Synthesis and anti-hepatitis B activity of new substituted uracil and thiouracil glycosides

Article abstract of DOI:10.1007/s12272-010-0601-y

A number of N- and S-substituted uracil and thiouracil glycosides were synthesized by coupling reaction of 5,6-dibenzyle pyrimidine derivatives with the corresponding acetobromosugar. The synthesized compounds were tested for their antiviral activity agai

Full text of DOI:10.1007/s12272-010-0601-y

Arch Pharm Res Vol 33, No 6, 797-805, 2010
DOI 10.1007/s12272-010-0601-y
Synthesis and Anti-Hepatitis B Activity of New Substituted Uracil and
Thiouracil Glycosides
Mohammed T. Abdel-Aal
Chemistry Department, Faculty of Science, Menoufia University, Shebin El-Kom Egypt
(Received September 3, 2009/Revised February 3, 2010/Accepted February 21, 2010)
A number of
N- and S-substituted uracil and thiouracil glycosides were synthesized by cou-
pling reaction of 5,6-dibenzyle pyrimidine derivatives with the corresponding acetobromo-
sugar. The synthesized compounds were tested for their antiviral activity against hepatitis
B virus. Plaque reduction infectivity assay was used to determine virus count reduction as
a result of treatment with tested compounds which showed moderate to high antiviral activ-
ities.
Key words: Pyrimidines, Glycosides, Thioglycosides, Antiviral activity
al., 1995; Nugent et al., 1998) due to their incorpora-
tion into polynucleic acids (Holland et al., 1958) and
INTRODUCTION
Pyrimidines have been recognized as important therefore act as potential inhibitors of protein and
heterocyclic compounds due to their diverse biological polynucleic acid syntheses (Abbot et al., 1978). On the
activities such as Tie-2 kinase inhibitors (Matloobi other hand, nucleoside analogs are structurally,
and Kappe, 2007), HIV-1 inhibitor (Gadhachanda et metabolically, and pharmacodynamically related agents
al., 2007), anti-malarial (Agarwal et al., 2005), secre- that have diverse biological actions including modifi-
tive adenosine A1 receptor antagonist (Chang et al., cations of both the glycon and aglycon parts, have
2004), antibacterial (Hegab et al., 2007), anticancer stimulated extensive research as biological inhibitors
(Capdeville et al., 2002), analgesic (Zaki et al., 2006), (El Ashry et al
cardiovascular (Atwal, 1998), anti-allergic (Ozeki et compounds substituted with benzyl moieties have
al
, 1989) activities. The thio analogues of pyrimidine been reported to exhibit high in vitro antiviral activity
bases, including 2-thiouracil, are minor components of against both DNA and RNA viruses (Diana et al.,
-RNA. Their -, - or S,N-disubstituted analogs have 1977, 1978; Kelley et al., 1990; Malik et al., 2006; Kim
., 2000a, 2000b, 2007a, 2007b). Many
.
t
S N
shown therapeutic effects including antiviral (Larson et al., 2008). The presence of hydrophobic residues
et al., 1983; Holly 1987; Rashad et al., 2008) and lining the compound binding pocket of enzymes has
antitumor (Chu et al
al., 1986) activities. Furthermore, the glycosylthio allyl/benzyl/alkyl groups at suitable sites of nucleo-
heterocycles (Brajeswar and Walter, 1984; Kuhn et al., bases which could facilitate the or -CH interac-
1990; Blanc-Muesser et al , 1992; Mereyala and tions with the amino acid residues of the enzyme
Gurijala, 1993; El Ashry et al., 2005, 2006) are com- (Nisho et al., 1988; Smerdon et al., 1994; Ding et al.,
ponents of -RNA. Their -, - or S,N-disubstituted 1995; Spence et al., 1995). The presence of these
., 1984; Lin and Liu, 1985; Park et necessitated the development of new derivatives with
π-π
π
.
t
S
N
analogs have shown therapeutic properties, especially moieties generally enhances the potency profile of lead
antiviral, anti-thyroid and antitumor activities drug molecules (Pontikis et al., 1997; El-Brollosy et
(Niedzwicki et al., 1982; Hawthorne, 1993; Tanaka et al., 2002). It was envisaged that the presence of hydro-
phobic moieties at C-5 and C-6 of pyrimidine might
enhance the anti-hepatitis B virus (HBV) activities of
Correspondence to: Mohammed T. Abdel-Aal, Chemistry
the resulting compounds. We have been interested in
Department, Faculty of Science, Menoufia University, Shebin
the attachment of carbohydrate moieties to heterocycles
and in the synthesis of nucleoside analogues in order
El-Kom Egypt
E-mail: mohammedabdelaal65@gmail.com
797

798
M. T. Abdel-Aal
to find new biologically active leads (Abdel Aal et al
.,
3310 (NH), 1675 (C=O). 1H NMR (DMSO-d6, 300
2003, 2006, 2008). Owing to the above facts, the aim of MHz):
δ 4.46 (s, 2H, CH2), 4.78 (s, 2H, CH2), 7.11 (m,
the present work is the synthesis and evaluation of 3H, Ar-H), 7.31 (m, 3H, Ar-H), 7.52 (m, 2H, Ar-H),
the anti-HBV activity of new dibenzyl substituted 7.70 (m, 2H, Ar-H), 11.56 (s, 1H, NH), 12.89 (s, 1H,
uracil and thiouracil
thioglycoside analogous.
