Synthesis of various fused pyrimidine rings and their pharmacological and antimicrobial evaluation

Article abstract of DOI:10.2298/JSC130528016M

Various fused pyrimidines, such as furo[2,3-d]pyrimidine, triazolo- [1,5-a]pyrimidine and tetrazolo[1,5-a]pyrimidine, were synthesized in the reactions of thioxopyrimidine-6(1H)-ones with ethyl chloroacetate (under different reaction conditions), thiourea and sodium nitrite. Pyrimidine thiones reacted with POCl3/PCl5 to give the chloro derivatives which reacted with sodium azide and thiourea to give tetrazolo[1,5-c]pyrimidines, and pyrimido pyrimidines. Thioxopyrimidine-6(1H)-ones reacted with benzylamine to give pyrrolo[2,3-d]pyrimidinethiones. Theoretical calculation using MIDO/3, Fukui indices and the heat of formation of some compounds were carried out. The pharmacological and antimicrobial activities of some of the synthesized products were also evaluated.

Full text of DOI:10.2298/JSC130528016M

J. Serb. Chem. Soc. 79 (9) 1059–1073 (2014)
UDC 547.853+547.78:615.27/.28+615.277
JSCS–4647
Original scientific paper
Synthesis of various fused pyrimidine rings and their
pharmacological and antimicrobial evaluation
*
MOUNIR A. SALEM , MAGDA I. MARZOUK and NAGLAA F. MAHMOUD
Heterocyclic Synthetic Laboratory, Chemistry Department, Faculty of Science, Ain Shams
University Cairo, Egypt
(Received 28 May 2013, revised 13 February, accepted 4 March 2014)
Abstract: Various fused pyrimidines, such as furo[2,3-d]pyrimidine, triazolo-
[1,5-a]pyrimidine and tetrazolo[1,5-a]pyrimidine, were synthesized in the
reactions of thioxopyrimidine-6(1H)-ones with ethyl chloroacetate (under
different reaction conditions), thiourea and sodium nitrite. Pyrimidine thiones
reacted with POCl3/PCl5 to give the chloro derivatives which reacted with
sodium azide and thiourea to give tetrazolo[1,5-c]pyrimidines, and pyrimido
pyrimidines. Thioxopyrimidine-6(1H)-ones reacted with benzylamine to give
pyrrolo[2,3-d]pyrimidinethiones. Theoretical calculation using MIDO/3, Fukui
indices and the heat of formation of some compounds were carried out. The
pharmacological and antimicrobial activities of some of the synthesized pro-
ducts were also evaluated.
Keywords: fused pyrimidine; thiazoles; pyrimidopyrimidines; antitumor; anti-
oxidants; antimicrobial.
INTRODUCTION
Heterocyclic compounds are of great importance in the synthesis of pharma-
1–4
cologically active compounds. Pyrimidine nuclei are the active core of various
bioactive molecules and are best known as the heterocyclic core of nucleic acid
bases.
In general, heterocyclics encompassing a pyrimidine moiety have found
5–7
8–13
applications in a wide spectrum of biological
and therapeutic areas.
Such
14,15
a ring system is often incorporated into drugs designed as anticancer,
antipyretic, anti-inflammatory,
antifungal, antibacterial and anti-psoriasis agents. Some derivatives are act-
anti-
16
17
18,19
20
21
viral, antihypertensive, analgesic,
22
22
23
24
ive on the blood circulatory system, stimulate skin preparative regeneration and
increase the efficacy of antibiotic therapy of Staphylococcus and proteus-infected
25
wounds. As part of ongoing interest in the synthesis of heterocyclic com-
*Corresponding author. E-mail: salemmai1947@yahoo.com
doi: 10.2298/JSC130528016M
1059
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1060
SALEM, MARZOUK and MAHMOUD
pounds, convenient syntheses of 5-substituted 2-thioxo-4-aryl-1,2,3,6-tetrahydro-
pyrimidin-6-ones and 4-aryl-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-car-
26
bonitrile were reported. The study presented herein dealt with the efficient syn-
thesis of a variety of fused pyrimidine derivatives, and an investigation of their
biological activities.
RESULTS AND DISCUSSION
Chemistry
The hydrolysis of nitriles is generally considered one of the best methods for
the preparation of carboxylic acids. The hydrolysis of nitriles proceeds in the
distinct steps under acid or base treatment to achieve amides and carboxylic
acids. The hydrolysis of the 5-cyano group in 2-thioxopyrimidine 1a in basic
medium was unsuccessful due to the low reactivity of the group. However, the
hydrolysis was successfully achieved with 70 % sulfuric acid as the weakly
nucleophilic nitrile was activated by protonation using H SO to make it more
2
4
electrophilic. This facilitates the reaction to give the corresponding 2-thioxopy-
rimidine-5-carboxylic acid (1g), Scheme 1. The structure of compound 1g was
confirmed by spectroscopic data, the IR spectrum revealed the presence of ν at
OH
–1
1
3416 cm , the H-NMR spectrum showed a band at δ 11.0 ppm for the acidic
OH and the C-NMR spectrum showed the presence of a carboxylic C=O at δ
13
165.2 ppm and the absence of C≡N.
