Synthesis and anticancer effects of some novel pyrazolo[3,4-d]pyrimidine derivatives by generating reactive oxygen species in human breast adenocarcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2011.01.013

A series of novel substituted pyrazolo[3,4-d]pyrimidines (compounds 2-12) were synthesized starting with pyrimidinone derivative 1. Their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7) cell lines has been investigated and most of the te

Full text of DOI:10.1016/j.ejmech.2011.01.013

European Journal of Medicinal Chemistry 46 (2011) 1019e1026
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer effects of some novel pyrazolo[3,4-d]pyrimidine
derivatives by generating reactive oxygen species in human breast
adenocarcinoma cells
,
b
b
Aymn E. Rashad a , Abeer E. Mahmoud , Mamdouh M. Ali
*
a Photochemistry Department, National Research Center, Dokki, Cairo, Egypt
b Biochemistry Department, Division of Genetic Engineering and Biotechnology, National Research Center, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel substituted pyrazolo[3,4-d]pyrimidines (compounds 2e12) were synthesized starting
with pyrimidinone derivative 1. Their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7)
cell lines has been investigated and most of the tested compounds exploited potent cytotoxic activity
against MCF-7 cell lines comparable to the activity of the commonly used anticancer drug cisplatin.
Received 9 November 2010
Received in revised form
3 January 2011
Accepted 11 January 2011
Available online 15 January 2011
Treatment of MCF-7 cells with increased doses (2, 5, 10, 20 mg/ml) of the tested compounds revealed that
the activity of superoxide dismutase and the level of hydrogen peroxide were significantly increased,
while the activities of catalase and glutathione peroxidase and the levels of reduced glutathione were
significantly lowered compared with control MCF-7 cells. In general, acyclic nucleoside derivative 4
revealed the highest anticancer activity among the other tested compounds.
Keywords:
Pyrazolo[3,4-d]pyrimidines
S-Alkyl derivatives
Nucleosides
Ó 2011 Elsevier Masson SAS. All rights reserved.
Reactive oxygen species
MCF-7
1. Introduction
persuaded goals of contemporary medicinal chemistry [12]. Cyto-
toxic drugs remain the mainstay of cancer chemotherapy and are
As part of our ongoing research program on heterocyclic
compounds which may serve as leads for designing novel chemo-
therapeutic agents, we were particularly interested in pyrazoles
and fused pyrazolopyrimidines [1e3]. Pyrazolo[3,4-d]pyrimidines
and their related fused heterocycles are considered to be an
important chemical synthon of various physiological significance
and pharmaceutical utility. They are of considerable chemical and
pharmacological importance because of their structural similarities
with purine and many of their derivatives were reported to possess
variety of biological effects including antiviral [4], antimicrobial [5],
anti-inflammatory [6], and xanthine oxidase inhibitor [7] activities.
Moreover, many pyrazolo[3,4-d]pyrimidines contributed to the
quest for an ultimate antitumor chemotherapeutic agent [8e10]
especially when heterocyclic moieties reported to possess anti-
tumor activity, such as thienopyrimidine [11], which could be
incorporated into the pyrazolopyrimidine nucleus.
being administrated with novel ways of therapy such as inhibitors
of signals and therefore it is important to discover novel cytotoxic
agents with spectra of activity and toxicity that differ from those of
current agents.
Reactive oxygen species (ROS), such as superoxide anions
(Oꢀ2 ) and hydrogen peroxide (H2O2) are normal byproducts of
cellular aerobic metabolism and superoxide anions are
secondarily converted to H2O2 by the superoxide dismutase
(SOD). H2O2 has a dual role in cellular homeostasis and low
levels of intracellular H2O2 stimulate cell growth [13], whereas
high levels of H2O2 lead to cell senescence and apoptosis [14].
Several anticancer agents like 5-fluorouracil [15], organo-
platinum compounds [16], and anthracyclines [17], increase
levels of ROS. Tumor cells have higher levels of reactive oxygen
species than normal cells and are therefore more sensitive to
the additional oxidative stress generated by anticancer agents
[18]. Cells also contain a variety of free radical scavenging
systems that protect them from the effects of drugs that
generate oxygen free radicals such as catalase and glutathione
peroxidase are enzymes that detoxify H2O2 [19].
On the other hand, the development of potent and effective
antineoplastic drugs has become one of the most intensely
As a result of many extensive investigations in the field of anti-
cancer drugs, different types of anticancer chemicals have appeared
* Corresponding author. Fax: þ20 20337 0931.
E-mail address: aymnelzeny@yahoo.com (A.E. Rashad).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.013

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A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
such as anthracylines and cisplatin that induce apoptotic cell death
in cancer cells via generation of active oxygen species [20].
In the light of the aforementioned facts, and as a continuation to
our investigations on the synthesis of biologicallyactive heterocyclic
compounds [21e23], we were prompted to synthesize some new
heterocyclic pyrazolo[3,4-d]pyrimidine thionucleoside analogs by
introducing various substituents at 4-position (such as alkyl, ary-
lalkyl, and sugar moieties) in an attempt to improve the anticancer
profiles of these products on human breast adenocarcinoma cell line
MCF-7, with special attention to the causal relationship of reactive
oxygen species induction in the cells.
Similarly, when derivative 8 was treated with b-D-glycopyranose
pentaacetate in the presence of SnCl4, it afforded product identical in
all respects to the thioglycoside 11. The 1H NMR spectrum of
compound 11 showed a doublet at 6.26 ppm (J ¼ 8.50 Hz) assigned to
the anomeric proton of the glucose moiety with a diaxial orientation
of H-10 and H-20 indicating the
b-configuration. The other protons of
the glucopyranose ring resonate at 4.08e5.55 ppm, while the four
acetoxy groups appear as four singlets in the 1.97e2.06 ppm region
providing further verification of the structure [23].
