New pyrimidinone and fused pyrimidinone derivatives as potential anticancer chemotherapeutics

Article abstract of DOI:10.1002/ardp.201200119

A series of novel substituted pyrimidinones and fused pyrimidinones (compounds 3-18) were synthesized starting with oxiranylmethanone 2. The in vitro cytotoxicity against a human breast adenocarcinoma (MCF-7) cell line was investigated and most of the tested compounds showed potent cytotoxic activity against the MCF-7 cell line comparable to the activity of the commonly used anticancer drug cisplatin. Treatment of MCF-7 cells with increasing doses (2, 5, 10, and 20 μg/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, derivatives 11 and 16 revealed the highest anticancer activity among the tested compounds.

Full text of DOI:10.1002/ardp.201200119

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
1
Full Paper
New Pyrimidinone and Fused Pyrimidinone Derivatives
as Potential Anticancer Chemotherapeutics
Aymn E. Rashad1,2, Ahmed H. Shamroukh1,3, Nabil M. Yousif1, Mowafia A. Salama1, Hatem S. Ali4,
Mamdouh M. Ali5, Abeer E. Mahmoud5, and Mahmoud El-Shahat1
1 National Research Center, Photochemistry Department, Dokki, Cairo, Egypt
2 Faculty of Science and Human Studies, Chemistry Department, Hurraiymla, Shaqra University,
Saudi Arabia
3 Faculty of Science, Chemistry Department, Hail University, Saudi Arabia
4 Department of Food Science and Nutrition, College of Food Science and Agriculture, King Saud University,
Saudi Arabia
5 National Research Center, Division of Genetic Engeneering and Biotechnology, Biochemistry Department,
Dokki, Cairo, Egypt
A series of novel substituted pyrimidinones and fused pyrimidinones (compounds 3–18) were
synthesized starting with oxiranylmethanone 2. The in vitro cytotoxicity against a human breast
adenocarcinoma (MCF-7) cell line was investigated and most of the tested compounds showed
potent cytotoxic activity against the MCF-7 cell line comparable to the activity of the commonly used
anticancer drug cisplatin. Treatment of MCF-7 cells with increasing doses (2, 5, 10, and 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, derivatives 11 and 16 revealed the highest anticancer activity among the tested
compounds.
Keywords: Anticancer / Fused pyrimidinones / MCF-7 cells / Oxiranylmethanone / Pyrimidinone
Received: March 25, 2012; Revised: April 28, 2012; Accepted: April 29, 2012
DOI 10.1002/ardp.201200119
Introduction
prostate, and breast cancer [3–10]. Because of the aforemen-
tioned pharmacological potential of this class of compounds,
our research group has been involved in synthesizing and
testing the pharmacological activities of pyrimidinone and
fused pyrimidinone derivatives, hoping that they could have
some chemical and biological interest [4, 5, 14, 15, 26, 27].
Pyrimidinone and fused pyrimidinone derivatives are well
known for their pharmacological properties [1, 2]. These com-
pounds have therapeutic applications, as anticancer [3–10],
interferon inducer [11], antiviral [12–15], anti-hypertensive
[16, 17], hypoglycaemic [18, 19], anticonvulsive [20], anti-hista-
minic [21], analgesic, and antiinflammatory drugs [22–25]. As
cited above, the pyrimidinone and their analogue derivatives
are present in many anticancer agents. In fact, it has been
reported that these substances were proven to have significant
activities against different types of human cancers, namely:
leukemia, lung, liver, colon, CNS, melanoma, ovarian, renal,
Results and discussion
Chemistry
(2E)-1,3-Bis(4-chlorophenyl)-2-propen-1-one (1) was synthe-
sized by the Claisen–Schmidt condensation method [28],
which was performed with hydrogen peroxide in alkaline
medium to produce (4-chlorophenyl)[3-(4-chlorophenyl)-
oxiranyl]methanone (2) (Scheme 1). Analytical and spectral
data of compound 2 are in agreement with the proposed
structure (cf. Experimental section). Compound 2, as a typical
epoxide, was utilized as a key starting material in the syn-
thesis of many pyrimidinone derivatives. Thus, heating a
Correspondence: Dr. Aymn E. Rashad, National Research Center,
Photochemistry Department, Dokki, 12311 Cairo, Egypt. Faculty of
Science and Human Studies, Chemistry Department, Hurraiymla, Shaqra
University, Saudi Arabia
E-mail: aymnelzeny@yahoo.com
Fax: þ20 20337 0931
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
A. E. Rashad et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Scheme 1. Synthesis of compounds 2–5.
mixture of compound 2 with different nitrogen nucleophiles
(such as thiourea, urea, and thiosemicarbazide) produced
pyrimidinone derivatives 3–5, respectively (Scheme 1). The
latter compounds showed correct values in elemental
Condensation of compound 3 with bromoacetic acid in a
mixture of glacial acetic acid/acetic anhydride produced
thiazolo[3,2-a]pyrimidine-3,6-dione derivative 8 [29]. The pro-
posed structure for the favorable isomer was established on
1
analysis as well as compatible IR spectral data. The H NMR
spectrum (DMSO-d6) of compound 3, as a representative
example, showed the expected signals corresponding to
the aromatic protons and two types of exchangeable protons
corresponding to two types of NH groups; besides, two
doublets appear at d 3.09 and 3.49 ppm, each corresponding
to a pyrimidine proton. The 13C NMR spectrum of compound
–
–
–
3 showed the expected signals for C O at 175.82, C S at
–
182.08, aromatic carbons at 127.83–133.74, and two signals
in the SP3 carbon region at 42.88 and 71.37 ppm. The appear-
ance of the latter signals in 13C NMR spectrum and the two
1
signals at d 3.09 and 3.49 ppm in H NMR spectrum of com-
pound 3 suggests that the produced pyrimidine ring is in fact
alicyclic not aromatic and prefers to be in chair form. The two
protons at C-4 and C-6 can undergo long range coupling and
they both appear as doublets (cf. Experimental section).
Methylation of the sodium salt of compound 3 with methyl
iodide gave 4,6-bis(4-chlorophenyl)-2-(methylthio)pyrimidin-
5(4H)-one (6) (Scheme 2). The structure of the aforementioned
compound was confirmed on the basis of its elemental and
spectral data. Its 1H NMR spectrum showed singlet signal at d
2.62 ppm of the CH3 protons, while the IR spectrum revealed
absorption band at 1713 cmꢀ1 for C O group and the
–
–
absence of absorption bands due to (2NH). The MS gave the
molecular ion peak at m/z (%) ¼ 362 (90), besides the isotopic
pattern of two chlorine atoms (cf. Experimental section).
The hydrazide derivative 7, as key compound for further
reactions, was prepared through the reaction of the S-methyl
derivative 6 or thioxo-pyrimidinone derivative 3 with hydra-
zine hydrate in refluxing ethanol. The product revealed
–
absorption bands for NH, NH , and C O groups in the IR
–
2
spectrum, and its 1HNMR spectrum showed signals at d 3.98
and 6.69 ppm due to NH2 and NH (D2O exchangeable).
The MS gave the molecular ion peak at m/z (%) ¼ 346 (26)
(cf. Experimental section).
Scheme 2. Synthesis of compounds 6–11.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Pyrimidinone and Fused Pyrimidinone as Anticancer Agents
3
the basis of its spectral data, since the IR spectrum showed
–
absorption bands assignable to the (2C O) groups and its
–
1H NMR spectrum revealed a signal at d 4.01 ppm for (CH2). In
addition, the 13C NMR spectrum showed signals at d 34.50,
–
171.42, and 185.68 ppm accountable for the (CH ) and (2C O)
2
–
groups, respectively (cf. Experimental section).
