Synthesis and Anti-Hepatitis B Virus Activity of Glucosylated 2-O-Ethyluracils

Article abstract of DOI:10.1007/s00706-002-0575-7

Summary. 2-O-Ethyluracils were silylated with HMDS and condensed in the presence of TMS-triflate with β-D-glucose pentaacetate to give the corresponding β-nucleosides. Alternatively, these could be synthesized by nucleoside coupling of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide with the sodium salts of 2-O-ethyluracils, which were deprotected with saturated ammonia in methanol. 6′-O-Tosylate nucleoside derivatives were prepared by treating of the latter with tosyl chloride in anhydrous pyridine. The compounds thus obtained were treated with sodium azide in anhydrous DMF to afford the corresponding 6′-azido nucleoside derivatives, which can also be prepared by treatment with sodium azide in the presence of carbon tetrabromide and triphenylphosphine in anhydrous DMF. Nucleophilic displacement of the 6′-tosyloxy group by morpholine gave 6′-deoxy-6′- morpholino nucleosides. The reduction of the azido group of the 6′-azido nucleosides using triphenylphosphine in pyridine afforded the 6′-amino analogues. Glucosylated 2-O-ethyluracils showed moderate activity against HBV.

Full text of DOI:10.1007/s00706-002-0575-7

Monatshefte f€ur Chemie 134, 929–939 (2003)
DOI 10.1007/s00706-002-0575-7
Synthesis and Anti-Hepatitis B Virus
Activity of Glucosylated 2-O-Ethyluracils
ꢀ
Adel A.-H. Abdel-Rahman
Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koam, Egypt
Received September 16, 2002; accepted (revised) October 15, 2002
Published online April 24, 2003 # Springer-Verlag 2003
Summary. 2-O-Ethyluracils were silylated with HMDS and condensed in the presence of TMS-triflate
with ꢀ-D-glucose pentaacetate to give the corresponding ꢀ-nucleosides. Alternatively, these could be
synthesized by nucleoside coupling of 2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl bromide with the
sodium salts of 2-O-ethyluracils, which were deprotected with saturated ammonia in methanol. 60-O-
Tosylate nucleoside derivatives were prepared by treating of the latter with tosyl chloride in anhydrous
pyridine. The compounds thus obtained were treated with sodium azide in anhydrous DMF to afford
the corresponding 60-azido nucleoside derivatives, which can also be prepared by treatment with
sodium azide in the presence of carbon tetrabromide and triphenylphosphine in anhydrous DMF.
Nucleophilic displacement of the 60-tosyloxy group by morpholine gave 60-deoxy-60-morpholino
nucleosides. The reduction of the azido group of the 60-azido nucleosides using triphenylphosphine
in pyridine afforded the 60-amino analogues. Glucosylated 2-O-ethyluracils showed moderate activity
against HBV.
Keywords. Nucleosides, convergent synthesis of; 2-O-Ethyluracils; Human immunodeficiency virus;
Glycosides; Oligonucleotides.
Introduction
Hepatitis B virus (HBV) causes acute and chronic hepatitis, which affects nearly
300 million people worldwide [1]. Chronic infection with HBV has been asso-
ciated with a high risk for the development of primary hepatocellular carcinoma
[2]. In such carriers vaccination is not an effective therapy and alpha interferon has
demonstrated some promise [3]. Effective antiviral therapy against HBV infection
has not been fully developed. Studies have been hampered by the extremely narrow
host range and limited access to experimental culture systems. Nucleoside ana-
logues and, particularly, the unnatural L-configuration have emerged as potential
anti-HBV agents with more promising pharmacological and toxicological profiles
[4] than their D-counterparts. Thus, 20-fluoro-5-methyl-ꢀ-L-arabino furanosyluracil
(ꢀ-L-FMAU) is considered as a clinical candidate for treatment of chronic HBV
ꢀ
E-mail: adelnassar63@hotmail.com

930
A. A.-H. Abdel-Rahman
Fig. 1. Potential anti-HBV agents
infections and is undergoing preclinical toxicology studies [5]. The 20,30-dideoxy-
ꢀ-L-cytidine (ꢀ-L-ddC) and its 5-fluoro analogue (ꢀ-L-FddC) demonstrated
equally potent activity against HBV in vitro, having the same ED50 value of
0.01 mM. The unusual group of nucleosides such as L-SddC [(–)-BCH-189] in
which the 30-CH2 group has been replaced by a hetero-atom [6] exhibits potent
anti-HBV and HIV activity in vitro. In this direction, we were successful in synthe-
sizing a series of 2-O-alkylated 2-uracil nucleoside derivatives [7, 8] in order to
find new antiviral agents.
Results and Discussion
Synthesis
The 2-O-ethyluracils (1a–d) were prepared as described by Hilbert and Jansen [9].
Silylation of the nucleobase with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) was
carried out according to Vorbr€uggen et al. [10] by refluxing the nucleobase in
HMDS in the presence of catalytic amounts of ammonium sulfate. Coupling of
ꢀ-D-glucose pentaacetate (2) with the silylated derivatives of 1a–d was carried out
under the Vorbr€uggen conditions [11] to afford the ꢀ-anomers 4a–d. Alternatively,
4a–d could be synthesized by treatment of 2-O-ethyluracils (1a–d) with NaH in
DMF at 70–80ꢁC followed by addition of 2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyra-
nosyl bromide (3). In all cases the yields were in the range of 75–92% after
purification by column chromatography on silica gel with ether:petroleum ether
(1:1, v=v). Treatment of the nucleoside derivatives 4a–d with saturated ammonia in
methanol resulted in complete deprotection of the hydroxyl groups and the result-
ing products were purified by column chromatography on silica gel using 5–10%
MeOH in CHCl3 as eluent to give 5a–d in 92–97% yields (Scheme 1).
