Synthesis of some new pyrimidine derivatives and evaluation of their anticancer and antibacterial activities

Article abstract of DOI:10.1007/s11164-012-0597-7

Starting from the reaction of ethyl cyanoacetate with thiourea and the appropriate aldehydes, a series of new pyrimidine derivatives were prepared. Ten selected pyrimidine derivatives were subjected to a screening system for the investigation of their ant

Full text of DOI:10.1007/s11164-012-0597-7

Res Chem Intermed (2013) 39:821–841
DOI 10.1007/s11164-012-0597-7
Synthesis of some new pyrimidine derivatives
and evaluation of their anticancer and antibacterial
activities
•
Omar A. Fathalla Neama A. Mohamed
•
•
Walaa S. El-Serwy Hala F. AbdelHamid
•
•
Sherein I. Abd El-Moez Abdel-mohsen M. Soliman
Received: 25 March 2012 / Accepted: 9 May 2012 / Published online: 29 May 2012
Ó Springer Science+Business Media B.V. 2012
Abstract Starting from the reaction of ethyl cyanoacetate with thiourea and the
appropriate aldehydes, a series of new pyrimidine derivatives were prepared. Ten
selected pyrimidine derivatives were subjected to a screening system for the
investigation of their antitumor potency against liver (HEPG2) cell line. The anti-
tumor activity results indicated that most of the selected pyrimidine derivatives
showed moderate growth inhibition activity against the tested cell line, but with
varying intensities in comparison to the known anticancer drugs: 5-fluorouracil and
doxorubicin. Some of the synthesized compounds were also tested for their anti-
microbial activity against bacteria as well as fungal isolates.
Keywords Pyrimidine derivatives Á Cytotoxic activity Á Liver HEPG2 cancer
cell lines Á Antibacterial activity
Introduction
The pyrimidine moiety with some substitution shows promising antitumor activity, as
there are large numbers of pyrimidine-based antimetabolites. An early metabolite
prepared was 5-fluorouracil (5-FU) [1]. The biological profiles of this new generation of
pyrimidine represent good progress with regard to the older compounds [2]. A
pyrimidine derivative followed by 5-thiouracil also exhibits some useful antineoplastic
O. A. Fathalla Á N. A. Mohamed Á W. S. El-Serwy Á A. M. Soliman (&)
Therapeutical Chemistry Department, National Research Center, Dokki, Cairo, Egypt
e-mail: solimanmohsen@yahoo.com
H. F. AbdelHamid
Chemistry of Pesticides Department, National Research Center, Dokki, Cairo, Egypt
S. I. Abd El-Moez
Microbiology and Immunology Department, National Research Center, Dokki, Cairo, Egypt
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activities [3]. Thiouracils are also used as therapeutic agents in anticancer treatment [4],
and their nucleosideanalogsare foundint-RNAamongmanyprokaryotes[5, 6], and are
also used as anticancer and thyroid drugs [7–9]. After the invention of 5-FU as an
antimetabolite of uracil [10], it has become one of the most widely used antineoplastic
agents. Analogously, some 5-halogenated thiouracils [11] have been synthesized and
screened for anticancer activity. It has been reported that the tested compounds have
comparable activity to that of uracil [12–14]. From 1961 to 2006, different research
laboratories investigated the anticancer activity of some 5-substituted-2-thiouracils and
reported that the tested compounds were found to inhibit DNA synthesis [15–18]. We
report here the synthesis of novel thiouracil derivatives based on the diverse medicinal
uses and biological activities of thiouracil as anticancer drugs [19–24]. Based on these
findings, the present work aimed to synthesize a new group of pyrimidine compounds
incorporated with different heterocycles as a trial so that the resulting compounds would
have better biological activity as antiproliferative agents in the field of liver cancer.
Experimental
Chemistry
All melting points are uncorrected and were taken on electro-thermal capillary
melting point apparatus. Infrared spectra were performed on JASCO FT/IR-6100
1
using KBr discs. H-NMR spectra was obtained on Jeol 270 MHz and on Jeol sx
500 MHz spectrometers using TMS as an internal standard. The mass spectra were
recorded on a Jeol JMS-AX 500. All reactions were followed and checked by TLC
(aluminum sheets) using chloroform–methanol. These (9:1 v/v) eluents and the
plates were sprayed with iodine.
General procedure for the preparation of compounds 1a, b
A mixture of thiourea (0.1 mol), ethyl cyanoacetate (0.1 mol), and the appropriate
aldehydes, namely 4-hydroxybenzaldehyde and 2-hydroxybenzaldehyde, were
stirred in sodium ethoxide solution for 48 h, and then the reaction mixtures were
poured into ice-water. After acidification with dilute HCl, the precipitate was
filtered off, dried under suction, then crystallized from the proper solvent to give
compounds 1a, b.
6-(4-Hydroxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1a)
Crystallized from acetic acid, yellow crystals, m.p. 168 °C, yield 90 %. Analysis:
for C11H7N3O2S, M. Wt. 245.25, calcd: C, 53.87; H, 2.88; N, 17.13. Found: C,
53.44; H, 2.30; N, 17.02. IR (KBr, cm-1): 1,721 (C=O), 2,228 (–C:N), 3,018 (NH
of thiouracil), and 3,289 (OH). 1H-NMR (DMSO-d6, d ppm): 5 (1H, s, OH,
exchangeable with D2O), 6.9–7.9 (4H, m, aromatic), and 8.1, 13 (2H, s, 2NH of
thiouracil, exchangeable with D2O), MS: (m/z) & 245 (10 %).
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823
6-(2-Hydroxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1b)
Crystallized from dimethylformamide, yellow crystals, m.p. 220 °C, yield 90 %.
Analysis: for C11H7N3O2S, M. Wt. 245.25, calcd: C, 53.87; H, 2.88; N, 17.13.
Found: C, 53.60; H, 2.75; N, 17.01. IR (KBr, cm-1): 1,680 (C=O), 2,225 (–C:N),
3,300 (NH of thiouracil), and 3,400 (OH). 1H-NMR (DMSO-d6, d ppm): 5.4 (1H, s,
OH, exchangeable with D2O), 6.8–7.3 (4H, m, aromatic), and 8.0, 12 (2H, s, 2NH of
thiouracil, exchangeable with D2O). MS: (m/z) & 245 (100 %).
General procedure for the preparation of compounds 2a, b
A mixture of 1a, b (0.01 mol) and phosphorus pentachloride (0.01 mol) in phosphorus
oxychloride (20 mL) was heated on a steam bath for 3 h and the reaction mixture
poured gradually on to crushed ice. The precipitate was filtered off, dried under
vacuum, then crystallized from the proper solvent to give compounds 2a, b.
4-Chloro-6-(4-hydroxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (2a)
Crystallized from dimethylformamide, brown yellow crystals, m.p. 215 °C, yield
90 %. Analysis: for C11H6ClN3OS, M. Wt. 263.70, calcd: C, 50.10; H, 2.29; N,
15.93. Found: C, 49.85; H, 2.09; N, 15.78. IR (KBr, cm-1): 1,691 (C=N), 2,231
1
(–C:N), 3,330 (NH of thiouracil), and 3,400 (OH). H-NMR (DMSO-d6, d ppm):
5.2 (1H, s, OH, exchangeable with D2O), 6.7–7.4 (4H, m, aromatic), and 12 (1H,
s, NH of thiouracil, exchangeable with D2O). MS: (m/z) & 263 (0.30 %).
4-Chloro-6-(2-hydroxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (2b)
Crystallized from acetic acid, brown crystals, m.p. 254 °C, yield 80 %. Analysis:
for C11H6ClN3OS, M. Wt. 263.70, calcd: C, 50.10; H, 2.29; N, 15.93. Found: C,
49.75; H, 2.01; N, 15.15. IR (KBr, cm-1): 1,692 (C=N), 2,226 (–C:N), 3,200 (NH
of thiouracil), and 3,310 (OH). 1H-NMR (DMSO-d6, d ppm): 5.6 (1H, s, OH,
exchangeable with D2O), 6.9–7.6 (4H, m, aromatic), and 12.5 (1H, s, NH of
thiouracil, exchangeable with D2O). MS: (m/z) & 263 (7 %).
General procedure for the preparation of compounds 3a, b
A mixture of 2a, b (0.01 mol) and hydrazine hydrate (0.01 mol) in methanol
(10 mL) was stirred for 8 h. The precipitate was filtered off, dried under suction
then crystallized from the proper solvent to give compounds 3a, b.
4-Hydrazinyl-6-(4-hydroxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (3a)
Crystallized from acetic acid, brown crystal, m.p. 245 °C, yield 85 %. Analysis: for
C11H9N5OS, M. Wt. 259.28, calcd: C, 50.95; H, 3.50; N, 27.01. Found: C, 50.55; H,
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2.86; N, 26.54. IR (KBr, cm-1): 2,211 (–C:N), 3,127 (Sec NH), 3,299, 3,400 (Prim
1
NH), and 3,410 (OH). H-NMR (DMSO-d6, d ppm): 2, 2.2 (3H, s, Sec NH, Prim
NH, exchangeable with D2O), 5.3 (1H, s, OH, exchangeable with D2O), 6.6–7.4
(4H, m, aromatic), and 13 (1H, s, NH of thiouracil, exchangeable with D2O). MS:
m/z & 259 (5 %).
4-Hydrazinyl-2-thioxo-2,6-dihydropyrimido[5,4-c]quinolin-5(1H)-one (3b)
Crystallized from benzene, yellow crystals, m.p. [ 300 °C, yield 80 %. Analysis:
for C11H9N5OS, M. Wt. 259.28, calcd: C, 50.95; H, 3.50; N, 27.01. Found: C, 50.65;
H, 2.79; N, 26.74. IR (KBr, cm-1): 1,669 (C=O), 3,281 (Sec NH), 3,310, 3,405
1
(Prim NH). H-NMR (DMSO-d6, d ppm): 2.3, 2.4 (3H, s, Sec NH, Prim NH,
exchangeable with D2O), 7.1–7.9 (4H, m, aromatic), 8 (1H, s, Sec NH,
exchangeable with D2O), and 13.4 (1H, s, NH of thiouracil, exchangeable with
D2O). MS: [M ? 2]? m/z & 261 (1.89 %).
General procedure for the preparation of compounds 4a–c
A mixture of 3a, b and 3c [25] (0.01 mol) and 2-(bis(methylthio)methylene)
malononitrile (0.01 mol) in methanol (20 mL) and triethylamine (1 mL) was
refluxed for 8–12 h. The precipitate was filtered off, dried under suction, then
crystallized from the proper solvent to give compounds 4a–c.
4-[5-Amino-4-cyano-3-(methylsulfanyl)-1H-pyrazol-1-yl]-6-(4-hydroxyphenyl)-2-
thioxo-1,2-dihydropyrimidine-5-carbonitrile (4a)
Crystallized from acetic acid, brown crystals, m.p. [ 300 °C, yield 65 %. Analysis:
for C16H11N7OS2, M. Wt. 381.43, calcd: C, 50.38; H, 2.91; N, 25.70. Found: C,
50.15; H, 2.36; N, 25.54. IR (KBr, cm-1): 2,220, 2,228 (2–C:N), 3,290 (NH of
thiouracil), 3,330, 3,410 (Prim NH), and 3,420 (OH). 1H-NMR (DMSO-d6, d ppm):
2.5 (3H, s, SCH3), 5.1 (1H, s, OH, exchangeable with D2O), 6.6–7.4 (4H, m,
aromatic), 8 (2H, s, Prim NH, exchangeable with D2O), and 13.4 (1H, s, NH of
thiouracil, exchangeable with D2O). MS: m/z & 381 (2 %).
5-Amino-3-(methylsulfanyl)-1-(5-oxo-2-thioxo-1,2,5,6-tetrahydropyrimido[5,4-c]
quinolin-4-yl)-1H-pyrazole-4-carbonitrile (4b)
Crystallized from dimethylformamide, brown crystals, m.p. 266 °C, yield 55 %.
Analysis: for C16H11N7OS2, M. Wt. 381.43, calcd: C, 50.38; H, 2.91; N, 25.70.
Found: C, 50.05; H, 2.36; N, 25.14. IR (KBr, cm-1): 1,700 (C=O), 2,228 (–C:N),
3,281 (Sec NH), 3,330, 3,400 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2.6 (3H, s,
SCH3), 7.2–8.2 (4H, m, aromatic), 7.9, 8.1 (3H, s, Sec NH, Prim NH, exchangeable
with D2O), and 13.5 (1H, s, NH of thiouracil, exchangeable with D2O). MS:
m/z & 381 (13 %).
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Synthesis and evaluation of pyrimidine derivatives
825
4-[5-Amino-4-cyano-3-(methylsulfanyl)-1H-pyrazol-1-yl]-6-(2-methoxyphenyl)-2-