The presence of an active methylene group in compound 8
could be confirmed by condensation with benzaldehyde in
glacial acetic acid/acetic anhydride mixture in the presence
of anhydrous sodium acetate to produce 2-benzylidine
derivative 9 (Scheme 2). The latter compound was prepared
directly, via a one-pot reaction, by refluxing compound 3 with
bromoacetic acid and benzaldehyde in glacial acetic acid/ace-
tic anhydride mixture to give a product identical in all aspects
with compound 9. Moreover, the structure of compound 9
could be confirmed by heating with hydroxyl amine hydro-
chloride in glacial acetic acid/acetic anhydride mixture to
give isoxazolo[50,40:4,5][1,3]thiazolo[3,2-a]pyrimidin-7(8H)-one
derivative 10 [30] (Scheme 2). The structures of the latter
compounds were confirmed on the basis of their analytical
and spectral data, where the IR spectrum of compound 9
–
revealed absorption bands characteristic of two C O groups,
–
and its 1H NMR spectrum showed the presence of benzylic-H.
–
While the absence of one C O group in the IR spectrum as well
–
as the benzylic-H signal in the 1H NMR spectrum of compound
10 confirmed its structure (cf. Experimental section).
Mannich reaction on compound 3, isolate the 3-substituted
product which proved to possess structure of 4,6-bis(4-chloro-
phenyl)-1-(piperidin-1-ylmethyl)-2-thioxo-tetrahydro-pyrimidin-
–
5(6H)-one 11 (Scheme 2). The presence of C S in the IR and
–
13C NMR spectra of compound 11 revealed that the site of
attack was on the N- and not S-atom [31] (cf. Experimental
section).
Compound 4 was alkylated with ethyl bromoacetate, in the
presence of anhydrous potassium carbonate to give the ethyl
ester derivative 12 (Scheme 3). Analytical and spectral data of
this compound is in agreement with the proposed structure
(cf. Experimental section). In particular, the IR spectrum
Scheme 3. Synthesis of compounds 12–18.
–
showed absorption bands assignable to (2C O) groups. The
–
absence of the NH-signals and the presence of singlet, triplet
and quartet signals at d 1.16, 4.08, and 4.89 ppm, for O–CH2
and OCH2CH3 protons, respectively, in the 1H NMR spectrum
confirmed its structure. The O-alkylation at C2- pyrimidine
librium is affected by many factors: pH, temperature, the
–
nature of the substituents around the (C N), and the used
–
solvent [32–34]. This equilibrium can be shifted in either
direction by controlling these factors, and the IR spec-
troscopy is helpful in revealing which form is predominant,
since the azido structure can show a characteristic band in
the region n 2100–2200 cmꢀ1. Thus, nitrosation of the hydra-
zino group of compound 7 afforded 5,7-bis(4-chlorophenyl)-
tetrazolo[1,5-a]pyrimidin-6-one 13. Analytical and spectral
data are in agreement with the proposed structure (cf.
Experimental section). In particular, the IR spectrum of com-
pound 13 did not show absorption frequency indicative of
the azido group. Compound 13 was reduced to 2-amino-4,6-
–
was confirmed by the absence of one of the (C O) groups of
–
the pyrimidine ring in the 13C NMR spectrum.
In an attempt to prepare the acid hydrazide derivative
from the reaction of compound 12 with hydrazine hydrate
in ethanol under reflux temperature, it afforded an un-
expected product identical in all aspects with compound 7
(cf. Experimental section).
Heterocyclic azides, especially azidomethines, can exist in
equilibrium with their tetrazolo tautomers and this equi-
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
A. E. Rashad et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 1. Effect of treatment at various concentrations (5, 12.50, 25,
or 50 mg/mL) of prepared compounds on MCF-7 cell cytotoxicity
(IC50) as measured with the MTT method
bis(4-chlorophenyl)-pyrimidin-5(4H)-one 14 when heated
with zinc dust in glacial acetic acid (cf. Experimental
section). The IR spectrum of 14 showed bands for NH2
group and its 1H NMR spectrum showed signals at 6.86
(s, 2H, NH2, D2O exchangeable). Also, the mass spectrum
showed the molecular ion peak Mþ at m/z 331 as the base
peak as well as the presence of isotopic pattern of two
chlorine atoms.
Compounds Concentration
in (mg/mL)
% of cell
viability
IC50
(mg/mL)
Cisplatin
0
5
100
45
25
7
4.00
18.00
15.00
13.00
11.00
10.00
8.00
12.50
25
50
0
Moreover, heating of compound 7 with carbon disulphide
in the presence of ethanolic potassium hydroxide afforded
5,7-bis(4-chlorophenyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-
a]pyrimidin-6-one 15. The structure of the latter compound
was confirmed on the basis of its spectral data (cf.
Experimental section), since the IR spectrum showed
4
2
100
64
57
40
18
100
75
54
36
9
100
62
51
32
14
100
70
45
25
9
100
67
42
16
6
100
57
36
22
15
5
12.50
25
50
0
5
–
absorption bands assignable to the (NH) and (C S) groups
–
5
and its 1H NMR spectrum revealed a signal at d 10.03 ppm for
12.50
25
50
0
(NH) (D2O exchangeable).
On the other hand, treatment of compound 7 with chloro-
acetyl chloride at room temperature gave 3-chloromethyl-5,7-
bis(4-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyrimidin-6-one 16
(Scheme 3). The structure of compound 16 was established
according to its elemental and spectroscopic data (cf.
Experimental section).
8
5
12.50
25
50
0
10
11
15
5
12.50
25
50
0
Also, refluxing compound 7 with acetic anhydride or
benzoyl chloride afforded 5,7-bis(4-chlorophenyl)-3-methyl-
[1,2,4]triazolo[4,3-a]pyrimidin-6-one 17 or 5,7-bis(4-chloro-
phenyl)-3-phenyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-one 18,
respectively (Scheme 3). The spectral data of the latter com-
pounds are in agreement with the assigned structures (cf.
Experimental section).
5
12.50
25
50
0
5
12.50
25
50
Anticancer activity of prepared compounds
The newly synthesized compounds were screened for their
in vitro cytotoxicity and growth inhibitory activities against
the human breast carcinoma cell line MCF-7, in comparison
with the activity of the known anticancer agent cisplatin as a
reference drug. The cytotoxicity of the tested compounds was
expressed by median growth inhibitory concentration (IC50
)
which required producing 50% cytotoxic effect against MCF-7
cells after 24 h exposure to the tested compounds and the
screening results are compiled in Table 1 and Fig. 1.
The results indicated that six compounds (2, 5, 8, 10, 11,
and 15) were the most active compounds used in this study
(Fig. 1).
From Table 1, the tested compounds showed anticancer
activity with IC50 values ranging from 8.00 to 18.00 mg/mL. It
is clear from the obtained data that the comparison of the
cytotoxicity against MCF-7 cell line of the tested compounds
has shown that the growth inhibitory potency follows
the order 15 > 11 > 10 > 8 > 5 > 2 and reaching strong
correlation with that of cisplatin (IC50: 4 mg/mL) in the case
of compound 16 (IC50: 8.00 mg/mL).
Figure 1. Effect of treatment at various concentrations (5, 12.50,
25, or 50 mg/mL) of prepared compounds on MCF-7 cell cytotoxicity
(IC50).
