Synthesis of substituted thieno[2,3-d]pyrimidine-2,4-dithiones and their S-glycoside analogues as potential antiviral and antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.060

Previously, we synthesized and evaluated several thienopyrimidine derivatives containing heterocyclic ring substituents linked to the pyrimidine-2-thione nucleus at C-2 by a two- to four-atom spacer as potential anti-HIV-1 and antimicrobial agents. Also, from the literature, S-substituted pyrimidin-4-ones A and B exhibited interesting anti-HIV-1 activity. To further investigate the synthesis, tools and biological activities, we synthesized several new thienopyrimidine derivatives derived from thieno[2,3-d]-pyrimidine- 2,4-dithione (3a,b) The compounds were designed to comprise the heterocyclic substituents directly linked to the thienopyrimidines nucleus at C-2. Moreover, various related triazolo[4,3-a]benzothieno[2,3-d]pyrimidines derived from 2-thioxothienopyrimidine were also prepared as isosteres. Among the synthesized derivatives 3-18, the compounds 3a, 8a, 10a, 13a and 14a were showing complete inhibition at 128 mg/mL or less.

Full text of DOI:10.1016/j.ejmech.2010.05.060

European Journal of Medicinal Chemistry 45 (2010) 4026e4034
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of substituted thieno[2,3-d]pyrimidine-2,4-dithiones and their
S-glycoside analogues as potential antiviral and antibacterial agents
,
Hend N. Hafez a, Hoda A.R. Hussein a, Abdel-Rahman B.A. El-Gazzar b
*
a Photochemistry Department (Heterocyclic& Nucleosides Group), National Research Centre, 12622 Dokki, Cairo, Egypt
b Al-Imam Muhammad Ibn Saud, Islamic University, Faculty of Science, Department of Chemistry, P.O. Box: 90950 Riyadh 11623, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously, we synthesized and evaluated several thienopyrimidine derivatives containing heterocyclic
ring substituents linked to the pyrimidine-2-thione nucleus at C-2 by a two- to four-atom spacer as
potential anti-HIV-1 and antimicrobial agents. Also, from the literature, S-substituted pyrimidin-4-ones A
and B exhibited interesting anti-HIV-1 activity. To further investigate the synthesis, tools and biological
activities, we synthesized several new thienopyrimidine derivatives derived from thieno[2,3-d]-pyrim-
idine-2,4-dithione (3a,b) The compounds were designed to comprise the heterocyclic substituents
directly linked to the thienopyrimidines nucleus at C-2. Moreover, various related triazolo[4,3-a]ben-
zothieno[2,3-d]pyrimidines derived from 2-thioxothienopyrimidine were also prepared as isosteres.
Among the synthesized derivatives 3e18, the compounds 3a, 8a, 10a, 13a and 14a were showing
complete inhibition at 128 mg/mL or less.
Received 31 January 2010
Received in revised form
25 May 2010
Accepted 26 May 2010
Available online 2 June 2010
Keywords:
Thieno[2,3-d]pyrimidine-2,4-thione
Triazolothienopyrimidine
S-Glycosidothieno[2,3-d]pyramidine
Antiviral
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antimicrobial activities
1. Introduction
3,4-dihydro-4-oxopyrimidines (S-dihydro-(alkyloxy)-benzyl-ox-
ypyrimidines) (A) that received a great deal of attention for their
Acquired immunodeficiency syndrome (AIDS) was identified by
Centers for Disease Control and Prevention (CDC) in June 1981.
Since then, it has been widely spread in the world and seriously
affects healthy conditions of human being, HIV-1 reverse tran-
scriptase (RT) [1] played a critical role in the process of HIV
replication and has been identified as one of the main targets for
anti-HIV drug discovery [2].
Two classifications of RT inhibitors have been studied: nucleo-
side reverse transcriptase inhibitors (NRTIs) and non-nucleoside
transcriptase inhibitors (NNRTIs). NNRTIs, being less toxic than
NRTIs, had received great attention [3e5]. Three NNRTIs nevirapine
[6], delavirdine [7], and efavirenz [8] have been approved by FDA.
Not only being effective RT inhibitors, these three NNRTIs also acted
as the key components of the combination therapy [9e11].
However, NNRTIs can easily induce drug-resistant variants of HIV-1,
which have also been seen in the treatment with other chemo-
therapeutic agents. New anti-HIV agents with novel structures or
mechanism(s) of action are still in demand [12,13].
anti-HIV-1, characterized by the presence of a
b-carbonyl group
on the C-2 side chain (Fig. 1) [15,16]. These compounds exhibited
significant anti-HIV activity and more interesting, they might
interfere with a target that differed from HIV RT, or act on RT in
a way that is different from typical NNRTIs. To further optimize
the structure of S-DABO derivatives and improve the anti-HIV
functionality, we designed two series of thienopyrimidine and its
S-glycoside derivatives based on previously investigated scaffolds
A and B (Fig. 1) [17]. These novel chemical entities were char-
acterized by (i) introduction of a heteroaromatic ring at C-6, C-5
fused pyrimidines; (ii) introduction of a triazole ring at C-2, C-3
fused pyrimidines; (iii) the presence of S-glycosides substituent
at the end of C-2 side chain to investigate the importance of the
glycosyl moiety, which might favor the enhance of intact
molecular structure to the binding pocket of the target due to the
increasing flexibility. Herein, we report the synthesis, antiviral
and antimicrobial evaluation, and the structureeactivity rela-
tionship (SAR) of these compounds.
A previous study [14] described a variety of series of 6-
naphthylmethyl substituted 2-(alkylthio)-5-alkyl-6-arylmethyl-
On the basis of the above considerations, original nucleoside
analogues directed upon reverse transcriptase still arouse consid-
erable interest [18]. In the hope of elucidating and/or finding better
therapeutic agents, S-glycosidothieno[2,3-d]-pyrimidine analogues
seem to be one of the recommended targets and also an extension
of our work on pyrimidines [19], C-nucleosides [20,21] and thieno
* Corresponding author. Tel.: þ202 373 18830; fax: þ202 333 70931.
E-mail address: profelgazzar@yahoo.com (A.-R.B.A. El-Gazzar).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.060

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
4027
spectrum of 3a and 2-methylthiothieno[2,3-d]pyrimidine (10a)
indicated that the site of the alkylation is the sulfur atom at C-2 and
not the nitrogen atom (Fig. 2).
The synthetic route we used for the preparation of 2-S-(b-D-
glycopyranosyl/or furanosyl)-thieno[2,3-d]pyrimidine is outlined
in Scheme 3. The thieno[2,3-d]pyrimidines 3a,b were converted
into their potassium salts 9a,b by using of KOH in acetone and
stirring at room temperature for long time with 2,3,4,6-tetra-O-
acetyl-
a-D-glucopyranosyl bromide (11b) or 2,3,4,6-tetra-O-acetyl-
Fig. 1. Structure of S-DABSs.
a-D-galactopyranosyl bromide (11c), yielding the S-glycosylated
nucleosides 17aed in good yields. Thin layer chromatography
(chloroform:methanol, 7:3) indicated the purity of the compounds.
