase kinase-3b (GSK-3b) inhibitors,21,22 xanthine oxidase
inhibitors23 or through modulating oxygen stress in cancer cells.24
Stimulated by the successful applications of pyrazolo[3,4-
d]pyrimidines and aroylhydrazones as antitumor agents, our
objective was to synthesize a new class of pyrazolo[3,4-d]pyrim-
idines endowed with the structural elements of hydrazones, hop-
ing that the hybrid of these pharmacophoric features would
produce enhanced antitumor activity. During a previous study,25
6-methyl-1-phenyl-5-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimi-
din-4(5H)-one 2 was found to exploit potent cytotoxic activity
against MCF-7 cell line. Since the antitumor activity associated
with hydrazones has been attributed to the presence of the
CONH–N@CH pharmacophore with active azomethine proton,3
therefore a new series of N1-(4-chlorophenyl) pyrazolo[3,4-d]
⇑
Corresponding author. Tel.: +20 2 33144107/0101690694; fax: +20 2 2362
8426.
E-mail address: hanankadry2005@yahoo.com (H.H. Kadry).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.036

G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
6809
lizing enzymes as well as the levels of the oxidative stress param-
eters in MCF-7 cells were estimated.
N
O
N
R
N
O
N
2. Results and discussion
2.1. Chemistry
N
N
N
H
NHAr
F3C
N
2
1
The synthetic approaches adopted to obtain the target com-
pounds are outlined in Schemes 1–3. Hydrolysis of ethyl 5-ami-
339
with
R1
no-1-(4-chlorophenyl)-1H-pyrazole-4-carboxylate
R2
R3
alcoholic sodium hydroxide followed by neutralization afforded
the corresponding carboxylic acid 4,40 which was then refluxed
with acetic anhydride to give 1-(4-chlorophenyl)-6-methylpyraz-
olo[3,4-d][1,3]oxazin-4(1H)-one 5. Condensation of the pyrazolox-
azine 5 with hydrazine hydrate (99%) afforded the 5-amino
pyrazolo[3,4-d]pyrimidine derivative 6. The latter compound was
reacted with certain substituted aromatic aldehydes to produce
the corresponding Schiff’s bases 7a–e (Scheme 1). IR spectra of
compounds 7a–e showed disappearance of absorption bands for
(NH2). Also, 1H NMR spectra revealed singlet signal resonating at
d 8.62–10.04 ppm for (N@CH) proton. According to literature,41
the presence of singlet downfield (CH@N) signal accounts for for-
mation of E-isomers exclusively.
O
N
R1
N
R2
R3
N
O
N
N
N
X
N
N
HN
H
NHCOCH3
7a-e
13a-d
X= ---
X= SO2
17a-d
Cl
Figure 1. Strategies for the design of the newly synthesized compounds.
pyrimidine hydrazones 7a–e was designed in which the 5-quin-
oline ring in compound 2 was replaced with 5-arylideneamino
moiety (Fig. 1). Furthermore, arylsulfonylimidazolidinone26,27
and arylsufonylpyrazole28 derivatives were claimed to exhibit
excellent activity against a broad spectrum of human tumor cell
lines. Accordingly, a second series of N1-(4-chlorophenylsulfo-
nyl)pyrazolo[3,4-d]pyrimidine hydrazones 13a–d was synthe-
sized to explore the influence of incorporating sulfonyl group
on the antitumor activity. In an attempt to prepare the N1-phen-
ylsulfonyl and N1-4-methylphenylsulfonyl analogues of 13a–d,
only the 4-(arylidene)hydrazinecarbonylpyrazole congeners
17a–d were obtained. The cytotoxic activity of the produced pyr-
azoles 17a–d was evaluated due to structural similarity with the
lead compound 1 (Fig. 1).
Oxygen is the essential molecule for all aerobic organisms,
and plays predominant role in ATP generation, namely, oxidative
phosphorylation. During this process, reactive oxygen species
(ROS), such as superoxide anions (OÅ2À) and hydrogen peroxide
(H2O2) are produced as by-products,29 under physiological condi-
tions; cells have a series of defense systems to counteract these
reactive insults. Such defense systems include intracellular
superoxide dismutase (SOD) that converts OÅ2À to H2O2, catalase
(CAT) and glutathione peroxidase (GSH-Px) that eliminate H2O2,
in addition to free radical scavenging compounds like glutathi-
one (GSH).29,30 The balance of ROS formation and antioxidative
defense level is crucial to cell survival and growth.29 On the con-
trary, disturbance of this balance can produce oxidative stress.
This state of oxidative stress can result in injury to all the
important cellular components like proteins, DNA and membrane
lipids which can cause cell death.31–33
One of the principal objectives of the present work was the syn-
thesis of N1-arylsulfonylpyrazolo[3,4-d]pyrimidine derivatives
(Scheme 2). The ethyl carboxylate intermediates 10a–c were pre-
pared by the cyclocondensation of ethyl ethoxymethylenecyanoac-
etate 8 with 4-(un) substituted phenylsulfonyl hydrazines 9a–c in
refluxing acetonitrile. Reacting 10a–c with acetic anhydride
yielded the desired acetylated derivative 11 in case of 4-chloro-
phenylsulfonyl derivative only. Hydrazinolysis of 11 furnished
the respective acid hydrazide 12. Coupling of 12 with different aro-
matic aldehydes, followed by in situ intramolecular cyclization
yielded the pyrazolo[3,4-d]pyrimidine derivatives 13a–d. While,
the reaction of acid hydrazide 12 with 3,4,5-trimethoxybenzoyl
chloride afforded compound 14. The structures of 13a–d and 14
were established on the basis of their elemental analyses and spec-
tral data. IR spectra of 13a–d were characterized by the absence of
(NH and NH2) bands and the presence of two sharp peaks around
1303.88–1323.17 and 1126.43–1186.86 cmÀ1 for (SO2). Their 1H
NMR spectra showed singlet downfield (CH@N) signals and two
doublets at d 7.14–7.65 and 7.65–7.93 ppm for 4-chlorophenyl
protons. It is worthy to mention that the lack of (NH) bands in
the IR spectra and exchangeable (NH) signals in the 1H NMR
spectra asserted the production of the cyclic pyrazolo[3,4-d]
pyrimidines 13a–d. In contrast, the IR spectral data for compound
14 exhibited characteristic absorption bands for (NH) at 3317.56
cmÀ1 and (SO2) at 1327.03 and 1157.29 cmÀ1. Its 1H NMR spectrum
showed a typical AB system of 4-chlorophenyl along with single
exchangeable signal at d 10.48 ppm corresponding to the amide
NH, which affirmed cyclization.
On the other hand, the acetylation of 5-amino-N1-phenylsulfo-
nyl and 5-amino-N1-(4-methylphenylsulfonyl)pyrazoles 10a,b
produced ethyl 5-acetamido-1H-pyrazole-4-carboxylate 1542 as a
result of hydrolytic cleavage of the SO2–N bond. Treatment of the
ester 15 with hydrazine hydrate furnished the acid hydrazide 16.
Subsequent condensation of 16 with various aromatic aldehydes
in refluxing glacial acetic acid for 24 h in an attempt to obtain
the cyclized pyrazolo[3,4-d]pyrimidine derivatives was not suc-
cessful. Instead, the 4-(arylidene)hydrazinecarbonylpyrazoles
17a–d were isolated (Scheme 3). The structure of compounds
17a–d was supported by analytical and spectral data. IR spectra
of compounds 17a–d displayed no absorption bands of (SO2)
group, while those derived from (NH) and (C@O) were observed.