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Pyrimidinone and Fused Pyrimidinone as Anticancer Agents
5
Table 2. Effect of treatment with the prepared compounds on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GSH-Px), as well as the levels of reduced glutathione (GSH) and hydrogen peroxide (H2O2) in MCF-7 cells
Compounds
SOD
(U/mg protein)
CAT
(U/mg protein)
GSH-Px
(U/mg protein)
GSH
(nmol/mg protein)
H2O2
(nmol/mg protein)
DMSO (control)
Cisplatin
2
5
8
10
11
15
40.30 ꢁ 4.75
130.80 ꢁ 15.65a
50.00 ꢁ 5.20b
7.60 ꢁ 0.70
2.96 ꢁ 0.22a
7.36 ꢁ 0.75b
5.90 ꢁ 0.60b
4.90 ꢁ 0.42b
3.64 ꢁ 0.56a
3.20 ꢁ 0.30a
2.30 ꢁ 0.26a
9.30 ꢁ 1.00
4.40 ꢁ 0.40a
8.40 ꢁ 0.80b
7.60 ꢁ 0.80b
6.90 ꢁ 0.68a
6.30 ꢁ 0.66a
5.20 ꢁ 0.48a
4.80 ꢁ 0.50a
40.00 ꢁ 5.00
21.60 ꢁ 2.40a
36. 00 ꢁ 2.40b
35. 20 ꢁ 3.30b
32.11 ꢁ 3.12a
29. 00 ꢁ 3.00a,b
22. 60 ꢁ 2.40a
19. 60 ꢁ 1.60a
15.70 ꢁ 1.60
47.50 ꢁ 5.70a
25.00 ꢁ 2.60a,b
27.60 ꢁ 2.80a,b
30.80 ꢁ 3.30a,b
34.00 ꢁ 3.20a,b
43.40 ꢁ 4.15a
50.00 ꢁ 4.80a
78.84 ꢁ 9.30a,b
84.00 ꢁ 8.40a,b
90.60 ꢁ 9.70a,b
110.30 ꢁ 11.20a
120.00 ꢁ 12.00a
a
b
Data are expressed as means ꢁ SE of six separate experiments. and is significant difference from control and cisplatin groups,
respectively, at p <0.05.
To elucidate the mechanism by which the prepared com-
pounds exert their antitumor activities, we estimated the
activities of the free-radical-metabolizing enzymes including
superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GSH-Px) as well as the levels of the oxidative
stress parameters including hydrogen peroxide (H2O2), nitric
oxide (NO), and reduced glutathione (GSH) in MCF-7 cells
treated with the prepared compounds, as well as the effect
of these compounds on the levels of total protein and nucleic
acids.
production of ROS. As shown in Table 3, the levels of total
protein and nucleic acids were significantly lower than those
of control, while the level of NO was significantly higher in
MCF-7 cells treated with most compounds as compared to
control cells. The highest activity was found for compound
15, 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.
The antitumor activities of these compounds were
accompanied by increases in SOD activities of tumor-treated
cells compared to control cells. This means that these
compounds can cause H2O2 production and 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 are
lowered in groups treated with the prepared compounds
compared to control cells. Consequently, the excess H2O2
produced in tumor cells with the compounds cannot be
removed. In other words, the accumulation of H2O2 and
other free radicals in tumor cells should be the cause of
As shown in Table 2, general treatment of the cells with
different compounds and cisplatin (at 1/10 of the IC50 values)
resulted in a significant increase in the activity of SOD and
level of H2O2 higher than those of control accompanied
with a significant depletion in the activity of CAT, GSH-Px,
and the level of GSH. These changes were in the order of
15 > 11 > 10 > 8 > 5 > 2 which is in accordance with the
order of cytotoxicity activity of the tested compounds, indi-
cating an increase in the cellular levels of reactive oxygen
species (ROS). These results indicate that the antitumor effect
of the present compounds may be exerted at least partly by
Table 3. Effect of prepared compounds on the level of total protein, nucleic acids (RNA and DNA), and nitric oxide (NO) in MCF-7 cells
Compounds
Protein
RNA
DNA
NO
(mmol/mg protein)
(mg/106 cells)
(mg/106 cells)
(mg/106 cells)
DMSO (control) 110.50 ꢁ 12.30
15.30 ꢁ 1.60
3.40 ꢁ 0.40a
13.20 ꢁ 12.70b
8.50 ꢁ 0.67a,b
6.80 ꢁ 0.60a,b
5.00 ꢁ 0.48a,b
3.40 ꢁ 0.33a
2.90 ꢁ 0.28a
8.50 ꢁ 0.80
1.90 ꢁ 0.16
Cisplatin
2
33.60 ꢁ 3.70a
90.20 ꢁ 8.80b
87.30 ꢁ 7.60a,b
80.00 ꢁ 8.20a,b
57.60 ꢁ 6.00a,b
45.20 ꢁ 4.60a,b
30.50 ꢁ 3.60a
2.50 ꢁ 0.30a
7.50 ꢁ 0.73b
6.50 ꢁ 0.65a,b
5.80 ꢁ 0.60a,b
5.30 ꢁ 0.50a,b
4.60 ꢁ 0.38a,b
3.00 ꢁ 0.27a
4.20 ꢁ 0.37a
2.70 ꢁ 0.30a,b
3.00 ꢁ 0.34a,b
3.20 ꢁ 0.40a,b
3.80 ꢁ 0.36a
4.20 ꢁ 0.45a
4.80 ꢁ 0.46a
5
8
10
11
15
The values are expressed as mean ꢁ SE of six separate experiments. a and b is significant difference from control and cisplatin groups,
respectively, at p <0.05.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
A. E. Rashad et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
tumor cell killing. Thus, the results of the present study
are consistent with the hypothesis that the prepared
compounds exert their antitumor effects because they
produce ROS. Moreover, from our data it is observed that
the compounds having higher SOD activity with lowering
CAT and GSH-Px activities are those having high antitumor
activity.
compounds may come as potent anticancer agents in the
modern clinical trials.
Experimental
Chemistry
All melting points are uncorrected and were measured using
Electrothermal IA 9100 apparatus (Electrothermal, Essex, UK). IR
spectra were recorded as potassium bromide pellets on a Perkin-
Elmer 1650 spectrophotometer (Perkin-Elmer, Norwalk, CT,
USA). 1H NMR and 13C NMR spectra were determined on a
Jeol-Ex-300 NMR spectrometer (JEOL, Tokyo, Japan) and chemical
shifts were expressed as parts per million, ppm (d values) against
TMS as internal reference. Mass spectra were recorded on a VG
2AM-3F mass spectrometer (Thermo Electron Corporation, USA).
Microanalyses were operated using Mario El Mentar apparatus,
Organic Microanalysis Unit, and the results were within the
accepted range (ꢁ0.20) of the calculated values. 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 [28].
The results showed that treatment with these compounds
leads to an increase in the level of NO and according to Blanco
[35] an increase in NO level leads to apoptosis (programmed
cell death), whereas an increase in ROS leads to necrosis (cell
death), so, the way a tumor cell dies reflects the radical
balance in the system.
In addition, our results showed that the increase in the NO
level was accompanied with depletion of the levels of total
protein and nucleic acids compared to control and this can
be explained by several cytotoxic effects, including reactions
with proteins and nucleic acids. The main targets of NO in
proteins are the SH group [36] and Fe of active sites [37]. In the
nucleus, NO has been shown to cause mutations of genes [38]
and inhibition of DNA repair enzymes [39] and to mediate
DNA strand breaks [40]. NO has been shown to mediate
apoptosis [41].
