Synthesis, biological and medicinal significance of S-glycosido-thieno[2,3-d]-pyrimidines as new anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2009.12.056

Several 2-thioglycosides were prepared. Glycosylation of 2-thioxo-thieno[2,3-d]-pyrimidines 5a,b with 1-bromo-2,3,5-tri-O-acetyl-α-d-arabinofuranosyle 7, 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl and galacto-pyranosyl bromide 8a,b gave the protected β-d-nuclosides 10a,b and 13a-d in high yields, which were transformed to deacetylated derivatives 14a,b and 15a-d. The structures of the compounds were elucidated by spectral and elemental analysis. Anti-inflammatory and Analgesic activities screening of the new compounds (at a dose of 100?mg/kg body weight) utilizing in vivo acute carrageenan-induced paw oedema standard method exhibited that the deacetylated derivatives 14a,b and 15a-d possess highly promising activities.

Full text of DOI:10.1016/j.ejmech.2009.12.056

European Journal of Medicinal Chemistry 45 (2010) 1485–1493
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological and medicinal significance of
S-glycosido-thieno[2,3-d]-pyrimidines as new anti-inflammatory
and analgesic agents
,
a
b
Hend N. Hafez a , Abdel-Rahman B.A. El-Gazzar , Galal A.M. Nawwar
*
a Photochemistry Department, Heterocyclic & Nucleosides Unit, National Research Center, 12622 Dokki, Cairo, Egypt
b Green Chemistry Department, National Research Center, 12622 Dokki, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 2-thioglycosides were prepared. Glycosylation of 2-thioxo-thieno[2,3-d]-pyrimidines 5a,b with
1-bromo-2,3,5-tri-O-acetyl- -arabinofuranosyle 7, 2,3,4,6-tetra-O-acetyl- -glucopyranosyl and gal-
acto-pyranosyl bromide 8a,b gave the protected -nuclosides 10a,b and 13a–d in high yields, which
were transformed to deacetylated derivatives 14a,b and 15a–d. The structures of the compounds were
elucidated by spectral and elemental analysis. Anti-inflammatory and Analgesic activities screening of
the new compounds (at a dose of 100 mg/kg body weight) utilizing in vivo acute carrageenan-induced
paw oedema standard method exhibited that the deacetylated derivatives 14a,b and 15a–d possess
highly promising activities.
Received 30 April 2009
Received in revised form
14 December 2009
Accepted 23 December 2009
Available online 13 January 2010
a-
D
a-D
b-D
Keywords:
N-Amino-2-thioxo-thieno[2,3-d]pyrimidin-
4-one
Ó 2010 Elsevier Masson SAS. All rights reserved.
S-Glycosido-thieno[2,3-d]-pyramidine
Anti-inflammatory
Analgesic activities
1. Introduction
other strategy is the inhibition of inducible nitric oxide synthase
(iNOS), which contributes to acute and chronic inflammation [7–9].
Rheumatic diseases are the most prevalent causes of disability
in Western countries, and non-steroidal anti-inflammatory drugs
(NSAIDs) are still the most commonly used remedies. NSAIDs cause
several serious adverse effects; the most important one is gastric
injury that might later cause gastric ulceration and renal injury [1].
Attempts to develop non-steroidal anti-inflammatory drugs that
are devoid of classical NSAID toxicity, especially gastrointestinal
injury, follow several strategies. One of which is selective cyclo-
oxygenase-2 inhibition (COX-2) [2,3]. Although agents that inhibit
COX-2 while sparing COX-1 represented a new attractive thera-
peutic development, they also gave rise to some of the side effects
seen with traditional dual COX inhibitors (NSAIDs), namely, effects
on the kidney that might manifest as an increased incidence of
hypertension, edema and associated clinical states [4–6]. Therefore,
selective COX-2 inhibitors may not be the proper strategy to over-
come the damaging effects of conventional NSAIDs which are used
for the chronic inflammatory diseases in a long term basis. The
In addition, it is known that bacterial infections often produce
pain and inflammation. In normal practice, two groups of agents
(chemotherapeutic, analgesic and anti-inflammatory) are prescribed
simultaneously. Unfortunately, none of the drugs possesses these
three activities in a single component. Therefore, our aim is to find
a compound having dual effect analgesic, anti-inflammatory and
anti-microbial activities. While searching for such a compound, we
have found that 2-thioxo-thieno[2,3-d]-pyrimidine ring is one of the
moieties on which studies have been concentrated. In our laboratory,
we have designed and synthesized some 2-thioxo-thieno[2,3-
d]pyrimidine and pyridopyrimidine derivatives in the search for new
non-steroidal anti-inflammatory agents [10–23]. A considerable
number of the prepared compounds have been found to have
analgesic-anti-inflammatory activity comparable to or higher than
that of indomethacin. 2-thioxopyrimidines is also a isostere of
purines and promizole whose anti-microbial activities have been
extensively investigated and performed [24,25]. All of these have
made us think that pyrimidines are promising compounds for
finding a drug which has analgesic, anti-inflammatory and anti-
microbialaction.
Pyrimidines have a long and distinguished history extending
from the days of their discovery as important constituents of
* Corresponding author. Tel.: þ202 373 18830; fax: þ202 333 70931.
E-mail address: hendnagah2000@yahoo.com (H.N. Hafez).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.056

1486
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
nucleic acids to their current use in the chemotherapy of AIDS.
Recently, pyrimidines derivatives have generated wide-spread
interest due to their antiviral properties (Fig. 1); 5-Iododeoxyur-
idine (A) is an antiviral agent of high selectivity. 5-Trifluromethyl-
20-deoxyuridine (B) has been found useful against infections
Compounds 5a,b was found to be useful for the syntheses of the
interesting S-glycosides. As a model experiment the alkylation of 5a
was carried out by the reaction of one equivalent of methyliodide
with the potassium salt 6a generated in situ by the reaction of 5a
with alcoholic potassium hydroxide. The structure of the new
2-methylthio-quinazoline 7 was confirmed by all spectroscopic data.
The 13C NMR spectrum as an example, reveled that the corre-
resistant to IDU therapy. Ara-A 9-b-D-arabinofuranosyl adenine (C),
a relatively new antiviral drug, is effective against herpes infections
of eye, brain and skin. It is especially effective against IDU-resistant
herpes virus [26]. Also, Retrovir (AZT-16, D) is a potent inhibitor of
the in vivo replication and cytopathic effects of HIV and has been
recently approved for use against AIDS and severe ARC [27].
There are a large number of pyrimidine-based antimetabolites.
They are usually structurally related to the endogenous substrates
that they antagonize. The structural modification may be on the
pyrimidine ring or on the pendant sugar groups. One of the early
metabolites prepared was 5-fluorouracil [28]. Other active pyrim-
idines and thieno[2,3-d]pyrimidines nucleosides have recently
been synthesized as antiviral against HIV-1 and Herpes simplex
Virus (HSV-1) [29–31]. However, thioglycosides of thieno[2,3-d]-
pyrimidine are not known in the literature.
sponding signal of the C-2 (C–SCH3) appeared at
d 159 ppm. The
chemical shifts in the 13C NMR spectrum of the 2-thioxo- (5a) and
2-methylthio-thieno[2,3-d]pyrimidine (7) indicated that the site of
the alkylation is the sulfur atom rather than the nitrogen atom
(Scheme 2, Table 1).