Deprotection of 11 with methanolic ammonia afforded the free
thioglycoside 12 (Scheme 1). The 1H NMR spectrum of the latter
compound showed the anomeric proton as a doublet at
(J ¼ 8 Hz) indicative for the -configuration, and the signals of the
other six carbon-bonded glucose protons appear as multiplets at
3.10e4.25, while the signals of the four hydroxyl groups of the
glucose moiety are observed at 4.52e5.34 (exchangeable with
D2O). The 13C NMR spectrum of 12 is characterized by the absence
d 6.12
2. Results and discussion
b-D
2.1. Chemistry
d
d
In recent reports from our laboratory, we described the prepa-
ration of some novel functionalized azine nucleosides with prom-
ising antitumor activity [11,24]. These common features
encouraged us to develop a new straightforward route for synthesis
of pyrazolo[3,4-d]pyrimidine thionucleoside analogs in an attempt
to improve the anticancer profiles of these products. Thus, heating
of compound 1 [25] in phosphorus oxychloride, gave the corre-
sponding 4-chloropyrazolo[3,4-d]pyrimidine derivative 2. The
structure of the latter compound was confirmed on the basis of its
elemental analysis and spectral data (Section 3, Scheme 1). In
particular, the IR spectrum of compound 2 indicated the absence of
absorption bands assignable to the NH, C]O and the mass spec-
trum showing the isotopic pattern due to the presence of chlorine
atom (Section 3).
of the C]S signal and the presence of a signal at
d
85.49 corre-
61.18, 67.64,
sponding to the C-10 atom. Another five signals at
d
69.59, 71.38 and 74.81 are assigned to C-60, C-40, C-20, C-30 and C-50,
respectively (Section 3).
2.2. Anticancer activity of prepared compounds
Prepared compounds (1, 2, 3, 4, 5, 6, and 10) were screened in
vitro using MCF-7 cell line. Cells exposed to different concentra-
tions (2, 5, 10 and 20 mg/ml) of the tested compounds exhibited
a dose-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), revealing preservation of
viability in the presence of the tested compounds (Fig. 1). The doses
of the tested compounds were selected based on the preliminary
studies.
The tumor cell line showed normal growth in our culture system
and DMSO did not seem to have any noticeable effect on cellular
growth. A gradual decrease in viability of cancer cells was observed
with increasing concentration of the tested compounds, in a dose-
dependent inhibitory effect. The median growth inhibitory
concentration (IC50) after 24 h was for compounds 1, 2, 3, 4, 5, 6, and
Pyrazolo[3,4-d]pyrimidine-5(4H)-thione derivative
3
was
prepared by treatment of the 4-chloroderivative 2 with thiourea in
refluxing ethanol. Analytical and spectral data of compound 3 are in
agreement with the proposed structure, particularly the presence
of C]S signal in the IR and 13C NMR spectra confirmed its structure.
Moreover, the 1H NMR spectra showed a singlet for NH proton at
d
13.76 ppm which was removed on deuteration (Section 3).
Treatment of compound 3 with various alkyl halides like: ethyl
iodide, 2-bromoethyl methyl ether, 2-bromoethyl ethyl ether or
ethyl bromoacetate in alcoholic potassium hydroxide solution, gave
a series of S-substituted pyrazolo[3,4-d]pyrimidine derivatives 4e7
in good to excellent yields. The IR spectra of the latter compounds
indicated the absence of the NH and C]S signals and their 1H NMR
and 13C NMR spectra revealed the presence of ethyl, methoxyethyl,
ethoxyethyl, and ethoxycarbonylmethyl signals, respectively
(Section 3).
10 was 4.40, 7.00, 8.00, 3.00, 4.30, 6.40, and 9.40 mg/ml, respectively.
It is clear from the data that the comparison of the cytotoxicity
against MCF-7 cell line (Fig. 1) of the tested compounds has shown
that the growth inhibitory potency follows the order,
4 > 5 > 1 > 6 > 2 > 3 > 10. Compound 4 was the best compound
exerting a significant cytotoxic effect on MCF-7 cells compared with
cisplatin (the commonly used anticancer drug).
There is equilibrium between a free radical (FR)/reactive oxygen
species (ROS) formation and endogenous antioxidant defense
mechanisms, but if this balance is disturbed, it 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. In recent years,
increasing experimental and clinical data has provided compelling
evidences for the involvement of oxidative stress in large number of
pathological states including carcinogenesis [27]. Oxidative stress
however, is not always detrimental but selective oxidative stress
sometimes is desirable and can be utilized therapeutically also.
There are numerous drugs which are known to act by the mecha-
nism of oxidative stress and can be utilized therapeutically like
chloroquin, quinine, mefloquine, primaquin, artemisinin [28] and
cisplatin [29] etc. Recently, new therapeutic strategies that take
advantage of increased reactive oxygen species or inhibition of
endogenous antioxidant defense, hence producing a state of
oxidative stress selectively in cancer cells have gained importance.
To elucidate the mechanism by which the prepared compounds
exert their antitumor activities, we estimated the activities of the
In addition, heating of compound 3 with hexamethyldisilazane
(HMDS) in the presence of ammonium sulfate gave the trime-
thylsilylthiopyrimidine derivative
treated with 1-O-acetyl-2,3,5-tri-O-benzoyl-
8
which was subsequently
-ribofuranose in
b-D
the presence of SnCl4 according to the method of Niedballa and
Vorbrüggen [26] to afford the corresponding S-riboside 9 (Scheme
1). The 1H NMR spectrum of compound 9 showed a doublet at
0
0
d
6.29 ppm (J1 ,2 ¼ 4.40 Hz) assigned to the anomeric proton
indicating the -configuration [24] and the other sugar protons
resonate at
3.95e4.58 ppm. The 13C NMR spectrum of 9 is char-
acterized by the absence of the C]S signal and the presence of
a signal at
97.87 ppm corresponding to the C-10 atom. Another
b
d
d
four signals at d
61.66, 70.15, 71.32 and 79.93 are assigned to C-50, C-
30, C-20, and C-40, respectively (Section 3).
Debenzoylation of the blocked riboside 9 was achieved in
methanolic ammonia to afford the free 1-b-D-ribofuranosyl deriv-
ative 10. The IR and IH NMR spectra of the latter compound showed
the presence of hydroxyl groups and its 13C NMR spectrum
providing further verification of the structure (Section 3).

