6-Amino-2-thio- and 6-aminouracils as precursors for the synthesis of antiviral and antimicrobial methylenebis(2-thiouracils), tricyclic pyrimidines, and 6-alkylthiopurine-2-ones

Article abstract of DOI:10.1007/s00706-007-0753-8

Several derivatives of 5-arylmethylenebis(1-methyl-6-amino-2-thiouracils) and 5-aryldipyrimidopyridines were prepared by stirring of 6-amino-1-methyl-2- thiouracil and 6-amino-1-benzyluracil with different aromatic aldehydes in ethanol in the presence of

Full text of DOI:10.1007/s00706-007-0753-8

Monatshefte fu¨r Chemie 139, 161–168 (2008)
DOI 10.1007/s00706-007-0753-8
Printed in The Netherlands
6-Amino-2-thio- and 6-Aminouracils as Precursors for the Synthesis
of Antiviral and Antimicrobial Methylenebis(2-thiouracils), Tricyclic
Pyrimidines, and 6-Alkylthiopurine-2-ones
Shaker Youssif1;y;ꢀ and Sahera F. Mohamed2
1
Medical Chemistry Division, Faculty of Medicine, University of Jazan, Jazan, Saudi Arabia
2
Department of Chemistry, Faculty of Education for Girls, Jazan, Saudi Arabia
Received May 6, 2007; accepted June 24, 2007; published online November 23, 2007
# Springer-Verlag 2007
Summary. Several derivatives of 5-arylmethylenebis(1-
cleic acids and their constituents have gained special
methyl-6-amino-2-thiouracils) and 5-aryldipyrimidopyridines
were prepared by stirring of 6-amino-1-methyl-2-thiouracil
and 6-amino-1-benzyluracil with different aromatic aldehydes
in ethanol in the presence of HCl or refluxing with AcOH.
On the other hand, 6-alkylthio-3,9-dimethylpurine-2-ones were
synthesized by the alkylation of 3,9-dimethyl-6-thioxanthine
which was prepared by treatment of 3,9-dimethylxanthine
with P2S5. The structures of the novel compounds were char-
acterized by spectroscopic methods. The effects of the novel
compounds on both HAV and HSV type 1 were investigated.
Also, some compounds showed inhibitory effects on Gram-
positive and Gram-negative bacteria as well as yeast and
fungi.
significance since some of them exhibit considerable
cytostatic and antiviral activities [6]. In particular,
sulfur-containing analogous of purine bases, such
as 6-mercaptopurine and its derivatives, have been
widely used as drugs in cancer chemotherapy [7].
Unfortunately, few data are available to evaluate
the putative altered function of nucleic acids in cells
treated with thiopurines. Several reports have de-
scribed effects of 6-thioguanine on RNA metabolism
and protein synthesis, which may be a result of in-
corporation of the drug into RNA [8]. It has been
reported that 6-mercaptopurine reduced the trans-
forming activity. Bacillus subtilis UTH-8505 has
been grown in the presence of 6-thioguanine and
6-mercaptopurine. DNA isolated from these and con-
trol cultures were used to transform B. subtilis T3, a
strain lacking tryptophan synthetase, to prototrophy.
6-Mercaptopurine exposure decreased the transform-
ing activity by 80 and 6-thioguanine by 20% [9].
Due to the importance of N-substituted 6-amino-
2-thiouracils with respect to their antitumor activity,
and in extension to our work [10, 11], we look to
prepare new fused thiouracils and study their antimi-
crobial and antiviral activities. Herpes simplex virus,
HSV (Herpesviridae is a family of enveloped DNA
viruses that occurs in man, cold–blooded vertebrates,
and in vertebrates. HSV-1 and HSV-2 are the most
common causes of genital ulceration in developed
Keywords. 6-Amino-1-methyl-2-thiouracil; 5-Arylmethyl-
enebis(1-methyl-6-amino-2-thiouracils); 5-Aryldipyrimido-
pyridines; 6-Alkythiopurines; Antiviral and antimicrobial
activity.
Introduction
In 1943, Astwood discovered the high antithyroid
activity and low toxicity of 2-thiouracil [1] and many
derivatives of this compound have been prepared and
tested for physiological activity [2–5]. Amongst a
variety of compounds known to interfere with the
metabolism in proliferating cells, antagonists of nu-
y
On leave from Chemistry Department, Faculty of Science,
Zagazig University, Zagazig, Egypt
ꢀ
Corresponding author. E-mail: syoussifzg@yahoo.com

162
S. Youssif and S. F. Mohamed
countries. In the United States only, approximately
500000 new cases of herpes are reported each year.