thioxo-1,2-dihydropyrimidine-5-carbonitrile (4c)
Crystallized from benzene, orange crystals, m.p. [ 300 °C, yield 60 %. Analysis:
for C17H13N7OS2, M. Wt. 395.46, calcd: C, 51.63; H, 3.31; N, 24.79. Found: C,
50.65; H, 2.70; N, 24.34. IR (KBr, cm-1): 2,210, 2,225 (2–C:N), 3,175 (NH of
thiouracil), 3,315, 3,430 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2.8 (3H, s,
SCH3), 3.6 (3H, s, OCH3), 6.9–7.9 (4H, m, aromatic), 8.1 (2H, s, Prim NH,
exchangeable with D2O), and 12.5 (1H, s, NH of thiouracil, exchangeable with
D2O). MS: m/z & 395 (9 %).
General procedure for the preparation of compounds 5a–c
A mixture of 4a–c (0.01 mol) and trimethylorthoformate (15 mL) in acetic
anhydride (10 mL) was heated under reflux for 2 h. The solid obtained was
recrystallized from the proper solvent to give 5a–c.
Ethyl{4-cyano-1-[5-cyano-6-(4-hydroxyphenyl)-2-thioxo-1,2-dihydropyrimidin-4-yl]
-3-(methylsulfanyl)-1H-pyrazol-5-yl}imidoformate (5a)
Crystallized from benzene, white crystals, m.p. [ 300 °C, yield 70 %. Analysis: for
C19H15N7O2S2, M. Wt. 437.49, calcd: C, 52.16; H, 3.46; N, 22.41. Found: C, 51.05; H,
2.90; N, 22.14. IR (KBr, cm-1): 2,220, 2,228 (2–C:N), 3,281 (Sec NH), and 3,320
(OH). 1H-NMR (DMSO-d6, d ppm): 1.2 (3H, t, CH3), 2.4 (3H, s, SCH3), 3.4 (2H, q,
CH2), 5.3 (1H, s, OH, exchangeable with D2O), 6.4–7.7 (4H, m, aromatic), 7.5 (1H, s,
CH), and13(1H, s, NH ofthiouracil, exchangeablewith D2O). MS: m/z & 437 (10 %).
Ethyl[4-cyano-3-(methylsulfanyl)-1-(5-oxo-2-thioxo-1,2,5,6-
tetrahydropyrimido[5,4-c]quinolin-4-yl)-1H-pyrazol-5-yl]imidoformate (5b)
Crystallized from dimethylformamide, brown crystals, m.p. [ 300 °C, yield 65 %.
Analysis: for C19H15N7O2S2, M. Wt. 437.49, calcd: C, 52.16; H, 3.46; N, 22.41.
Found: C, 51.75; H, 3.26; N, 21.94. IR (KBr, cm-1): 1,645 (C=O), 2,212 (–C:N),
3,433 (Sec NH). 1H-NMR (DMSO-d6, d ppm): 1.2 (3H, t, CH3), 2.5 (3H, s, SCH3),
3.3 (2H, q, CH2), 7.1–8.2 (4H, m, aromatic), 7.1 (1H, s, CH), and 8.3, 12.5 (2H, s,
2Sec NH, exchangeable with D2O). MS: m/z & 437 (17 %).
Ethyl{4-cyano-1-[5-cyano-6-(2-methoxyphenyl)-2-thioxo-1,2-dihydropyrimidin-4-yl]
-3-(methylsulfanyl)-1H-pyrazol-5-yl}imidoformate (5c)
Crystallized from benzene, brown crystals, m.p. 184 °C, yield 60 %. Analysis: for
C20H17N7O2S2, M. Wt. 451.52, calcd: C, 53.20; H, 3.79; N, 21.71. Found: C, 53.05;
H, 3.36; N, 21.14. IR (KBr, cm-1): 2,213, 2,228 (2–C:N), 3,418 (Sec NH).
1H-NMR (DMSO-d6, d ppm): 1.1 (3H, t, CH3), 2.4 (3H, s, SCH3), 3.2 (2H, q, CH2),
3.7 (3H, s, OCH3), 6.9–7.2 (4H, m, aromatic), 7.3 (1H, s, CH), and 12 (1H, s, NH of
thiouracil, exchangeable with D2O). MS: m/z & 451 (20 %).
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General procedure for the preparation of compounds 6a–c
A mixture of 5a–c (0.01 mol) and hydrazine hydrate (0.01 mol) in methanol
(10 mL) was stirred for 8 h. The precipitate was filtered off, dried under suction,
then crystallized from the proper solvent to give compounds 6a–c.
4-[4-Hydrazinyl-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-6-(4-
hydroxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6a)
Crystallized from benzene, brown crystals, m.p. [ 300 °C, yield 55 %. Analysis:
for C17H13N9OS2, M. Wt. 423.47, calcd: C, 48.22; H, 3.09; N, 29.77. Found: C,
47.95; H, 2.66; N, 29.14. IR (KBr, cm-1): 2,215 (–C:N), 3,281 (Sec NH), 3,330,
3,400 (Prim NH), and 3,300 (OH). 1H-NMR (DMSO-d6, d ppm): 2.4 (3H, s, SCH3),
2 (2H, s, Prim NH, exchangeable with D2O), 5.3 (1H, s, OH, exchangeable with
D2O), 6.9–7.8 (4H, m, aromatic), 8.1 (1H, s, CH), and 4.2, 13.2 (2H, s, 2 Sec NH,
exchangeable with D2O). MS: m/z & 423 (16 %).
4-[4-Hydrazinyl-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-2-thioxo-
2,6-dihydropyrimido[5,4-c]quinolin-5(1H)-one (6b)
Crystallized from acetic acid, brown crystals, m.p. [ 300 °C, yield 70 %. Analysis:
for C17H13N9OS2, M. Wt. 423.47, calcd: C, 48.22; H, 3.09; N, 29.77. Found: C,
47.75; H, 2.88; N, 29.14. IR (KBr, cm-1): 1,670 (C=O), 3,433 (Sec NH), 3,310,
3,400 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2.4 (3H, s, SCH3), 2.1 (2H, s, Prim
NH, exchangeable with D2O), 7.1–8.3 (4H, m, aromatic), 8.2 (1H, s, CH), and 4,
8.1, 13.2 (3H, s, 3Sec NH, exchangeable with D2O). MS: m/z & 423 (75 %).
4-[4-Hydrazinyl-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-6-(2-
methoxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6c)
Crystallized from benzene, brown crystals, m.p. 262 °C, yield 75 %. Analysis: for
C18H15N9OS2, M. Wt. 437.50, calcd: C, 49.42; H, 3.46; N, 28.81. Found: C, 49.11; H,
2.87; N, 27.24. IR (KBr, cm-1): 2,210 (–C:N), 3,201 (Sec NH) 3,300, 3,440 (Prim
NH). 1H-NMR (DMSO-d6, d ppm): 2.6 (3H, s, SCH3), 2.2 (2H, s, Prim NH,
exchangeable with D2O), 3.8 (3H, s, OCH3), 6.7–7.2 (4H, m, aromatic), 8.2 (1H, s,
CH), and 4, 12.2 (2H, s, 2Sec NH, exchangeable with D2O). MS: m/z & 437 (50 %).
General procedure for the preparation of compounds 7a–c
A mixture of 1a, b and 1c [25] (0.01 mol) and hydrazine hydrate (0.01 mol) in
methanol (10 mL) was heated under refluxed for 5 h. The precipitate was filtered
off, dried under suction, then crystallized from the proper solvent to give
compounds 7a–c.
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827
2-Hydrazinyl-4-(4-hydroxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (7a)
Crystallized from dimethylformamide, brown crystal, m.p. 156 °C, yield 80 %.
Analysis: for C11H9N5O2, M. Wt. 243.22, calcd: C, 54.32; H, 3.73; N, 28.79. Found:
C, 53.55; H, 3.16; N, 28.54. IR (KBr, cm-1): 1,700 (C=O), 2,211 (–C:N), 2,814
1
(Sec NH), 3,227, 3,312 (Prim NH), and 3,310 (OH). H-NMR (DMSO-d6, d ppm):
2.2, 2.4 (3H, s, Sec NH, Prim NH, exchangeable with D2O), 5.6 (1H, s, OH,
exchangeable with D2O), 6.9–7.8 (4H, m, aromatic), and 12.5 (1H, s, NH of
thiouracil, exchangeable with D2O). MS: m/z & 243 (6 %).
2-Hydrazinylpyrimido[5,4-c]quinoline-4,5(3H,6H)-dione (7b)
Crystallized from benzene, yellow crystals, m.p. 219 °C, yield 60 %. Analysis: for
C11H9N5O2, M. Wt. 243.22, calcd: C, 54.32; H, 3.73; N, 28.79. Found: C, 53.85; H,
3.66; N, 27.94. IR (KBr, cm-1): 1,669, 1,721 (2C=O), 3,334 (Sec NH), 3,270, 3,433
1
(Prim NH). H-NMR (DMSO-d6, d ppm): 2.4, 2.5 (3H, s, Sec NH, Prim NH,
exchangeable with D2O), 7.2–8.2 (4H, m, aromatic), 8.9 (1H, s, Sec NH,
exchangeable with D2O), and 12 (1H, s, NH of thiouracil, exchangeable with
D2O). MS: [M ? 2]? m/z & 245 (15 %).
2-Hydrazinyl-4-(2-methoxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (7c)
Crystallized from benzene, yellow crystals, m.p. 128 °C, yield 65 %. Analysis: for
C12H11N5O2, M. Wt. 257.24, calcd: C, 56.03; H, 4.31; N, 27.22. Found: C, 55.65; H,
3.86; N, 26.74. IR (KBr, cm-1): 1,721 (C=O), 2,225 (–C:N), 3,200 (Sec NH),
3,290, 3,335 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2, 2.6 (3H, s, Sec NH, Prim
NH, exchangeable with D2O), 3.8 (3H, s, OCH3), 6.9–7.7 (4H, m, aromatic), and 13
(1H, s, NH of thiouracil, exchangeable with D2O). MS: m/z & 257 (10 %).
General procedure for the preparation of compounds 8a–c
A mixture of 7a–c (0.01 mol) and 2-(bis(methylthio)methylene)malono nitrile
(0.01 mol) in methanol (20 mL) and triethylamine (1 mL) was refluxed for 8–12 h.
The precipitate was filtered off, dried under suction, then crystallized from the
proper solvent to give compounds 8a–c.
2-[5-Amino-4-cyano-3-(methylsulfanyl)-1H-pyrazol-1-yl]-4-(4-hydroxyphenyl)-6-
oxo-1,6-dihydropyrimidine-5-carbonitrile (8a)
Crystallized from acetic acid, brown crystals, m.p. [ 300 °C, yield 60 %. Analysis:
for C16H11N7O2S, M. Wt. 365.36, calcd: C, 52.60; H, 3.03; N, 26.84. Found: C,
52.15; H, 2.76; N, 25.94. IR (KBr, cm-1): 1,700 (C=O), 2,220, 2,227 (2–C:N),
3,190 (NH of thiouracil), 3,230, 3,390 (Prim NH), and 3,400 (OH). 1H-NMR
(DMSO-d6, d ppm): 2.7 (3H, s, SCH3), 5.3 (1H, s, OH, exchangeable with D2O),
6.6–7.2 (4H, m, aromatic), 8.1 (2H, s, Prim NH, exchangeable with D2O), and 12.9
(1H, s, NH of thiouracil, exchangeable with D2O). MS: m/z & 365 (5 %).
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5-Amino-1-(4,5-dioxo-3,4,5,6-tetrahydropyrimido[5,4-c]quinolin-2-yl)-3-(methyl
sulfanyl)-1H-pyrazole-4-carbonitrile (8b)
Crystallized from benzene, brown crystals, m.p. 239 °C, yield 65 %. Analysis: for
C16H11N7O2S, M. Wt. 365.36, calcd: C, 52.60; H, 3.03; N, 26.84. Found: C, 52.25; H,
2.86; N, 26.14. IR (KBr, cm-1):1,645, 1,700 (2C=O), 2,207 (–C:N), 3,169 (Sec NH),
3,281, 3,300 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2.5 (3H, s, SCH3), 7.1–7.9 (4H,
m, aromatic), 8.2, 8.4 (3H, s, Sec NH, Prim NH, exchangeable with D2O), and 13 (1H,
s, NH of thiouracil, exchangeable with D2O). MS: m/z & 365 (29 %).
2-[5-Amino-4-cyano-3-(methylsulfanyl)-1H-pyrazol-1-yl]-4-(2-methoxyphenyl)-6-
oxo-1,6-dihydropyrimidine-5-carbonitrile (8c)
Crystallized from dimethylformamide, orange crystals, m.p. [ 300 °C, yield 60 %.
Analysis: for C17H13N7O2S, M. Wt. 379.39, calcd: C, 53.82; H, 3.45; N, 25.84.
Found: C, 53.25; H, 3.16; N, 25.14. IR (KBr, cm-1): 1,715 (C=O), 2,210, 2,220 (2–
C:N), 3,270 (NH of thiouracil), 3,335, 3,410 (Prim NH).1H-NMR (DMSO-d6, d
ppm): 2.4 (3H, s, SCH3), 3.8 (3H, s, OCH3), 6.7–7.8 (4H, m, aromatic), 7.7 (2H, s,
Prim NH, exchangeable with D2O), and 13.5 (1H, s, NH of thiouracil, exchangeable
with D2O). MS: m/z & 379 (34 %).
General procedure for the preparation of compounds 9a–c
A mixture of 8a–c (0.01 mol) and trimethylorthoformate (15 mL) in acetic
anhydride (10 mL) was heated under reflux for 2 h. The solid obtained was
recrystallized from the proper solvent to give 9a–c.
Ethyl{1-[5-cyano-4-(4-hydroxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl]-4-iso
cyano-3-(methylsulfanyl)-1H-pyrazol-5-yl}imidoformate (9a)
Crystallized from benzene, brown crystals, m.p. [ 300 °C, yield 70 %. Analysis:
for C19H15N7O3S, M. Wt. 421.43, calcd: C, 54.15; H, 3.59; N, 23.27. Found: C,
53.85; H, 3.36; N, 23.04. IR (KBr, cm-1): 1,669 (C=O), 2,220, 2,229 (2–C:N),
1
3,190 (Sec NH), and 3,267 (OH). H-NMR (DMSO-d6, d ppm): 1.1 (3H, t, CH3),
2.6 (3H, s, SCH3), 3.6 (2H, q, CH2), 5.4 (1H, s, OH, exchangeable with D2O),
6.6–7.8 (4H, m, aromatic), 7.4 (1H, s, CH), and 13.4 (1H, s, NH of thiouracil,
exchangeable with D2O). MS: m/z & 421 (19 %).
Ethyl[1-(4,5-dioxo-3,4,5,6-tetrahydropyrimido[5,4-c]quinolin-2-yl)-4-isocyano-3-
(methylsulfanyl)-1H-pyrazol-5-yl]imidoformate (9b)
Crystallized from benzene, brown crystals, m.p. 170 °C, yield 75 %. Analysis: for
C19H15N7O3S, M. Wt. 421.43, calcd: C, 54.15; H, 3.59; N, 23.27. Found: C, 53.75;
H, 3.26; N, 22.94. IR (KBr, cm-1): 1,645, 1,742 (2C = O), 2,210 (–C:N), 3,412
1
(Sec NH). H-NMR (DMSO-d6, d ppm): 1.3 (3H, t, CH3), 2.5 (3H, s, SCH3), 3.5
123