A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
1021
Cl
S
O
N
POCl3, heat
N
NH
thiourea, EtOH,
heat
NH
N
N
N
N
R
N
N
R
N
N
R
2
3
1
HMDS, (NH4)2SO4
heat
KOH, EtOH,
R1X
heat
SSiMe3
SR1
N
N
N
N
N
N
N
N
R
R
8
N gas, SnCl ,
dry CH2Cl2
2
4
4 -7
OAc
BzO
AcO
O
OAc
O
OAc
R1
X
I
CH2CH3
4:
5:
6:
7:
OAc
OBz
OBz
OAc
S
BzRibose
N
Br
Br
Br
CH2CH2OCH
3
S
AcGlucose
N
CH CH OCH CH
3
2
2
2
CH COOCHCH
3
2
2
N
N
N
R
N
N
N
R
9
11
MeOH/NH3 gas
MeOH/NH3 gas
r.t.
r.t.
S
Ribose
N
S
Glucose
N
N
N
N
N
R
N
N
R
12
10
HO
HO
O
O
OH
OH
N
Ribose
Glucose
=
R
=
=
OH
OH
S
N
Me
OH
Scheme 1. Synthesis routes of compounds 2e12.
free radical-metabolizing enzymes superoxide dismutase (SOD),
catalase (CAT), and glutathione peroxidase (GSH-Px), as well as the
levels of glutathione (GSH) and H2O2 in MCF-7 cells in the presence
or absence of the tested compounds comparing to cisplatin.
As shown in Table 1, in general treatment of the cells with
different compounds resulted in dose-dependent increase in the
activity of SOD and the level of H2O2 higher than those of control
accompanied with dose-dependent depletion in the activity of CAT
and GSH-Px, as well the level of GSH. These changes were in the
orderof 4 > 5 >1 >6 > 2 > 3 >10 which is in the accordancewith the
order of cytotoxicity activity of the tested compounds
(4 > 5 >1 >6 > 2 > 3 >10 ) (Fig.1). The highest activity was found for
compound 4 and 5, 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 which showed the highest antitumor
activity. These results indicate that the antitumor effect of the
present compounds may be exerted at least partly by production of
H2O2 and the H2O2 produced should be rapidly removed through the
activation of CAT and GSH-Px. The present results show that activi-
ties of CAT and GSH-Px and the level of reduced GSH are lowered in
groups treated with the prepared compounds (in dose-dependent
manner) compared with the control group (Table 1). Consequently,
the excess H2O2 produced in tumorcells with the compounds cannot
be removed and the accumulation of H2O2 in tumor cells should be
partly the cause of tumor cell killing. Thus the results of the present
study are consistent with the hypothesis that the prepared

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A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
110
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
Compound 4
25
Compound 1
Compound 5
Compound 2
Compound 6
Compound 3
Compound 10
Cisplatin
Fig. 1. Effect of different concentrations (2, 5, 10 and 20 mg/ml) of the prepared compounds (1, 2, 3, 4, 5, 6, and 10) on MCF-7 cells growth measured by MTT assay.
compounds exert their antitumor effects through production of
reactive ROS. Determination of intracellular GSH levels suggested
that a concomitant reduction of CAT, GSH-Px and GSH was necessary
to allow ROS-mediated growth inhibitory of malignant cells. A rapid
decrease of GSH can happen by non-oxidative loss of glutathione in
stress-induced apoptosis via GSH extrusion and might trigger the
downstreamevents of apoptosis byleaving cells unprotectedagainst
thiol oxidation and free radical production [30].
However, there is a concern for selective production of oxidative
stress in cancer cells only, without exhibiting significant cytotox-
icity in the normal cells. Moreover, there is a need of conducting
larger, adequately powered clinical trials to prove therapeutic
efficacy and selectivity of these potential anticancer agents acting
by oxidative stress mechanisms.
Moreover, structural activities correlation of the obtained
results revealed that, replacement of the oxo group in compound 1
with chloro or thioxo (compounds 2 and 3) decrease the anticancer
effects. While, addition of substituted alkyl or arylalkylthio moie-
ties to pyrimidine ring (compounds 4 and 5) increase the anticancer
activity relative to the parent pyrazolopyrimidine derivatives
(compounds 1, 2, and 3). Meanwhile, addition of sugar moieties
(compounds 10) to the pyrazolopyrimidine derivative 3 decreases
the anticancer activity. In general, the acyclic nucleoside of pyrazolo
[3,4-d]pyrimidine derivative 4 revealed the highest anticancer
activity among the other tested compounds.
In addition as shown in Table 2, nucleic acid (DNA and RNA)
concentrations were significantly lowered in MCF-7 cells treated
with all compounds in dose-dependent manner as compared to
control and cisplatin treated groups. These changes were in the
order of 4 > 5 >1 >6 > 2 > 3 > 10. These results were confirmed by
the fact that most chemotherapeutic agents cause cells to over
generate ROS, so they are capable of inducing oxidative damage to
DNA and subsequent apoptosis [31]. The cascade of signals medi-
ating apoptosis often involves a ROS intermediate messenger, and
ROS can short-circuit the pathway, bypassing the need for upstream
signals for cell suicide. Recently, Huang et al. [32] observed that
selective inhibition of SOD kills human cancer cells but not normal
cells, suggesting that regulation of free radical-producing agents
may also have important clinical applications. This mechanism for
the effects of ROS-generating anticancer agents is only beginning to
be understood, as previously the mechanism of most anticancer
agents was believed to be due mainly to direct interaction with DNA
and interference with DNA regulatory machinery (e.g., top-
oisomerases, helicases) and to initiation of DNA damage via
production of ROS [33]. The most important target for ROS in the
carcinogenesis process probably is DNA. Damage to DNA can take
many forms, ranging from specifically oxidized purine and pyrimi-
dine bases to DNA lesions such as strand breaks, sister chromatid
exchanges, and the formation of micronuclei [34]. The highly reac-
tive hydroxyl radicals ($OH) are able to react with nucleotide bases
(as well as with the deoxyribose chain), forming a variety of modi-
fied bases; among which 8-hydroxyguanine (8-OHdG) [35]. Iron
atoms that are bound to DNA can interact with highly diffusible, but
relatively unreactive H2O2 in a metal-catalyzed Fenton-type reac-
tion to produce $OH-mediated DNA damage and strand breaks [36].