In order to compare 13C NMR data, 13C chemical shifts of new
compounds 5a, 7 and 13a together with those previously reported
for compounds 5c and 5d are shown in Scheme 2. The NMR study of
compound 5a was carried out in DMSO-d6 solution because this
compound is very insoluble in CDCl3. 13C chemical shifts of
compound 5a were assigned by comparing the chemical shifts of
the corresponding 2-thioxo, 2-oxo-derivatives 5c and 5d. On the
other hand, the correct assignment for C-2 in compound 7 and 13a
was checked by comparison with the corresponding chemical shifts
of related derivatives A and B previously reported [35,36], (see
Scheme 2).
2. Results and discussion
As can be seen in Table 1 and Scheme 2, the chemical shift of C-2
is shifted downfield about 25 ppm by changing an oxo group
(compounds 5c) by a thioxo group (compounds 5d and 5a about
174 ppm). Comparing 13C NMR spectra of unsubstituted derivative
5a and S-derivative 7 (recorded both in DMSO-d6), it is interesting
to point out whilst the C-2 signal appears shifted 25 ppm to up-field
in 7, as expected for the change of an N–C]S to N]C–S-group.
NMR spectra of soluble compounds in CDCl3 13a–d were recorded
in this solvent. In order to compare 13C chemical shifts of
compounds 7 and 13a, the 13C NMR spectrum of 13a was also
recorded in DMSO-d6 showing both spectra only slight or no
chemical shift differences. One possible explanation, of this
anomalous fact, could be the existence of compound 13a mainly in
the form S-glycosides (see Scheme 3).
2.1. Chemistry
On the basis of the above considerations, original nucleoside
analogs directed upon reverse transcriptase still aroused consid-
erable interest [32]. In the hope of elucidating and/or finding better
therapeutic agents, S-glycosido-thieno[2,3-d]pyrimidine analogs
seem to be one of the recommended targets and extension of our
work on pyrimidines [33], C-nucleosides [13,22] and thieno[2,3-
d]pyrimidines [10–12]. Also, due to the data of S-glycosides are
relatively few, all this promoted us to research and developed in the
synthesis of these types of nucleosides.
With respect tothe previous studies carried out in ourlaboratory,
5-acetyl and/or 5-ethylcarboxylate-3-ethyl-(2-amino-4-methyl-
thiophene)carboxylate precursors [34] have been regarded as
promising intermediates to produce 2-thioxo-thieno[2,3-d]pyrimi-
dine with some structural analogies with natural nucleobases. The
interaction of acetylacetone with ethyl cyanoacetate and sulfur
metal in absolute ethanol in the presence of diethylamine led to
ethyl thiophene-3-carboxylate 1b. The hydrazide 3b obtained by
refluxing of ethylcarboxylate 1b with hydrazine hydrate in ethanol.
2-Thioxo-thieno[2,3-d]pyrimidin-4-ones (5a,b) were produced by
two way. The first: action of carbon disulfide on hydrazide 3a,b and
the second: action of dimethylsulfate and carbon disulfide on
5-acetyl and/or 5-ethylcarboxylate-3-ethyl(2-amino-4-methyl-
thiophene)carboxylate in dimethylsulfoxide followed by hydrazine
hydrate (99%) as shown in Scheme 1.
The synthetic route we used for the preparation of 2-S-(b-D-gly-
copyranosyl/or furanosyl)-thieno[2,3-d]pyrimidine is outlined in
Scheme 3. The heterocycle thieno[2,3-d]pyrimidines 5a,b was con-
verted into its potassium salt 6a,b with used of KOH in acetone and
was stirred at room temperature for long time with 2,3,4,6-tetra-O-
acetyl-a-D-glucopyranosyl bromide (8b) or 2,3,4,6-tetra-O-acetyl-a-
D-galacto-pyranosyl bromide (8c), afforded the S-glycosylated
nucleosides 13a–d in good yields (68–78%). Thin layer chromatog-
raphy (chloroform:methanol, 8:2) indicated the formation of the
pure compounds. The structures of the S-glycosides were confirmed
by elemental analysis and spectral data (IR, 1H NMR, 13C NMR). The
1H NMR spectrum of compound 13a as an example, showed the
O
O
N
O
N
O
H3C
F3C
I
N
N
NH
NH
HN
NH
O
O
O
N
H
O
N
H
HO
HO
HO
O
HO
O
O
O
HO
H
H
H
N3
OH
OH
OH
H
A
B
C
D
Fig. 1.

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
1487
C2H5O
R
H3C
CONHNH2
NH2
CN
DEA
S
+
O
CH3
H2NHNOC
S
H3C
R
COOEt
NH2
1a
2a
O
O
(a)
NH2NH2
S
H3C
R
CONHNH2
NH2
1a,b
H3C
R
COOEt
S
H3C
COOEt
S
(b)
S
-
+
3a,b
S Na
S
N
H
R
N
H
SCH3
S
CS2
a, R = COOC2H5
b, R = COCH3
4a,b
NH2NH2
O
H3C
R
NH2
S
H3C
COOEt
N
(a) CS2, NaOH, DMSO
(b) (CH3)2SO4
S
S
N
H
R
N
H
NHNH2
S
5a,b
Scheme 1. Synthetic route of thioxo-thieno[2,3-d]pyrimidines
anomeric proton of the glucose moiety as a doublet at
with a coupling constant J ¼ 10.68 Hz indicating -configuration of
the anomeric center. The other protons of the gluco-pyranose ring
resonated at 3.95–5.10 ppm, while the four acetoxy groups
appeared as four singles at
1.90, 2.00, 2.05 and 2.10 ppm. The 13C
d
5.69 ppm
In addition to the above synthetic methods a so called ‘‘one-pot’’
protocol of the silyl-Hilbert-Johnson reaction was described [37]. This
‘one-pot’ protocol was used by Wolfe for the synthesis of b-D-ribo-
nucleosides [38]. In this procedure, the nucleobase was silyated with
HDMS in presence of (NH4)2SO4 in MeCN and glycosylated with
b
d
d
NMR revealed the absence of the thione carbon atom at about
1,2,3,4,6-penta-O-acetyl-a-D-glyco-pyranose in presence of SnCl4.
174 ppm and a resonance of –N]C–N– carbon atom (C-2) at
Here, MeCN was used as a solvent and HMDS (Hexamethyldisilane) as
glycosylation catalyst (Vorbru¨ggen conditions). The nucleobases were
silyated and directly glycosylated in one step. Townsend applied this
procedure for the synthesis of toyocamycin [39]. We used the ‘one-
d
158 ppm was indicated to the chemical shift of the corresponding
carbon atom (Scheme 3). The signals at 166.5, 169.2, 169.3,
169.5 ppm are due to the four acetoxy carbonyl atoms (4C]O), and
the four signals around 20.29–20.36 ppm are assigned to the
acetate methyl carbon atoms. Also, the six signals at 67.78, 69.82,
d
d
pot’ method for the synthesisd of the 2-(20,30,40,60-tetra-O-acetyl-
b-D-
d
glycopyranosylthio)-thieno[2,3-d]pyrimidin-4-one analogs 13a,b.
The nucleobases 5a,b were silylated with HMDS in anhydrous MeCN
at room temperature in presence of ammonium sulfate and then
reacted with 1,2,3,4,6-penta-O-acetyl-a-D-glyco-pyranose (Scheme
3). This afforded the glycosylated compounds 13a,b (in 54% yields)
after working up.
72.54, 75.01, 81.94 and 98.68 ppm were assigned to C-50, C-60, C-40, C-
20, C-30 and C-10, respectively. Moreover, the IR spectra of compounds
13 revealed the absence of the stretching signal of a thione group.
Similarly, the reaction of heterocycle base 5a,b with 1-bromo-2,3,5-
tri-O-acetyl-a-D-arabinofuranosyle (8a) furnished the S-glycosated
product 10a,b. The structures assignment of this product is based on
their elemental analysis and the spectral data (see Experimental).
