Synthesis and antiviral evaluation of some new pyrazole and fused pyrazolopyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.06.054

Some novel substituted pyrazole and pyrazolo[3,4-d]pyrimidine derivatives 2, 4, 8, and 9 were synthesized. Also, some acyclic S-nucleosides of pyrazolo[3,4-d]pyrimidine derivatives 10-13 were prepared via reaction of pyrazolo[3,4-d]pyrimidine-4(3H)-thione derivative 9 with some acyclic sugars. Moreover, the N-nucleoside derivative 14 was prepared via reaction of compound 8 with glucosamine hydrochloride. The antiviral evaluation of some selected new products showed that they have promising antiviral activity against hepatitis-A virus (HAV) and herpes simplex virus type-1 (HSV-1).

Full text of DOI:10.1016/j.bmc.2008.06.054

Bioorganic & Medicinal Chemistry 16 (2008) 7102–7106
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral evaluation of some new pyrazole and fused
pyrazolopyrimidine derivatives
a
a
b
Aymn E. Rashad a, , Mohamed I. Hegab , Randa E. Abdel-Megeid , Jehan A. Micky ,
*
Farouk M. E. Abdel-Megeid a
a Photochemistry Department, National Research Centre, Dokki, Cairo, Egypt
b Chemistry Department, Faculty of Science, Alazhar University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Some novel substituted pyrazole and pyrazolo[3,4-d]pyrimidine derivatives 2, 4, 8, and 9 were synthe-
sized. Also, some acyclic S-nucleosides of pyrazolo[3,4-d]pyrimidine derivatives 10–13 were prepared
via reaction of pyrazolo[3,4-d]pyrimidine-4(3H)-thione derivative 9 with some acyclic sugars. Moreover,
the N-nucleoside derivative 14 was prepared via reaction of compound 8 with glucosamine hydrochlo-
ride. The antiviral evaluation of some selected new products showed that they have promising antiviral
activity against hepatitis-A virus (HAV) and herpes simplex virus type-1 (HSV-1).
Received 9 June 2008
Revised 26 June 2008
Accepted 27 June 2008
Available online 2 July 2008
Keywords:
Pyrazole
Ó 2008 Elsevier Ltd. All rights reserved.
Pyrazolo[3,4-d]pyrimidine
S- and N-nucleosides
Antiviral activity
1. Introduction
sides with fewer side effects than those observed for the already
known derivatives.
The pyrazole ring has attracted great attention1 as it has be-
come fairly accessible and it shows diverse properties. These het-
erocyclic compounds are not only interesting from a viewpoint of
biological activities as potential inhibitors of HIV,2 herbicides,3
bactericides,4 and analgesic drugs,5 but also important and useful
as starting materials for the synthesis of other fused heterocyclic
pyrazolo[3,4-d]pyrimidine derivatives of considerable chemical
and pharmacological importance such as purine analogs.6,7 Several
substituted pyrazolo[3,4-d]pyrimidine derivatives have xanthine
oxidase inhibitor activity7,8 like allopurinol which was first synthe-
sized by Robins in 1956,9 and is still the drug for the treatment of
hyperuricemia and gouty arthritic disease.7–10 Also, it was re-
ported11 that some pyrazolo[3,4-d]pyrimidine derivatives demon-
strated significant antimicrobial,12 antimycobacterial activity,13
antitumor,14 and antiviral activity.15 On the other hand, few cyclic
pyrazolo[3,4-d]pyrimidine nucleosides were reported in the litera-
ture,16 while other acyclic fused pyrimidine nucleosides were re-
ported to be potent anti-herpetic drugs with selective activity
against herpes simplex virus (HSV-1 and HSV-2)17,18 and varicella
zoster virus (VZV).18 However, there is still a great need and inter-
est to prepare more active cyclic and acyclic pyrimidine nucleo-
The biological activity and structure–activity relationship
(SAR) of some newly synthesized compounds were evaluated
in comparison to those of amantadine and acyclovir, and these
compounds were found to possess potent antiviral activities
(see Figs. 1 and 2).
