Synthesis and in vitro antitumor activity of new substituted thiopyrimidine acyclic nucleosides and their thioglycoside analogs

Article abstract of DOI:10.1080/15257770902946165

Some new thiopyrimidine acyclic nucleosides and thioglycoside derivatives 3a-c, 4a-c, 6a,b, and 7a,b were synthesized. The cytotoxicity and antitumor evaluation of all prepared compounds have been tested in vitro against Ehrlich's ascites carcinoma cell line and their activity against glutathione peroxidase and catalase were reported. The role of the prepared compounds as free radical regulators and the therapeutic antitumor effect of a balanced generation of free radicals are discussed. Compounds 2, 3b, 3c, 4a, and 4c inhibited significantly in a dose dependent manner the growth of Ehrlich ascites carcinoma cells while the other compounds did not show any antitumor activity even at higher concentrations.

Full text of DOI:10.1080/15257770902946165

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Synthesis and In Vitro Antitumor Activity
of New Substituted Thiopyrimidine
Acyclic Nucleosides and Their
Thioglycoside Analogs
Wael A. El-Sayed a , Aymn E. Rashad a , Samir M. Awad b & Mamdouh
M. Ali c
a Photochemistry Department, National Research Center, El Dokki,
Cairo, Egypt
b Faculty of Pharmacy, Helwan University, Cairo, Egypt
c Biochemistry Department, Division of Genetic Engineering and
Biotechnology, National Research Center, Cairo, Egypt
Version of record first published: 29 May 2009.
To cite this article: Wael A. El-Sayed , Aymn E. Rashad , Samir M. Awad & Mamdouh M. Ali (2009):
Synthesis and In Vitro Antitumor Activity of New Substituted Thiopyrimidine Acyclic Nucleosides and
Their Thioglycoside Analogs, Nucleosides, Nucleotides and Nucleic Acids, 28:4, 261-274
To link to this article: http://dx.doi.org/10.1080/15257770902946165
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Nucleosides, Nucleotides and Nucleic Acids, 28:261–274, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770902946165
SYNTHESIS AND IN VITRO ANTITUMOR ACTIVITY OF NEW
SUBSTITUTED THIOPYRIMIDINE ACYCLIC NUCLEOSIDES
AND THEIR THIOGLYCOSIDE ANALOGS
Wael A. El-Sayed,1 Aymn E. Rashad,1 Samir M. Awad,2 and
Mamdouh M. Ali3
1Photochemistry Department, National Research Center, El Dokki, Cairo, Egypt
2Faculty of Pharmacy, Helwan University, Cairo, Egypt
3Biochemistry Department, Division of Genetic Engineering and Biotechnology, National
Research Center, Cairo, Egypt
Some new thiopyrimidine acyclic nucleosides and thioglycoside derivatives 3a-c, 4a-c, 6a,b,
2
and 7a,b were synthesized. The cytotoxicity and antitumor evaluation of all prepared compounds
have been tested in vitro against Ehrlich’s ascites carcinoma cell line and their activity against
glutathione peroxidase and catalase were reported. The role of the prepared compounds as free
radical regulators and the therapeutic antitumor effect of a balanced generation of free radicals are
discussed. Compounds 2, 3b, 3c, 4a, and 4c inhibited significantly in a dose dependent manner
the growth of Ehrlich ascites carcinoma cells while the other compounds did not show any antitumor
activity even at higher concentrations.
Keywords Thiopyrmidines; acyclic nucleosides; thioglycosides; free radicals; antitumor
activity
INTRODUCTION
Pyrimidines have been recognized as important heterocyclic com-
pounds due to their diverse biological activities such as Tie-2 ki-
nase inhibitors,[1] HIV-1 inhibitor,[2] antimalarial,[3] secretive adeno-
sine A1 receptor antagonist,[4] antibacterial,[5] anticancer,[6] analgesic,[7]
cardiovascular,[8] and antiallergic[9] activities. The thio analogues of pyrim-
idine bases, including 2-thiouracil, are minor components of t-RNA. Their
S-, N - or S,N -disubstituted analogs have shown therapeutic properties,
Received 16 December 2008; accepted 18 March 2009.
Address correspondence to Wael A. El-Sayed, Photochemistry Department, National Research
Center, El-Behoose St., El Dokki, Cairo, Egypt. E-mail: waelshendy@gmail.com
261