Structures of the S-glycosides were confirmed by elemental anal-
yses and spectral data (IR, 1H, 13C NMR). The 1H NMR spectrum of
compound 17a as an example, showed the anomeric proton of the
[2,3-d]pyrimidines [22e24]. Since the data of S-glycosides are
relatively few, also promoted us to make research and developed in
the synthesis of these types of nucleosides.
glucose moiety as a doublet at
J ¼ 10.72 Hz indicating -configuration of the anomeric center.
Other protons of the glucopyranose ring resonated at
3.97e5.13 ppm, while the four acetoxy groups appeared as four
singlets at
1.94e2.14 ppm. The 13C NMR spectrum revealed the
absence of the thione C-2 atom at about 174.2 ppm and a resonance
of eN]CeNe carbon atom (C-2) at 158 ppm (Fig. 2). The signals
in the region 168.3e170.9 ppm are due to the four acetoxy
carbonyl atoms (4C]O), and the four signals around
22.19e22.59 ppm are assigned to the acetate methyl carbon
60.23, 65.22, 67.68, 69.32, 75.63,
d 5.94 ppm with a coupling constant
2. Results and discussion
b
With respect to the previous studies carried out in our labora-
tory, 3-(2-amino-thiophene)-carbonitrile derivative precursors
[25] have been regarded as promising intermediates to produce
thieno[2,3-d]pyrimidine-2,4-dithione derivatives 3a,b with some
structural analogies with natural nucleobases. The interaction of
acyclic ketone with malononitrile and sulfur element in absolute
ethanol in the presence of diethylamine led to 3-(2-amino-thio-
phene)-carbonitrile derivatives 1a,b. The pyrimidine-2,4-dithione
derivatives 3a,b were obtained by refluxing of 1a,b with carbon
disulfide in dry pyridine (85e99% yield) as shown in Scheme 1.
The 1,3-dipolar cycloaddition of the nitrileimines (generated in
situ from hydrazonoyl chlorides 4aed and triethylamine in dry
chloroform) to thienopyrimidine-2,4-dithione derivatives 3a,
d
d
d
d
d
atoms. Also, the five signals at
d
87.59 ppm were assigned to C-60, C-30, C-20, C-40, C-50 and C-10,
respectively. Moreover, the IR spectra of compounds 13 revealed
the absence of the vibration band of a thione group. Similarly, the
reaction of heterocycle base 3a,b with 1-bromo-2,3,5-tri-O-acetyl-
a-D-arabinofuranose (11a) furnished the S-glycosated product 13a,
b
afforded novel 1,3-disubstituted triazolo[1,2,4]thieno[2,3-d]
b. The structure assignments of this product are based on its
elemental analysis and the spectral data (see Experimental).
In addition to the above synthetic methods a so called “one-
pot” protocol of the silyl-HilberteJohnson reaction was described
[26]. This ‘one-pot’ protocol was used by Wolfe for the synthesis
pyrimidine derivatives 8aed in good yields (70e82%) under dry
conditions (Scheme 2). This cycloaddition is chemoselective as it
occurs only on C]S at C-2 of compound 3 furnishing exclusively
the triazol[1,2,4]thieno[2,3-d]pyrimidines 8 after H2S elimination.
The mechanism of the reaction may proceed via the cycloaddition
of one mole of hydrazonoyl chloride 4 (via electron reach nitrogen
of the dipole) to the C-2 of compound 3 (Scheme 2). The spectro-
scopic analyses (IR, NMR, MS) conformed the structure 8 and not
structure 5.
of
was silylated with HDMS in presence of (NH4)2SO4 in MeCN and
glycosylated with 1,2,3,4,6-penta-O-acetyl- -glycopyranose in
b-D-ribonucleosides [27]. In this procedure, the nucleobase
a-D
presence of SnCl4. Here, MeCN was used as a solvent and HMDS
(hexamethyldisilane) as glycosylation catalyst (Vorbrüggen
conditions). The nucleobases were silylated and directly glyco-
sylated in one step. Townsend applied this procedure for the
synthesis of toyocamycin [28].
Compounds 3a,b were found to be useful for the syntheses of
the interesting S-glycosides. As a model experiment the alkylation
of 3a was carried out by the reaction of 1 mol equiv of methyl iodide
with the potassium salt 9a (generated in situ by the reaction of 3a
with alcoholic potassium hydroxide). Structure of the new 2-
methylthiothieno[2,3-d]pyrimidines 10 was confirmed by spec-
troscopic data. The 13C NMR spectrum as an example, revealed that
We used the ‘one-pot’ method for the synthesis of the
(20,30,40,60-tetra-O-acetyl-
b-D-glycopyranosylthio)-thieno[2,3-d]py-
rimidin-4-one analogues 17a,b. The nucleobases 3a,b were silylated
with HMDS in anhydrous MeCN at room temperature in presence of
the signal of the C-2 (CeSCH3) appeared at d
159 ppm. The 13C NMR
Scheme 1.

4028
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
Scheme 2.
ammonium sulfate and then reacted with 1,2,3,4,6-penta-O-acetyl-
-glycopyranose (Scheme 3). This afforded the glycosylated
2.1. In vitro anti-herpes simplex-1 virus (HSV-1) evaluation
a-D
compounds 17a,b (in 54% yields) after working up.
The synthesized compounds were tested for their in vitro anti-
viral activity against herpes simplex-1 virus (HSV-1) grown on Vero
African green monkey kidney cells. The antiviral, antimitotic and
antibiotic aphidicolin was used as a control [29]. Antiviral activity is
defined as confluent, relatively unaltered monolayers of stained
Vero cell treated with HSV-1. The cytotoxic activity of the tested
compounds was performed using Vero cell culture [29]. Cytotoxicity
was estimated as the concentration that caused approximately 50%
loss of the monolayer present around the plaques caused by HSV-1
(Table 1). An improved plague reduction assay for antiviral activity
was used to test the compounds. Plague reduction assay typically
Deacetylation of S-nucleosides 13a,b and 17aed proceeded
smoothly via methanolic ammonia treatment to afford the free
nucleosides 14a,b and 18aed in good to excellent yields
(Scheme 3). The 1H NMR data of the compounds 14 and 18 revealed
the absence of the acetyl protons in the region
appearance of the D2O-exchangeable OH protons in the region
4.60e5.56. The IR data of the compound 14a as a typical example
d 1.90e2.20 and
d
showed also the absence of the acetyl carbonyl function around
1700 cmꢀ1 and the appearance of the characteristic OH band at
3500 (br) cmꢀ1
.
Fig. 2. 13C NMR for some C]S and CeS analogues.

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
4029
Scheme 3.
used a monolayer of cultured host-cells which were allowed to bind
virus, then overplayed with a layer of medium thickened with agar
or another thickener. Samples to be tested were either incorporated
into the thickened layer or absorbed in a paper disc laid on the
thickened layer. Abou-Karam and Shier [29] modified this approach
to allow the production of acceptable HSV-1 plaques without the use
of a thickening. A serial dilution of samples in 96-plates was used to
estimate the end-point concentration for antiviral agents [30]. In the
same time, this assay retains the ability to estimate the cytotoxicity
which is reflected as reduction in the size or number of viral plaques
in a cell monolayer. Among the 13 tested compounds, compounds
14a and 14b showed marginal activity as it reduced the number of
the plagues by 30% and 24% at a minimum antiviral concentration
Table 1
The cytotoxic and anti-HSV-1 activities of the synthesized compounds and the
antiviral antibiotic aphidicolin.