Reaction of 1a and c with hydrazine hydrate afforded the corresponding
2-hydrazino derivative 1h by nucleophilic substitution of the SH or CH S group,
3
respectively.
Masumoto et al. reported that treatment of 1-phenylpyrazolin-5-one with a
base afforded a mixture of three adducts through carbon, nitrogen and oxygen
27
anions. On the other hand, some pyrimidinethiones in the presence of K CO /
2
3
28–30
/dry acetone yielded S-alkylated and/or S-and N-dialkylated products.
Thi-
enopyridin-2-one bearing a cyano group at the α-position to oxygen, reacted with
ethyl chloroacetate in the presence of sodium ethoxide to give the oxygen
alkylated derivative as the sole product, which cyclized to afford the corres-
31,32
ponding furothiopyridine.
The reaction of 1a and b with ethyl chloroacetate in the presence of a base
depended on the reaction conditions. Furthermore, it was found that treatment of
1a and b with ethyl chloroacetate in dry acetone, as a polar aprotic solvent, under
reflux conditions in the presence of anhydrous K CO afforded the S-alkylated
2
3
products 2a and b (Scheme 1). The N-alkylated product (I) and the correspond-
ing cyclic product 7-aryl-2,3-dihydro-3,5-dioxo-5H-thiazolo[3,2-a]pyrimidine-6-
-carbonitrile (II) were not isolated.
To account for these results, theoretical calculations using MIDO/3 and
Fukui indices were performed. As expected, the predicted electron density of the
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FUSED PYRIMIDINES
1061
O
Y
Ar
EtO
N
O
N
H
I
S
N
Ar
1a,b
Y
Ar
N
O
S
anhyd K2CO3
dry acetone
O
O
O
N
H
S
N
Y
Y
O
O
HN
ClCH2COOCH2CH3
2a,b
II
R
N
1
Ar
O
O
1a,b,e,f
a) y = C N
b) y = C N
C) y = COCH3
d) y = COOEt
O
Y
a) R = SH,Y = C N
b) R = SH,Y = C N
N
C2H5ONa
Ethanol
S
N
Ar
c) R = SCH3,Y = C N
d) R = SCH3,Y = C N
e) R = SH,Y = COCH3
f) R = SH,Y = CO2CH2CH3
g) R = SH,Y = COOH
h) R = NHNH2,Y = C N
H
3a-d
NaOC2H5 / fusion 170-180°C
O
O
OEt
O
OEt
O
1a,b,e,f
N
N
Y
O
C2H5ONa
S
S
N
N
fusion 170-180°C
Ar
Ar
H
H
4d
4
a) Y =NH2
b) Y =NH2
c) Y =CH3
d) Y =OH
Scheme 1. Synthesis of compounds 2a and b; 4a and d; Ar = C6H3-3,4-(O–CH2–O) for 1a, c,
g and h, 2a, 3a and 4a; Ar = C6H3-3,4-(OCH3)2 for 1b, d, e, f, 2b, 3b–d and 4b–d.
oxygen atom (–0.298 and –3.05 for 1a and b, respectively) is larger than that of
the sulfur atom (–0.172 and –0.188 for 1a and b, respectively). However, since
the size of the atom plays a significant role in the extent of its nucleophilicity, the
S atom is more nucleophilic than the O atom. Consequently, the reaction pro-
ceeded via S-alkylation and afforded the thermodynamically more stable pro-
–1
ducts 2a (ΔH = –82.03 kcal* mol , ΔE = E
–E
= 8.048 eV) and 2b
f
LUMO HOMO
–E
–1
((ΔH = –83.667 kcal mol , ΔE = E
2a was also confirmed by C-NMR spectroscopy, which evidenced the
= 8.45 eV). The structure of
f
LUMO HOMO
13
presence of S-CH COOC H by the appearance of a signal at δ 159.98 ppm,
2
2 5
which is attributable to –C=S, and not at δ ≈ 180 ppm.