The 60-O-tosylate nucleoside derivatives 6a–d were prepared in 80–85% yields
by treating of 5a–d with tosyl chloride in dry pyridine at 0ꢁC, which were treated
with sodium azide in anhydrous DMF at 80ꢁC to afford the corresponding 60-azido
derivatives 7a–d in 80–90% yields. Compounds 5a–d were allowed to react with
sodium azide in the presence of carbon tetrabromide and triphenylphosphine in
anhydrous DMF at room temperature to give 7a–d in 91–94% yields.
Nucleophilic displacement of the 60-tosyloxy group in 6a–d by morpholine at
reflux temperature gave 60-deoxy-60-morpholino nucleosides 8a–d in 64–78%
yields.

Synthesis and Anti-Hepatitis B Virus Activity
931
Scheme 1
Scheme 2

932
A. A.-H. Abdel-Rahman
Table 1. Inhibition of HBV replication by selected compounds
Compd.
Inhibition=%
Cytotoxicity=%
5a
5b
5c
5d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
60.11
66.50
70.50
72.13
65.15
78.07
30.11
29.50
78.11
79.33
26.30
68.33
69.00
25.00
80.15
23.18
02.00
02.50
08.00
09.01
07.00
01.22
03.50
03.50
01.25
02.01
04.15
10.00
11.00
04.00
01.99
04.50
Following the procedures reported in Ref. [12] on the utility of the azido group
as a synthon for a terminal amino group in an oligonucleotide, we reduced the
azido group of compounds 7a–d using triphenylphosphine in pyridine and obtained
the 60-amino analogues 9a–d in 75–80% yields (Scheme 2).
Testing
Preliminary viral screening against HBV indicated that compounds 5a, 5b, 7b, 8a,
8b, and 9c were found to be active against HBV replication with IC50 ¼ 83, 83, 80,
and 88 ꢂM and CC50>90, 93, 88, and 95 ꢂM. Compounds 7c, 7d, 8c, 9b, and 9d
showed moderate viral replication inhibition and low cytotoxicity, while com-
pounds 5c, 5d, 7a, 8d, and 9a showed high inhibition with high cytotoxicity
(Table 1).
Conclusions
The glucosylated 2-O-ethyluracils can be synthesized either by condensation of
the silylated bases with ꢀ-D-glucose pentaacetate or by alkylation of the sodium
salt of the base derivatives with 2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl bro-
mide. 50-Azido and 50-amino derivatives were also synthesized in order to increase
the number of tested compounds screened for anti-HBV activity.
Experimental
Pyridine was distilled from CaH2 and stored over molecular sieves. Other solvents were purified
according to the standard procedures. TLC was performed on plastic plates Silica Gel 60 F254
(E. Merck, layer thickness 0.2 mm). The detection was achieved by treatment with a solution of

Synthesis and Anti-Hepatitis B Virus Activity
933
15% H2SO4 in methanol and heating at 150ꢁC. Concentrations were performed on a rotary evaporator
at a temperature below 40ꢁC. 1H and 13C NMR spectra were recorded on a Bruker AC 250 FT NMR
spectrometer at 250 MHz for 1H NMR and 62.9 MHz for 13C NMR with TMS as an internal standard.
EIMS and FABMS spectra were recorded with a Finnigen MAT 312=AMD. Infrared spectra were
recorded in KBr pellets on a Pye Unicam SP-1025 spectrometer. The microanalyses were performed at
¨
the microanalytical unit, Universitat Konstanz, Germany. Viral screening against HBV was conducted
at the National Liver Institute, Menoufia University, Egypt.
General Procedure for Preparation of 4a–d
Method A. A mixture of 2-O-ethyluracil derivatives 1a–d (5mmol), 60mg of (NH4)2SO4 and 40cm3
of 1,1,1,3,3,3-hexamethyldisilazane was refluxed (140ꢁC) overnight. The obtained clear solution was
cooled and the solvent was removed in vacuo. The resulting residue was dissolved in 15cm3 of
anhydrous acetonitrile and a solution of 1.25 g of ꢀ-D-glucose pentaacetate (2) (3.2mmol) in
15cm3 of anhydrous acetonitrile was added while stirring. The mixture was cooled to À 30ꢁC and
a solution of 0.75cm3 of TMS triflate (3.9mmol) in 5 cm3 of anhydrous acetonitrile was added
dropwise during 5 minutes. The mixture was stirred for 1 h at À 30ꢁC and then at room temperature
for 1 h. The mixture was diluted with 200 cm3 of CH2Cl2, washed with 150 cm3 of a cold sat. aq.
NaHCO3 solution, 3Â100 cm3 of cold H2O and dried over Na2SO4. The solvent was removed in vacuo
and the residue was chromatographed on silica gel with ether:petroleum ether (1:1, v=v) to afford 4a–d
in 78–92% yields.
Method B. 2-O-Ethyluracil derivatives 1a–d (5mmol) were suspended in 25cm3 of anhydrous
DMF at room temperature. To this suspension 0.26 g of NaH (50%, 5 mmol) were added and the
mixture was stirred at 70–80ꢁC for 0.5 h and cooled to room temperature. 2,3,4,6-Tetra-O-acetyl-ꢁ-D-
glucopyranosyl bromide (3) (2.26 g, 5.5 mmol) was added, the mixture was stirred at room temperature
for 3 h until the starting material was consumed (TLC) and then filtered. The residue resulting from
evaporation of the filtrate under reduced pressure was purified by silica gel column chromatography
with ether:petroleum ether (1:1, v=v) to afford 4a–d.