3. Experimental
3.1. Chemistry
All melting points are uncorrected and measured using Electro-
thermal IA 9100 apparatus (Shimadzu, Japan). IR spectra were
recorded as potassium bromide pellets on a PerkineElmer 1650
spectrophotometer, National Research Center, Cairo, Egypt. 1H NMR
and 13C NMR spectra were determined on a Jeol-Ex-400 NMR
spectrometer and chemical shifts were expressed as part per
million; (d values, ppm) against TMS as internal reference (Organic
Chemistry, Chemnitz University of Technology, Chemnitz,
Germany). Mass spectra were recorded on EI þ Q1 MSLMR UPLR,
National Research Center, Cairo, Egypt. Microanalyses were oper-
ated using Mario Elmentar apparatus, Organic Microanalysis Unit,
National Research Center, Cairo, Egypt. Column Chromatography
was performed on (Merck) Silica gel 60 (particle size
0.06e0.20 mm). Follow up of the reactions and checking the purity
of the compounds was made by TLC on silica gel-precoated
aluminum sheets (Type 60 F254, Merck, Darmstadt, Germany).
Compound 1 was prepared according to a reported method [25].
2.3. Conclusion
3.1.1. 4-Chloro-1-(9-methyl-5,6-dihydronaphtho[10,20:4,5]thieno
[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine (2)
Compound 1 (0.01 mol) was heated at reflux temperature in
phosphorus oxychloride (20 mL) for 2 h. The solution was then
In conclusion, the present results suggest that oxidative stress
sometimes can be utilized therapeutically and potential anticancer
drugs acting by this mechanism may prove novel in future.

A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
1023
Table 1
Effect of treatment with different concentrations (2, 5,10 and 20
peroxidase (GSH-Px), as well as the levels of reduced glutathione (GSH) and hydrogen peroxide (H2O2) in MCF-7 cells.
m
g/ml) of the prepared compounds on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione
Treatment (
Control
mg/ml)
SOD U/mg protein
CAT U/mg protein
GSH-Px U/mg protein
GSH nmol/mg protein
H2O2 nmol/mg protein
27.61 ꢂ 3.72
7.90 ꢂ 0.67
11.20 ꢂ 1.36
39.82 ꢂ 4.20
12.50 ꢂ 1.54
Cisplatin
2
5
10
20
131.22 ꢂ 15.60
160.00 ꢂ 17.00
390.50 ꢂ 40.80
470.60 ꢂ 55.00
2.80 ꢂ 0.30
2.00 ꢂ 0.25
1.40 ꢂ 0.15
1.20 ꢂ 0.11
4.40 ꢂ 0.42
3.00 ꢂ 0.35
2.11 ꢂ 0.25
1.40 ꢂ 0.18
18. 30 ꢂ 2.00
16.20 ꢂ 1.60
13.50 ꢂ 1.80
11.20 ꢂ 1.30
38.00 ꢂ 4.11
58.80 ꢂ 6.80
77.00 ꢂ 8.00
84.80 ꢂ 9.60
1
2
5
10
20
95.60 ꢂ 10.00
126.20 ꢂ 13.20
240.40 ꢂ 27.70
290.00 ꢂ 31.00
4.00 ꢂ 0.50
3.60 ꢂ 0.38
2.80 ꢂ 0.30
2.30 ꢂ 0.27
6.90 ꢂ 0.70
5.50 ꢂ 0.62
3.11 ꢂ 0.40
2.65 ꢂ 0.25
27. 90 ꢂ 2.30
18.66 ꢂ 1.90
17.70 ꢂ 0.18
16.00 ꢂ 1.70
32.00 ꢂ 2.80
40.60 ꢂ 4.70
52.00 ꢂ 5.30
60.30 ꢂ 6.50
2
2
5
10
20
80.20 ꢂ 8.80
90.60 ꢂ 10.70
160.40 ꢂ 18.00
200.70 ꢂ 24.00
5.80 ꢂ 0.70
4.90 ꢂ 0.52
4.20 ꢂ 0.56
3.65 ꢂ 0.41
8.00 ꢂ 0.92
7.60 ꢂ 0.80
7.00 ꢂ 0.73
6.70 ꢂ 0.72
35. 80 ꢂ 4.00
29.50 ꢂ 0.34
27.20 ꢂ 0.38
25.00 ꢂ 2.60
28.23 ꢂ 2.50
25.45 ꢂ 2.90
37.85 ꢂ 3.90
52.33 ꢂ 6.11
3
2
5
10
20
77.90 ꢂ 8.00
85.40 ꢂ 8.70
6.33 ꢂ 0.70
5.36 ꢂ 0.62
4.90 ꢂ 0.57
4.00 ꢂ 0.53
8.30 ꢂ 0.90
7.90 ꢂ 0.82
7.70 ꢂ 0.83
6.82 ꢂ 0.78
39. 90 ꢂ 4.10
33.12 ꢂ 3.40
29.11 ꢂ 2.95
27.00 ꢂ 3.00
24.20 ꢂ 3.00
21.60 ꢂ 2.90
30.50 ꢂ 4.00
46.37 ꢂ 5.12
120.80 ꢂ 14.70
180.60 ꢂ 19.00
4
2
5
10
20
125.00 ꢂ 14.00
150.25 ꢂ 16.50
360.00 ꢂ 37.50
385.00 ꢂ 39.00
3.60 ꢂ 0.30
2.90 ꢂ 0.35
1.76 ꢂ 0.19
1.55 ꢂ 0.20
5.60 ꢂ 0.60
4.25 ꢂ 0.50
3.00 ꢂ 0.37
2.20 ꢂ 0.24
20.11 ꢂ 2.25
19.16 ꢂ 2.20
17.00 ꢂ 1.80
14.80 ꢂ 1.80
35.00 ꢂ 3.70
55.00 ꢂ 5.50
60.50 ꢂ 7.00
80.00 ꢂ 8.60
5
2
5
10
20
105.00 ꢂ 12.20
135.70 ꢂ 16.12
310.25 ꢂ 33.00
310.00 ꢂ 35.60
3.90 ꢂ 0.42
3.40 ꢂ 0.37
3.00 ꢂ 0.35
2.10 ꢂ 0.26
6.00 ꢂ 0.75
5.65 ꢂ 0.66
4.00 ꢂ 0.50
2.90 ꢂ 0.42