(MAC) of 0.29 and 0.21
mM/L with lowest cytotoxicity effect (IC50
Compound
% Reduction in
number in plaques
Minimum antiviral
Cytotoxicity
(IC50
M/La
>1.50 M/L). The rest of the compounds did not show any inhibitory
m
conc (MAC)
m
M/L
) m
effect against HSV-1 (Table 1). The results also revealed that the
synthesized compounds showed different levels of cytotoxicity. The
highest cytotoxicity was observed in compounds 3a, 3b, 8d and 8h
(IC50 <0.1 mM/L), which containing dithions (2C]S) in positions 2
and 4 in compounds 3a and 3b, also compounds 8d and 8h which are
Aphidicolin
3a
3b
8d
8h
10a
10b
13a
13b
14a
14b
17a
18a
18d
100
B
B
B
B
B
B
B
B
30
24
B
B
B
0.02
B
B
B
B
B
B
B
B
0.29
0.21
B
B
B
0.58
0.07
0.09
0.09
0.07
0.64
0.45
1.39
1.15
>1.70
>1.46
0.56
0.61
0.38
halogen containing derivatives. On the other hand, the deprotected
S-glycoside, specially S-
derivatives 14a, 14b where found to possess the lowest cytotoxic
effect (IC50 >1.5 M/L).
b-D-arabinofuranosyl thienopyrimidine
m
2.2. In vitro anti-human immunodeficiency virus-1 (HIV-1)
evaluation
B: 0% reduction in number of viral plaques.
IC50: The concentration of drug that caused 50% loss of the monolayer present
around the plaques.
a
The procedure used to evaluate the anti-HIV-1 potency is
designed to detect agents acting at any stage of the virus

4030
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
reproductive cycle [31]. The assay involves the killing of T4
lymphocytes by HIV-1; compounds that interfere with viral activ-
ities will protect cells from cytolysis. The median effective
concentration (EC50) of the tested compounds using infected cells
was compared with their cytotoxic effect (IC50) in uninfected
cultures. Infected and uninfected cultures were incubated without
test compounds to serve as controls. Zidovudine (AZT) treated
cultures were also used as positive controls. The screening results
(Table 2) revealed that the 2,4-dithiones derivatives 3a and 3b
exhibited marginal activity against HIV-1 with EC50 6.5 and
towards P. putida and the activity is enhanced in case of arabino-
furanosyl group at position of C-2. Further, the MIC of 14a,b towards
P. putida is 64 mg/mL whereas ampicillin was inactive up to
256 mg/mL.
3. Experimental
All starting materials, solvents, and reagents were very pure
grade and used as purchased. Chromatography solvents were HPLC
grade and used without further purification. Flash column chro-
matography was performed on Merck KGaA Silica gel 60 F254
(particle size 0.040e0.063 mm) unless otherwise stated. Reactions
were monitored by thin layer chromatography (TLC) using Merck
KGaA precoated glass plates (silica gel 60 F254). Melting points were
determined on the Electrothermal 9100 melting point apparatus
(Electrothermal, UK) and are uncorrected. The IR spectra (KBr) were
recorded on an FT-IR NEXCES spectrophotometer (Shimadzu,
Japan). The 1H NMR spectra were measured with a Jeol ECA
500 MHz (Japan) in DMSO-d6 or CDCl3 and chemical shifts were
5.25 mM/L, respectively. Although the effective concentrations of
compounds 3a and 3b are greatly higher than that of AZT, they
seemed to be less cytotoxic. The obtained results proved that non-
substituted of the thieno[2,3-d]pyrimidines nucleus are essential
for antiviral activity. The weak activity of compounds 3a and 3b and
the absence of activity in the other compounds may be attributed to
the lack of substitution in position 2 of the thieno[2,3-d]-pyrimi-
dines nucleus as compared with the active HEPT derivatives.
recorded in
d ppm relative to TMS. Mass spectra (EI) were run at
2.3. Antibacterial activity
70 eV with a Finnigan SSQ 7000 spectrometer (Thermo-Instrument
System Incorporation, USA). The biological medium components
were obtained from Sigma Chem. Co., St. Louis, MO, USA. The
pharmacological evaluations of the products were carried out in
Pharmacological Unit Pharmacology department, (NCI, Cairo
University, Egypt). The starting materials, 2-amino-4,5,6,7-tetra-
hydrobenzo-/and or 4,5,6,7,8-pentahydrocyclohepta-thiophen-3-
carbonitrile (1a,b) were prepared according to the literature
procedures [32,33]. Table 4 shows the characterization data of
compounds 3e18.
The results of antibacterial studies are given in Table 3. Among
the synthesized derivatives 3e18, compounds 3a, 8a, 10a, 13a and
14a were showing complete inhibition at 128 mg/mL or less. The
rest of the compounds showed incomplete inhibition.
Among the 2,4-thieno[2,3-d]pyrimidine-dithiones (3a,b), 2-S-
methyl-thieno-[2,3-d]pyrimidine-4-thione
(10a,b)
and
tri-
azolothienopyrimidenes (8a,e) compound 3a completely inhibited
Escherichia coli at 64 mg/mL but it could inhibit Staphylococcus
aureus and Pseudomonas putida at 128 mg/mL. Introducing
a diphenyl-triazolo group at C-2eC-3 ring (8a) made it relatively
ineffective towards E. coli but found to be active against other two
strains. Compound 10a with an S-methyl group at C-2 in pyrimidine
ring exhibited activity similar to compound 8a. Any other substi-
tution at the positions C-2eN-3 of the pyrimidine ring and C-4eC-5
of the thiophene ring system retarded the efficiency of the resulting
compounds. The acetylated glycosides 17a,b resulted in decreased
activity when compared to 3a but placing a acetylated arabino-
furanosyl group at C-2 in pyrimidine ring on thieno[2,3-d]pyrimi-
dine resulted in potent compounds 13a,b when compared to all
other compounds. De-acetylated S-glycosides compounds in thie-
nopyrimidine resulted in 14a,b and 18a,b, which were active
3.1. Synthesis of substituted thieno[2,3-d]pyrimidine-2,4(1H,3H)-
dithiones (3a,b)
General procedure. To a solution of o-aminonitrile 1a or 1b
(0.01 mol) in pyridine (10 mL), carbon disulfide (0.05 mol) was
added and the mixture was heated on a water-bath for 6e8 h. After
cooling, ethanol was added and the separated solid was collected
by filtration, washed with ether to give 3a,b in good yield.
3.1.1. Synthesis of 3a
From 1a, as yellow crystals; IR (cmꢀ1
,
n
): 3424 (br, NH), 2923 (CH
alkyl), 1230, 1235 (2CS); 1H NMR (DMSO-d6,
d
, ppm): 1.67e1.69 (m,
4H, 2CH2), 2.96 (m, 2H, CH2), 3.34 (m, 2H, CH2), 13.0, 14.0 (2br, 2H,
2NH, D2O-exchangeable); 13C NMR: 22.23, 22.49, 25.43, 26.59
(4CH2), 126.5, 130.8, 135.8, 148.3 (carbon of the thiophene ring),
174.2 (C]S), 180.0 (C]S); Its MS (m/z), 254 (Mþ, 100).