1-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)-2-ethoxypyrimidin-4(1H)-one
(4a, C20H26N2O11)
1
Yield: Method A 1.35g (90%), Method B 1.99 g (85%), foam; H NMR (DMSO-d6): ꢃ ¼ 1.40 (t,
0
0
00
0
0
J ¼ 6.9 Hz, CH3), 2.02, 2.03, 2.04, 2.07 (4s, 4COCH3), 4.09 (dd, J6 ,6 ¼ 2.0, J5 ,6 ¼ 12.2Hz, 6 -H),
0
0
4.22 (m, 50-H), 4.32–4.42 (m, 60-H, OCH2), 5.06 (t, J3 ,4 ¼ 9.5 Hz, 4 -H), 5.16 (t, J2 ,3 ¼ 9.2 Hz, 2 -H),
0
0
0
0
0
0
0
0
0
0
5.58 (t, J2 ,3 ¼ 9.3 Hz, 3 -H), 6.44 (d, J1 ,2 ¼ 8.0 Hz, 1 -H), 6.66 (d, J ¼ 5.5 Hz, 5-H), 8.47
(d, J ¼ 5.5 Hz, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 14.05 (CH3), 20.08, 20.19 (4COCH3), 61.39
(C-60), 62.95 (OCH2), 67.78 (C-20), 70.19 (C-30), 71.18 (C-40), 71.82 (C-50), 92.66 (C-10), 101.32
(C-5), 160.81 (C-6), 164.19 (C-2), 168.57 (C-4), 168.88, 169.12, 169.35, 169.74 (4COCH3) ppm;
FABMS (DMSOþ 3-nitrobenzyl alcohol): m=z ¼ 471 (M þ Hþ ).
1-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)-2-ethoxy-5-methylpyrimidin-4(1H)-one
(4b, C21H28N2O11)
Yield: Method A 1.42 g (92%), Method B 2.07g (88%), foam; 1H NMR (DMSO-d6): ꢃ ¼ 1.42
0
00
(t, J ¼ 7.0 Hz, CH3), 1.77 (s, CH3), 2.02, 2.03, 2.05, 2.07 (4s, 4COCH3), 4.07 (dd, J6 ,6 ¼ 2.0,
J5 ,6 ¼ 12.1 Hz, 60-H), 4.25 (m, 50-H), 4.35 (m, 60-H), 4.53 (q, J ¼ 7.1Hz, OCH2), 5.05
0
0
0
0
0
0
0
0
0
0
0
(t, J3 ,4 ¼ 9.5 Hz, 4 -H), 5.17 (t, J2 ,3 ¼ 9.2 Hz, 2 -H), 5.55 (t, J2 ,3 ¼ 9.6 Hz, 3 -H), 6.40
(d, J1 ,2 ¼ 8.0 Hz, 1 -H), 8.37 (s, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 13.65 (CH3), 14.05 (CH3),
0
0
0
20.09, 20.19 (4COCH3), 61.35 (C-60), 65.15 (OCH2), 67.79 (C-20), 70.16 (C-30), 71.13 (C-40), 71.80

934
A. A.-H. Abdel-Rahman
(C-50), 92.63 (C-10), 117.99 (C-5), 134.95 (C-6), 160.19 (C-2), 167.17 (C-4), 168.83,
169.13, 169.30, 169.76 (4COCH3) ppm; FABMS (DMSOþ 3-nitrobenzyl alcohol): m=z ¼ 485
(Mþ Hþ ).
1-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)-5-bromo-2-ethoxypyrimidin-4(1H)-one
(4c, C20H25BrN2O11)
1
Yield: Method A 1.38g (79%), Method B 2.05 g (75%), foam; H NMR (DMSO-d6): ꢃ ¼ 1.38 (t,
0
0
00
0
0
J ¼ 6.9 Hz, CH3), 2.02, 2.03, 2.04, 2.07 (4s, 4COCH3), 4.06 (dd, J6 ,6 ¼ 2.0, J5 ,6 ¼ 12.2Hz, 6 -H),
4.20 (m, 50-H), 4.38 (m, 60-H), 4.54 (q, J ¼ 7.0Hz, OCH2), 5.09 (t, J3 ,4 ¼ 9.5 Hz, 4 -H), 5.19
0
0
0
(t, J2 ,3 ¼ 9.0 Hz, 2 -H), 5.53 (t, J2 ,3 ¼ 9.1 Hz, 3 -H), 6.47 (d, J1 ,2 ¼ 8.1 Hz, 10-H), 8.37 (s, 6-H)
0
0
0 0
0
0
0
0
13
ppm; C NMR (DMSO-d6): ꢃ ¼ 14.06 (CH3), 20.08, 20.20 (4COCH3), 61.36 (C-60), 65.25 (OCH2),
67.75 (C-20), 70.22 (C-30), 71.19 (C-40), 71.87 (C-50), 92.73 (C-10), 139.30 (C-5), 159.31 (C-6), 165.59
(C-2), 168.87 (C-4), 168.86, 169.19, 169.34, 169.77 (4COCH3) ppm; FABMS (DMSO þ 3-nitrobenzyl
alcohol): m=z ¼ 549, 551 (Mþ Hþ ).
1-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)-2-ethoxy-5-iodo-pyrimidin-4(1H)-one
(4d, C20H25IN2O11)
1
Yield: Method A 1.48g (78%), Method B 2.29 g (77%), foam; H NMR (DMSO-d6): ꢃ ¼ 1.40 (t,
J ¼ 6.9 Hz, CH3), 2.02, 2.03, 2.04, 2.09 (4s, 4COCH3), 4.04 (dd, J6 ,6 ¼ 2.1, J5 ,6 12.1 Hz, 60-H), 4.20
0
00
0
0
(m, 50-H), 4.41 (m, 60-H), 4.58 (q, J ¼ 7.0 Hz, OCH2), 5.01 (t, J3 ,4 ¼ 9.5 Hz, 4 -H), 5.11 (t,
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,3 ¼ 9.2 Hz, 2 -H), 5.58 (t, J2 ,3 ¼ 9.3 Hz, 3 -H), 6.48 (d, J1 ,2 ¼ 8.0 Hz, 1 -H), 8.37 (s, 6-H) ppm;
13C NMR (DMSO-d6): ꢃ ¼ 14.06 (CH3), 20.08, 20.17 (4COCH3), 61.41 (C-60), 65.25 (OCH2), 67.71
(C-20), 70.23 (C-30), 71.15 (C-40), 71.79 (C-50), 92.61 (C-10), 138.19 (C-5), 156.21 (C-6), 165.99 (C-2),
168.87 (C-4), 168.89, 169.15, 169.35, 169.75 (4COCH3) ppm; FABMS (DMSO þ 3-nitrobenzyl
alcohol): m=z ¼ 597 (Mþ Hþ ).
