Synthesis of some new pyrazolo[3,4-d]pyrimidine derivatives of expected anticancer and radioprotective activity

Article abstract of DOI:10.1016/j.ejmech.2009.09.039

On the account of the reported anticancer activity of pyrazolo[3,4-d]pyrimidines, a new series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and tested for in-vitro anticancer activity against Ehrlich Ascites Carcinoma (EAC) cell line. Moreove

Full text of DOI:10.1016/j.ejmech.2009.09.039

European Journal of Medicinal Chemistry 45 (2010) 171–178
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some new pyrazolo[3,4-d]pyrimidine derivatives of expected
anticancer and radioprotective activity
,
b
a
Mostafa M. Ghorab a , Fatma A. Ragab , Saleh I. Alqasoumi ,
*
Ahmed M. Alafeefy c, Sarah A. Aboulmagd d
a Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Medicinal Chemistry Department, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
d Department of Drug Radiation Research, National Centre for Radiation Research and Technology, Atomic Energy Authority, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
On the account of the reported anticancer activity of pyrazolo[3,4-d]pyrimidines, a new series of pyr-
azolo[3,4-d]pyrimidine derivatives were synthesized and tested for in-vitro anticancer activity against
Ehrlich Ascites Carcinoma (EAC) cell line. Moreover, one of the target products was evaluated for in-vivo
radioprotective activity.
The reaction of o-aminoester 1 with benzylamine in presence of triethylorthoformate yielded the cor-
responding 5-benzylpyrazolopyrimidine derivative 2. The N-amino derivative 3 was used to synthesize
new derivatives of pyrazolopyrimidines 4–7. The corresponding 1,3,4-oxadiazolopyrazolopyrimidine
derivatives 12 and 14 were obtained via reaction of compound 9 with reagent 10 and/or triethylortho-
formate. Thiophosgenation of compound 1 furnished the corresponding 5-isothiocyanate derivative 15,
which was reacted with o-phenylenediamine, thiosemicarbazide and anthranilic acid to give benzimi-
dazolopyrazolopyrimidine, 17, pyrazolotriazolopyrimidine, 19 and pyrazolopyrimidobenzoxazine, 20
respectively. The structures of the synthesized compounds were confirmed by microanalyses, IR, NMR, and
mass spectral data.
Received 18 September 2008
Received in revised form
17 September 2009
Accepted 25 September 2009
Available online 30 September 2009
Keywords:
Pyrazolo[3,4-d]pyrimidines
Anticancer
Radioprotective activity
Compounds 2 and 9 showed intermediate anticancer activity compared to doxorubicin as positive
control with IC50 values of 90 and 100
significant radioprotective effect.
m
g/ml, respectively. On the other hand, compound 5 showed
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
much difference in the basic structure of pyrazolopyrimidines and
purines where a pyrazole ring replaces the imidazole ring of
purines [2,8–11]. In the light of the aforementioned facts, and as
a continuation of our investigations on the synthesis of biologically
active heterocyclic compounds [4,6,7,15–22], we were prompted to
synthesize some new pyrazolo[3,4-d]pyrimidine derivatives to
evaluate their potential anticancer and radioprotective activities.
Recently, considerable attention has been devoted to the
construction of new derivatives of pyrazolo[3,4-d]pyrimidines on
the account of their reported biological activities [1–14].
In the literature, several methods have been described for the
elaboration of substituted pyrazolo[3,4-d]pyrimidines [1–7], which
as a class have been reported to have anticancer and antileukemic
activity. Different mechanisms account for the cytotoxic effect of
this class of compounds, where they have been reported to act as
cyclin dependent kinase inhibitors [11], tyrosine kinase inhibitors
[12], potent xanthine oxidase inhibitors [13] and/or adenosine
receptor antagonists [14].
2. Results and discussion
2.1. Chemistry
The compounds were designed in the aim of exploring their
anticancer and radioprotective activities. The sequence of reactions
followed in the synthesis of the target compounds is illustrated in
Schemes 1–3.
Additionally, pyrazolo[3,4-d]pyrimidines are considered as
biologically active isomeric purine analogues, since there is no
This paper describes a facile one step synthesis of 5-benzyl-1-
phenyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one (2) [23] via
reaction of 5-amino-1-phenyl-1H-pyrazole-4-carboxylic acid ethyl
* Corresponding author. Tel.: þ20 966 534292860; fax: þ20 966 (01) 4670560.