Table 2
The cytotoxic and anti-HIV-1 activities of some compounds 3e18 and the antiviral
drug zidovudine (AZT).
Compound
IC50
(
m
M/L)
EC50
6.5
5.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(
m
M/L)
TI50 (IC50/EC50)
3.1.2. Synthesis of 3b
From 1b, as yellow crystals; IR (cmꢀ1
,
n
): 3424 (br, NH), 2931
3a
3b
8d
8h
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>15.38
>19.05
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
50.86
(CH alkyl), 1235, 1242 (2CS); 1H NMR (DMSO-d6,
4H, 2CH2), 1.75 (m, 2H, CH2), 2.64 (m, 2H, CH2), 3.21 (m, 2H, CH2),
12.60, 13.20 (2br, 2H, 2NH, D2O-exchangeable); Its MS (m/z), 268
(Mþ, 100).
d, ppm): 1.56 (m,
10a
10b
13a
13b
14a
14b
17a
18a
18d
AZT
3.2. Synthesis of 1,3-disubstituted-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]substituted thieno[2,3-d]pyrimidine-5-thiones (8aeh)
General procedure. A mixture of 3a,b (0.01 mol) and hydrazonoyl
chlorides 4aed (0.01 mol) was stirred under reflux in dry chloro-
form (30 mL) and 4 drops of triethylamine for 12 h. The solvent was
evaporated under reduced pressure. The solid produced was
washed three times by 30 mL methanol, and crystallized to produce
(8aeh).
35.6
0.7
IC50: 50% inhibitory concentration (the molar concentration of drug that caused 50%
inhibition of cell growth). EC50: 50% effective concentration (the molar concentra-
tion of drug that caused 50% protection against HIV cytopathic effect). TI50: thera-
peutic index.

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
4031
Table 3
MIC (mg/mL) and the zone of inhibition (in mm) values of various thieno-, triazolothieno- and S-glycosido-thieno[2,3-d]pyrimidines in gram positive and gram negative
bacteria.
Compound
E. coli
S. aureus
P. putida
Zone of inhibition (mm)
Zone of inhibition (mm)
Zone of inhibition (mm)
MIC (mg/mL)
128 mg/mL
64 mg/mL
MIC (mg/mL)
128 mg/mL
64 mg/mL
MIC (mg/mL)
128 mg/mL
64 mg/mL
3a
3b
8a
8b
64
64
128
64
>3
>3
>3
>3
>3
>3
>3
>3
<1
>3
>3
>2
>2
<1
<1
>5
>3
>3
<1
>3
<1
<1
<1
>3
<1
>2
>2
<1
>2
<1
<1
>5
128
>128
128
>128
128
128
128
64
>128
>128
128
>128
>128
>128
>128
16
>3
>2
>3
>2
>3
>3
>3
>3
<1
>2
>3
<1
>2
<1
<1
>5
<1
>2
<1
>2
>2
<1
>2
>3
<1
>2
>2
<1
>2
<1
<1
>5
128
128
128
128
128
128
128
128
>128
64
128
>128
128
128
>128
>256
>3
>3
>3
>3
>3
>3
>3
>3
<1
>3
>3
>2
>3
>3
<1
0
<1
>2
<1
>2
<1
<1
<1
<1
<1
>3
>2
>2
>2
<1
<1
0
8c
128
128
128
64
>128
128
128
>128
>128
>128
>128
16
10a
10b
13a
13b
14a
14b
17a
17b
18a
18b
Ampicillin
3.2.1. 1,3-Diphenyl-6,7,8,9-tetrahydro[1,2,4]triazolo[4,3-a]
benzothieno[2,3-d]pyrimidine-5-thione (8a)
3.2.3. 3-Ethylcarboxylate-1-(4-tolyl)-6,7,8,9-tetrahydro[1,2,4]
triazolo[4,3-a]benzothieno-[2,3-d]pyrimidine-5-thione (8c)
It was obtained from 3a and chloro-(4-tolylhydrazono)-ethyl-
It was obtained from 3a and N-phenylbenzenecarbohydrazonoyl
chloride 4a as white crystals; IR (cmꢀ1
): 3039 (CH aryl), 2921 (CH
alkyl), 1645 (C]N), 1238 (CS); 1H NMR (DMSO-d6,
, ppm): 1.52 (m,
,
n
acetate 4c as white crystals; IR (cmꢀ1
,
n
): 3031 (CH aryl), 2926 (CH
d
alkyl), 1705 (CO), 1245 (CS), 1645 (C]N); 1H NMR (DMSO-d6,
d,
4H, 2CH2), 1.69 (m, 2H, CH2), 2.62 (m, 2H, CH2), 6.96e7.11 (m, 4H,
phenyl), 7.37e7.49 (m, 6H, phenyl); Its MS (m/z), 414 (Mþ, 39).
ppm): 1.26e1.29 (t, 3H, CH3), 1.59 (m, 4H, 2CH2), 2.65 (s, 3H, CH3),
2.83 (m, 2H, CH2), 3.31 (m, 2H, CH2), 4.06e4.11 (q, 2H, CH2),
7.11e7.16 (d, 2H, phenyl), 7.49e7.52 (d, 2H, phenyl); Its MS (m/z),
424 (Mþ, 43).
3.2.2. 3-Acetyl-1-(4-nitrophenyl)-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]benzothieno[2,3-d]-pyrimidine-5-thione (8b)
It was obtained from 3a and 2-oxo-N-(4-nitrophenyl)-propane
3.2.4. 3-Acetyl-1-(4-chlorophenyl)-6,7,8,9-tetrahydro[1,2,4]triazolo
[4,3-a]benzothieno[2,3-d]pyrimidine-5-thione (8d)
hydrazonoyl chloride 4b as yellow powder; IR (cmꢀ1
,
n
): 3022 (CH
aryl), 2924 (CH alkyl), 1723 (CO), 1234 (CS), 1650 (C]N); 1H NMR
(DMSO-d6, , ppm): 1.68 (m, 4H, 2CH2), 2.74 (s, 3H, CH3), 2.89 (m,
It was obtained from 3a and 2-oxo-N-(4-chlorophenyl)-propane
d
hydrazonoyl chloride 4d as yellow powder; IR (cmꢀ1
,
n
): 2998 (CH
aryl), 2920 (CH alkyl), 1711 (CO), 1654 (C]N), 1242 (CS); 1H NMR
(DMSO-d6, , ppm): 1.59 (m, 4H, 2CH2), 2.67 (s, 3H, CH3), 2.85 (m,
2H, CH2), 3.28 (m, 2H, CH2), 7.06e7.10 (d, 2H, phenyl), 7.65e7.70 (d,
2H, phenyl); Its MS (m/z), 425 (Mþ, 68).
d
2H, CH2), 3.27 (m, 2H, CH2), 7.06e7.11 (d, 2H, phenyl), 7.46e7.52 (d,
2H, phenyl); Its MS (m/z), 414 (Mþ, 65).
Table 4
3.2.5. 1,3-Diphenyl-6,7,8,9,10-petaahydro[1,2,4]triazolo[4,3-a]
cycloheptathieno[2,3-d]-pyrimidine-5-thione (8e)
Characterization data of compounds 3e18.
Comp. No.