General Procedure for Preparation of 5a–d
A saturated solution of ammonia in methanol (25 cm3) was added dropwise while stirring to a solution
of 0.5 g of 4a–d in 5 cm3 of methanol at 0ꢁC. The mixture was stirred at room temperature for 2 h and
the solvent was removed in vacuo. The residue was chromatographed on silica gel with 5–10% MeOH
in CHCl3 as eluent to give 5a–d.
1-D-Glucopyranosyl-2-ethoxypyrimidin-4(1H)-one (5a, C12H18N2O7)
1
Yield: 0.30 g (95%); mp 168–170ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.31 (t, J ¼ 6.9 Hz, CH3), 3.26–3.31
(m, 30-H, 40-H, 50-H, 60-H), 3.48 (m, 60-H), 3.64 (m, 20-H), 4.31 (q, J ¼ 6.9 Hz, OCH2), 4.51 (t,
J ¼ 5.7 Hz, 60-OH), 4.99 (d, J ¼ 5.1 Hz, 40-OH), 5.08 (d, J ¼ 3.8 Hz, 30-OH), 5.31 (d, J ¼ 4.6 Hz, 20-
OH), 5.71 (d, J1 ,2 ¼ 7.5 Hz, 1 -H), 6.58 (d, J ¼ 5.5 Hz, 5-H), 8.35 (d, J ¼ 5.5Hz, 6-H) ppm; 13C NMR
(DMSO-d6): ꢃ ¼ 14.14 (CH3), 60.45 (C-60), 62.73 (OCH2), 69.44 (C-40), 72.54 (C-20), 76.51 (C-50),
77.57 (C-30), 96.17 (C-10), 101.59 (C-5), 160.12 (C-6), 164.30 (C-2), 169.70 (C-4) ppm; MS:
m=z ¼ 302 (Mþ ).
0
0
0
1-D-Glucopyranosyl-2-ethoxy-5-methylpyrimidin-4(1H)-one (5b, C13H20N2O7)
1
Yield: 0.32 g (97%); mp 210–213ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.39 (t, J ¼ 7.0 Hz, CH3), 1.75 (s,
CH3), 3.29–3.33 (m, 30-H, 40-H, 50-H, 60-H), 3.45 (m, 60-H), 3.60 (m, 20-H), 4.30 (q, J ¼ 6.9 Hz,

Synthesis and Anti-Hepatitis B Virus Activity
935
OCH2), 4.54 (brs, 60-OH), 5.01 (brs, 40-OH), 5.12 (brs, 30-OH), 5.33 (brs, 20-OH), 5.73 (d,
J1 ,2 ¼ 7.3 Hz, 1 -H), 8.27 (s, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 13.50 (CH3), 14.05 (CH3),
60.43 (C-60), 62.79 (OCH2), 69.55 (C-40), 72.52 (C-20), 76.58 (C-50), 77.67 (C-30), 96.27 (C-10),
117.98 (C-5), 134.90 (C-6), 161.00 (C-2), 167.04 (C-4) ppm; MS: m=z ¼ 316 (Mþ ).
0
0
0
1-D-Glucopyranosyl-5-bromo-2-ethoxypyrimidin-4(1H)-one (5c, C12H17BrN2O7)
1
Yield: 0.32 g (93%); mp 188–190ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.39 (t, J ¼ 6.9 Hz, CH3), 3.16–3.28
(m, 30-H, 40-H, 50-H, 60-H), 3.44 (m, 60-H), 3.61 (m, 20-H), 4.30 (q, J ¼ 6.9Hz, OCH2), 4.51 (brs,
0
60-OH), 4.92 (brs, 40-OH), 5.05 (brs, 30-OH), 5.26 (brs, 20-OH), 5.68 (d, J1 ,2 ¼ 7.4 Hz, 1 -H), 8.30 (s,
0
0
13
6-H) ppm; C NMR (DMSO-d6): ꢃ ¼ 14.01 (CH3), 60.41 (C-60), 62.71 (OCH2), 69.48 (C-40), 72.52
(C-20), 76.56 (C-50), 77.60 (C-30), 96.19 (C-10), 139.25 (C-5), 159.40 (C-6), 166.39 (C-2), 168.79 (C-4)
ppm; MS: m=z ¼ 380, 382 (Mþ ).
1-D-Glucopyranosyl-2-ethoxy-5-iodopyrimidin-4(1H)-one (5d, C12H17IN2O7)
1
Yield: 0.33 g (92%); mp 223–225ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.37 (t, J ¼ 6.9 Hz, CH3), 3.22–3.37
(m, 30-H, 40-H, 50-H, 60-H), 3.67 (m, 60-H), 3.70 (m, 20-H), 4.36 (q, J ¼ 6.9Hz, OCH2), 4.88–5.01 (m,
20-OH, 30-OH, 40-OH, 60-OH), 5.67 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 8.30 (s, 6-H) ppm; 13C NMR (DMSO-d6):
ꢃ ¼ 14.08 (CH3), 60.47 (C-60), 62.76 (OCH2), 69.50 (C-40), 72.55 (C-20), 76.56 (C-50), 77.66 (C-30),
96.27 (C-10), 138.29 (C-5), 156.19 (C-6), 165.90 (C-2), 168.75 (C-4) ppm; MS: m=z ¼ 302 (Mþ ).