The 1H NMR spectra of compounds 17b–d in DMSO-d6 displayed
Tumor cells have higher levels of ROS and are frequently more
deficient in most crucial antioxidative enzymes than normal cells,
therefore they are more vulnerable to additional oxidative
stress.34,35 Accordingly, a unique antitumor strategy named ‘oxida-
tive therapy’ was developed by delivering excess oxidative stress
or disrupting antioxidative defense system in cancer cells.36,37
Many conventional anticancer drugs like camptothecin, doxorubi-
cin, cisplatin and anthracyclines exhibit antitumor activity by gen-
erating ROS.38 In addition, several pyrazolo[3,4-d]pyrimidines and
hydrazones are known to induce apoptotic death in cancer cells via
generation of reactive oxygen species.7,8,24 Hence, the effect of the
prepared compounds on the activities of the free-radical-metabo-

6810
G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
O
O
O
OH
O
OC2H5
N
N
N
N
NH2
NH2
N
N
N
ii
i
Cl
iii
Cl
Cl
5
4
3
R1
R2
R3
O
N
O
NH2
N
N
N
7a R1 =R2 = R3 = H
7b R1 = H, R2 = OCH3, R3 = H
N
N
N
N
iv
N
R
1 = OCH3, R2 = OH, R3 = H
7c
7d R1 = OCH3, R2 = OCH3, R3 = H
7e R1 =R2 = R3 = OCH3
6
Cl
7a-e
Cl
Scheme 1. Reagents and conditions: (i) NaOH, CH3OH, reflux 10 h; (ii) acetic anhydride, reflux, 4 h; (iii) NH2NH2ÁH2O, n-butanol, reflux 24 h; (iv) Ar-CHO, glacial acetic acid,
reflux 16 h.
O
OC2H5
NH2
NHNH2
O
O
S
O
N
N
O
OC2H5
N
S
i
O
C2H5O
+
9a, 10a R = H
R
8
9b, 10b
R = CH3
9a-c
9c, 10c R = Cl
R1
R
R2
10a-c
O
R3
O
O
N
NHNH2
NHCOCH3
N
OC2H5
N
O
N
N
S
N
NHCOCH3
N
O
iv
iii
N
S
N
S
O
O
O
O
Cl
Cl
Cl
13a-d
H3CO
12
OCH3
OCH3
11
H
N
O
13a R1 =R2 = R3 = H
N
13b R1 = H, R2 = OCH3, R3 = H
O
N
13c
R
1 = OCH3, R2 = OCH3, R3 = H
N
N
S
13d R1 =R2 = R3 = OCH3
O
O
Cl
14
Scheme 2. Reagents and conditions: (i) CH3CN, reflux 6 h; (ii) acetic anhydride, reflux,4 h; (iii) NH2NH2ÁH2O, n-butanol, reflux 24 h; (iv) Ar-CHO, glacial acetic acid, reflux
24 h; (v) (OCH3)3C6H2COCl, dry benzene, stir room temperature 3 h.

G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
6811
O
OC2H5
NH2
O
O
N
O
NHNH2
OC2H5
N
S
ii
i
O
N
N
NHCOCH3
NHCOCH3
N
H
N
H
15
16
iii
R
i
R1
10a, b
R2
R3
10a
R = H
10b R = CH3
O
O
N
OC2H5
NHCOCH3
N
H
N
N
H
N
NHCOCH3
N
S
O
O
17a-d
17a R1 =R2 = R3 = H
17b R1 = H, R2 = OCH3, R3 = H
17c
R
1 = OCH3, R2 = OCH3, R3 = H
17d R1 =R2 = R3 = OCH3
R
Scheme 3. Reagents and conditions: (i) acetic anhydride, reflux, 4 h; (ii) NH2NH2ÁH2O, n-butanol, reflux 24 h; (iii) Ar-CHO, glacial acetic acid, reflux 24 h.
The cytotoxic activities are expressed by median growth inhib-
itory concentration (IC50) and provided in Table 1. From the results,
it is evident that all the tested compounds displayed potent to
moderate growth inhibitory activity, in particular compound 7e
O
O
OC2H5
OC2H5
N
O
N
NHCOCH3
NHCOCH3
N
S
N
S
(IC50 = 7.60
lM) was found to be more potent and efficacious than
O
O
O
cisplatin (IC50 values 13.29
lM).
Although the number of tested compounds in this study is lim-
ited, some structural features that are important for explanation of
their cytotoxic effects can be referred. In general, the pyrazolo[3,4-
d]pyrimidine containing compounds 7a–e and 13a–d were more
potent than those possessing pyrazole ring 17a–d. Moreover, it
was envisioned that the incorporation of sulfonyl functionality be-
tween pyrazolo[3,4-d]pyrimidine scaffold and 4-chlorophenyl
moiety in 13a–d may increase the anticancer activity of com-
pounds 7a–e. Results in Table 1 illustrated that, adding sulfonyl
group had inconsistent effect on activity. While, compound 13a al-
most showed the same activity as 7a, compounds 13b–d exhibited
decline in potency compared with the congeneric compounds 7b–
e, probably due to lower stability of the pyrazole-sulfone linkage.
In addition, to verify the hypothesis that integrating an active azo-
methine proton to the pyrazolo[3,4-d]pyrimidine motif would be
profitable to the cytotoxic activity, compound 14 having an amide
linkage was prepared. As expected, the replacement of azomethine
linker in 13d by an amide linker in 14 led to significant decrease in
Cl
10c
Cl
Figure 2. Effect of electron withdrawing chlorine.
a mixture of tautomers, since the proton of cyclic NH is mobile be-
tween the two annular nitrogen atoms.42 Moreover, the 1H NMR
spectra confirmed the production of the uncyclized pyrazoles
where three pairs of exchangeable singlet signals derived from
amide (NH) at d 10.26–10.64, hydrazide (NH) at d 11.10–11.60
and pyrazole (NH) at d 12.84–13.15 were detected.
A point of interest in this investigation was the different behav-
ior of various 5-amino-N1-arylsulfonylpyrazoles 10a–c during
acetylation with acetic anhydride. The stability of N1-(4-chloro-
phenylsulfonyl)pyrazole derivative 10c to acid hydrolysis could
be attributed to the presence 4-chloro substituent. Presumably,
the presence of an electron withdrawing chlorine atom on the phe-
nyl ring draws electrons away from the sulfonyl group. Therefore
the neighboring nitrogen can feed its lone pair of electrons into
the sulfonyl to form a dipolar resonance structure (Fig. 2). The lat-
ter is resistant to nucleophilic attack as reflected by their resistance
to hydrolysis. Apparently, this resonance stabilization is not fa-
vored in case of phenyl and 4-methylphenyl analogues 10a,b.
activity (IC50 = 27.80 and 35.77 lM, respectively).
From the other point of view, it has been reported that the pres-
ence of electron donating substituent as methoxy or ethoxy group
seems to be a contributing factor for high antitumor activity.44,45
Therefore, the effect of introducing single or multiple methoxy sub-
stituents to the arylidene moiety on the activity of the synthesized
compounds has been explored. The results indicated that the posi-
tion and number of methoxy groups contributed to the growth
inhibitory activity. Increasing the number of methoxy groups on
the arylidene motif was accompanied by enhancement of antiprolif-
erative activity and reached the highest activity with the most po-
2.2. In vitro cytotoxic activity
tent trimethoxybenzylidene derivative 7e (IC50 = 7.60 lM).