M.p. [ꢁC]
Yield [%]/solvent
Mol. formulaa (mol.wt.)
C10H10N2S3 (254.4)
C11H12N2S3 (268.4)
C23H18N4S2 (414.5)
It was obtained from 3b and N-phenylbenzene-carbohy-
drazonoyl chloride 4a as white crystals; IR (cmꢀ1
,
n
): 3042 (CH aryl),
3a
3b
8a
8b
8c
8d
8e
8f
315e317
279e281
200e202
165e167
170e172
180e182
162e164
175e177
210e212
150e152
148e150
239e241
250e252
237e239
171e173
135e137
188e190
110e112
201e203
131e133
211e213
167e169
196e198
180e182
85/Dioxane
76/Ethanol
68/Ethanol
72/Ethanol
70/Ethanol
80/Ethanol
76/Ethanol
72/Ethanol
68/Ethanol
73/Ethanol
86/Dioxane
79/Ethanol
65/benzene
61/benzene
58/n-hexane
54/n-hexane
64/benzene
61/benzene
60/benzene
66/benzene
58/benzene
49/benzene
53/benzene
51/benzene
2918 (CH alkyl), 1640 (C]N), 1237 (CS); 1H NMR (DMSO-d6,
d
,
ppm): 1.58 (m, 4H, 2CH2), 1.73 (m, 2H, CH2), 2.68 (m, 2H, CH2), 3.19
(m, 2H, CH2), 6.95e7.01 (m, 4H, phenyl), 7.25e7.29 (m, 6H, phenyl);
Its MS (m/z), 428 (Mþ, 55).
C19H15N5S2O2 (425.5)
C21H20N4S2O2 (424.5)
C19H15N4S2OCl (414.9)
C24H20N4S2 (428.9)
C20H17N5O3S2 (439.5)
C22H22N4S2O2 (438.5)
C20H17N4OS2Cl (428.9)
C11H12N2S3 (268.4)
3.2.6. 3-Acetyl-1-(4-nitrophenyl)-6,7,8,9,10-pentahydro[1,2,4]
triazolo[4,3-a]cycloheptathieno[2,3-d]pyrimidine-5-thione (8f)
It was obtained from 3b and 2-oxo-N-(4-nitrophenyl)-propane
8g
8h
10a
10b
13a
13b
14a
14b
17a
17b
17c
17d
18a
18b
18c
18d
hydrazonoyl chloride 4b as yellow powder; IR (cmꢀ1
,
n
): 3013 (CH
aryl), 2919 (CH alkyl), 1703 (CO), 1638 (C]N), 1231 (CS); 1H NMR
(DMSO-d6, , ppm): 1.61 (m, 4H, 2CH2), 1.76 (m, 2H, CH2), 2.64 (m,
C
12H14N2S3 (282.4)
C21H24N2S3O7 (512.6)
C22H26N2S3O7 (526.6)
C15H18N2S3O4 (386.4)
C16H20N2S3O4 (400.4)
C24H28N2S3O9 (584.6)
C25H30N2S3O9 (598.7)
d
2H, CH2), 2.81 (s, 3H, CH3), 3.17 (m, 2H, CH2), 6.96e6.98 (d, 2H,
phenyl), 7.27e7.31 (d, 2H, phenyl); Its MS (m/z), 439 (Mþ, 38).
C
24H28N2S3O9 (584.6)
3.2.7. 3-Ethylcarboxylate-1-(4-tolyl)-6,7,8,9,10-pentahydro[1,2,4]
triazolo[4,3-a]cycloheptathieno[2,3-d]pyrimidine-5-thione (8g)
It was obtained from 3b and chloro-(4-tolylhydrazono)-ethyl-
C25H30N2S3O9 (598.7)
C16H19N2S3O5 (415.5)
C17H21N2S3O5 (429.5)
C16H19N2S3O5 (415.5)
C17H21N2S3O5 (429.5)
acetate 4c as white crystals; IR (cmꢀ1
,
n
): 3036 (CH aryl), 2920 (CH
alkyl), 1720 (CO), 1619 (C]N), 1237 (CS); 1H NMR (DMSO-d6,
ppm): 1.23e1.27 (t, 3H, CH3), 1.55 (m, 4H, 2CH2), 1.75 (m, 2H, CH2),
d
,
a
Analysis for C, H, N and the results were within ꢂ0.4% of the theoretical values.

4032
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
2.66 (m, 2H, CH2), 2.75 (s, 3H, CH3), 3.19 (m, 2H, CH2), 4.09e4.16
(q, 2H, CH2), 7.13e7.19 (d, 2H, phenyl), 7.67e7.73 (d, 2H, phenyl); Its
MS (m/z), 438 (Mþ, 32).
furnish crude nucleosides which were purified by column chro-
matography (30% ethylacetate in ether) to afford the pure
thioglycosides.
3.2.8. 3-Acetyl-1-(4-chlorophenyl)-6,7,8,9,10-pentahydro[1,2,4]
triazolo[4,3-a]cycloheptathieno[2,3-d]pyrimidine-5-thione (8h)
It was obtained from 3b and 2-oxo-N-(4-chlorophenyl)-propane
3.4.1. 2-(20,30,50-Tri-O-acetyl-
b-D-Arabinofuranosylthio)-5,6,7,8-
tetrahydrobenzothieno[2,3-d]pyrimidine-4-thione (13a)
It was obtained from 3a and (2,3,5-tri-O-acetyl-
a
-
D
-arabino-
): 3430 (br,
NH), 2985 (CH alkyl), 1752 (3CO), 1230 (CS); 1H NMR (DMSO-d6,
hydrazonoyl chloride 4d as yellow powder; IR (cmꢀ1
,
n
): 3023 (CH
aryl), 2927 (CH alkyl), 1711 (CO), 1640 (C]N), 1233 (CS); 1H NMR
(DMSO-d6, , ppm): 1.58 (m, 4H, 2CH2), 1.73 (m, 2H, CH2), 2.65 (m,
furanosyl)-bromide (11a) as yellow powder; IR (cmꢀ1
, n
d
,
d
ppm): 1.67 (m, 4H, 2CH2), 1.95, 1.99, 2.01 (3s, 9H, 3CH3CO), 2.61 (m,
2H, CH2), 2.95 (m, 2H, CH2), 4.07 (m, 1H, H-40), 4.13 (m, 2H, H-50,
H-500), 5.26 (m, 1H, H-30), 5.34 (m, 1H, H-20), 6.72 (d, 1H, J ¼ 3.67 Hz,
H-10), 13.00 (br, 1H, NH); 13C NMR: 20.83 (CH2), 22.16, 22.20, 22.56
(3CH3), 22.70, 24.90, 27.98 (3CH2), 61.49 (C-50), 66.20 (C-30), 66.45
(C-20), 67.32 (C-40), 85.53 (C-10), 125.8, 130.4, 132.7, 148.3 (carbon of
the thiophene ring), 158.2 (CeS), 168.1, 169.5, 170.6 (3C]O), 180.0
(C]S); Its MS (m/z), 512 (Mþ, 28%).
2H, CH2), 2.78 (s, 3H, CH3), 3.18 (m, 2H, CH2), 7.16e7.19 (d, 2H,
phenyl), 7.36e7.40 (d, 2H, phenyl); Its MS (m/z), 428 (Mþ, 82).