262
W. A. El-Sayed et al.
especially antiviral, antithyroid and antitumor activities[10–12] due to their
incorporation into polynucleic acids[13] and therefore act as potential
inhibitors of protein and polynucleic acid syntheses.[14] On the other hand,
nucleoside analogs are structurally, metabolically, and pharmacodynami-
cally related agents that have diverse biological actions and therapeutic ef-
fects including antiviral[15–17] and antitumor[18–20] activities. Furthermore,
the glycosylthio heterocycles[21–23] and acyclic nucleoside[23–25] analogues
including modifications of both the acyclic glycon and aglycon parts have
stimulated extensive research as biological inhibitors.[26–29] The impor-
tance of substituted hydroxylalkyl chain conformation in the interaction
of acyclic nucleosides with enzymes has been demonstrated.[30] Moreover,
the 5-substituted derivatives of pyrimidine acyclonucleoside analogs have
exhibited pronounced inhibitory properties with respect to uridine phos-
phorylase and have enhanced antitumor action.[19,20]
Owing to the above facts, the aim of the present work is to synthesize
new substituted thiopyrimidine nucleobases and their acyclic nucleoside
analogs as well as evaluation of the cytotoxicity of these compounds in vitro
against Ehrlich’s ascites carcinoma cell line. In addition, their activity against
glutathione peroxidase and catalase was studied. The role of the prepared
compounds as free radical regulators and the therapeutic antitumor effect
of a balanced generation of free radicals were discussed.
RESULTS AND DISCUSSION
Reaction of cinnamaldehyde, ethyl cyanoacetate and thiourea in
ethanol afforded 1,2,3,4-tetrahydro-4-oxo-6-styryl-2-thioxopyrimidine-5-
carbonitrile (2). Reaction of compound 2 with methyl iodide, ethyl iodide,
chloroethyl methyl ether afforded the alkylthio derivatives 3a-c, respectively.
1
The H NMR spectrum of compound 3a, as a representative example,
showed the presence of the methyl group at δ 2.77 ppm, the aromatic
proton signals at δ 7.47–7.75 ppm in addition to the peak corresponding to
the NH group at δ 8.33 ppm. Its 13C NMR spectrum showed the presence
of the methyl group, the aromatic carbon atoms in addition to the C=O
and C=N groups. The absence of the peak corresponding to C=S group
indicates that the alkylation had taken place at the sulfur atom to give the
alkythio derivatives.
In order to synthesize a number of N -substituted acyclic nucleo-
side analogs, we used 1,6-dihydro-2-(methylthio)-6-oxo-4-styrylpyrimidine-5-
carbonitrile (3a) as a nucleobase. Thus, reaction of 3a with chloroacetalde-
hyde dimethylacetal, chloroethyl methyl ether or 3-chloropropane-1,2-diol
gave the corresponding N -acyclic nucleoside analogs 4a-c (see Scheme 1).
The IR spectrum of compound 4a, as a representative example, showed the
1
characteristic peaks for the C=O and CN groups and its H NMR showed