Deacetylation of S-nucleosides 10a,b and 13a–d proceeded
smoothly via methanolic ammonia treatment to afford the free
nucleosides 14a,b and 15a–d in good to excellent yields (Scheme 3).
The 1H NMR data of the compounds 14 and 15 revealed the absence
of the acetyl protons and appearance of the D2O exchangeable OH
O
O
O
protons at
example showed also the absence of the acetyl function and the
appearance of the characteristic OH’s band at 3360 (br) cmꢀ1
d 3.10–3.50. The IR data of the compound 14a as a typical
4
4
4
5
5
H3C
R
6
5
NH
N
NH2
S
NH
.
8
8
2
7
2
8
2
N
H
S
S
N
H
N
H
O
174 ppm
175.7 ppm
5c
5d
150.1 ppm
5a
2.2. Pharmacology
O
4
H3C
R
5
6
Analgesic activities of the resulting compounds were investigated
by p-benzoquinone-induced writhing test [40], which is a well
established method of testing the analgesic activity of compounds
and sufficiently sensitive to detect the effect of analgesics which are
less active than aspirin. Anti-inflammatory activities of the
compounds were assessed by utilizing carrageenan-induced hind
paw edema model [41]. Since the carrageenan edema has been used
in the development of indomethacin, many researchers adapted this
procedure for screening potential anti-inflammatory compounds.
O
N
NH2
4
H3C
R
5
6
N
NH2
2
7
8
N
S
S
AcO
7
8
2
H
S
N
SCH3
O
OAc
13a
7
158 ppm
OAc
159 ppm
OAc
Scheme 2. C-2 Chemical shifts of 2-thioxopyrimidines 5a, 7 and 13a.

1488
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
Table 1
13C NMR chemical shifts of the bases and S-Glycosides.
Compd. CH3
OCH2 CO(Es/Ac)
59.83 165.6
CO(4) C-2
C-5
C-6
C-7
C-8
C(10) C(20) C(30) C(40) C(50) C(60)
5aa
5ba
7a
14.83, 17.07
19.89, 20.78
13.98, 20.07
14.69, 14.86, 16.62,
16.79, 20.56
162.2 174.5 113.4 120.4 139.3 153.5
165.7 175.3 118.9 121.7 141.3 149.8
167.2 159.0 123.6 128.5 146.7 152.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
190.2
60.26 189.6
60.01 167.7, 168.2,
169.4, 169.9
10aa
164.2 158.9 105.5 124.1 142.9 156.3 83.68 68.40 68.09 67.62 67.48
10bb
13aa
17.67, 18.94, 21.02, 25.40
–
168.1, 168.4, 169.3, 169.9 163.7 158.2 116.5 133.2 140.6 155.9 88.45 77.14 81.45 76.88 67.35
–
14.31, 16.26, 20.29, 20.32, 59.62 166.5, 169.2, 169.3,
20.35, 20.36 169.5, 169.9
14.62, 16.76, 20.26, 20.28, 59.68 166.7,168.9, 169.2,
164.5 160.1 105.8 122.4 142.8 156.9 98.68 75.01 81.94 72.54 67.78 69.82
163.2 159.3 109.8 124.8 141.5 156.5 92.50 73.45 80.78 70.89 66.57 69.36
163.8 159.6 117.6 125.4 143.6 157.9 87.66 77.11 81.38 74.25 68.33 68.79
13ba
13cb
13db
20.30, 20.33
169.7, 170.3
17.61, 18.87, 20.66, 2075,
20.84, 25.36
–
–
166.3, 168.5, 169.6,
169.8, 170.7
17.62, 18.68, 20.64,
20.75, 20.85
165.7, 166.5, 168.4, 169.2, 162.5 159.3 115.7 123.6 142.9 156.5 90.06 77.38 81.95 74.90 66.34 67.87
170.3
14aa
14ba
15ac
15bc
15cc
15dc
13.90, 19.32
20.21, 21.51
14.00, 20.73
14.08, 19.68
20.34, 21.96
20.27, 22.06
60.73 167.5
– 185.9
61.02 166.8
60.91 168.3
165.3 157.4 118.7 126.3 140.8 154.8 85.79 69.48 67.29 66.55 68.21
164.8 158.6 120.4 126.9 141.6 156.2 84.41 68.37 68.43 67.08 67.79
162.5 159.8 117.9 125.9 143.0 153.8 85.64 73.44 77.27 70.50 77.63 61.53
167.5 158.4 118.6 128.2 142.6 155.0 87.36 75.54 76.93 71.38 77.87 62.08
163.3 157.9 119.0 127.5 141.9 153.9 86.76 74.29 77.35 70.89 78.20 61.75
162.9 156.9 116.9 126.8 145.3 156.1 89.78 74.12 78.03 70.58 77.47 60.59
–
–
–
–
188.4
186.0
Es, CO-Ester; Ac, CO-Acetyl.
a
Measured in DMSO-d6.
Measured in CDCl3.
Measured in DMSO-d6þD2O.
b
c
Carrageenan-induced edema is a non-specific inflammation
maintained by the release of histamine, 5-hydroxytryptamine,
kinins and later by prostaglandins [42]. The inhibitory effect of acid
NSAIDs, such as indomethacin, is usually weak in the first phase
(1–2 h), in contrast with their strong inhibition in the second phase
(3–4 h) [43]. Good inhibition of the second phase of carrageenan-
induced edema was observed for the compounds tested, suggesting
that they interfere with prostaglandin synthesis (Table 2).
However, the inhibitory effects of 14a and 14b reached to signifi-
cant values after 360 min. Among the compounds examined in this
study, the compounds 14a,b, 15a–c possessed the most prominent
and consistent activity. Compounds 14a,b, 15a,c deserve attention
and may be considered for further evaluation.
3. Exprimental
The obtained pharmacological results indicate that among the
compounds, the derivatives 5a,b, which did not carry the S-glyco-
sides moiety on the second position, was found weaker than that
bearing S-glycosides residue on this position and some of the title
compounds possess a good analgesic activity coupled with notable
anti-inflammatory properties. Moreover, all compounds showed
a remarkable gastric tolerance except 13d. Some preliminary
conclusions can be drawn as follows: As shown in Table 2, most
compounds showed potent inhibitory activities between 31.2 and
53.3% on 100 mg/kg dose. When substituents were taken into
consideration within them, it was determined that substituted
3-aminothieno[2,3-d]pyrimidines bearing S-glycosides (14a,b and
15a–d) had a stronger inhibitory effect on analgesic, anti-inflam-
matory activity than that of the acetylated-S-glycosides derivatives
(10a,b and 13a–d). According to this, we suggested the S-glycoside
with free hydroxyl groups was an important structural character-
istic of the analgesic-anti-inflammatory action of these derivatives.
Then the effect of the substitutes on the 3-aminothieno[2,3-
d]pyrimidines was investigated. It was clear that compounds
3.1. Chemistry
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 spectro-
photometer (Shimadzu, Japan). The 1H NMR spectra were
measured with a Jeol ECA 500 MHz (Japan) in DMSO-d6 or CDCl3
and chemical shifts were recorded in
d ppm relative to TMS. Mass
spectra (EI) were run at 70 eV with a Finnigan SSQ 7000 spec-
trometer (Thermo-Instrument System Incorporation, USA). The
purity of the compounds was checked on Aluminium plates coated
with silica gel (Merck). The Pharmacological evaluations of the
products were carried out in Pharmacological Unit Pharmacology
Department (NRC, Cairo, Egypt). The starting compounds 1a, 3a, 5a
were prepared according to previously reported procedures
[34,44].