Synthesis and evaluation of pyrimidine derivatives
829
(2H, q, CH2), 7.1–8.1 (4H, m, aromatic), 7.5 (1H, s, CH), and 8.4, 13.5 (2H, s, 2Sec
NH, exchangeable with D2O). MS: m/z & 421 (12 %).
Ethyl{1-[5-cyano-4-(2-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl]-4-iso
cyano-3-(methylsulfanyl)-1H-pyrazol-5-yl}imidoformate (9c)
Crystallized from acetic acid, brown crystals, m.p. [ 300 °C, yield 60 %. Analysis:
for C20H17N7O3S, M. Wt. 435.45, calcd: C, 55.16; H, 3.93; N, 22.52. Found: C,
55.05; H, 3.36; N, 22.14. IR (KBr, cm-1): 1,700 (C=O), 2,211, 2,220 (2–C:N),
3,330 (Sec NH). 1H-NMR (DMSO-d6, d ppm): 1.2 (3H, t, CH3), 2.3 (3H, s, SCH3),
3.1 (2H, q, CH2), 3.5 (3H, s, OCH3), 6.6–7.1 (4H, m, aromatic), 7.4 (1H, s, CH), and
12.5 (1H, s, NH of thiouracil, exchangeable with D2O). MS: m/z & 435 (40 %).
General procedure for the preparation of compounds 10a–c
A mixture of 9a–c (0.01 mol) and hydrazine hydrate (0.01 mol) in methanol
(10 mL) was stirred for 8 h. The precipitate was filtered off, dried under suction,
then crystallized from the proper solvent to give compounds 10a–c.
2-[4-Hydrazinyl-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-4-(4-
hydroxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (10a)
Crystallized from benzene, brown crystals, m.p. 207 °C, yield 80 %. Analysis: for
C17H13N9O2S, M. Wt. 407.40, calcd: C, 50.12; H, 3.22; N, 30.94. Found: C, 49.95;
H, 3.06; N, 30.44. IR (KBr, cm-1): 1,700 (C=O), 2,220 (–C:N), 3,181 (Sec NH),
3,230, 3,332 (Prim NH), and 3,400 (OH). 1H-NMR (DMSO-d6, d ppm): 2.6 (3H, s,
SCH3), 2.2 (2H, s, Prim NH, exchangeable with D2O), 5.5 (1H, s, OH, exchangeable
with D2O), 6.7–7.5 (4H, m, aromatic), 8.3 (1H, s, CH), and 4.1, 13.5 (2H, s, 2Sec
NH, exchangeable with D2O). MS: m/z & 407 (40 %).
2-[4-Hydrazinyl-3-(methylsulfanyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]
pyrimido[5,4-c]quinoline-4,5(3H,6H)-dione (10b)
Crystallized from benzene, brown crystals, m.p. 241 °C, yield 80 %. Analysis: for
C17H13N9O2S, M. Wt. 407.40, calcd: C, 50.12; H, 3.22; N, 30.94. Found: C, 49.75;
H, 2.88; N, 30.64. IR (KBr, cm-1): 1,670, 1,710 (2C=O), 3,330 (Sec NH), 3,228,
1
3,345 (Prim NH). H-NMR (DMSO-d6, d ppm): 2.5 (3H, s, SCH3), 2 (2H, s, Prim
NH, exchangeable with D2O), 7.2–8.2 (4H, m, aromatic), 8.4 (1H, s, CH), and 4.5,
8.3, 13.5 (3H, s, 3Sec NH, exchangeable with D2O). MS: m/z & 407 (60 %).
2-[4-Hydrazinyl-3-(methylsulfanyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-4-(2-
methoxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (10c)
Crystallized from benzene, brown crystals, m.p. [ 300 °C, yield 75 %. Analysis:
for C18H15N9O2S, M. Wt. 421.43, calcd: C, 51.30; H, 3.59; N, 29.91. Found: C, 50.11;
H, 3.36; N, 29.24. IR (KBr, cm-1): 1,660 (C=O), 2,228 (–C:N), 3,301 (Sec NH)
3,336, 3,422 (Prim NH). 1H-NMR (DMSO-d6, d ppm): 2.5 (3H, s, SCH3), 2 (2H, s, Prim
123