Genital herpes is life long and may result in painful
and recurrent lesions and systemic complications.
However, the contribution of asymptomatic viral
shedding to the genital transmission of herpes in the
population may account for as much as 50 to 90% of
cases [12]. Also it has been reported that HSV-1 and
HSV-2 are neurotropic viruses which may spread to
the brain during primary or recurrent infections.
HSV-1 is the most usual cause of sporadic encepha-
litis, except in neonates, with a mortality rate of 75%
and severe permanent sequelae in survivors [13].
Hepatitis AVirus (HAV) is classified with the entero-
virus group of the picornaviridae family. HAV has
a single molecule of RNA surrounded by a small
(27 nm diameter) protein capsid. HAV causes the
disease Hepatitis A, it is usually a mild illness char-
acterized by sudden onset of fever, malaise, nausea,
and abdominal discomfort. HAV is excreted in feces
of infected people and can produce clinical diseases
when susceptible individuals consume contaminated
water or food.
Results and Discussion
Chemistry
In the presence of hydrochloric acid at room temper-
ature or refluxing with glacial acetic acid, stirring of
1-methyl-6-amino-2-thiouracil (1) in absolute etha-
nol with different aromatic aldehydes afforded the
5-arylmethylenebis(6-amino-1-methyl-2-thiouracils)
2–7 in 44–69% yield and starting material. Under
Scheme 1

6-Amino-2-thio- and 6-Aminouracils as Precursors
163
Scheme 2
1
the same conditions the treatment of 6-amino-1-
benzyluracil (8) with different aromatic aldehydes
afforded the cyclic pyrido[2,3-d][6,5-d]dipyrimi-
dines 9–14 in 36–49% yield and starting material
as shown in Scheme 1. An explanation for the for-
mation of the open-chain compounds 2–7 may come
from the presence of sulfur in position 2, which de-
activates the uracil ring, increases the ꢀ-bond char-
acter of C¼NH2, and prevents cyclization contrary to
the case of oxygen in compounds 9–14. 1H NMR for
compounds 2–7 showed the absence of ꢁ ¼ 4.53 ppm
characteristic for CH-5 of uracil 1 and presence of
ꢁ ¼ 7.56–7.59 ppm characteristic for 2NH2 at C-6,60.
13C NMR showed ten signals for compound 2. The
1H NMR spectrum for compounds 9–14 showed
the absence of ꢁ ¼ 7.56–7.59 ppm characteristic for
NH2-6 and presence of ꢁ ¼ 11.23–11.98 ppm charac-
teristic for NH-10.
It has been reported that 6-thioguanine acts as
an inhibitor of the growth of Lactobacillus casei
[14, 15], embryonic tissue [16], and a number of
neoplasms, and 2-alkylthiopurines act as amplifiers
of the antibiotic activity of phleomycin [17]. In ad-
dition, 6-mercaptopurines and their analogues have
been a synthetic target for therapeutic areas of can-
cer [18], viral infections [19] in CNS, as ligands
for benzodiazepine receptors [20], and it is clinically
used in the treatment of leukaemia [18]. We look to
synthesize 6-mercaptopurine analogues which might
exhibit biological activities. In this work, we used
3,9-dimethylxanthine (15) [21, 22] as a starting ma-
terial. Several 6-alkylthio-3,9-dimethylpurine-2-ones
17–20 were prepared by refluxing 15 with phospho-
rous pentasulfide in dry pyridine to afford 6-thiox-
anthine 16. This was followed by the treatment with
different alkyl halides, such as methyl, ethyl, and
butyl iodide, and benzyl bromide in 2 N NaOH
affording 17–20 as shown in Scheme 2. The struc-
tures of compounds 17–20 were corroborated by 1H
NMR and UV spectra. H NMR for compound 17
showed the disappearance of the signal of the NH-1
proton at ꢁ ¼ 12.3 ppm for compound 16 and the
presence of a signal at ꢁ ¼ 4.08 ppm characteristic
of S–CH3-6.