E-mail address: mmsghorab@yahoo.com (M.M. Ghorab).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.039

172
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
O
N
d
NH2
N
Ph
N
N
N
N
Ph
N
S
N
N
N
NH2
3
N
N
e
N
N
N
Ph
N
N
N
Ph
5
4
c
O
O
N
NH2
O
b
N
N
N
N
NH2
N
Ph
1
Ph
3
a
CHPh
O
O
N
O
N
N
f
N
N
N
Ph
Ph
N
N
Ph
N
N
N
6
Ph
2
S
N
NH
N
g
N
N
Ph
7
Scheme 1. Reagents: a, benzylamine/TEOF/decaline; b, hydrazine hydrate/TEOF; c, P2S5/pyridine; d, P2S5/xylene; e, pyridine; f, 4-Benzylidene-2-phenyl-4H-oxazol-5-one;
g, thiourea.
ester (1) [24] with benzylamine in the presence of triethylortho-
formate (Scheme 1). Thionation of the 5-amino derivative (3) [25]
by treatment with phosphorous pentasulfide in pyridine gave the
unexpected pyrazolopyrimidotetrazine (4) rather than the expec-
ted mono thione derivative (5). The structure of compound 4 was
confirmed by elemental analysis (sulfur free), IR spectrum which
showed the absence of (NH2) and (C]S) bands also, 1H NMR
characteristic features at 1690 cmꢀ1 (C]O) and at 1577 cmꢀ1
(C]N), 1H NMR (DMSO-d6) revealed signals at
values 7.7–8.3 ppm
revealed
d
for 17 aromatic protons. The mass spectrum of
6
a molecular ion peak m/z at 458 (Mþ, 100%) as the base peak which
underwent fragmentation processes and clearly supported the
structure.
The study was extended to synthesize the fused azole contain-
ing 1,2,4-triazole nucleus containing the thione moiety with
potential biological activity [6,15,16]. Thus, compound 7 was
prepared, via a new method in our laboratory, under fusion
conditions of compound 3 with thiourea to furnish the triazole
thione derivative 7 [26]. Structure of compound 7 was supported in
the basis of elemental analysis, IR, 1H NMR and mass spectral data.
Its IR spectrum exhibited the absence of (C]O) band and the
(DMSO-d6) revealed signals at d values 7.5–8.0 ppm for 14 aromatic
protons. The mass spectrum showed a molecular ion peak m/z at
418 (Mþ, 100%) as the base peak. On repeating the same reaction in
xylene the mono thione derivative 5 was obtained based on the IR
data which showed the presence of (NH2) and (C]S) bands, 1H
NMR (DMSO-d6) revealed signals at
NMR signal at values of 210 ppm for C]S functionality, in addition
d C
values 5.7 ppm for NH2, 13
d
to the mass spectrum which revealed a molecular ion peak m/z at
243 (Mþ,100%) as the base peak. Compound 4 was also obtained via
refluxing of compound 5 in pyridine for a long time.
As a point of interest in this investigation it was found that the
N-amino derivative 3 was reacted with oxazolone derivative in dry
pyridine to give imidazole derivative (6). The structure of 6 was
deduced from elemental analysis and spectral studies. The IR
spectrum of 6 exhibited the absence of (NH2) bands and presence of
presence of NH band at 3300 cmꢀ1 1H NMR showed signals at
.
7.5–8.1 ppm corresponding to five aromatic protons. Mass spec-
trum of 7 revealed a molecular ion peak m/z at 268 (Mþ, 7.51 %),
with a base peak m/z at 77.
1,3,4-Oxadiazolopyrazolopyrimidine derivative (12) was obtai-
ned via reaction of 5-amino derivative 9 [25] with methyl 4-(N-
(bis(methylthio)methylene)sulfamoyl)phenylcarbamo
dithioate
10 [27] in dimethylformamide in presence of triethylamine

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
173
O
O
NH2
O
NHNH2
N
a
N
N
N
Ph
8
NH2
N
Ph
N
H
9
c
b
SCH3
SCH3
HN
SO2N
S
10
SCH3
O
N
O
H
N N
S
N CH OC2H5
H
N
O
S
O
SCH3
SCH3
N
N
N
N
N
Ph
O
H
N
Ph
N
O
H
11
13
- H2S
-C2H5OH
O
O
N
O
N
O
SCH3
SCH3
N
N
N
H
SO2N
N
N
N
Ph
N
N
Ph
N
12
14
Scheme 2. Reagents: a, urea/decaline; b, methyl 4-(N-(bis(methylthio)methylene)sulfamoyl)-phenylcarbamodithioate 10/dimethylformamide/triethylamine; c, triethylorthoformate.
(Scheme 2). This reaction proceeded through initial formation of
intermediate 11 followed by intramolecular cyclization with the
elimination of hydrogen sulfide (lead acetate paper).
The structure of compound 12 was proved on the basis of
elemental analysis, IR, NMR and mass spectral data, its IR spectrum
showed the presence of (NH) band at 3334 cmꢀ1 and (SO2) bands at
1370 and 1145 cmꢀ1, 1H NMR revealed signals for SCH3 at 1.6 ppm
and NH at 9.4 ppm. The mass spectrum of compound 12 revealed
a molecular ion peak m/z at 527 (Mþ, 2.72%), with a base peak
at 156.