0
0
0
General Procedure for Preparation of 6a–d
p-Toluenesulfonyl chloride (1.71 g, 10 mmol) was added to an ice-cooled solution of 10mmol of 5a–d
in 50 cm3 of anhydrous pyridine and left to stand overnight at 4ꢁC. The pyridine was removed in vacuo
and the residue was purified by column chromatography using 10% MeOH in CHCl3 as eluent to give
6a–d.
1-(6-O-p-Tolylsulfonyl-ꢀ-D-glucopyranosyl)-2-ethoxypyrimidin-4(1H)-one
(6a, C19H24N2O9S)
1
Yield: 3.74 g (82%); mp 189–190ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.31 (t, J ¼ 6.9 Hz, CH3), 2.49 (s,
CH3), 3.30–3.40 (m, 30-H, 40-H, 50-H), 3.60 (m, 60-H), 3.68 (m, 20-H), 4.39 (q, J ¼ 6.9 Hz, OCH2),
0
5.02–5.12 (m, 20-OH, 30-OH, 40-OH), 5.66 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 6.55 (d, J ¼ 5.5 Hz, 5-H), 7.39 (d,
J ¼ 7.8 Hz, Harom), 7.78 (d, J ¼ 7.8 Hz, Harom), 8.33 (d, J ¼ 5.5 Hz, 6-H) ppm; 13C NMR (DMSO-d6):
ꢃ ¼ 14.10 (CH3), 26.26 (CH3), 62.75 (OCH2), 65.40 (C-60), 69.33 (C-40), 72.64 (C-20), 76.44 (C-50),
77.51 (C-30), 96.10 (C-10), 101.63 (C-5), 127.88, 129.80, 132.50, 145.00 (Carom), 160.06 (C-6), 164.38
(C-2), 169.71 (C-4) ppm; FABMS (DMSOþ 3-nitrobenzyl alcohol): m=z ¼ 457 (Mþ Hþ ).
0
0
1-(6-O-p-Tolylsulfonyl-ꢀ-D-glucopyranosyl)-2-ethoxy-5-methylpyrimidin-4(1H)-one
(6b, C20H26N2O9S)
1
Yield: 3.99 g (85%); mp 190–193ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.41 (t, J ¼ 7.0 Hz, CH3), 1.75 (s,
CH3), 2.45 (s, CH3), 3.39–3.43 (m, 30-H, 40-H, 50-H), 3.65 (m, 60-H), 3.64 (m, 20-H), 4.32 (q, J ¼ 6.9
0
Hz, OCH2), 5.00 (brs, 40-OH), 5.10 (brs, 30-OH), 5.23 (brs, 20-OH), 5.79 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 7.33
(d, J ¼ 7.8 Hz, Harom), 7.81 (d, J ¼ 7.8 Hz, Harom), 8.20 (s, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 13.43
(CH3), 14.01 (CH3), 26.21 (CH3), 62.70 (OCH2), 66.03 (C-60), 69.50 (C-40), 72.44 (C-20), 76.63 (C-50),
77.57 (C-30), 96.33 (C-10), 117.93 (C-5), 127.80, 129.80, 132.53, 145.12 (Carom), 134.92 (C-6), 161.09
(C-2), 167.24 (C-4) ppm; FABMS (DMSOþ 3-nitrobenzyl alcohol): m=z ¼ 471 (Mþ Hþ ).
0
0

936
A. A.-H. Abdel-Rahman
1-(6-O-p-Tolylsulfonyl-ꢀ-D-glucopyranosyl)-5-bromo-2-ethoxypyrimidin-4(1H)-one
(6c, C19H23BrN2O9S)
1
Yield: 4.33 g (81%); mp 178–180ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.44 (t, J ¼ 6.9 Hz, CH3), 2.51 (s,
CH3), 3.26–3.37 (m, 30-H, 40-H, 50-H), 3.64 (m, 60-H), 3.51 (m, 20-H), 4.41 (q, J ¼ 6.9 Hz, OCH2),
0
4.99–5.09 (m, 20-OH, 30-OH, 40-OH), 5.75 (d, J1 ,2 ¼ 7.2 Hz, 1 -H), 7.30 (d, J ¼ 7.8 Hz, Harom), 7.80 (d,
J ¼ 7.8 Hz, Harom), 8.35 (s, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 14.01 (CH3), 26.20 (CH3), 62.76
(OCH2), 65.81 (C-60), 69.40 (C-40), 72.56 (C-20), 76.66 (C-50), 77.69 (C-30), 96.34 (C-10), 127.81,
129.77, 132.55, 145.19 (Carom), 139.32 (C-5), 159.88 (C-6), 166.45 (C-2), 168.83 (C-4) ppm; FABMS
(DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 535, 637 (Mþ Hþ ).
0
0
1-(6-O-p-Tolylsulfonyl-ꢀ-D-glucopyranosyl)-2-ethoxy-5-iodopyrimidin-4(1H)-one
(6d, C19H23IN2O9S)
1
Yield: 4.65 g (80%); mp 199–203ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.38 (t, J ¼ 6.9 Hz, CH3), 2.50 (s,
CH3), 3.25–3.36 (m, 30-H, 40-H, 50-H), 3.73 (m, 60-H), 3.73 (m, 20-H), 4.41 (q, J ¼ 6.9Hz, OCH2),
0
4.98–5.11 (m, 20-OH, 30-OH, 40-OH), 5.71 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 7.35 (d, J ¼ 7.8 Hz, Harom), 7.77 (d,
J ¼ 7.8 Hz, Harom), 8.30 (s, 6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 14.05 (CH3), 26.30 (CH3), 62.77
(OCH2), 65.45 (C-60), 69.59 (C-40), 72.66 (C-20), 76.77 (C-50), 77.96 (C-30), 96.43 (C-10), 127.77,
129.87, 132.59, 145.23 (Carom), 138.37 (C-5), 156.34 (C-6), 165.90 (C-2), 168.70 (C-4) ppm; FABMS
(DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 583 (Mþ Hþ ).