Pharmacology
The antiviral activities of the synthesized compounds
are shown in Table 1. The first group of the com-
pounds 2–7 has various virucidal effects. Their ac-
tivities were modified by substituent variations, and
certain trends are apparent. The unsubstituted com-
pound 2 showed a virucidal effect with 45% at con-
centration of 20 ꢂg=cm3 against HSV-1, where
a methoxy substituted, R1 ¼ OCH3 (5), showed no
change in the antiviral activity against HSV but de-
creased the activity against HAV-27. A halogen sub-
stituent, R2 ¼ F (3), showed also lower activity
against both HSV and HAV-27 than 2. Concurrent
a methoxy substitution, R ¼OCH3 (4), resulted in
the best activity with a concentration of 20 ꢂg=cm3
against HSV-1 with a higher inhibition percentage of
63%. Secondly, compounds of 5-aryldipyrimidopyri-
dines 9–14 showed promising results. The unsub-
stituted compound 9 showed moderate antiviral
activity of 44.5%, while substitution with methoxy,
R1¼OCH3 as in 12, and with halogen, R and R2¼Cl
(14), resulted in disappearance of the virucidal activ-
ity of 0% against HSV-1. The most promising ac-
tivity appeared with OH substituted compound 13
where the virucidal activity was 95.7 and 100% at
concentrations of 10 and 20 ꢂg=cm3, its EC50 was
<5 ꢂg=cm3. This compound is comparable with the
first antivirally active nucleoside analogue [23]. It
has been reported that 2-amino-7-[(1,3-dihydroxy-
2-propoxy)methyl]purine (S2242) represents the first
antivirally active nucleoside analogue with the side
chain attached to the N-7 position of the purine ring.

164
S. Youssif and S. F. Mohamed
Table 1. Antiviral activities of the 5-arylmethylenebis(6-amino-1-methyl-2-thiouracils) 2–7 and 5-aryldipyrimidopyridines 9–14
Compound
Ar
HAV-27
% of virucidal effect
HSV-1
% of virucidal effect
50%
inhibitory
concentration
50%
inhibitory
concentration
10 ꢂg=cm3
20 ꢂg=cm3
10 ꢂg=cm3
20 ꢂg=cm3
S2242
2
ND
>20
–
0.1–0.2
>20
>20
9.25
22.22
>20
>20
>20
>20
20
C6H5
C6H4-4-F
0
0
0
0
0
26
6
23
0
36
43
43
40
0
33
10
36
ND
ND
54
31
ND
0
45
32
63
45
3
4
5
6
7
9
10
11
12
13
14
C6H4-2-OCH3
C6H4-3-OCH3
C6H4-4-OH
C6H3-2,4-Cl
C6H5
–
–
>20
>20
23.25
>20
–
27
6
35.5
44.5
31
50
0
36
23
0
23
0
0
C6H4-4-F
27
27
0
95.7
0
C6H4-2-OCH3
C6H4-3-OCH3
C6H4-4-OH
C6H3-2,4-Cl
>20
–
–
<5
–
100
0
36
–
ND, Not determined. Each value is the mean of triplicate assays
Compound S2242 strongly inhibits the in vitro rep-
lication of both herpes simplex viruses, HSV-1 and
HSV-2.
inhibitory effect of 6 extended until the concentra-
tion of 5 ꢂg=cm3 and for 7 to 10 ꢂg=cm3 demonstrat-
ing the high activity of these two compounds against
B. subtilis. These results are comparable with the
reported data [24] for other nucleoside analogues,
6-(3-ethyl-4-methylanilino)uracil (EMAU) and 6-
[(3,4-trimethylene)anilino]uracil (TMAU), which
showed inhibitory effect against B. subtilis BD54
with MIC of 9 ꢂg=cm3. Compounds 17–20 showed
Concerning the antibacterial and antifungal activ-
ity, all synthesized compounds were tested against
Gram-positive and -negative bacteria, yeast, and
fungi. The first group of the synthesized compounds
2–7 showed various activities dependent on sub-
stitution. The unsubstituted compound 2 showed no
effect on both Gram-positive and -negative bacteria
whereas compounds 2–5 showed a moderate activity
against both C. tropicalis and A. niger. The substitu-
tion R2 ¼ F resulted in compound 3 with a broad
spectrum of antimicrobial activity. It showed a good
antimicrobial activity against all tested microor-
ganisms. The other important observation was the