3.3. Preparation of the alkylated S-methyl (10a,b)
General procedure. To a solution of 3a,b (0.01 mol) in aqueous
potassium hydroxide (0.01 mol) in distilled water (5 ml) was added
a solution of methyl iodide in acetone (40 ml). The reaction mixture
was stirred at room temperature for 24 h (under TLC control). The
solvent was evaporated under reduced pressure and the crude
product was filtered off and washed with distilled water to remove
KBr formed. The product was dried, and crystallized from the
proper solvent.
3.4.2. 2-(20,30,50-Tri-O-acetyl-
b-D-arabinofuranosylthio)-6,7,8,9,10-
pentahydrocycloheptathieno-[2,3-d]pyrimidine-4-thione (13b)
It was obtained from compound 3b and (2,3,5-tri-O-acetyl-
a-D-
arabinofuranosyl)-bromide (11a) as yellow powder; IR (cmꢀ1
,
n
):
3426 (br, NH), 2992 (CH alkyl), 1750 (3CO), 1240 (CS); 1H NMR
(DMSO-d6, , ppm): 1.55 (m, 4H, 2CH2), 1.71 (m, 2H, CH2), 1.94, 1.98,
d
3.3.1. Compound 10a
2.00 (3s, 9H, 3CH3CO), 2.65 (m, 2H, CH2), 3.19 (m, 2H, CH2), 4.00 (m,
1H, H-40), 4.14 (m, 2H, H-50, H-500), 5.29 (m, 1H, H-30), 5.40 (m, 1H,
H-20), 6.67 (d, 1H, J ¼ 3.67 Hz, H-10), 12.20 (br, 1H, NH); 13C NMR:
19.78, 20.80 (2CH2), 21.56, 22.10, 22.46 (3CH3), 22.75, 24.93, 28.15
(3CH2), 61.43 (C-50), 66.29 (C-30), 66.55 (C-20), 67.38 (C-40), 85.59
(C-10),125.7,130.9,133.2,148.4 (carbon of the thiophene ring),158.6
(CeS), 168.3, 169.1, 170.4 (3C]O), 181.0 (C]S); Its MS (m/z), 526
(Mþ, 36%).
Compound 10a was obtained from 3a, as yellow crystals; IR
, n
(cmꢀ1 ): 3440 (br, NH), 2918 (CH alkyl), 1230 (CS); 1H NMR
(DMSO-d6,
d, ppm): 1.62e1.65 (m, 4H, 2CH2), 2.23 (s, 3H, SCH3), 2.89
(m, 2H, CH2), 3.27 (m, 2H, CH2), 12.00 (br, 1H, NH, D2O-exchange-
able); 13C NMR: 14.38 (SCH3), 22.36, 22.43, 25.41, 26.67 (4CH2),
125.3, 131.6, 134.6, 149.5 (carbon of the thiophene ring), 158.2
(CeS), 180.0 (C]S); Its MS (m/z), 268 (Mþ, 100).
3.3.2. Compound 10b
3.4.3. 2-(20,30,40,60-Tetra-O-acetyl-
b-D-glucopyranosylthio)-5,6,7,8-
Compound 10b was obtained from 3b, as yellow crystals; IR
tetrahydrobenzothieno-[2,3-d]pyrimidine-4-thione (17a)
(cmꢀ1
,
n
): 3420 (br, NH), 2917 (CH alkyl), 1240 (CS); 1H NMR
It was obtained from compound 3a and (2,3,4,6-tetra-O-acetyl-
(DMSO-d6,
d
, ppm): 1.53 (m, 4H, 2CH2), 1.73 (m, 2H, CH2), 2.19 (s,
a
-
D
-glucopyranosyl)-bromide (11b) as a pale yellow powder; IR
(cmꢀ1 ): 3400 (br, NH), 2938 (CH alkyl), 1732 (CO), 1245 (CS); 1H
NMR (DMSO-d6, , ppm): 1.60e1.64 (m, 4H, 2CH2), 1.94, 2.02, 2.11,
3H, SCH3), 2.67 (m, 2H, CH2), 3.23 (m, 2H, CH2), 12.10 (br, 1H, NH,
, n
D2O-exchangeable); Its MS (m/z), 282 (Mþ, 100).
d
2.14 (4s,12H, 4CH3CO), 2.83 (m, 2H, CH2), 3.19 (m, 2H, CH2), 3.97 (m,
1H, H-50), 4.20 (m, 2H, H-60, H-600), 4.33 (m, 1H, H-40), 4.97 (t, 1H,
H-20), 5.13 (t, 1H, J ¼ 9.57 Hz, H-30), 5.94 (d, 1H, J ¼ 10.72 Hz, H-10),
11.00 (br, H, NH); 13C NMR: 20.74 (CH2), 22.19, 22.24, 22.37, 22.59
(4CH3), 22.75, 24.93, 27.86 (3CH2), 60.23 (C-60), 65.22 (C-30), 67.68
(C-20), 69.32 (C-40), 75.63 (C-50), 87.59 (C-10), 125.3, 129.8, 133.1,
149.6 (carbon of the thiophene ring), 158.6 (CeS), 168.3, 169.7,
170.2, 170.9 (4C]O), 180.4 (C]S); Its MS (m/z), 584 (Mþ, 23%).
3.4. Preparation of the acetylated S-nucleosides (13a,b) and
(17aed)
Method A. To a solution of 3a,b (0.01 mol) in aqueous potassium
hydroxide (0.01 mol) in distilled water (5 ml) was added a solution
of 1-bromo-2,3,5-tri-O-acetyl-
a-D-arabinofuranose (11a) or 2,3,4,6-
tetra-O-acetyl- -gluco-/or galactopyranosyl bromide (11b,c)
a-D
(0.015 mol) in acetone (40 ml). The reaction mixture was stirred at
room temperature for 24 h (under TLC control). The solvent was
evaporated under reduced pressure at 40 ꢁC, and the crude product
was filtered off and washed with distilled water to remove KBr
formed. The product was dried, and crystallized from the proper
solvent.
Method B. Compound 3a,b (0.01 mol) was stirred under reflux,
and dry conditions in 50 ml hexamethyldisilane (HMDS) in the
presence of ammonium sulfate (0.01 mol) for 50e60 h. The clear
solution formed was cooled and the solvent was evaporated in
vacuo to give the silylated compound 7 as yellow oil. The latter oil
was dissolved in acetonitrile (10 ml) and was added to a solution of
3.4.4. 2-(20,30,40,60-Tetra-O-acetyl-
b-D-glucopyranosylthio)-
6,7,8,9,10-pentahydrocycloheptathieno[2,3-d]pyrimidine-4-thione
(17b)
It was obtained from compound 3b and (2,3,4,6-tetra-O-acetyl-
a-
D
-glucopyranosyl)-bromide (11b) as a pale yellow powder; IR
(cmꢀ1 ): 3442 (br, NH), 2943 (CH alkyl), 1730 (CO), 1238 (CS); 1H
NMR (DMSO-d6, , ppm): 1.51 (m, 4H, 2CH2), 1.69 (m, 2H, CH2),
, n
d
1.92, 1.99, 2.02, 2.11 (4s, 12H, 4CH3CO), 2.65 (m, 2H, CH2), 3.21 (m,
2H, CH2), 3.84 (m, 1H, H-50), 4.09 (m, 2H, H-60, H-600), 4.26 (m, 1H,
H-40), 4.88 (t, 1H, H-20), 5.23 (t, 1H, J ¼ 9.61 Hz, H-30), 5.94 (d, 1H,
J ¼ 10.59 Hz, H-10), 12.10 (br, 1H, NH, D2O-exchangeable); Its MS
(m/z), 598 (Mþ, 25%).