830
O. A. Fathalla et al.
NH, exchangeable with D2O), 3.5 (3H, s, OCH3), 6.8–7.4 (4H, m, aromatic), 8 (1H, s,
CH), and 4.4, 12 (2H, s, 2Sec NH, exchangeable with D2O). MS: m/z & 421 (60 %).
Anticancer testing
Measurement of potential cytotoxicity by SRB assay
The selected pyrimidine derivatives 2a, 1a, 3c [25], 7c, 6b, 10b, 9b, 4c, 5c, and 6c
were subjected to a screening system for evaluation of their antitumor activity
against liver HEPG2 cancer cell lines in comparison to the known anticancer drugs:
5-FU and doxorubicin (DOX) (Table 1).
Potential cytotoxicity of the selected pyrimidine derivatives was tested using the
method of Skehan et al. [26] as follows.
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment
with the compound(s) to allow attachment of cells to the wall of the plate. Different
concentrations of the compound under test (0, 1, 2.5, 5, 10 lg/mL) were added to the
cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer
cells were incubated with the compound(s) for 48 h at 37 °C and in an atmosphere of
5 % CO2. Cultures were then fixed with trichloroacetic acid and stained for 30 min
with 0.4 % (wt/vol) sulforhodamine B (SRB) dissolved in 1 % acetic acid. Unbound
dye was removed by four washes with 1 % acetic acid, and protein-bound dye was
extracted with 10 mM unbuffered tris base [tris(hydroxymethyl)aminomethane] for
the determination of optical density in a computer-interfaced, 96-well microtiter plate
reader. The SRB assay results were linear with the number of cells and with values for
cellular protein measured by both the Lowry and Bradford assays at densities ranging
from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio
at 564 nm was approximately 1.5 with 1,000 cells per well. The relation between
surviving fraction and drug concentration is plotted to get the survival curve of both
cancer cell lines after the specified compound.
Table 1 Effect of some
Compound
IC50 (lg/mL)
selected newly synthesized
compounds on liver carcinoma
cell line (HEPG2)
5-Fluorouracil
5
Doxorubicin
3.56
9.6
2a
1a
3c
6.58
8.1
7a
6b
10b
9b
4c
6.85
6.38
9.33
8.26
7.11
3.56
5.23
5c
IC50: dose of the compounds
which reduces survival to 50 %
6c
123