The cytotoxic activity of the newly synthesized compounds was
evaluated against MCF-7 cancer cell line using Sulforhodamine B
(SRB) colorimetric assay, in comparison with cisplatin as a refer-
ence drug.43
However, compounds bearing unsubstituted benzylidene 7a, 13a,
17a demonstrated higher activity compared to those having a single
p-methoxy group 7b, 13b, 17b. This suggested that m-methoxy sub-

6812
G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
Table 1
In vitro cytotoxic activities of the synthesized compounds against MCF-7 cancer cell line
R1
R1
R2
R3
R2
R3
R1
R2
R3
O
N
R1
O
H
N
N
O
N
R2
R3
N
N
O
O
N
N
N
N
N
N
N
N
N
N
H
N
O S O
N
O S O
N
H
NHCOCH3
7a-e
13a-d
17a-d
14
Cl
Cl
Cl
Compound
R1
R2
R3
IC50 (lM)
Cisplatin
225
7a
7b
7c
—
—
H
H
—
—
H
OCH3
OH
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
H
—
—
H
H
H
H
OCH3
H
H
13.29 1.08
1.53 1.12
23.09 1.50
38.59 3.02
19.76 1.57
25.01 1.43
7.60 0.71
22.44 1.48
41.93 2.53
33.61 1.86
27.80 2.05
35.77 2.19
42.04 3.35
61.73 3.90
37.12 2.69
32.10 2.35
OCH3
7d
7e
OCH3
OCH3
H
13a
13b
13c
13d
14
17a
17b
17c
17d
H
OCH3
OCH3
OCH3
H
H
OCH3
OCH3
H
OCH3
OCH3
H
H
H
OCH3
OCH3
OCH3
OCH3
Data are expressed as means SE of three separate experiments.
stituent seems to be more favorable than p-methoxy substituent for
antitumor activity. The cytotoxic ability of this class of compounds
appears to depend on the position of methoxy group rather than
its electronic effect. This assumption could be supported by the fact
that replacing the p-methoxy in 7d by p-hydroxy in 7c—that has the
same electronic characters—was accompanied by increase in po-
It should be mentioned that, the changes in the activities of the
free-radical-metabolizing enzymes and oxidative stress parame-
ters were in the order of 7e > 7c > 7a > 13d > 17d > 7b which is in
accordance with the order of cytotoxic activity of the tested com-
pounds. The highest activity was found for the most potent antitu-
mor compound 7e, which resulted in the highest SOD activity and
H2O2 and low activities of CAT and GSH-Px as well as GSH level
than the other tested compounds. The consistency between cyto-
toxic activity and biochemical assay results indicated that the anti-
tumor effect of the present compounds may be exerted at least
partly by production of ROS.
Moreover, results in Table 3 illustrated that, treatment with
these compounds led to significant increase in the level of NO.
There is a growing body of evidence indicating that NO is able to
induce apoptosis by helping to dissipate the membrane potential
of mitochondria making it more permeable.46 In addition; the ele-
vated level of NO was accompanied with depletion in the levels of
total protein and nucleic acids compared to control. This can be ex-
plained by several cytotoxic effects that include reaction of NO
with proteins and nucleic acids. The main targets of NO in proteins
are the thiol group47 and iron of active sites.48 In the nucleus, NO
has been shown to cause gene mutation,49 to inhibit DNA repair
enzymes,50 and to mediate DNA strand breaks.51 Moreover, Bien-
venu et al.,52 reported that most chemotherapeutic agents cause
cells to over-generate ROS and thus, are capable of inducing apop-
tosis, and causing oxidative damage to DNA and proteins. The cas-
tency (IC50 = 25.01 and 19.76 lM, respectively).
2.3. Biochemical assays
To elucidate the mechanism by which the new compounds ex-
ert their antitumor activities, six compounds of various levels of
antiproliferative activity were selected to estimate their ability to
induce oxidative stress in cancer cells. We estimated the activities
of the free-radical-metabolizing enzymes including SOD, CAT and
GSH-Px as well as the levels of the oxidative stress parameters
including H2O2, nitric oxide (NO) and reduced GSH in MCF-7 cells
treated with the prepared compounds. Additionally, the effect of
these compounds on the levels of total protein and nucleic acids
was determined.
As shown in Table 2, in general treatment of the cells with differ-
ent compounds or cisplatin (at the 1/10 of IC50 values)24 resulted in
a significant increase in the activity of SOD and the level of H2O2
higher than those of control, accompanied with a significant de-
crease in the activity of CAT and GSH-Px, and depletion in GSH level.
This means that the antitumor activity of these compounds was
accompanied with high activity of SOD with subsequent increase
in H2O2 production. The produced H2O2 should be rapidly removed
through the activation of CAT and GSH-Px. The present results
showed that activities of CAT and GSH-Px and the level of reduced
GSH were lowered in groups treated with the prepared compounds
compared to control cells. Consequently, the excess H2O2 produced
in tumor cells with the compounds can not be removed. In other
words, the accumulation of H2O2 and other free radicals in tumor
cells should be partly the cause of tumor cell killing.
cade of signals mediating apoptosis often involves
a ROS
intermediate messenger, and ROS can short circuit the pathway,
bypassing the need for upstream signals for cell suicide. Later,
Huang et al.37 reported that regulation of free radical-producing
agents may also have important clinical applications. This mecha-
nism for the effects of ROS generating anticancer agents has been
understood recently, as previously the mechanism of most antican-
cer agents was believed to be mainly due to direct interaction with
DNA, interference with DNA regulatory machinery (e.g., topoi-

G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
6813
Table 2
Effect of treatment with the prepared compounds on the activities of SOD, CAT, GSH-Px, as well as the levels of reduced GSH and H2O2 in MCF-7 treated cells
Treatment (
l
g/ml)
SOD U/mg protein
CAT U/mg protein
GSH-Px U/mg protein
GSH nmol/mg protein
H2O2 nmol/mg protein
Control (DMSO)
Cisplatin
7a
7b
7c
7e
13d
17d
40.30 4.75
130.80 15.65a
95.25 11.00a
50.00 5.20b
7.60 0.70
2.96 0.22a
3.82 0.42a
7.40 0.85b
3.50 0.40a
2.20 0.24a
4.30 0.36a,b
5.85 0.60b
9.30 1.00
4.40 0.40a
6.00 0.60a
8.00 0.82b
5.40 0.60a
4.85 0.62a
6.80 0.62a
7.33 0.85b
40.00 5.00
21.60 2.40a
30.40 3.20b
36.30 2.45a
23.50 2.20a
17.80 0.20a,b
34.20 2.70a
30.11 3.32a
15.70 1.60
47.50 5.70a
30.00 2.80a,b
24.80 2.90a
39.40 3.65a,b
53.30 4.80b
27.80 3.00a
25.60 2.40a,b
100.30 11.70a
110.00 12.90a
80.00 9.22a,b
75.44 9.20a,b
a
b
Data are expressed as means SE of six separate experiments. and are significant differences from control and cisplatin groups respectively at (p <0.05).
Table 3
Effect of the prepared compounds on the level of total protein, nucleic acids (RNA and DNA) and NO in MCF-7 treated cells
Compounds
Protein (
l
g/106 cells)
RNA (
l
g/106 cells)
DNA (
l
g/106 cells)
NO (lmol/mg protein)
Control (DMSO)
Cisplatin
7a
7b
7c
7e
13d
17d
110.50 12.30
33.60 3.70a
55.40 6.00a,b
80.20 8.40a,b
42.60 4.72a,b
27.50 2.60a
78.30 8.60a,b
85.80 8.40a,b
15.30 1.60
3.40 0.40a
5.00 0.48a,b
12.00 1.73b
3.20 0.28a
2.60 0.28a,b
7.22 0.60a,b
8.80 0.75a,b
8.50 0.80
1.90 0.16
2.50 0.30a
5.11 0.55a,b
7.00 0.73a,b
4.50 0.52a,b
3.00 0.36a
5.30 0.71a,b
6.10 0.45a,b
4.20 0.37a
3.00 0.33a,b
2.90 0.40a,b
3.60 0.37a
4.40 0.45a
2.80 0.70a,b
3.00 0.34a,b
a
b
The values are expressed as mean SE of six separate experiments. and are significant differences from control and cisplatin groups respectively at (p <0.05).
somerases, helicases) and the initiation of DNA damage via produc-
tion of ROS.53
cer agents, there is concern for selective production of oxidative
stress in cancer cells only that initiates the need for further
investigation.