Synthesis of (4-chlorophenyl)[3-(4-chlorophenyl)-
oxiranyl]methanone (2)
Hydrogen peroxide (5 mL, 30%) was added portionwise to a
mixture of 1,3-bis(4-chloro-phenyl)-propenone 1 (0.01 mol) in
acetone (50 mL) and methanol (15 mL) containing NaOH (1 g)
at 20–258C with stirring. The reaction mixture was left
overnight, then cold water was added and the precipitated solid
was filtered off, washed with cold water, and crystallized from
ethanol to give compound 2. Yield 73%; m.p. 103–1058C.
Bienvenu et al. [42] reported that most chemotherapeutic
agents cause cells to overgenerate ROS and, thus, are capable
of inducing cell death by causing oxidative damage to
DNA, proteins, and lipids. The cascade 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. Recently, Huang et al. [43]
reported that regulation of free radical-producing agents
may also have important clinical applications. This mechan-
ism 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 regu-
latory machinery (e.g., topoisomerases and helicases).
Morever, the initiation of DNA damage via production of
ROS was reported [44].
IR spectrum (KBr, n, cmꢀ1): 1676 (C O). 1H NMR spectrum
–
–
(CDCl3, d ppm): 4.05 (s, 1H, epoxy-H), 4.17 (s, 1H, epoxy-H),
7.26–7.89 (m, 8H, Ar-H). 13C NMR spectrum (CDCl3, d ppm)
showed signals at: 58.67 (b-C), 60.95 (a-C), 127.13–135.06
þ
þ
–
(12 Ar-C), 191.93 (C O). MS, m/z (%): (296, 4M , 2%), (294, 2M ,
–
13%), (292, Mþ, 21%). Anal. Calcd. for C15H10Cl2O2 (293.15):
C, 61.46; H, 3.44; Cl, 24.19. Found: C, 61.38; H, 3.33; Cl, 24.02.
General procedure for the synthesis of 3–5
A mixture of compound 2 (0.01 mol) and (thiourea, urea, and
thiosemicarbazide) (0.01 mol) in ethanolic potassium hydroxide
(2 g in 100 mL ethanol) was refluxed for 4 h. The solvent was
evaporated and the formed precipitate was washed several times
with acidified cold water, filtered off, and recrystallized from the
proper solvent to give compounds 3–5.
Conclusion
In conclusion, the present results suggest that the synthes-
ized compounds possess significant antitumor activity com-
parable to the activity of the commonly used anticancer
drug, cisplatin, and they exert their antitumor activities
by regulating free radicals production by increasing the
activity of SOD and depletion of intracellular GSH level,
CAT, GSH-Px activities, accompanied with high production
of hydrogen peroxide, NO, and other free radicals causing
tumor cell death, as monitored by reduction in the synthesis
of protein and nucleic acids. The results revealed that these
4,6-Bis(4-chlorophenyl)-2-thioxo-tetrahydropyrimidin-5-
one (3)
Benzene; yield 63%; m.p. 169–1718C. IR spectrum (KBr, n, cmꢀ1):
3219, 3135 (2NH), 1745 (C O), 1250 (C S); 1H NMR spectrum
–
–
–
–
(DMSO-d6,
d
ppm): 3.09 (d, J ¼ 10 Hz, 1H, pyrimidine-H),
3.49 (d, J ¼ 10 Hz, 1H, pyrimidine-H), 7.15–7.98 (m, 8H, Ar-H),
10.79 (s, 1H, NH, D2O exchangeable), 11.64 (s, 1H, NH, D2O
exchangeable).13C NMR spectrum (DMSO-d6, d ppm): 42.88
–
(CH), 71.37 (CH), 127.83–133.74 (12Ar-C), 175.82 (C O), 182.08
þ
–
(C S). MS, m/z (%): (354, 4M , 16%), (352, 2M , 68%), (350,
þ
–
–
Mþ, 100%). Anal. Calcd. for C16H12Cl2N2OS (351.26): C, 54.71;
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Pyrimidinone and Fused Pyrimidinone as Anticancer Agents
7
H, 3.44; Cl, 20.19; N, 7.98; S, 9.13. Found: C, 54.63; H, 3.38;
Cl, 19.93; N, 7.79; S, 8.98.
(C O). 1H NMR spectrum (DMSO-d6, d ppm): 3.98 (bs, 2H, NH2,
–
–
D2O exchangeable), 6.69 (s, H, NH, D2O exchangeable), 7.21–7.50
(m, 9H, 8Ar-H þ pyrimidine-H). MS, m/z (%): (350, 4Mþ, 3%), (348,
2Mþ, 14%), (346, Mþ, 26%). Anal. Calcd. for C16H12Cl2N4O (347.21):
C, 55.35; H, 3.48; Cl, 20.42; N, 16.14. Found: C, 55.22; H, 3.25;
Cl, 20.53; N, 16.21.
4,6-Bis(4-chlorophenyl)tetrahydropyrimidine-2,5-dione (4)
Carbon tetrachloride; yield 64%; m.p. 191–1938C. IR spectrum
(KBr, n, cmꢀ1): 3176 (br 2NH), 1716 (2C O); 1H NMR spectrum
–
–
(DMSO-d6, d ppm): 3.05 (d, J ¼ 9.9 Hz, 2H, 2 pyrimidine-H), 7.20–
7.93 (m, 8H, Ar-H), 10.81 (s, 2H, 2NH, D2O exchangeable). 13C NMR
Synthesis of 5,7-bis(4-chlorophenyl)-7H-thiazolo[3,2-a]-
pyrimidine-3,6-dione (8)
spectrum (DMSO-d6, d ppm): 71.66 (2CH), 127.27þ–132.88 (12Ar-C),
þ
–
–
–
180.05 (C O), 181.84 (C O). MS, m/z (%): (338, 4M , 3%), (336, 2M ,
–
A mixture of compound 3 (0.01 mol) with bromoacetic acid
(0.01 mol) in glacial acetic acid (30 mL)/acetic anhydride
(15 mL) in the presence of fused anhydrous sodium acetate
(2 g) was refluxed for 3 h. The solution was cooled, gradually
poured onto cold water, and the formed precipitate was washed
several times with water, filtered off, and recrystallized from
acetic acid to give compound 8. Yield 57%; m.p. 231–2338C.
15%), (334, Mþ, 24%). Anal. Calcd. for C16H12Cl2N2O2 (335.19):
C, 57.33; H, 3.61; Cl, 21.15; N, 8.36. Found: C, 57.63; H, 3.58;
Cl, 21.03; N, 8.20.