1,2,3,4,6-penta-O-acetyl-a-D-glucopyranose (9) in acetonitrile
(5 ml) followed by addition of SnCl4 (1.8 ml). The reaction mixture
was stirred at room temperature for 16e20 h (under TLC control).
The mixture was poured into saturated sodium bicarbonate solu-
tion and extracted the thioglycosides by diethylether þ ethylacetate
(1:1, 100 ml). Evaporate the solvent under reduced pressure to
3.4.5. 2-(20,30,40,60-Tetra-O-acetyl-
b-D-galactopyranosylthio)-
5,6,7,8-tetrahydrobenzothieno[2,3-d]pyrimidine-4-thione (17c)
It was obtained from compound 3a and (2,3,4,6-tetra-O-acetyl-
a-D ,
-galactopyranosyl)-bromide (11c) as yellow powder; IR (cmꢀ1

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
4033
n
): 3996 (br, NH), 2987 (CH alkyl), 1728 (CO), 1241 (CS); 1H NMR
(DMSO-d6, , ppm): 1.56e1.61 (m, 4H, 2CH2), 1.97, 2.01, 2.13, 2.17
10), 124.9, 130.7, 135.3, 149.8 (carbon of the thiophene ring), 159.2
d
(CeS), 181.4 (C]S) Its MS (m/z), 415 (Mþ, 22%).
(4s, 12H, 4CH3CO), 2.81 (m, 2H, CH2), 3.12 (m, 2H, CH2), 3.99 (m, 1H,
H-50), 4.19 (m, 2H, H-60, H-600), 4.39 (m, 1H, H-40), 4.89 (t, 1H, H-20),
5.19 (t, 1H, J ¼ 9.62 Hz, H-30), 6.02 (d, 1H, J ¼ 10.68 Hz, H-10), 11.30
(br, H, NH); Its MS (m/z), 584 (Mþ, 35%).
3.5.4. 2-(
pentahydrocycloheptathieno[2,3-d]-pyrimidine-4-thione (18b)
It obtained from 17b; IR (cmꢀ1
): 3480 (br s, OH), 3290 (br, NH),
2989 (CH alkyl),1239 (CS); 1H NMR (DMSO-d6,
, ppm): 1.55 (m, 4H,
2CH2), 1.72 (m, 2H, CH2), 2.62 (m, 2H, CH2), 3.19 (m, 2H, CH2), 3.78
(m, 1H, H-50), 3.98 (m, 2H, H-60, H-600), 4.29 (m, 1H, H-40), 4.52 (br, H,
D2O-exchangeable OH), 4.77 (t, 1H, H-20), 5.00 (br s, 1H, D2O-
exchangeable OH), 5.10 (t, 1H, J ¼ 9.62 Hz, H-30), 5.17 (d, 1H,
J ¼ 4.8 Hz, D2O-exchangeable OH), 5.60 (br, H, D2O-exchangeable
OH), 6.09 (d, 1H, J ¼ 10.56 Hz, H-10), 11.00 (br, H, NH); Its MS (m/z),
429 (Mþ, 30%).
b-D-Glucopyranosylthio)-6,7,8,9,10-
,
n
d
3.4.6. 2-(20,30,40,60-Tetra-O-acetyl-
b
-D
-galactopyranosylthio)-
6,7,8,9,10-pentahydrocycloheptathieno[2,3-d]pyrimidine-4-thione
(17d)
It was obtained from compound 3b and (2,3,4,6-tetra-O-acetyl-
a
-
D
-galactopyranosyl)-bromide (11c)as yellowpowder;IR (cmꢀ1
,
n
):
3425 (br, NH), 2939 (CH alkyl), 1728 (2CO), 1246 (CS); 1H NMR
(DMSO-d6, , ppm): 1.55 (m, 4H, 2CH2), 1.70 (m, 2H, CH2), 1.96, 2.03,
d
2.07, 2.13 (4s,12H, 4CH3CO), 2.67 (m, 2H, CH2), 3.19 (m, 2H, CH2), 3.92
(m, 1H, H-50), 4.07 (m, 2H, H-60, H-600), 4.19 (m, 1H, H-40), 4.87 (t, 1H,
H-20), 5.29 (t, 1H, J ¼ 9.78 Hz, H-30), 5.99 (d, 1H, J ¼ 10.64 Hz, H-10),
12.25 (br, 1H, NH, D2O-exchangeable); Its MS (m/z), 598 (Mþ, 31%).
3.5.5. 2-(
[2,3-d]pyrimidine-4-thione (18c)
It obtained from 17c; IR (cmꢀ1
2993 (CH alkyl),1238 (CS); 1H NMR (DMSO-d6,
b-D-Galactopyranosylthio)-5,6,7,8-tetrahydrobenzothieno
,
n): 3510 (br s, OH), 3390 (br, NH),
d
, ppm): 1.66 (m, 4H,
3.5. Synthesis of diacetylated 2-(
pyrimidin-4-thiones (14a,b) and (18aed)
b
-
D
-glycosidylthio)-thieno[2,3-d]
2CH2), 2.89 (m, 2H, CH2), 3.21 (m, 2H, CH2), 3.89 (m, 1H, H-50), 4.02
(m, 2H, H-60, H-600), 4.32 (m, 1H, H-40), 4.91 (t, 1H, H-20), 4.60 (br, H,
D2O-exchangeable OH), 5.02 (br s, 1H, D2O-exchangeable OH), 5.04
(d, 1H, J ¼ 4.8 Hz, D2O-exchangeable OH), 5.13 (t, 1H, J ¼ 9.54 Hz,
H-30), 5.56 (br, H, D2O-exchangeable OH), 6.11 (d, 1H, J ¼ 10.62 Hz,
H-10), 10.30 (br, H, NH); Its MS (m/z), 415 (Mþ, 27%).
General procedure. Acetylated compound 13a,b or 17aed
(1.0 mmol) was dissolved in methanolic ammonia (saturated with
NH3 at 0 ꢁC, 100 ml). The reaction mixture was stirred overnight
and then heated the reaction mixture for 1 h at 120e130 ꢁC. The
mixture was then cooled and the solvent was evaporated to provide
the crude nucleoside. Purification by heating the crude in n-hexane
(100 ml, three times) provided 14a,b or 18aed as yellow solid.
Crystallization from methanol gave a pale yellow powder.