Synthesis and In Vitro Antitumor Activity
263
two OCH3 groups in addition to the CH2 signal as doublet which agreed
with the assigned structure.
On the other hand, treatment of compound 2 with 2,3,4,6-tetra-O-acetyl-
α-D-galacto- or glucopyranosyl bormide (5a,b) afforded the corresponding
thioglycosides (6a,b) (see Scheme 1). Their IR spectra showed characteristic
absorption bands in the carbonyl frequency region corresponding to the
1
O-acetyl groups and the H NMR spectra showed the anomeric proton
of the sugar moiety in the range δ 5.75–5.80 ppm as doublet, with a
coupling constants equal to 9.4 and 9.5 Hz for 6a and 6b, respectively
indicating the β-orientation of the thioglycosidic bond. The attachment of
the glycosyl residues to the sulfur rather than to the nitrogen has been
supported by the value of the chemical shift of the anomeric protons
which otherwise should appear at a lower field. The anomeric proton of
β-N -glucosides having an adjacent C=S, was reported to appear at higher
chemical shift (δ 6.9–7.2 ppm) due to the anisotropic deshielding effect of
the C=S.[31–34] Furthermore, the 13C NMR spectrum of 6a showed a signal
at δ 78.14 corresponding to the anomeric C-1 which also confirmed the
β-configuration. The absence of a peak corresponding to the C=S group
indicates that the attachment of the sugar at the sulfur atom.
When compounds 6a,b were treated with methanolic ammonia at 0◦C,
the deacetylated thioglycoside derivatives 7a,b were obtained in moderate
yields. The IR spectra of the deacetylated thioglycoside 7a,b showed char-
acteristic absorption bands corresponding to the OH groups for 7a and 7b,
1
respectively and their H NMR agreed with the assigned structures. (See
Scheme 1.)
Antitumor Activity
The tested compounds were screened in vitro using a single tumor
(Ehrlich ascites carcinoma cells). The results of the present study indicated
that compounds 2, 3b, 3c, 4a, and 4c could inhibit the growth of Ehrlich
ascites carcinoma cells significantly in culture, while the other compounds
did not show any antitumor activity even at higher concentrations.
As shown in Table 1, compounds 2, 3b, 3c, 4a, and 4c inhibited in
dose dependent manner the growth of Ehrlich ascites carcinoma cells. It
is observed that there was gradual decrease in the viability with increasing
the concentration in a dose dependent type. These compounds were found
to inhibit 50% proliferation of EAC cells in short term culture at con-
centrations of 13.7, 12.3, 6.2, 13, and 23 µg/ml, respectively. Compounds
increased the NO and TBARS concentrations in the culture medium in dose
dependent manner.
GSH-Px and CAT data for compounds 2, 3b, 3c, 4a, and 4c are given in
Table 1, which showed that these compounds were GSH-Px inhibitors, and
also prevent H2O2 degradation by inhibiting catalase activity. On the other

264
W. A. El-Sayed et al.
SCHEME 1 Synthesis route of substituted thiopyrimidine derivatives 2–7.
hand, reactive oxygen species (ROS), including hydroxyl radicals (·OH),
superoxide anion (O2·−), hydrogen peroxide (H2O2) and nitric oxide (NO)
are very transient species due to their high chemical reactivity that leads to
lipid peroxidation and oxidation of some enzymes, and a massive protein
oxidation and degradation.[33,36]
Our results revealed that, compounds 4a and 4c were more active
anticancer compounds than compounds 2, 3b, and 3c. The activity of

265

266
W. A. El-Sayed et al.
compounds 4a and 4c is due to an increase in NO level, which leads to
apoptosis, whereas in compounds 2, 3b, and 3c, an increase in oxygen
radical level leads to necrosis.[37]
The regulation of the glutathione peroxidase and catalase enzymes may
affect free radical balance in tumor. Glutathione peroxidase may scavenge
various peroxides, while catalase causes hydrogen peroxide degradation.[37]
Compounds 2, 3b, 3c, 4a, and 4c revealed inhibition of glutathione per-
oxidase and catalase activities. Compounds 4a and 4c demonstrated the
greatest inhibition of glutathione peroxidase and catalase enzymes.
The mechanism of action of these compounds can be explained by the
fact that these compounds may catalyze the conversion of superoxide and
hydrogen peroxide to free radical species that attack cellular membranes,
proteins and DNA causing cell damage.[38]
Moreover, Huang et al.[39] observed that selective inhibition of superox-
ide dismutase 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., topoiso-
merases, helicases) and to the initiation of DNA damage via production of
ROS.[40]
CONCLUSION
According to the obtained results, there is a correlation between the
efficiency in generating free radicals and the ability of the tested compounds
to suppress the growth of cancer cells, and we propose that the antitumor
effect of the tested compounds stems from the fact that they are able to
regulate the free radical balance inside the cells. Furthermore, structure-
activity relationship showed that the increase in NO level in compounds 4a
and 4c leading to apoptosis give rise to their high antitumor activity with
respect to other tested compounds.
EXPEREMINTAL
Chemistry
All melting points are uncorrected and measured using Electro-thermal
IA 9100 apparatus (Shimadzu, Japan). IR spectra were recorded as potas-
sium bromide pellets on a Perkin-Elmer 1650 Spectrophotometer, National
1
Research Centre, Cairo, Egypt. H NMR and 13C NMR spectra were de-
termined on a Jeol-Ex-300 NMR spectrometer and chemical shifts were
expressed as part per million; ppm (δ values) against TMS as internal