On the other hand, refluxing 1a and b with ethyl chloroacetate in ethanol and
sodium ethoxide gave the corresponding O-alkylated products 3a and b, respect-
ively, Scheme 1. It should be noted that, although 3a and b were predicted to be
–1
thermodynamically less stable, where the (ΔH of 3a is –55.95 kcal mol and
f
–1
ΔE = E
–E
= 7.018 eV, and that of 3b is –56.595 kcal mol and ΔE =
LUMO HOMO
*1 kcal = 4184 J
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1062
= E
SALEM, MARZOUK and MAHMOUD
–E
= 7.44 eV, and in addition, the keto (lactam) form is more
LUMO HOMO
–1
stable than the enol (lactim) form by ca.13 kcal mol , the formation of the
O-alkylated products 3a and b where the O atom with its higher electron density
acted as the nucleophile were facilitated by the presence of the high boiling point,
polar protic solvent and the strengthening of the employed base. In addition,
analysis of the local reactivity of 1b through an evaluation of the Fukui indices
–
+
indicated that the Fukui indices for the S atom were f = 0.643, f = 0.133 and
q = –0.589, while for the O atom they were f = 0.026, f = 0.046 and q =
N
k
k
–
+
k
k
N
= 0.527. The presence of electron withdrawing group (C≡N) in the α-position to
the carbonyl of the amide also acts as an effective factor in the reaction pathway
leading to the formation of 3a and b. In order to confirm this assumption, 1e and
f were prepared and allowed to react under reflux with ethyl chloroacetate in
ethanol and sodium ethoxide, whereby the O-alkylated products 3c and d were
isolated in a good yield as the sole products.
The ethyl 5-substituted-4-aryl-2-thioxo-1,2-dihydrofuro[2,3-d]pyrimidine-6-
-carboxylates 4a–d were obtained via either fusion of 1a, b, e and f with ethyl
chloroacetate at 170–180 °C in the presence of sodium ethoxide or cyclization of
the open chain products 3a–d under similar reaction conditions (Scheme 1).
Treatment of 1a with thiourea in n-butanol afforded the triazolo pyrimidine 6
1
without isolation of the intermediate 5 (Scheme 2). The H-NMR spectrum of 6
showed the lack of an NH group adjacent to the C=O.
33
It was of interest to condense 1a with 4-fluorobenzaldehyde and also with
34
p-benzoquinone, to obtain the bis(pyrimidine-5-carbonitriles) 7 and 9 in 73 %
and 68 % yield, respectively (Scheme 2).
It was found that the reaction of 1h with aqueous sodium nitrite solution in
the presence of acetic acid at 5 °C afforded the corresponding unisolated diazo-
nium salt followed by cyclization to give the tetrazolo pyrimidine 10 in 52 %
1
yield (Scheme 2). The IR spectra and the H-NMR bands showed the absence of
13
the NH group and the appearance of a signal at δ 151.37 ppm in the C-NMR
2
spectrum indicated the formation of the tetrazolo ring.
Treatment of 2-thioxopyrimidines 1a, b and d with a mixture of phosphorus
oxychloride and phosphorus pentachloride afforded the corresponding 4-chloro-
2-thioxopyrimidines 11a–c in 62–74 % yield (Scheme 3). The structures of com-
13
pounds 11b and c were confirmed by their C-NMR spectra, which showed
signals for –C–Cl at δ 153.80 and 158.8 ppm, respectively, rather than at δ ≈ 166
ppm, which is attributable to –C=O. Such compounds were utilized for the
synthesis of several new fused heterocyclic systems bearing 2-thioxo-pyrimidin-
4(1H)-one moieties as an active core of bioactive molecules with expected anti-
35–37
microbial and pharmacological activities.
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FUSED PYRIMIDINES
1063
Scheme 2. Synthesis of compounds 5–10; Ar = C6H3-3,4-(O–CH2–O) for 6, 7, 9 and 10.
Treatment of 11a with bi-functional nucleophilic substrates, i.e., sodium
azide and thiourea, afforded the tetrazolo pyrimidine 13, and pyrimido pyri-
midine 15, respectively (Scheme 3).
Reaction of 2-thioxopyrimidines 1a and b with benzylamine in n-butanol
afforded the pyrrolo pyrimidine-2-thiones 17a and b, respectively, via the for-
mation of the intermediate 16.
Biological activities
Antimicrobial, anticancer and antioxidant activities of some compounds
were investigated using standard methods and the results were compared with
those of standard drugs.
Antimicrobial activity
It was observed that some of the thirteen tested compounds showed good
activities against Gram-positive and Gram-negative bacteria, and the fungi Can-
dida albicans and Aspergillus niger. Compounds 3b, 3d, 4a and 11a showed
inhibition towards all the tested organisms (Table I).
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1064
SALEM, MARZOUK and MAHMOUD
Scheme 3. Synthesis of compounds 11a–c, 13, 15 and 17a and b; Ar = C6H3-3,4-(O–CH2–O)
for 11a and c, 13, 15 and 17a; Ar = C6H3-3,4-(OCH3)2 for 11b and 17b.
The following points were noticed. On comparison between the compounds
1a, 1c and 1g, it was noticed that compound 1a did not inhibit any of the tested
organisms, while conversion of the C=S group to CSCH or CN group into
3
COOH groups in compounds 1c and 1g, respectively, resulted in activity of 1c
against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
and of 1g against S. aureus, P. aeruginosa and C. albicans.