23. 60 ꢂ 2.90
20.95 ꢂ 2.80
18.75 ꢂ 2.00
15.60 ꢂ 1.66
30.75 ꢂ 3.86
45.77 ꢂ 5.90
58.50 ꢂ 6.00
66.80 ꢂ 7.50
6
2
5
10
20
85.70 ꢂ 9.14
115.20 ꢂ 12.00
190.25 ꢂ 21.00
250.60 ꢂ 29.35
5.20 ꢂ 0.65
4.50 ꢂ 0.60
4.00 ꢂ 0.37
3.20 ꢂ 0.35
7.35 ꢂ 0.85
7.00 ꢂ 0.80
6.00 ꢂ 0.65
5.25 ꢂ 0.60
30. 00 ꢂ 3.75
25.00 ꢂ 2.70
20.25 ꢂ 2.90
18.60 ꢂ 2.00
30.30 ꢂ 2.60
30.50 ꢂ 3.00
40.00 ꢂ 4.70
55.00 ꢂ 6.25
10
2
5
10
20
70.40 ꢂ 9.10
80.20 ꢂ 8.60
6.85 ꢂ 0.70
6.00 ꢂ 0.62
5.70 ꢂ 0.68
5.35 ꢂ 0.63
9.00 ꢂ 1.22
6.60 ꢂ 0.70
6.00 ꢂ 0.65
5.40 ꢂ 0.60
43.35 ꢂ 5.00
40.12 ꢂ 4.24
33.11 ꢂ 3. 80
30.00 ꢂ 3.60
21.23 ꢂ 2.60
28.45 ꢂ 3.00
35.85 ꢂ 4.00
40.33 ꢂ 4.13
110.00 ꢂ 12.40
160.60 ꢂ 18.00
Data are expressed as means ꢂ S.E. of four separate experiments. All values for prepared compounds and cisplatin were significantly different at P < 0.05 vs control.
cooled and poured with stirring onto ice and the solid that formed
was filtered off, washed several times with water, dried and puri-
fied on silica gel column using n-hexane: ethyl acetate (4:1) as an
eluent to give compound 2.
Yield 79%, m.p. 255e257 ꢁC. IR (KBr,
n
, cmꢀ1): 3127 (NH), 1221
(C]S).1H NMR (DMSO-d6,
d
ppm): 2.50 (s, 3H, CH3), 2.84e2.87 (m,
4H, 2CH2), 7.16e7.24 (m, 5H, 3AreH þ C3eH, C6eH), 8.04 (d,
J ¼ 7.60 Hz, 1H, AreH), 13.76 (s, 1H, NH, D2O exchangeable). 13C
NMR (DMSO-d6): 21.39 (CH3), 25.19 (CH2), 30.26 (CH2),
125.83e163.75 (16 AreC), 178.89 (C]S). EIMS, m/z (%): 402 (Mþ,
49). Anal. calcd. for C20H14N6S2 (402.50): C 59.68, H 3.51, N 20.88, S
15.93. Found: C 59.54, H 3.47, N 21.06, S 15.85.
Yield 65%, m.p. 142e144 ꢁC. IR (KBr,
NMR (DMSO-d6,
n
, cmꢀ1): 1526 (C]N). 1H
d
ppm): 2.79 (s, 3H, CH3), 2.93e2.98 (m, 4H, 2CH2),
7.21e7.34 (m, 5H, 3AreH þ C3eH, C6eH), 8.35 (d, J ¼ 8 Hz, 1H,
AreH,). EIMS, m/z (%): 406 (Mþ, 37Cl, 39.98), 404 (Mþ, 35Cl, 100).
Anal. calcd. for C20H13ClN6S (404.88): C, 59.33; H, 3.24; Cl, 8.76; N,
20.76; S, 7.92. Found: C, 59.51; H, 3.36; Cl, 8.81; N, 20.48; S, 8.11.
3.1.3. General procedure for the synthesis of compounds 4e7
To a solution of dry ethanol (20 mL) containing KOH (0.01 mol),
compound 3 (0.01 mol) was added, then the reaction mixture was
stirred at room temperature for 5 min, then ethyl iodide, 2-bro-
moethyl methyl ether, 2-bromoethyl ethyl ether or ethyl bromoa-
cetate (0.02 mol) was added and the reaction mixtures were stirred
at 70 ꢁC for 3, 4, 3 and 2 h, respectively. After evaporation under
reduced pressure, the residues were purified on silica gel column
3.1.2. 1-(9-Methyl-5,6-dihydronaphtho[10,20:4,5]thieno[2,3-d]
pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(3H)-thione (3)
Compound 2 (0.01 mol) and thiourea (0.01 mol) were heated at
reflux temperature in dry ethanol (40 mL) for 4 h. After cooling the
solid product was collected and recrystallized from dioxane to give
compound 3.

1024
A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
idine (5). Yield 75%, m.p. 217e219 ꢁC. IR (KBr,
n
, cmꢀ1): 1682 (C]
Table 2
N). 1H NMR (DMSO-d6,
d
ppm): 2.75 (s, 3H, CH3), 2.90e2.98 (m, 4H,
Effect of treatment with different concentrations (2, 5, 10 and 20
prepared compounds on the levels of DNA and RNA as compared with control in
MCF-7 cell line.
mg/ml) of the
2CH2), 3.39 (s, 3H, OCH3), 3.54e3.64 (m, 4H, 2CH2), 7.21e7.32 (m,
5H, 3AreH þ C3eH, C6eH), 7.77 (d, J ¼ 8 Hz, 1H, AreH). 13C NMR
Treatment (
m
g/ml)
DNA (
m
g/106 cells)
RNA (m
g/106 cells)
(DMSO-d6,
d ppm): d 25.14 (CH3), 25.36 (CH2), 29.13 (CH2), 29.86
Control
6.80 ꢂ 0.70
11.50 ꢂ 0.70
(SCH2), 58.65 (OCH3), 70.95 (CH2O), 126.06e160.58 (17AreC); Anal.
calcd. for C23H20N6OS2 (460.58): C, 59.98; H, 4.38; N, 18.25; S, 13.92.