On the other hand 1,3,4-oxadiazolopyrazolopyrimidinone (14)
was obtained via reaction of compound 9 with triethylortho-
formate as one carbon cyclizing reagent [21,22]. Compound 14 was
produced via initial formation of intermediate 13 followed by
intramolecular cyclization with elimination of ethanol. The struc-
ture of 14 was elucidated from elemental analysis and spectral data.
Its IR spectrum showed the absence of NH2 absorption band and
the presence of band at 1681 cmꢀ1 (C]O). 1H NMR spectrum of
compound 14 exhibited signals at 7.5–8.0 ppm (aromatic and
oxadiazole protons) and 8.4 ppm (CH pyrazole). The mass spectrum
of 14 exhibited a molecular ion peak m/z at 253 (Mþ, 100%), as
a base peak.
Although the chemistry of methyl-2-isothiocyanatobenzoate
has been investigated intensively [28–31], the number of publica-
tions on the synthesis and reactivity of heterocyclic isothiocyanates
is limited [32]. This prompted us to undertake the synthesis of
5-isothiocyanato-1-phenyl-1H-pyrazole-4-carboxylic acid ethyl
ester (15) from 1 [24] and thiophosgene (Scheme 3). The fact that
compound 15 contains two reactive groups at ortho positions
makes it a versatile intermediate, which through reactions with
some nucleophiles furnished some heterocyclic systems analogous
to purine, known with its potential anticancer activity [8–11]. The
structure of compound 15 was established on the basis of elemental
analysis, IR, NMR and mass spectral data. Its IR spectrum showed
the presence of (N]C]S) band at 2150 cmꢀ1 and no NH2 bands. 1H
NMR spectrum exhibited the presence of a triplet at 1.7 ppm for the
methyl group and a quartet at 4.2 ppm due to the methylene group,
also, it was confirmed by 13C NMR spectrum which revealed the
presence of N]C]S carbon signal at 140 ppm. The mass spectrum
of 15 exhibited a molecular ion peak m/z at 273 (47.7%), with a base
peak at 185.
Recently, particular importance has been attributed to
compounds containing an aromatic nucleus with imidazole, sulfur
and/or thione moieties as radioprotective and antimitotic agents
[6,15,16]. Thus interaction of compound 15 with o-phenylenedi-
amine in chloroform afforded the pyrazolopyrimidobenzimidazole
derivative (17). This reaction proceeded through initial formation of
the intermediate 16 followed by intramolecular cyclization and loss
of hydrogen sulfide (lead acetate paper). Also interaction of 15 with
thiosemicarbazide in dimethylformamide containing triethylamine
led to the formation of pyrazolotriazolopyrimidine derivative (19).
This reaction proceeded via initial formation of intermediate 18
followed by intramolecular cyclization and loss of hydrogen sulfide
(lead acetate paper). The IR spectrum of 17 did not contain
(N]C]S) band, also, its 1H NMR spectrum revealed the absence of
triplet-quartet pattern of the ethyl group. The N]C]S and ethyl
signals were also absent in 13C NMR spectra. The mass spectrum of
17 showed a molecular ion peak m/z at 301 (Mþ, 0.11%), with a base

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
O
O
N
N
NH 2
Ph
1
a
O
O
O
N
NH
O
c
N
b
N
S
N
Ph
N
H
N
N
S
NH 2
S
N
Ph
N
C
S
N
Ph
N
H
NH 2
15
16
18
- H 2
S
d
- H 2
S
O
O
O
H
N
N
N
N
S
N
N
N
N
N
Ph
N
H
S
N
Ph
N
N
H
N
N
H
O
HO
Ph
17
19
O
N
N
N
N
Ph
O
O
20
Scheme 3. Reagents: a, thiophosgene; b, o-phenylenediamine/chloroform; c, thiosemicarbazide/dimethylformamide/triethylamine; d, anthranilic acid/dioxane.
peak at 227. The IR spectrum of 19 revealed the absence of
(N]C]S) band and the presence of (2NH) bands at 3330,
2.3. Radioprotective activity
3300 cmꢀ1
.
1H NMR spectrum showed 2 NH signals at 8.1 and
Radiation exposure significantly increases lipid peroxidation
(LPx), such increase seems to be due to the inactivation of scav-
enger enzymes induced by reactive oxygen species (ROS). Oxidative
stress occurs in living organisms when the production of ROS
exceeds the ability of organisms to prevent their accumulation
[33,34]. Such elevation of LPx is accompanied by decline in GSH
content and in the activity of related antioxidant enzyme SOD.
