Design, synthesis of new pyrimidine derivatives as anticancer and antimicrobial agents

Article abstract of DOI:10.1080/00397911.2017.1332223

A new series of 6-aryl-5-cyano thiouracil derivatives were synthesized. Cyanouracil 1 was condensed with monochloroacetic acid and different aldehydes to give thiazolopyrimidine 2. On the other hand, treatment of cyanouracil 1 with 2-chloro-N-substituted-phenylac etamide afforded 4. Hydrazinolysis of 6 afforded the hydrazino derivatives 7 which upon reaction with different electrophilic reagents such as acetic anhydride, benzoyl chloride, and carbon disulfide yielded pyrimidine derivatives 8–15. Some of the new derivatives were explored for their antimicrobial activities. Compounds 7 and 9 have a promising activity, relatively equipotent to the reference drug. All of the new synthesized compounds were tested in vitro for their antiproliferative activities against HePG-2 and MCF-7 cell lines. Compounds 7, 9, and 2d displayed potent growth inhibitory effect toward the two cell lines more than the standard drug 5-FU. Furthermore, a docking study of the most active compounds was performed with thymidylate synthase enzyme.

Full text of DOI:10.1080/00397911.2017.1332223

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Design, Synthesis of New Pyrimidine Derivatives as
Anticancer and Antimicrobial Agents
Mohamed M. Mohamed, Ali K. Khalil, Eslam M. Abbass & Abeer M. El-Naggar
To cite this article: Mohamed M. Mohamed, Ali K. Khalil, Eslam M. Abbass & Abeer M. El-Naggar
(2017): Design, Synthesis of New Pyrimidine Derivatives as Anticancer and Antimicrobial Agents,
Synthetic Communications, DOI: 10.1080/00397911.2017.1332223
To link to this article: http://dx.doi.org/10.1080/00397911.2017.1332223
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Date: 28 May 2017, At: 06:44

Design, synthesis of new pyrimidine derivatives as anticancer and antimicrobial
agents
Mohamed M. Mohamed1, Ali K. Khalil1, Eslam M. Abbass1, Abeer M. El-Naggar1
1Chemistry Department, Faculty of science, Ain shams University, Abbassia, Cairo
11566, Egypt
Corresponding to author: Abeer M. El-Naggar, Chemistry Department, Faculty of
Science, Ain Shams University, Abassia 11566, Cairo, Egypt. E-mail:
elsayedam@sci.asu.edu.eg
Received 07 March 2017.
Abstract
A new series of 6-aryl-5-cyano thiouracil derivatives were synthesized. Cyanouracil 1
was condensed with monochloroacetic acid and different aldehydes to give
thiazolopyrimidine 2. On the other hand, treatment of cyanouracil 1 with
2‑chloro‑N‑substituted‑phenylac etamide afforded 4. Hydrazinolysis of 6 afforded the
hydrazino derivatives 7 which upon reaction with different electrophilic reagents such as
acetic anhydride, benzoyl chloride and carbon disulfide yielded pyrimidine derivatives 8-
15. Some of the new derivatives were explored for their antimicrobial activities.
Compounds 7 and 9 have a promising activity, relatively equipotent to the reference drug.
All of the new synthesized compounds were tested in vitro for their anti-proliferative
activities against HePG-2 and MCF-7 cell lines. Compounds 7, 9 and 2d displayed potent
growth inhibitory effect toward the two cell lines more than the standard drug 5-FU.
Furthermore, a docking study of the most active compounds was carried out with
thymidylate synthase enzyme.
1

GRAPHICAL ABSTRACT
KEYWORDS: antimicrobial, docking, pyrimidines, thiazolopyrimidines,
triazolopyrimidine
INTRODUCTION
Dihydropyrimidines occupy a unique place in medicinal chemistry due to their wide
application as drug and drug-intermediates possessing diverse pharmacological activities
including antitumor, analgesic, antineoplastic, cardiovascular and antiallergic activities.[1-
5] Similarly, the related thiouracil therapeutic derivatives have been known as potential
antiviral, antioxidant, anticancer and antimicrobial agents.[6-9] Moreover, literature survey
revealed that the thiouracil carbonitrile ring system has occupied a marked position in the
design and synthesis of novel chemotherapeutic agents with remarkable antitumor and
antimicrobial and HCV inhibiting activities Figure 1. [10-14]
It is well established that uracil derivatives exert their anti-cancer activity through
inhibition of folate metabolism, which is considered as an important target for the
development of new anticancer agents due to its role in the biosynthesis of nucleic acid
precursors. [15] The inhibition of folate dependent enzymes such as thymidylate synthase,
2