Found: C, 59.82; H, 4.41; N, 18.43; S, 13.76.
Cisplatin
2
5
10
20
3.20 ꢂ 033
2.80 ꢂ 0.33
2.23 ꢂ 0.24
1.11 ꢂ 0.14
4.70 ꢂ 0.50
3.43 ꢂ 0.39
3.11 ꢂ 0.35
2.30 ꢂ 0.26
3.1.3.3. 4-(2-Ethoxyethylsulfanyl)-1-(9-methyl-5,6-dihydronaphtho[10,
20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine
(6). Yield 57%, m.p.164e166 ꢁC. IR (KBr, , cmꢀ1): 1685 (C]N). 1H
n
1
NMR(DMSO-d6,
d
ppm):1.25(t, J¼ 7.50Hz, 3H,OCH2CH3), 2.70(s, 3H,
2
5
10
20
4.20 ꢂ 0.43
3.80 ꢂ 0.39
3.00 ꢂ 0.33
2.70 ꢂ 0.28
7.60 ꢂ 0.70
6.40 ꢂ 0.62
5.81 ꢂ 0.60
5.25 ꢂ 0.55
CH3), 2.89e3.00 (m, 4H, 2CH2), 3.42 (q, J ¼ 7.50 Hz, 2H, OCH2CH3),
3.59 (t, J ¼ 7.50 Hz, 2H, SCH2), 3.69 (t, J ¼ 7.50 Hz, 2H, CH2O), 7.21e7.38
(m, 5H, 3AreH þ C3eH, C6eH), 7.98 (d, J ¼ 8.0 Hz,1H, AreH).13C NMR
(DMSO-d6, d ppm):14.35 (CH3), 23.49 (CH3), 24.22(CH2), 30.05(CH2),
2
45.35 (SCH2), 66.69 (OCH2), 68.22 (CH2O),126.60e158.43 (17 AreC).
Anal. calcd. for C24H22N6OS2 (474.61): C 60.74, H 4.67, N 17.71, S 13.51.
Found: C 60.65, H 4.63, N 17.58, S 13.68.
2
5
10
20
4.85 ꢂ 0.50
4.46 ꢂ 0.42
4.00 ꢂ 0.42
3.85 ꢂ 0.41
8.75 ꢂ 0.72
7.50 ꢂ 0.50
6.70 ꢂ 0.43
6.50 ꢂ 0.18
3.1.3.4. 4-(2-Ethoxycarbonymethylsulfanyl)-1-(9-methyl-5,6-dihydr
onaphtho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]
3
2
5
10
20
5.30 ꢂ 0.20
5.00 ꢂ 0.52
4.76 ꢂ 0.48
4.00 ꢂ 0.41
9.11 ꢂ 0.92
7.90 ꢂ 0.7
pyrimidine (7). Yield 62%, m.p.138e140 ꢁC. IR (KBr,
n
, cmꢀ1): 1685
ppm): 1.25 (t, J ¼ 7.50 Hz,
(C]N), 1715 (C]O). 1H NMR (DMSO-d6,
d
7.00 ꢂ 0.63
6.80 ꢂ .0.65
3H, OCH2CH3), 2.65 (s, 3H, CH3), 2.83e2.91 (m, 4H, 2CH2), 4.17 (q,
J ¼ 7.50 Hz, 2H, OCH2CH3), 4.64 (s, 2H, SCH2), 7.18e7.25 (m, 5H,
3AreH þ C3eH, C6eH), 7.82 (d, J ¼ 8.0 Hz, 1H, AreH). 13C NMR
4
2
5
10
20
3.80 ꢂ 0.40
3.10 ꢂ 0.33
2.76 ꢂ 0.30
1.82 ꢂ 0.21
5.50 ꢂ 0.60
4.32 ꢂ 0.53
4.00 ꢂ 0.52
3.61 ꢂ 0.35
(DMSO-d6,
d ppm): 14.12 (CH3), 24.95 (CH3), 25.04 (CH2), 29.79
(CH2), 32.84 (OCH2), 61.64 (SCH2), 126.18e160.43 (17AreC), 166.93
(C]O). Anal. calcd. for C24H20N6O2S2 (488.59): C 59.00, H 4.13, N
17.20, S 13.13. Found: C 59.15, H 4.25, N 17.17, S 13.18.
5
2
5
10
20
4.00 ꢂ 0.46
3.44 ꢂ 0.36
2.96 ꢂ 0.30
2.50 ꢂ 0.28
6.20 ꢂ 0.65
5.72 ꢂ 0.60
5.21 ꢂ 0.61
4.80 ꢂ 0.50
3.1.4. 4-(20,30,50-Tri-O-benzoyl-
b-D-ribofuranosylsulfanyl)-1-(9-
methyl-5,6-dihydronaphtho [10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-
1H-pyrazolo[3,4-d]pyrimidine (9)
Compound 2 (0.386 g, 1 mmol) was refluxed by stirring under
anhydrous condition for 12 h with hexamethyldisilazane (60 mL)
and ammonium sulfate (0.02 g). The obtained clear solution was
evaporated under reduced pressure and the resulting trimethylsi-
lylated pyrimidine 8 was dissolved in dry 1,2-dichloromethane
6
2
5
10
20
4.60 ꢂ 0.48
4.20 ꢂ 0.50
3.75 ꢂ 0.37
3.00 ꢂ 0.33
7.90 ꢂ 0.82
7.00 ꢂ 0.68
6.50 ꢂ 0.65
6.20 ꢂ 0.70
(40 mL) and a solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribo-
10
2
5
10
20
furanose (1 mmol) and SnCl4 (1.6 mL) in dry 1,2-dichloromethane
(10 mL) was then added dropwise with stirring under inert atmo-
sphere (nitrogen gas) until the reaction was judged complete by TLC
(6 h). The reaction mixture was poured into saturated NaHCO3
solution and extracted with CHCl3 (2 ꢃ 30 mL). The extracts were
collected and dried over anhydrous Na2SO4, filtered and concen-
trated producing the crude nucleoside that was purified on silica gel
column using n-hexane: ethyl acetate (4:1) as an eluent to give 9.