substitution with Cl, which resulted in the only an-
tibacterial compound 7. Compounds 4–6 showed
inhibition activity only against the Gram-positive
bacterium B. subtilis. The second group of the
synthesized compounds showed no activity against
C. tropicalis. On the other hand, compound 10
showed activity against B. subtilis, whereas E. coli
was affected only by 13 and A. niger was affected
only by 14. These results showed that the second group
of the synthesized compounds has poor antimicrobi-
al activity and the most promising compounds were
6 and 7 which showed the best inhibitory effect
against B. subtilis. Thus the MIC was determined
for both compounds and the results showed that the
Table 2. Antimicrobial activity of the newly synthesized
compounds 2–19
Compound
Diameter of the inhibition zone=mm
B. subtilis E. coli C. tropicalis A. niger
2
3
4
5
6
0.0
11.0
12.0
10.0
14.0
13.0
0.0
8.0
0.0
0.0
0.0
0.0
8.0
0.0
0.0
0.0
11.0
0.0
0.0
0.0
0.0
8.0
10.0
12.0
12.0
8.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0
10.0
8.0
10.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.0
8.0
0.0
0.0
0.0
7
9
10
11
12
13
14
17
18
19
20
0.0
0.0
10.0
15.0
12.0
10.0
10.0
10.0
10.0
10.0

6-Amino-2-thio- and 6-Aminouracils as Precursors
165
5-(2-Methoxyphenyl)methylenebis(6-amino-1-methyl-2-
a mild antimicrobial activity, i.e. they are non-
promising as synthesis targets.
thiouracil) (4, C18H20N6O3S2)
Yield 1.72g (62%); mp 270ꢁC; yellowish crystals; IR (KBr):
Depending on the above mentioned results, it
could be concluded that both compound 13 as potent
antiviral and 3 as a broad-spectrum antimicrobial
could be considered to be viable choice for further
investigations. Of course, the compounds would
need to show superiority in other factors, such as
bioavailability, oral absorption, and toxicity.
ꢃꢀ¼ 3370 (NH2), 3273 (NH2), 1630 (C¼O), 1610 (NH2)
1
cmꢂ1; H NMR (DMSO-d6): ꢁ ¼ 12.15 (s, 2NH), 7.59 (bs,
2NH2), 7.14–7.08 (m, 2H-arom), 6.87–6.81 (m, 2H-arom),
5.41 (s, CH), 3.78 (s, 2NCH3), 3.62 (s, OCH3) ppm; 13C
NMR (DMSO-d6): ꢁ ¼ 28.3 (CH), 39.2 (CH3), 55.4 (OCH3),
91.3, 112.4, 117.8, 126.4, 128.2, 129.8, 131.1, 131.9, 162.4,
171.6 ppm.
5-(3-Methoxyphenyl)methylenebis(6-amino-1-methyl-2-
thiouracil) (5, C18H20N6O3S2)
Experimental
Yield 1.89g (69%); mp 278ꢁC; yellowish crystals; IR (KBr):
ꢃꢀ¼ 3350 (NH2), 3265 (NH2), 1628 (C¼O), 1617 (NH2)
Melting points were determined with an electro thermal Mel-
Temp II apparatus. IR spectra were obtained in the solid state
in form of KBr discs using a Perkin-Elmer model 1430 spec-
trometer. The UV spectra were determined with Perkin Elmer,
Lambda 5 or 15 spectrometers. 1H and 13C NMR spectra were
recorded on a Varian Mercury 300 MHz spectrometer in
DMSO-d6 as the solvent and TMS as internal standard.
Elemental analyses were performed at the Micro-analytical
Centre, Cairo University, Giza, Egypt, and were found to be
matching the calculated values properly.
1
cmꢂ1; H NMR (DMSO-d6): ꢁ ¼ 12.25 (s, 2NH), 7.57 (bs,
2NH2), 7.16–7.11 (m, 3H-arom), 6.71 (s, 1H-arom), 5.48 (s,
CH), 3.77 (s, OCH3), 3.67 (s, 2NCH3) ppm.
5-(4-Hydroxyphenyl)methylenebis(6-amino-1-methyl-2-
thiouracil) (6, C17H18N6O3S2)
Yield 1.75g (44%); mp 276ꢁC; yellowish crystals; IR (KBr):
ꢃꢀ¼ 3640 (br, OH), 3400 (NH2), 3290 (NH2), 1638 (C¼O),
1600 (NH2) cmꢂ1; 1H NMR (DMSO-d6): ꢁ ¼ 12.24 (s, 2NH),
7.57 (bs, 2NH2), 7.13–6.97 (m, 2H-arom), 6.72–6.69 (m,
2H-arom), 6.63 (s, OH), 5.48 (s, CH), 3.78 (s, 2NCH3)
ppm; 13C NMR (DMSO-d6): ꢁ ¼ 28.3 (CH), 39.2 (CH3),
91.5, 113.1, 117.6, 126.5, 128.3, 131.2, 162.32, 171.4 ppm.