Comparing the esters 2a and 2b, it was noticed that compound 2a showed
activity against S. aureus, E. coli and P. aeruginosa only, while compound 2b
exhibited activity against all the tested organisms, indicating that the presence of
the 3,4-dimethoxy phenyl group in 2a was more effective than the 1,3-benzo-
dioxole group in 2b.
The (aryloxy)acetate compounds 3b and 3d showed activity against all the
tested organisms, while the (aryloxy)acetate 3a showed activity against S.
aureus, E. coli and P. aeruginosa only; this indicated that the presence of the 3,4-
-dimethoxyphenyl group in 3b and 3d is more effective than the 1,3-benzodioxole
group in 3a.
The furocarboxylate compound 4a showed activity against all the tested
organisms while 4c showed activity against S. aureus, E. coli, P. aeruginosa and
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FUSED PYRIMIDINES
1065
C. albicans. 4d exhibited activity against Bacillus subtilis, S. aureus, C. albicans
and A. niger; this indicated that the NH group in 4a is more effective than the
2
CH and OH groups in 4c and 4d, respectively.
3
TABLE I. Antimicrobial activity for some of the products; the well diameter was 1 cm (100
μL of each one was tested); St = standard, that is chloramphenicol at 1 mg mL-1 for Gram-
positive bacteria, cefalexin for Gram-negative bacteria at 1 mg mL-1, fluconazole for A. niger
at 1 mg mL-1 and flucoral for C. albicans at 1 mg mL-1
Inhibition zone diameter, mm
Compound
B. subtilis S. aureus E. coli P. aeruginosa C. albicans A. niger
1a
1c
1g
2a
2b
3a
3b
3d
4a
4c
4d
7
–
–
–
–
24
–
22
24
20
–
20
–
–
24
23
19
21
26
26
28
21
22
26
–
–
21
–
–
14
18
21
23
24
23
26
24
23
–
–
–
23
–
–
–
19
21
22
22
24
–
–
–
–
–
–
18
26
18
24
26
22
20
–
–
20
22
20
–
26
26
20
23
–
22
32.5
24
18
32.5
11a
St
20
32.5
26
32.5
22
28
Compound 7 exhibited activity against P. aeruginosa and A. niger. Con-
version of C=O group in 1a to a chlorine atom in 11a resulted in inhibition of all
the tested organisms.
Pharmacological activity
Antitumor activity using the in vitro Ehrlich ascites assay. Cancer still con-
tinues to be a major health problem worldwide. The development of new anti-
cancer therapeutic agents is one of the fundamental goals in medicinal chemistry.
Compound 4d proved to have the highest cytotoxic activity, 100 % mortality
with 5-flurouracil as a standard, followed by 4b, 11b, 1b and 6, having mortal-
–1
ities of 97.5, 93.5, 91 and 84.1 %, respectively, at 100 μg mL . Compounds 1e,
1a and 11a exhibited medium activity, while compounds 17a, 1f, 10 and 2b
showed low activity. The variations of inhibition of the Ehrlich antitumor activity
with concentration of the test compounds are listed in Table II.
Antioxidant activity using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) inhibition. Twelve compounds were tested for antioxidant activity
as reflected in their ability to inhibit oxidation in rat brain and kidney homo-
genates, Table II. Compounds 4d, 11b, 6, 4b and 1b showed a similar antioxidant
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1066
SALEM, MARZOUK and MAHMOUD
activity to ascorbic acid, used as standard. Compounds 1e, 1a, 10 and 11a exhi-
bited moderate antioxidant activity, while 1f, 17a and 2b showed lower activity.
TABLE II. Antitumor (dead, %) and antioxidant activities (ABTS method) for some of the
products
Antitumor activity
Antioxidant activity
Bleomycin dependent-
DNA damage
c / µ mL-1
Compound
100
68.9
91
50
37
43
25 Absorbance Inhibition, %
20
18
1a
1b
1e
1f
2b
4b
4d
6
0.213
0.038
0.201
0.447
0.485
0.056
0.033
0.049
0.22
0.300
0.045
0.453
–
59.4
89.1
61.7
14.8
7.6
89.3
93.7
91.4
56.4
42.8
92.8
13.7
–
0.118
0.079
0.124
0.140
0.173
0.090
0.086
0.089
0.129
0.138
0.084
0.159
–
79.8 38.6 21.1
45.7 28.6
33.1 28.6
14
14
97.5
63
36.7
100 100 98.2
84.1 11.4 25.6
36.8 19.1 11.6
10
11a
11b
17a
Sta
ABTS
56
93.5
30
48
17.2
21.4
48.2 27.3 13.8
98
–
62.7
–
37
–
0.525
0.029
–
94.5
–
Ascorbic acidb
a
–
–
–
0.093
b
5-Flurouracil was used as standard for the antitumor activity; standard for the antioxidant activity
Bleomycin-dependent DNA damage. The bleomycins are a family of glyco-
38
peptide antibiotics that are routinely used as antitumor agents. The bleomycin
assay has been adopted for assessing the pro-oxidant activity of food antioxi-
dants. The antitumor antibiotic bleomycin binds iron ions and DNA. If the
3+
2+
samples to be tested are able to reduce bleomycin–Fe to bleomycin–Fe ,
DNA degradation in the system will be stimulated, resulting in a positive test for
pro-oxidant activity. DNA degradation is accompanied by the formation of a
product similar to malondialdehyde. L-ascorbic acid was used as the reducing
3+
2+
agent to reduce Fe to Fe . Twelve compounds were selected for bleomycin-
dependent DNA-damage testing (Table II).