Synthesis and In Vitro Antitumor Activity
267
reference (Faculty of Science, Cairo University, Cairo, Egypt). Microanalyses
were operated using Mario Elmentar apparatus, Organic Microanalysis
Unit, National Research Centre, Cairo, Egypt and the results were within
the accepted range of the calculated values. Column chromatography was
performed on (Merck) Silica gel 60 (particle size 0.06–0.20 mm). Antitumer
activity evaluation was carried out at Biochemistry Department, Division of
Genetic Engineering and Biotechnology, National Research Center, Cairo,
Egypt.
1,2,3,4-Tetrahydro-4-oxo-6-styryl-2-thioxopyrimidine-5- carbonitrile (2).
To a solution of cinnamaldehyde (6.60 g, 50 mmol) and thiourea (3.81 g, 50
mmol) in ethanol (75 mL) was added ethylcyanoacetate (5.66 g, 50 mmol)
and the mixture was refluxed for 6 hours. The solvent was concentrated
under reduced pressure and water (100 mL) was added. The resulting solid
was filtered, washed with ice cold water and then recrystallized from ethanol
to afford 10.3 g compound of 2 (81%); m.p. 262–263◦C; IR (KBr): 3395
(NH), 2212 (CN), 1668 (C=O); 1H NMR (DMSO-d6, 300 Mz), δH: 10.12 (s,
1 H, NH), 8.33 (s, 1 H, NH), 8.07 (d, 1 H, J 6.5 Hz, CH), 7.72 (m, 2 H,
Ar-2H), 7.45 (m, 3 H, Ar-3H), 7.29 (d, 1 H, J 6.5 Hz, CH). Analysis calcd.
for C13H9N3OS: C, 61.16; H, 3.55; N, 16.46. Found: C, 60.95; H, 3.49; N,
16.35%.
1,6-Dihydro-2-(alkylthio)-6-oxo-4-styrylpyrimidine-5-carbonitrile
(3a,b,c). To a well stirred solution of compound 2 (2.55 g, 10 mmol)
and potassium hydroxide (0.56 g, 10 mmol) in alcoholic mixture of ethanol
(10 mL) and water (20 mL) was added methyl iodide or ethyl iodide (10
mmol mole). The reaction mixture was stirred at 60◦C for 4 hours then
cooled and the precipitated solid was filtered, washed with water and
crystallized from ethanol to give compounds 3a or 3b in 77–79% yields.
1,6-Dihydro-2-(methylthio)-6-oxo-4-styrylpyrimidine-5-carbonitrile (3a). Pale
yellow powder (77%); m.p. 294–296◦C; IR (KBr): 3395 (NH), 2213 (CN),
1
1667 (C=O); H NMR (DMSO-d6, 300 Mz), δH: 8.33 (s, 1 H, NH) 8.05 (d,
1 H, J 6.5 Hz, CH), 7.75 (m, 2 H, Ar-2H), 7.47 (m, 3 H, Ar-3H), 7.21 (d, 1
H, J 6.5 Hz, CH), 2.77 (s, 3 H, SCH3). 168.79 (pyrimidyl-C), 163.24 (C=N),
160.50 (C=O), 142.04 (CH), 134.50 (Ar-C), 129.14 (Ar-2C), 128.68 (Ar-2C),
128.47 (Ar-C), 121.56 (CH), 115.10 (CN), 93.09 (pyrimidyl-C), 13.32 (CH3).
Analysis calcd. for C14H11N3OS: C, 62.43; H, 4.12; N, 15.60. Found: C, 62.22;
H, 3.91; N, 15.88%.
1,6-Dihydro-2-(ethylthio)-6-oxo-4-styrylpyrimidine-5-carbonitrile (3b). Pale
yellow powder (79%); m.p. 288–289◦C; IR (KBr): 3396 (NH), 2212 (CN),
1670 (C=O); 1H NMR (DMSO-d6, 300 Mz), δH: 8.07 (s, 1 H, NH), 8.01 (d,
1 H, J 6.5 Hz, CH), 7.76 (m, 2 H, Ar-2H), 7.47 (m, 3 H, Ar-3H), 7.22 (d,
1 H, J 6.5 Hz, CH), 4.01 (q, 2 H, J 3.5 Hz, CH2), 1.43 (t, 3 H, J 3.5 Hz,
CH3). Analysis calcd. for C15H13N3OS: C, 63.58; H, 4.62; N, 14.83. Found:
C, 63.75; H, 63.75; N, 14.46%.