Synthesis and evaluation of pyrimidine derivatives
831
Biochemical Analysis
Animals
Male albino mice weighing 18–20 g were used in the present study. Mice were
divided into three main groups as follows:
1-Group (1) Untreated or control group (five mice).
2-Group (2) Divided into two subgroups (five mice for each subgroup) and treated
with 5-FU or DOX as reference anticancer drugs.
3-Group (3) Divided into eight subgroups (five mice for each subgroup) and
treated with the selected pyrimidine derivatives 2a, 1a, 3c [25], 7c,
6b, 10b, 9b, 4c, 5c, and 6c.
Treatment
Group (1) Each mouse was given a single intraperitoneal injection of 0.1 mL
DMSO.
Group (2) Each mouse was given a single intraperitoneal injection of 0.1 mL
containing 12 mg/kg body weight 5-FU or DOX dissolved in sterile
water.
Group (3) Each mouse was given a single intraperitoneal injection of 0.1 mL
containing 12 mg/kg body weight of the s selected pyrimidine derivatives
2a, 1a, 3c [25], 7c, 6b, 10b, 9b, 4c, 5c, and 6c, respectively, dissolved in
DMSO. Blood was collected after 7 days from all mice groups. The
biochemical effects of the selected pyrimidine derivatives 2a, 1a, 3c [25],
7c, 6b, 10b, 9b, 4c, 5c, and 6c on some liver enzymes such as aspartate
and alanine amino transferases (AST and ALT) [27] and alkaline
phosphatase (ALP) [28], were done using blood auto analyzer (Olympus
AV 400, Japan) (Table 2). Moreover, albumin [29], globulins [30] and
creatinine [31], total lipids [32], cholesterol [33], triglycerides [34], and
bilirubin [35] in serum of mice were evaluated in comparison to 5-FU and
DOX (Tables 3 and 4). Statistical analysis of the results was performed
using Chi-square values (SPSS computer program).
Table 2 Biochemical effects of treatment of tested compounds with 5-FU and DOX, on serum ALT,
AST, and ALP in mice
Mice groups Biochemical parameters
Alanine amino transferase Aspartate amino transferase Alkaline phosphatase
Mean SD ALT (IU/mL) Mean SD AST (IU/mL) Mean SD ALP (k.k./dL)
Control
5-FU
P\
43.5 2.03
51.47 9.02
0.001
108.32 4.19
130.431 8.92
0.001
18.70 1.10
25.485 ? 6.03
0.001
DOX
59.26 12.03
147.226 16.34
30.317 5.14
123