3.5.6. 2-(
pentahydrocycloheptathieno[2,3-d]-pyrimidine-4-thione (18d)
It obtained from 17d; IR (cmꢀ1
): 3530 (br s, OH), 3315 (br, NH),
2979 (CH alkyl), 1255 (CS); 1H NMR (DMSO-d6,
, ppm): 1.58 (m, 4H,
b-D-Galactopyranosylthio)-6,7,8,9,10-
,
n
d
2CH2), 1.70 (m, 2H, CH2), 2.65 (m, 2H, CH2), 3.20 (m, 2H, CH2), 3.83
(m, 1H, H-50), 3.89 (m, 2H, H-60, H-600), 4.18 (m, 1H, H-40), 4.86 (t, 1H,
H-20), 4.63 (br, H, D2O-exchangeable OH), 5.03 (br s, 1H, D2O-
exchangeable OH), 5.09 (d, 1H, J ¼ 4.8 Hz, D2O-exchangeable OH),
5.16 (t, 1H, J ¼ 9.70 Hz, H-30), 5.49 (br, H, D2O-exchangeable OH),
6.12 (d, 1H, J ¼ 10.65 Hz, H-10), 10.20 (br, H, NH); Its MS (m/z), 429
(Mþ, 28%).
3.5.1. 2-(
[2,3-d]pyrimidine-4-thione (14a)
It obtained from 13a; IR (cmꢀ1
2974 (CH alkyl),1253 (CS); 1H NMR (DMSO-d6,
b-D-Arabinofuranosylthio)-5,6,7,8-tetrahydrobenzothieno
,
n
): 3520 (br s, OH), 3370 (br, NH),
, ppm): 1.68 (m, 4H,
d
2CH2), 2.90 (m, 2H, CH2), 3.23 (m, 2H, CH2), 3.76 (m, 2H, H-50, H-500),
4.08 (m, 1H, H-40), 4.79 (t, 1H, H-20), 5.11 (t, J ¼ 5.40 Hz, J ¼ 4.95 Hz,
OHeC(50)), 5.18 (d, J ¼ 4.45 Hz, OH-C(30)), 5.39 (d, J ¼ 5.96 Hz, OHeC
(20)), 5.63 (t, 1H, J ¼ 9.78 Hz, H-30), 6.94 (d, 1H, J ¼ 5.64 Hz, H-10),
10.20 (br, H, NH); 13C NMR: 19.90, 22.76, 24.93, 27.92 (4CH2), 60.89
(C-50), 65.37 (C-30), 67.56 (C-20), 69.39 (C-40), 87.47 (C-10), 125.6,
131.2, 134.4, 149.1 (carbon of the thiophene ring), 158.5 (CeS), 179.9
(C]S); Its MS (m/z), 386 (Mþ, 35%).
3.6. In vitro anti-herpes simplex-1 virus (HSV-1)
Samples were prepared by dissolving in DMSO and diluting
aliquots into sterile culture medium before preparing serial dilution
and placed in microtiter trays. Microtiter trays with confluent
monolayer cultures of Vero cells were inverted, the medium shaken
out, and replaced with serial dilutions of sterile extracts in triplicate
3.5.2. 2-(
pentahydrocycloheptathieno[2,3-d]-pyrimidine-4-thione (14b)
It obtained from 13b; IR (cmꢀ1
): 3500 (br s, OH), 3410 (br, NH),
2986 (CH alkyl),1254 (CS); 1H NMR (DMSO-d6,
, ppm): 1.53 (m, 4H,
b-D-Arabinofuranosylthio)-6,7,8,9,10-
in 100 mL medium followed by tittered virus in 100 mL medium
,
n
containing 10% (v/v) calf serum in each well. In each tray, the last
row of wells was reserved for controls that were not treated with
compounds or not treated with virus. The tray were cultured and
incubated at 37 ꢁC in 5% CO2 atmosphere for 6 h. The trays were
inverted onto a pad of paper towels, the remaining cells rinsed
carefully with medium, and fixed with 3.7% (v/v) formaldehyde in
saline for 20 min. The fine cells were rinsed with water, and
examined visually. Antiviral activity is identified as confluent,
relatively unaltered monolayers of stained Vero cells treated with
HSV-1. Cytotoxicity was estimated as the concentration that caused
approximately 50% loss of the monolayer present around the pla-
ques caused by HSV-1 (Table 1).
d
2CH2),1.67(m, 2H, CH2), 2.59 (m, 2H, CH2), 3.15 (m, 2H, CH2), 3.80 (m,
2H, H-50, H-500), 4.12 (m, 1H, H-40), 4.67 (t, 1H, H-20), 5.09 (t,
J ¼ 5.42 Hz, J ¼ 4.96 Hz, OHeC(50)), 5.19 (d, J ¼ 4.43 Hz, OHeC(30)),
5.33 (d, J ¼ 5.94 Hz, OHeC(20)), 5.66 (t,1H, J ¼ 9.60 Hz, H-30), 6.89 (d,
1H, J ¼ 5.67 Hz, H-10), 10.80 (br, H, NH); Its MS (m/z), 400 (Mþ, 42%).
3.5.3. 2-(
[2,3-d]pyrimidine-4-thione (18a)
It obtained from 17a; IR (cmꢀ1
2987 (CH alkyl),1250 (CS); 1H NMR (DMSO-d6,
b-D-Glucopyranosylthio)-5,6,7,8-tetrahydrobenzothieno
,
n
): 3490 (br s, OH), 3385 (br, NH),
, ppm): 1.64 (m, 4H,
d
2CH2), 2.87 (m, 2H, CH2), 3.18 (m, 2H, CH2), 3.86 (m, 1H, H-50), 4.08
(m, 2H, H-60, H-600), 4.29 (m,1H, H-40), 4.55 (br, H, D2O-exchangeable
OH), 4.87 (t, 1H, H-20), 5.02 (br s, 1H, D2O-exchangeable OH), 5.11 (t,
1H, J ¼ 9.57 Hz, H-30), 5.14 (d, 1H, J ¼ 4.8 Hz, D2O-exchangeable OH),
5.52 (br, H, D2O-exchangeable OH), 6.02 (d, 1H, J ¼ 10.56 Hz, H-10),
10.60 (br, H, NH); 13C NMR: 19.98, 21.88, 23.79, 26.76 (4CH2), 61.53
(C-60), 66.31 (C-30), 68.34 (C-20), 68.94 (C-40), 77.81 (C-50), 89.78 (C-
3.7. In vitro anti-human immunodeficiency virus-1 (HIV-1)
Compounds were prepared for assay by dissolving in DMSO then
diluted 1:100 in cell culture medium before preparing serial dilution
and placed in microtiter trays. T4 lymphocytes (CEM cell line) were
added and after a brief interval (1 min or more) HIV-1 was added

4034
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 4026e4034
resulting in a 1:200 final dilution of each of the tested compounds.
Cultures were incubated at 37 ꢁC in 5% CO2 atmosphere for 6 days.
Tetrazolium salt XTT was added to all cells and cultures were incu-
bated to allow formazan color development by virally infected cells.
Individual wells were analyzed spectrophotometrically to quanti-
tative formazan production and, in addition, were viewed micro-
scopically for detection of viable cells. Results were compared with
controls and zidovudine (AZT) treated wells as a positive control and
a determination about activity was made as a percentage protection
of T4 cells against HIV-1 cytopathic effect (Table 2).
antibacterial and antifungal testing. The authors also are grateful to
the staff of the Department of Health and Human Services, National
Cancer Institute (NCI), Cairo University, Egypt for conducting the
antiviral screening of the newly synthesized compounds.
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The authors wish to thank Dr. A. El-Azazy, Department of
Microbiology, National Research Center, Egypt for performing the