268
W. A. El-Sayed et al.
2-(2-Methoxyethylthio)-1,6-dihydro-6-oxo-4-styrylpyrimidine-5-carbonitrile
(3c): 2.25 g (72% yield); m.p. 289–290◦C; IR (KBr): 3410 (NH), 2206 (CN),
1657 (C=O); 1H NMR (DMSO-d6, 300 Mz), δH: 8.21 (s, 1 H, NH), 7.74 (d,
1 H, J 6.5 Hz, CH), 7.42 (m, 2 H, Ar-2H), 7.39 (m, 3 H, Ar-3H), 7.28 (d, 1
H, J 6.5 Hz, CH), 3.40 (t, 2 H, J 5.4 Hz, CH2), 3.27 (s, 3 H, OCH3), 3.25 (t,
2 H, J 5.4 Hz, CH2). Analysis calcd. for C16H15N3O2S: C, 61.32; H, 4.82; N,
13.41. Found: C, 60.96; H, 4.61; N, 13.09%.
General Procedure for the Synthesis of Compounds 4a and 4b
To a well stirred solution of compound 3a (2.69 g, 10 mmol) and sodium
hydride (0.25 g, 10 mmol) in N,N -dimethylformamide (15 mL) was added
chloroacetaldehyde dimethylacetal or chloroethyl methyl ether (0.01 mole).
The reaction mixture was stirred at room temperature for 10 hours and then
poured on ice-cold water. The precipitated solid was filtered, washed with
water and recrystallized from ethanol to give compounds 4a,b in 71–76%
yields.
1,6-Dihydro-1-(2,2-dimethoxyethyl)-2-(methylthio)-6-oxo-4-styrylpyrimidine-5-
carbonitrile (4a). Pale yellow powder (76%); m.p. 288–290◦C; IR (KBr):
1
2215 (CN), 1667 (C=O); H NMR (DMSO-d6, 300 Mz), δH: 8.07 (d, 1 H, J
6.5 Hz, CH), 7.60 (m, 2 H, Ar-2H), 7.37 (m, 3 H, Ar-3H), 7.24 (d, 1 H, J 6.5
Hz, CH), 4.58 (t, 1 H, J 5.8 Hz, CH), 4.52 (d, 2 H, J 5.8 Hz, CH2), 3.41 (s, 3
H, OCH3), 3.39 (s, 3 H, OCH3), 2.57 (s, 3 H, CH3). 168.33 (pyrimidyl-C),
164.11 (C=N), 162.55 (C=O), 143.24 (CH), 135.25 (Ar-C), 129.18 (Ar-2C),
128.33 (Ar-2C), 128.14 (Ar-C), 123.08 (CH), 116.12 (CN), 96.24 (CH),
92.15 (pyrimidyl-C), 49.88 (2OCH3), 46.95 (CH2), 14.32 (CH3). Analysis
calcd. for C18H19N3O3S: C, 60.49; H, 5.36; N, 11.76. Found: C, 60.71; H,
5.48; N, 11.57%.
4,5-Dihydro-5-(2-methoxyethyl)-6-(methylthio)-4-oxo-2-styrylpyridine-3-
carbonitrile (4b). Pale yellow powder (71%); m.p. 258–259◦C; IR (KBr):
2215 (CN), 1654 (C=O); 1H NMR (DMSO-d6, 300 Mz), δH: 8.07 (d, 1 H, J
6.5 Hz, CH), 7.60 (m, 2 H, Ar-2H), 7.37 (m, 3 H, Ar-3H), 7.24 (d, 1 H, J 6.5
Hz, CH), 3.82 (t, 2 H, J 5.6 Hz, CH2), 3.73 (t, 2 H, J 5.6 Hz, CH2), 3.37 (s,
3 H, OCH3), 2.62 (s, 3 H, CH3). Analysis calcd. for C17H17N3O2S: C, 62.36;
H, 5.23; N, 12.83. Found: C, 62.05; H, 5.15; N, 13.11%.
1,6-Dihydro-1-(2,3-dihydroxypropyl)-2-(methylthio)-6-oxo-4-styrylpyrimidine-5-
carbonitrile (4c): To a stirred solution of compound 3a (2.69 g, 10 mmol)
and potassium hydroxide (0.56 g, 10 mmol) in ethanol (25 mL) was added
3-chloropropane-1,2-diol (1.11 g, 10 mmol) and the reaction mixture was
heating under reflux for 3 hours. The solvent was concentrated under
reduced pressure then cooled and the precipitated solid was filtered, and
crystallized from ethanol to give 2.34 g of compound 4c as yellow powder
(71% yield); m.p. 287–289◦C; IR (KBr): 3380 (OH), 2216 (CN), 1662
(C=O); 1H NMR (DMSO-d6, 300 Mz), δH: 8.11 (d, 1 H, J 6.5 Hz, CH), 7.57