3.1.1. 5-Acetyl-3-ethyl(2-amino-4-methylthiophene)
carboxylate (1b)
bearing 2-(
b
-D-ribofuranosylthio) residues (14a, 14b) displayed the
According to Gewald et al. [44] and the developed method,
best activity. By contrast, 2-(
b
-D
-glucopyranosyl-thio)-/or 2-(
b-D-
a
mixture of acetylacetone (10 mmol), ethylcyano-acetate
galactopyranosyl-thio)- (15a–d) analogs were less active, which
can be hypothesized that the larger subsistent causes decreasing of
the activity for this series may be detrimental to the overall activity.
Among the compounds It appeared that presence of an ethyl-
carboxylate substituent in position-6 of the 3-aminothieno[2,3-d]-
pyrimidine nucleus led to more active compounds (14a, 15a,b)
compared with acetyl substituted derivatives in the same position
of 3-aminothieno[2,3-d]pyrimidine (14b, 15c,d). A quiet similar
pattern of anti-inflammatory activity was observed with that of
analgesic activity. Although not significant, inhibitory ratios for all
compounds were above 30% for the last two measurements.
(10 mmol), sulfur (10 mmol) and diethylamine (10 mmol) was
heated (70 ꢁC) under stirring in absolute ethanol for 4 h, then leave
the mixture for 24 h at 0 ꢁC. The formed solid was collected by
filtration, washed with ethanol (20 mL), dried and crystallized from
absolute ethanol, as yellow crystals in a 88% yield, m.p. 160–162 ꢁC;
IR (cmꢀ1
NMR (DMSO-d6,
CH3), 2.69 (s, 3H, CH3), 4.32 (q, 2H, J ¼ 7.0 Hz, OCH2) 6.79 (br, 2H,
NH2, D2O exchangeable); 13C NMR:
14.35 (CH3), 16.88 (CH3), 30.19
,
n
); 3424 (br, NH2), 2937 (CH alkyl), 1745, 1718 (2CO); 1H
d, ppm): 1.37 (t, 3H, J ¼ 7.0 Hz, CH3), 2.42 (s, 3H,
d
(CH3), 60.25 (CH2), 109.15, 120.78, 146.58, 166.87 (4C of thiophene
ring), 190.23 (CO), 203.41 (CO); Its MS (m/z), 227 (Mþ, 100);

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
1489
O
O
O
N
H3C
R
NH2
S
H3C
R
H3C
R
NH2
NH2
N
CH3I
N
N
KOH
-
+
S
N
H
S K
S
N
S
SCH3
7
6a,b
5a,b
AcO
O
Br
5a, R = COOC2H5
5b, R = COCH3
OAc
AcO
R1
AcO
O
Acetone
OAc
8a
Br
R2
OAc
Silylation
O
8b,c
AcO
R1
R2
OAc
NH2
H3C
O
O
OAc
N
O
H
H3C
R
NH2
S
N
H3C
NH2
OAc
S
R
N
S
9a,b
N
AcO
S
N
O
AcOR1
SSiMe3
R
S
N
OAc
O
11a,b
OAc
9a, R1 = H, R2 = OAc
9b, R1 = OAc, R2 = H
AcO
10a,b
R2
13a-d
OAc
ammonia
methanol
O
O
O
N
H3C
NH2
H3C
NH2
S
N
NH2
H3C
R
N
N
R
S
S
N
R
S
N
AcO
S
S
HO
H
O
R1
HO
O
O
HO
OAc
OAc
OH
OAc
R2
OH
HO
14a,b
15a-d
12a,b
13a, R = COOC2H5 , R1 = H, R2 = OAc
13b, R = COOC2H5 , R1 = OAc, R2 = H
13c, R = COCH3 , R1 = H, R2 = OAc
13d, R = COCH3 , R1 = OAc, R2 = H
15a, R = COOC2H5 , R1 = H, R2 = OH
15b, R = COOC2H5 , R1 = OH, R2 = H
15c, R = COCH3 , R1 = H, R2 = OH
15d, R = COCH3 , R1 = OH, R2 = H
14a, R = COOC2H5
14b, R = COCH3
Scheme 3. Synthetic route of acetylated-S-glycoside and S-glycosides of thieno[2,3-d]pyrimidines.
Table 2
Percent analgesic activity and inhibition of carrageenan paw edema (CPE) of the compounds 5a,b and 10–15.
Anti-inflammatory Activity
Comp. No
Swelling in thickness (ꢂ10ꢀ2 mm) ꢃ SEM (% inhibition)
Analgesic Activity
Ratio of ulceration
90 min
180 min
270 min
360 min
Number of writhin ꢃ
SEM (% Inhibition)
Control
5a
5b
10a
10b
13a
13b
13c
13d
14a–3a
14b–3b
15a–3c
15b–3f
15c–3d
15d–3g
ASA
41.7 ꢃ 4.13
47.7 ꢃ 4.36
54.2 ꢃ 4.95
59.0 ꢃ 5.31
44.3 ꢃ 3.84
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
2/6
0/6
0/6
1/6
0/6
0/6
0/6
2/6
–
41.2 ꢃ 4.03 (1.2)
44.8 ꢃ 4.31 (6.1)
28.3 ꢃ 2.06 (32.1)
29.8 ꢃ 2.14 (28.5)
36.2 ꢃ 2.69 (13.2)
30.5 ꢃ 2.64 (26.9)
31.0 ꢃ 3.13 (25.7)
41.2 ꢃ 3.54 (14.9)
28.3 ꢃ 2.06 (32.1)
29.8 ꢃ 2.14 (28.5)
29.3 ꢃ 2.91 (29.7)
30.5 ꢃ 2.64 (26.9)
31.0 ꢃ 3.13 (25.7)
35.7 ꢃ 2.80 (14.4)
–
44.8 ꢃ 4.31 (6.1)
44.0 ꢃ 4.39 (7.8)
30.7 ꢃ 2.51 (35.6)*
33.5 ꢃ 1.98 (29.8)
40.7 ꢃ 2.64 (14.7)
35.0 ꢃ 2.81 (26.6)
36.3 ꢃ 3.15 (23.9)
44.0 ꢃ 4.39 (7.8)
30.7 ꢃ 2.51 (35.6)*
33.5 ꢃ 1.98 (29.8)
32.8 ꢃ 3.06 (31.2)
35.0 ꢃ 2.81 (26.6)
36.3 ꢃ 3.15 (23.9)
39.8 ꢃ 2.70 (16.6)
–
49.7 ꢃ 4.84 (8.3)
49.7 ꢃ 4.84 (8.3)
34.7 ꢃ 2.14 (35.9)**
38.7 ꢃ 1.89 (28.6)*
43.2 ꢃ 2.77 (20.3)
37.7 ꢃ 2.63 (30.4)*
38.0 ꢃ 2.5 (29.9)*
48.5 ꢃ 4.29 (10.5)
34.7 ꢃ 2.14 (35.9)**
38.7 ꢃ 1.89 (28.6)*
35.8 ꢃ 2.04 (33.9)*
37.7 ꢃ 2.63 (30.4)*
38.0 ꢃ 2.5 (29.9)*
44.7 ꢃ 2.94 (17.5)
–
54.0 ꢃ 4.80 (8.5)
54.0 ꢃ 4.80 (8.5)
36.0 ꢃ 2.98 (38.9)**
38.0 ꢃ 1.79 (35.6)**
45.8 ꢃ 2.09 (22.3)
40.5 ꢃ 2.80 (31.4)*
38.8 ꢃ 1.70 (34.2)**
51.3 ꢃ 4.14 (13.1)
36.0 ꢃ 2.98 (38.9)**
38.0 ꢃ 1.79 (35.6)**
38.7 ꢃ 2.42 (34.4)**
40.5 ꢃ 2.80 (31.4)*
38.8 ꢃ 1.70 (34.2)**
46.8 ꢃ 3.46 (20.7)
–
33.7 ꢃ 1.94 (23.9**)
28.5 ꢃ 2.41 (35.7**)
20.7 ꢃ 1.33 (53.3***)
22.7 ꢃ 2.52 (48.8***)
27.5 ꢃ 2.75 (37.9*)
25.2 ꢃ 2.10 (43.1***)
24.2 ꢃ 1.56 (45.4***)
38.2 ꢃ 2.95 (13.8)
20.7 ꢃ 1.33 (53.3***)
22.7 ꢃ 2.52 (48.8***)
21.7 ꢃ 2.03 (51.0***)
25.2 ꢃ 2.10 (43.1***)
24.2 ꢃ 1.56 (45.4***)
28.5 ꢃ 2.41 (35.7**)
21.3 ꢃ 1.80 (51.9***)
–
INDO
30.7 ꢃ 4.18 (29.5)*
33.0 ꢃ 3.38 (30.6)*
35.3 ꢃ 3.31 (41.9)**
34.2 ꢃ 3.11 (42.7)***
Aspirin (ASA); Indomethacin (INDO); *p < 0.05, **p < 0.01, ***p < 0.001.

