Inhibition of hepatitis B virus (HBV) replication by pyrimidines bearing an acyclic moiety: Effect on wild-type and mutant HBV

Article abstract of DOI:10.1021/jm058271d

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The main clinical limitation of a current antiviral drug for HBV, lamivudine, is the emergence of drug-resistant viral strains upon prolonged therapy. A group of 5-, 6-, or 5,6-substituted acyclic pyrimidine nucleosides with a 1-[(2-hydroxyethoxy)methyl] moiety were synthesized and evaluated for antiviral activities. The target compounds were prepared by the reaction of silylated uracils possessing a variety of substituents at the C-5 or C-6 positions or both with 1,3-dioxolane in the presence of potassium iodide and chlorotrimethylsilane by a convenient and single-step synthesis. Among the compounds tested, 5-chloro and 5-bromo analogues possessing an acyclic glycosyl moiety were the most effective and selective antiviral agents in the in vitro assays against wild-type duck HBV (EC₅₀ = 0.4-2.2 and 3.7-18.5 μM, respectively) and human HBV-containing 2.2.15 cells (EC₅₀ = 4.5-45.4 and 18.5-37.7 μM, respectively). These compounds were also found to retain sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and double mutations (L180M/M204V). The compounds investigated did not show cytotoxicity to host HepG2 and Vero cells, up to the highest concentration tested. The results presented here confirm and accentuate the potential of acyclic pyrimidine nucleosides as anti-HBV agents and extend our previous observations. We herein report the capability of acyclic pyrimidine nucleosides to inhibit the replication of both wild-type and drug-resistant mutant HBV.

Full text of DOI:10.1021/jm058271d

J. Med. Chem. 2006, 49, 2049-2054
2049
Inhibition of Hepatitis B Virus (HBV) Replication by Pyrimidines Bearing an Acyclic Moiety:
Effect on Wild-Type and Mutant HBV
Wassila Semaine,†,‡ Monika Johar,† D. Lorne J. Tyrrell,§ Rakesh Kumar,*,† and B. Agrawal*,‡
Department of Laboratory Medicine and Pathology, 1-41 Medical Sciences Building, and Department of Surgery and Department of Medical
Microbiology and Immunology, Faculty of Medicine and Dentistry, UniVersity of Alberta, Edmonton, Alberta T6G 2H7, Canada
ReceiVed October 5, 2005
Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The main clinical
limitation of a current antiviral drug for HBV, lamivudine, is the emergence of drug-resistant viral strains
upon prolonged therapy. A group of 5-, 6-, or 5,6-substituted acyclic pyrimidine nucleosides with a 1-[(2-
hydroxyethoxy)methyl] moiety were synthesized and evaluated for antiviral activities. The target compounds
were prepared by the reaction of silylated uracils possessing a variety of substituents at the C-5 or C-6
positions or both with 1,3-dioxolane in the presence of potassium iodide and chlorotrimethylsilane by a
convenient and single-step synthesis. Among the compounds tested, 5-chloro and 5-bromo analogues
possessing an acyclic glycosyl moiety were the most effective and selective antiviral agents in the in vitro
assays against wild-type duck HBV (EC50 ) 0.4-2.2 and 3.7-18.5 µM, respectively) and human HBV-
containing 2.2.15 cells (EC50 ) 4.5-45.4 and 18.5-37.7 µM, respectively). These compounds were also
found to retain sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and double
mutations (L180M/M204V). The compounds investigated did not show cytotoxicity to host HepG2 and
Vero cells, up to the highest concentration tested. The results presented here confirm and accentuate the
potential of acyclic pyrimidine nucleosides as anti-HBV agents and extend our previous observations. We
herein report the capability of acyclic pyrimidine nucleosides to inhibit the replication of both wild-type
and drug-resistant mutant HBV.
Introduction
Hepatitis B virus (HBV) infection is one of the leading causes
of death due to infectious diseases worldwide. There are
approximately 400 million people with chronic HBV infection,
with an annual global death toll of 1.2 million per year.1,2
Chronic HBV infected people are 10 times more numerous than
HIV (human immunodeficiency virus) patients. An HBV
infected mother can pass the infection to her nonvaccinated
infant at the time of birth.3,4 Neonatal exposure to HBV results
in chronic HBV infection in 90% of the cases, whereas adults
Figure 1.
exposed to HBV result in chronic infection in only 10% of the
cases.3 In the chronic carriers, 25-40% of the people develop
demonstrated to be well-tolerated, to reduce serum HBV DNA
liver cirrhosis and hepatocellular carcinoma leading to significant
levels significantly, and to lead to seroconversion in 20-27%
mortality.
of the patients treated for 12 weeks at a daily dose of 30 mg/
Until recently, the major therapeutic option for HBV carriers
day or greater.1,9 However, primary limitations of adefovir
was R-interferon. However, its use is limited because the success
therapy are dose-related side effects such as nephrotoxicity,
lactic acidosis, and severe hepatomegaly with steatosis, and
rate is low and serious side effects are observed.5,6 Lamivudine
[(-)-â-L-2′,3′-dideoxy-3′-thiacytidine, (-)3TC, 1a, Figure 1]
cessation of therapy may result in serious hepatitis.10 In clinical
has been clinically used for the treatment of HBV infection for
trials, after the discontinuation of adefovir, 25% of patients had
a few years. It is orally effective and well tolerated and shows
exacerbations of hepatitis (alanine aminotransferase (ALT)
marked inhibition of HBV DNA levels, loss of HBeAg (hepatitis
elevation of 10-times the upper limit of normal levels or greater)
B e antigen), seroconversion (defined as HBeAg-negative,
antibodies to HBeAg-positive, and HBV-DNA-negative) and
and there were even fatalities.10
Treatment with lamivudine or adefovir in HBV infected
patients leads to quantitative decrease in HBV replication.
However, after cessation of treatment, virus DNA levels rebound
to baseline pretreatment or even higher levels.7,11,12 Therapy with
lamivudine and adefovir inhibits cytoplasmic HBV replication
but has no direct effect on HBV covalently closed circular DNA
(cccDNA).11,12 However, continuous and complete suppression
of HBV DNA replication may deplete cccDNA.13 Short-term
therapy with HBV DNA synthesis inhibitors cannot deplete the
pool of cccDNA, and this could be one reason for rapid rebound
of viral replication after cessation of therapy.14 Continuous long-
clinical improvements in the liver inflammation and histology
in the majority of patients.7,8 Recently, adefovir dipivoxyl
[9-[2-[bis[(pivaloyloxy)methoxy]phosphinyl]methoxy]-ethyl]-
adenine, 1b] has also been approved as another antiviral agent
for the treatment of chronic HBV infection. Adefovir was
* Corresponding author. Phone: (780) 492-7545. Fax: (780) 492-7521.
E-mail: rakesh.kumar@ualberta.ca (R.K.). Phone: (780) 492-0929. Fax:
(780) 492-9828. E-mail: bagrawal@ualberta.ca (B.A.).
† Department of Laboratory Medicine and Pathology.
‡ Department of Surgery.
§ Department of Medical Microbiology and Immunology.
10.1021/jm058271d CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/01/2006