Additionally LPx can be initiated by hydrogen abstraction from lipid
molecules by lipid radiolytic products. This leads to permeability
8.3 ppm. 13C NMR showed C]S signal at 191 ppm. The mass
spectrum of compound 19 showed a molecular ion peak at 284 (Mþ,
100%) as a base peak.
Finally, reaction of compound 15 with anthranilic acid in
dimethylformamide containing triethylamine afforded pyr-
azolopyrimidobenzoxazine derivative 20 (Scheme 3). The IR spec-
trum of 20 showed the presence of (2C]O) bands at 1690,
1680 cmꢀ1, 1H NMR spectrum exhibited signals at 7.3–8.1 ppm for
10 aromatic protons and the mass spectrum of 20 revealed
a molecular ion peak m/z at 330 (Mþ, 0.1%), with a base peak at 86.
Table 1
In-vitro cytotoxic activity of some new synthesized compounds.
2.2. In-vitro anticancer activity
Compd. no.
Non-viable cells (%)
Concentration (
g mlꢀ1
IC50
(
m
g/ml)a
m
)
Doxorubicin, the reference drug used in this study, is one of the
most effective antitumor agents used to produce regressions in
acute leukemias, Hodgkin’s disease and other lymphomas. The
relationship between surviving ratio and drug concentration was
plotted to obtain the survival curve of Ehrlich Ascites Carcinoma
(EAC) cell line. The response parameter calculated was IC50 value
which corresponds to the compound concentration causing 50%
mortality in net cells.
All the newly synthesized compounds were evaluated for their
in-vitro cytotoxic activity except for compounds 4, 19 which were
difficult to solubilize. Compounds 2 and 9 showed some activity
lower than the reference drug doxorubicin (CAS-23214-92-8)
(Table 1).
100
75
50
25
2
5
6
7
9
12
14
17
80
10
0
30
50
20
0
0
30
100
20
10
0
10
30
10
0
0
30
75
10
10
0
10
20
10
0
0
10
55
10
10
10
10
10
10
10
10
10
20
90
>100b
>100b
>100b
100
>100b
>100b
>100b
>100b
52
20
Doxorubicin
a
IC50 value: corresponds to the compound concentration causing 50% mortality
in net cells.
b
IC50 > 100 mg/ml is considered inactive.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
175
changes, secondary alteration in membrane proteins and other
sequences [35,36].
(Varian, Fort Collins, USA). Chemicals used are supplied from Sigma–
Aldrich (SIGMA–ALDRICH Chemie GmbH, Steinheim, Germany).
2.3.1. Effect of compound 5 and/or
peroxidation and antioxidant status
g
-irradiation on lipid
3.1.1. 5-Benzyl-1-phenyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-
4-one (2)
Several compounds containing a thione and/or an amino group
revealed radioprotective activity [6,15,16]. On this basis, compound
5 containing both the abovementioned groups was selected to be
evaluated as a possible radioprotective agent.
A mixture of compound 1 (9.24 g, 0.04 mol) [24] and benzylamine
(4.2 g, 0.04 mol) in decaline (8.28 g, 0.06 mol) and triethylortho-
formate (15 ml) was refluxed for 18 h, the reaction mixture was
cooled and filtered, the obtained solid was crystallized from ethanol.
Yield, 84 %; m.p. 169–171 ꢂC; IR (KBr, cmꢀ1): 3055 (CH arom.), 2956,
2.3.1.1. Glutathione level in blood (GSH). As summarized in Table 2,
a very high significant depletion in GSH level was observed in blood
of irradiated mice compared to their corresponding controls.
Treatment with compound 5 prior to irradiation ameliorated
radiation-induced GSH depletion in blood to a great extent.
2866 (CH aliph.), 1688 (C]O), 1576 (C]N). 1H NMR (CDCl3)
d: 5.2 [s,
2H, CH2], 7.2–8.0 [m, 11H, CH pyrazole þ Ar-H], 8.28 [s, 1H, CH
pyrimidine]. MS, m/z (%): 302 [Mþ] (100). Anal. Calcd. For C18H14N4O:
C, 71.52; H, 4.64; N, 18.54. Found: C, 71.70; H, 4.45; N, 18.82.
3.1.2. Bis-1,1’-diphenyl-pyrazolo[3,4-d]pyrimido
2.3.1.2. Superoxide dismutase (SOD) activity in blood. Results in
[3,4-c][1,2,4,5]tetrazine (4)
Table 2, showed a very high significant inhibition in SOD activity in
3.1.2.1. Method (A). A mixture of compound 3 [25] (2.27 g, 0.01 mol)
and phosphorous pentasulfide (2.66 g, 0.012 mol) in pyridine (20 ml)
was refluxed for 12 h, the reaction mixture was concentrated and
allowed to cool down, the solid obtained was crystallized from
ethanol. Yield, 64%; m.p. >360 ꢂC; IR (KBr, cmꢀ1): 3108 (CH arom.),
blood of animals exposed to
compound 5 and exposed to
g
-irradiation. Mice treated with
g
-irradiation revealed a non signifi-
cant change in SOD activity in blood when compared to the control
level.