1490
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
C10H13NO3S (227.2); Requires (Found): C, 52.84 (52.81); H, 5.76
(5.75); N, 6.16 (6.14).
285 (Mþ, 100%); C10H11N3O3S2 (285.3); Requires (Found): C, 42.09
(42.07); H, 3.89 (3.85); N, 14.73 (14.71).
3.1.2. 5-Acetyl-2-amino-3-carbohydrazide-4-methylthiophene (3b)
A suspension of dry compound 1b (10 mmol) in hydrazine
hydrate (80%) (5 mL) was stirred under gentle reflux. The insoluble
solid dissolved within 10 min with copious evolution of hydrogen
sulfide to form a clear solution. After 30 min when the solid product
started separating out, heating was continued for 4 h. The reaction
mixture was then allowed to cool to room temperature. The solid
was filtered, washed with ethanol, dried and crystallized from
3.1.4.2. 3-Amino-6-acetyl-5-methyl-2-thioxo-thieno[2,3-d]pyrimidin-4-
one (5b). It was obtained from 4b, orange crystals, yield 85%, m.p.
254–257 ꢁC, IR (cmꢀ1
,
n
); 3423–3273 (br, NH, NH2), 2944 (CH alkyl),
1669, 1609 (2CO); 1H NMR (DMSO-d6,
d, ppm): 2.28 (s, 3H, CH3), 2.67
(s, 3H, CH3), 7.19 (br, 2H, NH2), 7.54 (br, 1H, NH), 9.30 (br, 1H, NH); Its
MS (m/z), 255 (Mþ, 100%); C9H9N3O2S2 (255.3); Requires (Found): C,
42.34 (42.31); H, 3.55 (3.58); N, 16.46 (16.44).
Method B. To a warmed ethanolic sodium hydroxide solution
(0.40 g in 50 mL ethanol), compound 3b (10 mmol) and carbon
disulfide (excess 5 mL) were added. The mixture was heated under
reflux for 15 h. The reaction mixture was allowed to cool to 0 ꢁC, the
deposited precipitate was filtered off, washed by water (20 mL),
dried and crystallized from dioxane as dark yellow crystals.
ethanol; as yellow crystals in a 78% yield, m.p. 112–114 ꢁC; IR (cmꢀ1
,
n
); 3426–3156 (br, NH, NH2), 2966 (CH alkyl), 1693, 1637 (2CO); 1H
NMR (DMSO-d6, d, ppm): 2.40 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.55
(br, 2H, NH2, D2O exchangeable), 6.79 (br, 2H, NH2, D2O
exchangeable), 8.05 (br, 1H, NH, D2O exchangeable); Its MS (m/z),
213 (Mþ); C8H11N3O2S (213.2); Requires (Found): C, 45.05 (45.07);
H, 5.20 (5.17); N, 19.70 1 (19.69).
3.1.5. 3-Amino-5-methyl-6-ethylcarboxylate-2-methyllthio-
thieno[2,3-d]pyrimidin-4-one (7)
3.1.3. Methyl-N-(4-methyl-5-substituted-3-carboxy-
To a warmed ethanolic KOH solution prepared by dissolving
(10 mmol) of KOH in 50 mL (ethanol) was added each of compound
5a (10 mmol), the heating was continued for 30 min and the
mixture was allowed to cool to room temperature, and the proper
methyliodide (12 mmol) was added. The mixture was stirred under
reflux for 5 h, then cool to room temperature, poured into cold
water (100 mL). The solid product precipitated was filtered off
washed with 100 mL water. The product was dried and crystallized
from dioxane as a yellow powder, yield 85%, m.p. 219–221 ꢁC, IR
thiophene)dithiocarbamate (4a,b)
General procedure; To a vigorously stirred solution of 2-amino-3-
carboxy-thiophene (1a,b) (20 mmol) in dimethylsulfoxide (10 mL)
at room temperature, carbon disulfide (1.98 g, 26 mmol) and
aqueous sodium hydroxide (1.2 mL, 20 mol solution) were added
simultaneously over 30 min, the stirring was continued for further
30 min. Dimethylsulfate (2.5 g, 20 mmol) was added drop wise to
the reaction mixture with stirring at 5–10 ꢁC, it was further stirred
for 2 h and poured into ice-water, the solid obtained was filtered,
dried and crystallized from ethanol.
(cmꢀ1
,
n
); 3385 (br, NH2), 2936 (CH alkyl), 1680, 1665 (2CO); 1H
, ppm): 1.28 (t, 3H, CH3), 2.20 (s, 3H, CH3), 2.38 (s,
NMR (DMSO-d6,
d
3H, SCH3), 3.67 (q, 2H, OCH2), 8.10 (br, 2H, NH2); Its MS (m/z), 299
(Mþ, 100%); C11H13N3O3S2 (299.3); Requires (Found): C, 44.13
(44.09); H, 4.37 (4.40); N, 14.03 (13.98).
3.1.3.1. Methyl N-(4-methyl-3,5-dicarboxyethyl-thiophene)dithio-
carbamate (4aꢀ).1 It was obtained from 1a, yield 87%, m.p. 116–
118 ꢁC, IR (cm
, n); 3426 (br, NH), 2985 (CH alkyl), 1708, 1663
(2CO); 1H NMR (DMSO-d6,
d
, ppm): 1.36 (t, 3H, J ¼ 7.00 Hz, CH3),
3.1.6. Preparation of the acetylated-S-nucleosides (10a,b)
and (13a–d)
Method A: To a solution of 5a,b (0.01 mol) in aqueous potassium
hydroxide (0.01 mol) in distilled water (5 mL) was added a solution
1.38 (t, 3H, J ¼ 7.02 Hz, CH3), 1.44 (s, 1H, SH), 2.72 (s, 3H, SCH3), 2.77
(s, 3H, CH3), 4.31 (q, 2H, J ¼ 7.00 Hz, CH2), 4.41 (q, 2H, J ¼ 7.02 Hz,
CH2), 7.25 (br, 2H, NH2), 9.30 (br, 1H, NH) (NH, NH2, D2O
exchangeable); Its MS (m/z), 347 (Mþ, 100%); C13H17NO4S3 (347.4);
Requires (Found): C, 44.93 (44.87); H, 4.93 (4.88); N, 4.03 (4.06).