2050 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Semaine et al.
Scheme 1a
a Reagents: (i) Bis(trimethylsilyl)acetamide, dry acetonitrile; (ii) 1,3-dioxolane, potassium iodide, trimethyl chlorosilane, 25 °C; (iii) quenched with
MeOH and neutralized with NaHCO3.
term therapy with an array of potent nontoxic individual anti-
HBV agents or a combination of anti-HBV agents may lead to
continuous inhibition of viral DNA synthesis resulting eventually
in the depletion of the cccDNA pool. Depletion of cccDNA
can be achieved, as has been shown with penciclovir (PCV,
1c) in duck HBV (DHBV)-infected hepatocytes.15
primary duck hepatocytes infected with DHBV in vitro.32-34
Given the novelty of their structures and in effort to further
explore the structure-activity relationships (SARs) in this series
of promising antivirals, we now report synthesis and anti-HBV
activity of various 5-, 6- or 5,6-substituted analogues of 1-[(2-
hydroxyethoxy)methyl]uracils (17-31). In these studies, we note
that 5-halo analogues 17 and 18 in particular display potent and
selective anti-HBV activity against duck HBV, wild-type human
HBV, and lamivudine-resistant HBV in vitro.
A significant clinical problem with antiviral therapy in chronic
HBV and liver transplant patients has been the emergence of
resistance to lamivudine.13,16-22 Genotypic resistance emerges
in 14-32% of patients within the first 12 months of lamivudine
therapy, increasing to 40% within 2 years of treatment and 57%
by the 3rd year.8,23-25 Initially, resistance to lamivudine was
identified with mutations in a conserved functional epitope of
HBV DNA polymerase in the YMDD motif in lamivudine-
treated patients.18 Subsequently, it was found that resistance to
lamivudine occurs most often in the conserved YMDD motif
of the nucleotide-binding site of the viral polymerase.20,26-28
Single nucleotide changes at codon 204 of the reverse tran-
scriptase domain of the polymerase result in the substitution of
either valine or isoleucine for methionine (M204V or M204I).
The valine substitution and occasionally the isoleucine substitu-
tion are accompanied by an additional upstream mutation at
codon 180, where a methionine is substituted for a leucine
(L180M).26 Adefovir, lobucavir, and penciclovir (purine nucle-
otide and nucleoside analogues) have been shown to be active
against lamivudine-resistant HBV.29,30 Therefore, nucleoside
analogues that have sugar ring systems that are modified or
acyclic may retain activity against lamivudine-resistant mu-
tants.29 This permits the possibility that lamivudine-resistant
HBV could be treated with other nucleoside agents singly or in
combination. It is therefore important to continue research to
develop new antiviral strategies to better combat wild-type and
mutant HBV infections.
Chemistry
The target compounds 5-, 6-, or 5,6-substituted 1-[(2-
hydroxyethoxy)methyl]uracils (17-31) were achieved by a
simple and convenient one-step synthesis using iodomethyl
[(trimethylsilyl)oxy]ethyl ether, prepared in situ from 1,3-
dioxolane and trimethyl chlorosilane, instead of acetoxyethyl
acetoxymethyl ether as alkylating reagent.31,35,36 By this method,
steps involving synthesis of the acyclic chain (2-acetoxyethyl
acetoxymethyl ether) and deprotection of the acetyl group were
eliminated. Thus uracils 2-16 silylated with bis(trimethylsilyl)-
acetamide in dry acetonitrile were reacted with trimethylchlo-
rosilane, potassium iodide, and 1,3-dioxolane at room temper-
ature for 16-24 h to yield the desired compounds 17-31
(Scheme 1). In 1H NMR, the chemical shifts of NCH2 protons
in 6-substituted and 5,6-disubstituted derivatives (27-30) were
observed at δ 5.12-5.36, a similar region as those observed
for 5-substituted derivatives (17-21, 24, and 25, δ 5.07-5.28),
providing evidence for the of N-1 alkylation in compounds 27-
30. It is expected that N-3 alkylated product would have resulted
in the NCH2 protons being at much lower field than those in
the N-1 alkylated product due to the electron-withdrawing
properties of the two adjacent carbonyl functionalities. Further,
the site of alkylation of the acyclonucleosides 27-30 was
established as N-1 by UV spectra determined with solutions at
neutral and basic pH where no or little shift in λmax was observed
(experimental data).
Acyclic pyrimidine nucleosides have previously been shown
to have antiviral activity against herpes simplex virus.31 We have
recently reported pyrimidine nucleosides with acyclic structures
as selective inhibitors of DHBV.32-34 This new class of
molecules displayed strong inhibition of HBV replication in