Results in Table II showed that compounds 1b, 4d, 4b, 6 and 11b have the
ability to protect DNA from the damage induced by bleomycin. On the other
hand, the rest of the compounds exhibited weak activities. By comparing the
obtained results of the investigated compounds to their structures the following
structure–activity relationships were postulated; i) compounds 1b, 4b, 4d, 6 and
11b were more potent than ascorbic acid, which may be attributed to the presence
of the thioxopyrimidine moiety in 1b, 4b, 4d, and 11b, and the thioxo triazolo
moiety in 6; ii) compounds 4b and 11b were less potent than 1b, which may be
due to the replacement of the C=O and C≡N moieties into ethyl aminofuran-
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FUSED PYRIMIDINES
1067
carboxylate in compound 4b and C=O into chlorine atom in compound 11b; iii)
compound 6 was more potent than compound 1a, which may be attributable to
the presence of the thioxotriazolo moiety.
EXPERIMENTAL
Chemistry
Chemicals were obtained from Alfa Aesar and used as provided. Melting points were
measured on a Gallenkanp or a Griffin melting point apparatus. The infrared absorption
spectra were measured on a Pye Unicam SP 2000 infrared spectrophotometer using the KBr
wafer technique. The EI-MS spectra were determined using an AE1 MS 902 mass spectro-
1
meter. The H-NMR and 13C-NMR spectra were recorded on a Bruker AC-300 instrument at
300 and 75 MHz, respectively, using DMSO-d6 as the solvent. Chemical shifts are expressed
as δ / ppm and TMS was used as an internal standard. All the spectral measurements were
performed at the Micro analytical Center of Cairo University, Egypt, the Micro analytical
Center of Ain Shams University or the Main Defense Chemical Laboratory. Elemental anal-
ysis were realized at the Faculty of Science, Ain Shams University, using a Perkin–Elmer
2400 C, H and N elemental analyzer and satisfactory analytical data (±0.3 %) were obtained
for all compounds. The antimicrobial activities were determined at Al-Azhar University,
Faculty of Science, Fermentation Biotechnology and Applied Microbiology (Ferm-BAM)
Center, Egypt. The pharmacological activities were performed at the Pharmacology
Department, Faculty of Pharmacy, Mansoura University, Egypt. The completion of chemical
reactions was monitored by TLC. Compounds 1a–f were prepared according to the litera-
ture.26
The physical, analytic and spectral data for the prepared compounds are given in
Supplementary Material to this paper.
Synthesis of 6-(1,3-benzodioxol-5-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxylic acid (1g). A mixture of 1a (2.73 g, 10.0 mmol) and sulfuric acid (15 mL, 70 %) was
heated under reflux for 3 h. The reaction mixture was left to cool and then poured onto cold
water. The obtained solid was filtered off, dried and recrystallized from benzene. Pale brown
crystals, Yield: 53 %
Synthesis of 4-(1,3-benzodioxol-5-yl)-2-hydrazino-6-oxo-1,6-dihydropyrimidine-5-carbo-
nitrile (1h). To a solution of 1a or 1c (0.01 mol) in n-butanol (40 mL), hydrazine hydrate
(0.75 mL, 0.015 mol, 98 %) was added and the reaction mixture was heated under reflux for 6
h. The solvent was evaporated and the obtained solid was collected by filtration, washed with
water, dried and then recrystallized from methanol. Brown crystals, yield: 65 % from 1a and
72 % from 1c.
General procedure for the synthesis of (dihydropyrimidin-2-ylthio)acetates 2a and b. A
mixture of 1a or 1b (0.01 mol), ethyl chloroacetate (1.3 mL, 0.012 mol) and anhydrous
K2CO3 (5.0 g, 40 mmol) in dry acetone (60 mL) was refluxed for 18 h and then filtered while
hot, concentrated to half its volume and poured onto cold water. The obtained solid was
filtered off, dried and recrystallized from a suitable solvent to afford 2a and 2b, respectively.
2a: brown crystals (acetic acid), yield: 71 %, and 2b: orange crystals (toluene/few drops of
light petroleum 60–80 °C), yield: 72 %.
General procedure for the synthesis of (dihydropyrimidin-4-yloxy)acetates 3a–d. A mix-
ture of 1a, 1b, 1e or 1f (0.01 mol) and ethyl chloroacetate (1.3 mL, 0.012 mol) in sodium
ethoxide solution (0.46 g of Na in 20 mL of absolute ethanol) was heated under reflux for 2 h.