In summary, the present results suggest that the synthesized
compounds possess significant antitumor activity comparable to
the activity of commonly used anticancer drug, cisplatin and they
exert their antitumor activities by modulating free radicals produc-
tion through increasing the activity of SOD and depletion of intra-
cellular reduced GSH level, CAT and GSH-Px activities,
accompanied with high production of H2O2, NO and other free rad-
icals causing tumor cells death, as monitored by reduction in the
synthesis of protein and nucleic acids.
4. Experimental
4.1. Chemistry
Melting points are uncorrected and determined in one end
open capillary tubes using Gallen Kamp melting point apparatus
MFB-595-010M (Gallen Kamp, London, England). Microanalysis
was carried out at Micro-analytical Unit, Faculty of Science, Cairo
University, the regional center for microbiology and biotechnol-
ogy, Al-Azhar University and Organic Microanalyses Section,
Central Laboratory, National Research Center. Infrared Spectra
were recorded on Schimadzu FT-IR 8400S spectrophotometer
(Shimadzu, Kyoto, Japan), and expressed in wave number
(cmÀ1), using potassium bromide discs. The NMR spectra were
recorded on a Varian Mercury VX-300 NMR spectrometer. 1H
spectra were run at 300 MHz and 13C spectra were run at
75.46 MHz in deuterated chloroform (CDCl3) or dimethylsulfox-
ide (DMSO-d6). Chemical shifts are quoted in d and were related
to that of the solvents. Mass spectra were recorded using Hew-
lett Packard Varian (Varian, Polo, USA) and Shimadzu Gas Chro-
matograph Mass spectrometer-QP 1000 EX (Shimadzu, Kyoto,
Japan). TLC were carried out using Art.DC-Plastikfolien, Kieselgel
60 F254 sheets (Merck, Darmstadt, Germany), the developing
solvents were benzene/methanol (4:1) and the spots were
visualized at 366, 254 nm by UV Vilber Lourmat 77202 (Vilber,
Marne La Vallee, France).
3. Conclusion
Three series of N1-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidines
7a–e,
N1-(4-chlorophenylsulfonyl)pyrazolo[3,4-d]pyrimidines
13a–d and 4-(hydrazinecarbonyl) pyrazoles 17a–d endowed with
arylidene moiety were synthesized. The new compounds were
evaluated for their cytotoxic activity against MCF-7 breast cancer
cells compared to cisplatin as reference drug. Some structural
features were found to be beneficial to the antitumor activity of
such compounds. In particular, the pyrazolo[3,4-d]pyrimidine
containing compounds 7a–e and 13a–d were more potent than
those possessing pyrazole ring 17a–d. Also, substitution with N1-
(4-chlorophenyl) on the pyrazolo[3,4-d]pyrimidine scaffold in
7a–e was more favorable than N1-(4-chlorophenylsulfonyl) in
13a–d. Meanwhile, increasing the number of methoxy groups on
the arylidene substituent was accompanied by enhancement of
antitumor activity and reached the highest activity with the most
potent trimethoxybenzylidene derivative 7e (IC50 = 7.60 lM).
Moreover, compounds 7a–c, 7e, 13d and 17d were evaluated
for their ability to induce oxidative stress in cancer cells. Estimat-
ing the activities of the free-radical-metabolizing enzymes and the
levels of the oxidative stress parameters suggested that the antitu-
mor effect of the present compounds may be exerted by modulat-
ing free radicals production within the tumor cell. Although, the
biological screening results of the tested compounds could offer a
promising framework that may lead to discovery of potent antican-
Compounds 339 and 440 were obtained according to the re-
ported procedures.
4.1.1. 1-(4-Chlorophenyl)-6-methylpyrazolo[3,4-d][1,3]oxazin-
4(1H)-one (5)
A mixture of 5-amino-1-(4-chlorophenyl)-1H-pyrazole-4-car-
boxylic acid 4 (2.37 g, 0.01 mol) and acetic anhydride (5 ml) was
heated under reflux for 4 h. The reaction mixture was cooled to

6814
G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
room temperature. The formed solid was filtered, dried and crys-
tallized from methanol.
for C20H16ClN5O3 (409.83): C, 58.61; H, 3.94; N, 17.09. Found: C,
59.00; H, 4.24; N, 17.00.
Mp 147–148 °C; yield: 68%. IR
m
max/cmÀ1: 3132.40 (CH aro-
matic), 1778.37 (C@O). 1H NMR (CDCl3) d ppm: 2.55 (s, 3H, CH3),
7.51 (d, 2H, H-3 and H-5 of Cl-C6H4, J = 8.7 Hz), 7.99 (d, 2H, H-2
and H-6 of Cl-C6H4, J = 8.7 Hz), 8.19 (s, 1H, H-3 pyrazole). Anal.
Calcd for C12H8ClN3O2 (261.66): C, 55.08; H, 3.08; N, 16.06. Found:
C, 55.27; H, 3.16; N, 16.19.
4.1.3.4. (E)-1-(4-Chlorophenyl)-5-(3,4-dimethoxybenzylidenea-
mino)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(7d).
Mp 252–254 °C; yield: 70%. IR m
max/cmÀ1: 3050.00 (CH
aromatic), 3000.00, 2954.95 (CH aliphatic), 1705.07 (C@O). 1H
NMR (DMSO-d6) d ppm: 2.48 (s, 3H, CH3), 3.84 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 7.13 (d, 1H, H-5 of (OCH3)2–C6H3, J = 8.4 Hz),
7.45 (d, 1H, H-6 of (OCH3)2–C6H3, J = 8.4 Hz), 7.57 (s, 1H, H-2 of
(OCH3)2–C6H3), 7.66 (d, 2H, H-3 and H-5 of Cl-C6H4, J = 8.7 Hz),
8.15 (d, 2H, H-2 and H-6 of Cl-C6H4, J = 8.7 Hz), 8.38 (s, 1H, H-3 pyr-
azole), 8.70 (s, 1H, N@CH). Anal. Calcd for C21H18ClN5O3 (423.85):
C, 59.51; H, 4.28; N, 16.52. Found: C, 59.20; H, 4.55; N, 16.24.
4.1.2. 5-Amino-1-(4-chlorophenyl)-6-methyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (6)
A mixture of 1-(4-chlorophenyl)-6-methylpyrazolo[3,4-d][1,3]
oxazin-4(1H)-one
5 (2.62 g, 0.01 mol) and hydrazine hydrate
(0.60 ml, 0.012 mol) in n-butanol (20 ml) was heated under reflux
for 24 h. The reaction mixture was concentrated in vacuum then
cooled. The separated solid was filtered and crystallized from
ethanol.
4.1.3.5. (E)-1-(4-Chlorophenyl)-6-methyl-5-(3,4,5-trimethoxyb-
enzylideneamino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
Mp 257–259 °C; yield: 52%. IR
m
max/cmÀ1: 3309.85, 3275.13
(7e).