1-Amino-4,6-bis(4-chlorophenyl)-2-thioxo-
tetrahydropyrimidin-5(6H)-one (5)
ꢀ
1
–
IR spectrum (KBr, n, cm 1): 1742, 1719 (2C O); H NMR spectrum
Benzene, yield 52%; m.p. 179–1818C. IR spectrum (KBr, n, cmꢀ1):
–
(DMSO-d6, d ppm): 3.27 (d, J ¼ 6.1 Hz, 1H, pyrimidine-H), 3.34
(d, J ¼ 6.3 Hz, 1H, pyrimidine-H), 4.01 (s, 2H, CH2), 7.38–7.77
(m, 8H, Ar-H). 13C NMR spectrum (DMSO-d6, d ppm) showed
signals at: 34.50 (CH2), 60.02 (CH), 70.05 (CH), 129.03–132.40
1
3294 (NH ), 3165 (NH), 1721 (C O), 1287 (C S); H NMR spectrum
–
–
–
–
2
(DMSO-d6, d ppm): 3.12 (d, J ¼ 10 Hz, 1H, pyrimidine-H), 3.79
(d, J ¼ 10 Hz, 1H, pyrimidine-H), 6.02 (bs, 1H, NH2, D2O exchange-
able) 7.16–7.66 (m, 8H, Ar-H), 9.01 (bs, 1H, NH, D2O exchange-
able). 13C NMR spectrum (DMSO-d6, d ppm): 43.50 (CH), 72.14 (CH),
–
–
(Ar-C), 155.23 (C8‘), 171.42 (C O), 185.68 (C O). MS, m/z (%):
–
–
(394, 4Mþ, 2%), (392, 2Mþ, 5%), (390, Mþ, 12%). Anal. Calcd. for
C18H12Cl2N4O2S (391.28): C, 55.25; H, 3.09; Cl, 18.12; N, 7.16;
S, 8.18. Found: C, 55.08; H, 2.97; Cl, 18.19; N, 7.23; S, 8.30.
–
–
123.88–135.94 (12Ar-C), 176.24 (C O), 182.00 (C S). MS, m/z (%):
–
–
(369, 4Mþ, 2%), (367, 2Mþ, 14%), (365, Mþ, 19%). Anal. Calcd. for
C16H13Cl2N3OS (366.27): C, 52.47; H, 3.58; Cl, 19.36; N, 11.47;
S, 8.75. Found: C, 52.20; H, 3.31; Cl, 19.44; N, 11.56; S, 8.52.
Synthesis of 2-benzylidine-5,7-bis(4-chlorophenyl)-5H,7H-
thiazolo[3,2-a]pyrimidine-3,6-dione (9)
Method A: A mixture of compound 8 (0.01 mol) and benzaldehyde
(0.01 mol) in acetic anhydride (30 mL) was refluxed for 3 h. The
solution was cooled, gradually poured onto cold water, and
the formed precipitate was filtered off and recrystallized from
glacial acetic acid to give compound 9.
Synthesis of 4,6-bis(4-chloro-phenyl)-2-(methylthio)-
pyrimidin-5(4H)-one (6)
A mixture of compound 3 (0.01 mol) and methyl iodide
(0.01 mol) in ethanolic sodium ethoxide (prepared by dissolving
2 g of Na in 100 mL dry ethanol) was refluxed for 4 h. The
reaction mixture was then cooled and poured onto ice-cold
water. The solid product obtained after acidification with hydro-
chloric acid (37%) was filtered off, washed several times with
water, and recrystallized from ethanol to give compound 6. Yield
Method B: A mixture of compound 3 (0.01 mol), bromoacetic
acid (0.01 mol), and benzaldehyde (0.01 mol) in a mixture of
glacial acetic acid (30 mL)/acetic anhydride (15 mL) in the
presence of anhydrous sodium acetate (2 g) was refluxed for
5 h. The solution was cooled, gradually poured onto cold
water, and the formed precipitate was washed several times
with water, filtered off, and recrystallized from glacial acetic
acid to give a compound identical in all aspects with compound 9
obtained from Method A. Yield (66% from Method A, 47%
from Method B); m.p. 257–2598C. IR spectrum (KBr, n, cmꢀ1):
57%; m.p.136–1388C. IR spectrum (KBr, n, cmꢀ1): 1713 (C O);
–
–
d ppm): 2.62 (s, 3H, CH3),
1H NMR spectrum (DMSO-d6,
7.60–8.35 (m, 9H, 8Ar-H þ pyrimidine-H). MS, m/z (%):
(366, 4Mþ, 10%), (364, 2Mþ, 61%), (362, Mþ, 90%). Anal. Calcd.
for C17H12Cl2N2OS (363.27): C, 56.21; H, 3.33; Cl, 19.52; N, 7.71; S,
8.83. Found: C, 55.99; H, 3.19; Cl, 19.63; N, 7.88; S, 8.91.
1758, 1720 (2C O); 1H NMR spectrum (DMSO-d6, d ppm): 2.92
–
–
Synthesis of 4,6-bis(4-chlorophenyl)-2-
hydrazinylpyrimidin-5(4H)-one (7)
Method A: A mixture of compound 3 (0.01 mol) and hydrazine
hydrate (0.02 mol, 99%) was refluxed for 2 h. The resulting solid
after cooling was collected by filtration and recrystallized from
ethanol to give compound 7.
(d, J ¼ 6.1 Hz, 1H, pyrimidine-H), 3.74 (d, J ¼ 6.33 Hz, 1H,
pyrimidine-H), 7.13–7.89 (m, 13H, Ar-H), 8.46 (s, 1H, benzylic-H).
MS, m/z (%): (482, 4Mþ, 0.1%), (480, 2Mþ, 1%), (478, Mþ, 3%). Anal.
Calcd. for C25H16Cl2N4O2S (479.39): C, 62.64; H, 3.36; Cl, 14.79;
N, 5.84; S, 6.67. Found: C, 62.39; H, 3.50; Cl, 14.52; N, 5.99;
S, 6.55.
Method B: A mixture of compound 6 (0.01 mol) and hydrazine
hydrate (0.011 mol, 99%) in ethanol (20 mL) was refluxed for 4 h.
The resulting solid was collected by filtration and recrystallized
from ethanol to give compound 7.
Method C: A solution of compound 12 (0.01 mol) in ethanol
(50 mL) and hydrazine hydrate (0.011 mol, 99%) was refluxed for
6 h. After cooling, the separated solid was collected and recrystal-
lized from ethanol to give 7 identical in all aspects (m.p. and
mixed m.p.) with compounds obtained from Methods A and B.
Yield (78% from Method A, 59% from Method B, 62% Method C),
m.p. 210–2128C. IR (KBr, n, cmꢀ1): 3422–3245 (br NH2, NH), 1715
Synthesis of 6,8-bis(4-chlorophenyl)-3-phenyl-6H-
isoxazolo[50,40:4,5][1,3]thiazolo[3,2-a]pyrimidin-7(8H)-one
(10)