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1068
SALEM, MARZOUK and MAHMOUD
Most of the solvent was evaporated and then the remaining acidified with cold diluted HCl
(20 mL, 2 M). The oily residue obtained was extracted with ethyl acetate (3×50 mL) and dried
over anhydrous MgSO4. The solid obtained after evaporation of the solvent was filtered off,
dried and recrystallized from a suitable solvent to give 3a–d. 3a: brown crystals (acetic acid),
yield: 57 %; 3b: orange crystals (toluene/light petroleum, 60–80 °C, 1:1 V/V), yield: 61 %; 3c:
yellow crystals (acetic acid), yield: 70 %; 3d: pale brown crystals (acetic acid), yield: 64 %.
General procedure for the synthesis of furo[2,3-d]pyrimidine-6-carboxylates 4a–d. A
mixture of 1a, 1b, 1e or 1f (0.01 mol) and ethyl chloroacetate (1.30 mL, 0.012 mol, 99 %) in
sodium ethoxide solution (0.92 g of Na in 40 mL of absolute ethanol) was fused at 170–180
°C for 3 h. The reaction mixture was left to cool, poured onto ice water and acidified with HCl
(15 mL, 2 M). The product was extracted with ethyl acetate (3×40 mL), dried over anhydrous
MgSO4 and most of the solvent was evaporated. The obtained solid was filtered off, dried and
recrystallized from an appropriate solvent to give 4a–d. 4a: brown crystals (methanol), yield:
63 %; 4b: brown crystals (ethanol), yield 61 %; 4c: brown crystals (ethanol), yield 66 %; 4d:
brown crystals (toluene), yield 55 %.
Synthesis of authentic samples of 4a and c. A mixture of 3a or 3c (0.01 mol) and sodium
ethoxide (0.46 g of Na metal in 20 mL of absolute ethanol) was heated at 170–180 °C for 1 h.
The reaction mixture was left to cool, poured onto ice water and then acidified with HCl (15
mL, 2 M). The solid obtained was filtered off, dried and recrystallized from a proper solvent
to yield 4a or 4c.
Synthesis of 5-(1,3-benzodioxol-5-yl)-7-oxo-2-thioxo-1,2,3,7-tetrahydro[1,2,4]triazolo-
[1,5-a]pyrimidine-6-carbonitrile (6). A mixture of 1a (2.73 g, 10.0 mmol) and thiourea (0.94
g, 0.012 mol, 97 %) in n-butanol (40 mL) was heated under reflux for 4 h. Most of the solvent
was evaporated and the obtained solid was filtered off, washed with water, dried and recrys-
tallized from ethanol. Brown crystals, yield: 76 %.
Synthesis of 3,3′-[(4-fluorophenyl)methylene]bis[6-(1,3-benzodioxol-5-yl)-4-oxo-2-thi-
oxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile] (7). A mixture of 1a (5.46 g, 20.0 mmol)
and 4-fluorobenzaldehyde (1.27 g, 10.0 mol) in acetic anhydride (15 mL) in the presence of a
catalytic amount of anhydrous K2CO3 was heated at 70–80 °C for 3.5 h. The solid obtained
after cooling was filtered off, dried and then recrystallized from ethanol. White crystals, yield:
73 %.
Synthesis of 3,3′-(3,6-dioxocyclohexa-1,4-diene-1,4-diyl)bis[6-(1,3-benzodioxol-5-yl)-4-
-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile] (9). A mixture of 1a (5.46 g, 0.02
mol) and p-benzoquinone (1.30 g, 0.012 mol) in aqueous acetone (80 mL, 50 %) was stirred
for 6 h at room temperature. The reaction mixture was kept overnight and then poured onto
ice/water. The obtained solid was filtered off, washed with hot water, dried and recrystallized
from ethanol/toluene (2:1 by volume). Pale brown crystals, yield: 68 %.
Synthesis of 5-(1,3-benzodioxol-5-yl)-7-oxo-1,7-dihydrotetrazolo[1,5-a]pyrimidine-6-car-
bonitrile (10). An aqueous solution of sodium nitrite (1.07 g, 15.0 mol) in water (10 mL) was
added to a stirred cold (0 °C) solution of 1 h (2.7 g, 10 mmol) in acetic acid (30 mL). The
reaction mixture was stirred at 0 °C for 3 h, poured onto ice/saturated NaHCO3 (40 mL) and
the product was extracted with ethyl acetate (60 mL). The organic layer was dried over
anhydrous MgSO4 and half of the solvent was removed. The obtained solid was filtered off,
and recrystallized from dioxane. Brown crystals, yield: 52 %.