which catalyzes the reductive methylation of deoxyuridylate (dUMP) to thymidylate
(dTMP) has also been recognized as an interesting target for drug discovery. [16] In the
light of the aforementioned facts, this work aims to design and synthesize of a new series
of thiouracil carbonitrile derivatives hoping that they could have some chemical and
biological interest.
RESULTS AND DISCUSSION
Chemistry
In connection with our program aiming to the synthesize and evaluate the biological
activity of fused heterocycles,[17-19] we have tried to designed and synthesized a novel
series of biologically active pyrimidines derivatives and evaluated their anticancer
activity. Thus, thioxopyrimidine 1 was condensed with chloroacetic acid and different
aromatic aldehydes in a mixture of acetic acid and acetic anhydride in the presence of
fused sodium acetate to yield thiazolopyrimidine derivatives 2a-h (Scheme 1).
The structures of compounds 2a-h were confirmed by their analytical and spectral data.
Thus the IR spectrum of 2 showed the disappearance of NH band and presence of
characteristic absorption band corresponding to the vibrational coupling of carbonyl
groups around 1769-1671cm-1 which elucidate the rout of cyclization, this direction of
cyclization may be due to the steric hindrance of anisyl group moiety.[20, 21] 1H-NMR
spectra for this group exhibited a singlet signal corresponding to the ethylinic protons at
the range 7.07-8.46 ppm. Further evidence was gained from their mass spectra that
showed the correct molecular ion peaks beside some of abundant peaks (cf.
3

experimental). The mechanistic pathway for the transformation of 1 to 2 is represented in
(Scheme 2).
Compound 1 was allowed to react with the appropriate
2‑chloro‑N‑substituted‑phenylacetamide (3a-c) in the presence of potassium carbonate,
in dimethylformamide (DMF) to yield the target compounds 2-((5-cyano-4-(4-
methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(4-aryl) acetamide and 3-((4-
chloro-2-methylphenyl )amino)-7-(4-methoxyphenyl)-5-oxo-5H-thiazolo[3, 2-
a]pyrimidine-6-carbonitrile (4a-c) in good yield. Synthesis of the intermediate and target
compounds was accomplished according to the steps depicted in (Scheme 1). The
reaction proceeded via nucleophilic substitution reaction followed by cyclization in case
of 4c. IR spectra of 4a,b revealed the existence of bands in the frequency range 3293-
3308 and 1654-1711 cm-1 corresponding to NH2, NH and C=O groups, respectively. 1H-
NMR spectra comfirmed the structure of compounds 4a-c. where spectrum of 4a showed
signals at 4.16, 10.41 ppm corresponding to CH2 and two NH group, respectively.
Meanwhile, 4b spectrum showed signals at 4.19, 10.67, 13.78 ppm corresponding to
CH2, NH and NH2 groups, respectively. However, 1H-NMR spectrum of 4c exhibited a
singlet signal at 9.63 corresponding to NH which supported the suggested structure. On
the other hand, N-alkylation of 1 with p-toluene sulphonyl chloride produced 6-(4-
methoxyphenyl)-4-oxo-2-thioxo-1-tosyl-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (5)
which was carried out by stirring in dry DMF utilizing potassium carbonate as base
catalyst. The IR spectra of compound 5 showed the disappearance of NH bands and the
appearance of characteristic absorption bands corresponding to SO2 at 1167 cm-1. The
4

1H-NMR spectra of compound 5 revealed two singlet signals at 2.69, 13.11 ppm
assignable to CH3 and NH groups respectively. On the other hand, Compound 1 was
alkylated with ethylchloroacetate, in the presence of anhydrous potassium carbonate to
give the ethyl ester derivative 6. Analytical and spectral data were in agreement with the
proposed structure. The IR spectrum showed signals at 3301, 1735, and 1660 cm-1
corresponding to NH, (CO ester) and (CO) amide respectively. The 1H-NMR spectrum of
6 revealed signals at 1.08, 4.05 and 13.7 ppm for CH3, CH2 and NH respectively. The
structure of compound 6 was established chemically by the reaction with hydrazine
hydrate in boiling ethanol afforded 2-hydrazinyl-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 7 (Scheme 1). The hydrazine derivative 7 was also
isolated by treating 1 with hydrazine hydrate under the same reaction condition. The
structure of compound 7 was corroborated by spectroscopic data.
The IR spectrum showed bands at 3324, 3277 and 1685 cm-1 attributed to NH2, NH and
C=O groups respectively. The 1H NMR spectrum of compound 7 showed signals at 3.49
and 8.19 attributed to NH2, NH and at 10.15 for NH (amide) groups, respectively. A
plausible mechanism for the formation of the hydrazine derivative 7 is outlined in
(scheme 3). Hydrazino pyrimidines can be considered as key starting precursor for
synthesis of new pyrimidine derivatives which might have chemotherapeutic and
biological activity. So, the hydrazino derivative 7 was reacted with some electrophilic
reagents such as arylidine malononitrile, formic acid, acetic anhydride, cyclopentanone,
benzoyl chloride and cabon disulphide. Thus, when the hydrazino derivative 7 was
subjected to react with arylidene in pyridine, it afforded the Schiff base product 8. The
5