5.35 ꢂ 0.50
5.16 ꢂ 0.52
4.86 ꢂ 0.47
4.45 ꢂ 0.45
9.50 ꢂ 0.70
8.90 ꢂ 0.90
7.70 ꢂ 0.73
7.22 ꢂ 0.74
Data are expressed as means ꢂ S.E. of four separate experiments. All values for
prepared compounds and cisplatin were significantly different at P < 0.05 vs control.
using chloroform: methanol (9:1) as an eluent to give compounds
4, 5, 6 or 7, respectively.
Yield 68%, m.p.210e212 ꢁC. IR (KBr,
NMR (DMSO-d6, ppm): 2.74 (s, 3H, CH3), 2.80e2.94 (m, 4H, 2CH2),
n
, cmꢀ1): 1710 (C]O). 1H
d
3.1.3.1. (4-Ethylsulfanyl)-1-(9-methyl-5,6-dihydronaphtho[10,20:4,5]
thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine
Yield 86%, m.p. 194e196 ꢁC. IR (KBr, , cmꢀ1):1691 (C]N). 1H NMR
(DMSO-d6,
3.95 (m, 1H, 40-H), 4.18e4.58 (m, 4H, 50-H2, 20-H, 30-H), 6.29 (d,
0
0
(4).
J1 ,2 ¼ 4.40 Hz, 1H, 10-H), 7.19e7.56 (m, 18H, AreH), 7.78e8.10 (m,
n
3H, AreH þ C3eH, C6eH). 13C NMR (DMSO-d6,
d ppm): 23.89 (CH3),
d
ppm): 1.37 (t, J ¼ 7.50 Hz, 3H, NCH2CH3), 2.76 (s, 3H,
25.67 (CH2), 30.27 (CH2), 61.66 (C-50), 70.15 (C-30), 71.32 (C-20),
79.93 (C-40), 97.87 (C-l0), 124.75e140.83 (35 Ar–C), 165.02, 169.09,
169.57 (3C]O). Anal. calcd. for C46H34N6O7S2 (846.95): C 65.24, H
4.05, N 9.92, S 7.57. Found: C 65.19, H 3.97, N 10.12, S 7.62.
CH3), 2.88e2.99 (m, 4H, 2CH2), 3.32 (q, J ¼ 7.20 Hz, 2H, NCH2CH3),
7.21e7.38 (m, 5H, 3AreH þ C3eH, C6eH), 7.78 (d, J ¼ 8.0 Hz, 1H,
AreH). 13C NMR (DMSO-d6,
d ppm): 14.26 (CH3), 24.64 (CH3), 25.10
(CH2), 25.35 (CH2), 29.86 (SCH2), 123.24e166.54 (17AreC). Anal.
calcd. for C22H18N6S2 (430.56): C 61.37, H 4.21, N 19.52, S 14.89.
Found: C 61.54, H 4.29, N 19.41, S 14.74.
3.1.5. 4-(1- b-D-Ribofuranosylsulfanyl)-1-(9-methyl-5,6-dihydronap
htho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyr
imidine (10)
3.1.3.2. 4-(2-Methoxyethylsulfanyl)-1-(9-methyl-5,6-dihydronaph-
tho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrim
Dry ammonia gas was passed into a solution of protected
nucleoside 9 (1 mmol) in dry methanol (20 mL) at 0 ꢁC for 0.5 h,

A.E. Rashad et al. / European Journal of Medicinal Chemistry 46 (2011) 1019e1026
1025
then the reaction mixture was stirred at room temperature until
the reaction was judged complete by TLC (5 h). The resulting
mixture was then concentrated under reduced pressure at 40 ꢁC to
afford a solid residue that was purified on silica gel column using
chloroform: methanol (4:1) as an eluent to give 10.
The estimation of in vitro tumor cell growth inhibition was
assessed by incubating 0.65 ꢃ 105 MCF-7 cells in 1 ml phosphate
buffer saline with varying concentrations of the prepared
compounds and cisplatin (commonly used anticancer drug) at 37 ꢁC
for 24 h in CO2 atmosphere. Cells were cultured for 24 h to assure
total attachment. Afterward, the tested compounds were added to
the cells. Cell survival was evaluated at the end of the incubation
period with MTT colorimetric assay. In all the cellular experiments,
results were compared with untreated cells.
Yield 54%, oil. IR (KBr, n
, cmꢀ1): 3440e3230 (OH),1645 (C]N).1H
NMR (DMSO-d6, d ppm): 2.46 (s, 3H, CH3), 2.65e2.80 (m, 4H, 2CH2),
4.14e4.40 (m, 3H, 40-H, 50-H2), 4.50e4.62 (m, 2H, 20-H, 30-H), 4.86
(m,1H, OH, D2O exchangeable), 4.98 (m,1H, OH, D2O exchangea0ble),
0
0
5.10 (m,1H, OH, D2O exchangeable), 6.64 (d, J1 ,2 ¼ 5.20 Hz,1H,1 -H),
7.18e7.31 (m, 6H, 4AreH þ C3eH, C6eH). 13C NMR (DMSO-d6,
3.2.3. Cytotoxicity assay
d
ppm): 25.15 (CH3), 25.67 (CH2), 29.89 (CH2), 64.25 (C-50), 72.26 (C-
Effect of the prepared compounds on the growth of MCF-7 cells
was estimated by MTT colorimetric assay according to Mosmann
[37]. This assay is based on the selective ability of living cells to
reduce the yellow soluble salt of MTT to a purple-blue insoluble
formazan precipitate. The viable cell number is proportional to the
production of formazan salts. The crystals of formazan were dis-
solved in DMSO and the optical density was measured spectro-
photometrically (Microplates reader, Asys Hitech, Austria).
Cells (0.65 ꢃ 105 cells/well) were plated separately in a sterile
30), 75.14 (C-20), 81.45 (C-40), 92.16 (C-l0), 115.52e150.15 (17AreC).