of 2,3,4,6-tetra-O-acetyl-
a
-D-gluco-/or galacto-pyranosyl bromide
(8b,c) or 1-bromo-2,3,5-tri-O-acetyl-
a-D
-arabinofuranosyle (8a)
(15 mmol) 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 5a,b (10 mmol) was stirred under reflux,
and dry conditions in 50 mL hexamethyldisilane (HMDS) in the
presence of ammonium sulfate (10 mmol) for 50–60 h. The clear
solution formed was cooled and the solvent was evaporated in vacuo
to give the silylated compound 7 as yellow oil. The letter oil was
dissolved in acetonitrile (10 mL) and was added to a solution of
3.1.3.2. Methyl N-(4-methyl-5-acetyl-3-carboxyethyl-thiophene)dithio-
carbamate (4b). It was oꢀb1tained from 1b, yellow crystals, yield 91%,
m.p. 134–137 ꢁC, IR (cm
, n); 3407 (br, NH), 2985 (CH alkyl), 1678,
1665 (2CO); 1H NMR (DMSO-d6,
d
, ppm): 1.36 (t, 3H, J ¼ 7.01 Hz,
CH3), 2.42 (s, 3H, CH3), 2.68 (s, 3H, CH3), 2.70 (s, 3H, SCH3), 4.30 (q,
2H, J ¼ 7.01 Hz, CH2), 6.60 (br, 1H, NH, D2O exchangeable); Its MS
(m/z), 317 (Mþ, 100%); C12H15NO3S3 (317.4); Requires (Found): C,
45.40 (45.37); H, 4.76 (4.73); N, 4.41 (4.39).
3.1.4. 3-Amino-6-ethyl(5-methyl-2-thioxo-thieno[2,3-d]pyrimidin-
4-one)carboxylate or 6-acetyl-5-methyl-2-thioxo-thieno[2,3-d]-
pyrimidin-4-one (5a,b)
1,2,3,4,6-penta-O-acetyl-a-D-gluco-pyranose (9) in acetonitrile
General procedure; To a solution of each of 4a,b (10 mmol) in
ethanol 30 mL was treated with hydrazine hydrate (10 mmol, 99%)
and refluxed on a water bath until the methylmercaptan evolution
ceases (8 h). After cooling, the solid obtained was filtered, dried and
recrystallized from ethanol/acetone mixture.
(5 mL) followed by addition of SnCl4 (1.8 mL). The reaction mixture
was stirred at room temperature for 16–20 h (under TLC control).
The mixture was poured into saturated sodium bicarbonate solution
and extracted the thioglycosides by diethylether þ ethylacetate (1:1,
100 mL). Evaporate the solvent under reduced pressure to furnish
crude nucleosides which were purified by column chromatography
(30% ethylacetate in ether) to afford the pure thioglycosides.
3.1.4.1. 3-Amino-6-ethyl(5-methyl-2-thioxo-thieno[2,3-d]pyrimidin-4-
one)carboxylate (5a). It was oꢀb1tained from 4a, brown crystals, yield
75%, m.p. 192–194 ꢁC, IR (cm
, n); 3425 (br, NH), 2927 (CH alkyl),
1695, 1676 (2CO); 1H NMR (DMSO-d6,
d, ppm): 1.28 (t, 3H, J ¼ 7.0 Hz,
3.1.6.1. 3-Amino-5-methyl-6-ethyl[2-(20,30,50-tri-O-acetyl-
b-D-arabino-
furanosyl-thio)-thieno[2,3-d]pyrimidine-4-one]carboxylate (10a). It
was obtained from compound 5a (10 mmol) and 1-bromo-2,3,5-tri-
CH3), 2.44 (s, 3H, CH3), 4.19 (q, 2H, J ¼ 7.04 Hz, CH2), 7.54 (br, 2H,
NH2), 9.30 (br, 1H, NH), (NH, NH2, D2O exchangeable); Its MS (m/z),

H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
1491
O-acetyl-
a
-
D
-arabinofuranosyle (8a) (10 mmol); as yellow powdꢀe1r,
3.1.6.6. 3-Amino-6-acetyl-5-methyl-2-(20,30,40,60-tetra-O-acetyl-
galactopyranosyl-thio)-thieno[2,3-d]pyrimidin-4-one (13d). It was
obtained from compound 5b (10 mmol) and 2,3,4,6-tetra-O-acetyl-
-galacto-pyranosyl bromide (8c) (10 mmol); as a pale yellow
powder, crystallized from n-hexane, yield 71%, m.p. 130–132 ꢁC, IR
(cmꢀ1
); 3433 (br, NH2), 2963 (CH alkyl), 1750 (4CO acetyl), 1688
(CO acetyl),1630 (COimide); 1H NMR (CDCl3,
, ppm): 1.97, 2.01, 2.04,
2.09(4s,12H, 4CH3CO), 2.16(s, 3H, CH3), 2.34 (s, 3H, CH3), 3.93(m,1H,
H-50), 4.10 (m, 2H, H-60, H-600), 4.76 (m, 1H, H-40), 5.31 (m, 1H, H-20),
5.44 (t, 1H, J ¼ 9.58 Hz, H-30), 5.63 (d,1H, J ¼ 10.72 Hz, H-10), 7.40 (br,
2H, NH2); Its MS (m/z), 585 (Mþ, 21%); C23H27N3O11S2 (585.5);
Requires (Found): C, 47.17 (47.18); H, 4.65 (4.63); N, 7.17 (7.14).
b-D-
crystallized from n-hexane; yield 67%, m.p. 140–142 ꢁC, IR (cm
,
n
); 3431 (br, NH2), 2978 (CH alkyl), 1752 (4CO), 1671 (CO imide); 1H
NMR (DMSO-d6,
d
, ppm): 1.24 (t, 3H, J ¼ 7.46 Hz, CH3), 1.96–2.04 (3s,
a-D
9H, 3CH3CO), 2.46 (s, 3H, CH3), 3.78 (q, 2H, J ¼ 7.46 Hz, OCH2), 3.95
(m, 1H, H-40), 4.20 (m, 2H, H-50, H-500), 5.25 (m, 1H, H-30), 5.51 (m,
1H, H-20), 5.55 (d, 1H, J ¼ 3.65 Hz, H-10), 8.10 (br, 2H, NH2); Its MS
(m/z), 543 (Mþ, 28%); C21H25N3O10S2 (543.5); Requires (Found): C,
46.40 (46.41); H, 4.63 (4.58); N, 7.73 (7.69).
,
n
d
3.1.6.2. 3-Amino-6-acetyl-5-methyl-2-(20,30,50-tri-O-acetyl-
b-D-ara
binofuranosyl-thio)-thieno[2,3-d]pyrimidin-4-one (10b). It was
obtained from compound 5b (10 mmol) and 1-bromo-2,3,5-tri-O-
acetyl-
crystallized from pet-ether 40–60, yield 85%, m.p. 199–201 ꢁC, IR
(cmꢀ1
); 3426 (br, NH2), 2929 (CH alkyl), 1747 (3CO acetyl), 1680
CO acetyl, 1625 (CO imide); 1H NMR (DMSO-d6,
, ppm): 2.00 (s, 3H,
CH3), 2.05, 2.06, 2.09 (3s, 9H, 3CH3CO), 2.33 (s, 3H, CH3), 3.84 (m,
1H, H-40), 4.79 (m, 2H, H-50, H-500), 5.34 (m,1H, H-30), 5.38 (m,1H, H-
20), 5.80 (d, 1H, J ¼ 3.72 Hz, H-10), 8.00 (br, 2H, NH2); Its MS (m/z),
513 (Mþ, 23%); C20H23N3O9S2 (513.5); Requires (Found): C, 46.78
(46.75); H, 4.51 (4.48); N, 8.18 (8.19).