conversion of 7 to 8 could be visualized on the basis of the nucleophilic attack by
nitrogen of the hydrazino group at the electron deficiency β- carbon of the arylidene
followed by elimination of malononitrile molecule. (C.f. Scheme 5).
IR of 8, showed bands at 3223, 1658 cm-1 corresponding to NH, C=O groups,
respectively. 1H-NMR of 8 revealed signals at 3.79, 3.82, 8.10, 12.19 and 12.37
corresponding to 2 OCH3, olifinic CH and 2NH groups. A strong evidence for the
structure of 8 was gained chemically by reacting the hydrazine derivative 7 with p-
methoxybenzaldehyde under the same condition. Refluxing of hydrazino derivative 7
with acetic anhydride afforded the mono acetyl derivative 9. The spectral data of
compound 9 was in agreement with the assigned structure. IR spectrum displayed bands
at 3226, 1630-1660 cm-1 corresponding to NH, 2(CO) groups, respectively. 1H-NMR
showed signals at 1.89, 10.19 ppm corresponding to CH3, NH groups, respectively. 2-(2-
cyclopentylidenehydrazinyl)-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-
carbonitrile 10 was obtained by refluxing compound 7 with cyclopentanone in ethanol.
Its 1H-NMR showed multiplet signals at (1.65-1.96) ppm due to CH2 groups of
cyclopentanone moiety (cf. Experimental part).
However, stirring of 2-Chloro-N-(4-chlorophenyl)acetamide with hydrazino derivative 7
afforded N-(4-chlorophenyl)-2-(2-(5-cyano-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyrimidin-2-yl)hyda-zinyl) acetamide 11. IR spectrum of 11 showed signals at
3317, 1700, 1649 cm-1 corresponding to NH and two C=O groups, respectively. 1H-NMR
of 11 showed signals at 3.85, 4.86 and 8.76, 10.63 ppm for OCH3, CH2 and 4NH groups,
6

respectively. Diazotization of compound 7 resulted in the formation of
tetrazolopyrimidine derivative 12. The structure of 12 was elucidated by the spectral data.
IR showed absorption bands at 3221 and 1651 cm-1 due to NH and CO groups,
respectively and disappearance of NH2 group. In addition to 1H-NMR exhibited signals at
3.83 and 5.96 ppm attributed to OCH3 and NH groups, respectively.
Treatment of the hydrazino derivative 7 with formic acid afforded triazolopyrimidone
derivative 13 (Scheme 6). IR spectrum was in agreement with the predicted structure as it
showed the disappearance of NH2 bands. In addition, 1H-NMR spectrum revealed signals
at 9.23 ppm for olefinic CH. Triazolopyrimidone derivative 14 was obtained by stirring 7
with benzoyl chloride in dry DMF with anhydrous potassium carbonate. The structure of
compound 14 was characterized by the presence of bands 3267 and 1685cm−1 for the
amide groups NH and C=O groups. Moreover, 1H-NMR spectra exhibited a singlet band
at 8.65 ppm for NH group. Refluxing of compound 7 with carbon disulphide in pyridine
afforded triazolopyrimidone derivative 15. The formation of 15 is assumed to proceed via
nucleophilic addition of nucleophilic amino group of the hydrazino moiety to C=S group
of carbon disulfide followed by cyclization and elimination of H2S molecule. (Scheme 7)
IR spectrum of 15 exhibited signals at 3421, 1679 due to twoNH and C=O groups,
respectively. In addition, an absorption band was revealed at 1452 cm-1 corresponding to
C=S group. Moreover, 1H NMR showed two singlet signals at 9.30 and 9.80 ppm
attributed to two NH groups.
BIOLOGICAL ACTIVITY
7

Antimicrobial Studies
Some of the newly synthesized compounds 1, 2, 4, 5, 7, 9 and 13 were assayed in vitro
for their antimicrobial activity against two gram negative bacteria: namely, Pseudomonas
aeruginosa and Escherichia coli, and two gram positive bacteria: Staphylococcus aureus
and Bacillis subtilis and two fungal species, namely: Aspergillus flavus and Candida
albicans. The fungicide Colitrimazole and the bactericides Ampicillin were used as
references to evaluate the potency of the tested compounds under the same conditions. As
a parameter of antimicrobial activity, the values of the zone of inhibition (mm) and %
activity index were determined. The results are depicted in Table 1, (Figure 2).
It has been observed that compounds 2d, 7 and 9 possess a pronounced antimicrobial
activity against Staphylococcus aureus, Bacillis Subtilis, Escherichia coli, Candida
albicans and Aspergillus flavus compared to the reference drugs, while, compounds 2c,
2e and 2h showed exerted moderate activity. Compounds (1, 2b, 2f, 4c, 4a, and 5)
showed low activity. However, compounds 2a, 4c, 4b and 13 were either inactive or
weakly active against the tested microorganisms. Hydrazino derivative 7 and
acetohydrazide derivative 9 caused a pronounced inhibition effect against Gram-positive
(Staphylococcus aureus, Bacillus cereus), Gram-negative (Escherichia coli) bacteria and
fungi (Candida albicans and A. flavus). On the other hand, increased antibacterial activity
was achieved by cyclization to thiazolopyrimidine. However, cyclization to
triazolopyrimidine as in 13 and alkylation in 4(a-c) either decreased or diminished
antimicrobial activity, respectively.
8