N δ 69.33, 72.54 (2CH2),
-glycosides as well as their NH). 13C NMR (DMSO-d6):
107.78-150.82 (12Ar-C), 52.10 (C-6), 152.90 (C-5), 164.14
(C-4), 172.98 (C=S). Anal. calcd. for C18H16N2OS
[308.40]: C, 70.10; H, 5.23; N, 9.08. Found: C, 69.89;
H, 5.18; N, 8.82.
MATERIALS AND METHODS
Melting points were determined using a Büchi ap-
paratus. IR spectra (KBr) were recorded with a Bruker-
Vector22 instrument (Bruker, Bremen). 1H NMR
spectra were recorded with a Varian Gemini spectro-
5,6-Dibenzyl-1-(
pyrimidine-2,4(1
General procedure. To a solution of the pyrimidine
O
H
-acetyl-
,3H)-dione (4a-c)
β-D-glycopyranosyl)
meter at 300 MHz and 200 MHz with TMS as internal derivative 2a (1 mmol) and triethylamine (0.2 mL) in
standard. Chemical shifts were reported in scale DMF (10 ml) was added a solution of the correspond-
(ppm) relative to TMS as a standard and the coupling ing -acetyl- -glycopyranosyl bromide (3a-c) (10
constants ( values) are given in Hz. The progress of mmol). The reaction mixture was stirred at room
δ
O
α-D
J
the reactions was monitored by TLC using aluminum temperature until reaction was judged complete by
silica gel plates 60 F245. EImass spectra were recorded TLC using chloroform/methanol 99.5:0.5. The solvent
with a HP D5988 A 1000 MHz instrument (Hewlett, was evaporated under reduced pressure at 40oC and
Packard). Antiviral activity against HBV was tested at the residue was washed with distilled water, dried,
the Liver Institute, Menoufia University, Shebien El- and crystallized from ethanol to afford 4a-c in 72-77%
Kom Egypt. Compounds 3a-c were synthesized from yields.
the corresponding sugars according to a reported
procedure (Scheurer and Smith, 1954).
5,6-Dibenzyl-1-(2,3,4,6-tetra-
pyranosyl)- pyrimidine-2,4(1
White solid (0.48 g, 77%), m.p. 150-151oC; IR (KBr)
1740 (C=O), 1675 (C=O). 1H NMR (DMSO-d6, 300 MHz):
1.88, 1.91, 1.97, 2.06 (4s, 12H, 4 CH3), 4.05 (m, 1H,
= 11.4 Hz, = 3.5 Hz, 1H, H-6'), 4.16
= 11.4 Hz, = 2.8 Hz, 1H, H-6''), 4.58 (s, 2H,
) (1.48g, 5 mmol) and the CH2), 4.85 (s, 2H, CH2), 4.96 (m, 1H, H-4'), 5.22 (m,
reaction mixture was heated under reflux for 10-12 h 1H, H-3'), 5.31 (t, = 9.6 Hz, 1H, H-2'), 5.87 (d, J1',2'
O
H
-acetyl-
,3H)-dione (4a)
β-D-galacto-
ν
Synthesis of 5,6-dibenzylpyrimidine derivatives
(2a,b)
General procedure. To a mixture of urea or thiourea
δ
(5 mmol), calcium hydroxide (0.37 g, 5 mmol) and H-5'), 4.16 (dd,
absolute ethanol (25 mL), was added ethyl 2-benzyl-3- (dd,
oxo-4-phenylbutanoate (
J
J
J
J
1
J
=
(TLC). The solvent was concentrated under reduced 10.2 Hz, 1H, H-1'), 7.05 (m, 3H, Ar-H), 7.33 (m, 3H,
pressure and the precipitated solid was filtered off Ar-H), 7.51 (m, 2H, Ar-H), 7.76 (m, 2H, Ar-H), 12.15
and crystallized from ethanol to afford 2a,b in 79-81% (s, 1H, NH). 13C NMR (DMSO-d6):
δ 19.21, 19.76,
yields.
20.08, 20.28 (4CH3), 62.89 (C-6'), 64.24 (C-4'), 68.69
(C-3'), 71.22 (C-2'), 71.87 (C-5'), 73.15, 74.87 (2CH2),
92.33 (C-1'), 107.82-150.79 (12Ar-C), 152.43 (C-6),
153.25 (C-5), 159.15 (C-2), 164.43 (C-4), 168.05, 168.87,
5,6-Dibenzylpyrimidine-2,4(1H,3H)-dione (2a)
White solid (1.15 g, 79%), m.p. 150-151oC; IR (KBr)
ν
3315 (NH), 1672 (C=O). 1H NMR (DMSO-d6, 300 MHz): 169.88, 170.12 (4 C=O). Anal. calcd. for C32H34N2O11
δ
4.44 (s, 2H, CH2), 4.75 (s, 2H, CH2), 7.02 (m, 3H, Ar- [622.62]: C, 61.37; H, 5.50; N, 4.50. Found: C, 61.12;
H), 7.27 (m, 3H, Ar-H), 7.48 (m, 2H, Ar-H), 7.70 (m, H, 5.38; N, 4.37.