a
-
D
-arabinofuranosyle (8a) (10 mmol) as yellow powder,
3.1.7. General procedure for deacetylation of compounds 10a,b
and 14a–d
,
n
To a stirred solution of compounds 10a,b and 13a–d (1 mmol) in
methanol (10 mL) was added portion wise NaOMe 0.054 g (1 mmol)
in anhydrous methanol (10 mL) by saturated ammonia gas at room
temperature and the solution was stirred overnight. After evapo-
ration of solvent in vacuo, H2O (10 mL) was added and the mixture
was extracted several times with CH2Cl2 to remove the ester
formed during the deprotection. To the resulting aqueous solution
was added an ion exchange resin (Dowex 50W ꢂ 2, Hþ form),
previously washed with methanol. After stirring for tin minutes,
the solution was filtered, evaporated in vacuo and the residue was
flashed chromatographed on silica gel with the gradient 0–10%
methanol in chloroform to give compounds 14a,b and 15a–d. Also,
purification by heating the crude in n-hexane (100 mL, three times)
and crystallization from methanol gave a pale yellow powder.
d
3.1.6.3. 3-Amino-5-methyl-6-ethyl[2-(20,30,40,60-tetra-O-acetyl-
b-D-glu-
copyranosyl-thio)-thieno[2,3-d]pyrimidin-4-one]carboxlate (13a). It
was obtained from compound 5a (10 mmol) and 2,3,4,6-tetra-O-
acetyl-
yellow powder, yield 70%, m.p. 186–188 ꢁC, IR (cmꢀ1
NH), 2938 (CH alkyl), 1742 (CO), 1686 (CO), 1667 CO; 1H NMR
(DMSO-d6,
, ppm): 1.30 (t, 3H, J ¼ 7.52 Hz, CH3), 1.90, 2.00, 2.05,
a-D
-glucopyranosyl bromide (8b) (10 mmol); as a pale
,
n); 3435 (br s,
d
2.10 (4s, 12H, 4CH3CO), 2.63 (s, 3H, CH3), 3.85 (q, 2H, J ¼ 7.52 Hz,
CH2), 3.95 (m, 1H, H-50), 4.15 (m, 2H, H-60, H-600), 4.30 (m, 1H, H-40),
4.95 (t, 1H, H-20), 5.10 (t, 1H, J ¼ 9.59 Hz, H-30), 5.69 (d, 1H,
J ¼ 10.68 Hz, H-10), 8.00 (br,2H, NH2); Its MS (m/z), 615 (Mþ, 13%);
331 (Mþ ꢀ C10H11N3O3S2, 36%), 285 (Mþ þ 1 ꢀ C14H19O9, 30%);
C24H29N3O12S2 (615.6); Requires (Found): C, 46.82 (46.79); H, 4.75
(4.72); N, 6.82 (6.79).
3.1.7.1. 3-Amino-6-ethyl[5-methyl-2-(
thieno[2,3-d]-pyrimidin-4-one]carboxylate (14a). It obtained from
10a. yield 44%, m.p.183–185 ꢁC, IR (cmꢀ1
); 3465 (br, NH), 3360 (br
s, OH’s), 2961 (CH alkyl), 1685, 1662 (2CO); 1H NMR (DMSO-d6,
b-D-arabinofuranosyl-thio)-
,
n
d
,
ppm): 1.22 (t, 3H, J ¼ 7.50 Hz, CH3), 2.40 (s, 3H, CH3), 3.56 m, H-
C(50), 3.80 (q, 2H, J ¼ 7.50 Hz, OCH2), 3.88 (m, H-C(40), 4.05 (m, H-
C(30), 4.23 (m, H-C(20)), 5.04 (t, J ¼ 5.30 Hz, J ¼ 4.92 Hz, OH-C(50),
5.15 (d, J ¼ 4.31 Hz, OH-C(30), 5.36 (d, J ¼ 5.91 Hz, OH-C(20), 6.04 (d,
J ¼ 5.63 Hz, H-C(10), 9.20 (br, 2H, NH2); Its MS (m/z), 417 (Mþ, 19%);
C15H19N3O7S2 (417.4); Requires (Found): C, 43.15 (43.12); H, 4.59
(4.55); N, 10.07 (10.09).
3.1.6.4. 3-Amino-5-methyl-6-ethyl[2-(20,30,40,60-tetra-O-acetyl-
b-D-gal-
actopyranosyl-thio)-thieno[2,3-d]pyrimidin-4-one]carboxlate (13b). It
was obtained from compound 5a (10 mmol) and 2,3,4,6-tetra-O-
acetyl-
powder, crystallized from n-hexane, yield 68%, m.p. 109–111 ꢁC, IR
(cmꢀ1
); 3420 (br s, NH), 2951 (CH alkyl), 1739 (CO), 1689 (CO), 1664
CO; 1H NMR (DMSO-d6,
, ppm): 1.26 (t, 3H, J ¼ 7.49 Hz, CH3), 1.89,
a-D-galacto-pyranosyl bromide (8c) (10 mmol) as yellow
3.1.7.2. 3-Amino-6-acetyl-5-methyl-2-(
thieno[2,3-d]-pyrimidin-4-one (14b). It obtained from 10b. yield
39%, m.p. 231–233 ꢁC, IR (cmꢀ1
); 3420 (br, NH), 3350 (br s,
OH’s), 2928 (CH alkyl), 1700, 1672 (2CO); 1H NMR (DMSO-d6,
ppm): 2.04 (t, 3H, CH3), 2.29 (s, 3H, CH3), 3.50 m, H-C(50), 3.81 m,
H-C(40), 4.02 m, H-C(30), 4.26 (m, H-C(20)), 5.01 (t, J ¼ 5.32 Hz,
J ¼ 4.88 Hz, OH-C(50), 5.12 (d, J ¼ 4.35 Hz, OH-C(30), 5.33 (d,
J ¼ 5.94 Hz, OH-C(20), 6.07 (d, J ¼ 5.60 Hz, H-C(10), 8.40 (br, 2H,
NH2); Its MS (m/z), 387 (Mþ, 31%); C14H17N3O6S2 (387.4); Requires
(Found): C, 43.40 (43.37); H, 4.42 (4.39); N, 10.85 (10.78).
b-D-arabinofuranosyl-thio)-
,
n
d
, n
2.03, 2.05, 2.11 (4s, 12H, 4CH3CO), 2.59 (s, 3H, CH3), 3.68 (q, 2H,
J ¼ 7.49 Hz, CH2), 3.98 (m, 1H, H-50), 4.20 (m, 2H, H-60, H-600), 4.22 (m,
1H, H-40), 5.11 (t, 1H, H-20), 5.31 (t, 1H, J ¼ 9.59 Hz, H-30), 5.54 (d, 1H,
J ¼ 10.68 Hz, H-10), 7.98 (br, 2H, NH2); Its MS (m/z), 615 (Mþ, 23%); 331
(Mþ ꢀ C10H11N3O3S2, 48%), 285 (Mþ þ 1 ꢀ C14H19O9, 28%);
C24H29N3O12S2 (615.6); Requires (Found): C, 46.82 (46.76); H, 4.75
(4.71); N, 6.82 (6.84).
d,
3.1.6.5. 3-Amino-6-acetyl-5-methyl-2-(20,30,40,60-tetra-O-acetyl-
copyranosyl-thio)-thieno[2,3-d]pyrimidin-4-one (13c). It was obtained
from compound 5b (10 mmol) and 2,3,4,6-tetra-O-acetyl- -gluco-
b
-
D
-glu-
3.1.7.3. 3-Amino-6-ethyl[5-methyl-2-(b-D-glucopyranosyl-thio)-thieno-
[2,3-d]pyrimidin-4-one]carboxylateꢀ1(15a). It obtained from 13a.