0
0
General Procedure for Preparation of 7a–d
Method A. Carbon tetrabromide (0.97 g, 3 mmol) was added to a mixture of 2 mmol of 5a–d, 0.68 g of
triphenylphosphine (2.6mmol) and 0.39 g of NaN3 (6mmol) in 10cm3 of anhydrous DMF. The
mixture was stirred at room temperature for 48h, was quenched by addition of 5 cm3 of methanol,
and the solvent was removed in vacuo. The residual oil was applied to a column of silica and eluted
with 6% MeOH in CHCl3 to give 7a–d.
Method B. A mixture of 5 mmol of 6a–d and 0.32g of NaN3 (5mmol) in 25 cm3 of anhydrous
DMF was heated for 2 h at 80ꢁC (TLC). The solvent was removed under reduced pressure and the
residue was triturated with ice–H2O. A white precipitate was collected by filtration, washed with ice–
H2O and ether and dried. The products were crystallized from absolute ethanol to give 7a–d.
1-(6-Azido-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethoxypyrimidin-4(1H)-one
(7a, C12H17N5O6)
Yield: Method A 0.60 g (92%), Method B 1.39 g (85%); mp 193–195ꢁC; IR (KBr): ꢄꢀ¼ 3415 (OH),
2119 (CN3) cmÀ 1; FABMS (DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 328 (Mþ Hþ ).
1-(6-Azido-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethyl-5-methylpyrimidin-4(1H)-one
(7b, C13H19N5O6)
Yield: Method A 0.64 g (94%), Method B 1.53 g (90%); mp 205–207ꢁC; IR (KBr): ꢄꢀ¼ 3430 (OH),
2127 (CN3) cmÀ 1; FABMS (DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 342 (Mþ Hþ ).
1-(6-Azido-6-deoxy-ꢀ-D-glucopyranosyl)-5-bromo-2-ethoxypyrimidin-4(1H)-one
(7c, C12H16BrN5O6)
Yield: Method A 0.75 g (93%), Method B 1.68 g (83%); mp 266–268ꢁC; IR (KBr): ꢄꢀ¼ 3423 (OH),
2129 (CN3) cmÀ 1; FABMS (DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 406, 408 (Mþ Hþ ).

Synthesis and Anti-Hepatitis B Virus Activity
937
1-(6-Azido-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethoxy-5-iodopyrimidin-4(1H)-one
(7d, C12H16IN5O6)
Yield: Method A 0.82 g (91%), Method B 1.80 g (80%); mp 279–282ꢁC; IR (KBr): ꢄꢀ¼ 3410 (OH),
2125 (CN3) cmÀ 1; FABMS (DMSO þ 3-nitrobenzyl alcohol): m=z ¼ 454 (Mþ Hþ ).
General Procedure for Preparation of 8a–d
A solution of 1 mmol of 6a–d in 4.35cm3 of morpholine (50 mmol) was stirred under reflux for 13h.
The solvent was removed in vacuo and the residue was chromatographed on silica with 5% MeOH in
CHCl3 to remove the impurities and then with 10–15% MeOH in CHCl3 as eluent to afford 8a–d.
1-(6-Deoxy-6-morpholino-ꢀ-D-glucopyranosyl)-2-ethoxypyrimidin-4(1H)-one
(8a, C16H25N3O7)
1
Yield: 0.28 g (75%), foam; H NMR (DMSO-d6): ꢃ ¼ 1.33 (t, J ¼ 6.9 Hz, CH3), 2.55 (m, 60-H), 2.80
(m, 2NCH2), 3.39 (m, 50-H), 3.60 (m, 20-H), 3.70–3.80 (m, 30-H, 40-H, 2OCH2), 4.30 (q, J ¼ 6.9 Hz,
0
0
0
OCH2), 4.70 (brs, 3OH), 5.40 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 6.60 (d, J ¼ 5.5 Hz, 5-H), 8.33 (d, J ¼ 5.5 Hz,
6-H) ppm; 13C NMR (DMSO-d6): ꢃ ¼ 14.10 (CH3), 54.30 (2NCH2), 61.25 (C-60), 62.73 (OCH2), 66.60
(2OCH2), 66.70 (C-50), 69.84 (C-40), 73.56 (C-20), 76.97 (C-30), 96.90 (C-10), 101.67 (C-5), 160.43
(C-6), 164.31 (C-2), 169.60 (C-4) ppm; MS: m=z ¼ 371 (Mþ ).
1-(6-Deoxy-6-morpholino-ꢀ-D-glucopyranosyl)-2-ethoxy-5-methylpyrimidin-4(1H)-one
(8b, C17H27N3O7)
1
Yield: 0.30 g (78%); mp 145–147ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.42 (t, J ¼ 6.9 Hz, CH3), 1.79 (s,
CH3), 2.65 (m, 60-H), 2.77 (m, 2NCH2), 3.43 (m, 50-H), 3.56 (m, 20-H), 3.70–3.85 (m, 30-H, 40-H,
0
0
0
2OCH2), 4.36 (q, J ¼ 6.9 Hz, OCH2), 4.77 (brs, 3OH), 5.44 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 8.30 (s, 6-H) ppm;
13C NMR (DMSO-d6): ꢃ ¼ 13.56 (CH3), 14.11 (CH3), 54.36 (2NCH2), 61.45 (C-60), 62.77 (OCH2),
66.80 (2OCH2), 66.66 (C-50), 69.74 (C-40), 73.50 (C-20), 76.99 (C-30), 96.96 (C-10), 118.05 (C-5),
135.11 (C-6), 161.11 (C-2), 166.90 (C-4) ppm; MS: m=z ¼ 385 (Mþ ).
