Synthesis, biological evaluation and molecular docking studies of some pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.05.028

Some novel pyrimidine-5-carbonitrile derivatives bearing various substituent have been synthesized. The structures of target compounds were confirmed by elemental analysis and spectral data. Some selected members of the newly synthesized compounds were investigated for their cytotoxic potency against certain human tumor cell lines. Five representative active anticancer compounds 6a, 6c, 6d, 17a and 18a were subjected to docking using MOE program on the 3D structure of two enzymes, namely; thymidylate synthase and dihydrofolate reductase. The antimicrobial activities of the synthesized compounds were tested against Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri and Candida albicans. Compounds 2c, 7a and 9c showed broad spectrum antimicrobial activity.

Full text of DOI:10.1016/j.ejmech.2013.05.028

Accepted Manuscript
Synthesis, biological evaluation and molecular docking studies of some pyrimidine
derivatives
Ahmed M. Fargualy, Nargues S. Habib, Khadiga A. Ismail, Ahmed M.M. Hassan,
Marwa T.M. Sarg
PII:
S0223-5234(13)00337-1
10.1016/j.ejmech.2013.05.028
EJMECH 6206
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 March 2013
Revised Date: 28 April 2013
Accepted Date: 22 May 2013
Please cite this article as: A.M. Fargualy, N.S. Habib, K.A. Ismail, A.M.M. Hassan, M.T.M. Sarg,
Synthesis, biological evaluation and molecular docking studies of some pyrimidine derivatives, European
Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.028.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis of some pyrimidine analogues as a biologically active backbone with the
hope to go a step forward in the field of anticancer and antimicrobial treatment.
O
OH
H
O
N
S
N
N
NC
O
R
OH
O
O
N
N
R
S
N
O
- epichlorohydrin
- morpholin
N
N
R
S
N
- POCl3
NC
N
NC
- epichlorohydrin
- POCl3
- morpholine
- morpholine
- epichlorohydrin
H
N
H
O
S
N
O
N
S
N
ClCH2CH2N(CH2CH3)2.HCl
- CH3I
NC
NH
NC
R
R
- CH3I
- morpholine or piperidine
or N-benzylpiperazine
H
-NH2NH2.H2O
O
N
N
NH2
O
N
N
R1
N
ethyl cyanoacetate
NC
- POCl3
- morpholine or piperidine
or N-benzylpiperazine
N
O
N
N
N
R2
R
NC
CH2CN
diethyl malonate
N
NC
O
R
R1
N
O
O
N
R
S
R2
O
O
N
R
N
N
NH
NC
H
N
NC

ACCEPTED MANUSCRIPT
Highlights
• The design and synthesis of novel
pyrimidine derivatives.
• Compounds were investigated for
anticancer and antimicrobial activities.
• Docking was performed for the five
most active anticancer compounds.

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and molecular docking studies of some
pyrimidine derivatives
Ahmed M. Fargualya, Nargues S. Habiba, Khadiga A. Ismailb*, Ahmed M. M. Hassana
Marwa T. M. Sargc
aDepartment of Pharmaceutical chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
bDepartment of Pharmaceutical chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
cDepartment of Pharmaceutical chemistry, Faculty of Pharmacy, Elazhar University, Cairo, Egypt
A B S T R AC T
Some novel pyrimidine-5-carbonitrile derivatives bearing various substituent have been
synthesized. The structures of target compounds were confirmed by elemental analysis and
spectral data. Some selected members of the newly synthesized compounds were investigated for
their cytotoxic potency against certain human tumor cell lines. Five representative active
anticancer compounds 6a, 6c, 6d, 17a and 18a were subjected to docking using MOE program on
the 3D structure of two enzymes, namely; thymidylate synthase and dihydrofolate reductase. The
antimicrobial activities of the synthesized compounds were tested against Staphylococcus aureus,
Pseudomonas aeruginosa, Shigella flexneri and Candida albicans. Compounds 2c, 7a and 9c
showed broad spectrum antimicrobial activity.
Keywords:
Pyrimidine Pyrimidine-5-carbonitrile Anticancer Antimicrobial Docking and Molecular modeling.
*Corresponding author: Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt. 21215.
Fax : 002-03-4873273
E-mail address : kadiga99@yahoo.com
1. Introduction
Pyrimidine ring is the building unit of DNA and RNA which explains the fact that
pyrimidine derivatives exhibit diverse pharmacological activities. The most pronounced of which
are anticancer [1-9], antiviral [10-12] especially anti-HIV [13-15], antimalarial [16-18]
antimicrobial [19-20] and anti-inflammatory [21-22]. They also show activity against
gonadotropin releasing hormone receptors [23-25] as well herbicidal activity targeting
acetohydroxyacid synthase, which catalyze the first common step in branched-chain amino acid
biosynthesis [ 26-28]. Furthermore, many pyrimidine-5-carbonitrile derivatives proved to exhibit
potent anticancer [29,30] as well as antimicrobial activities [29,31,32]. Patients with neoplastic
disorders are mostly subjected to microbial infections. The coadminstration of multiple drugs for
treating patients suffering from cancer disease accompanied with microbial infections might
inflect some added health problems especially in patients with impaired liver and/or kidney
functions. Therefore, the concept of monotherapy by a single drug which possesses dual utility
might be advantageous from both therapeutic as well as cost-effective stand points.
Consequently, our efforts were devoted to synthesize and investigate further innovative
pyrimidine analogues with dual function; anticancer/ antimicrobial,. Aligned with this scope,
some pyrimidine-5-carbonitriles, bearing biologically active functionalities were adopted to be
synthesized (schemes 1, 2 and 3). Thioethers were found to show enhanced antimicrobial
activities [33,34]. Utilizing the same goal, it was designed to synthesize various thioethers having
different side chains with the hope to go a step forward in the field of anticancer and
antimicrobial treatment. Synthesis of compounds 5a-c were adopted on the rationale that some
chloropyrimidines were found to possess anticancer activity [35]. Furthermore, our interest was
extended to investigate anticancer and antimicrobial activities of chlorosubstituted pyrimidines
bearing 2,3-epoxypropyl functionality 16a,c. Phosphochloridate 4, although obtained
unexpectedly, yet its preparation was not beyond the scope of our interest due to its structural
similarity to the anti-HIV cyclic phosphotriesters [36].
1

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SAR study of the 2,4,6-trisubstituted pyrimidines revealed that 4-morpholino substitution
appeared to exhibit important anti-proliferative activity leading to the most potent anticancer
compounds[37]. However, replacing 4-morpholino with various linear or cyclic alkylamino
substituents led to decrease in activity[37]. Encouraged by the foretasted findings and in a search
for new and efficient agents, it was designed to synthesize 4-substituted amino-2-
thioxopyrimidines 6a-i and 2-substituted amino-6-oxopyrimidines 11a-f. Moreover, it was
desired to incorporate both morpholine functionality together with either 2,3-epoxypropyl or 2-
hydroxypropyl substituents onto the substituted pyrimidine core 17a,b and 18a,b respectively, in
an attempt to improve efficacy by increasing one more bioactive moiety. The fact that hydrazino
derivatives are capable of exhibiting anticancer activity by alkylating DNA through a free radical
intermediate [38] prompted us to synthesize 2-hydrazinopyrimidines 12a-c. The latter was also
considered as key intermediate to incorporate other biologically active ring systems fused or
attached to pyrimidine core.
The integrity for the structures of the newly synthesized compounds would be substantiated by
microanalyses, IR, 1H-NMR, 13C-NMR and MS data.
Furthermore, molecular modeling concept was adopted in an attempt to gain a better insight on
the molecular interactions. Molecular docking studies were designed to help in understanding the
mode of action of the active compounds through their interactions with the active sites of the
proposed enzymes.
2. Results and discussion
2.1. Chemistry
Synthesis of the intermediate and target compounds was performed according to the
reactions outlined in schemes 1-3. In scheme 1, the starting compound 6-Substituted-4-oxo-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles 1a-c were prepared by adopting a previous
reported literature procedure [29]. Alkylation of these pyrimidine thione derivatives 1a-c
following El-Meligie, S. et al. method[39], where an aqueous solution of the potassium salt of the
pyrimidine thione 1a-c was stirred at room temperature with an equimolar amount of
epichlorohydrin to yield the desired compounds 2a-c. IR spectra of compounds 2a-c showed the
characteristic absorption bands of the epoxy C–O–C function at 1239-1237, 1002-854, 772-755
cm–1 in addition to bands corresponding to C–S–C group at 1287-1281, 1078-1075 cm–1. 1H-NMR
spectra of compounds 2a-c revealed a doublet of doublet integrated for one proton at 3.10-3.47
ppm corresponding to cis hydrogen of epoxy CH2, a doublet of doublet integrated for one proton
at 3.40 – 3.52 ppm assigned for trans hydrogen of epoxy CH2, a multiplet at 3.80-4.48 ppm
assigned for CH epoxy proton, two doublet of doublets at 2.84-4.31 ppm corresponding to the S–
CH2 protons. Opening of oxirane ring was achieved by adopting El-Meligie, S. et al. procedure
[39], in which the epoxide derivative 2a,c was refluxed with morpholine in absolute ethanol to
produce 2-hydroxy-3-morpholinopropylthiopyrimidine derivative, 3a,b. IR spectra of
compounds 3a,b showed the characteristic absorption bands corresponding to OH and NH
functionalities at 3558-3413, 3456-3211, 3437-3140 cm–1 respectively. Besides, two bands at
1234-1180 and 1043-1030 cm-1 attributed to C–O–C function of the morpholine ring. They also
showed bands at 1147-1137 cm–1 corresponding to the aliphatic C–N function, 1109-1108 cm–1
1
corresponding to secondary alcoholic group. H-NMR spectrum of compound 3b showed a
multiplet at 2.99-3.01 ppm attributed to S-CH2 protons, two multiplets at 3.60-3.64 and 3.66-3.72
ppm corresponding to the morpholine C3,5, C2,6 protons respectively; a multiplet at 4.09-4.10 ppm
attributed to CH-CH2-N protons, two deuterium oxide exchangeable singlets at 6.78 and 8.09
ppm corresponding to NH and OH protons respectively; in addition to the signals due to furan
protons. Oxiran-2-ylmethylthio 2a was allowed to react with phosphorous oxychloride in order to
yield the corresponding chloro derivative. However, the reagent not only introduced the chlorine
atom in the 4-position of the reactant but also forced the oxirane ring to be opened, forming the
cyclic phosphochloridate ring, furnishing compound 4. IR spectrum of compound 4 showed the
absorption bands corresponding to P=O function at 1250 cm–1, C–Cl function at 766 cm–1 and P–
Cl function at 696 cm–1 [40]and lacked the absorption bands due to C=O and C–O–C functions.
1H-NMR spectrum of compound 4 revealed two multiplets at 1.06-1.10, 4.40-4.51 ppm attributed
to CH2O and CHO protons of cyclic phosphochloridate moiety respectively [41]. Chlorination of
pyrimidine thione derivatives 1a-c were achieve by refluxing the precursors 1a-c with excess
2

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phosphorous oxychloride followed by cooling. The cold solution was poured on crushed ice with
vigorous stirring, left in refrigerator overnight to obtain the chloro compounds 5a-c. IR spectra of
5a-c lacked the stretching absorption bands due to C=O group and displayed stretching absorption
1
bands due to C–Cl functionality at 873-856 cm–1. H-NMR spectrum of compound 5c showed
two doublets at 6.85 and 7.84 ppm, attributed to C4 and C3 protons of the furan ring respectively.
It also revealed a singlet at 8.17 ppm corresponding to furan C5 proton and a deuterium oxide
exchangeable singlet at 13.07 ppm integrated for one proton, attributed to NH function.
Replacement of the chloro function with secondary amines was reported to be achieved by heating
the chloro compound together with the amine with or without catalyst [42-44]. The use of excess
amine was also reported [45-47]. In the present work, the precursors 5a-c were fused with two
equivalents of the secondary amine on a sand bath at 160ºC, where the second mole of the amine
is supposed to act as a proton abstractor. The residue was triturated with 10% acetic acid to get rid
of the excess amine leaving the product in a pure form, which was then crystallized from the
appropriate solvent. IR spectra of compounds 6a-i revealed the absence of the absorption bands
due to C–Cl function and showed the absorption bands due to the secondary amine moieties at
1
their expected frequencies. H-NMR spectrum of compound 6a revealed the presence of three
triplets at 3.62, 3.67 and 3.78 ppm corresponding to C5, C3 and C2,6 protons of the morpholine
1
moiety. Furthermore, H-NMR spectrum of compound 6f showed four multiplets attributed to
the piperidine protons; at 1.45-1.51 ppm due to C4 protons, 1.53-1.63 ppm due to C3,5 protons,
1
2.60-2.62 ppm corresponding to C6 protons and 2.84-2.86 ppm due to C2 protons. H-NMR
spectrum of compound 6g revealed a multiplet at 3.70-3.90 ppm integrated for ten protons of the
piperazine and CH2 moieties and another multiplet at 7.20-7.80 ppm integrated for ten protons of
the two phenyl groups. Compounds 7a,b were prepared by stirring the pyrimidine thione
derivatives 1a,b with the hydrochloride salt of diethylaminoethyl chloride in aqueous potassium
hydroxide solution. However, alkylation using anhydrous potassium carbonate in dry
dimethylformamide was invalid. IR spectra of compounds 7a,b lacked the absorption bands due
to N–C=S functionality and displayed the stretching absorption bands due to C–S–C function at
1297, 1051-1046 cm–1and C-N aliphatic at 1206-1181 and 1108-1103 cm-1. 1H-NMR spectrum of
compound 7b showed two triplets at 0.87 and 0.92 ppm due to two methyl groups, a triplet at 1.15
ppm due to S–CH2 protons, a triplet at 2.33 ppm corresponding to N–CH2 protons and a quartet at
2.37 ppm due to the four protons of N(CH2–CH3)2 moiety. In addition to a deuterium oxide
exchangeable singlet at 4.07 ppm attributed to NH function. The scope of our investigation was
extended to record the effect of S-alkylation with substituted acetanilide derivatives 8a,b on the
biological activity of the synthesized compounds 9a-d. In scheme 2 compounds 9a-d were
achieved via the reaction of the pyrimidine-2-thione derivatives 1a,c with 4-substituted
chloroacetanilide derivatives 8a,b and anhydrous potassium carbonate in dry dimethylformamide.
It is to be noted down that 4-substituted chloroacetanilide derivatives 8a,b , in their turn, were
prepared by treating a solution of the appropriate aniline derivative in a mixture of
dimethylformamide and tetrahydrofuran with chloroacetyl chloride according to reported
procedure [48,49]. IR spectra of compounds 9a-d lacked the absorption bands characteristic for
N–C=S function and displayed the absorption bands characteristic for C–S–C function at 1296-
1293, 1096 cm–1. 1H-NMR spectrum of compound 9b showed two doublets at 2.70 and 2.85 ppm
each integrated for one proton assigned for S–CH2 protons, a singlet at 3.67 ppm due to OCH3
protons, two doublets at 6.84 and 7.42 ppm due to the p-methoxyphenyl C2,6 and C3,5 protons
respectively. In addition to two deuterium oxide exchangeable singlets at 4.12 and 10.23 ppm
1
attriubuted to pyrimidine NH and p-methoxyphenyl NH amide protons respectively. H-NMR
spectrum of compound 9c showed a singlet at 3.78 ppm assigned for the S–CH2 protons, two
doublets at 7.28 and 7.54 ppm attributed to the p-chlorophenyl C2,6 and C3,5 protons respectively.
It also revealed two deuterium oxide exchangeable singlets at 4.07 and 10.96 ppm corresponding
to the pyrimidine NH and p-chlorophenyl NH amide protons respectively. Methyl thio derivatives
10a-c were prepared by stirring the thione derivatives 1a-c with two equivalents of methyl iodide
in presence of anhydrous potassium carbonate in dry dimethylformamide according to our
previously reported method [29]. IR spectra of the title compounds 10a-c lacked bands due to
NH and N–C=S functions, whereas displayed absorption bands due to C–S–C function at 1283-
1
1228 and 1094-1088 cm–1. H-NMR spectrum of compound 10c showed a singlet at 2.65 ppm
3