5-Arylmethylenebis(6-amino-1-methyl-2-thiouracils) 2–7
and 5-Substituted 1,9-Dibenzyl-3,5,7,10-tetrahydro-2,4,6,8-
tetraoxopyrido[2,3-d][6,5-d]dipyrimidines 9–14
Method A: To a solution of 1.96 mmol 6-amino-1-methyl-2-
thiouracil (1) [25] or 1.96 mmol 6-amino-1-benzyluracil (8)
[26] in hot absolute ethanol, 0.98 mmol of an appropriate
aromatic aldehyde was added in presence of 1.0 cm3 conc.
HCl. The reaction mixture was stirred for 1.5–2.5 h at room
temperature. The formed product was filtered off, washed with
ethanol, dried, and recrystallized from 50% acetic acid.
Method B: A mixture of 3.2 mmol 1 or 8 and 1.6 mmol of
an appropriate aromatic aldehyde in 15 cm3 glacial acetic acid
was heated under reflux for 2–7 h. After cooling the formed
product was filtered off and recrystallized from 50% acetic acid.
5-(2,4-Dichlorophenyl)methylenebis(6-amino-1-methyl-2-
thiouracil) (7, C17H16Cl2N6O2S2)
Yield 1.60g (53%); mp 272ꢁC; yellowish crystals; IR (KBr):
ꢃꢀ¼ 3410 (NH2), 3280 (NH2), 1635 (C¼O), 1607 (NH2)
1
cmꢂ1; H NMR (DMSO-d6): ꢁ ¼ 12.33 (s, 2NH), 7.56 (bs,
2NH2), 7.44–6.83 (m, 3H-arom), 5.41 (s, CH), 3.78 (s,
2NCH3) ppm.
1,9-Dibenzyl-5-phenyl-3,5,7,10-tetrahydro-2,4,6,8-
tetraoxopyrido[2,3-d][6,5-d]dipyrimidine (9, C29H23N5O4)
Yield 1.13g (49%); mp 268ꢁC; white crystals; IR (KBr):
5-Phenylmethylenebis(6-amino-1-methyl-2-thiouracil)
(2, C17H18N6O2S2)
ꢃꢀ¼ 3120 (NH), 1680 (C¼O) cmꢂ1
;
1H NMR (DMSO-d6):
Yield 1.8 g (47%); mp 270ꢁC; yellow crystals; IR (KBr):
ꢁ ¼ 11.99 (bs, NH), 10.95 (s, 2NH), 7.40–7.05 (m, 15H-
arom), 5.48 (s, CH), 5.14 (s, 2CH2) ppm; 13C NMR
(DMSO-d6): ꢁ ¼ 34.16 (CH), 43.19 (CH2), 124.71, 125.42,
126.35, 127.39, 128.29, 128.52, 128.46, 130.25, 131.77,
133.28, 140.22, 149.80 ppm.
ꢃꢀ¼ 3380 (NH2), 3270 (NH2), 1630 (C¼O), 1612 (NH2)
1
cmꢂ1; H NMR (DMSO-d6): ꢁ ¼ 12.25 (s, 2NH), 7.59 (bs,
2NH2), 7.22–7.19 (m, 2H-arom), 7.14–7.10 (m, 3H-arom),
5.52 (s, CH), 3.78 (s, 2NCH3) ppm; 13C NMR (DMSO-d6):
ꢁ ¼ 28.21 (CH), 39.4 (CH3 (1)), 89.7, 112.2, 117.8, 126.5,
128.3, 131.2, 162.5 (2C¼O), 171.8 (2C¼S) ppm.