In Vitro Anti-Proliferative Activity
Anticancer activity screening of the newly synthesized compounds was measured in vitro
utilizing two different human cancer cell lines namely, hepatocellular carcinoma (HePG-
2), mammary gland (MCF-7), using sulforhodamine B (SRB) colorimetric assay as
described by Skehan et al.[22] Due to the structure similarity between 5-FU (Figure 3) and
thiouracil derivatives, 5-FU was selected as a standard reference anticancer agent. The
chemosensitivity responses of cell lines to 5-FU and analogues are presented in Table 2.
The results obtained indicated that the tested compounds exhibited good, moderate or
weak anti-proliferative activities against the tested cell lines. In general, the compounds
7, 9, 2d and 2e were found to be the most potent derivatives against the two cell lines.
Compound 7 was 0.62 and 0.62 times as active as 5-FU against HepG2 (IC50 = 25.5 µM)
and MCF-7 (IC50 = 23.9 µM), respectively. Compound 9 it was 0.66 and 0.72 times as
active as 5-FU against HepG2 (IC50 = 25.7 µM) and MCF-7 (IC50 = 25.7 µM),
respectively. Compound 2d it was 0.62 and 0.70 times as active as 5-FU against HepG2
(IC50 = 24.1 µM) and MCF-7 (IC50 = 27.7 µM), respectively. Compound 2e it was 1.06
and 0.58 times as active as 5-FU against HepG2 (IC50 = 40.9 µM) and MCF-7 (IC50 =
22.4 µM), respectively.
Compound 2g it was 0.60 times as active as 5-FU against HepG2 (IC50 = 23.4 µM).
However, it was possessed moderate anti-proliferative activities against MCF-7 cell.
Compound 2h it was 0.96 times as active as 5-FU against MCF-7 (IC50 = 36.9 µM).
However, it was possessed moderate anti-proliferative activities against HepG2 cell.
9

Compound 15 it was 0.90 times as active as 5-FU against HepG2 (IC50 = 34.9 µM) and
possessed moderate anti-proliferative activities against MCF-7 cell. The compounds (2a-
c), 2f, (4a-c), 6, 5, 8, 10, 11, 12, 13 and 14 were found to possess either moderate or weak
anti-proliferative activities against the two cell lines .
Similarity in structures between 5-FU and thiouracil derivatives may suggest possible
similarity in the mechanism of action between these drugs. 5-FU induces cell kill mainly
via destructing DNA synthesis by inhibiting thymidylate synthase which is the enzyme
catalyses the conversion of deoxyuridylate to deoxythymidylate.[23] Molecular docking
comparing the binding affinity of 5-FU and thiouracil derivatives was therefore
conducted.
Molecular Docking
Based on the fact that 5-fluorouracil derivatives and their structurally related compounds
like thiouracil carbonitrile derivatives, are well known to inhibit thymidylate synthase,
we decided to carry out a molecular docking study of the highest biologically active three
newly synthesized thiouracil carbonitrile derivatives into the binding site of thymidylate
synthase (PDB id1JU6) which was retrieved from PDB bank http://www.rcsb.org/pdb
using 5-FU as a reference for docking results. The results of docking study were reported
as TS binding free energy (ΔG). The negative values of free energies refer to spontaneity
of bindings Table 3.
10

The docking results revealed that, the highest binding compound to TS was 7 with
binding energy of -42.23 Kcal/mol. Compound 7 formed six hydrogen bonds with the
amino acid residues of thymidilate synthase. Carbonyl group at the 4-position of
pyrimidine moiety was anchored by hydrogen bonding interactions with His 256. Cyano
group at the 5-position of pyrimidine moiety was involved in a hydrogen bonding
interaction with ASP218 between N of the subitituted NH NH2 group at the 2-position of
thiopyrimidine ring of the ligand and the amino acids Ala312 and Val 313 and as well as
between N at NH-NH2 of the substituted group at the 2-position of thiopyrimidine ring
and the amino acid Try 258 of the enzyme Figure 4.
On the other hand, compound 9 exhibited promising TS inhibitory activity (IC50 = 25.73±
2.22 µM) and TS inhibition % = 0.72). Introduction of acetyl moiety of the compound 9
is likely important for increasing the affinity for the receptor. It showed hydrogen bonds
with the amino acid Asn226, Arg50, Asn112, Gln214, and Asp218. (Figure 5).
The proposed binding mode of compound 2d showed affinity value of -39.19 kcal/mol. It
showed one hydrogen bond interaction between S at fused bicyclic system and the amino
acid Ser216, Oxygen atom at thiazolidine moiety formed interaction with the amino acid
Arg50. In addition, It showed one hydrogen bond interaction between OCH3 at 4-position
of phenyl moiety and Ans226 Figure 6.
EXPERIMENTAL PART
Chemistry
11