2H, Ar-H), 11.55 (s, 1H, NH), 12.14 (s, 1H, NH). 13C
NMR (DMSO-d6):
(12Ar-C), 152.12 (C-6), 152.92 (C-5), 160.05 (C-2), anosyl)pyramid-ine-2,4(1
164.24 (C-4). Anal. calcd. for C18H16N2O2 [292.33]: C, White solid (0.46 g, 75%), m.p. 147-148oC; IR (KBr)
73.95; H, 5.52; N, 9.58. Found: C, 73.77; H, 5.42; N, 1738 (C=O), 1677 (C=O). 1H NMR (DMSO-d6, 300 MHz):
δ
69.15, 72.21 (2CH2), 107.23-150.15 5,6-Dibenzyl-1-(2,3,4,6-tetra-
O
-acetyl-
β-D-glucopyr-
H,3
H)-dione (4b)
ν
9.71.
δ
1.89, 1.92, 1.97, 2.07 (4s, 12H, 4 CH3), 4.02 (m, 1H,
H-5'), 4.15 (dd, = 11.2 Hz, = 3.5 Hz, 1H, H-6'), 4.17
)- (dd, = 11.2 Hz, = 2.8 Hz, 1H, H-6''), 4.57 (s, 2H,
CH2), 4.84 (s, 2H, CH2), 4.97 (m, 1H, H-4'), 5.24 (m,
1H, H-3'), 5.30 (t, = 9.6 Hz, 1H, H-2'), 5.88 (d, J1',2'
J
J
5,6-Dibenzyl-2-thioxo-2,3-dihydropyrimidin-4(1
H
J
J
one (2b)
White solid (1.25 g, 81%), m.p. 150-151oC; IR (KBr)
ν
J
=

Anti-Hepatitis B Activity of Pyrimidine Glycosides
799
10.4 Hz, 1H, H-1'), 7.11 (m, 3H, Ar-H), 7.38 (m, 3H, 159.30 (C-2), 164.83 (C-4). Anal. calcd. for C24H26N2O7
Ar-H), 7.53 (m, 2H, Ar-H), 7.78 (m, 2H, Ar-H), 12.18 [454.47]: C, 63.43; H, 5.77; N, 6.16. Found: C, 63.29;
(s, 1H, NH). 13C NMR (DMSO-d6):
20.09, 20.32 (4CH3), 62.88 (C-6'), 64.24 (C-4'), 68.69
δ 19.24, 19.76, H, 5.68; N, 6.02.
(C-3'), 71.24 (C-2'), 71.88 (C-5'), 73.21, 74.90 (2CH2), 5,6-Dibenzyl-1-(β-D-glucopyranosyl)]pyrimidine-
92.35 (C-1'), 107.84-150.82 (12Ar-C), 152.52 (C-6), 153.29 2,4(1
(C-5), 159.12 (C-2), 164.42 (C-4), 168.18, 168.88, 169.88, White solid (0.39 g, 87%), m.p. 190-191oC; IR (KBr)
170.18 (4 C=O). Anal. calcd. for C32H34N2O11 [622.62]: 3422-3405 (OH), 1670 (C=O). 1H NMR (DMSO-d6, 300
C, 61.37; H, 5.50; N, 4.50. Found: C, 61.14; H, 5.41; N, MHz): 4.05 (m, 2H, H-6',6''), 4.11-4.18 (m, 2H, H-
4',5'), 4.25 (m, 1H, H-3'), 4.59 (s, 2H, CH2), 4.87 (s, 2H,
CH2), 4.90 (t, = 9.8 Hz, 1H, H-2'), 4.96 (m, 1H, OH),
-xylopyranosyl) 5.12 (d, = 6.2 Hz, 1H OH), 5.17 (m, 1H, OH), 5.22 (t,
= 5.8 Hz, 1H, OH), 5.85 (d, J1',2' = 9.8 Hz, 1H, H-1'),
7.14 (m, 3H, Ar-H), 7.36 (m, 3H, Ar-H), 7.50 (m, 2H,
1742 (C=O), 1676 (C=O). 1H NMR (DMSO-d6, 300 MHz): Ar-H), 7.77 (m, 2H, Ar-H), 12.52 (s, 1H, NH). 13C NMR
1.89, 1.95, 2.04 (4s, 9H, 3 CH3), 4.10 (dd, = 10.8 (DMSO-d6): 66.15 (C-6'), 68.32 (C-4'), 70.56 (C-3'),
Hz, = 3.4 Hz, 1H, H-5'), 4.17 (m, 1H, H-5''), 4.55 (s, 71.89 (C-2'), 72.45 (C-5'), 74.28, 75.14 (2CH2), 92.98
2H, CH2), 4.81 (s, 2H, CH2), 4.97 (m, 2H, H-3',4'), 5.32 (C-1'), 108.60-150.80 (12Ar-C), 152.20 (C-6), 152.95 (C-
(dd, = 9.4 Hz, = 9.8 Hz, 1H, H-2'), 5.88 (d, J1',2' 5), 159.32 (C-2), 164.82 (C-4). Anal. calcd. for
H,3H)-dione (5b)
ν
δ
4.36.
J
5,6-Dibenzyl-1-(2,3,4-tri-
O
-acetyl-
)-dione (4c)
White solid (0.39 g, 72%), m.p. 152-153oC; IR (KBr)
β
-
D
J
pyrimidine-2,4(1H,3H
J
ν
δ
J
δ
J
J
J
=
9.8 Hz, 1H, H-1'), 7.10 (m, 3H, Ar-H), 7.32 (m, 3H, Ar- C24H26N2O7 [454.47]: C, 63.43; H, 5.77; N, 6.16. Found:
H), 7.53 (m, 2H, Ar-H), 7.77 (m, 2H, Ar-H), 12.10 (s, C, 63.30; H, 5.69; N, 6.05.