Anal. calcd. for C25H22N6O4S2 (534.62): C 56.17, H 4.15, N 15.72, S
12.00. Found: C 56.39, H 4.17, N 15.81, S 11.86.
3.1.6. 4-(20,30,40,60-Tetra-O-acetyl-
b-D-glucopyranosylsulfanyl)-1-(9
-methyl-5,6-dihydronaphtho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)
-1H-pyrazolo[3,4-d]pyrimidine (11)
The same procedure as in preparation of compound 9 was per-
formed, but 1,2,3,4,6-penta-O-acetyl-
was used to give product 11 from 3.
b
-
D
-glucopyranose (1 mmol)
flat bottom 96-well microplate (Falcon), and treated with 20
different concentration of tested compounds and cisplatin (2, 5, 10
or 20
g/ml), for 24 h at 37 ꢁC, in a humidified 5% CO2 atmosphere.
After incubation, media were removed and 40 l MTT solution/well
were added and incubated for an additional 4 h. MTT crystals were
solubilized by adding 200 l of DMSO/well and plate was shacked
ml of
Yield 60%, m.p.191e193 ꢁC. IR (KBr,
n
, cmꢀ1): 1715 (C]O). 1H
m
NMR (DMSO-d6,
d
ppm): 1.97e2.06 (4s, 12H, 4CH3CO), 2.56 (s, 3H,
m
CH3), 2.82e2.94 (m, 4H, 2CH2), 4.08e4.12 (m, 2H, 60-H2), 4.29 (m,
1H, 50-H), 4.96e5.13 (m, 2H, 40-H, 30-H), 5.55 (m, 1H, 20-H), 6.26 (d,
m
J1 ,2 ¼ 8.50 Hz, 1H, 10-H), 7.18e7.24 (m, 6H, 4AreH þ C3eH, C6eH).
gently for 10 min at room temperature. The results were deter-
mined photometricaly using microplate ELISA reader and the
absorbance was 570 nm. Data were expressed as the percentage of
relative viability compared with untreated cells. Percentage of
relative viability was calculated and cytotoxic concentration was
expressed by half maximal inhibitory concentration (IC50). IC50
calculations were performed using Microsoft Excel and Microcal
Origin software for PC. The MCF-7 cells were harvested and
homogenates were prepared in saline using a tight pestle homog-
enizer until complete cells disruption for further biochemical
analysis.
0
0
13C NMR (DMSO-d6,
d ppm): 20.49e20.62 (5CH3), 24.95 (CH2),
29.92 (CH2), 61.04 (C-60), 67.32 (C-40), 69.33 (C-20), 70.31 (C-30),
70.65 (C-50), 90.91 (C-10), 127.17e152.34 (17AreC), 169.41e170.48
(4C]O). Anal. calcd. for C34H32N6O9S2 (732.80): C 55.73, H 4.40, N
11.47, S 8.75. Found: C 55.92, H 4.37, N 11.62, S 8.56.
3.1.7. 4-( b-D-Glucopyranosylsulfanyl)-1-(9-methyl-5,6-dihydronap
htho[10,20:4,5] thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyr
imidine (12)
The same procedure as in preparation of compound 10 was used
to give product 12 from 11.
Yield 74%, oil. IR (KBr,
n
, cmꢀ1): 3500e3200 (OH), 1612 (C]N).
3.2.4. Biochemical assays
1H NMR (DMSO-d6,
d
ppm): 2.56 (s, 3H, CH3), 2.86e2.98 (m, 4H,
Antioxidant enzyme activities and the level of both reduced
glutathione (GSH) and hydrogen peroxide (H2O2) were expressed in
cell lysates as a function of total cellular protein which was deter-
mined according to Lowry et al. [38]. The activities of superoxide
dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-
Px) were determined as described by method of Paglia and
Valentine [39], Aebi [40], Marklund and Marklund [41], respec-
tively. The levels of GSH and H2O2 were determined using the
method of Ellman [42] and Wolf [43], respectively. Nucleic acids
(DNA and RNA) and total protein were precipitated and measured
in cell homogenates. Total DNA was extracted and assayed
according to the method described by Zhou et al. [44], total RNA
was extracted and assayed according to the method adopted from
the procedure provided by Hybaid/AGS (Germany).
2CH2), 3.10e3.40 (m, 3H, 60-H2, H-50), 4.10e4.25 (m, 3H, H-40, H-30,
H-20), 4.52 (m, 1H, OH, D2O exchangeable), 5.03 (m, 1H, OH, D2O
exchangeable), 5.17 (m, 1H, OH, D2O exchangeable), 5.34 (m, 1H,
OH, D2O exchangeable), 6.12 (d, J1 ,2 ¼ 8.0 Hz, 1H, H-10), 6.94e7.26
0
0
(m, 6H, 4AreH þ C3eH, C6eH). 13C NMR (DMSO-d6,
d ppm): 22.68
(CH3), 24.95 (CH2), 29.66 (CH2), 61.18 (C-60), 67.64 (C-40), 69.59 (C-
20), 71.38 (C-30), 74.81 (C-50), 85.49 (C-10), 128.17e149.50 (17AreC);
Anal. calcd. for C26H24N6O5S2 (564.65): C 55.31, H 4.28, N 14.88, S
11.36. Found: C 55.49, H 4.37, N 14.75, S 11.42.
3.2. Materials and methods
3.2.1. Chemicals
Dimethyl sulfoxide (DMSO) and MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) were purchased from Merck
(Darmstadt, Germany). All other chemicals and reagents used were
of analytical grade.
3.2.5. Statistical analysis
The results are reported as Mean ꢂ Standard error (S.E.) for at
least four times experiments. Statistical differences were analyzed
according to followed by one way ANOVA test followed by student’s
t test wherein the differences were considered to be significant at
p < 0.05.
3.2.2. Cell culture
The MCF-7 human breast cell line was maintained in Dulbecco’s
modified Eagle’s medium DMEM0 supplemented with 10% heat
inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and
Acknowledgment
streptomycin (100 m
g/ml) at 37 ꢁC in humidified atmosphere con-
taining 5% CO2. MCF-7 cells at a concentration of 0.50 ꢃ 106 were
Dr. Aymn E. Rashad is grateful to Prof. Klaus Banert, Chemnitz
University of Technology, Germany for facilities, help, and support.
grown in a 25 cm2 flask in 5 ml of complete culture medium.

1026
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