All melting points were measured on a Gallenkamp melting point apparatus and were
uncorrected. The IR spectra were recorded on a Pye-Unicam SP-3-300 infrared
spectrophotometer (KBr dicks) and expressed in wave number (cm-1). 1H NMR spectra
were run at 400 MHz, on a Varian Mercury VX-300 and BrukerAvance III NMR
spectrometer respectively, while 13C NMR spectra were run at 75 MHz. TMS was used as
an internal standard in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (δ)
are quoted in ppm. The abbreviations used are as follows: s, singlet; d, doublet; m,
multiplet. All coupling constant (J) values are given in hertz. The mass spectra were
recorded on Shimadzu GCMS-QP-1000EX mass spectrometer at 70 eV. Elemental
analyses were performed on CHN analyzer and all compounds were within ± 0.4 of the
theoretical values. The reactions were monitored by thin-layer chromatography (TLC)
using TLC sheets coated with UV fluorescent silica gel Merck 60 F254 plates and were
visualized using UV lamp and different solvents as mobile phases. All reagents and
solvents were purified and dried by standard techniques. All the newly synthesized
compounds gave satisfactory elemental analysis. Compounds 1 [14], 3(a-c) [24, 25] are
previously reported.
General Procedure For The Preparation Of Compounds 2a-H
A mixture of 1 (10 mmol) and different aldehydes namely, 4-methoxybenzaldehyde,
thiophene- carboxaldehyde, 3,4-dimethoxybenzaldehyde, 4-chlorobenzaldehyde, 4-
nitrobenzaldehyde, benz- aldehyde, 3,4-dichlorobenzaldehyde and salycialdehyde (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 5-8 h. The solution was cooled,
12

gradually poured onto cold water, and the formed precipitate was washed several times
with water, filtered off, and recrystallized from ethanol to afford pure product 2a-h.
2-(4-Methoxybenzylidene)-7-(4-Methoxyphenyl)-3,5-Dioxo-3,5-Dihydro-2H-Thiazo
Lo[3,2-A]Py Rimidine-6-Carbonitrile (2a)
Yield 78 %; yellow crystal; m.p. 291-293 oC (EtOH); IR (cm-1) ν: 3040 (CH aromatic),
2845 (CH aliphatic), 2213 (CN), 1751, 1685 (CO). 1H-NMR (400 MHz, DMSO-d6) δ
(ppm): 3.85 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.14 (d, 2H, Ar-H, J = 8.8 Hz), 7.16 (d,
2H, Ar-H, J = 7.2 Hz), 7.73 (d, 2H, Ar-H, J = 8.8Hz), 8.06 (d, 2H, Ar-H, J = 7.2 Hz),
8.12 (s, 1H, CH ethylenic). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 56.3 (2), 99.5,
115.2 (4), 118.6 (2), 128.7 (2), 129.2, 130.2, 133.8, 133.4, 145.4, 161.7 (2), 166.7, 167.6,
168.8, 170.3; MS (m/z): 417 (M+, 19%), 164 (100 %); Anal. Calcd for: C22H15N3O4S
(417.44): C, 63.30; H, 3.62; N, 10.07; Found: C, 63.07; H, 3.50; N, 9.96 %.
General Procedure For The Preparation Of Compounds 4a-C
To a suspension of (1) (0.01 mol) in dry DMF (20 ml) containing anhydrous K2CO3 (0.01
mol), 2‑chloro‑N‑substituted‑phenylacetamide (0.01 mol) (3a-c) was added. The
reaction mixture was stirred at room temperature for 8-10 h, coold, poured onto crushed
ice, and then acidified with dilute acetic acid. The obtained precipitate was filtered,
washed with H2O, dried, and recrystallized from ehanol to afforded 4a-c.
N-(4-Chlorophenyl)-2-(5-Cyano-4-(4-Methoxyphenyl)-6-Oxo-1,6-Dihydropyrimidin-2-
Ylthio) Acetamide (4a)
13