* Corresponding author. Tel.: +20 27319 969; fax: +20 20337 0931.
E-mail address: aymnelzeny@yahoo.com (A.E. Rashad).
Figure 1. Effect of some novel pyrazole and fused pyrazolopyrimidine derivatives
on HAV (MBB cell culture strain) in comparison with amantadine (A#) as a control.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.054

A. E. Rashad et al. / Bioorg. Med. Chem. 16 (2008) 7102–7106
7103
ing benzoylthiourea derivative 2. The structure of compound 2
was confirmed by the elemental analysis and spectral data (see
Section 4). Alkaline hydrolysis of the latter compound with so-
dium hydroxide did not afford only product assigned to the
structure of compound 3, but also afforded the cyclized 4-amin-
opyrazolo[3,4-d]pyrimidine-6(5H)-thione derivative 4. Moreover,
compound 3 could be obtained from the reaction of 5 with
ethyl-(ethoxymethylene)cyanoacetate 6, which is identical in all
respects (physical and spectral data) with that obtained previ-
ously (Scheme 1).
On the other hand, compound 719 was converted to its corre-
sponding 4-chloro derivative 8, when heated with phosphorus
oxychloride under reflux. The mass spectrum of compound 8 gave
fragments showing the isotopic pattern due to the presence of
chlorine atom (see Section 4).
Also, on heating compound 719 with phosphorus pentasulfide17
or compound 8 with thiourea in ethanol,20 the reaction afforded 1-
(5,6-dihydronaphtho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-
pyrazolo[3,4-d]pyrimidine-4(3H)-thione (9). Analytical and spec-
tral data are in agreement with the proposed structure of
compound 9, particularly the absence of C@O and the presence
of C@S signals in the IR and 13C NMR spectra (see Section 4).
Since the synthesis of acyclovir, as one of the most potent anti-
herpetic drug by Schaffer et al. in 1978,21 many attempts have
been directed by nucleoside chemists to prepare a lot of relative
compounds with various side chains and glycons17,21–23; however,
Figure 2. Effect of some novel pyrazole and fused pyrazolopyrimidine derivatives
on HSV-1 (MBB cell culture strain) in comparison with acyclovir (C*) as a control.
2. Discussion
2.1. Chemistry
The reaction of 5-amino-1-(5,6-dihydronaphtho[10,20:4,5]thie-
no[2,3-d]pyrimidin-11-yl)-1H-pyrazole-4-carbonitrile (1)12 with
benzoyl isothiocyanate (generated in situ) gave the correspond-
Scheme 1.

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A. E. Rashad et al. / Bioorg. Med. Chem. 16 (2008) 7102–7106
acyclonucleosides of pyrazolo[3,4-d]pyrimidine derivatives were
rarely reported in the literature.24 Thus, in continuation of our pre-
vious work17,22,24–26 in preparing various cyclic and acyclic nucleo-
sides of different heterocyclic compounds, we describe in this
report the synthesis of some S- and N-nucleosides related to pyraz-
olo[3,4-d]pyrimidine derivatives. So, when the potassium salt (gen-
erated in situ) of compound 9 (which then exists in the thiol
tautomeric form under the condition of the reaction) was treated
with 2-chloroethyl methyl ether, chloroacetaldehyde dimethylace-
tal, 2-chloroehanol, and 2-(2-chloroethoxy)ethanol afforded the
corresponding S-acyclic nucleosides 10, 11, 12, and 13, respectively.
The absence of C@S signals in 13C NMR spectra of compounds 10 and
11 confirmed that the site of the attack was on the S- and not on the
N- atom. Moreover, the presence of methoxyethyl and dimethoxy-
ethyl protons and the absence of the NH signal in 1H NMR spectra
of compounds 10 and 11 confirmed their structures. Similarly, the
IR spectra of compounds 12 and 13 revealed signals indicative for
the OH groups, while their 1H NMR spectra revealed signals indica-
tive for the OH and CH2 groups (see Section 4).