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due to S-CH3 protons, while N-CH3 protons appeared at 3.38 ppm which overlapped with the
signals of the solvent. Replacement of S-methyl function with an amine moiety ,was achieved by
fusion of methylsulfide derivatives 10a-c with two equivalents of the selected amine at 170 ºC,
followed by treatment with ethanol to remove the excess amine leaving the desired compounds
11a-f, in a pure form which was then crystallized. IR spectra of compounds 11a-f lacked the
absorption bands due to C–S–C function and displayed the absorption bands characteristic for the
C–N aliphatic function. 1H-NMR spectrum of compound 11b revealed the presence of multiplets
at 2.32-2.33, 2.60-2.61 and 3.61-3.75 ppm due to morpholine protons, and a singlet at 4.05 ppm
attributed to N-CH3 protons. 1H-NMR spectrum of compound 11e showed a singlet at 2.88 ppm
due to N-CH3 protons, a multiplet at 3.40-3.50 ppm attributed to piperazine protons, a singlet at
3.64 ppm corresponding to N-CH2 protons and a multiplet at 7.30-7.44 ppm corresponding to
phenyl and benzyl C3,5 protons. Furthermore, two triplets at 7.50 and 7.54 ppm attributed to C4
protons of both benzyl and phenyl respectively and two doublets at 7.51 and 7.80 ppm due to C2,6
protons of benzyl and phenyl moieties respectively. 2-Hydrazino derivatives 12a-c were prepared
by hydrazinolysis of the 2-methylthio precursors 10a-c in ethanol according to reported conditions
[29]. IR spectra of compounds 12a-c lacked the absorption bands due to C–S–C function and
1
displayed the absorption bands corresponding to NH function at 3458-3114 cm–1. H-NMR
spectrum of compound 12c lacked the characteristic singlet of methyl sulfide protons while
revealed the presence of two deuterium oxide exchangeable singlets at 9.84 and 9.86 ppm each
integrated for half proton due to NH indicating amino-imino tautomerism. It also showed a
deuterium oxide exchangeable singlet at 10.33 ppm integrated for two protons corresponding to
NH2 function. Pyranopyrazole derivative 13a,b was obtained upon refluxing hydrazino derivatives
12a,c with excess diethyl malonate. IR spectra of compounds 13a,b revealed the presence of
absorption bands attributed to carbonyl functions at 1746-1744, 1662-1658 cm–1 and absorption
bands due to C–O–C function at 1284-1222, 1082-1037 cm–1. 1H-NMR spectrum of compound
13b showed a singlet at 3.43 ppm attributed to pyranopyrazole CH2 protons, a singlet at 3.46 ppm
due to N-methyl protons and a deuterium oxide exchangeable singlet at 10.42 ppm due to NH
function. The mass spectrum of compound 13b revealed a molecular ion peak m/z at 367[M+]
absent, with a base peak m/z at 158 and 157(100). In the present investigation, fusion of the
hydrazino compound 12a with two equivalents of ethyl cyanoacetate gave rise to the desired
cyanomethyltriazolo derivative 14, in addition to another product which was found to be ethyl 3-
iminopropanoate derivative 15. Formation of 15 can be postulated on the fact that one molecule of
ethyl cyanoacetate reacts with the substrate through its ester functionality, while the other
molecule reacts via its nitrile group. IR spectrum of compound 14 lacked the absorption bands
due to NH function and showed absorption bands corresponding to C ≡N, C=O, C=N and C=C
1
functions at their expected frequencies; H-NMR spectrum of compound 14 revealed the
presence of two doublets at 3.15 and 3.53 ppm attributed to the cyanomethyl protons, a singlet at
3.81 ppm due to N-methyl protons. The mass spectrum of compound 14 revealed a molecular ion
peak m/z at 291[M++1](6), with a base peak m/z at 69(100). IR spectrum of compound 15
showed the absorption bands due to NH function at 3336 cm–1, C ≡N at 2224 cm–1 and an ester
carbonyl function at 1727 cm–1. It also showed C–O–C at 1292, 1017 cm–1. On the other hand, 1H-
NMR spectrum of compound 15 displayed a triplet at 1.18 ppm and a quartet at 4.11 ppm due to
the ethyl ester protons. It also revealed two singlets at 3.37 and 3.56 ppm corresponding to
CH2CN and CH2CO protons respectively. The mass spectrum of compound 15 revealed a
molecular ion peak m/z at 421[M+] absent, with a base peak m/z at 198(100). Scheme 3 shows the
preparation of dimorpholino derivatives 18a,b. This was achieved through two different
precursors. In the first method, starting with 2-(oxiran-2-yl)methylthio-4-chloropyrimidine
derivatives 16a,b, which were prepared by treating a solution of chloropyrimidine thione
derivative 5a,c in DMF with two equivalents of aqueous sodium hydroxide solution followed by
dropwise addition of an equivalent amount of epichlorohydrin. IR spectra of compounds 16a,b
lacked the absorption bands corresponding to NH and N–C=S functions, while revealed the
absorption bands corresponding to C–S–C function at 1266-1255, 1085-1081 cm–1, epoxy C–O–C
function at 1266-1255, 1036-1030, 779-760 cm–1 and absorption band due to C–Cl function at
885-873 cm–1. In addition to absorption bands due to CH2, C ≡N, C=N and C=C at their expected
frequencies.1H-NMR spectrum of compound 16a showed two multiplets at 2.30-2.34 and 2.59-
4

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2.63 ppm attributed to S-CH2 protons. It also revealed the oxirane protons as two multiplets at
2.68-2.71 and 4.02-4.10 ppm corresponding to methine and methylene protons respectively.
16a,b, were separately refluxed with four equivalents of morpholine in dry dioxane. It is assumed
that one mole of morpholine is used to open the oxirane ring system and the other replaces the 4-
chloro functionality. While the other two moles are supposed to act as catalysts, facilitating the
reaction. The second method, involved refluxing 2-(oxiran-2-yl)methylthio-4-morpholino-
pyrimidine derivatives 17a,b with two equivalents of morpholine in dry dioxane. 17a,b were
fruitfully achieved by treating 4-morpholinopyrimidine thione derivatives 6a,c with
epichlorohydrin in alkaline medium. IR spectra of compounds 17a,b lacked the absorption bands
due to N–C=S function, while displayed absorption bands of C–S–C function at 1285-1257, 1069-
1067 cm–1 and epoxy C–O–C function at 1255-1229, 1028-1022, 783-772 cm–1. It also showed
absorption bands due to C–N aliphatic function of morpholine at 1209-1206, 1115-1114 cm–1. 1H-
NMR spectrum of compound 17a revealed the presence of signals corresponding to epoxypropyl
side chain as two multiplets, at 2.59-2.60 ppm integrated for two protons due to OCH2 moiety, and
at 4.03-4.06 ppm integrated for one proton attributed to OCH proton. Signals corresponding to S–
CH2 protons were overlapped with the solvent signals. Compounds 18a,b prepared by the
aforementioned two methods were found to be identical, as revealed by TLC, m.p. and mixed m.p.
IR spectra of compounds 18a,b lacked absorption bands due to C–Cl and epoxy C–O–C
1
functionalities, while revealed a broad band at 3438-3407 cm–1 due to alcoholic function.. H-
NMR spectrum of compound 18a revealed signals corresponding to the two morpholine moieties
interpreted as two triplets at 4.47 and 4.50 ppm corresponding to C3,5 protons of S-
propylmorpholine and C3,5 protons of 4-morpholino moieties respectively. A multiplet integrated
for eight protons at 4.55-4.66 ppm attributed to C2,6 protons of both morpholine moieties. It also
showed a deuterium oxide exchangeable singlet at 8.75 ppm due to alcoholic proton.
2.2. Biological evaluation
2.2.1. In-vitro anticancer screening:
In the current protocol, 27 compounds were submitted to the NCI and were tested initially at a
single high dose (10 µM) in the full NCI 60 cell panel. Results of the tested compounds were
reported as a mean graph of the percent growth of treated cells, and presented as percentage
growth inhibition caused by the test compounds (table 1). 4-Oxo-2-thioxopyrimidine derivative
1c showed promising anticancer activity against many cell lines. Replacing the 4-oxo moiety with
4-morpholino group (6a, 6c, 17a & 18a) resulted in marked increase in activity against several
cell lines. Moreover, replacement of 4-oxo moiety with 1-piperidinyl group in compound 6d
resulted in slight increase in activity. On the other hand, replacement of 4-oxo with the bulky N-
benzylpiperazine moiety 6g, 6h and 6i diminished the anticancer activity. Furthermore, alkylation
of the 2-thio group with an ethyl diethylamino function (7a), p-chloroacetanilides (9c & 9d),
methyl group (10c), oxiranylmethyl group (17a) and hydroxypropylmorpholino moiety (18a)
showed increase in activity against several cell lines. Replacement of 2-thioxo moiety with
different amines did not increase the activity as much, this is contradictory to the marked increase
in activity in case of 4-amino substitution, where compounds 11b, 11c and 11d showed good
activity against two Melanoma cell lines. Moreover, 2-hydrazino compound 12c showed good
activity against Colon cancer HCC-2998. Compound 17a satisfied the threshold inhibition criteria
and passed forwards for evaluation in the full panel 5-dose in-vitro anti-tumor screen. The three
response parameters GI50, TGI and LC50 values in µM/l of compound 17a in the full panel, 5-dose
assay are presented in table 2. The GI50, TGI and LC50 values revealed that compound 17a
exhibited potential growth inhibition activity against most of the tested subpanel tumor cell lines.
It showed promising activity toward several cell lines of Non-Small Cell Lung cancer especially
EKVX, HOP-62, HOP-92, NCI-H23, and NCI-H322M. Concerning Colon cancer, it was most
active against HCC-2998 and HCT-15 cell lines. It also showed promising activity toward CNS
cancer SF-295 and SNB-75 cell lines. Moreover, it exhibited good activity against all Melanoma
cell lines. However, it was most active against three Ovarian cancer cell lines; namely IGROV1,
OVCAR-3 and OVCAR-5. It was most active against Renal cancer especially 786-0, A498 and
CAKI-1 cell lines. Finally, it was highly active against all Breast cancer cell lines especially
MCF7, HS 578T, BT-549 and T-47D cell lines. However, the compound was non-selective
5