1,9-Dibenzyl-5-(4-fluorophenyl)-3,5,7,10-tetrahydro-2,4,6,8-
tetraoxopyrido[2,3-d][6,5-d]dipyrimidine (10, C29H22FN5O4)
Yield 1.09g (45%); mp 278ꢁC; white crystals; IR (KBr):
5-(4-Fluorophenyl)methylenebis(6-amino-1-methyl-2-
thiouracil) (3, C17H17FN6O2S2)
ꢃꢀ¼ 3120 (NH), 1670 (C¼O) cmꢂ1
;
1H NMR (DMSO-d6):
Yield 1.8 g (45%); mp 286ꢁC; yellow crystals; IR (KBr):
ꢃꢀ¼ 3420 (NH2), 3315 (NH2), 1665 (C¼O), 1614 (NH2)
ꢁ ¼ 11.53 (s, NH), 10.93 (s, 2NH), 7.39–6.96 (m, 14H-arom),
5.24 (s, CH), 5.12 (s, 2CH2) ppm; 13C NMR (DMSO-d6):
ꢁ ¼ 33.98 (CH), 43.27 (CH2), 125.18, 127.27, 128.43, 128.67,
129.19, 129.52, 131.20, 133.62, 133.78, 138.38, 149.90,
151.17 ppm.
cmꢂ1
;
1H NMR (DMSO-d6): ꢁ ¼ 12.25 (bs, 2NH), 7.59
(bs, 2NH2), 7.16–7.11 (tt, J ¼ 5.7, 2.7 Hz, 2H-arom),
7.04–6.98 (t, J ¼ 9 Hz, 2H-arom), 5.48 (s, CH), 3.77 (s,
2NCH3) ppm.

166
S. Youssif and S. F. Mohamed
nol): lmax (") ¼ 340.8 (4.30), 255 (3.68), 224 (3.94), 205
1,9-Dibenzyl-5-(2-methoxyphenyl)-3,5,7,10-tetrahydro-
2,4,6,8-tetraoxopyrido[2,3-d][6,5-d]dipyrimidine
(11, C30H25N5O5)
1
(4.17) nm (molꢂ1 dm3 cmꢂ1); H NMR (DMSO-d6): ꢁ ¼ 12.3
(s, NH-1), 7.70 (s, CH-8), 3.90 (s, NCH3-9), 3.62 (s, NCH3-
3) ppm.
Yield 1.10g (45%); mp 226ꢁC; yellowish crystals; IR (KBr):
ꢃꢀ¼ 3170 (NH), 1660 (C¼O) cmꢂ1
;
1H NMR (DMSO-d6):
6-Alkylmercapto-3,9-dimethylxanthines 17–20
ꢁ ¼ 11.62 (s, NH), 10.78 (s, 2NH), 7.41-7.23 (m, 10H-arom),
7.12-7.09 (t, 1H-arom), 6.94–6.91 (d, 1H-arom), 6.84–6.82 (d,
J ¼ 6.0 Hz, 1H-arom), 6.71–6.67 (t, J ¼ 6.0 Hz, 1H-arom),
5.43 (s, CH), 5.13 (s, 2CH2), 3.62 (s, OCH3) ppm; 13C NMR
(DMSO-d6): ꢁ ¼ 33.41 (CH), 43.16 (CH2), 54.62 (OCH3),
114.11, 125.28, 126.61, 127.13, 127.35, 128.60, 128.96,
129.43, 129.72, 131.15, 133.61, 149.86, 157.09 ppm.
3,9-Dimethyl-6-thioxanthine (0.5g) was dissolved in 6.0 cm3
2 N NaOH and an excess of alkyl halides, 7.5mmol methyl,
ethyl, butyl iodide, and 2.5mmol benzyl bromide were added
dropwise with stirring for 2 h. The reaction mixture was
cooled in an ice bath and acidified using acetic acid till pH
adjusted 5–6. The mixture was evaporated in vacuo to a half
amount and the formed precipitate was collected, washed with
water, and recrystallized from methanol.
1,9-Dibenzyl-5-(3-methoxyphenyl)-3,5,7,10-tetrahydro-
2,4,6,8-tetraoxopyrido[2,3-d][6,5-d]dipyrimidine
(12, C30H25N5O5)
3,9-Dimethyl-6-methylthiopurine-2-one (17, C8H10N4OS)
Yield 0.42 g (79%); mp 263ꢁC; UV (methanol): lmax (") ¼
Yield 1.0 g (41%); mp 252ꢁC; yellow crystals; IR (KBr):
1
273.0 (3.92), 213.0 (4.32) nm (molꢂ1 dm3 cmꢂ1); H NMR
ꢃꢀ¼ 3130 (NH), 1655 (C¼O) cmꢂ1
;
1H NMR (DMSO-d6):
ꢁ ¼ 11.23 (s, 2NH), 8.06 (bs, NH), 7.38–7.24 (m, 10H-arom),
7.08–7.02 (t, 1H-arom), 6.56–6.58 (tt, 3H-arom), 5.41 (s,
CH), 5.13 (s, 2CH2), 3.60 (s, OCH3) ppm.