Yield 77 %; white crystal; m.p. 268-270 oC (EtOH); IR (cm-1) ν: 3308 (NH), 3083 (CH
aromatic), 2841 (CH aliphatic), 2222 (CN), 1683 (CO amide), 1654 (CO amide), 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 3.78 (s, 3H, OCH3), 4.16 (s, 2H, CH2), 6.83 (d,
2H, Ar-H, J = 8.4 Hz), 7.34 (d, 2H, Ar-H, J = 8.4 Hz), 7.54 ( d, 2H, Ar-H, J = 8.0 Hz),
7.88 (d, 2H, Ar-H, J = 8.0 Hz), 10.41 (br. s, 2H, 2NH); 13C-NMR (100 MHz, DMSO-d6)
δ (ppm): 35.4, 55.4, 91.4, 113.6 (2), 116.1, 120.4 (2), 126.9, 127, 128.7 (2), 130.8 (2),
127.9, 161, 162, 164.9, 165.3, 166.1; MS (m/z): 426 (M+, 25 %), 428 (M++2, 8.32 %),
126 (100 %); Anal. Calcd for: C20H15ClN4O3S (426.88): C, 56.27; H, 3.54; N, 13.12;
Found: C, 56.08; H, 3.49; N, 13.00 %.
Formation Of 2-(2-(4-Methoxybenzylidene)Hydrazinyl)-4-(4-Methoxyphenyl)-6-Oxo-
1,6-Dihydro Pyrimidine-5-Carbonitrile (8).
A mixture of 7 (10 mmol) and 2-(4-methoxybenzylidene)malononitrile (10 mmol) was
refluxed in pyridine (20 mL)for 8 hr. The reaction mixture was cooled, acidified with
dilute acetic acid and the solid obtained was filtered off, dried and then recrystallized
form ethanol to give 8.
Yield 75 %; brown crystal; m.p. 296-297 oC (EtOH); IR (cm-1) ν: 3223 (NH), 3168 (CH
aromatic), 2835 (CH aliphatic), 2208 (CN), 1658 (CO), 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 3.79 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.96 (d, 2H, Ar-H, J = 8.0 Hz), 7.06 (d,
2H, Ar-H, J = 8.4 Hz), 7.87 (d, 2H, Ar-H, J = 8.0 Hz), 7.94 (d, 2H, Ar-H, J = 8.4 Hz),
8.10 (s, 1H, CH), 12.19 (s, 1H, NH); 12.37 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-
d6) δ (ppm): 57.2 (2), 95.0, 114.3 (2), 116.4 (2), 118.8, 128.1, 129.5 (2), 132.8, 135.5,
14

136.1, 145.4, 160.8, 162.9, 164.7, 171.2, 174.4; MS (m/z): 375 (M+, 5 %), 226 (38 %),
107 (100 %); Anal. Calcd for: C20H17N5O3 (375.38): C, 63.99; H, 4.56; N, 18.66; Found:
C, 63.65; H, 4.62; N, 18.59 %.
Formation Of 5-(4-Methoxyphenyl)-7-Oxo-3,7-Dihydrotetrazolo[1,5-A]Pyrimidine-6-
Carbonitril (12).
Compound 7 (10 mmol) was dissolved in (10 mL) concentrated hydrochloric acid and
then (7 mL) of sodium nitrite (10 %) was added dropwise with stirring over a period of 2
hours at 0 oC. The obtained yellow solid was filtered off, dried and recrystallized from
ethanol to give 12 as yellow crystals.
Yield 77 %; yellow crystal; m.p. 237-238 oC (EtOH); IR (cm-1) ν: 3223 (NH), 3076 (CH
aromatic), 2836 (CH aliphatic), 2222 (CN), 1651 (CO), 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 3.83 (s, 3H, OCH3), 5.96 (s, 1H, NH), 7.08 (d, 2H, Ar-H, J = 8.0 Hz), 7.78 (d,
2H, Ar-H, J = 8.0 Hz). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 55.2, 94.4, 117.1 (2),
118.9, 128.4 (2), 130.3, 132.5, 164.1, 167.5, 171.6; MS (m/z): 268 (M+, 20 %), 225 (100
%); Anal. Calcd for: C12H8N6O2 (268.23): C, 53.73; H, 3.01; N, 31.33; Found: C, 53.61;
H, 2.92; N, 31.25 %.
Formation Of 7-(4-Methoxyphenyl)-5-Oxo-1,5-Dihydro-[1,2,4]Triazolo[4,3-
Α]Pyrimidine-6-Ca-Rbonitrile (13).
A mixture of 7 (10 mmol) and formic acid (20 mL) was refluxed for 8-10 h. the solid was
filtered off, dried and then recrystallized from ethanol to give 13. Yield 80 %; yellow
15