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toward almost all types of cancer but it exhibited moderate selectivity towards Renal cancer with
selectivity ratio 3.22 (table 3).
2.2.2. Antimicrobial screening:
Antimicrobial activities of the newly synthesized compounds were tested against
Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri and Candida albican. using
the cup diffusion technique [50]. Further evaluation was then carried out on compounds showing
reasonable inhibition zones to determine their minimal inhibitory concentration (MIC) values
using the two fold serial dilution method [51]. The results illustrated in tables 4 and 5 revealed
that from the tested compounds, non had superior antibacterial activity than the standard
antibiotics. Moreover, compounds 2c, 6i, 7a, 9c, 15, 18a and 18b displayed antifungal activity
against C.albicans. However, most of them proved to possess nearly 50% of the activity of
Clotrimazole, while compounds 6i and 9c were found to exhibit 75% of its activity. In a study of
SAR, the 4-oxo-2-thioxopyrimidine derivative 1c showed nearly 60% activity as that of
Ofloxacin against the gram negative Sh.flexneri. The oxiranylmethylthio derivatives 2a-c
enhanced the activity against Sh.flexneri. However, the 4-(2-furyl) derivative 2c exhibited
additional activity toward S.aureus, P.auregionsa as well as C.albicans. On the other hand, ring
opening of the oxiranylmethylthio derivatives to yield compounds 3a,b or the treatment of 2a with
phosphorus oxychloride to yield the phosphochloridate derivative 4 diminished the antimicrobial
activity. 4-Chloropyrimidines 5a,c elicited activity against Gram negative bacteria, while their 4-
substituted aminopyrimidines 6a-i showed promising activity of which compounds 6c, 6d, 6e and
6g were the most active; nearly 60% that of Ofloxacin. Furthermore, compound 6i exhibited both
antibacterial and antifungal activities. Moreover, alkylation of the 2-thioxo function with
diethylaminoethyl group, compound 7a showed promising broad spectrum activity against the test
organisms. The N-substituted-2-substituted thioacetamido derivatives 9a-d showed variable
antimicrobial activities depending on the substitution on the p-substitution on the phenyl ring. The
4-OCH3 derivatives 9a and 9b showed 60% activity that of Ofloxacin against Sh.flexneri while the
4-chloro derivatives 9c and 9d were equipotent with Ampicillin and half the activity of Ofloxacin
against S.aureus and P.aureginosa respectively. In addition, compound 9c showed 75% activity
that of Clotrimazole against the fungus C.albicans. Methylthio derivative 10c exerted 50% the
activity of Ampicillin against S.aureus. Replacement of S-alkyl by amino functionality; 11a-f did
not improve the activity. However, only compounds 11c and 11e showed activity against
Sh.flexneri and P.aeruginosa. Moreover, the hydrazino compound 12c exhibited antimicrobial
activity against Sh.flexneri its potency reached 60% that of Ofloxacin. While introduction of
pyranopyrazole moiety at the 2-position of the pyrimidine ring did not enhance the antimicrobial
activity where only 13a showed activity against P. auerginosa. Cyclization of
hydrazinopyrimidine derivative 12a into the corresponding cyanomethyltriazolopyrimidine 14,
did not improve the activity, while its open chain counter-part 15 exhibited activity reaching to
60% that of Clotrimazole against C.albicans. Introduction of an oxiranylmethylthio side chain to
either chloropyrimidines 5a,c or 4-morpholinopyrimidines 6a,c furnishing compounds 16a,b and
17a,b respectively, resulted in diminished activity. Nevertheless, compound 16a retained the
activity of its precursor against Sh.flexneri. On the other hand, dimorpholinopyrimidine
derivatives 18a,b elicited activity about 60% that of Clotrimazole against C.albicans.
Computer aided drug design:
Five representative active anticancer compounds 6a, 6c, 6d, 17a and 18a were subjected to
docking using Molecular Operating Environment (MOE) program [52] on the 3D structure of two
enzymes, namely; thymidylate synthase (TS)(1BID) and dihydrofolate reductase (DHFR). The
study also shows the enzymes interactions with their substrates; 2'-deoxyuridine-5'-
monophosphate (DUMP ) and methotrexate(MTX), respectively.
Docking on the active site of thymidylate synthase:
MOE docking studies of the inhibitors was performed using thymidylate synthase co-crystallized
with the substrate DUMP (PDB ID: 1BID) as a template.
Docking of DUMP into TS active site revealed that several interactions were considered to be
responsible for the observed affinity of the compound. As it acts as a hydrogen bond acceptor
with the backbone Asp 169 residue and the side chain residues; Arg 166, Ser 167, Asn 177, His
207 and Tyr 209 as well as hydrogen bond donor with the side chain residues; Asn 177, His 207
6

ACCEPTED MANUSCRIPT
and Tyr 209. In addition to hydrophobic interactions with: Arg 21, Cys 146, His 147, Gln 165,
Arg 166, Ser 167, Cys 168, Asp 169, Gly 173, Asn 177, His 207 and Tyr 209 as shown in fig. (1).
Docking of compound 6a into TS active site revealed that several molecular interactions were
considered to be responsible for the observed affinity, as the N of the cyano function acts as a
hydrogen bond acceptor with the side chain residues; His 207 (3.47Aº) and Tyr 209 (3.20Aº) with
a strength of 11.2% and 17.2%; respectively. In addition to many hydrophobic interactions
between the phenyl moiety as well as the morpholine group and the sulphur atom with the
following residues; Asp 20, Arg 21, Leu 143, Ala 144, Cys 146, Arg 166, Ser 167, His 207 and
Tyr 209 as shown in fig. (2).
Docking of compound 6c into TS active site revealed the presence of hydrogen bond interaction
between the N of the cyano group of the compound as it acts as hydrogen bond acceptor with the
side chain residue Tyr 209 (3.22Aº) with a strength of 16.5%, in addition to hydrophobic
interactions between the furan ring, morpholine moiety and sulphur atom with the following
amino acid residues: Asp 20, Arg 21, Trp 83, Leu 143, Ala 144, Cys 146, Arg 166 and Try 209 as
shown in fig. (3).
Docking of compound 6d into TS active site illustrated the interaction of the NH group of the
compound as a hydrogen bond donor with the side chain residue Glu 58
(2.77 Aº) at a strength
of 3% while the pyrimidine N-3 acted as a hydrogen bond acceptor with the side chain residue Tyr
94 (3.34Aº) at a strength of 6.8%. This beside many hydrophobic interactions concerning the
piperidine moiety, phenyl ring and cyano function with the following amino acid residues: Glu58,
Phe 62, Ile 79, Trp 80, Tyr 94, Leu 163, Ala 144, Cys 146, His 147 and Arg 166 as shown in fig.
(4).
Docking of compound 17a into TS active site revealed the presence of hydrogen bond interaction
between the N-1 of pyrimidine, as it acts as a hydrogen bond acceptor, and the side chain Asn 177
residue (2.92Aº) with a strength of 23.6%. There is also hydrophobic interactions between the
morpholine moiety, CN function and benzene ring with many amino acid residues: Ser 54, Ile 55,
Glu 58, Ile 79, Cys 146, Gln 165, Ser 167, Cys 168, Asp 169, Leu 172, Gly 173, Phe 176 and Asn
177as shown in fig. (5).
Docking of compound 18a into TS active site revealed the presence of several molecular
interactions which were considered to be responsible for the observed affinity of the compound;
the hydrogen atom of the hydroxyl group acts as a hydrogen bond donor to the side chain residue
Glu 58 (3.64 Aº) with a strength of 23.7%, also the oxygen atom of the hydroxyl group acts as a
hydrogen bond donor and acceptor with His 147 residue (3.16Aº) with a strength 17.1%.
Moreover the oxygen atom of morpholine moiety and N-1 of pyrimidine both acts as hydrogen
bond acceptor to Trp 83 and Asn 177 residues at a strength of 13.9% and 11.3%; respectively. In
addition to many hydrophobic interactions between pyrimidine C4-morpholine, cyano function,
phenyl group and the following amino acid residues: Glu 58, Ile 79, Trp 80, Trp 83, Cys 146, His
147, Gln 165, Ser 167, Cys 168, Asp 169, Leu 172, Gly 173, Phe 176 and Asn 177 as shown in
fig. (6).
Docking on the active site of dihydrofolate reductase (DHFR):
Docking simulation study of the synthesized compounds 6a, 6c, 6d, 17a and 18a
MOE docking studies of the inhibitors were performed using dihydrofolate reductase co-
crystallized with methotrexate (PDB ID: 4DFR) as a template.
Docking of MTX into DHFR active site revealed that hydrogen bond interactions beside
hydrophobic interactions were considered to be responsible for the observed affinity as it acts as a
hydrogen bond donor to the backbone Ile 5 and Ile 94 residues and the side chain Asp 27 residue.
It also acts as a hydrogen bond accepter to Arg 52 and Arg 57 residues. This beside many
hydrophobic interactions with various amino acid residues: ILe 5, Ala 6, Ala7, Asp 27, Leu 28,
Phe 31, Lys 32, Ser 49, Ile 50, Arg 52, Leu 54, Arg 57, Ile 94, Tyr 100 and Thr 113 as shown in
fig. (7).
Docking of compound 6a into DHFR active site revealed the presence of hydrogen bond
interaction between the N of the cyano function as it acts as hydrogen bond acceptor and the
backbone Ala 7 residue (2.66Aº) with a strength of 98.6% in addition to many hydrophobic
interactions among morpholine moiety and the sulphur atom and the following amino acid
7

ACCEPTED MANUSCRIPT
residues: Ile 5, Ala 6, Ala 7, Ile 14, Gly 15, Asp 27, Phe 31, Thr 46, Ile 50, Ile 94 and Tyr 100 as
shown in fig. (8).
Docking of compound 6c into DHFR active site revealed the presence of strong hydrogen bond
interactions with the N of the cyano moiety as it acts as a hydrogen bond acceptor with the
backbone Ala 7 residue (2.68Aº) at a strength of 90.5% as well as with the side chain Tyr 100
residue (3.10 Aº) with a strength of 20.4%. Besides, many hydrophobic interactions between the
morpholine moiety, thioxo function and the pyrimidine ring with: Ile 5, Ala 6, Ala 7, Ile 14, Gly
15, Asp 27, Phe 31, Thr 46, Gly 96 and Tyr 100 as shown in fig. (9).
Docking of compound 6d into DHFR active site revealed the presence of hydrogen bond
interaction between the N of cyano function and side chain Tyr 100 residue (3.45 Aº) as it acts as
a hydrogen bond acceptor with a strength of 7.1%. It also showed several hydrophobic
interactions between the thioxo function, phenyl ring and pyrimidine carbons with: Ile 5, Ala 6,
Ala7, Gly15, Met 16, Glu 17, Asn 18, Met 20, Trp 22, Asp 27, Phe 31, Ile 94 and Tyr 100 as show
in fig. (10).
Docking of compound 17a into DHFR active site illustrated the presence of strong hydrogen
bond interactions between N of cyano group as it acts as a hydrogen bond acceptor with the
backbone Ala 7 residue (2.72Aº) with a strength of 70.5%. In addition to the oxygen of the
morpholinyl group which acts as a hydrogen bond acceptor with the side chain Thr 46 residue
(2.70Aº) at a strength of 45.4%. Besides, the hydrophobic interactions among the morpholine ring,
oxiranylmethylthio function as well as the pyrimidine carbons and several amino acid residues
namely; Ile 5, Ala 6, Ala7, Asp 27, Trp 30, Phe 31,
Thr 46, Ile 50, Leu 54, Ile 94, Gly 95, Gly
96 and Tyr 100 as shown in fig. (11).
Docking of compound 18a into DHFR active site revealed the presence of strong hydrogen bond
interaction between the N of the cyano moiety, as it act as a hydrogen bond acceptor, and the
backbone Ala 7 residue (2.84Aº) with a strength of 77.9%. This besides, many hydrophobic
interactions involving the S-propylmorpholine moiety, the 4-morpholino group, the phenyl ring as
well as the cyano carbon and the following amino acid residues: Ala 7, Ile 14, Gly 15, Asp 27,
Leu 28, Phe 31, Thr 46, Ser 49, Ile 50, Gly 96, Gly 97 and Tyr 100 fig.(12).
2.
Conclusion
The design and synthesis of novel pyrimidine derivatives bearing various substituents or
heterocyclic moieties joined to the pyrimidine ring system. Some selected members of the newly
synthesized compounds were investigated for their cytotoxic potency against certain human tumor
cell lines as well as their antimicrobial activities. Compound 17a satisfied the threshold inhibition
criteria and passed forwards for evaluation in the full panel, 5-dose in-vitro anti-tumor screen.
Although most compounds were either devoid or of weak antimicrobial activity, compounds 2c
and 7a were found to exhibit broad spectrum activity. Their potency against S.aureus was nearly
half or equal to Ampicillin respectively, about 50% as potent as Ofloxacin against P.aeruginosa
and 60% as potent against Sh.flexneri, beside exhibiting 62% the activity of Clotrimazole against
C.albicans. Docking was performed for the five most active anticancer compounds 6a, 6c, 6d, 17a
and 18a on the two enzymes; thymidylate synthase and dihydrofolate reductase in a trial to predict
their mode of action as anticancer agents. Compounds 17a and 18a exhibited strong interactions
with dihydrofolate reductase enzyme.
4. Experimental
4.1. Chemistry
Melting points were determined in open glass capillaries on a Griffin-Kamp melting point
apparatus and are uncorrected. IR spectra were recorded, for potassium bromide discs, on a
1
Perkin-Elmer 1430 Infrared spectrophotometer. H-NMR spectra were determined either on Joel
spectrometer (500 MHz) at the central laboratory, Faculty of science, Alexandria University, or on
Varian Mercury VX-300 NMR spectrometer, Faculty of science, Cairo University. Data are
reported as δ values (ppm) relative to trimethylsilane(TMS) as an internal standard. The type of
signal is indicated by one of the following letters: s = singlet,d = doublet, dd = doublet of doublet,
t = triplet, q = quartet, m = multiplet. 13C-NMR spectrum was recorded on Joel spectrometer at
the central laboratory, Faculty of science, Alexandria University . Mass spectra were carried out
8