1596 (C]N). 1H NMR (CDCl3) : 7.5–8.0 [m, 14H, Ar-H]. 13C NMR
d
2.3.1.3. Lipid peroxidation content (MDA) in plasma. As shown in
(CDCl3) d: 109.2, 122.5, 128.3, 132.8, 140.7, 142.0, 145.1, 151.8, 164.2.
Table 2, mice exposed to
g-irradiation showed a high significant
MS, m/z (%): 418 [Mþ] (100). Anal. Calcd. For C22H14N10: C, 63.16; H,
elevation in Malondialdehyde (MDA) level in plasma compared
with the control values. Treatment with compound 5 prior to
irradiation exhibited a remarkable reduction in MDA levels in blood
as compared with irradiated group.
From the above findings and from results of Table 2, it is obvious
that treatment with compound 5 reduced LPx, and ameliorated to
a great extent GSH content and the activity of SOD in blood of mice
3.35; N, 33.49. Found: C, 63.37; H, 3.31; N, 33.76.
3.1.2.2. Method (B). A solution of 5 (2.43 g, 0.01 mol) in pyridine
(20 ml) was refluxed for 24 h. The reaction mixture was poured into
ice cooled water and the obtained solid was crystallized from
ethanol (m.p. and m.m.p.).
exposed to
g-irradiation.
3.1.3. 5-Amino-1-phenyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidine-
4-thione (5)
A mixture of 3 (2.27 g, 0.01 mol) and phosphorous pentasulfide
(2.66 g, 0.012 mol) in xylene (20 ml) was heated under reflux for
3 h. The reaction mixture was filtered on hot, the solid separated
from the filtrate was crystallized from ethanol. Yield, 69%; m.p.
192–194 ꢂC; IR (KBr, cmꢀ1): 3260, 3240 (NH2), 3024 (CH arom.),
3. Experimental
3.1. Chemistry
1598 (C]N),1216 (C]S). 1H NMR (CDCl3)
[m, 7H, Ar-H]. 13C NMR (CDCl3)
d: 5.7 [s, 2H, NH2], 7.3–8.2
Melting points are uncorrected and were determined on a Stuart
melting point apparatus (Stuart Scientific, Redhill, UK). Elemental
analyses (C, H, N) were performed on Perkin–Elmer 2400 analyzer
(Perkin–Elmer, Norwalk, CT, USA) at Microanalytical Laboratories of
the Faculty of Science, Cairo University. All compounds were within
ꢁ0.4% of the theoretical values. The IR spectra (KBr) were measured
on (Pye Unicam SP 1000 IR spectrophotometer Thermoelectron,
Egelbach, Germany). 1H NMR spectra were obtained on a Bruker
proton NMR-Avance 300 (300, MHz) (Bruker, Munich, Germany),
using tetramethylsilane (TMS) as internal standard. Splitting
patterns were designated as follows: s: singlet; d: doublet; t: triplet;
m: multiplet. Mass spectra were run on Varian MAT 311-A70ev
d: 109.1, 123.3, 130.0, 132.6, 142.3,
144.1,145.0,169.2 and 210.0 ppm, MS, m/z (%): 243 [Mþ] (100). Anal.
Calcd. For C11H9N5S: C, 54.32; H, 3.70; N, 28.81. Found: C, 54.62; H,
3.55; N, 29.12.
3.1.4. 5-(4-Benzylidene-5-oxo-2-phenyl-4,5-dihydro-imidazol-
1-yl)-1-phenyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidine-4-one (6)
A mixture of compound 3 (2.27 g, 0.01 mol) and 4-benzylidene-
2-phenyl-4H-oxazol-5-one (2.49 g, 0.01 mol) in glacial acetic acid
(20 ml) containing fused sodium acetate (2 g) was refluxed for 3 h,
The reaction mixture was cooled and then poured onto cold water,
Table 2
Effect of compound 5 administration on blood glutathione (GSH) content, activity levels of superoxide dismutase (SOD) and plasma lipid peroxide concentrations (LPx) of
normal and irradiated mice.