Mp 232–234 °C; yield: 55%. IR m
max/cmÀ1: 3005.10 (CH
(NH2), 3101.45 (CH aromatic), 1693.50 (C@O). 1H NMR (DMSO-
d6) d ppm: 2.64 (s, 3H, CH3), 5.77 (s, 2H, NH2, exch. D2O), 7.61 (d,
2H, H-3 and H-5 of Cl-C6H4, J = 8.7 Hz), 8.13 (d, 2H, H-2 and H-6
of Cl-C6H4, J = 8.7 Hz), 8.34 (s, 1H, H-3 pyrazole). Anal. Calcd for
C12H10ClN5O (275.69): C, 52.28; H, 3.66; N, 25.40. Found: C,
52.60; H, 3.81; N, 25.22.
aromatic), 2943.37, 22831.50 (CH aliphatic), 1697.36 (C@O). 1H
NMR (CDCl3) d ppm: 2.75 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 3.95 (s,
6H, 2 OCH3), 7.14 (s, 2H, H-2 and H-6 of (OCH3)3–C6H2), 7.46 (d,
2H, H-3 and H-5 of Cl-C6H4, J = 8.7 Hz), 8.12 (d, 2H, H-2 and H-6
of Cl-C6H4, J = 8.7 Hz), 8.74 (s, 1H, H-3 pyrazole), 9.88 (s, 1H,
N@CH). Anal. Calcd for C22H20ClN5O4 (453.88): C, 58.22; H, 4.44;
N, 15.43. Found: C, 57.96; H, 4.28; N, 15.55.
4.1.3. General procedure for the synthesis of (E)-5-(substituted
benzylideneamino)-1-(4-chlorophenyl)-6-methyl-1H-pyrazolo
[3,4-d]pyrimidin-4(5H)-one (7a–e)
A mixture of 6 (2.75 g, 0.01 mol) and the appropriate aromatic
aldehyde (0.012 mol) in glacial acetic acid (10 ml) was heated un-
der reflux for 16 h. The reaction mixture was concentrated in vac-
uum then cooled. The obtained solid was filtered, dried and
crystallized from ethanol.
4.1.4. General procedure for the synthesis of ethyl 5-amino-1-
(4-substituted phenylsulfonyl)-1H-pyrazole-4-carboxylate
(10a–c)
A solution of ethyl ethoxymethylenecyanoacetate 8 (5.07 g,
0.03 mol) and the proper phenylsulfonylhydrazines 9a–c
(0.03 mol) in acetonitrile (50 ml) was heated under reflux for 6 h.
The reaction mixture was concentrated in vacuum then cooled.
The separated solid was filtered and crystallized from acetonitrile.
4.1.3.1. (E)-5-(Benzylideneamino)-1-(4-chlorophenyl)-6-methyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (7a).
Mp 230–
4.1.4.1. Ethyl 5-amino-1-(phenylsulfonyl)-1H-pyrazole-4-car-
boxylate (10a).
232 °C; yield: 75%. IR
m
max/cmÀ1: 3010.50 (CH aromatic), 1712.79
Mp 149–151 °C; yield: 93%. IR m :
max/cmÀ1
(C@O). 1H NMR (CDCl3) d ppm: 2.69 (s, 3H, CH3), 7.46–7.57 (m,
3H, H-3, H-4 and H-5 of C6H5), 7.89 (d, 2H, H-3 and H-5 of Cl-
C6H4, J = 8.4 Hz), 8.09–8.25 (m, 4H, H-2 and H-6 of C6H5 and H-2
and H-6 of Cl-C6H4), 8.91 (s, 1H, H-3 pyrazole), 10.04 (s, 1H,
N@CH). Anal. Calcd for C19H14ClN5O (363.80): C, 62.73; H, 3.88;
N, 19.25. Found: C, 62.83; H, 3.75; N, 19.45.
3437.15, 3309.85 (NH2), 3062.96 (CH aromatic), 2985.81 (CH ali-
phatic), 1693.50 (C@O), 1381.03, 1134.14 (SO2). 1H NMR (CDCl3)
d ppm: 1.31 (t, 3H, CH2CH3, J = 7.2 Hz), 4.21 (q, 2H, CH2CH3,
J = 7.2 Hz), 6.55 (s, 2H, NH2, exch. D2O), 7.52 (d, 2H, H-3 and H-5
of C6H5, J = 7.5 Hz), 7.64–7.69 (m, 2H, H-4 of C6H5 and H-3 of pyr-
azole), 7.98 (d, 2H, H-2 and H-6 of C6H5, J = 7.5 Hz). Anal. Calcd for
C
12H13N3O4S. H2O (313.33): C, 46.00; H, 4.83; N, 13.41. Found: C,
4.1.3.2. (E)-1-(4-Chlorophenyl)-5-(4-methoxybenzylideneami-
no)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(7b).
45.84; H, 4.62; N, 13.48.
Mp 244–246 °C; yield: 60%. IR m
max/cmÀ1: 3001.24 (CH
aromatic), 2954.95, 2830.50 (CH aliphatic), 1701.22 (C@O). 1H
NMR (DMSO-d6) d ppm: 1.97 (s, 3H, CH3), 3.84 (s, 3H, OCH3),
6.85 (d, 2H, H-3 and H-5 of OCH3-C6H4, J = 8.1 Hz), 7.06 (d, 2H,
H-3 and H-5 of Cl-C6H4, J = 8.4 Hz), 7.56 (d, 2H, H-2 and H-6 of
Cl-C6H4, J = 8.4 Hz), 7.82 (d, 2H, H-2 and H-6 of OCH3-C6H4,
J = 8.1 Hz), 8.63 (s, 1H, H-3 pyrazole), 9.73 (s, 1H, N@CH). Anal.
Calcd for C20H16ClN5O2 (393.83): C, 60.99; H, 4.09; N, 17.78. Found:
C, 61.20; H, 4.10; N, 17.70.
4.1.4.2. Ethyl 5-amino-1-(4-methylphenylsulfonyl)-1H-pyra-
zole-4-carboxylate (10b).
Mp 122–123 °C; yield: 90%. IR
m
max/cmÀ1
:
3460.30, 3267.41 (NH2), 3132.40 (CH aromatic),
2985.81 (CH aliphatic), 1693.50 (C@O), 1365.60, 1138.00 (SO2).
1H NMR (CDCl3) d ppm: 1.35 (t, 3H, CH2CH3, J = 7.2 Hz), 2.43 (s,
3H, CH3), 4.26 (q, 2H, CH2CH3, J = 7.2 Hz), 6.52 (s, 2H, NH2, exch.
D2O), 7.35 (d, 2H, H-3 and H-5 of CH3–C6H4, J = 8.1 Hz), 7.87 (d,
2H, H-2 and H-6 of CH3–C6H4, J = 8.4 Hz), 8.30 (s, 1H, H-3 pyrazole).
Anal. Calcd for C13H15N3O4S (309.34): C, 50.47; H, 4.89; N, 13.58.
Found: C, 50.92; H, 5.08; N, 13.67.
4.1.3.3. (E)-1-(4-Chlorophenyl)-5-(4-hydroxy-3-methoxybenzy-
lideneamino)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (7c).
Mp 261–263 °C; yield: 52%. IR m
max/cmÀ1: 3414.00
4.1.4.3. Ethyl 5-amino-1-(4-chlorophenylsulfonyl)-1H-pyrazole-
4-carboxylate (10c).