832
O. A. Fathalla et al.
Table 2 continued
Mice groups Biochemical parameters
Alanine amino transferase Aspartate amino transferase Alkaline phosphatase
Mean SD ALT (IU/mL) Mean SD AST (IU/mL) Mean SD ALP (k.k./dL)
P\
2a
0.001
0.001
0.001
80.7 19.09
0.001
162.17 34.5
0.001
38.58 12.61
0.001
P\
1a
38.9 8.9
n.s.
123.9 11.4
0.01
18.83 6.29
n.s.
P\
3c
39.56 6.7
n.s.
112.54 12.7
0.01
19.94 4.35
n.s.
P\
7a
46.21 4.17
n.s.
107.81 4.25
n.s.
21.94 3.4
0.01
P\
6b
53.7 10.08
0.001
142.3 29.7
0.001
45.42 10.41
0.001
P\
10b
P\
9b
81.34 27.3
0.001
151.52 45.6
0.001
43.7 8.36
0.001
33.42 7.05
n.s.
111.2 11.05
n.s.
18.32 3.07
n.s.
P\
4c
60.5 9.7
0.001
156.22 20.1
0.001
44.26 7.01
0.001
P\
5c
68.34 11.9
0.001
146.4 28.1
0.001
36.9 9.8
0.001
P\
6c
50.81 12.01
0.01
119 9.56
0.01
22.07 3.42
0.01
P\
Data are expressed as mean SD
P [ 0.05 insignificant, P \ 0.01 significant, P \ 0.001 highly significant, n.s. non-significant
Table 3 Biochemical effects of treatment with 5-FU and DOX, and some selected newly synthesized
compounds on serum total lipids, cholesterol, triglycerides, and bilirubin in mice
Mice groups Biochemical parameters
Total lipids (mg/dL) Cholesterol (mg/dL) Triglycerides (mg/dL) Bilirubin (mg/dL)
Control
5-FU
P\
323.41 27.1
378.2 31.4
0.001
94.32 13.5
105.9 11.7
0.001
108.7 16.8
126.5 19.4
0.001
0.63 0.04
0.75 0.10
0.001
DOX
P\
366.7 6.10
0.001
109.3 14.2
0.001
137.8 17.10
0.001
0.81 0.19
0.001
2a
363.6 29.3
0.001
116.4 8.3
0.01
98.4 10.6
n.s.
0.96 0.8
0.001
P\
1a
329.34 19.7
95.3 9.4
119.7 18.8
0.65 0.9
123

Synthesis and evaluation of pyrimidine derivatives
833
Table 3 continued
Mice groups Biochemical parameters
Total lipids (mg/dL) Cholesterol (mg/dL) Triglycerides (mg/dL) Bilirubin (mg/dL)
P\
3c
n.s.
n.s.
0.01
n.s.
331.63 17.5
n.s.
96.4 10.5
n.s.
118.6 19.70
0.01
0.68 0.11
n.s.
P\
7a
317.4 30.7
n.s.
93.24 19.53
n.s.
116.23 20.5
n.s.
0.51 0.08
0.01
P\
6b
372.8 37.6
0.001
110.3 17.8
0.01
93.6 9.5
n.s.
1.08 0.7
0.001
P\
10b
P\
9b
382.09 23.6
0.001
111.76 34.3
0.01
172.9 36.3
0.001
0.84 0.4
0.01
328.3 13.7
n.s.
96.8 17.2
n.s.
112.3 10.6
n.s.
0.67 0.01
n.s.
P\
4c
374.9 36.6
0.001
123.3 26.09
0.001
132.8 26.03
0.001
0.99 0.07
0.001
P\
5c
326.3 18.7
n.s.
95.6 14.9
n.s.
113.7 8.6
n.s.
0.61 0.04
n.s.
P\
6c
364.19 23.8
0.001
105.6 17.4
0.01
119.8 19.3
0.01
0.76 0.15
0.01
P\
Data are expressed as mean SD
P [ 0.05 insignificant, P \ 0.01 significant, P \ 0.001 highly significant, n.s. non-significant
Table 4 Biochemical effects of treatment with 5-FU and DOX, on serum albumin, globulin, and cre-
atinine in mice
Mice groups
Biochemical parameters
Albumin (mg/dL)
Globulin (mg/dL)
A/G ratio (mg/dL)
Creatinine (mg/dL)
Control
5-FU
P\
DOX
P\
2a
5.63 0.51
6.49 0.92
0.01
4.32 0.9
5.75 0.8
0.01
1.3
0.69 0.03
0.81 0.06
0.01
1.13
0.01
1.078
0.01
1.003
0.001
1.006
0.001
1.46
n.s.
6.37 0.85
0.01
5.91 0.63
0.01
0.78 0.04
0.01
7.1 0.3
0.01
7.62 0.76
0.01
0.78 0.03
0.01
P\
1a
7.2 0.61
0.01
6.62 0.86
0.01
0.8 0.1
0.01
P\
3c
5.97 0.34
n.s.
4.09 0.63
n.s.
0.65 0.09
n.s.
P\
7a
5.92 0.82
n.s.
5.12 0.9
n.s.
1.15
n.s.
0.73 0.04
n.s.
P\
123