ACCEPTED MANUSCRIPT
using either a Schimadzu GCMS-QP-1000EX mass spectrometer at 70 ev or HP5000988A mass
spectrometer at Faculty of science, Cairo University. Elemental analyses were performed on
Elementar Vario El III CHN analyzer (Germany) at the microanalytical unit, Faculty of science,
Cairo University. Phosphorous was detected by using oxygen fust technique followed by
measuring on Nanocolor 300D spectrophotometer at Faculty of science, Cairo University.
Reactions were monitored by thin-layer chromatography (TLC) on silica gel (60 GF254, Merck),
using glass plates. The spots were visualized by exposure to iodine vapour or UV-lamp at λ 254
nm for few seconds.
4.1.1. 6-Substituted-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5- carbonitriles (1a-c).
A mixture of thiourea (1.5 g, 20 mmole), ethyl cyanoacetate (2.6 g, 2.1 ml, 20 mmole),
appropriate aldehyde (20 mmole) and K2CO3 (2.8 g, 20 mmole) in EtOH 95% (50 ml) was
refluxed for 12h, then cooled. The obtained precipitate was filtered and washed with EtOH. The
product was then dissolved in hot H2O, filtered while hot and neutralized with gl.HOAc; where
upon the desired thione was obtained. It was filtered, washed with H2O, dried and crystallized
from DMF/H2O.
4.1.1.1. 4-Oxo-6-phenyl 2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1a)
C11H7N3OS (229.26); yield: 90%; mp: 290-291 ºC [26].
4.1.1.2. 6-(4-Nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5- carbonitrile (1b)
C11H6N4O3S (274.26); yield 76%; mp: 232-234 ºC [26].
4.1.1.3. 6-(2-Furyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1c)
C9H5N3O2S (219.22) ; yield 70%; mp: > 300 ºC. IR (KBr, cm–1): 3121 (NH); 3087 (CH furan);
≡
2232 (C N); 1674, 1661 (C=O); 1589, 1469 (C=N, δNH, C=C–Ar, C=C- furan); 1550, 1272,
1148, 1086 (N-C=S); 1410, 1326 (C–N lactam); 1224, 1039 (C–O–C); 767 (oop furan). 1H-NMR
of compound 1c (DMSO-d6) δppm: 6.94 (dd, 1H, J = 3.8, 1.9Hz, furan C4-H); 7.91 (d, 1H, J =
3.8Hz, furan C3-H); 8.25 (s, 1H, furan C5-H); 13.03 (s, 2H, 2NH, D2O exchangeable). Anal.
Calcd for C9H5N3O2S (219.22): C, 49.31; H, 2.30. Found C, 50.24; H, 2.8.
4.1.2. 4-Substituted-2-(oxiran-2-ylmethylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitriles
(2a-c).
The appropriate thione 1a-c (2 mmole) was suspended in H2O (30 ml) then treated with a solution
of KOH (0.22 g, 4 mmole) in 5 ml H2O. The suspension was stirred at R.T. for 30 min., where a
clear solution was obtained. Epichlorohydrin (0.19 g, 0.16 ml , 2 mmole) was added and stirring
was continued at R.T. for 6h, during which a precipitate was formed. It was filtered, washed with
H2O, dried and crystallized from the appropriate solvent
4.1.2.1 2-(Oxiran-2-ylmethylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile (2a).
Crystallized from DMF/H2O; yield: 76%; mp: 183ºC. IR (KBr, cm–1): 3412, 3264 (NH); 2224
≡
(C N); 1726, 1661 (C=O); 1599, 1516, 1500, 1489 (C=N, C=C), 1550 (δ NH); 1439, 1315 (C–N
lactam); 1239, 1002, 770 (C–O–C epoxy); 1281, 1076 (C–S–C). 1H-NMR of (DMSO-d6) δppm:
3.47 (dd, 1H, J = 9.9, 4.2Hz, epoxy CH2 cis hydrogen); 3.52 (dd, 1H, J = 9.9, 3.05Hz, epoxy CH2
trans hydrogen); .80-3.82 (m, 1H, epoxy CH); 4.06 (2 overlapping dd, 2H, J = 14.15, 6.45Hz, S–
CH2);7.45 (t, 1H, J = 6.9Hz, phenyl C4-H);7.64( dd, 1H, J = 11.5, 7.65Hz, phenylC3-H);7.74 (dd,
1H, J = 12.2, 6.9Hz, phenyl C5-H);7.88 (d, 1H, J = 7.65 Hz, phenyl C2-H);8.27 (s, 1H, NH, D2O
exchangeable). Anal. Calcd for C14H11N3O2S. H2O(303.34);C, 55.44; H, 4.32; N,13.85. Found C,
55.21; H, 4.45; N,13.36.
4.1.2.2.4-Nitrophenyl-2-(oxiran-2-ylmethylthio)-6-oxo-1,6-dihydropyrimidine-5carbonitrile(2b).
–1
≡
Crystallized from DMF/H2O ; yield: 71%; mp: 150ºC. IR (KBr, cm ): 3374 (NH); 2204 (C N);
1713, 1660 (C=O); 1591 (C=N, C=C); 1524, 1348 (NO2); 1239, 854, 755 (C–O–C epoxy); 1286,
1075 (C–S–C). H-NMR (DMSO-d6) δppm: 2.68-2.71 (m, 2H, OCH2 epoxy); 2.84-2.87 (m, 2H,
S–CH2);3.80-4.00 (m, 1H, CH epoxy); 6.69 (s, 1H, NH, D2O exchangeable); 7.55-8.50 (m, 4H, 4-
NO2-C6H4). Anal. Calcd for C14H10N4O4S(330.32); C, 50.91; H, 3.05. Found C, 50.98; H, 2.85.
4.1.2.3.4-(2-Fury)l-2-(oxiran-2-ylmethylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitriles (2c).
Crystallized from DMF/EtOH ; yield: 37%; mp: 190ºC. IR (KBr, cm–1): 3389 (NH); 3137 (CH
≡
furan); 2221 (C N); 1726, 1662 (C=O); 1586, 1518, 1500 (C=N, C=C); 1550 (δ NH); 1312 (C–
N lactam); 1237, 989, 772 (C–O–C epoxy); 1287, 1078 (C–S–C); 1177, 1022 (C–O–C furan); 772
1
(oop furan). H-NMR (DMSO-d6) δppm: 3.10 (dd, 1H, J = 12.25, 3.83Hz, epoxy CH2 cis
9

ACCEPTED MANUSCRIPT
hydrogen); 3.40 (dd, 1H, J = 12.2, 2.25 Hz, epoxy CH2 trans hydrogen); 4.28 (dd, 1H, J =14.15,
4.97 Hz, S–CH2);4.31 (dd, 1H, J = 14.15, 4.97Hz, S–CH2);4.44-4.48 (m, 1H, epoxy CH); 5.76 (s,
1H, NH, D2O exchangeable); 6.75 (dd, 1H, J = 3.8, 1.9Hz, furan C4–H); 7.42 (d, 1H, J = 3.8Hz,
furan C3–H); 7.99 (s, 1H, furan C5–H). Anal. Calcd for C12H9N3O3S(276.29); C, 52.36; H, 3.30;
N,15.26 Found C, 52.35; H, 2.95; N,15.60.
4.1.3. 4-Substituted-2-(2-hydroxy-3-morpholinopropylthio)-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitriles (3a,b).
The epoxide derivative 2a,c (2 mmole) was suspended in absolute EtOH, whereafter morpholine
(0.35 g, 0.35 ml, 4 mmole) was added. The reaction mixture was refluxed for 72h, allowed to
cool, and filtered. The obtained product was triturated with pet. ether 40-60 to yield pale yellow
solid which was crystallized from DMF/H2O.
4.1.3.1. 2-(2-Hydroxy-3-morpholinopropylthio)-6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-
carbonitrile (3a).
–1
≡
Yield: 23%; mp: 210ºC. IR (KBr, cm ): 3558, 3456, 3437 (NH, OH); 2205 (C N); 1716, 1659
(C=O); 1586, 1501 (C=N, δNH, C=C); 1310 (C–N lactam); 1245, 1073 (C–S–C); 1180, 1043 (C–
O–C); 1137 (C–N aliphatic); 1108 (2º OH). Anal. Calcd for C18H20N4O3S. 11/2H2O (399.46) ;C,
54.12; H, 5.80; Found: C, 54.56; H, 5.35.
4.1.3.2. 2-(2-Hydroxy-3-morpholinopropylthio)-4-(2-furyl)-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitrile (3b).
Yield: 41%; mp: 250ºC. IR (KBr, cm–1): 3413, 3211, 3140 (OH, NH); 3074 (CH furan); 2206
≡
(C N); 1690, 1645 (C=O); 1587, 1515, 1485 (C=N, δNH, C=C); 1305 (C–N lactam); 1256,
1030 (C–S–C); 1234,1030 (C–O–C); 1147 (C–N aliphatic); 1109 (2º OH); 761 (oop furan). H-
1
NMR (DMSO-d6) δppm: 2.99 – 3.01(m, 2H, S–CH2, under DMSO); 3.60-3.64 (m, 4H,
morpholine C3, 5–H);3.66-3.72 (m, 4H, morpholine C2, 6–H); 4.09–4.10 (m, 3H, CH, CH2-N);6.56-
6.69 (m, 1H, furan C4-H);6.78 (br.s, 1H, NH, D2O exchangeable)7.21 (d, 1H, J = 3.05Hz, furan
C3-H);7.84 (s, 1H, furan C5-H);8.09 (s, 1H, OH, D2O exchangeable). Anal. Calcd for
C16H18N4O4S. 11/2H2O( 371.41);C, 51.17;H,5.16;Found: C,52.15; H,5.20.
4.1.4. 4-Chloro-2-[(2-chloro-2-oxo-1,3,2-dioxaphospholan-4-yl)thio]-6-phenyl-pyrimidine-5-
carbonitrile(4)
A mixture of epichlorohydrin derivative 2a (0.61 g, 2 mmole) and POCl3 (10 ml) was heated
under reflux in an oil bath for 12 h. The reaction mixture was left to cool; poured onto crushed ice
and kept in refrigerator overnight. The obtained product was filtered washed with H2O, left to dry
and crystallized from EtOH/H2O.
–1
≡
Yield: 56%; mp: 120ºC; IR (KBr, cm ): 2227 (C N); 1605, 1544, 1505 (C=N, C=C); 1250
(P=O); 1250, 1074 (C–S–C); 1027 (C–O–P); 766 (C-Cl); 696 (P-Cl). 1H-NMR (DMSO-d6) δppm:
1.06-1.10 (m, 2H, CH2O); 3.91-4.30 (m, 2H, S–CH2); 4.40 – 4.51 (m, 1H, CHO); 7.53-7.74 (m,
3H, Phenyl C3, 5-H); 7.84-7.92 (m, 2H, phenyl C2, 6-H). Anal. Calcd for C14H10Cl2N3O3PS
4,
(402.19): N,10.45;P,7.70; Found: 10.54; 7.22. EI-Mass spectrum of compound 4 m/z (relative
abundance %):401(M+.) (44); 400 (51); 326 (39); 325 (44); 324 (54); 303 (17); 302 (61); 289 (59);
288 (100); 270 (32); 269 (22); 268 (81); 255 (22); 252 (56); 209 (37); 200 (37); 186(24); 185
(66); 184 (37); 183(73); 182 (49); 180 (51); 141 (83); 140 (56); 127 (37); 116 (49); 115 (46); 114
(61); 113 (32); 111 (44); 110 (20); 109 (22); 105 (37); 104 (49); 103 (61); 101 (51); 100 (27); 99
(44); 95 (34); 89 (34); 88(27); 87 (49); 86 (32); 82 (76); 77 (88); 76 (95); 75 (63); 74 (27); 73
(46); 72 (56); 70 (27); 63 (54); 61(42); 60 (51); 59 (81); 58 (34); 56 (15); 54 (49); 52 (27); 51(49);
50 (90).
4.1.5. 6-Substituted-4-chloro-2-thioxo-1,2-dihydropyrimidine-5-carbonitriles (5a-c)
A mixture of the appropriate thione derivative 1a-c (2 mmole) and POCl3 (5 ml) was heated under
reflux in an oil bath for 12 h. The reaction mixture was left to cool and poured onto crushed ice. It
was then left in refrigerator overnight. The obtained product was filtered, washed with H2O and
left to dry.
4.1.5.1. 4-Chloro-6-phenyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile(5a)
–1
≡
Yield: 84%; mp: 120ºC; IR (KBr, cm ): 3165 (NH); 2230 (C N); 1675, 1644 (C=N); 1591,
1505 (C=C); 1534, 1267, 1143, 1000 (δ-NH, N-C=S); 1445, 1336 (C–N thiolactam); 873 (C-Cl).
Anal. Calcd for C11H6ClN3S.(247.03) (402.19):N,16.97; Found: 16.67.
4.1.5.2. 4-Chloro- 6-(4-nitrophenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (5b)
10

ACCEPTED MANUSCRIPT
Yield: 87%; mp: 300ºC; IR (KBr, cm–1):
N); 1677, 1660 (C=N); 1629, 1598(C=C); 1525 (δNH); 1525, 1348 (NO2);
≡
3384, 3109 (NH); 2224 (C
1525, 1260, 1145, 1012 (N–C=S); 1413, 1348 (C–N thiolactam); 856 (C–Cl).
Anal. Calcd for C11H5ClN4O2S.H2O(310.72):C,42.52; H,2.27; Found: 43.11;3.33.
4.1.5.3. 4-Chloro-6-(2-furyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (5c)
–1
≡
Yield: 95%; mp: 160ºC; IR (KBr, cm ): 3136 (NH); 2921 (CH furan); 2230 (C N); 1662, 1645
(C=N); 1579, 1489 (C=C); 1533, 1265, 1146, 1016 (δNH,
N–C=S); 1405, 1325 (C–N
thiolactam); 1225, 1067 (C–O–C); 866 (C-Cl); 778 (oop furan).1H-NMR (DMSO-d6) δppm: 6.85
(d, 1H, J = 3.05 Hz, furan C4–H); 7.84 (d, 1H, J = 3.85 Hz, furan C3–H); 8.17 (s, 1H, furan C5–H);
13.07 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C9H4ClN3OS.H2O: (255.68)N,16.43;
Found: 16.71.
4.1.6. 6-Substituted-4-substitutedamino-2-thioxo-1,2-dihydropyrimidine-5-carbonitriles
(6a-i).
The appropriate chloropyrimidine derivative 5a-c (2 mmole) together with the appropriate
secondary amine (4 mmole) were fused on a sand bath at 160-170ºC for 6h. The reaction mixture
was left to cool then triturated with 10% acetic acid. The obtained product was filtered, washed
with EtOH, left to dry and crystallized from the appropriate solvent.
4.1.6.1. 4-Morpholinyl-6-phenyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6a).
Crystallized from EtOH ; Yield: 80%; mp: 190ºC; IR (KBr, cm ): 3189 (NH); 2205 (C N);
–1
≡
1660, 1644 (C=N); 1607, 1518, 1491 (C=C); 1550 (δNH); 1585, 1284, 1117, 1003 (N-C=S); 1307
(C-N thiolactam); 1206 (C-N aliphatic); 1256, 1069 (C–O–C).1H-NMR (DMSO-d6) δppm: 3.62 (t,
2H, J = 4.6Hz, morpholine C5–H); 3.67 (t, 2H, J = 4.6Hz, morpholine C3–H); 3.78 (t, 4H, J =
4.6Hz, morpholine C2, 6–H); 7.47 (t, 2H, J = 7.65Hz, phenyl C3, 5–H); 7.52 (t, 1H, J = 7.65Hz,
phenyl C4–H); 7.77 (d, 2H, J = 7.65Hz, phenyl C2, 6–H).. Anal. Calcd for C15H14N4OS(298.36);
N,18.78; found,18.58.
4.1.6.2. 4-Morpholinyl-6-(4-nitrophenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6b).
Crystallized from DMF/EtOH; Yield: 43%; mp: above 300 ºC; IR (KBr, cm–1): 3374 (NH); 2201
≡
(C N); 1643 (C=N); 1601, 1517 (C=C) 1551, 1369( (NO2); 1551, 1261, 1182, 1003 (N–C=S);
1412 (C–N thiolactam); 1261, 1076
(C–O–C); 1121 (C–N aliphatic). Anal. Calcd for
C15H13N5O3S (391.40) N,17.89; found,17.45.
4.1.6.3. 6-(2-Furyl)-4-morpholinyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6c).
Crystallized from DMF/EtOH; Yield: 43%; mp: 180 ºC; IR (KBr, cm–1): 3410 (NH); 3114 (CH
≡
furan); 2200 (C N); 1627 (C=N); 1585, 1550, 1500 (δNH, C=C); 1585, 1289, 1115, 1004 (N–
C=S); 1438, 1309 (C–N thiolactam); 1260, 1068 (C–O–C); 1212 (C–N aliphatic); 757 (opp
furan). Anal. Calcd for C13H12N4O2S(288.32) N,19.43; found,19.66.
4.1.6.4. 6-Phenyl-4-(1-piperidinyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6d).
Crystallized from DMF/H2O;Yield:50%; mp: 185ºC; IR (KBr, cm ): 3412 (NH); 2193 (C N);
1643 (C=N); 1586, 1506, 1492 (δNH, C=C); 1549, 1272, 1130, 1027(N–C=S); 1306 (C–N
thiolactam);1212 (C–N aliphatic). Anal. Calcd for C16H16N4S N2H2O(332.42) N,16.85;
found,16.24.
–1
≡
4.1.6.5. 6-(4-Nitrophenyl)-4-piperidinyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6e).
Crystallized from DMF/EtOH; Yield: 52%; mp: above 300ºC; IR (KBr, cm–1): 3357, 3217 (NH);
≡
2193 (C N); 1690 (C=N); 1602, 1517 (δNH, C=C); 1536, 1369 (NO2); 1568, 1273, 1181,
1021 (N–C=S); 1311 (C–N thiolactam); 1214 (C–N aliphatic). Anal. Calcd for
C16H15N5O2S2H2O(377.42) N,18.56; found,18.00.
4.1.6.6. 6-(2-Furyl)-4-piperidinyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6f).
Crystallized from DMF/H2O; Yield: 93%; mp: 230 ºC; IR (KBr, cm–1): 3135 (NH); 3108 (CH
≡
furan); 2194 (C N); 1643 (C=N); 1500 (C=C); 1550, 1272, 1138, 991 (δNH, N–C=S); 1307
(C–N thiolactam); 1232,1026
(C–O–C); 1213 (C–N aliphatic); 781 (oop furan).1H-NMR
(DMSO-d6) δppm: 1.45-1.51 (m, 2H, piperidine C4–H); 1.53-1.63 (m, 4H, piperidine C3, 5–H);
2.60-2.62 (m, 2H, piperidine C6–H); 2.84-2.86 (m, 2H, piperidine C2–H); 3.80 (br.s, 1H, NH, D2O
exchangeable);6.68 (dd, 1H, J = 3.8, 2.3 Hz, furan C4–H); 7.34 (d, 1H, J = 3.8 Hz, furan C3–H);
7.93 (d, 1H, J = 1.8 Hz, furan C5–H). Anal. Calcd forC14H14N4OS(286.35)N, 19.57; found,19.88.
4.1.6.7. 4-N-Benzylpiperazinyl-6-phenyl-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6g).
11