Mp 162–164 °C; yield: 84%. IR mmax/
(OH), 3008.95 (CH aromatic), 2920.23, 2850.79 (CH aliphatic),
1708.93 (C@O). 1H NMR (DMSO-d6) d ppm: d 2.48 (s, 3H, CH3),
3.85 (s, 3H, OCH3), 6.94 (d, 1H, H-5 of OH-OCH3-C6H3, J = 7.8 Hz),
7.35 (d, 1H, H-6 of OH–OCH3–C6H3, J = 7.8 Hz), 7.55 (s, 1H, H-2 of
OH-OCH3-C6H3), 7.65 (d, 2H, H-3 and H-5 of Cl-C6H4, J = 9.0 Hz),
8.16 (d, 2H, H-2 and H-6 of Cl-C6H4, J = 9.0 Hz), 8.38 (s, 1H, H-3 pyr-
azole), 8.62 (s, 1H, N@CH), 10.05 (s, 1H, OH, exch. D2O). Anal. Calcd
cmÀ1: 3429.43, 3294.42 (NH2), 3066.82 (CH aromatic), 2985.81
(CH aliphatic), 1689.64 (C@O), 1384.89, 1130.29 (SO2). 1H NMR
(CDCl3) d ppm: 1.33 (t, 3H, CH2CH3, J = 7.2 Hz), 4.26 (q, 2H, CH2CH3,
J = 7.2 Hz), 6.52 (s, 2H, NH2, exch. D2O), 7.54 (d, 2H, H-3 and H-5 of
Cl-C6H4, J = 9.6 Hz), 7.95 (d, 2H, H-2 and H-6 of Cl-C6H4, J = 9.6 Hz),
8.50 (s, 1H, H-3 pyrazole). MS, m/z: 356 [M+ÀCH3]. Anal. Calcd for

G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
6815
C12H12ClN3O4S (329.76): C, 43.71; H, 3.67; N, 12.74; S, 9.72. Found:
C, 44.00; H, 4.00; N, 13.00; S, 9.52.
Cl-C6H4, J = 7.8 Hz), 8.61 (s, 1H, H-3 pyrazole), 9.90 (s, 1H, N@CH).
MS, m/z: 458 [M++1]. Anal. Calcd for C20H16ClN5O4S (457.89): C,
52.46; H, 3.52; N, 15.29. Found: C, 52.18; H, 3.69; N, 15.05.
4.1.5. Ethyl 5-acetamido-N-(4-chlorophenylsulfonyl-1H-
pyrazole-4-carboxylate (11)
4.1.7.3. (E)-1-(4-Chlorophenylsulfonyl)-5-(3,4-dimethoxybenzy-
lideneamino)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (13c).
A mixture of compound 10c (3.39 g, 0.01 mol) in acetic anhy-
dride (10 ml) was refluxed for 4 h. The reaction mixture was cooled
and filtered; the obtained solid was crystallized from absolute
ethanol.
Mp 196–198 °C; yield: 65%. IR m
max/cmÀ1: 3066.82
(CH aromatic), 2931.80, 2835.36 (CH aliphatic), 1662.64 (C@O),
1311.59, 1145.72 (SO2). 1H NMR (DMSO-d6) d ppm: 1.89 (s, 3H,
CH3), 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.95 (d, 1H, H-5 of
(OCH3)2–C6H3, J = 7.5 Hz), 7.15 (s, 1H, H-2 of (OCH3)2–C6H3), 7.64
(d, 1H, H-6 of (OCH3)2–C6H3, J = 7.8 Hz), 7.75 (d, 1H, H-3 and H-5
of Cl-C6H4, J = 8.1 Hz), 7.88 (d, 2H, H-2 and H-6 of Cl-C6H4,
J = 8.7 Hz), 8.42 (s, 1H, H-3 pyrazole), 10.72 (s, 1H, N@CH). 13C
NMR (DMSO) d: 20.5, 55.4, 55.5, 108.2, 111.2, 120.13, 120.5,
127.7, 129.2, 129.9, 130.6, 140.5, 141.6, 149.0, 149.8, 160.0,
168.0. MS, m/z: 488 [M+]. Anal. Calcd for C21H18ClN5O5S (487.92):
C, 51.69; H, 3.72; N, 14.35; S, 6.57. Found: C, 51.85; H, 3.85; N,
14.22; S, 6.61.
Mp 210–212 °C; yield: 60%. IR
m : 3294.13 (NH),
max/cmÀ1
3066.82 (CH aromatic), 2985.81 (CH aliphatic), 1710.0 (C@O),
1689.64 (C@O), 1276.88, 1114.86 (SO2). 1H NMR (CDCl3) d ppm:
1.38 (t, 3H, CH2CH3, J = 7.2 Hz), 2.30 (s, 3H, CH3), 4.37 (q, 2H,
CH2CH3, J = 7.2 Hz), 7.50 (d, 2H, H-3 and H-5 of Cl-C6H4,
J = 9.6 Hz), 7.84 (d, 2H, H-2 and H-6 of Cl-C6H4, J = 9.6 Hz), 7.88
(s, 1H, H-3 pyrazole), 9.62 (s, br, 1H, NHCOCH3 exch. D2O). MS,
m/z: 356 [M+ÀCH3]. Anal. Calcd for C14H14ClN3O5S (371.8): C,
45.23; H, 3.80; N, 11.30; S, 8.62. Found: C, 45.54; H, 3.95; N,
11.30; S, 8.60.
4.1.6. N-(4-Chlorophenylsulfonyl)-4-(hydrazinecarbonyl)-1H-
pyrazol-5-yl) acetamide (12)
4.1.7.4. (E)-1-(4-Chlorophenylsulfonyl)-5-(3,4,5-trimethoxyben-
zylideneamino)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (13d).
(CH aromatic), 2939.52 (CH aliphatic), 1635.64 (C@O), 1323.17,
1126.43 (SO2). 1H NMR (CDCl3) d ppm: 1.77 (s, 3H, CH3), 3.85 (s,
3H, OCH3), 3.92 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.89 (s, 2H, H-2
and H-6 of (OCH3)3-C6H2), 7.48 (d, 1H, H-3 and H-5 of Cl-C6H4,
J = 8.1 Hz), 7.82 (d, 2H, H-2 and H-6 of Cl-C6H4, J = 8.7 Hz), 7.93
(s, 1H, H-3 pyrazole), 9.88 (s, 1H, N@CH).) Anal. Calcd for
A mixture of 11 (3.71 g, 0.01 mol) and hydrazine hydrate
(0.60 ml, 0.012 mol) in n-butanol (20 ml) was refluxed for 24 h.
The reaction mixture was concentrated in vacuum then cooled.
The separated solid was filtered and crystallized from ethanol.
Mp 216–218 °C; yield: 90%. IR m
max/cmÀ1: 3032.10
Mp 184–186 °C; yield: 65%. IR
m
max/cmÀ1: 3410.15, 3302.13,
3178.89 (NH2 and NH), 3093.82 (CH aromatic.), 2924.09, 2821.21
(CH aliphatic), 1624.06 (br, 2 C@O), 1315.45, 1149.57 (SO2). 1H
NMR (DMSO-d6) d ppm: 2.42 (s, 3H, CH3), 4.20 (s, br, 2H, CONHNH2,
exch. D2O), 5.59 (s, br, 1H, CONHNH2, exch. D2O), 6.82 (d, 2H, H-3
and H-5 of Cl-C6H4, J = 8.7 Hz), 7.64 (d, 2H, H-2 and H-6 of Cl-C6H4,
J = 8.7 Hz), 7.84 (s, 1H, H-3 pyrazole), 8.89 (s, br, 1H, NHCOCH3
exch. D2O). MS, m/z: 359 [M++2]. Anal. Calcd for C12H12ClN5O4S
(357.77): C, 40.28; H, 3.38; N, 19.57; S, 8.96. Found: C, 40.28; H,
3.38; N, 19.75; S, 8.66.
C22H20ClN5O6S (517.94): C, 51.02; H, 3.89; N, 13.52. Found: C,
51.25; H, 4.06; N, 13.08.