834
O. A. Fathalla et al.
Table 4 continued
Mice groups
Biochemical parameters
Albumin (mg/dL)
Globulin (mg/dL)
A/G ratio (mg/dL)
Creatinine (mg/dL)
6b
6.87 0.49
0.01
6.86 0.8
0.01
1.02
0.001
1.13
0.01
1.12
n.s.
1.7 0.43
0.001
P\
10b
P\
9b
11.43 1.48
0.001
8.97 0.9
0.001
0.76 0.25
n.s.
5.62 0.68
n.s.
4.68 1.06
n.s.
0.68 0.08
n.s.
P\
4c
6.47 0.46
0.01
6.42 0.7
0.01
1.001
0.001
1.15
n.s.
0.77 0.05
0.01
P\
5c
5.92 0.81
n.s.
4.72 0.91
n.s.
0.84 0.6
0.01
P\
6c
7.73 0.52
0.01
6.25 0.82
0.01
1.23
0.01
0.84 0.06
0.01
P\
Data are expressed as mean SD
P [ 0.05 insignificant, P \ 0.01 significant, P \ 0.001 highly significant, n.s. non-significant
Antimicrobial testing
Antimicrobial evaluation of some synthesized compounds
Antimicrobial activities were carried out against highly pathogenic strains; two
Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus), two
Gram-negative bacteria (Salmonella Typhimurium, Escherichia coli) and one
mycotic strain (Candida albicans) isolated from animal origin. Agar disk diffusion
(qualitative method) and minimum inhibitory concentration (MIC) (quantitative
method) were used in this study. Fresh cultures were prepared in Muller Hinton
Broth for bacterial strains and Sabaroud Dextrose broth for mycotic strains. The
inoculated tubes were incubated at 37 and 28 °C for 24 h, respectively. Serial
dilutions were carried out for each strain, and dilution matching with 0.5
Mc-Farland was selected for screening of antimicrobial activities. Ciprofloxacin
100 lg/mL and fluconazole 100 lg/mL were used as reference drugs (Oxoid).
Determination of antimicrobial activity by Disk-diffusion method. [36]
Muller Hinton and Sabaroud Dextrose agar plates were prepared. Bacterial (Gram-
positive bacteria; L. monocytogenes and S. aureus and Gram-negative bacteria;
E. coli and S. Typhimurium) and fungal strains (C. albicans) matching with 0.5
Mc-Farland was spread onto the surface of the agar plates using sterile cotton swabs.
Isolates were isolated from mastitic cow milk and minced meat. For evaluation of
antibacterial activities, Whatman no. 1 filter paper disks were saturated with 50 lL
of the extract, others were saturated with 50 lL ciprofloxacin (100 lg/mL) and
123

Synthesis and evaluation of pyrimidine derivatives
835
others 50 lL DMSO as control negative. The same method was used for evaluation
of antimycotic activities using fluconazole (100 lg/mL). Disks were placed onto
inoculated agar plates and left for 1 h at 25 °C to allow a period of pre-incubation.
The plates were re-incubated at 37 and 28 °C for 24 h for bacterial and mycotic
strains, respectively. Then, the plates were observed for antimicrobial activities by
determining the diameters of the zones of inhibition for each of the samples. Tests
were run in triplicate for each strain to avoid any error.
Determination of MIC [37]
The microtiter dilution plate using 96-well sterile microplates is a quantitative method
used for evaluation of the antimicrobial activity of testing compounds. Initial
concentration of 100 % was used, then twofold serial dilutions of the compounds and
reference drugs (ciprofloxacin and fluconazole) were tested. Plates were inoculated
with 100 lL of tested strains (0.5 Mc-Farland, about 1 9 108 cells/mL) and incubated
at 37–28 °C for 24 h for bacterial and fungal strains, respectively. After incubation,
plates were examined visually for bacterial or fungal growth precipitation. The
experiment was repeated three times. The lowest concentration that showed complete
hindrance of growth was taken as MIC (Table 6).
Results revealed that compound 10b gives the highest antibacterial activity against
all tested strains with a mean zone of inhibition equal to 18.2 mm followed by
5c (7 mm) then 10a (7 mm) then 3c (6.6 mm) then 7b (6.6 mm) then 2a (6 mm)
then 3b (5.4 mm) then 6b (5.2 mm) then 5b (5.2 mm) then 9b (5 mm) then
4c (5 mm) then 6c (4.8 mm) then 2b (4.6 mm) then 7a (4.4 mm) then
4b (4.2 mm) then 8b (4.2 mm) and finally 1a (3.4 mm) as shown in Table 5. Also,
compounds 10b and 3c showed the highest results in (MIC) test as shown in Table 6.
Table 5 Agar well diffusion method showing antimicrobial activities of testing compounds compared
with reference drugs; the results are in mm
Strains samples
L. monocytogenes S. aureus E. coli S. Typhimurium C. albicans
(A)
(B)
(2)
(5)
(6)
2a
1a
3c
18 s
17
-ve
-ve
-ve
-ve
-ve
12
-ve
-ve
-ve
-ve
-ve
40 s
-ve
-ve
-ve
-ve
-ve
-ve
-ve
12
-ve
11
11
11
13
12
13
13
12
15
11
11
-ve
-ve
10
12
7a
6b
10b
9b
4c
-ve
15 s
14
11
-ve
12
13
-ve
-ve
-ve
-ve
-ve
12
-ve
-ve
10
12
5c
12
6c
12
-ve
12
3b
7b
5b
-ve
10 s
-ve
-ve
-ve
15
123

836
O. A. Fathalla et al.
Table 5 continued
Strains samples
L. monocytogenes S. aureus E. coli S. Typhimurium C. albicans
(A)
(B)
(2)
(5)
(6)
4b
-ve
-ve
-ve
-ve
11
10
11
10
-ve
40
–
-ve
-ve
-ve
-ve
-ve
40
10
11
12
13
-ve
50
–
-ve
-ve
-ve
12
-ve
–
8b
2b
10a
Control negative (DMSO) -ve
Ciprofloxacin 100 lg/mL
Fluconazole 100 lg/mL
30
–
–
38
Table 6 Minimum inhibitory concentration method showing antimicrobial activities of testing com-
pounds compared with reference drugs; the results are in lg/mL
Strains samples
L. monocytogenes S. aureus E. coli S. Typhimurium C. albicans
2a
1a
3c
12.5
12.5
100
-ve
100
50
-ve
-ve
-ve
-ve
-ve
50
-ve
-ve
-ve
-ve
-ve
50
100
-ve
100
100
100
50
-ve
-ve
100
100
-ve
100
-ve
-ve
100
-ve
100
-ve
-ve
-ve
-ve
-ve
50
7a
6b
10b
9b
4c
50
-ve
-ve
-ve
-ve
-ve
50
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
1.56
–
50
50
50
5c
50
50
6c
50
50
3b
7b
5b
4b
8b
2b
10a
-ve
-ve
-ve
-ve
-ve
-ve
-ve
25
100
100
100
100
50
25
100
100
100
100
-ve
1.56
–
50
Control negative (DMSO) -ve
-ve
0.78
–
-ve
–
Ciprofloxacin 100 lg/mL
Fluconazole 100 lg/mL
6.25
–
3.125
Results and discussion
Chemistry
Some new pyrimidine derivatives were synthesized via the reaction of ethyl
cyanoacetate with thiourea and the appropriate aldehydes namely 4-hydroxy
benzaldehyde, 2-hydroxybenzaldehyde and 2-methoxybenzaldehyde to give 1a, b,
123

Synthesis and evaluation of pyrimidine derivatives
837
and 1c [25] (Scheme 1). Compounds 1a, b, and 1c [25] were chlorinated to give the
chloro compounds 2a, b, and 2c [25] which then reacted with hydrazine hydrate
gave compounds 3a, b, and 3c [25] (Scheme 1).
Compounds 3a, b, and 3c [25] reacted with 2-(bis(methylthio)methylene)mal-
ononitrile to give compounds 4a–c, respectively, which reacted with trimethylor-
thoformate to give compounds 5a–c which then reacted with hydrazine hydrate to
give compounds 6a–c (Scheme 1).
O
Cl
S
CN
R
CN
R
HN
N
NaOEt
SCH3
POCl3
PCl5
RCHO
NCCH2COOC2H5
H2N
NH2
S
N
H
1 a-c
S
N
H
2 a-c
NC
NC
SCH3
N
H5C2O
N
N
H2N
H2N NH
H3C
S
S
CN
CN
N
N
CN
R
CN
CN
R
CH(OC2H5)3
N
H3C
NH2NH2
r.t
N
N
S
N
H
S
N
H
R
S
N
H
3 a,c
NH2NH2
r.t
4 a,c
5 a,c
NH2NH2
r.t
NC
SCH3
H2N NH
O
N
NHNH2
H2N
H3C
H3C
S
CN
N
O
SCH3
N
N
NH
S
CN
N
NH
S
N
H
N
N
S
N
S
N
H
CN
R
CH(OC2H5)3
N
3b
4b
N
H
NHNH2
NC
SCH3
SCH3
N
6 a,c
H5C2O
N
N
N
N
N
O
N
O
N
NH
N
NH
NH2NH2
r.t
S
N
H
S
N
H
5b
6b
OH
a,R =
HO
b,R =
H
3C
O
c,R =
Scheme 1
123