On the other hand, the reactivity of compound 8 toward N-
nucleophiles was investigated. So, when compound 8 was refluxed
with glucosamine hydrochloride in the presence of triethylamine,
the reaction afforded N-[1-(5,6-dihydronaphtho[10,20:4,5]thie-
no[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-gluco-
samine (14). The proposed structure of compound 14 was
elucidated on the basis of analytical and spectral data (see Section
4, Scheme 2).
include the three possibilities for antiviral activity; virucidal effect,
virus adsorption and effect on virus replication for both HAV and
HSV-1 at concentrations 10
ous that at a concentration of 10
l l
g/105 and 20 g/105 cells. It was obvi-
l
g/105 cells, enaminonitrile and
the thiourethane of the pyrazole derivative (compounds 1 and 2)
revealed the highest anti-HAV activity in comparison with amanta-
dine (A#) as a control. While at a concentration of 20 g/105 cells,
l
compounds 1 and 3 revealed the highest anti-HAV activity in com-
parison with the other tested compounds. On the other hand, at a
concentration of 10 l
g/105 cells, the thiourethane and S-acyclic
nucleosides (compounds 2, 10, and 11) revealed the highest anti-
HSV-1 activity in comparison with the other tested compounds.
While at a concentration of 20 l
g/105 cells, compounds 2 and 10
revealed the highest anti-HSV-1 activity in comparison with the
other tested compounds.
3. Conclusions
Structural activities correlation of the obtained results re-
vealed that at both concentrations the substituted pyrazole
derivatives (compounds 1–3) revealed higher anti-HAV activity
than the fused pyrazolopyrimidine derivatives (7 and 9), that
is, fusion of pyrimidine to the pyrazole ring decreases the anti-
HAV activity.
Moreover, at a concentration of 10 l
g/105 cells, addition of
fused pyrimidine to the pyrazole ring (compounds 7 and 9) slightly
increases the anti-HSV-1 activity. On the other hand, at concentra-
tion of 20 l
g/105 cells, the substituted pyrazole and fused pyrazol-
2.2. Antiviral bioassay
opyrimidine derivatives revealed higher anti-HSV-1 activity than
S-acyclic nucleoside derivatives. (compounds 11–13). In general,
at higher concentrations all tested compounds revealed lower anti-
viral activity than the controls (amantadine and acyclovir).
Plaque infectivity assay was carried out to test compounds 1, 2,
3, 7, 9 and 10–13 for antiviral activity. The test was performed to
Scheme 2.

A. E. Rashad et al. / Bioorg. Med. Chem. 16 (2008) 7102–7106
7105
using petroleum ether/ethyl acetate (3:1) as an eluent to give com-
pound 8. Yield 55%, mp 190–192 °C; IR
max 1526 cmꢀ1 of the C@N,
and absence of the C@O and NH groups; 1H NMR (DMSO-d6): d
4. Experimental
4.1. Chemistry
m
0
0
2.9–3.0 ppm (m, 4H, C5 –CH2 + C6 –CH2), 7.2–7.3 (m, 4H, 3Ar–
0
H + C3–H), 8.0 (s, 1H, C9 –H), 8.35 (d, 1H, Ar–H, J = 7.5 Hz), 8.9 (s,
4.1.1. General
1H, C6–H); EIMS, m/z (%): 392 (M+, 37Cl, 39.98), 390 (M+, 35Cl, 100).
Anal. Calcd for C19H11ClN6S (390.05): C, 58.39; H, 2.84; N, 21.50; S,
8.20. Found: C, 58.51; H, 2.61; N, 21.38; S, 8.41.
All melting points are uncorrected and measured using Electro-
thermal IA 9100 apparatus (Shimadzu, Japan). IR spectra were re-
corded as potassium bromide pellets on a Perkin-Elmer 1650 Spec-
trophotometer, National Research Centre, Cairo, Egypt. 1H NMR
and 13C NMR spectra were determined on a Jeol-Ex-300 NMR spec-
trometer, and chemical shifts were expressed as part per million;
ppm (d values) against TMS as internal reference (Faculty of Sci-
ence, Cairo University, Cairo, Egypt). Mass spectra were recorded
on EI + Q1 MSLMR UPLR, National Research Centre, Cairo, Egypt.