1-(6-Deoxy-6-morpholino-ꢀ-D-glucopyranosyl)-5-bromo-2-ethoxypyrimidin-4(1H)-one
(8c, C16H24BrN3O7)
Yield: 0.35 g (78%), yellow foam; 1H NMR (DMSO-d6): ꢃ ¼ 1.42 (t, J ¼ 6.9 Hz, CH3), 2.61 (m, 60-H),
2.67 (m, 2NCH2), 3.51 (m, 50-H), 3.50 (m, 20-H), 3.70–3.84 (m, 30-H, 40-H, 2OCH2), 4.33 (q,
J ¼ 6.9 Hz, OCH2), 4.79 (brs, 3OH), 5.54 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 8.31 (s, 6-H) ppm; 13C NMR
(DMSO-d6): ꢃ ¼ 14.06 (CH3), 54.42 (2NCH2), 61.67 (C-60), 62.92 (OCH2), 67.23 (2OCH2), 67.66
(C-50), 69.70 (C-40), 73.44 (C-20), 76.87 (C-30), 96.99 (C-10), 139.30 (C-5), 159.43 (C-6), 166.43 (C-2),
168.60 (C-4) ppm; MS: m=z ¼ 450, 452 (Mþ ).
0
0
0
1-(6-Deoxy-6-morpholino-ꢀ-D-glucopyranosyl)-2-ethoxy-5-iodopyrimidin-4(1H)-one
(8d, C16H24IN3O7)
1
Yield: 0.38 g (78%); mp 212–214ꢁC; H NMR (DMSO-d6): ꢃ ¼ 1.37 (t, J ¼ 6.9 Hz, CH3), 2.63 (m,
60-H), 2.59 (m, 2NCH2), 3.48 (m, 50-H), 3.59 (m, 20-H), 3.73–3.89 (m, 30-H, 40-H, 2OCH2), 4.41 (q,
J ¼ 6.9 Hz, OCH2), 4.72 (brs, 3OH), 5.74 (d, J1 ,2 ¼ 7.0 Hz, 1 -H), 8.30 (s, 6-H) ppm; 13C NMR
(DMSO-d6): ꢃ ¼ 14.04 (CH3), 54.41 (2NCH2), 61.40 (C-60), 62.63 (OCH2), 66.77 (2OCH2, C-50),
69.78 (C-40), 73.62 (C-20), 76.89 (C-30), 96.97 (C-10), 138.30 (C-5), 155.99 (C-6), 166.09 (C-2),
168.80 (C-4) ppm; FABMS (DMSOþ 3-nitrobenzyl alcohol): m=z ¼ 498 (Mþ Hþ ).
0
0
0

938
A. A.-H. Abdel-Rahman
General Procedure for Preparation of 9a–d
The azido derivatives 7a–d (1mmol) and 0.43g of triphenylphosphine (1.65 mmol) were dissolved in
30cm3 of pyridine and stirred at room temperature for 1 h. Concentrated NH4OH (25%) was then
added and the solution was stirred for 2 h. The solvent was removed in vacuo, 5 cm3 of H2O were
added, and triphenylphosphine and triphenylphosphine oxide were removed by filtration. The filtrate
was extracted with benzene and with ether to remove the residual triphenylphosphine and then con-
centrated to dryness. Recrystallization of the solid residue from absolute ethanol gave 9a–d.
1-(6-Amino-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethoxypyrimidin-4(1H)-one
(9a, C12H19N3O6)
Yield: 0.23 g (78%); mp 115–117ꢁC; IR (KBr): ꢄꢀ¼ 3455, 3415 (NH2) cmÀ 1; FABMS (DMSO þ
3-nitrobenzyl alcohol): m=z ¼ 302 (Mþ Hþ ).
1-(6-Amino-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethoxy-5-methylpyrimidin-4(1H)-one
(9b, C13H21N3O6)
Yield: 0.25 g (80%); mp 170–171ꢁC; IR (KBr): ꢄꢀ¼ 3460, 3419 (NH2) cmÀ 1; FABMS (DMSO þ
3-nitrobenzyl alcohol): m=z ¼ 316 (Mþ Hþ ).
1-(6-Amino-6-deoxy-ꢀ-D-glucopyranosyl)-5-bromo-2-ethoxypyrimidin-4(1H)-one
(9c, C12H18BrN3O6)
Yield: 0.28 g (76%); mp 193–195ꢁC; IR (KBr): ꢄꢀ¼ 3470, 3435 (NH2) cmÀ 1; FABMS (DMSO þ
3-nitrobenzyl alcohol): m=z ¼ 380, 382 (Mþ Hþ ).
1-(6-Amino-6-deoxy-ꢀ-D-glucopyranosyl)-2-ethoxy-5-iodopyrimidin-4(1H)-one
(9d, C12H18IN3O6)
Yield: 0.32 g (75%); mp 271–275ꢁC; IR (KBr): ꢄꢀ¼ 3450, 3425 (NH2) cmÀ 1; FABMS (DMSO þ
3-nitrobenzyl alcohol): m=z ¼ 428 (Mþ Hþ ).
Antiviral Assays
The hepatoplastoma cell line (Hep G2-2.2.15) was used to evaluate the antiviral effect of the tested
compounds against HBV [13]. The cells were incubated in growth medium (RPMI-1640, 10% heat-
inactivated fetal-calf serum (FCS)) and antibiotics at 37ꢁC, 5% CO2 with and without tested com-
pounds. The average production HBV virion DNA from cell cultures with addition of different
concentration of a tested compound was expressed relatively to HBV virion DNA in cultures without
the tested compound. Quantitative analysis of HBV-DNA was done using a semiquantitative nested
PCR followed by DIG PCR ELISA as previously described [14]. The cytotoxic effect of the com-
pounds was assessed by culturing the Hep G2-2.2.15 cells in the presence of compounds as described
for the viability of their cells and they were analyzed using a MTT-assay.
Acknowledgments
The author thanks DANIDA for the support of this work at Menoufia University through the project
‘‘Development of New Drugs Against Hepatitis’’ and Prof. Dr. R. R. Schmidt for spectroscopic
¨
measurements at Konstanz University. Thanks are also due to Mr. K. Hagele for measuring the
FABMS.