1H, NH). 13C NMR (DMSO-d6):
δ
19.24, 19.89, 20.32
(4CH3), 62.88 (C-5'), 64.25 (C-4'), 68.67 (C-3'), 71.22 (C- 5,6-Dibenzyl-1-(
2'), 73.20, 74.87 (2CH2), 92.42 (C-1'), 107.80-150.82 (1 ,3 )-dione (5c)
(12Ar-C), 152.51 (C-6), 153.28 (C-5), 159.20 (C-2), White solid (0.35 g, 84%), m.p. 190-191oC; IR (KBr)
164.28 (C-4), 168.86, 169.82, 170.18 (3 C=O). Anal. 3415-3405 (OH), 1670 (C=O). 1H NMR (DMSO-d6, 300
calcd. for C92H30N2O9 [550.20]: C, 63.27; H, 5.49; N, MHz): 4.05 (m, 2H, H-5',5''), 4.14 (m, 1H, H-4'), 4.22
5.09. Found: C, 63.14; H, 5.41; N, 5.89. (m, 1H, H-3'), 4.57 (s, 2H, CH2), 4.87 (s, 2H, CH2), 4.88
(t, = 9.8 Hz, 1H, H-2'), 4.94 (m, 1H, OH), 5.18 (m,
1H, OH), 5.21 (t, = 5.8 Hz, 1H, OH), 5.87 (d, J1',2'
9.8 Hz, 1H, H-1'), 7.14 (m, 3H, Ar-H), 7.35 (m, 3H, Ar-
General Procedure. Dry gaseous ammonia was passed H), 7.52 (m, 2H, Ar-H), 7.78 (m, 2H, Ar-H), 12.18 (s,
β-D-xylopyranosyl)pyrimidine-2,4
H
H
ν
δ
J
J
=
5,6-Dibenzyl-1-(
β-D-glycopyranosyl)pyrimidine-
2,4(1 ,3 )-dione (5a-c)
H
H
through a solution of a protected glycosides 4a-c (1 1H, NH). 13C NMR (DMSO-d6):
δ 66.14 (C-5'), 68.34
mmol) in dry methanol (20 mL) at 0oC for 0.5 h, and (C-4'), 70.58 (C-3'), 71.89 (C-2'), 74.30, 75.15 (2CH2),
then the mixture was stirred at 0oC for about 5 h. The 92.98 (C-1'), 108.60-150.80 (12Ar-C), 152.20 (C-6), 152.95
solvent was evaporated under reduced pressure at (C-5), 159.42 (C-2), 164.89 (C-4). Anal. calcd. for
40oC to give a solid residue, which was crystallized C23H24N2O6 [424.45]: C, 65.08; H, 5.70; N, 6.60. Found:
from ethanol.
C, 64.91; H, 5.59; N, 6.48.
5,6-Dibenzyl-1-(β-D-galactopyranosyl)pyrimidine-
5,6-Dibenzyl-2-[(
thio]-2,3dihydro-pyrimidin-4(1
General procedure To a solution of the pyrimidine
3412-3398 (OH), 1665 (C=O). 1H NMR (DMSO-d6, 300 thione 2b (1 mmol) in aqueous potassium hydroxide
MHz): 4.03 (m, 2H, H-6',6''), 4.12-4.18 (m, 2H, H-4',5'), [0.056 g, 1 mmol in distilled water (16 mL)] was added
4.24 (m, 1H, H-3'), 4.57 (s, 2H, CH2), 4.86 (s, 2H, CH2), a solution of the corresponding -acetyl- -glycopyr-
4.91 (t, = 9.8 Hz, 1H, H-2'), 4.98 (m, 1H, OH), 5.11 anosyl bromide (3a-c) (1 mmol) in acetone (30 mL).
(d, = 6.2 Hz, 1H, OH), 5.18 (m, 1H, OH), 5.22 (t, The reaction mixture was stirred at room temperature
O
-acetyl-
β
-
D
-glycopyranosyl)
2,4(1
H,3H)-dione (5a)
H)-one (6a-c)
White solid (0.38 g, 85%), m.p. 190-191oC; IR (KBr)
ν
.
δ
O
α-D
J
J
J =
5.8 Hz, 1H, OH), 5.85 (d, J1',2' = 9.8 Hz, 1H, H-1'), 7.10 until reaction was judged complete by TLC using chlo-
(m, 3H, Ar-H), 7.35 (m, 3H, Ar-H), 7.52 (m, 2H, Ar-H), roform/methanol 99.5:0.5. The solvent was evaporated
7.77 (m, 2H, Ar-H), 12.34 (s, 1H, NH). 13C NMR (DMSO- under reduced pressure at 40oC and the residue was
d6):
δ 64.63 (C-6'), 67.24 (C-4'), 70.14 (C-3'), 71.21 (C- washed with distilled water to remove potassium bro-
2'), 71.89 (C-5'), 74.25, 75.10 (2CH2), 92.97 (C-1'), mide formed. The product was dried, and crystallized
108.62-150.80 (12Ar-C), 152.18 (C-6), 152.92 (C-5), from ethanol.