4.1.8. (E)-1-(4-Chlorophenylsulfonyl)-5-(3,4,5-trimethoxyben-
zamido)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (14)
A mixture of 3,4,5-trimethoxybenzoic acid (2.12 g, 0.01 mol)
and thionyl chloride (5 ml) was refluxed gently for 1 h. The excess
thionyl chloride was removed under vacuum in water-bath. The
residue was azeotroped three times with dry benzene to remove
the last traces of thionyl chloride. The crude acid chloride was
dissolved in dry benzene (20 ml) and was added drop-wise with
stirring to a buffered solution of the hydrazide derivative 12
(3.57 g, 0.01 mol) in dry benzene (10 ml), keeping the temperature
at 10 °C. After addition was complete, stirring was continued at
room temperature for 3 h. The reaction mixture evaporated in vac-
uum and the residue was triturated with ice-water. The obtained
solid product—crude benzamide derivative 14—was filtered,
washed several times with water, dried and crystallized from abso-
lute ethanol.
4.1.7. General procedure for the synthesis of (E)-5-(substituted
benzylideneamino)-1-(4-chlorophenylsulfonyl)-6-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (13a–d)
A mixture of 12 (3.57 g, 0.01 mol) and the appropriate aldehyde
(0.012 mol) in glacial acetic acid (10 ml) were heated under reflux
for 24 h. The reaction mixture was concentrated in vacuum then
cooled. The obtained solid product was filtered, dried and crystal-
lized from acetic acid.
4.1.7.1. (E)-5-(Benzylideneamino)-1-(4-chlorophenylsulfonyl)-
6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(13a).
Mp 190–192 °C; yield: 92%. IR
m
max/cmÀ1: 3010.50 (CH
Mp 228–229 °C; yield: 81%. IR m : 3317.56 (NH),
max/cmÀ1
aromatic), 1693.20 (C@O), 1318.13, 1149.57 (SO2). 1H NMR (CDCl3)
d ppm: 1.56 (s, 3H, CH3), 7.14 (d, 2H, H-3 and H-5 of Cl-C6H4,
J = 8.7 Hz), 7.37–7.47 (m, 3H, H-3, H-4 and H-5 of C6H5), 7.65 (d,
2H, H-2 and H-6 of Cl-C6H4, J = 8.5 Hz), 7.76 (s, 1H, H-3 pyrazole),
7.79–7.84 (m, 2H, H-2 and H-6 of C6H5), 7.87 (s, 1H, N@CH). MS,
m/z: 427.86 [M+ÀCH3]. Anal. Calcd for C19H14ClN5O3S (427.86): C,
53.34; H, 3.30; N, 16.37; S, 7.49. Found: C, 53.49; H, 3.34; N,
16.67; S, 7.81.
3066.82 (CH aromatic), 2939.52 (CH aliphatic), 1681.93 (C@O),
1327.03, 1157.29 (SO2). 1H NMR (DMSO-d6) d ppm: 1.25 (s, 3H,
CH3), 3.72 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
6.89 (d, 2H, H-3 and H-5 of Cl-C6H4, J = 9.0 Hz), 7.25 (s, 2H, H-2
and H-6 of (OCH3)3-C6H2), 7.88 (d, 2H, H-2 and H-6 of Cl-C6H4,
J = 8.4 Hz), 8.85 (s, 1H, H-3 pyrazole), 10.48 (s, 1H, NH, exch.
D2O). 13C NMR (DMSO) d: 20.5, 55.8, 56.0, 60.0, 104.9, 106.5,
128.5, 129.3, 129.5, 129.9, 137.8, 139.0, 140.5, 141.5, 152.5,
152.6, 153.8, 165.5, 166.85). MS, m/z: 517 [M+ÀCH3]. Anal. Calcd
for C22H20ClN5O7S (533.94): C, 49.49; H, 3.78; N, 13.12. Found: C,
49.29; H, 3.56; N, 13.18.
4.1.7.2. (E)-1-(4-Chlorophenylsulfonyl)-5-(4-methoxybenzylide-
neamino)-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(13b).
Mp 202–204 °C; yield: 85%. IR m
max/cmÀ1: 3035.96 (CH
aromatic), 2924.09 (CH aliphatic), 1693.49 (C@O), 1303.88,
1168.86 (SO2). 1H NMR (CDCl3) d ppm: 1.26 (s, 3H, CH3), 3.90 (s,
3H, OCH3), 6.97 (d, 2H, H-3 and H-5 of OCH3–C6H4, J = 9.0 Hz),
7.02 (d, 2H, H-2 and H-6 of OCH3–C6H4, J = 9.0 Hz), 7.61 (d, 2H,
H-3 and H-5 of Cl-C6H4, J = 7.8 Hz), 7.83 (d, 2H, H-2 and H-6 of
4.1.9. Ethyl 5-acetamido-1H-pyrazole-4-carboxylate (15)42
A mixture of compound 10a or 10b (0.01 mol) and acetic anhy-
dride (5 ml) was heated under reflux for 4 h. The reaction mixture
was cooled to room temperature. The formed solid was filtered,
dried and crystallized from ethanol.

6816
G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
Mp 201–203 °C (reported mp 204 °C); yield: 68%. IR
m
max/cmÀ1
:
4.1.11.4. (E)-N-{4-[2-(3,4,5-Trimethoxybenzylidene)hydrazine-
carbonyl]-1H-pyrazol-5-yl}acetamide (17d). Mp 271–272 °C;
yield: 91%. IR
max/cmÀ1: 3294.42, 3132.40 (3 NH), 3089.96 (CH
3340.71, 3251.98 (2NH), 2978.09 (CH aliphatic), 1681.93, 1604.77
(C@O). 1H NMR (CDCl3) d ppm: 1.38 (t, 3H, CH2CH3, J = 7.2 Hz), 2.29
(s, 3H, CH3), 4.37 (q, 2H, CH2CH3, J = 7.2), 7.77 (s, 1H, H-3 pyrazole),
9.57 (s, 1H, NHCOCH3, exch. D2O), 11.85 (s, 1H, NH pyrazole, exch.
D2O).
m
aromatic), 2943.37, 2839.22 (CH aliphatic), 1689.64, 1651.07
(C@O). 1H NMR (DMSO-d6) d ppm: (mixture of tautomers) 2.20
(s, 3H, CH3), 3.70 (s, 3H, OCH3), 3.83 (s, 6H, 2 OCH3), 7.00 (s, 2H,
H-2 and H-6 of (OCH3)3–C6H2), 7.99, 8.19 (2s, 1H, H-3 pyrazole),
8.27, 8.35 (2s, 1H, N@CH), 10.40, 10.60 (2s, 1H, NHCOCH3, exch.
D2O), 11.40, 11.54 (2s, 1H, CONH–N@, exch. D2O), 12.88, 13.15
(2s, 1H, NH pyrazole, exch. D2O). MS, m/z: 362 [M+]. Anal. Calcd
for C16H19N5O5 (361.35): C, 53.18; H, 5.30; N, 19.38. Found: C,
52.95; H, 5.27; N, 19.64.
4.1.10. N-[4-(Hydrazinecarbonyl)-1H-pyrazol-5-yl]acetamide
(16)
A mixture of 15 (1.97 g, 0.01 mol) and hydrazine hydrate (0.60 ml,
0.012 mol) in n-butanol (20 ml) was heated under reflux for 24 h. The
reaction mixture was concentrated in vacuum then cooled. The sepa-
rated solid was filtered and crystallized from ethanol.