Synthesis and Anti-Hepatitis B Virus Activity
939
References
[1] Ayoola EA, Balayan MS, Deinhardt F, Gust I, Kureshi AW, Maynard JE, Nayak NC, Brodley
DW, Ferguson M, Melnick J, Purcell RH, Zuckerman AJ (1988) Bull World Health Org 66: 443
[2] a) Beasley RP, Hwang LY, Lin CC, Chien CS (1981) Lancet ii: 1129; b) Di Bisceglei AM, Rustgi
VK, Hoofnagle JH, Dusheik GM, Lotze MT (1988) Ann Intern Med 108: 390
[3] a) Pai SB, Liu SH, Zhu YL, Chu CK, Cheng YC (1996) Antimicrob Agents Chemother 40: 380;
b) Chirgwin JM, Przybla AF, Mac Donald RJ, Rutter WJ (1979) Biochemistry 18: 5294; c) Chu
CK, Ma TW, Shanmuganathan K, Wang C, Xiang Y, Pai SB, Yao GQ, Sommadossi JP, Cheng YC
(1995) Antimicrob Agents Chemother 39: 979; d) Colcacino JM, Malcolm SK, Jaskunas SR
(1994) Antimicrob Agents Chemother 38: 1997; e) Lader BA, Chesebro B, Richman DD (1990)
Antimicrob Agents Chemother 34: 436; f) Parker WB, Cheng YC (1994) J NIH Res 6: 57
[4] a)Beach JW, Jeong LS, AlvesAJ, PohlD, Chang CN, DoongSL, SchinaziRF, Cheng YC, ChuCK
(1992) J Org Chem 57: 2217; b) Condreay LD, Jansen RW, Powdrill TF, Johnson LC, Selleseth
DW, Paff MT, Daluge SM, Painter GR, Furman PA, Ellis MN, Averett DR (1994) Antimicrob
Agents Chemother 38: 616; c) Doong SL, Tsai CH, Shinazi RF, Liotta DC, Cheng YC (1991) Proc
Natl Acad Sci USA 88: 8495; d) Furman PA, Davis M, Liotta DC, Paff M, Frick LW, Nelson DJ,
Dornsife RE, Warster JA, Wilson LJ, Fyfe JA, Tuttle JV, Miller WH, Condreay L, Averett DR,
Schinazi RF, Painter GR (1992) Antimicrob Agents Chemother 36: 2686; e) Gosselin G, Schinazi
RF, SommadossiJP, MatheC, BergogneMC, AubertinAM, KirnA, ImbachJL(1994)Antimicrob
Agents Chemother 38: 1292; f) Kim HO, Shanmuganathan K, Alves AJ, Jeong LS, Beach JW,
Schinazi RF, Chang CN, Cheng YC, Chu CK (1992) Tetrahedron Lett 33: 6899; g) Lin TS, Luo
MZ, Pai SB, Dutschuman GE, Cheng YC (1994) J Med Chem 37: 798
[5] a) Ma TW, Pai SB, Zhu YL, Lin JS, Shanmuganathan K, Du J, Wang CG, Kim H, Newton MG,
Cheng YC, Chu CK (1996) J Med Chem 39: 2835; b) Ma TW, Lin JS, Newton MG, Cheng YC,
Chu CK (1997) J Med Chem 40: 2750
[6] a) Chu CK, Beach JW, Jeong LS, Choi BG, Comer FL, Alves AJ, Schinazi RF (1991) J Org Chem
56: 6503; b) Jeong LS, Alves AJ, Carrigan SW, Kim HO, Beach JW, Chu CK (1992) Tetrahedron
Lett 33: 595; c) Coates JAV, Cammack NS, Jenkinson HJ, Mutton IM, Pearson BA, Storer R,
Cameron JM, Penn CR (1992) Antimicrob Agents Chemother 36: 202; d) Norbeck DW, Spanton
S, Broder S, Mitsuya H (1989) Tetrahedron Lett 30: 6263; Chu CK, Ahn SK, Kim HO, Beach JW,
Alves AJ, Jeong LS, Islam Q, Van Roey P, Schinazi RF (1991) Tetrahedron Lett 32: 3791; e) Kim
HO, Ahn SK, Alves AJ, Beach JW, Jeong LS, Choi BG, Van Roey P, Schinazi RF (1992) J Med
Chem 35: 1987; f) Grove KL, Guo X, Liu SH, Gao Z, Chu CK, Cheng YC (1995) Cancer Res 55:
3008
[7] a) Abdel-Bary HM, Abdel-Rahman AAH, Pedersen EB, Nielsen C (1995) Monatsh Chem 126:
811; b) Zeid IF, Abdel-Rahman AAH (1999) J Chem Res (S): 192; (M): 977; c) Abdel-Rahman
AAH, Abdel-Aal MT (2001) J Chem Res (S): 262; (M): 730
[8] a) El Ashry ESH, Abdel-Rahman AAH, Rashed N, Rasheed HA (1999) Arch Pharm 332: 327;
b) El Ashry ESH, Abdel-Rahman AAH, Rashed N, Rasheed HA (1999) Pharmazie 54: 893
[9] Hilbert GE, Jansen EF (1935) J Am Chem Soc 57: 552
€
[10] Vorbruggen H, Krolikiewicz K, Bennua B (1981) Chem Ber 114: 1234
[11] Niedballa U, Vorbr€uggen H (1974) J Org Chem 39: 3654
[12] a) Mungall WS, Greene GL, Heavner GA, Letsinger RL (1975) J Org Chem 40: 1659;
b) Staudinger H, Hauser E (1921) Helv Chim Acta 4: 887
[13] Korba BE, Gerin JL (1992) Antiviral Res 19: 55
[14] Fouad T, Nielsen C, Brunn L, Pedersen EB (1998) Sc J Az Med Fac (GIRLS) 19: 1173
¨
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¨
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