ACCEPTED MANUSCRIPT
–1
≡
Crystallized from EtOH; Yield: 36 %; mp: 174 ºC; IR (KBr, cm ): 3396 (NH); 2201 (C N);
1659, 1643 (C=N); 1587, 1491 (δNH, C=C); 1537, 1261, 1181, 1027(N–C=S); 1209, 1101 (C–N,
tertiary amine).1H-NMR (DMSO-d6) δppm: 3.70-3.90 (m, 10H, piperazine and N–CH2); 4.49 (s,
1H, NH, D2O exchangeable);7.20-7.80 (m, 10H, two phenyl protons).Anal. Calcd for
C22H21N5S2H2O(435.54)N, 16.08; found, 16.00.
4.1.6.8. 2-N-Benzylpiperazinyl-6-(4-nitrophenyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile
(6h).
Crystallized from DMF/EtOH; Yield: 59 %; mp: above 300 ºC; IR (KBr, cm–1): 3356, 3213 (NH);
≡
2199 (C N); 1600, 1518 (C=N, C=C); 1530, 1345 (NO2); 1313 (C–N thiolactam); 1263, 1180,
999 (N–C=S); 1209, 1109 (C–N, tertiary amine). Anal.Calcdfor C22H20N6O2S2H2O(480.54)N,
17.49; found, 17.74. EI- Mass spectrum of compound 6h m/z (relative abundance %):369(25);
368(59); 341(8); 313(19); 311(8); 285(14); 278(4); 264(16); 257(15); 256(21); 252(4); 250(7);
241(10); 239(17); 236(22); 227(11); 213(21); 201(5); 199(13); 194(8); 185(20); 171(17); 166(9);
157(14); 152(15); 149(29); 147(17); 139(19); 138(15); 137(23); 135(20); 133(17); 129(40);
127(15); 125(30); 124(17); 123(32); 121(23); 119(16); 115(18); 113(18); 112(20); 111(50);
110(26); 109(42); 107(21); 105(20); 101(15); 99(26); 98(39); 97(77); 96(33); 95(58); 93(23);
91(21); 87(20); 86(10); 85(62); 84(42); 83(78); 82(30); 81(59); 79(20); 73(40); 71(69); 70(35);
69(100); 68(21); 67(39); 60(35); 57(73); 56(23); 55(76).
4.1.6.9. 4-N-Benzylpiperazinyl-6-(2-furyl)-2-thioxo-1,2-dihydropyrimidine-5-carbonitrile (6i).
Crystallized from DMF/EtOH; Yield: 25 %; mp: above 300 ºC; IR (KBr, cm–1): 3409 (NH), 3059
≡
(CH furan); 2200 (C N); 1643 (C=N); 1586, 1550, 1500 (δNH, C=C); 1568, 1278, 1179, 1028
(N–C=S); 1278, 1028 (C–O–C); 1358 (C–N thiolactam); 1108 (C–N, tertiary amine); 750 (oop
furan). Anal. Calcd for C20H19N5OS. EtOH (423.53);C,62.39,H,5.95,N, 16.54; found: C,62.29,
H,5.97, N,16.2.
4.1.7. 4-Substituted-2-[2-(diethylamino)ethylthio]-6-oxo-1,6-dihydropyrimidine-5-carbonitriles;
(7a,b).
To a suspension of the appropriate thione 1a,b (2 mmole) in aqueous KOH solution (0.34 g, l ml)
was added 2-(diethylamino)ethyl chloride.HCl (2 mmole, 0.34 g). The reaction mixture was
stirred at R.T. for 4h during which a precipitate was formed. The product was then filtered,
washed with H2O, left to dry then crystallized from DMF/H2O.
4.1.7.1 2-[2-(Diethylamino)ethylthio]-4-phenyl-6-oxo-1,6-dihydropyrimid-ine-5-carbonitrile
(7a).
–1
≡
Yield: 67%; mp: 217ºC; IR (KBr, cm ): 3412 (NH); 2206 (C N); 1660 (C=O); 1644 (C=N);
1550 (δNH); 1585, 1525 (C=C); 1297, 1051 (C-S–C); 1435, 1316 (C–N lactam); 1206, 1103 (C–
N aliphatic). Anal. Calcd for C17H20N4OS. 1/2 H2O(337.44) C 60.51,H 6.27,N 16.6;
found,C,61.22, H,6.51, N,16.22.
4.1.7.2 2-[2-(Diethylamino)ethylthio]-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimid-ine-5-
carbonitrile (7b).
–1
≡
Yield: 25%; mp: above 300ºC; IR (KBr, cm ): 3374 (NH); 2206 (C N); 1660 (C=O); 1643
(C=N); 1626, 1594, 1518 (δNH, C=C); 1551, 1346 (NO2); 1452 (C–N lactam); 1181, 1108 (C–N
aliphatic); 1046 (C–S–C).1H-NMR (DMSO-d6) δppm: 0.87 (t, 3H, J = 6.9 Hz, CH2-CH3); 0.92
(t, 3H, J = 6.9 Hz, CH2-CH3); 1.15 (t, 2H, J = 6.9 Hz, S-CH2); 2.33 (t, 2H, J = 6.9 Hz, N-CH2);
2.37 (q, 4H, J = 6.9 Hz, N-(CH2-CH3)2); 4.07 (s, 1H, NH, D2O exchangeable); 7.91 (d, 2H, J = 8.4
Hz, 4-NO2- C6H4-C2, 6-H); 8.28 (d, 2H, J = 8.4 Hz, 4-NO2-C6H4-C3, 5-H).Anal. Calcd for
C17H19N5O3S.1/2 H2O(382.44);C 53.39, H 5.27; found,C,53.43, H,5.13.
4.1.8 N-(4-Substitutedphenyl)-2-[(4-substituted-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-
yl)thio]acetamides(9a-d)
A mixture of the appropriate thione 1a,c (2 mmole), anhydrous K2CO3 (0.28 g, 2 mmole) and the
selected 4-substituted chloroacetanilide derivative 8a,b (2 mmol) in dry DMF (10 ml) was stirred
at R.T. for 6h. The reaction mixture was diluted with H2O and the obtained product was filtered,
washed with H2O and crystallized from DMF/H2O.
4.1.8.1 N-(4-Methoxyphenyl)-2-[(4-phenyl-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-
yl)thio]acetamides(9a)
12

ACCEPTED MANUSCRIPT
–1
≡
Yield: 88%; mp: 230ºC; IR (KBr, cm 3269 (NH); 2220 (C N); 1654 (C=O); 1614 (C=N); 1595,
1511 (C=C); 1557, 1533 (δNH); 1415, 1369 (C–N lactam); 1293 (C–S–C); 1255, 1162, 1024(C–
O–C). Anal. Calcd for C20H16N4O3S(392.43) C 61.21, H 4.11, N 14.28; found, C,60.87, H,3.57,
N,13.92.
4.1.8.2 N-(4-Methoxyphenyl)-2-[(4-(2-furyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-
yl)thio]acetamides(9b)
–1
≡
Yield: 85 %; mp: 230ºC; IR (KBr, cm ): 3267 (NH); 3130 (CH furan); 2223 (C N); 1691 1663
(C=O); 1588 (C=N); 1551 (δNH); 1411, 1330 (C–N lactam); 1294 (C–S–C); 1253, 1022(C–O–C);
779 (oop furan).1H-NMR (DMSO-d6) δppm: 2.70 (d, 1H, J = 1.55Hz, S–CH2);2.85 (d, 1H, J =
1.5Hz, S–CH2);3.67 (s, 3H, OCH3);4.12 (s, 1H, pyrimidine NH, D2O exchangeable); 6.74 (dd, 1H,
J = 3.8, 1.5Hz, furan C4–H); 6.84 (d, 2H, J = 9.2Hz, 4-OCH3–C6H4–C2, 6–H); 7.42 (d, 2H, J =
9.2Hz, 4-OCH3–C6H4–C3, 5–H); 7.54 (d, 1H, J = 3.05Hz, furan C3–H); 8.05 (s, 1H, furan C5–H);
10.23 (s, 1H, NH, D2O exchangeable). Anal. Calcd forC18H14N4O4S(382.39) C 56.54,H 3.69,N
14.68; found,C,55.81,H,2.77,N,14.25.
4.1.8.3 N-(4-Chlorophenyl)-2-[(4-phenyl-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-
yl)thio]acetamides(9c)
–1
≡
Yield: 88 %; mp: 240ºC; IR (KBr, cm ): 3379, 3272 (NH); 2195 (C N); 1655 (C=O); 1586,
1491 (C=N, C=C); 1554, 1535 (δNH); 1436, 1315 (C–N lactam); 1296, 1096 (C–S–C); 822 (C–
Cl).1H-NMR (DMSO-d6) δppm: 3.78 (s, 2H, S–CH2); 4.07 (s, 1H, pyrimidine NH, D2O
exchangeable); 7.28 (d, 2H, J = 8.4 Hz, 4-Cl-C6H4–C2, 6–H); 7.35 (t, 1H, J = 7.65 Hz, C6H5–C4–
H); 7.43 (t, 2H, J = 7.65Hz, C6H5–C3, 5–H); 7.54 (d, 2H, J = 8.4 Hz, 4-Cl-C6H4–C3, 5–H); 7.70 (d,
2H, J = 7.65Hz, C6H5–C2, 6–H); 10.96 (s, 1H, NH, D2O exchangeable). Anal. Calcd for
C19H13ClN4O2S.2H2O(432.88) N 12.94; found: 12.81.
4.1.8.4
yl)thio]acetamides(9d)
Yield: 68 %; mp: 245ºC; IR (KBr, cm ): 3436, 3318 (NH); 3126 (CH furan); 2226 (C N); 1689,
1657 (C=O); 1590, 1492 (C=N, C=C); 1538 (δNH); 1410, 1328 (C-N lactam); 1250, 1022 (C–
N-(4-Chloroxyphenyl)-2-[(4-(2-furyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-
–1
≡
O–C); 1096 (C–S–C), 823 (C–Cl); 762 (oop furan). Anal. Calcd C17H11ClN4O3S(386.81) C
52.79,H 2.87,N 14.48; found,C,53.24, H,2.83, N,14.75.
4.1.9 4-Substituted-1-methyl-2-methylthio-6-oxo-1,6-dihydropyrimidine-5-carbonitriles (10a-c)
A mixture of the appropriate thione derivative 1a-c (20 mmole), K2CO3 (4.1 g, 30 mmole) and
CH3I (5.68 g, 2.5 ml, 40 mmole) in dry DMF (10 ml) was stirred for 6 h at R.T. The reaction
mixture was diluted with H2O, filtered, washed with H2O and crystallized from the appropriate
solvent.
4.1.9.1 1-Methyl-2-methylthio-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitriles (10a)[29]
–1
≡
Crystallized from CH3OH, Yield: 85 %; mp: 240ºC; IR (KBr, cm ): 2928 (CH3); 2216 (C N);
1676 (C=O); 1647 (C=N); 1505 (C=C); 1373 (N–CH3), 1308 (S–CH3); 1228, 1088(C–S–C);
1415, 1344 (C–N lactam). C13H11N3OS(257.32).
4.1.9.2
1-Methyl-2-methylthio-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitriles
(10b)[29]
Crystallized from DMF/H2O; Yield: 82 %; mp: 242ºC; IR (KBr, cm–1): 2929 (CH3), 2218
≡
(C N); 1665 (C=O); 1601, 1519 (C=N, C=C); 1568, 1346 (NO2); 1412, 1316 (C-N lactam);
1369 (N–CH3); 1316 (S–CH3); 1283, 1089 (C–S–C). C13H10N4O3S(302.31)
4.1.9.3 4-(2-Furyl)-1-methyl-2-methylthio-6-oxo-1,6-dihydropyrimidine-5-carbonitriles (10c)
Crystallized from DMF; Yield: 93 %; mp: 270ºC; IR (KBr, cm–1): 3135 (CH furan); 2930 (CH3);
≡
2222 (C N); 1672 (C=O); 1628 (C=N); 1593; 1502 (C=C); 1412, 1340 (C–N lactam); 1365
(N–CH3); 1309 (S-CH3); 1234, 1094 (C–S–C); 1184, 1043 (C–O–C); 777 (opp furan). H-NMR
1
(DMSO-d6) δppm: 2.65 (s, 3H, S–CH3); 3.38 (s, 3H, N-CH3, under DMSO); 6.81 (dd, 1H, J = 3.8,
1.5Hz, furan C4–H); 7.55 (d, 1H, J = 3.8Hz, furan C3–H); 8.12 (s, 1H, furan C5–H).Anal. Calcd for
C11H9N3O2S(247.27) C 53.43,H 3.64,N 16.99,S 12.97; found C,53.03, H,3.73, N,16.50, S,12.41.
4.1.10 4-Substituted-2-substitutedamino-1-methyl-6-oxo-1,6-dihydro-pyrimidine-5-carbonitriles
(11a-f).
A mixture of the appropriate derivative of compound 10a-c (2 mmole) and the
selected
secondary amine (4 mmole) was fused at 160-170ºC for 12 h. The reaction mixture was then
13