Synthesis and In Vitro Antitumor Activity
269
(m, 2 H, Ar-2H), 7.31 (m, 3H, Ar-3H), 7.22 (d, 1H, J 6.5 Hz, CH), 4.57
(d, 2H, J 5.8 Hz, CH2), 4.53 (m, 1 H, CH), 4.32 (d, 1 H, J 5.8 Hz, OH),
4.22 (m, 2 H, CH2), 4.11 (t, 1 H, J 5.2 Hz, OH), 2.58 (s, 3 H, CH3). 168.22
(pyrimidyl-C), 163.14 (C=N), 163.52 (C=O), 142.70 (CH), 134.21 (Ar-C),
128.19 (Ar-2C), 128.24 (Ar-2C), 127.97 (Ar-C), 127.97 (Ar-C), 118.72 (CN),
91.18 (pyrimidyl-C), 72.78 (CH), 62.43 (CH2), 45.87 (CH2), 14.32 (CH3).
Analysis calcd. for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24. Found: C,
59.15; H, 5.11; N, 12.18%.
1,6-Dihydro-2-[(2,3,4,6-tetra-O-acetyl-β-D-glycopyranosyl)thio]-6-oxo-4-
styrylpyrimidine-5-carbonitrile (6a,b). General procedure: To a solution of
1,2,3,4-tetrahydro-4-oxo-6-styryl-2-thioxopyrimidine-5-carbonitrile (2) (2.55
g, 10 mmol) in aqueous potassium hydroxide [10 mmol in distilled water
(16 mL)] was added a solution of 2,3,4,6-tetra-O-acetyl-α-D-galacto (5a)
or gluco- (5b) pyranosyl bromide (10 mmol) in acetone (30 mL). The
reaction mixture was stirred at room temperature until reaction was judged
complete by TLC using chloroform/methanol 99.5:0.5. The solvent was
evaporated under reduced pressure at 40◦C and the residue was washed
with distilled water to remove potassium bromide formed. The product was
dried, and crystallized from ethanol.
1,6-Dihydro-2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)thio]-6-oxo-4-
styrylpyrimidine-5-carbonitrile (6a). Yellow powder (75%); m.p. 166–168◦C;
1
IR (KBr): 3398 (NH), 2210 (CN), 1740 (C=O), 1658 (C=O); H NMR
(DMSO-d6, 300 Mz), δH: 7.89 (s, 1 H, NH), 7.46 (m, 2 H, Ar-2H), 7.31 (m,
3 H, Ar-3H), 7.28 (d, 1 H, J 6.5 Hz, CH), 5.80 (d, 1 H, J 1,2 9.5 Hz, H-1),
5.24 (t, 1 H, J 2,3 9.6 Hz, H-2), 5.15 (dd, 1 H, J 2,3 9.6 Hz, J 3,4 9.3 Hz, H-3),
ꢁ
4.93 (t, 1 H, J 3,4 9.3 Hz, H-4), 4.10 (dd, 1 H, J 6,6 11.2 Hz, J 5,6 3.5 Hz, H-6),
4.05 (dd, 1 H, J 6,6 11.2 Hz, J 5,6 2.8 Hz, H-6ꢁ), 3.98 (m, 1 H, H-5), 1.96, 2.03,
2.11, 2.13 (4s, 12 H, 4CH3). 168.79 (pyrimidyl-C), 164.14, 164.32, 164.67,
165.35, 169.45 (5CO), 163.33 (C=N), (C=O), 142.11 (CH), 134.65 (Ar-C),
134.65 (Ar-C), 134.65 (Ar-C), 128.41 (Ar-C), 122.51 (CH), 116.15 (CN),
92.29 (pyrimidyl-C), 78.14 (C-1), 72.48 (C-2), 71.65 (C-3), 68.66 (C-4),
64.21 (C-5), 62.98 (C-6), 15.27, 15.23, 15.44, 15.65 (4CH3). Analysis calcd.
for C27H27N3O10S: C, 55.38; H, 4.65; N, 7.18. Found: C, 55.04; H, 4.42; N,
7.35%.
ꢁ
ꢁ
1,6-Dihydro-2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thio]-6-oxo-4-
styrylpyrimidine-5-carbonitrile (6b). Yellow powder (77%); m.p. 159–161◦C;
1
IR (KBr): 3305 (NH), 2208 (CN), 1735 (C=O), 1658 (C=O); H NMR
(DMSO-d6, 300 Mz), δH: 7.92 (s, 1 H, NH), 7.72 (d, 1 H, J 6.5 Hz, CH),
7.51 (m, 2 H, Ar-2H), 7.34 (m, 3 H, Ar-3H), 7.25 (d, 1 H, J 6.5 Hz, CH),
5.75 (d, 1 H, J 1,2 9.4 Hz, H-1), 5.27 (t, 1 H, J 2,3 9.5 Hz, H-2), 5.18 (dd, 1 H,
J 2,3 9.5 Hz, J 3,4 9.2 Hz, H-3), 4.62 (t, 1 H, J 3,4 9.2 Hz, H-4), 4.33 (m, 1 H,
ꢁ
ꢁ
H-5), 4.18 (dd, 1 H, J 6,6 11.4 Hz, J 5,6 3.5 Hz, H-6), 4.10 (dd, 1 H, J 6,6 11.4
Hz, J 5,6 2.8 Hz, H-6ꢁ), 1.93, 2.04, 2.11, 2.15 (4s, 12 H, 4CH3). Analysis calcd.
ꢁ
for C27H27N3O10S: C, 55.38; H, 4.65; N, 7.18. Found: C, 55.15; H, 4.36; N,
7.24%.