Groups
GSH mg/dl
SOD U/ml
LPx
m
mol/ml
Survival (%)a
Control
CMC
Radiation
Compound 5
Compd. 5 þ Rad
47.28 ꢁ 3.02
2.4 ꢁ 0.16
120.88 ꢁ 1.37
100
100
80
100
100
45.42 ꢁ 0.97 (ꢀ3.94%)
33.56 ꢁ 2.60** (ꢀ29.02%)
49.63 ꢁ 2.31 (þ4.95%)
51.84 ꢁ 0.97 (þ9.63%)
2.25 ꢁ 0.17 (ꢀ6.31%)
1.69 ꢁ 0.12** (ꢀ29.7%)
2.35 ꢁ 0.03 (ꢀ2.04%)
2.18 ꢁ 0.18 (ꢀ9.30%)
113.66 ꢁ 3.38 (ꢀ5.97%)
140.82 ꢁ 7.38* (þ16.50%)
127.32 ꢁ 11.77 (þ5.33%)
113.40 ꢁ 5.05 (ꢀ6.19%)
Each value is the mean of ten mice ꢁ SE. In parentheses: percentage of change from control group.
*High significant difference from control group at P < 0.01.
**Very high significant difference from control group at P < 0.001.
CMC: Carboxymethylcellulose.
a
percentage of live animals after 7 days from the radiation dose.

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
the solid obtained was crystallized from ethanol. Yield, 74%; m.p.
185–187 ꢂC; IR (KBr, cmꢀ1): 3090 (CH arom.), 2929, 2850 (CH
Anal. Calcd. For C13H11N3O2S: C, 57.14; H, 4.03; N, 15.38. Found: C,
57.32; H, 4.38; N, 15.62.
aliph.), 1690, 1680 (2C]O), 1577 (C]N). 1H NMR (CDCl3)
d: 7.7–8.3
[m, 18H, Ar-H þ CH]. MS, m/z (%): 458 [Mþ] (100). Anal. Calcd. For
C27H18N6O2: C, 70.74; H, 3.93; N, 18.34. Found: C, 70.50; H, 4.26; N,
18.63.
3.1.9. 1-Phenyl-4H-11H-pyrazolo[3,4-d]pyrimido
[2,1-b]benzo[d]imidazol-4-one (17)
A mixture of 15 (2.73 g, 0.01 mol) and o-phenylinediamine
(1.08 g, 0.01 mol) in CHCl3 (20 ml) was refluxed for 5 h. The solid
obtained was crystallized from ethanol. Yield, 80%; m.p. 213–
215 ꢂC; IR (KBr, cmꢀ1): 3306 (NH), 3029 (CH arom.), 1707 (C]O),
3.1.5. 7-Phenyl-7H-pyrazolo[4,3-e]-[1,2,4]triazolo
[1,5-c]pyrimidine-2-thione (7)
A mixture of compound 3 (2.27 g, 0.01 mol) and thiourea (0.76 g,
0.01 mol) was fused on an oil bath at 220 ꢂC for 30 min., the solid
obtained was crystallized from ethanol. Yield, 86%; m.p. 289–
291 ꢂC; IR (KBr, cmꢀ1): 3300 (NH), 3020 (CH arom.), 1559 (C]N),
1636 (C]N). 1H NMR (CDCl3)
azole], 8.3 [s, 1H, NH]. 13C NMR (CDCl3)
d
: 7.4–7.9 [m, 10H, Ar-H þ CH pyr-
d
: 110.1, 116.8, 118.2, 120.0,
121.8, 122.9, 125.3, 127.4, 130.7, 132.0, 141.1, 142.4, 143.2, 162.1, 167.0.
MS, m/z (%): 301 [Mþ] (0.11), 227 (100). Anal. Calcd. For C17H11N5O:
C, 67.77; H, 3.65; N, 23.26. Found: C, 67.51; H, 3.89; N, 23.50.
1221 (C]S). 1H NMR (DMSO-d6)
d: 7.5–8.1 [m, 5H, Ar-H], 8.5 [s, 1H,
CH pyrazole], 9.0 [s, 1H, CH pyrimidine], 9.4 [s, 1H, NH]. Anal. Calcd.
For C12H8N6S: C, 53.73; H, 2.99; N, 31.34. Found: C, 53.92; H, 3.34; N,
31.58.
3.1.10. 1-Phenyl-7-thioxo-1,6,7,8-tetrahydro-pyrazolo
[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-4-one (19)
A
mixture of compound 15 (2.73 g, 0.01 mol) and thio-
3.1.6. 7-(4-(Dimethylthio-imidebenzene-sulphonamide)amino)-
4H-1-phenyl-[1,3,4]-oxadiazolo[3,2-a]pyrazolo[3,4-d]pyrimidin-
4-one (12)
semicarbazide (0.91 g, 0.01 mol) in dimethylformamide (15 ml)
containing 3 drops of triethylamine was refluxed for 3 h till the
evolution of H2S gas has ceased (no black precipitate formed on
a lead acetate wetted paper), the reaction mixture was concen-
trated and allowed to cool down, the solid obtained was crystal-
lized from ethanol. Yield, 74%; m.p. >360 ꢂC; IR (KBr, cmꢀ1): 3330,
3300 (2NH), 3080 (CH arom.), 1680 (C]O), 1620 (C]N), 1250
A mixture of 9 (0.97 g, 0.004 mol) [25], and red HgO (0.87 g,
0.004 mol), was dissolved in dimethylformamide (15 ml). The dis-
solved mixture was added drop wise to a solution of 10 (1.46 g,
0.004 mol) [27] in dimethylformamide (15 ml) containing 3 drops
of triethylamine and maintained at 70 ꢂC in a water bath for 24 h.