General procedure for the synthesis of dihydropyrimidine-5-carbonitriles 11a–c. A mix-
ture of 1a, 1b or 1d (0.01 mol), phosphorus oxychloride (20 mL) and phosphorus penta-
chloride (1.5 g, 7.0 mmol) was heated on a water bath for 4 h. The reaction mixture was
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FUSED PYRIMIDINES
1069
poured onto cold water. The obtained solid was filtered off, washed several times with light
petroleum 60–80 °C, dried and recrystallized from a suitable solvent. 11a: brown crystals
(ethanol), yield: 74 %; 11b: brown crystals (ethanol), yield: 62 %; 11c: brown crystals
(ethanol), Yield: 64 %.
Synthesis of 7-(1,3-benzodioxol-5-yl)-5-thioxo-5,6-dihydrotetrazolo[1,5-c]pyrimidine-8-
-carbonitrile (13). To a stirred solution of 11a (2.9 g, 10 mmol) in DMF (30 mL), sodium
azide (0.76 g, 12 mmol) was added in portions and the mixture was stirred at room tem-
perature for 4 h. The mixture was poured onto ice water and the obtained solid was filtered
off, dried and then recrystallized from methanol. Yellow crystals, yield: 54 %.
Synthesis of 4-amino-5-(1,3-benzodioxol-5-yl)pyrimido[4,5-d]pyrimidine-2,7(1H,3H)-
-dithione (15). A mixture of 11a (2.8 g, 0.010 mol), thiourea (0.69 g, 0.012 mol) and few
drops of piperidine in n-butanol (40 mL) was heated at 130–140 °C for 4 h. The mixture was
left to cool to room temperature and poured onto cold HCl (40 mL, 2 M). The obtained solid
was filtered off, washed with water (3×30 mL), dried and recrystallized from n-butanol.
Yellow crystals, yield: 62 %.
Synthesis of 5-amino-4-aryl-6-phenyl-1,3-dihydro-2H-pyrrolo[2,3-d]pyrimidine-2-thiones
17a and b. A mixture of chloropyrimidine 1a or b (0.01 mol) and benzylamine (1.35 mL, 12.0
mmol) in n-butanol (40 mL) was heated under reflux for 4 h. The reaction mixture was
concentrated to half its volume and poured onto cold water. The obtained solid was filtered
off, dried and recrystallized from the proper solvent. 17a: brown crystals (DMF), yield: 72 %;
17b: brown crystals (n-butanol), yield: 67 %.
Biological and pharmaceutical activities
Antimicrobial activity. Some of the products were screened for their antimicrobial acti-
vity including Gram-positive bacteria (B. subtilis and S. aureus), Gram-negative bacteria (E.
coli and P. aeruginosa) and antifungal activity (C. albicans and A. niger).
Agar well diffusion method. The agar well diffusion method39 was used for the deter-
mination of the inhibition zone. DMSO was used as the solvent and the blank with appropriate
drugs as standards. The results are given in Table I. With a sterile loop, pure colonies of the
bacterial cultures were picked up. The colonies were suspended in 5 mL of sterile physio-
logical saline. Using sterile forceps, the wells containing the biomaterial were placed onto the
agar surface and gently pressed down to ensure contact. The plates were per-incubated for 1 h
in a refrigerator followed by incubation at 37 °C for 24 h.
Cell line and cell culture. Different concentrations of the tested compounds were pre-
pared (ED100, ED50 and ED25 as μg/ml DMSO). The amount of DMSO was adjusted to give a
final concentration of 0.1%. Ascites fluid was obtained from the peritoneal cavity of the donor
animal from (National cancer Institute, Cairo, Egypt) contain Ehrlich cell was as aseptically
aspirated. The cells were grown partially floating and attach in a suspension culture (RPMI
1660 medium, Sigma Chemical Co. St. Louis, USA), supplemented with 10% fetal bovine
serum 9GIBCO, UK). They were maintained at 37 °C in humidified atmosphere with 5% CO2
for 2 h. The viability in control experiments (DMSO only without drug) exceeded 95% as
determined by microscopical examination using a hemocytometer and trypan blue stain (stains
only the dead cells).40
MTT assay. Briefly, cells were seeded in a 96-well plate at a density of 1×104 cells well-1
as previously described.41-43 Drugs at different concentrations were added to each well and
cultured for 48 h, followed by incubation with 5 mg L-1 MTT (3-(4,5-dimethylthiazol-2-yl)-
-2,5-diphenyltetrazolium bromide) for 4 h, after which the supernatant was removed after
centrifugation. Finally, 100 µL of DMSO was added and the absorbance, A, at a wavelength
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1070
SALEM, MARZOUK and MAHMOUD
of 490 nm was measured by using an Elisa Reader EXL 800. The relative cell proliferation
inhibition rate (IR) is given by:




A490,exp
IR = 1−
×100



A490,control

where A490,exp and A490,control are the absorbances at 490 nm for the experimental and control,
respectively.