270
W. A. El-Sayed et al.
1,6-Dihydro-2-[(β-D-glycopyranosyl)thio]-6-oxo-4-styrylpyrimidine-5-
carbonitrile (7a,b). General procedure: Dry gaseous ammonia was passed
through a solution of a protected nucleoside 6a,b (10 mmol) in dry
methanol (20 mL) at 0◦C for 0.5 hours, and then the mixture was stirred at
0◦C for about 5 hours. The solvent was evaporated under reduced pressure
at 40◦C to give a solid residue, which was crystallized from ethanol to afford
the desired products.
1,6-Dihydro-2-[(β-D-galactopyranosyl)thio]-6-oxo-4-styrylpyrimidine-5-
carbonitrile (7a). Pale yellow powder (67%); m.p. 232–233◦C; IR (KBr): 3366
(NH), 3282 (NH), 2215 (CN), 1663 (C=O); 1H NMR (DMSO-d6, 300 Mz),
δH: 7.88 (s, 1 H, NH), 7.73 (d, 1 H, J 6.5 Hz, CH), 7.51 (m, 2 H, Ar-2H),
7.34 (m, 3 H, Ar-3H), 7.32 (d, 1 H, J 6.5 Hz, CH), 5.42 (d, 1 H, J 1,2 9.3 Hz,
H-1), 5.14 (t, 1 H, J 5.8 Hz, OH), 5.05 (m, 1 H, OH), 4.95 (d, 1 H, J 6.2 Hz,
OH), 4.82 (m, 1 H, OH), 4.44 (t, 1 H, J 2,3 9.2 Hz, H-2), 4.25 (t, 1 H, J 2,3 9.2
Hz, H-3), 3.92 (m, 1 H, H-4), 3.52 (m, 1 H, H-6ꢁ), 3.48 (dd, 1 H, J 6,6 11.4
ꢁ
Hz, J 5,6 3.4 Hz, H-6), 3.41 (m, 1 H, H-5). Analysis calcd. for C19H19N3O6S:
C, 54.67; H, 4.59; N, 10.07. Found: C, 54.37; H, 4.42; N, 10.22%.
1,6-Dihydro-2-[(β-D-glucopyranosyl)thio]-6-oxo-4-styrylpyrimidine-5-
carbonitrile (7b). Pale yellow powder (69%); m.p. 237–238◦C; IR (KBr): 3345
(NH), 3278 (NH), 2212 (CN), 1660 (C=O); 1H NMR (DMSO-d6, 300 Mz),
δH: 7.92 (s, 1 H, NH), 7.72 (d, 1H, J 6.5 Hz, CH), 7.53 (m, 2 H, Ar-2H),
7.38 (m, 3 H, Ar-3H), 7.35 (d, 1 H, J 6.5 Hz, CH), 5.39 (d, 1 H, J 1,2 9.4 Hz,
H-1), 5.17 (t, 1 H, J 5.8 Hz, OH), 5.11 (t, 1 H, J 6.4 Hz, OH), 4.92 (d, 1
H, J 6.2 Hz, OH), 4.79 (m, 1 H, OH), 4.47 (t, 1 H, J 2,3 9.5 Hz, H-2), 4.22
(t, 1 H, J 2,3 9.5 Hz, H-3), 3.94 (m, 1 H, H-4), 3.42 (m, 1 H, H-5ꢁ), 3.47 (m,
2 H, H-6,6ꢁ). Analysis calcd. for C19H19N3O6S: C, 54.67; H, 4.59; N, 10.07.
Found: C, 54.33; H, 4.29; N, 10.19%.
ANTITUMOR ACTIVITY
Chemicals
Dimethylsulphoxide (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.
Cell Line
Ehrlich’s ascites carcinoma cells (EAC) were obtained from the National
Cancer Institute, Cairo University, Egypt. The cells were maintained by
intraperitoneal inoculation of 1 × 106 viable cells in mice.
The estimation of in vitro tumor cell growth inhibition was assessed
by incubating 1 × 106 EAC cells in 1 ml phosphate buffer saline with
varying concentrations of the prepared compounds at 37◦C for 3 hours