Microanalyses were operated using Mario Elmentar apparatus, Or-
ganic Microanalysis Unit, National Research Centre, Cairo, Egypt,
and the results were within the accepted range of the calculated
values. Column Chromatography was performed on (Merck) Silica
gel 60 (particle size 0.06–0.20 mm).
4.1.5. 1-(5,6-Dihydronaphtho[10,20:4,5]thieno[2,3-d]pyrimidin-
11-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(3H)-thione (9)
(i) Compound 719 (0.01 mol) was heated at reflux temperature in
(20 ml) dry pyridine containing phosphorus pentasulfide (0.01 mol)
for 5 h. The solution was cooled and poured with stirring onto ice,
acidified with dil hydrochloric acid and the solid that formed was fil-
tered off, washed several times with water, dried and recrystallized
from dimethylformamide to give compound 9 in 75% yield.
(ii) Compound 8 (0.01 mol) and thiourea (0.01 mol) were
heated at reflux temperature in dry ethanol (40 ml) for 4 h. The so-
lid product was collected and recrystallized from dimethylform-
amide to give compound 9 in 70% yield, mp 245–247 °C; IR mmax
3127 (NH) and 1221 cmꢀ1 of the C@S group; 1H NMR (DMSO-
Compounds 1, 3, 5, 6,12 and 719 were prepared as reported in the
literature.
0
0
d6): d 2.8–2.9 ppm (m, 4H, C5 –CH2 + C6 –CH2), 7.1–7.3 (m, 5H,
4.1.2. N-Benzoyl-N0-[4-cyano-1-(5,6-dihydronaphtho[10,20:4,5]-
thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazol-5-yl]thiourea (2)
A mixture of benzoyl chloride (0.01 mol), and ammonium iso-
thiocyanate (0.01 mol) was heated at reflux temperature in dry
acetone (20 ml) for 15 min, then compound 1 was added and the
reaction mixture was heated at reflux temperature for 2 h, poured
into ice water and the deposited solid was filtered off, washed sev-
eral times with water, dried and recrystallized from dioxane to give
compound 2. Yield 59%, mp 240–242 °C; IR (KBr) mmax 3286, 3133
0
3Ar–H + C9 –H + C3–H), 8.0 (d, 1H, Ar–H, J = 9.0 Hz), 8.2 (s, 1H, C6–
H), 13.8 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6): d
24.6 (C-50), 29 (C-60), 127–157 (sp2 carbon atoms), 164 (C@S). Anal.
Calcd for C19H12N6S2 (388.47): C, 58.74; H, 3.11; N, 21.63; S, 16.51.
Found: C, 58.31; H, 3.51; N, 21.77; S, 16.21.
4.1.6. General procedure for the synthesis of 10, 11, 12, and 13
To a solution of dry ethanol (20 ml) containing KOH (0.01 mol),
compound 9 (0.01 mol) was added, then the reaction mixture was
stirred at room temperature for 15 min, then 2-chloroethyl methyl
ether, chloroacetaldehyde dimethylacetal, 2-chloroehanol, or 2-(2-
chloroethoxy)-ethanol (0.01 mol) was added and the reaction mix-
tures were stirred at 70 °C for 3, 4, 3, and 2 h, respectively. The
reaction mixtures were evaporated under reduced pressure and
the residues were purified on silica gel column to give compounds
10, 11, 12, and 13, respectively.