(DMSO-d6): ꢁ ¼ 7.68 (s, CH-8), 4.08 (s, SCH3), 3.43 (s,
NCH3-9), 3.37 (s, NCH3-3) ppm.
3,9-Dimethyl-6-ethylthiopurine-2-one (18, C9H12N4OS)
Yield 0.49 g (86%); mp 250ꢁC; UV (methanol): lmax (") ¼
1,9-Dibenzyl-5-(4-hydroxyphenyl)-3,5,7,10-tetrahydro-
2,4,6,8-tetraoxopyrido[2,3-d][6,5-d]dipyrimidine
(13, C29H23N5O5)
1
272.0 (3.97), 215.0 (4.21) nm (molꢂ1 dm3 cmꢂ1); H NMR
(DMSO-d6): ꢁ ¼ 7.68 (s, CH-8), 4.05 (q, SCH2), 3.41 (s,
NCH3-9), 3.36 (s, NCH3-3), 1.28 (t, CH3) ppm.
Yield 1.06g (45%); mp 258ꢁC; yellowish crystals; IR (KBr):
1
ꢃꢀ¼ 3620 (br, OH), 3190 (NH), 1680 (C¼O) cmꢂ1; H NMR
3,9-Dimethyl-6-butylthiopurine-2-one (19, C11H16N4OS)
Yield 0.53 g (82%); mp 214ꢁC; 1H NMR (DMSO-d6):
ꢁ ¼ 7.75 (s, CH-8), 4.11 (t, SCH2), 3.55 (s, NCH3-9), 3.37
(s, NCH3-3), 1.82–1.93 (m, 2CH2), 0.88 (t, CH3) ppm.
(DMSO-d6): ꢁ ¼ 10.88 (s, 2NH), 8.97 (s, OH), 7.63 (bs,
NH), 7.38–7.25 (m, 10H-arom), 6.80–6.83 (d, J ¼ 8.4 Hz,
2H-arom), 6.57–6.54 (d, J ¼ 8.7 Hz, 2H-arom), 5.37 (s,
CH), 5.16 (s, 2CH2) ppm; 13C NMR (DMSO-d6): ꢁ ¼ 33.36
(CH), 43.09 (CH2), 116.12, 118.72, 124.02, 125.32, 127.41,
128.62, 129.01, 129.22, 129.63, 131.52, 133.39, 150.17,
154.67 ppm.
6-Benzylthio-3,9-dimethylpurine-2-one (20, C14H14N4OS)
Yield 0.61 g (84%); mp 279ꢁC; 1H NMR (DMSO-d6):
ꢁ ¼ 7.82 (s, CH-8), 7.34–7.45 (m, 5H-arom), 4.21 (s, SCH2),
3.38 (s, NCH3-9), 3.36 (s, NCH3-3) ppm.
1,9-Dibenzyl-5-(2,4-dichlorophenyl)-3,5,7,10-tetrahydro-
2,4,6,8-tetraoxopyrido[2,3-d][6,5-d]dipyrimidine
(14, C29H21Cl2N5O4)
Pharmacology
Yield 0.94g (36%); mp 250ꢁC; yellow crystals; IR (KBr):
The newly synthesized compounds were tested for their an-
timicrobial activity against some microorganisms. The fol-
lowing microorganisms were used: HSV-1, HAV-27 viruses
(obtained from Environmental Virology Lab. Department of
Water Pollution Res., National Research Centre) and Hepatitis
A (cell culture adapted strain HAV-MBB, kindly provided
ꢃꢀ¼ 3175 (NH), 1687 (C¼O) cmꢂ1
;
1H NMR (DMSO-d6):
ꢁ ¼ 11.98 (s, NH), 11.06 (s, NH), 10.76 (s, NH), 7.43–7.17
(m, 13H-arom), 5.39 (s, CH), 5.20 (s, 2CH2) ppm.
3,9-Dimethylxanthine (15)
This compound was prepared according to the reported meth-
od [17, 18]; mp 336ꢁC (Ref. [17] 322ꢁC).