A mixture of compound 9 (0.01 mol) with hydroxyl amine hydro-
chloride (0.01 mol) in glacial acetic acid (30 mL) in the presence
of anhydrous sodium acetate (2 g) was refluxed for 7 h. The
solution was cooled, gradually poured onto cold water, and
the formed precipitate was washed several times with water,
filtered off, and recrystallized from dioxane to give compound
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
A. E. Rashad et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
10. Yield 48%; m.p. 253–2558C. IR spectrum (KBr, n, cmꢀ1): 1714
–
1H, pyrimidine-H), 2.86 (d, J ¼ 10.30 Hz, 1H, pyrimidine-H),
7.37–7.95 (m, 13H, Ar-H). MS, m/z (%): (495, 4Mþ, 1%),
(493, 2Mþ, 7%), (491, Mþ, 18%). Anal. Calcd. for
C25H15Cl2N3O2S (492.39): C, 60.98; H, 3.07; Cl, 14.40; N, 8.53; S,
6.51. Found: C, 60.71; H, 2.81; Cl, 14.33; N, 8.35; S, 6.67.
Synthesis of 2-amino-4,6-bis(4-chlorophenyl)pyrimidin-
5(4H)-one (14)
(C O); 1H NMR spectrum (CDCl3, d ppm): 2.55 (d, J ¼ 10.30 Hz,
–
A mixture of compound 13 (0.01 mol) and zinc dust (0.01 mol) in
glacial acetic acid (10 mL) was stirred at room temperature for
2 h, then heated in a water bath at 808C for 8 h. The reaction
mixture was poured into water, extracted with chloroform and
the extract was evaporated under reduced pressure. The formed
solid was recrystallized from methanol to give 14. Yield 53%,
m.p. 191–1938C. IR spectrum (KBr, n, cmꢀ1): 3390 (NH2), 1747
Synthesis of 4,6-bis(4-chlorophenyl)-1-(piperidin-1-
(C O). 1H NMR spectrum (DMSO-d6, d ppm): 6.86 (bs, 2H, NH2,
–
–
ylmethyl)-2-thioxo-tetra hydropyrimidin-5(6H)-one (11)
To a solution of compound 3 (0.01 mol) in dry ethanol (30 mL),
formaldehyde (1 mL, 40%) was added and the reaction mixture
was heated for 5 min. After cooling, piperidine (0.01 mol) was
added and the reaction mixture was stirred for 3 h at room
temperature. The formed solid was filtered off, dried and recrys-
tallized from ethanol to give compound 11. Yield 79%; m.p. 187–
D2O exchangeable), 7.57–7.91 (m, 9H, 8Ar-H þ pyrimidine-H).
MS, m/z (%): (335, 4Mþ, 9%), (333, 2Mþ, 68%), (331, Mþ, 100%).
Anal. Calcd. for C16H11Cl2N3O (332.19): C, 57.85; H, 3.34;
Cl, 21.34; N, 12.65. Found: C, 57.61; H, 3.17; Cl, 21.43; N, 12.78.
Synthesis of 5,7-bis(4-chlorophenyl)-3-thioxo-2,3-
dihydro[1,2,4]triazolo[4,3-a]pyrimidin-6-one (15)
1898C. IR spectrum (KBr, n, cmꢀ1): 3220 (NH), 1722 (C O), 1284
–
–
(C S); H NMR spectrum (DMSO-d , d ppm): 1.99–2.05 (m, 6H,
1
–
–
6
A solution of compound 7 (0.01 mol) in ethanolic potassium
hydroxide [prepared by dissolving potassium hydroxide
(0.01 mol) in ethanol (50 mL)], and carbon disulphide (10 mL)
was added. The reaction mixture was heated in a water bath at
808C for 10 h and then poured into water, neutralized by hydro-
chloric acid (37%). The formed solid was collected and recrystal-
lized from ethanol to give 15. Yield 52%, m.p. 163–1658C. IR (KBr,
piperidine-H), 3.27 (d, J ¼ 6.1 Hz, 1H, pyrimidine-H), 3.34
(m, 5H, 4 piperidine-H þ pyrimidine-H), 5.67 (S, 2H, NCH2N),
7.38–7.69 (m, 8H, Ar-H), 9.77 (s, 1H, NH, D2O exchangeable).
13C NMR spectrum (DMSO-d6, d ppm): 23.51 (CH2), 25.94 (CH2),
44.57 (CH2), 51.97 (NCH2N), 64.16 (CH), 69.70 (CH), 124.73–133.37
þ
–
–
(12Ar-C), 175.51 (C O), 184.79 (C S). MS, m/z (%): (451, 4M , 3%),
–
–
(449, 2Mþ, 20%), (447, Mþ, 33%). Anal. Calcd. for C22H23Cl2N3OS
(448.42): C, 58.93; H, 5.17; Cl, 15.81; N, 9.37; S, 7.15. Found: C,
58.87; H, 4.99; Cl, 15.94; N, 9.49; S, 6.92.
n, cmꢀ1): 3122 (NH), 1716 (C O), 1222 (C S). 1H NMR spectrum
–
–
–
–
(DMSO-d6, d ppm): 7.09–7.74 (m, 9H, 8Ar-H þ pyrimidine-H),
10.03 (s, H, NH, D2O exchangeable). MS, m/z (%): (392, 4Mþ,
10%), (390, 2Mþ, 72%), (388, Mþ, 100%). Anal. Calcd. for
C17H10Cl2N4OS (389.27): C, 52.46; H, 2.59; Cl, 18.22; N, 14.39;
S, 8.24. Found: C, 52.11; H, 2.63; Cl, 18.01; N, 14.45; S, 8.45.
Synthesis of [4,6-bis(4-chlorophenyl)-5-oxo-4,5-
dihydropyrimidin-2-yloxy]acetic acid ethyl ester (12)
A mixture of compound 4 (0.01 mol), ethyl bromoacetate
(0.01 mol), and anhydrous potassium carbonate (0.04 mol) in
dry acetone (30 mL) was refluxed for 20 h. The reaction mixture
was left overnight, poured into water and extracted with ether.
The ether layer was washed with water and dried over anhydrous
MgSO4 and the excess solvent was evaporated under reduced
Synthesis of 3-chloromethyl-5,7-bis(4-chlorophenyl)-
[1,2,4]triazolo[4,3-a]pyrimidin-6-one (16)
A solution of compound 7 (0.01 mol) and chloroacetyl chloride
(0.01 mol) in 20 mL of dry dioxane was allowed to stand over-
night at room temperature. The solid formed was filtered off,
dried and recrystallized from dioxane to give 16. Yield 60%, m.p.
pressure to give 12. Yield 71%, oil. IR (KBr, n, cmꢀ1): 1740, 1713
182–1848C. IR (KBr, n, cmꢀ1): 1741 (C O). 1H NMR spectrum
1
–
–
–
(2 C O). H NMR spectrum (DMSO-d , d ppm): 1.16 (t, J ¼ 6.9 Hz,
–
6
(DMSO-d6, d ppm): 5.22 (s, 2H, CH2), 7.16–7.98 (m, 9H,
8Ar-H þ pyrimidine-H). Anal. Calcd. for C18H11Cl3N4O (405.67):
C, 53.29; H, 2.73; Cl, 26.22; N, 13.81. Found: C, 53.22; H, 2.55; Cl,
26.30; N, 13.63.
3H, CH3CH2O), 4.08 (q, J ¼ 5.4 Hz, 2H, CH3CH2O), 4.89 (s, 2H,
CH2), 7.13–7.97 (m, 9H, 8Ar-H þ pyrimidine-H). 13C NMR spec-
trum (DMSO-d6, d ppm): 14.53 (CH3), 61.74 (CH2), 64.38 (OCH2CO),
–
67.81 (CH), 125.62–153.51 (14Ar-C), 167.44 (C O, ester), 185.12
þ
–
(C O). MS, m/z (%): (422, 4M , 2%), (420, 2M , 13%), (418, M , 19%).
þ
þ
–
–
Anal. Calcd. for C20H16Cl2N2O4 (419.27): C, 57.30; H, 3.85;
Cl, 16.91; N, 6.68. Found: C, 57.47; H, 3.61; Cl, 16.76; N, 6.74.
Synthesis of 5,7-bis(4-chlorophenyl)-3-methyl-[1,2,4]-
triazolo[4,3-a]pyrimidin-6-one (17)
A solution of compound 7 (0.01 mol) in acetic anhydride (10 mL)
was refluxed for 6 h. The reaction mixture was cooled and
poured into cold water. The separated solid was filtered off, dried
Synthesis of 5,7-bis(4-chlorophenyl)tetrazolo[1,5-a]-
pyrimidin-6-one (13)
and recrystallized from ethanol to give 17. Yield 78%, mp 196–
1988C. IR (KBr) n, cmꢀ1: 1725 (C O). H NMR spectrum (DMSO-d ,
To an ice-cold solution of compound 7 (0.01 mol) in glacial acetic
acid (10 mL), a solution of sodium nitrite [prepared by dissolving
sodium nitrite (0.01 mol) in water (3 mL)] was added dropwise in
an ice-bath. The reaction mixture was allowed to stand overnight
at room temperature and then was poured into water. The
formed solid was filtered off, washed with water, dried and
recrystallized from dioxane to give 13. Yield 57%, m.p. 240–
1
–
–
6
d ppm): 2.34 (s, 3H, CH3), 7.59–8.39 (m, 9H, 8Ar-H þ pyrimidine-H).