(2NH), 2204 (C„N), 1629 (C@O) and 1330 (C@S) cmꢀ1 1H NMR
;
0
0
(DMSO-d6): d 2.9–3.1 ppm (m, 4H, C5 –CH2 + C6 –CH2), 6.0 (s, 1H,
NH, D2O exchangeable), 6.4–7.4 (m, 9H, Ar–H), 7.6 (s, 1H, C3–H),
0
7.9 (s, 1H, NH, D2O exchangeable), 9.1 (s, 1H, C9 –H). Anal. Calcd
for C26H17N7OS2 (507.59): C, 61.52; H, 3.38; N, 19.32; S, 12.63.
Found: C, 61.72, H, 3.63, N, 19.18, S, 12.24.
4.1.3. General procedure for the synthesis of compounds 3
and 4
4.1.6.1. 4-(2-Methoxyethylsulfanyl)-1-(5,6-dihydronaphtho
Compound 2 (0.01 mol) was heated at reflux temperature in
20 ml of alcoholic sodium hydroxide solution (sodium hydroxide
1.68 g, 7 ml water) for 4 h, then evaporated under reduced pres-
sure, and the residue was purified on silica gel column using petro-
leum ether 40–60 °C/ethyl acetate (7:3) as an eluent to give
products 3 and 4. Compound 3 obtained here is identical in all re-
spects with that obtained in previous report.12
[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]
pyrimidine (10). Yield 65%, mp 217–219 °C; IR
mmax absence of the
NH and C@S signals; 1H NMR (DMSO-d6): d 2.9–3.0 ppm (m, 4H,
0
0
C5 –CH2 + C6 –CH2), 3.5 (s, 3H, OCH3), 4.5–4.6 (m, 4H, 2CH2), 7.1–
0
7.6 (m, 5H, 4Ar–H + C3–H), 7.9 (s, 1H, C9 –H), 8.5 (s, 1H, C6–H);
13C NMR (DMSO-d6) d 24.6 (C-50), 29 (C-60), 29.78 (OCH3), 58.89
(CH2), 71.21 (CH2), 127–157 (sp2 carbon atoms), 164 (C@S). Anal.
Calcd for C22H18N6OS2 (446.55): C, 59.17; H, 4.06; N, 18.82; S,
14.36. Found: C, 59.52; H, 4.41; N, 18.63; S, 14.06.
4.1.3.1. 4-Amino-1-(5,6-dihydronaphtho[10,20:4,5]thieno[2,3-d]-
pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidin–6(5H)-thione
(4). Yield 55%, mp 298–301 °C; IR
mmax 3400, 3200, and 3071 (NH2/
4.1.6.2. 4-(Dimethoxyethylsulfanyl)-1-(5,6-dihydronaphtho
[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]
pyrimidine (11). Yield 33%, oil; IR mmax 1591 cmꢀ1 of the C@N
group, and absence of the NH and C@S signals; 1H NMR
(DMSO-d6): d 2.05–2.1 ppm (m, 2H, CH2), 2.6 (t, 1H, CH), 2.9–3.0
NH) cmꢀ1 and disappearance of the C„N group; 1H NMR (DMSO-
0
0
d6): d 2.8–2.9 ppm (m, 4H, C5 –CH2 + C6 –CH2), 7.1–7.3 (m, 6H, C3–
0
H, 3Ar–H, NH2, D2O exchangeable), 8.1 (s, 1H, C9 –H), 8.35 (d, 1H,
Ar–H, J = 7.8 Hz), 12.4 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) d 24.4 (C-50), 29.3 (C-60), 129–156 (sp2 carbon atoms),
167.3 (C@S). Anal. Calcd for C19H13N7S2 (403.49): C, 56.56; H,
3.25; N, 24.30; S, 15.89. Found: C, 56.32; H, 3.61; N, 24.76; S, 15.36.