Cytotoxic activity. Ehrlich cells44-46 (Ehrlich ascites carcinoma, EAC) were derived from
the ascitic fluid from diseased mouse (purchased from the National Cancer institute, Cairo,
Egypt). The cells were grown in suspension culture, partly floating and partly attached, in
RPMI 1640 medium, supplemented with 10 % fetal bovine serum. They were maintained at
37 °C in a humidified atmosphere with 5 % CO2. The viability of the cells used in the control
experiments (DMSO only without drug) exceeded 95 %, as determined with trypan blue. The
test compounds were prepared initially at a concentration of 1 mg mL-1 DMSO.
Antioxidant activity screening assay (ABTS method). For each of the investigated com-
pounds,47,48 2 mL of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) solution
(60 µM) was added to 3 mL MnO2 suspension (25 mg mL-1), all prepared in 5 mL aqueous
phosphate buffer solution (pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered and the
absorbance of the resulting green blue solution (ABTS radical solution) at 734 nm was
adjusted to ca. 0.5. Then, 50 µl of a 2 mM solution of a test compound in spectroscopic grade
MeOH/phosphate buffer (1:1) was added. The absorbance was measured and the reduction in
the color intensity was expressed as inhibition percentage. L-Ascorbic acid was used as the
standard antioxidant (positive control). A blank sample was run without ABTS and using
MeOH/phosphate buffer (1:1) instead of a test compound. A negative control was run with
ABTS and MeOH/phosphate buffer (1:1) only.
Bleomycin-dependent DNA damage assay. To the reaction mixtures in a final volume of
1.0 mL, the following reagents at the final concentrations were added: DNA (0.5 mg mL-1),
bleomycin sulfate (0.05 mg mL-1), FeCl3 (0.025 mM), magnesium chloride (5 mM), KH2PO4–
–KOH buffer pH 7.0 (30 mM), and ascorbic acid (0.24 mM) or a test fraction diluted with
MeOH to give a concentration of 0.1 mg mL-1. The reaction mixtures were incubated in a
water bath at 37 °C for 1 h. At the end of the incubation period, 0.1 mL ethylenediamine-
tetraacetic acid (EDTA) (0.1 M) was added to stop the reaction (the iron–EDTA complex is
unreactive in the bleomycin assay).49 DNA damage was assessed by adding 1 mL 1 % (w/V)
thiobarbituric acid (TBA) and 1 mL of 25 % (V/V) hydrochloric acid followed by heating in a
water-bath maintained at 80 °C for 15 min. The chromogen formed was extracted into
1-butanol, and the absorbance was measured at 532 nm.
CONCLUSIONS
The type of the products from reactions of thioxopyrimidin-6(1H)-ones with
ethyl chloroacetate were found in dependence on the reaction conditions to afford
S-alkylated or O-alkylated or furo[2,3-d]pyrimidine products. Reactions of 2-thi-
oxo-4-chloropyrimidine with bi-functional nucleophiles provided a convenient
route for the synthesis of the corresponding tetrazolo[1,5-c]pyrimidine, pyri-
mido[4,5-d]pyrimidine and pyrrolo[2,3-d]pyrimidine. Some of the products exhi-
bited promising antimicrobial, antitumor and antioxidant activities.
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___________________________________________________________________________________________________________________________
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FUSED PYRIMIDINES
1071
SUPPLEMENTARY MATERIAL
The physical, analytic and spectral data for the prepared compounds are available
electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
И З В О Д
СИНТЕЗА РАЗЛИЧИТИХ КОНДЕНЗОВАНИХ ПИРИМИДИНА И ИСПИТИВАЊЕ
ФАРМАКОЛОШКЕ И АНТИМИКРОБНЕ АКТИВНОСТИ
MOUNIR A. SALEM, MAGDA I. MARZOUK и NAGLAA F. MAHMOUD
Heterocyclic Synthetic Laboratory, Chemistry Department, Faculty of Science,
Ain Shams University Cairo, Egypt
Синтетисана је група деривата пиримидина који садрже кондензоване прстенове,
као што су фуро[2,3-d]пиримидин, триазоло[1,5-a]пиримидин и тетразоло[1,5-a]пири-
мидин, реакцијом тиоксопиримидин-6(1H)-онa са етил-хлорацетатом, под различитим
реакционим условима. Пиримидин-тиони у реакцији са POCl3/PCl5 дају хлор-деривате
који реакцијом са натријум-азидом и тиоуреом дају као производе тетразоло[1,5-c]пири-
мидин и пиримидо-пиримидине. Тиоксопиримидин-6(1H)-он реагује са бензил-амином
и као производ даје пироло[2,3-d]пиримидинтионе. Извршена су израчунавања топлота
стварања једињења применом Фукуи индекaса. Испитана је фармаколошка и антимик-
робна активност неким од синтетисаних деривата.
(Примљено 28. маја 2013, ревидирано 13. фебруара, прихваћено 4. марта 2014)
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