800
M. T. Abdel-Aal
5,6-Dibenzyl-2-[(2,3,4,6-tetra-
pyranosyl)thio]-2,3-dihydropyrimidin-4(1
O
-acetyl-
β
-
D
-galacto-
C), 152.42 (C-6), 152.98 (C-5), 155.57 (C-2), 164.24 (C-
4), 169.65, 170.32, 171.50 (3C=O). Anal. Calcd. For
C27H28N2O7S [566.62]: C, 61.47; H, 5.34; N, 4.94.
Found: C, 61.32; H, 5.25; N, 4.79.
H)-one
(6a)
Yellow solid (0.52 g, 82%), m.p. 144-145oC; IR (KBr)
ν
1745 (C=O), 1678 (C=O); 1H NMR (CDCl3):
2.11, 2.14 (4s, 12H, 4 CH3), 4.03 (m, 1H, H-5'), 4.14 (dd,
δ 1.94, 2.07,
5,6-Dibenzyl-2-[(
hydropyrimidin-4(1
General Procedure. Dry gaseous ammonia was passed
= 9.6 Hz, through a solution of a protected glycosides 6a-c (1
J
= 9.6 Hz, 1H, H-2'), 5.70 mmol) in dry methanol (20 mL) at 0oC for 0.5 h, and
β
-
D
-glycopyranosyl)thio]-2,3-di-
J
J
= 11.2 Hz,
= 2.8 Hz, 1H, H-6'), 4.60 (s, 2H, CH2), 4.78 (s, 2H,
= 9.3 Hz, 1H, H-4'), 5.25 (dd,
= 9.3 Hz, 1H, H-3'), 5.37 (t,
J = 3.2 Hz, 1H, H-6'), 4.17 (dd, J = 11.2 Hz,
H)-one (7a-c)
CH2), 4.95 (t,
J
J
J
(d, 1H, J1',2' = 10.2 Hz, H-1'), 7.12 (m, 3H, Ar-H), 7.33 then the mixture was stirred at 0oC for about 4-5
(m, 3H, Ar-H), 7.56 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), hours (TLC). The solvent was evaporated under
12.05 (s, 1H, NH); 13C NMR (CDCl3): 19.30, 19.55, reduced pressure at 40oC to give a solid residue, which
20.19, 20.23, (4CH3), 62.72 (C-6'), 64.25 (C-4'), 68.76 (C- was crystallized from ethanol.
3'), 70.36, 71.18 (2CH2), 71.26 (C-2'), 71.94 (C-5'), 86.15
(C-1'), 108.89-151.12 (12Ar-C), 152.14 (C-6), 152.96 (C- 5,6-Dibenzyl-2-[(
5), 164.18 (C-4), 169.64, 170.30, 171.27, 171.49 (4C=O). hydropyrimidin-4(1
Anal. Calcd. For C32H34N2O10S [638.68]: C, 60.18; H, Yellow solid (0.43 g, 89%), m.p. 153-154oC; IR (KBr)
5.37; N, 4.39. Found: C, 60.02; H, 5.29; N, 4.27. 4.14
3462-3438 (OH), 1662 (C=O); 1H NMR (CDCl3):
β-D
-galactopyranosyl)thio]-2,3-di-
H)-one (7a)
ν
δ
(m, 2H, H-6',6''), 4.05 (m, 1H, H-5'), 4.64 (s, 2H, CH2),
4.82 (s, 2H, CH2), 4.88 (m, 2H, H-4', H-3'), 4.97 (t, 1H,
5,6-Dibenzyl-2-[(2,3,4,6-tetra-
anosyl)thio]-2,3-dihydropyrimidin-4(1H)-one (6b)
Yellow solid (0.53 g, 84%), m.p. 147-148oC; IR (KBr)
1742 (C=O), 1674 (C=O); 1H NMR (CDCl3):
1.92, 1.97, J1',2' = 9.8 Hz, 1H, H-1'), 7.14 (m, 3H, Ar-H), 7.35 (m,
2.04, 2.12 (4s, 12H, 4 CH3), 4.05 (m, 1H, H-5'), 4.16 3H, Ar-H), 7.56 (m, 2H, Ar-H), 7.81 (m, 2H, Ar-H),
(dd, = 11.2 Hz, = 3.2 Hz, 1H, H-6'), 4.18 (dd,
12.11 (s, 1H, NH); 13C NMR (CDCl3): 64.72 (C-6'),
11.2 Hz, = 2.8 Hz, 1H, H-6'), 4.57 (s, 2H, CH2), 4.78 64.25 (C-4'), 68.76 (C-3'), 70.39, 71.21 (2CH2), 71.26
(s, 2H, CH2), 4.98 (t, 1H, = 9.3 Hz, H-4'), 5.19 (dd, (C-2'), 71.94 (C-5'), 88.14 (C-1'), 109.12-151.82 (12Ar-
= 9.6 Hz, = 9.3 Hz, 1H, H-3'), 5.30 (t, = 9.6 Hz, 1H, C), 152.16 (C-6), 153.11 (C-5), 164.12 (C-4). Anal.
O-acetyl-β-D-glucopyr-
J
= 9.6 Hz, H-2'), 5.04 (m, 1H, OH), 5.14 (d,
J = 6.8 Hz,
ν
1H, OH), 5.18 (m, 1H, OH), 5.21 (m, 1H, OH), 5.70 (d,
δ
J
J
J =
J
J
J
J
J
H-2'), 5.71 (d, J1',2' = 10.2 Hz, 1H, H-1'), 7.12 (m, 3H, Calcd. For C24H26N2O6S [470.54]: C, 61.26; H, 5.57; N,
Ar-H), 7.34 (m, 3H, Ar-H), 7.55 (m, 2H, Ar-H), 7.78 5.95. Found: C, 61.14; H, 5.49; N, 5.81.