ACCEPTED MANUSCRIPT
triturated with EtOH, filtered, washed with EtOH and left to dry. The product was crystallized
from the appropriate solvent.
4.1.10.1.
2-(4-Mopholinyl)-1-methyl-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-
carbonitriles (11a)
–1
≡
Crystallized from DMF/EtOH; Yield: 30 %; mp: 155ºC; IR (KBr, cm ): 2205 (C N); 1690
(C=O); 1604, 1569 (C=N, C=C); 1533, 1363 (NO2); 1409, 1304 (C–N lactam); 1270, 1241, 1067
(C–O–C); 1112 (C-N aliphatic). Anal. Calcd for C16H15N5O4.2H2O(377.36),N 18.55; found
18.37.
4.1.10.2. 4-(2-Furyl)-2-mopholinyl-1-methyl-6-oxo-1,6-dihydro-pyrimidine-5-carbonitriles (11b)
Crystallized from DMF/H2O; Yield: 35 %; mp: abouve 300ºC; IR (KBr, cm–1): 2961 (CH furan);
≡
2209 (C N); 1660 (C=O); 1644 (C=N); 1568, 1551, 1517 (C=C); 1447, 1306 (C–N lactam);
1263, 1067 (C–O–C); 1112 (C–N aliphatic); 786 (oop furan). 1H-NMR (DMSO-d6) δppm: 2.32-
2.33, 2.60-2.61 (two m, 4H, morpholine C3, 5–H); 3.61-3.75(m,4H,morpholine C2, 6–H); 4.05 (s,
3H, N–CH3); 6.71-6.74 (m, 1H,furan C4–H);7.33 (d, 1H, J = 3.05Hz, furan C3–H);7.99 (s, 1H,
furan C5-H). Anal. Calcd forC14H14N4O3.H2O(304.31)C 55.26, H 5.29; found C,55.59, H,5.20. -
EI- Mass spectrum of compound 11b m/z (relative abundance %): 285(M-1)(21); 284(14);
265(22); 264(39); 262(19); 256(17); 239(39); 237(25); 236(50); 227(13); 213(18); 211(15);
185(23); 152(23); 149(29); 137(22); 129(53); 125(26); 123(30); 121(24); 115(32); 112(25);
111(52); 110(26); 109(37); 100(17); 98(61); 97(87); 96(34); 95(67); 87(34); 86(26); 85(51);
84(48); 83(73); 82(27); 81(63); 79(23); 73(39); 71(64); 69(71); 67(39); 60(19); 57(100); 56(36);
55(77).
4.1.10.3. 1-Methyl-6-oxo-4-phenyl-2-(1-piperidinyl)-1,6-dihydropyrimidine-5-carbonitriles (11c)
–1
≡
Crystallized from EtOH; Yield: 70%; mp: 230ºC; IR (KBr, cm ): 2207 (C N); 1663 (C=O);
1584, 1492 (C=N, C=C); 1447, 1307 (C–N lactam); 1212 (C– N aliphatic). Anal. Calcd
forC17H18N4O(294.35) C 69.37, H 6.16,N 19.03; found 68.63, 6.35, 18.47.
4.1.10.4.
(11d)
4-(2-Furyl)-1-methyl-6-oxo-2-(1-piperidinyl)-1,6-dihydro-pyrimidine-5-carbonitriles
Crystallized from DMF/H2O;Yield:96%; mp: 220ºC; IR (KBr, cm–1): 2931 (CH furan); 2198
≡
(C N); 1660 (C=O); 1590, 1568, 1487 (C=N, C=C); 1446 (C-N lactam); 1256, 1022 (C–O–C);
1215 (C–N aliphatic); 782 (oop furan). Anal. Calcd for C15H16N4O2(284.31)C 63.57, H 5.67 ;
found: C, 63.57, H,5.49.
4.1.10.5 2-N-Benzylpiperazinyl-1-methyl-6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitriles
(11e)
–1
≡
Crystallized from EtOH; Yield: 57 %; mp: 105ºC; IR (KBr, cm ): 2213 (C N); 1665 (C=O);
1582, 1568, 1495 (C=N, C=C); 1447, 1313 (C-N lactam); 1210 (C–N aliphatic).1H-NMR
(DMSO-d6) δppm; 2.88 (s, 3H, N–CH3); 3.40-3.50 (m, 8H, piperazine protons);3.64 (s, 2H, N–
CH2); 7.30-7.44 (m, 4H, C6H5 and benzyl C3, 5-H);7.50 (t, 1H, J = 7.65Hz, benzyl C4-H);7.51 (d,
2H, J = 7.65Hz, benzyl C2, 6-H);7.54 (t, 1H, J = 7.65Hz, C6H5-C4-H);7.80 (d, 2H, J = 7.65Hz,
C6H5-C2,6-H).Anal. Calcd for C23H23N5O.2H2O(421.50): N, 16.61; found: 16.64.
4.1.10.6.
2-N-Benzylpiperazinyl-4-(2-furyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitriles (11f)
Crystallized from DMF/H2O; Yield: 81 %; mp: 130ºC; IR (KBr, cm–1): 3112 (CH furan); 2218
≡
(C N); 1672 (C=O); 1595, 1568, 1490 (C=N, C=C); 1447, 1313 (C-N lactam); 1269, 1076 (C–
O–C); 1206 (C–N aliphatic); 746 (oop furan). Anal. Calcd for C21H21N5O2(375.42) C, 67.18, H,
5.67, N, 18.65; found C,66.74, H,5.65, N,18.53.
4.1.11
4-Substituted-2-hydrazino-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitriles (12a-
c)
To a suspension of the appropriate S-CH3 derivative 10a-c (20 mmole) in EtOH (20 ml),
hydrazine hydrate (1 ml, 0.1 mole) was added and the reaction mixture was heated under reflux
for 6 h. The obtained product was filtered, washed with EtOH and crystallized from the
appropriate solvent.
4.1.11.1 2-Hydrazino-1-methyl-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitriles
( 12a)[29]
14

ACCEPTED MANUSCRIPT
Crystallized from DMF/CH3Cl; Yield: 92 %; mp: 270ºC; IR (KBr, cm–1): 3458, 3316 (NH); 2221
≡
(C N); 1675 (C=O); 1643 (C=N); 1587, 1512 (C=C); 1542 (δNH). Anal. Calcd for C12H11N5O
(241.25).
4.1.11.2 2-Hydrazino-1-methyl-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitriles
(12b)[29]
Crystallized from DMF/H2O; Yield: 88 %; mp: 300ºC; IR (KBr, cm–1): 3442, 3301, 3114 (NH);
≡
2199 (C N); 1661 (C=O); 1646 (C=N); 1607, 1503 (C=C); 1552 (δNH); 1532, 1350 (NO2).
Anal. Calcd for C12H10N6O3(286.25).
4.1.11.3 4-(2-furyl)-2-hydrazino-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitriles
(12c)
Crystallized from DMF/H2O; Yield:70 %; mp: 264-265ºC; IR (KBr, cm–1): 3316, 3141 (NH);
≡
3121 (CH furan); 2213 (C N); 1659 (C=O); 1594, 1539 (C=N, δNH, C=C); 1411 (C–N lactam);
1467, 1380 (N–CH3); 1260; 1020 (C–O–C); 775 (opp furan).1H-NMR (DMSO-d6) 3.32 (s, 3H, N–
CH3); 6.75 (dd, 1H, J = 3.8, 1.5Hz, furan C4–H);7.38 (d, 1H, J = 3.8Hz, furan C3–H);8.05 (s, 1H,
furan C5–H);9.84, 9.86 (two s, each 1/2 H, 2NH tautomers, D2O exchangeable);10.33 (s, 2H, NH2,
D2O exchangeable). Anal. Calcd for C10H9N5O2(231.21)C 51.95, H 3.92; found: C,51.50, H,3.25.
4.1.12 4-Substituted-1-methyl-6-oxo-2-[3,4,6-trioxo-1,2,3,4,5,6-hexahydro-pyrano[2,3-
c]pyrazol-2-yl]-1,6-dihydropyrimidine-5-carbonitriles (13a,b).
The appropriate hydrazine derivative 12a,c (2 mmole) was heated under reflux with diethyl
malonate (3.18 g, 3 ml, 20 mmole) for 6h, whereupon needle crystals separated out. The reaction
mixture was left to cool, filtered and the obtained product was washed with EtOH and crystallized
from DMF/EtOH.
4.1.12.1 1-Methyl-6-oxo-4-phenyl-2-[3,4,6-trioxo-1,2,3,4,5,6-hexahydro-pyrano[2,3-c]pyrazol-2-
yl]-1,6-dihydropyrimidine-5-carbonitriles (13a).
Yield: 60 %; mp: 230ºC; IR (KBr, cm ): 3278 (NH); 2218 (C N); 1746, 1658 (C=O); 1625
(C=N); 1595, 1492 (C=C); 1525 (δNH); 1253, 1222, 1037 (C–O–C). Anal. Calcd for
C18H11N5O5(377.31) C 57.30, H 2.94, N 18.56; found 57.11, 3.07, 18.87.
–1
≡
4.1.12.2 4-(2-furyl)-1-methyl-6-oxo-2-[3,4,6-trioxo-1,2,3,4,5,6-hexahydro-pyrano[2,3-
c]pyrazol-2-yl]-1,6-dihydropyrimidine-5-carbonitriles (13b).
Yield: 76 %; mp: 234ºC; IR (KBr, cm ): 3281 (NH); 2986 (CH furan); 2225 (C N); 1744, 1662
–1
≡
(C=O); 1592, 1520, 1467 (C=N, C=C); 1566 (δNH); 1284, 1263, 1224, 1082, 1040 (C–O–C); 776
(opp furan). 1H-NMR (DMSO-d6) 3.43 (s, 2H, pyranopyrazole C5–H); 3.46 (s, 3H, N– CH3); 6.75
(dd, 1H, J = 3.8, 1.5Hz, furan C4–H); 7.41 (d, 1H, J = 3.05Hz, furan C3–H); 8.02 (s, 1H, furan C5–
H);10.42 (s, 1H, NH, D2O exchangeable). 13C-NMR (DMSO-d6) δppm:167.71 (pyranopyrazole
C6); 165.58 (pyranopyrazole C4); 161.48 (pyranopyrazole C3); 155.68 (pyrimidine C6); 155.55
(pyranopyrazole C7a); 151.0 (pyrimidine C2); 150.21 (pyrimidine C4); 147.93 (furan C2); 117.92
≡
(pyrimidine C5); 117.83 (pyranopyrazole C3a); 116.0 (C N);113.51 (furan C5); 100(furan C3);
82.27
(furanC4);61.35
(pyranopyrazole
C5);
41.09
(N–CH3).
Anal.
Calcd
forC16H9N5O6.1/2H2O(376.28) C 51.07, H 2.68; found 51.39, 2.18. EI- Mass spectrum m/z
(relative abundance %):349(68); 347(59); 172(64); 158(100); 157(100); 113(68); 78(59).
4.1.13 3-Cyanomethyl-8-methyl-7-oxo-5-phenyl-7,8-dihydro[1,2,4]triazolo[4,3-a]pyrimidine-6-
carbonitrile (14) And ethyl 3-[2-cyano-N'-(5-cyano-1-methyl-6-oxo-4-phenyl-1,6-dihydro-
pyrimidin-2-yl)acetohydrazido]-3-iminopropanoate(15)
The hydrazine derivative 12a (0.48 g, 2 mmole) was heated with ethyl cyanoacetate (0.45 g, 0.42
ml, 4 mmole) in an oil bath at 160 for 5h. The solid mass was triturated with EtOH, filtered and
washed with EtOH. The product was then crystallized from EtOH giving compound 14, while the
alcohol-insoluble part was found to be compound 15.
3-Cyanomethyl-8-methyl-7-oxo-5-phenyl-7,8-dihydro[1,2,4]triazolo[4,3-a]pyrimidine-6-
carbonitrile (14)
–1
≡
Yield: 26 %; mp: 185ºC; IR (KBr, cm 2978 (CH2, CH3); 2215 (C N); 1672 (C=O); 1626
(C=N); 1530, 1474 (C=C); 1343 (C–N lactam).1H-NMR (DMSO-d6) 3.51, 3.53 (two d, 2H, J =
3.05Hz, CH2CN);3.81 (s, 3H, N-CH3);7.46, 7.49 (two t, 2H, J = 8.4Hz, C6H5–C3, 5–H);7.61, 7.66
(two d, 2H, J = 7.65Hz, C6H5–C2, 6–H);7.73, 7.76 (two t, 1H, J = 6.1Hz, C6H5–C4–H). Anal. Calcd
for C15H10N6O.1 1/2 H2O C 56.78, H 4.13; found 56.97, 3.95.
15