¨
¨
by Prof. Dr. Verena Gauss-Muller, Lubeck University of
Medicine, Institute of Molecular Virology, Germany), B. sub-
tilis as Gram-positive and E. coli as Gram-negative bacteria,
C. tropicalis as yeast, and A. niger as fungus. All bacterial,
yeast and fungal strains were obtained from Microbial
Biotechnology department (NRC), Egypt.
6-Mercapto-3,9-dimethylxanthine (16, C7H8N4OS)
3,9-Dimethylxanthine (5.0g) and 15 g P2S5 were refluxed in
45cm3 dry pyridine for 4 h in an oil bath. The mixture was
evaporated under vacuum to dryness and the residue was
boiled with 160 cm3 water and filtered. The filtrate was evap-
orated in vacuo to a third of its volume and the solution was
leaved to stand overnight. The formed precipitate was collect-
ed by filtration, dried at 100ꢁC, and recrystallized from 50cm3
H2O to yield 0.38g yellowish-white needles (70%); Rf (SG)¼
0.47 in methanol:chloroform¼ 2:8; mp 308ꢁC; UV (metha-
Antiviral Bioassay
Preparation of the Synthesized Compounds Solutions
The solutions of the compounds were prepared by dissolving
100 mg each in 1 cm3 of 10% DMSO in water. The final
concentration was 100ꢂg=mm3 (stock solution). The stock
solutions thus prepared were sterilized by addition of anti-

6-Amino-2-thio- and 6-Aminouracils as Precursors
167
biotic-antimycotic mixture: 10000 U penicillin G sodium,
10000ꢂg streptomycin sulfate, and 250 ꢂg amphotericin B
or 50 ꢂg=cm3 Gentamicin. The cells used were derived from
African green monkey kidney (Vero) and were propagated in
Dulbecco as well as the D in DMEM (Minimum essential
medium) supplemented with 10% fetal bovine serum and
1% antibiotic-antimycotic mixture. The pH was adjusted to
7.2–7.4 with saturated NaHCO3 solution.
2) Preparation of Plates and Inoculation
Inoculum (0.5cm3) was added to 24 cm3 of agar malted media
(50ꢁC) and mixed by simple inversion. The malted medium
was poured into each 150mm sterile Petri plates and 4–6 mm
wells were cut into the harden seeded plates by pasteur pipette
and finally, the wells were filled with the various compounds
(stock solution, 5 mg=cm3 DMSO). The plates were incubated
at 37ꢁC for 24h for bacteria and yeast and for 48–72h for
fungi. The diameters of the inhibition zones (mm) were mea-
sured and recorded.
Plaque Reduction Assay for Rapid Screening of Antiviral Activity
Plaque reduction assay [27–29] was carried out as follows: a
6-well plate was cultivated with Vero cell culture (105 cell=
cm3) and incubated for 2 days at 37ꢁC. HSV-1 and HAV-27
were diluted to give 104 PFU=cm3 final concentrations
for each virus and mixed with the test compound with two
concentrations, 10ꢂg=cm3 and 20 ꢂg=cm3, and incubated
overnight at 4ꢁC. Growth medium was removed from the
multiwell plate and virus-compound mixture was inoculated
(100 mm3=well). After 1 h contact time, the inoculum was
aspirated and 3 cm3 MEM with 1% agarose were overlaid
the cell sheets. The plates were left to solidify and incubated
at 37ꢁC until the development of virus plaques. Cell sheets
were fixed in 10% formalin solution for 2 h and stained with
crystal-violet. Control virus and cells were treated identically
without chemical compound. Virus plaques were counted and
the percentage of reduction was calculated. A 50% inhibitory
concentration EC50 was calculated as the concentration re-
quired reducing virus yield by 50%, in the compounds-treated
cultures compared with the untreated ones.
Determination of the Minimum Inhibitory Concentration
(MIC)
Serial dilutions of the promising compounds were subjected
to MIC determination, they were prepared in the following
pattern: 1000 ꢂg=cm3, 200 ꢂg=cm3, 100ꢂg=cm3, 50ꢂg=cm3,
10 ꢂg=cm3, and 5 ꢂg=cm3. Bacillus subtilis was selected to be
the test organism because it was the most sensitive organism.
The different concentrations of each compound were tested
with the Modified Agar Diffusion Cylinder method as de-
scribed before.
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168
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