(m, 2H, Ar-H), 12.14 (s, 1H, NH); 13C NMR (CDCl3):
19.32, 19.55, 20.19, 20.24, (4CH3), 62.72 (C-6'), 64.26 5,6-Dibenzyl-2-[(
(C-4'), 68.76 (C-3'), 70.38, 71.25 (2CH2), 71.21 (C-2'), pyrimidin-4(1
71.90 (C-5'), 86.69 (C-1'), 108.89-151.14 (12Ar-C), 152.14 Yellow solid (0.42 g, 90%), m.p. 152-153oC; IR (KBr)
(C-6), 152.95 (C-5), 164.20 (C-4), 169.64, 170.32, 171.27, 3458-3436 (OH), 1662 (C=O); 1H NMR (CDCl3):
4.14
171.48 (4C=O). Anal. Calcd. For C32H34N2O10S [638.68]: (m, 2H, H-6',6''), 4.05 (m, 1H, H-5'), 4.64 (s, 2H, CH2),
C, 60.18; H, 5.37; N, 4.39. Found: C, 60.05; H, 5.30; N, 4.82 (s, 2H, CH2), 4.87 (m, 2H, H-4', H-3'), 4.98 (t,
4.31. 9.6 Hz, 1H, H-2'), 5.05 (m, 1H, OH), 5.15 (d, = 6.8
Hz, 1H, OH), 5.18 (m, 1H, OH), 5.22 (m, 1H, OH), 5.72
-xylopyranosyl) (d, J1',2' = 10.2 Hz, 1H, H-1'), 7.14 (m, 3H, Ar-H), 7.38
β
-glucopyranosyl)thio]-2,3-dihydro-
)-one (7b)
H
ν
δ
J
=
J
5,6-Dibenzyl-2-[(2,3,4-tri-O-acetyl-β-D
thio]-2,3-dihydropyrimidin-4(1H)-one (6c)
(m, 3H, Ar-H), 7.55 (m, 2H, Ar-H), 7.88 (m, 2H, Ar-H),
Yellow solid (0.45 g, 80%), m.p. 153-154oC; IR (KBr)
ν
12.14 (s, 1H, NH); 13C NMR (CDCl3): 65.32 (C-6'),
1
1740 (C=O), 1672 (C=O); H NMR (CDCl3):
δ 1.95, 66.25 (C-4'), 68.72 (C-3'), 70.42, 71.23 (2CH2), 71.27
2.08, 2.12 (3s, 9H, 3 CH3), 4.07 (m, 1H, H-4'), 4.15 (dd, (C-2'), 71.95 (C-5'), 88.62 (C-1'), 109.14-151.84 (12Ar-
= 11.4 Hz, = 2.8 Hz, 1H, H-5'), 4.18 (m, 1H, H-5''), C), 152.16 (C-6), 153.14 (C-5), 163.84 (C-4). Anal.
4.60 (s, 2H, CH2), 4.79 (s, 2H, CH2), 4.96 (t, = 9.3 Hz, Calcd. For C24H26N2O6S [470.54]: C, 61.26; H, 5.57; N,
1H, H-3'), 5.36 (t, = 9.3 Hz, 1H, H-2'), 5.72 (d, J1',2' 5.95. Found: C, 61.16; H, 5.40; N, 5.71.
9.8 Hz, 1H, H-1'), 7.11 (m, 3H, Ar-H), 7.35 (m, 3H, Ar-
H), 7.57 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), 12.14 (s, 5,6-Dibenzyl-2-[(
1H, NH); 13C NMR (CDCl3): 19.30, 19.55, 20.19 (3CH3), pyrimidin-4(1
)-one (7c)
62.72 (C-5'), 64.25 (C-4'), 68.76 (C-3'), 70.34, 71.22 Yellow solid (0.37 g, 85%), m.p. 155-156oC; IR (KBr)
(2CH2), 71.27 (C-2'), 87.68 (C-1'), 108.80-151.18 (12Ar- 3460-3441 (OH), 1667 (C=O); 1H NMR (CDCl3):
4.15
J
J
J
J
=
β-xylopyranosyl)thio]-2,3-dihydro-
H
ν
δ

Anti-Hepatitis B Activity of Pyrimidine Glycosides
801
(m, 2H, H-5',5''), 4.60 (s, 2H, CH2), 4.80 (s, 2H, CH2), DNA using 1
µ
mol/L of each of the following primers:
4.84 (m, 2H, H-4', H-3'), 4.90 (t,
5.02 (m, 1H, OH), 5.14 (d, = 6.8 Hz, 1H, OH), 5.20 and HCID-2 primer (5'-TTGGGGGAGGAGATTAGGTT-
(m, 1H, OH), 5.72 (d, J1',2' = 9.8 Hz, 1H, H-1'), 7.16 (m, 3'), in a reaction mixture containing 14 L extracted
3H, Ar-H), 7.34 (m, 3H, Ar-H), 7.53 (m, 2H, Ar-H), supernatant, 4 mmol/L MgCl2,10 mol/L DIG-11-dUTP
7.80 (m, 2H, Ar-H), 12.14 (s, 1H, NH); 13C NMR (CDCl3): (Roche), 190
mol/L dTTP, 200 mol/1 dATP, dGTP,
62.72 (C-5'), 64.25 (C-4'), 68.76 (C-3'), 70.59, 71.43 dCTP (Roche) 1.5 U Taq polymerase (Roche), in a total
J = 9.6 Hz, 1H, H-2'), HCID-1 primer (5'-GGAAAGAAGTCAGAAGGCA-3')
J
µ
µ
µ
µ
(2CH2), 71.26 (C-2'), 88.14 (C-1'), 109.16-151.79 (12Ar- volume 50 µL. PCR reaction conditions were: 32 cycles
C), 152.27 (C-6), 153.14 (C-5), 164.11 (C-4). Anal. of 10 min at 94oC, 30 s at 58oC and 30 s at 72oC with
Calcd. For C23H24N2O5S [440.51]: C, 62.71; H, 5.49; N, a 3 s increment for each cycle in a Perkin Elmer 480