crystal; m.p. 250-252 oC (EtOH); IR (cm_1) ν: 3408 (NH), 3111 (CH aromatic), 2848 (CH
aliphatic), 2219 (CN), 1701 (CO); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 3.84 (s, 3H,
OCH3), 7.07 (d, 2H, Ar-H, J = 8.0 Hz), 7.09 (s, 1H, NH), 7.87 (d, 2H, Ar-H, J = 8.0 Hz);
9.29 (s, 1H, CH); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 55.4, 82.3, 113.4, 113.7,
117.2, 128.3, 130.2, 130.4, 133.1, 150, 155.5, 161.7, 168,7; Anal. Calcd for: C13H9N5O2
(267.24): C, 58.43; H, 3.39; N, 26.21; Found: C, 58.29; H, 3.29; N, 26.12 %.
Biological Assay
Antimicrobial Activity
Materials And Methods
Chemical compounds were individually tested against a panel of two Gram-positive
bacteria (Staphylococcus aureus and Bacillus subtilis) and two Gram-negative bacteria
(Escherichia coli and Pseudomonas aeuroginosa) using Muller Hinton agar medium
(Oxoid). The anti-fungal activities of the compounds were tested against two fungi
(Candida albicans and Aspergillus flavus) using Sabouraud dextrose agar medium
(Oxoid). Each of the compounds was dissolved in DMSO and solution of the
concentration 1 mg /mL was prepared. Separately paper discs of Whatman filter paper
were prepared with standard size (5cm), were cut and sterilized in an autoclave [26]. The
paper discs soaked in the desired concentration of the complex solution were placed
aseptically in the petri dishes containing Muller Hinton agar medium (Oxoid) seeded with
Staphylococcus aureus , Bacillus subtilis , E. coli , Pseudomonas aeuroginosa and
Candida albicans , Aspergillus flavus. using Sabouraud dextrose agar medium (Oxoid).
The petri dishes were incubated at 36 c and the inhibition zones were recorded after 24 h
16

of incubation. Each treatment was replicated three times. The antibacterial activity of a
common standard antibiotic ampicillin and antifungal Colitrimazole was also recorded
using the same procedure as above at the same concentration and solvents. The % activity
index for the complex was calculated by the formula as under :
Zoneof inhibitionbytestcompound diametre
% Activity Index
100
Zoneof inhibitionbystandard diametre
Cytotoxicity Assay
Cell Line
The cytotoxic activity of fourteen compounds was tested against two human tumor cell
lines namely hepatocellular carcinoma HePG2 and mammary gland breast cancer MCF-
7. The cell lines were obtained from ATCC via Holding company for biological products
and vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil was used as a standard
anticancer drug for comparison.
Chemical Reagents
The reagents used were RPMI-1640 medium, MTT, DMSO, 5-fluorouracil (sigma co.,
St. Louis, USA), and Fetal Bovine serum (GIBCO, Paisley, UK).
MTT Assay
The different cell lines mentioned above were used to determine the inhibitory effects of
compounds on cell growth using the MTT assay.[27-29] This colorimetric assay is based on
the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative
by mitochondrial succinate dehydrogenase in viable cells. Cell lines were cultured in
17

RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml
penicillin and 100µg/ml streptomycin at 37 °C in a 5% CO2 incubator. The cell lines
were seeded in a 96-well plate at a density of 1.0x104 cells/well66 at 37 °C for 48 h under
5% CO2. After incubation the cells were treated with different concentration of
compounds and incubated for 24 h. After 24 h of drug treatment, 20 µl of MTT solution
at 5mg/ml was added and incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of
100 µl is added into each well to dissolve the purple formazan formed. The colorimetric
assay is measured and recorded at absorbance of 570 nm using a plate reader (EXL 800,
BioTech,Winoosky, VT,USA). The relative cell viability in percentage was calculated as
(A570 of treated samples/A570 of untreated sample) X 100.
Molecular Docking
All docking studies were performed using ‘Internal Coordinate Mechanics [Molsoft ICM
Discovery Studio]. A set novel thiopyrimidine derivatives, were compiled by us using
ChemDraw. 3D structures were constructed using Chem 3D ultra12.0 software
[Molecular Modeling and Analysis, Cambridge Soft Corporation, USA (2010)]. The
selected compounds were energetically minimized by using MOPAC (semi-empirical
quantum mechanics), ynternational coordinate and saved as MDL MolFile (*.mol). The
thymidylate synthase (PDB id 1JU6) which was retrieved from PDB bank
http://www.rcsb.org/pdb. All bound waters, ligands and cofactors were removed from the
protein prior to the docking process.
CONCLUSION
18

A series of novel cyanothiouracil derivatives (2-15) was synthesized from 6-aryl-cyano
thiouracils and 6-aryl-4-hydrazino-2-thioxo-1, 2-dihydropyrimidine-5-carbonitriles
derivatives. Antimicrobi al and anticancer studies were performed. Potent activities were
observed for some of the synthesized compounds.
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22