Mp 236–238 °C; yield: 60%. IR
m
max/cmÀ1: 3402.43, 3339.85,
4.2. Biological evaluation
3159.40 (NH2 and NH), 2974.23, 2804.50 (CH aliphatic), 1627.92
(br, 2C@O). 1H NMR (DMSO-d6) d ppm: 4.17 (s, 3H, CH3), 5.63 (s,
2H, br, CONHNH2, exch. D2O), 7.71 (s, 1H, H-3 pyrazole), 8.90 (s,
2H, CONHNH2 and NHCOCH3, exch. D2O), 11.68 (s, br, 1H, NH pyr-
azole, exch. D2O). Anal. Calcd for C6H9N5O2 (183.17): C, 39.34; H,
4.95; N, 38.23. Found: C, 39.64; H, 5.01; N, 37.98.
4.2.1. Materials and methods
4.2.1.1. Chemicals. Dimethylsulfoxide (DMSO), cisplatin and Sulfor
hodamine-B stain (SRB) were purchased from Merck (Darmstadt,
Germany). All other chemicals and reagents used in this study
were of analytical grade and purchased from Sigma-Aldrich chem-
ical Co. (St. Louis, MO, USA).
4.1.11. General procedure for the synthesis of (E)-N-[4-(2-
substituted benzylidenehydrazinecarbonyl)-1H-pyrazol-5-
yl]acetamide (17a–d)
A mixture of 16 (1.83 g, 0.01 mol) and the appropriate aldehyde
(0.012 mol) in glacial acetic acid (10 ml) was heated under reflux
for 24 h. The precipitate formed was filtered, dried and crystallized
from absolute ethanol.
4.2.1.2. Cell culture.
The MCF-7 cancer cell line was obtained
from the American Type Culture Collection (Rockville, MD, USA).
The tumor cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat inactivated fetal
calf serum (GIBCO), penicillin (100 U/ml) and streptomycin
(100 lg/ml) at 37 °C in humidified atmosphere containing 5%
CO2. MCF-7 cells at a concentration of 0.50 Â 106 were grown in
4.1.11.1. (E)-N-[4-(2-Benzylidenehydrazinecarbonyl)-1H-pyra-
a 25 cm2 flask in 5 ml of complete culture medium.
zol-5-yl]acetamide (17a).
m
Mp 247–248 °C; yield: 76%. IR
max/cmÀ1: 3290.56, 3190.26, 3143.97 (3 NH), 3024.38 (CH arom.),
4.2.2. In vitro cytotoxic assay43
2927.94 (CH aliph.), 1693.50, 1643.35 (C@O). 1H NMR (DMSO-d6) d
ppm: (mixture of tautomers) 2.16 (s, 3H, CH3), 7.39–7.46 (m, 3H,
H-3, H-4 and H-5 of C6H5), 7.65–7.71 (m, 2H, H-2 and H-6 of
C6H5), 8.11 (s, br, 1H, H-3 pyrazole), 8.33 (s, br, 1H, N@CH), 10.37
(s, br, 1H, NHCOCH3, exch. D2O), 11.15 (s, br, 1H, CONH–N@, exch.
D2O), 11.47 (s, br, 1H, NH pyrazole, exch. D2O). Anal. Calcd for
The cytotoxic activity was measured in vitro using the SRB
colorimetric assay using the method of Skehan.43 Cells were
inoculated in 96-well microtiter plate (104 cells/ well) for 24 h
before treatment with the compound(s) to allow attachment of
cell to the wall of the plate. Test compounds were dissolved in
DMSO and diluted with saline to the appropriate volume. Differ-
ent concentrations of the compound under test (0.1, 2.5, 5, and
10 mmol/ml) were added to the cell monolayer. Triplicate wells
were prepared for each individual dose. Monolayer cells were
incubated with the compound(s) for 48 h. at 37 °C and in atmo-
sphere of 5% CO2. After 48 h., cells were fixed, washed, and
stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic
acid. Unbound dye was removed by four washes with 1% acetic
acid, and attached stain was recovered with Tris–EDTA buffer.
Color intensity was measured in an ELISA reader. The relation
between surviving fraction and drug concentration is plotted to
get the survival curve for breast tumor cell line after the speci-
fied time. The concentration required for 50% inhibition of cell
C13H13N5O2 (271.27): C, 57.56; H, 4.83; N, 25.82. Found: C, 57.50;
H, 4.60; N, 25.53.
4.1.11.2. (E)-N-{4-[2-(4-Methoxybenzylidene)hydrazinecarbon-
yl]-1H-pyrazol-5-yl}acetamide (17b).
Mp 242–243 °C; yield:
86%. IR
m
max/cmÀ1: 3305.99, 3278.99, 3140.11 (3NH), 3035.96 (CH
aromatic), 2939.52, 2839.22 (CH aliphatic), 1689.64, 1639.49
(C@O). 1H NMR (DMSO-d6) d ppm: (mixture of tautomers) 2.19 (s,
3H, CH3), 3.80 (s, 3H, OCH3), 7.03 (d, 2H, 2H, H-3 and H-5 of OCH3–
C6H4, J = 7.2 Hz), 7.64 (d, 2H, H-2 and H-6 of OCH3-C6H4, J = 7.8 Hz),
7.95, 8.04 (2s, 1H, H-3 pyrazole), 8.23, 8.26 (2s, 1H, N@CH), 10.37,
10.64 (2s, 1H, NHCOCH3, exch. D2O), 11.10, 11.37 (2s, 1H, CONH–
N@, exch. D2O), 12.84, 13.12 (2s, 1H, NH pyrazole, exch. D2O). MS,
m/z: 301.10 [M+]. Anal. Calcd for C14H15N5O3 (301.30): C, 55.81; H,
5.02; N, 23.24. Found: C, 55.56; H, 4.75; N, 23.50.
viability (IC50
Table 1.
) was calculated and the results are given in
4.2.3. Biochemical assays
4.1.11.3. (E)-N-{4-[2-(3,4-Dimethoxybenzylidene)hydrazinecar-
The cells in culture medium were treated with 20 ll of 1/10 of
bonyl]-1H-pyrazol-5-yl}acetamide (17c).
Mp 232–233 °C;
IC50 values of the compounds or the standard reference drug, cis-
platin, then incubated for 24 h at 37 °C, in a humidified 5% CO2
atmosphere. The MCF-7 cells were harvested and homogenates
were prepared in saline using a tight pestle homogenizer until
complete cell disruption for further biochemical analysis.
yield: 80%. IR 3309.85, 3282.84, 3248.13 (NH),
m
max/cmÀ1
:
3051.39 (CH aromatic), 2997.38, 2873.94 (CH aliphatic), 1693.50,
1649.49 (C@O). 1H NMR (DMSO-d6) d ppm: (mixture of tautomers)
2.20 (3H, s, CH3), 3.81 (s, 6H, 2 OCH3), 7.02 (d, 1H, H-5 of (OCH3)2–
C6H3, J = 7.1 Hz), 7.19 (d, 1H, H-6 of (OCH3)2–C6H3, J = 7.4 Hz), 7.32
(s, 1H, H-2 of (OCH3)2-C6H3), 7.95, 8.10 (2s, 1H, H-3 pyrazole), 8.23,
8.27 (2s, 1H, N@CH), 10.26, 10.64 (2s, 1H, NHCOCH3, exch. D2O),
11.20, 11.42 (2s,1H, CONH–N@, exch. D2O), 12.95, 13.14 (2s, 1H,
NH of pyrazole, exch. D2O). Anal. Calcd for C15H17N5O4 (331.33):
C, 54.38; H, 5.17; N, 21.14. Found: C, 54.68; H, 4.88; N, 21.42.
4.2.3.1. Antioxidant enzyme assays.
The supernatant ob-
tained after centrifugation of cell homogenates was used for the
determination of enzymes activities of SOD, CAT and GSH-Px as de-
scribed by Paglia and Valentine,54 Aebi,55 Marklund and Markl-
und,56 respectively.

G. S. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 6808–6817
6817
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