ACCEPTED MANUSCRIPT
EI- Mass spectrum m/z (relative abundance %): 291(M++ 1) (6); 265(46); 264(38); 263(20)
239(9); 237(12); 236(20); 213(12); 211(11); 185(13); 163(10); 159(9), 149(19); 141(16); 137(21);
135(16); 133(16); 129(26); 127(22); 125(27); 123(30); 119(21); 111(48), 110(21); 109(40);
107(16); 105(20); 99(17); 98(24); 97(78); 96(30); 95(60); 93(19); 85(50); 84(29); 83(80); 82(26);
81(73), 79(21); 73(30); 71(67), 70(27); 69(100); 68(19); 67(40); 60(20); 57(96); 56(28); 55(92).
Ethyl-3-[2-cyano-N'-(5-cyano-1-methyl-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-
yl)acetohydrazido]-3-iminopropanoate(15)
Yield: 40 %; mp: above 300ºC; IR (KBr, cm ): 3336 (NH); 2952 (CH3, CH2); 2224 (C N);
–1
≡
1727 (C=O ester); 1692 (C=O); 1617 (C=N); 1597, 1570, 1500 (C=C); 1534 (δNH); 1441,
1332 (C–N lactam) 1292, 1017 (C–O–C).1H-NMR (DMSO-d6) 1.18 (t, 3H, J = 7.2 Hz, CH2-
CH3);3.37 (s, 2H, CH2–CN);3.56 (s, 2H, CH2–CO);3.83 (s, 3H, N–CH3);3.98 (s, 1H, NH, D2O
exchangeable); 4.11 (q, 2H, J = 7.2Hz, CH2-CH3);7.47-7.60 (m, 3H, C6H5-C3, 4, 5–H);7.79 (dd, 2H,
J = 7.9, 1Hz, C6H5-C2, 6–H).Anal. Calcd for C20H19N7O4(421.41) C 57.01, H 3.50, N 24.28; found
57.11, 3.24, 24.55. EI- Mass spectrum of compound 15 m/z (relative abundance %): 318(83);
317(61); 274(57); 273(57); 271(61); 213(65); 198(100); 159(57); 103(61); 96(87); 64(78); 63(65).
4.1.14 6-Substituted-2-[(oxiran-2-yl)methylthio]-4-chloropyrimidine-5-carbonitriles (16a,b)
To a solution of chloropyrimidine thione derivative 5a,c (2 mmole) in DMF (5 ml) was added
NaOH solution (0.16 g, 4 mmole) in 2 ml H2O. The reaction mixture was stirred at R.T. and
epichlorohydrin (0.19 g, 0.16 ml, 2 mmole) was added dropwise while stirring. Stirring was
continued at R.T. for 48h. The reaction mixture was then diluted with H2O and the obtained
product was filtered, washed with H2O and crystallized from the appropriate solvent.
4.1.14.1 2-[(Oxiran-2-yl)methylthio]-6-phenyl-4-chloropyrimidine-5-carbonitriles (16a)
Crystallized from EtOH; Yield: 48 %; mp: 100ºC; IR (KBr, cm ): 2912 (CH2); 2226 (C N);
–1
≡
1597 (C=N); 1567 (C=C); 1266, 1085 (C–S–C); 1266, 1036, 760 (epoxy C–O–C); 873 (C–Cl).1H-
NMR (DMSO-d6) 2.30-2.34 (m, 1H, S–CH2);2.59-2.63 (m, 1H, S–CH2);2.68-2.71 (m, 1H, O–
CH);4.02-4.10 (m, 2H, O–CH2);7.40-7.90 (m, 5H, C6H5 ).Anal. Calcd for C14H10ClN3OS(303.77)
C 55.36, H 4.32, N 13.83; found 56.84, 4.92, 14.06. EI- Mass spectrum of compound m/z (relative
abundance %): 285(14); 284(45); 283(18); 281(53); 279(48) 270(22); 264(40); 257(45); 254(34);
253(23); 251(33); 238(33); 237(35), 227(36); 223(18); 222(37); 221(21); 215(31); 214(73);
213(28); 209(29); 196(28); 182(35); 179(18), 176(13); 169(25); 155(30); 154(22); 145(20);
141(23); 130(26); 127(25); 116(21); 105(26); 104(100); 103(23); 89(35); 82(22); 77(60), 76(24);
73(21); 70(29), 59(26); 57(13); 51(50); 50(37).
4.1.14.2 6-(2-furyl)-2-[(oxiran-2-yl)methylthio]-4-chloropyrimidine-5-carbonitriles (16b)
Crystallized from DMF/H2O; Yield: 58 %; mp: above 300ºC; IR (KBr, cm–1): 3137 (CH furan);
≡
2919, 2849 (CH2); 2224 (C N); 1643 (C=N); 1581, 1529, 1513 (C=C); 1255, 1081 (C–S–C);
1255; 1030, 779 (furan and epoxy C–O–C); 885 (C–Cl); 779 (oop furan). Anal. Calcd for
C12H8ClN3O2S(293.73) C 49.07, H 2.75; found: C, 50.32, H,2.49.
4.1.15 6-Substituted-2-[(oxiran-2-yl)methylthio]-4-morpholinopyrimidine-5-carbonitriles
(17a,b).
To solution of 4-morpholinopyrimidine derivative 6a and 6c separately (2 mmole) in DMF (5 ml)
was added aqueous NaOH solution (0.16 g, 4 mmole). The reaction mixture was stirred at R.T. for
1h, then epichlorohydrin (0.19 g, 0.16 ml, 2 mmole) was added and stirring was continued for 48
h. The reaction mixture was then diluted with H2O and the obtained product was filtered, washed
with H2O and crystallized from DMF/H2O.
4.1.15.1. 2-[(Oxiran-2-yl)methylthio]-6-phenyl-4-morpholinopyrimidine-5-carbonitriles (17a).
–1
≡
Yield: 70 %; mp: 198-200ºC; IR (KBr, cm ): 2920, 2850 (CH2); 2207 (C N); 1638 (C=N);
1585, 1519, 1492 (C=C); 1285, 1069 (C–S–C); 1267, 1055 (C-O-C); 1255, 1028, 772 (epoxy C–
O–C); 1206, 1115 (C–N aliphatic).1H-NMR (DMSO-d6) 2.59-2.60 (m, 2H, OCH2 epoxy);3.20-
3.40 (m, 2H, S-CH2 under DMSO);3.62 (t, 2H, J = 4.6Hz, morpholine C3–H);3.67 (t, 2H, J =
4.6Hz, morpholine C5–H);3.78 (t, 4H, J = 4.6Hz, morpholine C2, 6–H);4.03-4.06 (m, 1H, CH,
epoxy);7.46 (t, 2H, J = 7.6Hz, C6H5-C3, 5–H);7.50 (t, 1H, J = 7.6Hz, C6H5-C4–H);7.73 (d, 2H, J =
7.6Hz, C6H5-C2, 6–H).Anal. Calcd for C18H18N4O2S.H2O(372.45) N 15.04; found 14.86. EI- Mass
spectrum m/z (relative abundance %):294(100); 95(44); 85(41); 73(9); 72(16) 71(3); 70(75);
55(47).
16

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4.1.15.2. 6-(2-Furyl)-2-[(oxiran-2-yl)methylthio]-4-morpholinopyrimidine-5-carbonitriles(17b).
–1
≡
Yield: 62 %; mp: 190; IR (KBr, cm ): 3111 (CH furan); 2920, 2850 (CH2); 2201 (C N); 1640
(C=N); 1587, 1561, 1530 (C=C); 1257, 1067 (C–S–C); 1229; 1022, 783 (morpholine, furan and
epoxy C–O–C); 1209, 1114 (C–N aliphatic); 759 (opp furan).Anal. Calcd for
C16H16N4O3S(344.40) N 16.27; found 16.51. EI- Mass spectrum m/z (relative abundance
%):342(22); 341(87); 340(21); 312(27) 311(61); 310(51); 298(37); 296(75); 284(100); 283(28);
280(25); 252(16), 199(21); 156(21); 144(27); 143(34); 117(13); 115(14) 88(15); 86(21); 85(20);
83(12); 71(10); 58(15), 56(21); 55(30).
4.1.16
6-Substituted-2-(2-hydroxy-3-morpholinopropylthio)-4-morpholino-pyrimidine-5-
carbonitriles; (18a,b)
Method A: To a suspension of 2-(oxiran-2-yl)methylthio-4-chloropyrimidine derivative 16a,b (2
mmole) in dry dioxane (5 ml), morpholine (0.7 g, 0.7 ml, 8 mmole) was added and the reaction
mixture was heated under reflux for 24 h. The reaction mixture was concentrated, allowed to cool,
then diluted with H2O. The obtained product was filtered and crystallized from the appropriate
solvent.
Method B: The 2-(oxiran-2-yl)methylthio-4-morpholinopyrimidine derivatives 17a,b (2 mmole)
were allowed to react separately with morpholine (0.35 g, 0.35 ml, 4 mmole) in dry dioxane (5
ml) by heating under reflux for 12 h. The reaction mixture was concentrated, left to cool and
diluted with H2O. The obtained product was filtered, washed with H2O, left to dry and crystallized
from the appropriate solvent.
4.1.16.1 2-(2-Hydroxy-3-morpholinopropylthio)-6-phenyl-4-morpholinopyrimidine-5-
carbonitriles; (18a)
Crystallized from EtOH/H2O; Yield: 34 % (method A), 78% (method B); mp: 175ºC; IR (KBr,
–1
≡
cm ): 3438 (OH br. band); 2920, 2850 (CH2, CH); 2205 (C N); 1644 (C=N); 1585, 1520, 1492
(C=C); 1284, 1069 (C–S–C); 1256, 1023 (C–O–C); 1207, 1118 (C–N aliphatic).1H-NMR
(DMSO-d6) 3.27-3.32 (m, 2H, S–CH2);3.68-3.70 (m, 1H, CH); 4.08-4.12 (m, 2H, CH2–N);4.47 (t,
4H, J = 4.43 Hz, C3, 5 morpholine of S-propyl side chain));4.50 (t, 4H, J = 4.43 Hz, morpholine C3,
of 4-morpholino);4.55-4.66 (m, 8H, morpholine C2, 6–H)8.26-8.38(m, 3H, C6H5-C3, 4, 5–H);8.60
5
(d, J = 7.65 Hz, 2H, C6H5-C2, –H);8.75 (s, 1H, OH, D2O exchangeable).Anal. Calcd for
6
C22H27N5O3S.H2O(459.58)N 15.24; found 14.72. EI- Mass spectrum (relative abundance
%):433(7); 429(36); 351(84); 350(100) 321(46); 320(92); 308(21); 306(53); 295(21); 294(78);
293(21); 292(20), 262(35); 249(13); 248(12); 236(24); 235(18); 224(10) 223(15); 219(15);
209(16); 208(27); 180(21); 154(24), 153(32); 152(15); 129(18); 127(14); 91(13); 86(26); 77(53);
56(26), 55(15); 51(18).
4.1.16.2 6-(2-Furyl)-2-(2-hydroxy-3-morpholinopropylthio)-4-morpholino-pyrimidine-5-
carbonitriles; (18b)
Crystallized from DMF/H2O; Yield: 86 % (method A), 90% (method B %; mp: above 300ºC; IR
–1
≡
(KBr, cm 3407 (OH br. band); 2919 (CH furan); 2850 (CH2, CH); 2206 (C N); 1658 (C=N);
1562, 1502 (C=C); 1264, 1066 (C–S–C); 1264, 1022 (morpholine and furan C–O–C); 1110 (C–N
aliphatic); 754 (opp furan). Anal. Calcd for C20H25N5O4S.2H2O(467.55) C 51.38,H 6.25; found: C,
50.84, H,6.28. EI- Mass spectrum (relative abundance %): 257(27); 112(7); 111(29); 109(9)
99(9); 98(14); 97(42); 95(30); 85(46); 83(52); 82(18); 81(19), 71(45); 70(15); 69(35); 68(10);
67(20); 58(17) 57(100); 56(29); 55(67).
4.2 Antimicrobial activity
All compounds were preliminarily evaluated for their in-vitro antibacterial activity against
Staphylococcus aureus (ATCC 6538P) as a representative of Gram-positive; Pseudomonas
aeruginosa (ATCC 9027) and Shigella flexneri (ATCC 15391) as representatives of Gram-
negative bacteria. The compounds were also evaluated for their in-vitro antifungal activity against
Candida albicans (ATCC 2091). Their inhibition zones using the cup diffusion technique [50]
were measured. Compounds showing reasonable inhibition zones were subjected to determine
their minimal inhibitory concentration (MIC) values using the two fold serial dilution method[51].
Ampicillin and Ofloxacin (Tarivid (R), Sanofi-Aventis) were used as standard antibacterial agents,
while Clotrimazole (Canesten (R), Bayer) was used as a standard antifungal agent. Dimethyl
17