6.36. Found: C, 62.54; H, 5.32; N, 6.24.
thermal cycler (Perkin Elmer). The PCR product was
detected by DIG-ELISA assay (Roche). The optical
density (OD) from DNA of the test compound was
Antiviral activity
The synthesized compounds were tested for their compared to that of the blank culture.
antiviral activity against hepatitis B virus (HBV)
using the HepG2.2.2.15-cell line (Sells et al., 1987), a
human hepatoplastoma cell line producing HBV viral
particles (Korba and Gerin, 1992).
Cytotoxicity assay
3-(3,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Sigma) is a colorless substrate that is
The cell line was maintained in RPMI-1640 (Glutamax) transferred to a colored product by any living cell but
(Gibco BRL Life Technologies) culture medium con- not by dead cells or tissue culture medium. The
taining 100 IU/mL nystatin (Gibco BRL Life Technolo- cytotoxic effect of the compounds was accessed by
gies), 380
µg/mL G418 (genetecin) (Gibco BRL Life culturing the Hep G2-2.2.15 cells in the presence of
Technologies) and 10% fetal calf serum (FCS) (Gibco compounds using a MTT-assay (Fouad et al., 1998).
BRL Life Technologies). The transferred HepG2.2.2.15
cells were kept in a tissue culture flask at 37oC and
Calculation of IC50, CC50 and SI values
5% CO2. Subcultures were set up after a week by try-
The 50% inhibitory concentration (IC50) of an anti-
psination [(10% versin/trypsin (Biochrome KG)] and viral drug was determined by plotting the DNA con-
transferred to a 96-well tissue culture plate. Serial tent of the serial dilutions of the tested compound
dilutions of the test compounds were added to the cell versus the concentration of this compound. The 50%
suspension and incubated for 6 d at 37oC and 5% CO2. cytotoxic effect (CC50) was calculated from the average
The antiviral activity and cytotoxic effect of the test viability of the cells in proportion to the concentration
compound was estimated by comparing the DNA con- of the drug. The selective index (SI) could be calcul-
tent in the culture supernatant and the viability of the ated as CC50/IC50.
cells with the test compounds to those of HepG2.2.2.15
cells with no compounds added to their supernatant
(blank cells). The drug lamivudine [(4-amino-1-[(2 ,5 )-
RESULTS AND DISCUSSION
R
S
2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydro-
pyrimi-din-2-one), GlaxoSmithKline] which is a potent
Chemistry
In this investigation, new uracil and thiouracil nu-
selective inhibitor of HBV replication has been used cleobases substituted with two hydrophobic residues
as a standard positive control. Each compound was at positions 5 and 6 in the heterocyclic ring as well as
tested in triplicate.
their
N- and S-glycosides were synthesized. Thus,
reaction of ethyl 2-benzyl-3-oxo-4-phenylbutanoate (
1)
with urea or thiourea in ethanol in the presence of
DNA extraction
DNA extraction was done by incubating 10
µ
L of calcium hydroxides afforded the corresponding 5,6-
diluted supernatant with 10
37oC for 1 h, then carefully adding 9.6
followed by addition of 90
L of Tris-EDTA (TE) buffer 1H NMR spectra and elemental analysis which agreed
µ
L of 0.2 M NaOH at dibenzyl uracil or thiouracil derivatives 2a
, b respec-
µL of 0.2 M HCl tively. The structures of 2a were confirmed by IR,
, b
µ
[(2-amino-2-(hydroxymethyl)-1,3-propanediol-EDTA)
(Gibco BRL Life Technologies)].
with the assigned structures. Thus, the IR spectra of
2a and 2b showed characteristic absorption bands for
the NH groups in addition to absorption bands corres-
1
ponding to the carbonyl groups and the H NMR
PCR-ELISA detection of HBV DNA
The DNA content in the cell culture supernatant spectra agreed with the assigned structures (see ex-
was determined by PCR amplification of the HBV perimental part).

802
M. T. Abdel-Aal
When the 5,6-dibenzyl uracil derivative 2a was 3398
-
3422 cm-1 corresponding to the hydroxyl groups.
1
allowed to react with
O-acetylated-α-D-glycopyranosyl Their H NMR spectra revealed the absence of the
bromides 3a-c in DMF in presence of triethylamin acetyl-methyl signals and instead signals correspon-
(TEA) at room temperature the corresponding -gly- ding to the sugar hydroxyl groups appeared at 4.94
cosides 4a-c were obtained in 72-77% yields (Scheme 5.22 ppm.