The reaction mixture was filtered on hot and the filtrate was cooled
using an ice bath and poured onto (100 ml) H2O. The solid obtained
was crystallized from ethanol. Yield, 67%; m.p. 216–218 ꢂC; IR (KBr,
cmꢀ1): 3334 (NH), 3066 (CH arom.), 2950, 2855 (CH aliph.), 1706
(C]S). 1H NMR (CDCl3)
8.3 [2s, 2H, 2NH]. 13C NMR (CDCl3)
d
: 7.2–7.8 [m, 6H, Ar-H þ CH pyrazole], 8.1,
d
: 111.0, 122.3, 126.1, 131.7, 141.1,
142.3, 143.8, 160.5, 164.2, 191.0 (C]S). MS, m/z (%): 284 [Mþ] (100).
Anal. Calcd. For C12H8N6OS: C, 50.70; H, 2.82; N, 29.58. Found: C,
50.81; H, 2.66; N, 29.34.
(C]O),1613 (C]N),1370,1145 (SO2). 1H NMR (CDCl3)
d: 1.6 [s, 6H, 2
SCH3], 7.2–7.8 [m, 10H, Ar-H þ CH pyrazole], 9.4 [s, 1H, NH]. 13C
3.1.11. 1-Phenyl-4-H-pyrazolo[3,4-d]pyrimido
[2,1-b]benzo[1,3][d]oxazine-4,10-dione (20)
NMR (CDCl3)
d
: 16.1 (CH3), 111.1, 121.8, 124.4, 130.2, 133.9, 137.0,
142.1, 146.5, 154.2, 157.1, 161.7, 163.0 167.5, 168.3. MS, m/z (%): 527
[Mþ] (2.72), 156 (100). Anal. Calcd. For C21H17N7O4S3: C, 47.82; H,
3.23; N, 18.60. Found: C, 47.66; H, 3.52; N, 18.33.
A mixture of compound 15 (2.73 g, 0.01 mol) and anthranilic
acid (1.37 g, 0.01 mol) in dioxane (20 ml) containing 3 drops of
triethylamine was refluxed for 5 h. The reaction mixture was
concentrated and allowed to cool and the solid obtained was
crystallized from dioxane. Yield, 79%; m.p. 248–250 ꢂC; IR (KBr,
cmꢀ1): 3065 (CH arom.), 1690, 1680 (2C]O), 1590 (C]N). 1H NMR
3.1.7. 1-Phenyl[1,3,4]oxadiazolo[3,2-a]pyrazolo[3,4-d]pyrimidin-
4(1H)-one (14)
A solution of 9 (2.43 g, 0.01 mol) in triethylorthoformate (20 ml)
was heated under reflux for 3 h. The product obtained was crys-
tallized from ethanol. Yield, 71%; m.p. 235–237 ꢂC; IR (KBr, cmꢀ1):
(CDCl3)
d
: 7.3–8.1 [m, 10H, Ar-H þ CH pyrazole]. 13C NMR (CDCl3)
d:
112.0, 115.4, 120.2, 121.7, 125.0, 126.5, 129.2, 131.6, 134.0, 139.2,
140.5,141.2,142.0,161.6,164.5,165.0. MS, m/z (%): 330 [Mþ] (0.1), 86
(100). Anal. Calcd. For C18H10N4O3: C, 65.45; H, 3.03; N, 16.97.
Found: C, 65.26; H, 3.33; N, 16.65.
3050 (CH arom.), 1681 (C]O), 1585 (C]N). 1H NMR (DMSO-d6)
d:
7.5–8.0 [m, 6H, Ar-H þ CH oxadiazole], 8.4 [s, 1H, CH pyrazole]. 13C
NMR (DMSO-d6) d: 111.0, 123.3, 129.4, 132.7, 144.0, 145.3, 146.7,
157.2, 163.1, 168.0. MS, m/z (%): 253 [Mþ] (100). Anal. Calcd. For
C12H7N5O2: C, 56.92; H, 2.77; N, 27.67. Found: C, 56.60; H, 2.57; N,
27.92.