0
0
(m, 4H, C5 –CH2 + C6 –CH2), 3.45 (s, 6H, 2OCH3), 7.2–7.8 (m, 6H,
4Ar–H + C3–H + C9 –H), 9.2 (s, 1H, C6–H); 13C NMR (DMSO-d6): d
0
19 (C-50), 23.5 (C-60), 30.0 (CH2), 40.0 (2OCH3), 58 (CH), 127–145
(sp2 carbon atoms). Anal. Calcd for C23H20N6O2S2 (476.58): C,
57.96; H, 4.23; N, 17.63; S, 13.46. Found: C, 57.72; H, 4.52; N,
17.36; S, 13.77.
4.1.4. 4-Chloro-1-(5,6-dihydronaphtho[10,20:4,5]thieno[2,3-d]
pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidine (8)
Compound 719 (0.01 mol) was heated at reflux in phosphorus
oxychloride(20 ml) for 2 h. The solutionwas cooledand pouredwith
stirring onto ice and the solid that formed was filtered off, washed
several times with water, dried and purified on silica gel column
4.1.6.3. 4-(2-Hydroxyethylsulfanyl)-1-(5,6-dihydronaphtho
[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]
pyrimidine (12). Yield 53%, oil; IR mmax 3430–3300 cmꢀ1 of OH,

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A. E. Rashad et al. / Bioorg. Med. Chem. 16 (2008) 7102–7106
1591 cmꢀ1 of the C@N group, and absence of the NH and C@S sig-
4.2.4. Cytotoxicity assay
1
0
0
nals; H NMR (DMSO-d6): d 2.5–2.6 ppm (m, 4 H, C5 –CH2 + C6 –
Cytotoxicity was assayed for both dimethyl sulfoxide (DMSO)
and the tested compounds. Serial dilutions were prepared and
inoculated on Vero cells grown in 96-well tissue culture plates.
The maximum tolerated concentration (MTC) for each compound
was determined by both cell morphology and cell viability by
staining with Trypan blue dye.
CH2), 2.91–2.95 (m, 2H, CH2), 3.6–3.8 (m, 2H, CH2), 5.0 (br s, OH,
0
D2O exchangeable), 7.3–7.8 (m, 6H, 4Ar–H + C3–H + C9 –H), 9.2 (s,
1H, C6–H). Anal. Calcd for C21H16N6OS2 (432.52): C, 58.31; H,
3.73; N, 19.43; S, 14.83. Found: C, 58.75; H, 3.42; N, 19.12; S, 14.96.
4.1.6.4. 4-(Hydroxyethoxyethylsulfanyl)-1-(5,6-dihydro-naph
tho[10,20:4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazolo[3,4-d]
pyrimidine (13). Yield 47%, oil; IR mmax 3430–3300 cmꢀ1 of broad
OH, 1591 cmꢀ1 of C@N, and the absence of the NH and C@S signals;
1H NMR (DMSO-d6): d 2.1 ppm (t, 2H, CH2), 2.5–2.6 ppm (m, 4H,
4.2.5. Plaque reduction infectivity assay
A 6-well plate was cultivated with cell culture (105 cell/ml) and
incubated for 2 days at 37 °C. HSV-1 and HAV were diluted to give
104 PFU/ml final concentrations for each virus and mixed with the
C5 –CH2 + C6 –CH2), 2.91–2.95 (m, 2H, CH2), 3.6–3.8 (m, 2H, CH2),
5.0 (s, OH, D2O exchangeable), 7.3–7.8 (m, 6H, 4Ar–H + C3–
tested compound at 10
overnight at 4 °C. Growth medium was removed from the multiwell
plate and virus-compound mixture was inoculated (100 l/well).
l l
g/105 and 20 g/105 cells, and incubated
0
0
0
H + C9 –H), 9.2 (s, 1H, C6–H). Anal. Calcd for C23H20N6O2S2
l
(476.58): C, 57.96; H, 4.23; N, 17.63; S, 13.46. Found: C, 57.62; H,
4.65; N, 17.98; S, 13.03.