Synthesis and In Vitro Antitumor Activity
271
in CO2 atmosphere (Table 1). In all the cellular experiments, results were
compared with untreated cells.
In Vitro Cytotoxicity Assay
The effect of the prepared compounds on the growth of EAC was
estimated by MTT assay according to Mosmann.[41] The cytotoxic activity of
the compounds was determined by the MTT colorimetric assay. The MTT
colorimetric assay is based on the mitochondrial reduction of tetrazolium
salt by living cells. The viable cell number is proportional 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).
Cells (1 × 105 cells/well) were suspended in serum-free media. The cells
were plated separately in a sterile flat bottom 96-well microplate, and treated
with 20 µl of the drug with a final concentration as mentioned in Table 1,
for 24 hours at 37◦C, in a humidified 5% CO2 atmosphere. The control cells
without test compounds were cultured on separate plate. After incubation,
media were removed and 40 µl MTT solution/well were added and incu-
bated for an additional 4 hours. MTT crystals were solubilized by adding
200 µl of DMSO/well and plate was shacked at room temperature. The
results were determined photometrically using microplate ELISA reader
and the absorbance was 570 nm. The concentration of nitric oxide (NO),
and thiobarbituric acid reactive substances (TBARS) as indicator of lipid
peroxidation were determined according to Granger et al.[42]; Conrad et
al.[43] respectively. The activity of catalase (CAT) and glutathione peroxidase
(GSH-Px) were determined according to Aebi[44]; Paglia and Valentine,[45]
respectively.
Statistical Analysis
All experiments were repeated four times for each concentration and
the data was represented as Mean Standard error (S.E.) and transformed
to percentage of untreated cells.
Percentage of relative viability was calculated using the following equa-
tion:
(absorbance of treated cells)/Absorbance of control cells × 100.
Cytotoxic concentration was expressed by half maximal inhibitory con-
centration (IC50). IC50 calculations were performed using Microsoft Excel
and Microcal Origin software for PC.

272
W. A. El-Sayed et al.
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