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23

Table 1. Preliminary antibacterial activity for some of the synthesized compounds.
Compound
Diameter of inhibition zone (mm). % Activity index
E. coli P.aeuroginosa
S.
B.
C.
A.
aureus
subtilis
Albicans
flavus
1
7 (30.4)
NA
13 (54.2) 8 (36.4) 12 (50.0)
2 (8.3) NA 2 (8.3)
11 (45.8) 8 (36.4) 10 (41.7)
6 (26.1) 9 (36.0)
NA 5 (20.0)
2a
2b 7 (30.4)
5 (21.7) 9 (36.0)
2c
2d
2e
11
(47.8)
14
17 (70.8)
20 (83.3)
18 (75.0)
13 (54.2)
12 16 (66.7)
(54.5)
16 20 (83.3)
(72.7)
15 18 (75.0)
(68.2)
11 15 (62.5)
(50.0)
10 (41.7) 7 (31.8)
15 (65.2)
17 (73.9)
12 (52.2)
7 (30.4)
18
(72.0)
19
(60.9)
13
(76.0)
17
(56.5)
(68.0)
10
2f 8 (34.8)
(40.0)
2g 6 (26.1)
8 (33.3)
12 15 (62.5)
(54.5)
2 (9.1)
NA 4 (16.0)
2h
10
(43.5)
NA
14 (58.3)
11 (47.8)
16
(64.0)
4a
4c
4b
5
2 (8.3)
4 (16.7)
6 (25.0)
5 (20.8)
NA 3 (12.0)
3 (13.0) 7 (28.0)
2 (8.7)
NA
5 (20.8) 4 (18.2)
4 (16.7)
15(62.5)
2 (9.1)
NA
2 (8.0)
14
10
10 15 (62.5)
9 (39.1)
24

(43.5)
17
(45.4)
17 22 (91.7)
(77.3)
19 23 (95.8)
(56.0)
23
7
9
21 (87.5)
22 (91.7)
NA
18 (78.3)
17 (73.9)
(73.9)
18
(92.0)
21
(78.3)
NA
(86.4)
NA
(84.0)
NA
13
NA
NA
NA
Ampicillin 23 (100)
24 (100) 22 (100) 24 (100)
NA NA NA
NA
Colitrimazole
NA
23 (100) 25 (100)
*NA: No activity.
25

Table 2. Anti-proliferative activity toward HePG-2, MCF-7cell lines.
In vitro Cytotoxicity IC50 (µ molar/ml)a
Compound
HePG2
MCF-7
1
2a
2b
3c
2d
2e
2f
2g
2h
4a
4c
4b
5
89.90±4.20
84.12±4.17
86.14±3.92
67.92± 3.35
24.16±1.89
40.98±2.76
56.28±3.24
91.83±4.43
NA b
NA b
58.13 ± 2.99
27.24± 2.7
22.46± 2.95
49.35± 2.21
23.40± 2.94 52.3±3.24
60.93±3.71
NA b
36.90 ±2.14
NA b
98.08±4.90
NA b
93.31±4.82
NA b
31.55 ± 3.00
NA b
56.46± 2.11
NA
6
7
25.52±1.6
23.91±1.23
NA
8
NA
9
25.73 ±2.2
27.71± 2.7
NA b
10
11
12
NA b
NA b
NA b
NA b
NA b
26

13
14
15
NA b
NA b
NA b
NA b
34.92± 3.6
38.44±2.14
87.30±4.82
41.53±2.30
5- Fuc
aIC50: values are the mean ± S.D. of three separate experiments.
bNA: Compounds having IC50 value >100 µM.
c5- Flourouracil
27

Table 3. The docking energy scores of compounds 5-FU, 7, 9 and 2d with the amino acid
residues of the target enzyme thymidilate synthase forming hydrogen bonds.
Cpd.
No.
Docking score No. of Hydrogen
Amino acid residues – Atom of ligand
involved H-bond distance (A0)
Asp218: HN -- O8 2.1
(Kcal/mol)
- 18.87
bonds
4
5-FU
His256: HE2 -- O9 2.2
Ser216: OG -- H10 2.3
Try258: OH --H11 2.4
Try258: OH-- H27 2
7
-42.23
6
Ala312: O--H29 2.2
Val313: OCT2-- H29 2.5
Arg50: HH21-- N19 2
Asp218: HN -- N16 2.3
His256: HE2-- O17 2
9
-39.36
5
Asn226: OD1--H30 2
Arg50: HH21--O7 2.2
Asn112: HD22--O7 2
Gln214: HE21--O17 2.2
Asp218: HN -- O17 2.2
Lys47: HZ1-- O21 2.5
Arg50: HH11-- O21 1.8
2d
-39.19
4
Ser216: HG -- S18 2.4
28

Asn226: HD21-- O7 1.9
29