MS, m/z (%): (374, 4Mþ, 3%), (372, 2Mþ, 34%), (370, Mþ, 78%). Anal.
Calcd. for C18H12Cl2N4O (371.23): C, 58.24; H, 3.26; Cl, 19.10; N,
15.09. Found: C, 57.99; H, 3.40; Cl, 18.97; N, 14.98.
2428C. IR (KBr, n, cmꢀ1): 1732 (C O). 1H NMR spectrum (CDCl3,
Synthesis of 5,7-bis(4-chlorophenyl)-3-phenyl-[1,2,4]-
triazolo[4,3-a]pyrimidin-6-one (18)
A mixture of compound 7 (0.01 mol) and benzoyl chloride
(0.01 mol) with a catalytic amount of triethyl amine (2 drops)
in dry dioxane was refluxed for 8 h. The reaction mixture was
–
–
d ppm): 7.33–7.91 (m, 9H, 8Ar-H þ pyrimidine-H). MS, m/z (%):
(361, 4Mþ, 9%), (359, 2Mþ, 68%), (357, Mþ, 100%). Anal. Calcd. for
C16H9Cl2N5O (358.19): C, 53.65; H, 2.53; Cl, 19.80; N, 19.55. Found:
C, 53.47; H, 2.65; Cl, 19.61; N, 19.60.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Pyrimidinone and Fused Pyrimidinone as Anticancer Agents
9
cooled and the separated solid was filtered off, dried, and recrys-
tallized from dioxane to give 18. Yield 78%, mp 208–2108C. IR
using Microsoft Excel and Microcal Origin software for PC. After
that, the MCF-7 cells in culture medium were treated with 20 mL
of 1/10 of the IC50 values of the compounds and the standard
reference drug, cisplatin, then incubated for 24 h at 378C, in a
humidified 5% CO2 atmosphere. The cells were harvested and
homogenates were prepared in saline using a tight pestle hom-
ogenizer until complete cell disruption.
1
(KBr) n, cmꢀ1: 1722 (C O). H NMR spectrum (DMSO-d , d ppm):
–
–
6
7.24–7.96 (m, 14H, 13Ar-H þ pyrimidine-H). MS, m/z (%): (436,
4Mþ, 14%), (434, 2Mþ, 67%), (432, Mþ, 100%). Anal. Calcd. for
C23H14Cl2N4O (433.30): C, 63.76; H, 3.26; Cl, 16.36; N, 12.93.
Found: C, 63.89; H, 2.97; Cl, 16.48; N 12.65.
Biochemical assays
Anticancer activity
Materials and methods
The supernatant obtained after centrifugation of cell homogen-
ates was used for determination of enzyme activities of SOD, CAT,
and GSH-Px as described by Paglia and Valentine [46], Aebi [47],
and Marklund and Marklund [48], respectively.
Chemicals
Dimethylsulphoxide (DMSO), cisplatin, and MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were pur-
chased from Merck. All other chemicals and reagents used in
this study were of analytical grade and purchased from Sigma–
Aldrich Chemical Co. (St. Louis, MO, USA).
The levels of hydrogen peroxide (H2O2), NO, and GSH were
determined by the methods of Wolff [49], Montgomery and
Dymock [50], and Ellman [51], respectively.
Nucleic acids (DNA and RNA) and total protein were precipi-
tated and measured in cell homogenates. Total DNA was
extracted and assayed according to the method described by
Zhou et al. [52], total RNA was extracted and assayed according
to the method adopted from the method provided by Hybaid/
AGS (Germany), and total cellular protein was assayed by the
method of Lowry et al. [53].
Cell culture
The MCF-7 human breast 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 mg/mL) at 378C in humidified atmosphere containing 5%
CO2. MCF-7 cells at a concentration of 0.50 ꢂ 106 were grown in a
25 cm2 flask in 5 mL of complete culture medium.
The estimation of in vitro tumor cell growth inhibition was
assessed by incubating 0.65 ꢂ 105 MCF-7 cells in 1 mL phosphate
buffer saline with varying concentrations of the prepared com-
pounds and cisplatin (as þve control drug) at 378C for 24 h in
CO2 atmosphere. Cells were cultured for 24 h to assure total
attachment. Afterwards, 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 exper-
iments, results were compared with the untreated cells.
Statistical analysis
The results are reported as mean ꢁ standard error (SE) for at
least four replicate experiments. Statistical differences were ana-
lyzed by one-way ANOVA test followed by Student’s t-test wherein
the differences were considered to be significant at p <0.05.
The authors extend their appreciation to the Deanship of Scientific Research
at King Saud University for funding the work through the research group
project No. RGP-VPP-032.
The authors have declared no conflict of interest.
References
In vitro cytotoxicity assay
Effect of the prepared compounds on the growth of MCF-7 cells
was estimated by MTT colorimetric assay according to Mosmann
[45]. 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 pro-
portional to the production of formazan salts. The crystals of
formazan were dissolved in DMSO and the optical density was
measured spectrophotometrically (microplates reader, Asys
Hitech, Austria).
[1] D. J. Brown, in Comprehensive Heterocyclic Chemistry (Eds.: A. R.
Katritzky, C. W. Rees), Pergamon Press, Oxford, UK 1984,
Vol. 3, pp. 57–155.
[2] A. K. Singh, Talanta 1982, 29, 95–102.
[3] R. Dudhe, P. K. Sharma, P. Verma, A. Chaudhary, J. Adv. Sci.
Res. 2011, 2 (3), 10–17.
[4] A. E. Rashad, A. E. Mahmoud, M. M. Ali, Eur. J. Med. Chem.
2011, 46, 1019–1026.
Cells (1.00 ꢂ 106 cells/well) were plated separately in a sterile
flat bottom 96-well microplate (Falcon), and treated with 20 mL
of different concentrations of tested compounds and cisplatin
(5, 12.50, 25, or 50 mg/mL), for 24 h at 378C, in a humidified
5%ml MTT solution/well were added and incubated for an
additional 4 h. MTT crystals were solubilized by adding 200 mL
of DMSO/well and the plate was shaken gently for 10 min at
room temperature. The results were determined photometricaly
using microplate ELISA reader and the absorbance was 570 nm.
Data were expressed as the percentage of relative viability
compared with untreated cells.
[5] E. E. Flefel, M. A. Salama, M. El-Shahat, M. A. El-Hashash, A. F.
El-Farargy, Phosphorus, Sulfur Silicon Relat Elem. 2007, 182,
1739–1756.
[6] M. A. Ajam, B. P. R. Kumar, B. Suresh, Indian J. Pharm. Sci.
2008, 672–677.
[7] K. M. Amin, M. I. El-Zahar, M. M. Anwar, M. M. Kamel, M. H.
Mohamed, Acta Pol. Pharm. Drug Res. 2009, 66, 279–291.
[8] M. Banjanac, I. Tatic, Z. Ivezic, S. Tomi, J. Dumic,
Biotechnology 2009, 47 (3), 236–245.
[9] E. Nassar, J. Am. Sci. 2010, 6 (8), 463–471.