After 1 h of contact time, the inoculum was aspirated and 3 ml of
MEM with 1% agarose was overlaid the cell sheets. The plates were
left to solidify and incubated at 37 °C until the development of virus
plaques. Cell sheets were fixed in 10% formaline solution for 2 h and
stained with crystal violet stain. Control virus and cells were treated
identically without chemical compound. Virus plaques were
counted, and the percentage of reduction was calculated.27
4.1.7. N-[1-(5,6-Dihydronaphtho[10,20:4,5]thieno[2,3-d]
pyrimidin-11-yl)-1H-pyrazolo[3,4-d]pyrimidin]glucosamine (14)
A solution of compound 8 (0.01 mol) in dry ethanol (20 ml) was
treated with glucosamine hydrochloride (0.01 mol) in the presence
of triethylamine (2 drops) as a catalytic amount. The reaction mix-
ture was refluxed for 3 h, and the solid product was collected by fil-
tration on hot, dried, and recrystallized from dioxane to give
compound 14. Yield 45%, mp 224–226 °C; IR mmax 3250–3400
(NH + OH), 1745 cmꢀ1 (C@O); 1H NMR (DMSO-d6): d 2.9–3.3 ppm
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13. Ballell, L.; Field, R. A.; Chung, G. A. C.; Young, R. J. Bioorg. Med. Chem. Lett. 2007,
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1636.
0
0
(m, 8H, C5 –CH2 + C6 –CH2 + 4CH), 3.4–3.45 (m, 2 H, CH2O), 3.8 (s,
1H, OH, D2O exchangeable), 4.3–4.6 (m, 2H, 2OH, D2O exchange-
able), 4.9 (s, 1H, OH, D2O exchangeable), 5.9–5.95 (m, H, COCH),
6.0 (s, 1H, OH, D2O exchangeable), 6.6 (s, 1H, NH, D2O exchangeable),
0
7.1–7.6 (m, 5H, 4Ar–H + C3–H), 8.4 (s, 1H, C6–H), 8.9 (s, 1H, C9 –H).
Anal. Calcd for C24H23N7O5S (521.55): C, 55.27; H, 4.44; N, 18.80; S,
15.34. Found: C, 55.39; H, 4.53; N, 18.67; S, 15.71.
4.2. Antiviral bioassay
4.2.1. Preparation of synthetic compounds for bioassay
Hundred milligrams of each tested compound were dissolved in
1 ml of 10% DMSO in water. The final concentration was 100
(Stock solution). The dissolved stock solutions were decontami-
nated by addition of 50 g/ml antibiotic–antimycotic mixture
(10,000 U penicillin G sodium, 10,000 g streptomycin sulfates
and 250 g amphotericin B, PAA Laboratories GmbH, Austria).
lg/ll
l
l
l
17. Rashad, A. E.; Ali, M. A. Nucleosides Nucleotides 2006, 25, 17.
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Walker, R. T.; Balzarini, J.; Declercq, E. J. Med. Chem. 1991, 34, 349.
19. Rashad, A. E.; Hegab, M. I.; Abdel-Megeid, R. E.; Fathlla, N.; Abdel-Megeid, F. M.
E. Phosphorus, Sulfur, Silicon, Relat. Elem., 2008, submitted for publication..
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2007, 18, 274.
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Nucleosides Nucleotides 2007, 26, 379.
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M. Phytother. Res. 2004, 18, 30.
4.2.2. Cell culture
African green monkey kidney-derived cells (Vero) and human
hepatoma cell line (HepG2) were used. Cells were propagated in
Dulbeccos’ minimal essential medium (DMEM) supplemented with
10% foetal bovine serum, 1% antibiotic–antimycotic mixture. The
pH was adjusted at 7.2–7.4 by 7.5% sodium bicarbonate solution.
The mixture was sterilized by filtration through 0.2
nitrocellulose membrane.
lm pore size
4.2.3. Viruses
Herpes simplex virus type-1 (HSV-1) and hepatitis-A virus
(HAV, MBB-cell culture adapted strain) were obtained from Envi-
ronmental Virology Lab., Department of Water Pollution Research,
National Research Centre, Cairo, Egypt.