838
O. A. Fathalla et al.
O
O
O
CN
R
S
NH2NH2
Ref
HN
NaOEt
HN
NH
RCHO
NC-CH2COOC2H5
H2N
NH2
S
N
H
H2N
N
N
H
1 a-c
7 b
NH2NH2
O
Ref
H3C
H3C
S
S
CN
CN
O
H3C
H3C
S
CN
CN
CN
CN
HN
H2N
HN
S
N
N
R
NC
H2N
N
H
N
R
N
O
O
8 a,c
H3CS
HN
NH
7 a,c
H2N
N
N
NC
N
CH (OC2H5)3
H3CS
8 b
O
H5C2O
NC
CN
HN
CH (OC2H5)3
N
N
R
N
N
O
O
H5C2O
NC
H3CS
9 a,c
HN
NH
N
N
N
N
r.t NH2NH2
H3CS
9 b
O
N
NH2NH2
r.t
O
CN
R
HN
N
N
N
N
O
N
H
H2N
NH
HN
N
H3CS
N
N
N
N
10 a,c
N
H
H2N
OH
a,R =
c,R =
H3CS
HO
10 b
b,R =
H3C
O
Scheme 2
Compounds 1a, b, and 1c [25] were allowed to react with hydrazine hydrate to
give compounds 7a–c (Scheme 2). Compounds 7a–c reacted with 2-(bis(methyl-
thio) methylene)malononitrile to give compounds 8a–c, respectively, which reacted
with trimethylorthoformate to give compounds 9a–c which then reacted with
hydrazine hydrate to give compounds 10a–c (Scheme 2).
123

Synthesis and evaluation of pyrimidine derivatives
839
Anticancer activity
Result
Preliminary screening of the selected pyrimidine derivatives showed that all
selected compounds exhibited a moderate to strong growth inhibitory activity on the
tested cell line between 1 and 10 lg/mL concentrations in comparison to the known
anticancer drugs: 5-FU and DOX. Table 1 indicates the cytotoxic activity of the
newly synthesized pyrimidine derivatives 2a, 1a, 3c [25], 7c, 6b, 10b, 9b, 4c, 5c,
and 6c against liver HEPG2 cancer cell lines in comparison to the traditional
anticancer drugs: 5-FU and DOX. It can be deduced from the results that
compounds 5c and 6c were the most active and induced a reasonable growth
inhibition in a dose-dependent manner against HEPG2 when compared to 5-FU and
DOX (IC50 equals 3.56 and 5.23, while 5-FU and DOX were 5 and 3.56 lg/mL).
The effect of antitumor compounds on the biochemical parameters
Data obtained in Table 2 presents the liver enzymatic activities (ALT, AST, and
ALP) in serum of control and treated groups of mice. The results showed that the
values recorded for AST and ALT were significantly higher (P \ 0.001) with the
5-FU- and DOX-treated groups of mice than the control. On the other hand,
treatment with the new compounds 2a, 1a, 3c [25], 7c, 6b, 10b, 9b, 4c, 5c, and 6c
caused inverse effects, where some values recorded for AST and ALT were non-
significant (n.s.) or slightly higher (P \ 0.01) in comparison to control. Moreover,
the recorded data showed that ALP activities were significantly increased
(P \ 0.001) with the treatment of 5-FU and DOX while there were no significant
changes in ALP activities upon treatment with some of the new compounds. The
data listed in Table 3 demonstrate the comparison between the levels of total lipids,
cholesterol, triglycerides, and bilirubin in serum of treated mice and the control
group. It can be deduced from the present data that 5-FU and DOX caused a
significant increase in the level of these parameters while treatment with the
selected compounds showed moderate or no significant changes.
Table 4 represents a comparison between the levels of albumin, globulins, and
creatinine in serum of control and treated groups of mice. It is clear from the results
in the table that there was a slight increase in the level of albumin and creatinine and
globulins in the 5-FU and DOX treated groups of mice while there were moderate or
non-significant changes in the other treated groups.
Discussion
Cytotoxic drugs remain the mainstay of cancer chemotherapy and are being
administered with novel ways of therapy such as inhibitors of signals [38]. It is
therefore important to discover novel cytotoxic agents with spectra of activity and
toxicity that differ from those current agents. It is well known that chemotherapy
aims to destroy the cancer cells with various types of chemicals. The substances
123

840
O. A. Fathalla et al.
used are supposed to target mainly the cancer cells, and doses are calculated to
minimize the collateral damage to surrounding tissues, which nevertheless occurs
[39]. This kind of treatment increases the entropy of the organism, suppresses the
immune system, and forms a toxic cell environment which may destroy surrounding
healthy cells [40]. So it is important to minimize curing doses to the least amount
possible as well as trying to minimize the side effects of these drugs. For this, novel
derivatives of pyrimidine possessing a broader spectrum of antitumor activity and
fewer toxic side effects than 5-FU and DOX have been sought. The antitumor
activities of such compounds were assessed against HEPG2 cancer cell line in
comparison to the traditional anticancer drugs: 5-FU and DOX. Regarding the
antitumor activity study, some of the selected compounds showed reasonable
antitumor activity in comparison to 5-FU and DOX. Moreover, the study of the
induced biochemical parameters of the tested compounds in mice showed
insignificant differences relative to the control group, which indicates a moderate
margin of safety for the selected compounds. Comparable to 5-FU and DOX a dose
augmentation of compounds 5c and 6c, searching for possible higher potency,
seems, consequently, realizable without undesirable implications. Furthermore, the
selected compounds have important potential advantages over 5-FU and DOX
because of their lower toxicity and their ability to induce lower biochemical
parameters. These results are in agreement with Espinosa et al. [41] and
Kamalakannan and Venkappayya [42] who have reported novel derivatives of
5-FU possessing a broader spectrum of antitumor activity and fewer toxic side
effects than 5-FU.
Conclusion
As a part of our research on the related heterocyclic ring systems, and our attempts
to identify new target compounds for future development as antitumor drugs, we
previously reported the synthesis and biological of a series of substituted pyrimidine
derivatives that have various heterocyclic ring systems. These compounds were
selected for further structural modification in an effort to obtain more potent
compounds. The goal of this work in making these structural changes was to explore
the significance of the spacer on the respective biological activity as having
anticancer activity. The anticancer activity data indicate that compounds 5c and 6c
were the most active and induced a reasonable growth inhibition in a dose-
dependent manner against HEPG2 when compared to 5-FU and DOX (IC50 equals
3.56 and 5.23, while 5-FU and DOX were 5 and 3.56 lg/mL, respectively).
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