Cytotoxic concentration was expressed by half maximal
inhibitory concentration (IC50). IC50 calculations were performed
[10] V. Alagarsamy, U. S. Pathak, R. Revathi, Indian J. Heterocycl.
Chem. 2003, 12, 335–338.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
A. E. Rashad et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
[11] S. M. Vroegop, D. L. Chapman, D. E. Decker, L. A. Galinet, R. J.
Brideau, K. A. Ready, C. J. Dunn, S. E. Buxser, Int. J.
Immunopharmacol. 1999, 21, 647–662.
[28] J. P. Jasinski, R. J. Butcher, B. Narayana, K. Veena, H. S.
Yathirajan, Acta Cryst. 2009, E65, 2641–2642.
[29] S. M. Shrif, M. M. Yossef, Kh. M. Mobarak, A. M. Abdel-Fattah,
Tetrahedron 1993, 49, 9561.
[12] R. Saladino, U. Ciambecchini, G. Maga, P. Mastromarino, C.
Conti, M. A. Botta, Bioorg. Med. Chem. Lett. 2002, 10, 2143–
2153.
[30] A. B. A. El-Gazzar, M. M. Youssef, A. M. S. Youssef, A. A. Abu-
Hashem, F. A. Badria, Eur. J. Med. Chem. 2008, 44, 609–
6624.
[13] G. V. de Lucca, J. Liang, I. de Lucca, J. Med. Chem. 1999, 42,
135–152.
[31] A. I. Khodair, N. A. Al-Masoudi, J. Gesson, Nucleosides,
Nucleotides Nucleic Acids 2003, 22, 2061–2076.
[14] A. E. Rashad, A. H. Shamroukh, R. E. Abdel-Megeid, A.
Mostafa, A. Kandil, R. Elshesheny, M. A. Ali, K. Banert,
Eur. J. Med. Chem. 2010, 45, 5251–5257.
[32] H. Reimlinger, Chem. Ber. 1970, 103, 1900–1907.
[33] M. Tisler, Synthesis 1973, 123–136.
[15] A. E. Rashad, A. H. Shamroukh, R. E. Abdel-Megeid, A.
Mostafa, M. A. Ali, K. Banert, Nucleosides, Nucleotides Nuclic
Acid. 2010, 29, 809–820.
[34] C. J. Shishoo, K. S. Jain, J. Heterocycl. Chem. 1992, 29, 883–893.
[35] F. J. Blanco, R. L. Ochs, H. Schwarz, M. Lotz, Am. J. Pathol. 1995,
146, 75–85.
[16] C. A. Bernhart, F. B. Haudricourt, J. L. Assens, J. Gougat, C.
[36] Y. Molina, L. Vedia, B. McDonald, B. Reep, B. Brne, M.
Di Silvio, T. R. Billiar, J. Biol. Chem. 1992, 267, 24929–
24932.
`
Lacour, A. Roccon, C. Cazaubon, J. C. Breliere, G. Le Fur, D.
Nisato, Bioorg. Med. Chem. Lett. 1994, 4, 157–162.
[17] A. Salimbeni, R. Canevotti, F. Paleari, D. Poma, S. Caliari, F.
Fici, R. Cirillo, A. R. Renzetti, A. Subissi, L. Belvisi, G. Bravi, C.
Scolastico, A. Giachetti, J. Med. Chem. 1995, 38, 4806–4820.
[37] J. Hibbs, R. Taintor, Z. Vavrin, E. M. Rachlin, Biophys. Res.
Commun. 1988, 157, 87–94.
[38] M. J. Juedes, G. N. Wogan, Mutat. Res. 1996, 349, 51–61.
[18] G. R. Madhavan, R. Chakrabarti, R. K. Vikramadithyan, R. N.
Mamidi, V. Balraju, B. M. Rajesh, P. Misra, S. K. Kumar, B. B.
Lohray, V. B. Lohray, R. Rajagopalan, Bioorg. Med. Chem. 2002,
10, 2671–2680.
[39] M. Lepoivre, F. Fieschi, J. Coves, L. Thelander, M. Fontecave,
Biochem. Biophys. Res. Commun. 1991, 179, 442–448.
[40] K. Fehsel, A. Jalowy, S. Qi, V. Burkart, Diabetes 1993, 42, 496–
500.
[19] M. Yamaguchi, K. Wakasugi, R. Saito, Y. Adachi, Y.
Yoshikawa, H. Sakurai, A. Katoh, J. Inorg. Biochem. 2006,
100, 260–269.
[41] S. Dimmeler, A. M. Zeiher, Nitric Oxide 1997, 1, 275–281.
[42] P. Bienvenu, L. Caron, D. Gasparutto, J. F. Kergonou, EXS
1992, 62, 257–265.
[20] D. C. White, T. D. Greenwood, A. L. Downey, J. R. Bloomquist,
J. F. Wolfe, Bioorg. Med. Chem. 2004, 12, 5711–5717.
[43] P. Huang, L. Feng, E. A. Oldham, M. J. Keating, W. Plunkett,
Nature 2000, 407, 390–395.
[21] D. L. Temple, J. P. Yevich, R. R. Covington, C. A. Hanning, R. J.
Seidehamel, H. K. Mackey, M. J. Bartek, J. Med. Chem. 1979, 22,
505–510.
[44] D. A. Gewirtz, Biochem. Pharmacol. 1999, 57, 727–741.
[45] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
[22] A. Ranise, O. Bruno, F. Bondavalli, S. Schenone, M. D’Amico,
M. Falciani, W. Filippelli, F. Rossi, Farmaco 1994, 49, 551–558.
[46] E. D. Paglia, W. N. Valentine, J. Lab. Clin. Med. 1967, 70, 158–
169.
[23] H. I. Skulnick, J. H. Ludens, M. G. Wendling, E. M. Glenn,
N. A. Rohloff, R. J. Smith, W. Wierenga, J. Med. Chem. 1986, 29,
1499–1504.
[47] H. Aebi, in Method of Enzymatic Analysis, Academic Press, New
York 1984, Vol. 2, 673–684.
[48] S. Marklund, G. Marklund, Eur. J. Biochem. 1974, 47, 469–474.
[49] S. P. Wolff, Methods Enzymol. 1994, 233, 182–189.
[24] A. Ranise, O. Bruno, S. Schenone, F. Bondavalli, G. Falcone,
W. Filippelli, S. Sorrentino, Farmaco 1997, 52, 547–555.
[50] H. A. C. Montgomery, J. F. Dymock, Analyst 1961, 86, 414–
416.
[25] J. V. dos Anjos, R. M. Srivastava, J. H. Costa-Silva, L. Scotti,
M. T. Scotti, A. G. Wanderley, E. S. Leite, S. J. de Melo, F. J. B.
M. Junior, Molecules 2012, 17, 809–819.
[51] G. L. Ellman, Arch. Biochem. Biophys. 1959, 82, 70–77.
[52] T. Zhou, G. Zhou, W. Song, N. Eguchi, W. Lu, E. Lundin,
T. Jin, G. Nordberg, Toxicology 1999, 142, 1–13.
[26] A. E. Rashad, A. H. Shamroukh, R. E. Abdel-Megeid, W. A.
El-Sayed, Synth. Commun. 2010, 40, 1149–1160.
[53] O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J. Biol.
Chem. 1951, 193, 265–275.
[27] A. E. Rashad, A. H. Shamroukh, H. H. Sayed, S. M. Awad, N. A.
Abdel-Wahed, Synth. Commun. 2011, 41, 652–661.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com