1). The IR spectra of 4a-c showed absorption bands at On the other hand, reaction of the 5,6-dibenzyl thiour-
N
δ
-
1738-1742 cm-1 corresponding to acetyl carbonyl groups acil derivative 2b with
O-acetylated-α-D-glycopyranosyl
and absorption bands at 1675-1677 cm-1 for the amide bromide 3a-c in acetone in presence of potassium
carbonyl groups. The respective 1H NMR spectra hydroxide at room temperature afforded the corres-
1
showed the
ppm, the signals of the sugar protons at
ppm and the anomeric proton as a doublet at
5.87 ppm with coupling constant 9.8 10.4 Hz indi- 10.2 Hz indicating the
cating the
O
-acetyl-methyl groups at
δ
1.88
4.02
5.84- doublet at
-
-
2.07 ponding thioglycosides 6a-c in 80-84% yields. The H
δ
5.32 NMR spectra of 6a-c showed the anomeric proton as a
5.70-5.72 ppm with coupling constant 9.8
-configuration of the thiogly-
δ
δ
-
-
β
β
-configuration of the glycosidic bond. The cosidic (El-Sayed et al., 2008, 2009) bond. The attach-
13C NMR spectra showed characteristic signals for the ment of the glycosyl residues to the sulfur rather than
methyl and carbonyl groups. Treatment of 4a-c with to the nitrogen has been supported by the 13C NMR
methanolic ammonia solution at room temperature spectra of 6a-c which revealed the absence of a peak
afforded the deacetylated
N-glycosides 5a-c in 80-82% corresponding to the C=S group indicating that the
yields. The IR spectra of the deprotected glycosides attachment of the sugar has taken place at the sulfur
5a-c showed the characteristic absorption bands at atom. Furthermore, the 13C NMR spectra of 6a-c
Scheme 1. Synthesis of uracil and thiouracil glycosides

Anti-Hepatitis B Activity of Pyrimidine Glycosides
803
showed signal at
the anomeric carbons which also confirmed the
configuration. The thiol-thione tautomerism at
and C=S in thiouracil derivatives is well known to 3436
δ
86.15-87.68 ppm corresponding to ammonia solution at room temperature afforded the
deprotected thioglycoside 7a-c (Scheme 1). Their IR
-1 spectra showed the characteristic absorption bands at
3462 cm-1 corresponding to the hydroxyl groups.
β-
N
-
1
lead to alkylation at the sulfur atom. Furthermore, The H NMR spectra of 7a-c revealed the absence of
the chemical shift of C-4 in the 13C NMR spectra of the acetyl-methyl signals and instead signals corres-
6a-c
(
δ
146.18-164.24 ppm) is relatively close to C-4 in ponding to the sugar hydroxyl groups appeared at
δ
2b
(δ
164.14 ppm) indicating the presence of similar 5.02 5.22 ppm.
-
electronic environments and gives an evidence for
structures 6a-c. Deacetylation of 6a-c by methanolic
Antiviral activity
The synthesized compounds with carrying two hydro-
phobic residues at positions 5 and 6 in the pyrimidine
ring were tested for their antiviral activity against
hepatitis B virus (HBV) using the HepG2.2.2.15-cell
line, a human hepatoplastoma cell line producing
HBV viral particles (Korba and Gerin, 1992). The
results of our preliminary screening indicated that
Table I. Cytotoxic effect (CC50)a, inhibitory concentration
(IC50) and selectivity index (SI) of newly synthesized com-
pounds
HBV DNA
IC50 M]
Selectivity Index
SI
Compound
[
µ
Lamivudine
0.1
0.7
0.6
0.6
0.5
0.4
0.5
0.2
0.4
0.5
0.3
0.3
0.4
0.2
0.2
>1000
>142.8
>200.0
>166.6
>200.0
>250.0
>200.0
>500.0
>333.3
>200.0
>500.0
>333.3
>333.3
>500.0
>500.0
2a
2b
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
compounds 5b
viral activity with an effective concentration of 0.2
and a selectivity index >500.0, followed by compounds
6b and 6c. Compounds 4c 5c and 7a showed moderate
, 7b and 7c showed the highest anti-
µM
,
inhibitory activity against HBV (Tables I and II).
From the structure-activity relationship it is clear
that the attachment thiouracil derivatives attached to
glycopyranosyl sugar moieties through thioglycosidic
linkage showed higher antiviral activities against
HBV than the uracil derivatives with
N-glycosidic
bond. It can also be concluded that the free hydroxyl
glycosides have higher activity that their correspond-
ing acetylated analogous. Furthermore, the activity of
the the synthesized uracil and thiouracil glycosides
free hydroxyl glycopyranosyl moieties was higher than
their corresponding acetylated analogs.
aCC50(
µM) for all tested compounds is >100 µM.
Table II. Inhibition of HBV replication by tested com-
pounds
In conclusion, new
N- and S-substituted pyrimidine
glycoside derivatives were synthesized and some of
them showed moderate to high antiviral activity
against HBV. The free hydroxyl glycoside derivatives
showed higher antiviral activities against HBV than
their corresponding acetylated analogs.
HepG2
viable
cells
Concentration
M)
HBV DNA
In supernatant
Compound
(
µ
Lamivudine
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.25
0.28
0.17
0.17
0.13
0.17
0.13
0.83
0.17
0.13
0.21
0.21
0.17
0.83
0.83
1.00
0.35
0.56
0.56
0.19
0.41
0.19
0.90
0.41
0.19
0.63
0.63
0.41
0.90
0.90
2a
2b
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
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