ACCEPTED MANUSCRIPT
formamide was used as a blank. Compounds were dissolved in dimethylformamide in a
concentration of 1mg/ml. Each 100 ml of sterile molten agar (at 45ºC) received 1ml of 6 h. broth,
then the seeded agar was poured into sterile Petri dishes. Cups (8mm in diameter) were cut in the
agar. Each cup received 0.1 ml of the 1 mg/ml solution of the test compounds. Plates were then
incubated at 37ºC for 24 h. for bacteria and 48 h. for fungi. The resulting inhibition zones were
measured in mm diameter (tables 4). The test organisms were allowed to grow in their suitable
broth for 24 h. Two fold serial dilutions of the test compounds solution were prepared using the
proper sterile nutrient broth to obtain concentrations 500, 250, 125, 62.5, 31.25, 15.63, 7.82 and
3.91ꢀg/ml with the concentration of dimethylformamide not exceeding 2.5%. The tubes were then
inoculated with the test organisms; each 5ml received 0.1ml of the inoculums and were incubated
at 37ºC for 24 h. for bacteria and 48 h. for fungi. The tubes were observed for the presence or
absence of microbial growth. The minimum inhibitory concentration was determined visually in
which the non turbid tubes were observed and the lowest concentration giving no turbidity is the
minimum inhibitory concentration.
4.3 Computer aided drug design
All the molecular studies were carried out on an Intel pentium 1.6 GHz processor, 512 MB
memory with windows XP operating system using Molecular Operating Environment (MOE
2005.06; Chemical Computing Group, Montreal, Canada) as the computational software. All the
minimizations were performed with MOE until a RMSD gradient of 0.05 Kcal mol–1 A0-1 with
MMFF94X forcefield and the partial charges were automatically calculated. The coordinates of
the X-ray crystal structure of 2'-deoxyuridine-5'-monophosphate(DUMP) bound to thymidylate
synthase enzyme were obtained from Protein Data Bank (PDB ID: 1BID) as well as the
coordinates of the X-ray crystal structure of methotrexate (MTX) bound to dihydrofolate
reductase (DHFR) enzyme (PDB ID: 4DFR). Enzyme structures were checked for missing atoms,
bonds and contacts. Hydrogen atoms were added to the enzyme structure. Water molecules and
bound ligands were manually deleted. The ligand molecules were constructed using the builder
molecule and were energy minimized. The active site was generated using the MOE-Alpha site
finder. Dummy atoms were created from the obtained alpha spheres. Ligands were docked within
the thymidylate synthase and dihydrofolate reductase active sites using the MOE-Dock with
simulated annealing used as the search protocol and MMFF94X molecular mechanics forcefield
for 8000 interactions. The lowest energy conformation was selected and subjected to an energy
minimization using MMFF94X force field.
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21

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Table captions
Table 1: The mean growth percent, delta values and the percent growth inhibition against
some subpanel cell lines of the selected compounds
Table 2: GI50, TGI and LC50 of 5-Dose screening of compound 17a
Table 3: The MG-MID (GI50) µM/l and the selectivity ratio of compound 17a
Table 4: Inhibition zones (IZ) in mm diameter
Table 5: MIC values in µg/ml for some selected compounds and for reference drugs
Scheme captions
Scheme 1: Synthetic pathways for compounds 2 - 6
Scheme 2: Synthetic pathways for compounds 9 - 15
Scheme 3: Synthetic pathways to the dimorpholino compounds 18a,b.
Figure captions
Fig (1) Docking of DUMP in the active site of TS
Fig (2)Docking of compound 6a in the active site of TS
Fig (3) Docking of compound 6c in the active site of TS
Fig (4) Docking of compound 6d in the active site of TS
Fig (5) Docking of compound 17a in the active site of TS
Fig (6) Docking of compound 18a in the active site of TS
Fig (7) Docking of MTX in the active site of DHFR
Fig (8) Docking of compound 6a in the active site of DHFR
Fig (9) Docking of compound 6c in the active site of DHFR
Fig (10) Docking of compound 6d in the active site of DHFR
Fig (11) Docking of compound 17a in the active site of DHFR
Fig (12) Docking of compound 18a in the active site of DHFR
22

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Table 1: Mean growth percent, delta values and the percent growth inhibition against some subpanel cell lines of
the selected compounds
Comp.
No.
Mean
growth
percent
88.87
Delta
Panel
Subpanel cell lines (Growth inhibition percent)
1c
67.49
NonSmall Cell Lung Cancer NCI-H23 (78.62)
Breast Cancer
NCI/ADR-RES (74.23)
Melanoma
MALME-3M (55.56)
CNS Cancer
SNB-75 (42.8)
2a
3a
3b
5a
5c
6a
104.84
111.85
113.60
98.77
100.77
65.97
46.33
37.32
43.66
47.25
50.42
73.38
NonSmall Cell Lung Cancer
HOP-92 (41.49)
-
-
-
-
NonSmall Cell Lung Cancer
NonSmall Cell Lung Cancer
NonSmall Cell Lung Cancer
HOP-62 (47.36), HOP-92 (48.48)
HOP-62 (49.65)
EKVX (43.23), HOP-62 (48.67), HOP-92 (107.41), NCI-H522 (40.44)
Breast Cancer
Ovarian Cancer
Leukemia
BT-549 (42.26), HS578T (73.25), MCF7 (43.42), T-47D (62.72)
IGROV1 (70.9)
SR (46.94)
Renal Cancer
A498 (86.3), TK-10 (42.41)
Melanoma
Prostate Cancer
CNS Cancer
MALME-3M (103.74), SK-MEL-2 (58.19), UACC-62 (56.44)
PC-3 (52.98)
SF-295 (45.47), SNB-75 (84.88)
6c
55.91
76.95
NonSmall Cell Lung Cancer
EKVX (51.2), HOP-62 (64.25), HOP-92 (111.63), NCI-H460 (49.93),
NCI-H522 (50.41)
Colon Cancer
Breast Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Melanoma
HCC-2998 (121.04)
MCF7 (73.27), NCI/ADR-RES (59.76), T-47D (81.09)
IGROV1 (79.29), SK-OV-3 (45)
CCRF-CEM (50.39), RPMI-8226 (56.98), SR (82.19)
A498 (101.37), CAKI-1 (41.79), TK-10 (52.91)
M14 (60.23), MALME-3M (97.83), SK-MEL-2 (62.95),
UACC-62 (56.27)
Prostate Cancer
CNS Cancer
PC-3 (57.37)
SF-295 (52.6), SNB-75 (84.12)
6d
84.74
65.94
Non-SmallCell Lung Cancer HOP-92 (44.53), NCI-H23 (81.2)
Breast Cancer
Leukemia
Melanoma
-
NCI/ADR-RES (75.8)
SR (44.56)
MALME-3M (48.7)
-
HCC-2998 (71.82)
-
6g
6h
6i
109.51
104.68
105.38
107.37
42.02
76.50 Colon Cancer
30.33
-
7a
114.25 Colon Cancer
Breast Cancer
HCC-2998 (106.88)
NCI/ADR-RES (42.03)
HCC-2998 (84.43)
M14 (44.95)
9c
9d
108.90
99.24
96.40
96.43
93.33 Colon Cancer
Melanoma
69.50 NonSmall Cell Lung Cancer
Breast Cancer
94.78 Colon Cancer
Breast Cancer
44.17 NonSmall Cell Lung Cancer
Ovarian Cancer
HOP-62 (49.6)
NCI/ADR-RES (70.26)
HCC-2998 (98.38)
NCI/ADR-RES (48.76)
HOP-92 (44.28)
IGROV1 (40.1)
K-562 (41.8), RPMI-8226 (39.71)
RXF393 (47.74)
OVCAR-8 (47.1)
UACC-257 (69.8)
OVCAR-8 (42.4)
10c
11a
Leukemia
Renal Cancer
Ovarian Cancer
Melanoma
11b
11c
101.04
101.77
70.84
73.63 Ovarian Cancer
23

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Melanoma
71.40 Melanoma
54.90 Ovarian Cancer
Renal Cancer
SK-MEL-2 (69.76), UACC-257 (71.86)
11d
11e
100.66
100.28
SK-MEL-2 (70.74)
OVCAR-8 (54.62)
UO-31 (49.09)
NonSmall Cell Lung Cancer
12c
99.46
83.83
NCI-H23 (54.69)
Colon Cancer
Breast Cancer
HCC-2998 (84.37)
NCI/ADR-RES (55.88)
16a
17a
101.58
61.03
61.72 Ovarian Cancer
Melanoma
76.27 NonSmall Cell Lung Cancer
Breast Cancer
OVCAR-8(48.38)
UACC-257 (60.14)
HOP-62 (45.74), HOP-92 (81.74), NCI-H522 (42.1)
BT-549(88.59), HS578T(49.97), MCF7(55.16), MDA-MB-468(44.08),
T-47D (84.58)
Ovarian Cancer
Leukemia
IGROV1 (61.86), SK-OV-3 (71.93)
RPMI-8226 (52.1)
Renal Cancer
A498 (115.24), ACHN (40.76), CAKI-1 (61.43), RXF393 (68.55),
UO-31 (70.47)
Melanoma
Prostate Cancer
MALME-3M (74.29), UACC-62 (48.13)
PC-3 (63.91)
CNS Cancer
SF-295 (90.86), SNB-75 (65.92)
18a
54.02
83.53 NonSmall Cell Lung Cancer
HOP-62 (52.02), HOP-92 (69.26), NCI-H322M (47.68), NCI-H460(66)
Colon Cancer
Breast Cancer
HCT-116 (42.49)
BT-549(70.53), HS578T(93.81), MCF7(95.01), MDA-MB-435(46.98),
T-47D (74.01)
Ovarian Cancer
IGROV1 (129.51), OVCAR-3 (64.62), OVCAR-8 (66.57),
SK-OV-3 (58.89)
Renal Cancer
Melanoma
786-O (61.91), A498 (103.34), ACHN (45.6), CAKI-1
(62.65), SN12C (42.18), TK-10 (59.19), UO-31 (58.27)
M14 (40.12), MALME-3M (76.87), SK-MEL-2 (92.67), SK-MEL-28
(40.07), UACC-257 (82.93), UACC-62 (57.13)
PC-3 (43.49)
Prostate Cancer
CNS Cancer
SF-295 (56.39), SF-539 (43.39), SNB-75 (104.63)
18b
102.36
31.76
-
-
Table 2 GI50, TGI and LC50 of 5-Dose screening of compound 17a
17a (µM/l)
Panel-cell line
Leukemia :
GI50
TGI
LC50
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
> 100
34.1
> 100
28.9
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
91.8
14.1
33.3
> 100
Non-Small Cell Lung :
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
27.9
3.65
4.34
2.44
10.1
4.48
8.31
> 100
> 100
75.3
> 100
> 100
> 100
> 100
> 100
> 100
> 100
8.70
> 100
> 100
> 100
24

ACCEPTED MANUSCRIPT
NCI-H460
NCI-H522
10.3
72.5
> 100
> 100
> 100
> 100
Colon cancer :
HCC-2998
HCT-116
HCT-15
HT 29
KM 12
0.253
18.4
4.06
36.8
27.1
25.0
18.2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
SW-620
CNS cancer :
SF-268
SF-295
13.0
1.46
19.8
84.5
2.15
22.1
> 100
20.4
> 100
> 100
68.2
> 100
> 100
> 100
> 100
> 100
> 100
SF-539
SNB-19
SNB-75
U 251
> 100
Melanoma :
LOX IMVI
MALME-3M
M 14
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
6.48
> 100
> 100
> 100
98.3
> 100
>100
75.5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
2.56
11.9
5.45
17.0
50.7
3.12
Ovarian cancer :
IGROV 1
OVCAR-3
OVCAR-4
OVCAR-5
SK-OV-3
1.79
4.19
85.1
1.70
14.1
23.3
47.8
> 100
> 100
75.2
> 100
> 100
> 100
> 100
> 100
Renal cancer :
786-O
A498
2.70
1.06
3.23
0.635
16.4
21.3
5.28
> 100
31.4
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
ACHN
CAKI-1
SN12C
TK-10
4.63
7.57
UO-31
Prostate cancer :
PC-3
DU-145
22.1
13.4
> 100
> 100
> 100
> 100
Breast cancer :
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
3.92
11.8
10.0
1.15
6.71
3.22
3.59
4.82
> 100
> 100
> 100
6.09
> 100
> 100
22.5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
MDA-MB-468
> 100
25

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Table 3: The MG-MID (GI50) µM/l and the selectivity ratio of compound 17a
Fullpanel GI50
MG-MID
Subpanel tumor cell lines GI50 MG-MID (µM/l)
(µM/l)
(SI)
I
II
III
IV
V
VI
VII
5.18
VIII
17.75
(0.94)
IX
16.65
27.60
(0.60)
16.00
(1.04)
18.60
(0.90)
23.84
(0.70)
13.89
(1.20)
21.38
(0.78)
5.65
(3.22)
(2.95)
ILeukemia; IINon-Small Cell Lung cancer, IIIColon cancer;
IVCNS cancer; V Melanoma; VIOvarian cancer; VIIRenal cancer;
VIIIProstate cancer; IXBreast cancer
Table 4: Inhibition zones (IZ) in mm diameter:
Comp. No.
S. aureus
P. aureginosa
Sh. Flexneri
C.albicans
1c
2a
2b
2c
3a
3b
4
5a
5b
5c
6a
6b
6c
6d
6e
6f
6g
6h
6i
7a
9a
9b
9c
9d
10c
11a
11b
11c
11d
11e
11f
12c
13a
13b
14
16a
16b
17a
17b
18a
18b
15
16
16
17
18
16
16
17
18
16
18
18
16
16
16
17
16
18
16
20
18
16
16
22
18
17
16
17
17
16
18
17
16
19
16
17
16
17
17
16
16
18
21
22
17
24
18
18
19
22
17
17
17
18
22
20
23
17
20
17
17
21
20
20
17
18
17
17
17
24
19
17
17
20
17
17
19
22
18
18
17
18
17
22
22
22
25
22
22
23
22
22
22
22
22
22
22
22
22
22
22
29
25
22
23
30
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
24
25
15
15
18
15
15
15
15
15
15
15
15
15
15
15
18
15
15
18
18
15
15
20
18
19
15
15
15
15
17
15
15
15
15
15
15
15
15
15
15
15
26

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Ampicillin
Ofloxacin
Clotrimazole
Solvent
41
43
-
16
43
-
22
32
-
-
-
40
22
15
16
17
Table 5 : MIC values in µg/ml for some selected compounds and for reference drugs:
Comp. No.
S. aureus
P. aeruginosa
Sh. flexneri
C.albicans
1c
2a
2c
5a
5c
6a
6c
6d
6e
-
-
-
-
125
125
125
125
-
-
-
125
-
-
-
-
-
-
125
125
62.5
125
-
-
-
-
125
-
-
--
-
-
-
-
-
125
125
125
-
6f
125
6g
6i
7a
9a
9b
9c
9d
10c
11c
11e
12c
13a
15
-
125
125
125
-
125
-
125
125
125
-
-
3.91
3.91
-
-
3.91
3.91
125
-
-
-
-
-
-
-
-
125
125
-
-
125
-
-
-
-
-
125
125
-
-
-
-
125
-
125
-
125
-
125
125
-
-
-
-
125
-
125
125
16a
18a
18b
Ampicillin
125
-
-
-
125
-
3.91
-
-
Ofloxacin
-
-
1.95
-
7.81
-
-
Clotrimazole
12.5
27