3.2. Biological testing
3.2.1. Chemicals and facilities
3.1.8. 5-Isothiocyanato-1-phenyl-1H-pyrazole-4-carboxylic acid
ethyl ester (15)
All chemicals and reagents are supplied from Sigma–Aldrich
(SIGMA–ALDRICH Chemie GmbH, Steinheim, Germany). Facilities
A solution of 5-amino-1-phenyl-1H-pyrazole-4-carboxylic acid
ethyl ester 1 (2.31 g, 0.01 mol) [24] in chloroform (100 ml) was
added during 40 min. to a well stirred mixture of thiophosgene
(1.15 g, 0.01 mol), sodium hydrogen carbonate (1.26 g, 0.015 mol),
H2O (50 ml) and chloroform (20 ml). The mixture was stirred at
room temperature for further 90 min., the organic layer was
separated and washed twice with H2O, then dried over anhydrous
Na2SO4 and evaporated under reduced pressure to give a semisolid
yellowish mass. Yield, 68%; m.p. 50–52 ꢂC; IR (KBr, cmꢀ1): 3050 (CH
arom.), 2930, 2860 (CH aliph.), 2150 (NCS), 1720 (C]O), 1590
including animal house, biochemical equipments and g-irradiation
source have been made available by the National Centre for Radi-
ation Research and Technology (NCRRT), Cairo, Egypt. Whole body
g-irradiation was performed using Gamma cell-40 (Caesium-137
source – Canada).
3.2.2. Animals
Female Swiss albino mice weighing 25–30 g were used in this
study (The Holding Company for Biological Products and Vaccines,
VACSERA, Cairo, Egypt). Mice were housed at a constant tempera-
ture (24 ꢁ 2 ꢂC) with alternating 12 h light and dark cycles and fed
standard laboratory food and water. Tests were made in consider-
ation of the internationally valid guidelines. The National Centre for
Radiation Research and Technology (NCRRT), Cairo, Egypt is
(C]N). 1H NMR (CDCl3)
d
: 1.7 [t, 3H, CH3, J ¼ 6.7 Hz], 4.2 [q, 2H, CH2,
J ¼ 6.7 Hz], 7.2–7.7 [m, 5H, Ar-H], 8.1 [s, 1H, CH pyrazole]. 13C NMR
(CDCl3) d: 16.1 (CH3), 65.0 (CH2), 109.2, 122.5, 128.1, 133.5, 140.1,
144.6, 146.2, 147.3, 170.0. MS, m/z (%): 273 [Mþ] (47.7%), 185 (100%).

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 45 (2010) 171–178
177
concerned with biological and animal studies and has an approval
of an institution responsible for animal ethics.
3.2.5.4. Samples collection. Animals were fasted for 16 h prior to
sampling. Samples were collected after 7 days of the irradiation
dose. Whole blood was collected by heart puncture after light
anesthesia using heparinized syringes. One part was used for GSH,
and SOD estimations. The separated plasma from heparinized
blood was used for the determination of lipid peroxide as
malondialdehyde.
3.2.3. In-vitro anticancer activity
Ehrlich Ascites Carcinoma cells were obtained by needle aspi-
ration of ascetic fluid from the preinoculated mice under aseptic
conditions. Tumor cells suspension (2.5 ꢃ106 per ml) was prepared
in saline. Tested compounds solutions were prepared with various
dilutions by dissolving: 1, 0.75, 0.5 and 0.25 mg of the tested
compounds in a mixture of 0.7 ml dimethylsulfoxide (DMSO) and
0.3 ml saline.
3.2.5.5. Analytical procedures. Lipid peroxide level in plasma was
ascertained by the formation of MDA and measured as described by
Yoshioka et al. [38], GSH content was determined according to
Beutler et al. [39], SOD was quantized according to Minami et al.
[40].
In a set of sterile test tubes 0.8 ml saline, 0.1 ml of each of the
tested compounds solutions (corresponding to 100, 75, 50, 25 mg)
were mixed, then 0.1 ml of tumor cell suspension was added. The
test tubes were incubated at 37 ꢂC for 2 h. Trypan blue exclusion
test was carried out to calculate the percentage of non-viable cells
after 2 h of incubation [37]. The results of in-vitro cytotoxic activity
experiments are presented in (Table 2).
3.2.5.6. Statistical analysis. Student’s t test [41] was used for the
evaluation of the biochemical parameters.
Acknowledgement
3.2.4. Experimental tumor cells and tumor transplantation
A line of EAC was used in this study.
The parent line was kindly supplied by the National Cancer
Institute, Cairo University, Egypt. The tumor cells were maintained
by weekly intraperitoneal transplantation of cells.
The authors express their sincerest thanks to Prof. Dr. Eman
Noaman, Dept. of Radiation Biology, National Center for Radiation
Research and Technology, Atomic Energy Authority, Nasr City,
Cairo, Egypt, for performing the anticancer and radioprotective
activities.
3.2.5. Radioprotective activity
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