a
-D
yield 49%, m.p. 213–215 ꢁC, IR (cm
, n); 3425 (br, NH), 3345 (br s,
pyranosyl bromide (8b) (10 mmol); as a pale yellow pꢀo1wder, crystal-
OH’s), 2932 (CH alkyl), 1672, 1668 (2CO); 1H NMR (DMSO-d6, D2O
d,
lized from n-hexane; yield 78%, m.p. 186–189 ꢁC, IR (cm
,
n); 3450 (br,
ppm): 1.23 (t, 3H, J ¼ 7.3 Hz, CH3), 2.36 (s, 3H, CH3), 3.25 (dd, 2H, H-
60, H-600), 3.35–3.42 (m, 2H, H-20, H-30), 5.52 (dd, 1H, H-50), 3.71 (dd,
1H, H-40), 4.05 (q, 2H, J ¼ 7.04 Hz, OCH2), 4.59 (d, 1H, J ¼ 8.7 Hz, H-
10); Its MS (m/z), 447 (Mþ, 26%); C16H21N3O8S2 (447.4); Requires
(Found): C, 42.94 (42.87); H, 4.73 (4.69); N, 9.39 (9.36).
NH2), 2946 (CH alkyl), 1755 (4CO acetyl), 1694 (CO acetyl), 1626 (CO
imide); 1H NMR (CDCl3,
d, ppm): 1.97, 2.00, 2.01, 2.03 (4s,12H, 4CH3CO),
2.11 (s, 3H, CH3), 2.74 (s, 3H, CH3), 3.91 (m, 1H, H-50), 4.13 (m, 2H, H-60,
H-600), 5.12 (t, 1H, H-40), 5.34 (m, 1H, H-20), 5.36 (t, 1H, J ¼ 9.60 Hz, H-30),
5.65 (d,1H, J ¼ 11.0, H-10), 7.29 (br, 2H, NH2); Its MS (m/z), 585 (Mþ,
29%); C23H27N3O11S2 (585.5); Requires (Found): C, 47.17 (47.15); H, 4.65
(4.68); N, 7.17 (7.19).
3.1.7.4. 3-Amino-6-ethyl[5-methyl-2-(
b-D-galactopyranosyl-thio)-
thieno[2,3-d]pyrimidin-4-one]carboxylate (15b). It obtained from

1492
H.N. Hafez et al. / European Journal of Medicinal Chemistry 45 (2010) 1485–1493
13b. yield 51%, m.p. 151–153 ꢁC, IR (cmꢀ1
s, OH’s), 2926 (CH alkyl),1695, 1665 (2CO); 1H NMR (DMSO-d6, D2O,
,
n
); 3455 (br, NH), 3386 (br
prepared (0.5 mg/25
St. Louis, Missouri, U.S.A.) in physiological saline (154 mM NaCl)
into subplantar tissue of the right hind paw. As the control, 25 mL
mL) suspension of carrageenan (Sigma,
d
, ppm): 1.30 (t, 3H, J ¼ 7.00 Hz, CH3), 2.36 (s, 3H, CH3), 3.31 (dd, 2H,
H-60, H-600), 3.32–3.46 (m, 2H, H-20, H-30), 3.61 (dd, 1H, H-50), 3.76
(dd, 1H, H-40), 4.11 (q, 2H, J ¼ 7.02 Hz, CH2), 4.92 (d, 1H, J ¼ 9.01 Hz,
H-10); Its MS (m/z), 447 (Mþ, 22%); C16H21N3O8S2 (447.4); Requires
(Found): C, 42.94 (42.89); H, 4.73 (4.70); N, 9.39 (9.40).
saline solutions were injected into that of the left hind paw. Paw
oedema was measured in every 90 min during 6 h after induction of
inflammation. The difference in footpad thickness between the
right and left foot was measured with a pair of dial thickness gauge
callipers (Ozaki Co., Tokyo, Japan). Mean values of treated groups
were compared with mean values of a control group and analyzed
using statistical methods [41]. Indometacin (INDO) was used as
a reference compound.
3.1.7.5. 3-Amino-6-acetyl-5-methyl-2-(b-D-glucopyranosyl-thio)-
thieno[2,3-d]pyrimidin-4ꢀ-o1ne (15c). It obtained from 13c. yield 42%,
m.p. 201–203 ꢁC, IR (cm
, n); 3410 (br, NH), 3380 (br s, OH’s), 2940
(CH alkyl), 1710, 1664 (2CO); 1H NMR (DMSO-d6, D2O,
d, ppm): 2.19
(s, 3H, CH3), 2.28 (s, 3H, CH3), 3.50 (t, J ¼ 9.2 Hz, 1H, H-40), 3.46–3.54
(m, 2H, H-20, H-30), 3.75 (dd, J ¼ 6.1, 12.4 Hz, H-60), 3.93 (dd, J ¼ 1.70,
12.41 Hz, H-600), 5.19 (d, J ¼ 7.10 Hz, 1H, H-10); Its MS (m/z), 417 (Mþ,
18%); C15H19N3O7S2 (417.4); Requires (Found): C, 43.15 (43.11); H,
4.59 (4.55); N, 10.06 (10.08).
3.2.4. Gastric ulceration study
All the animals were sacrificed immediately after the last
measurement under ether anesthesia and stomachs were removed.
Then the stomachs were examined for lesions under a dissecting
microscope. Stomachs exhibiting one or more ulcers were consid-
ered positive.
3.1.7.6. 3-Amino-6-acetyl-5-methyl-2-(b-D-galactopyranosyl-thio)-
thieno[2,3-d]pyrimidin-4-oneꢀ1(15d). It obtained from 13d. yield
2961 (CH alkyl),1705,1668 (2CO); 1H NMR (DMSO-d6, D2O,
d, ppm):
2.16 (s, 3H, CH3), 2.30 (s, 3H, CH3), 3.38 (dd, 2H, H-60, H-600), 3.41–
3.58 (m, 2H, H-20, H-30), 3.68 (dd, 1H, H-50), 3.87 (dd, 1H, H-40), 5.09
(d, J ¼ 8.96 Hz, 1H, H-10); Its MS (m/z), 417 (Mþ, 14%); C15H19N3O7S2
(417.4); Requires (Found): C, 43.15 (43.13); H, 4.59 (4.53); N, 10.06
(10.03).
3.2.5. Statistical analysis of data
46%, m.p. 165–167 ꢁC, IR (cm
,
n
); 3436 (br, NH), 3390 (br s, OH’s),
Data obtained from animal experiments were expressed as
mean standard error (ꢃSEM). Statistical differences between the
treatments and the control were tested by two tailed Student’s
t-test. p < 0.05 was considered to be significant.
Acknowledgment
Authors thank Prof. Dr. W. El-Eraky Professor of pharmacology
and Head of Pharmaceutical Unit department, NRC, Cairo, Egypt, for
their helpful.
3.2. Pharmacological screening
3.2.1. Animals
Male Swiss albino mice (20–25 g) were obtained from the
animal house colony of the National Research Center, Cairo, Egypt.
The animals left for two days for acclimatization to animal room
conditions and were maintained on standard pellet diet and water
ad libitum [45]. The food was withdrawn on the day before the
experiment, but allowed free access of water. All procedures
involving animals were carried out in accordance with the guide for
the care and use of laboratory animals (National Academy of
Science of Egypt) and were approved by the Animals Studies
Committee at Washington University. Test samples and reference
compounds were suspended in 0.5% carboxymethyl cellulose and
administered to each mouse by using gastric gavages needle. The
control group animals, however, received same volume of dosing
vehicle. In the pharmacological studies, the animals were first
administered in 100 mg/kg (body weight) dose of the test drugs.
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