Inhibition of HBV Replication by Pyrimidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2051
Table 1. In Vitro Activity against Hepatitis B Virus and Toxicity of 5-, 6- or 5,6-Substituted Analogues of 1-[(2-Hydroxyethoxy)methyl]uracils
% inhibition
toxicity
CC50 (µM)
at10 µg/mLa
[EC50 (µM)] b,h
B1 cell line
M204I
mutant HBV
D88 cell line
L180M/ M204V
mutant HBV
DHBV primary
duck hepatocytes
2.2.15
wild-type HBV
HepG2
Vero
no.
R1
R2
X
cellsc,d
cellsd,e
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Br
Cl
I
NO2
NH2
C2H5
H
CF3
CH2OH
COOC2H5
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
S
89 [3.7-18.5]
94 [0.4-2.2]
50 [32.0]
53 [43.2]
65 [5.0]
45 [>47.8]
46 [>49.5]
76 [3.9-39.3]
55 [47.6]
53 [36.4]
60 [25]
60-73 [18.5-37.7]
82 [4.5-45.4]
f
67 [18.5-37.7]
73 [3.7-37.7]
>377
>454
>320
>432
>497
>478
>495
>393
>476
>364
>500
>409
>306
>408
>534
>377
>454
>320
>432
>497
>478
>495
>393
>476
>364
>500
>409
>306
>408
>534
69 [4.5-45.4]
70 [4.5-45.4]
f
30
30
20
30
53 [39.3]
0
20
30
0
30
25
f
0
30
54 [43.2]
50 [50.0]
40 [>47.8]
42 [>49.5]
40 [>47.8]
0
50 [39.3]
30
0
0
0
25
S
O
O
S
O
O
O
O
55-65 [19.6-39.3]
50-73 [23.8-47.6]
50-64 [18.2-36.4]
55-72 [25-50]
60 [40.9]
CH3
COOCH3
CH3
H
I
NO2
56 [40.9]
51 [30.6]
53 [30.6]
CH3
68 [20.4-40.8]
60-80 [20.4-40.8]
20
f
30
25 [>53]
25 [>53]
f
3-TCg
96 [0.04-0.2]
88 [2.1-4.4]
45 [>44]
80-90 [3.4-16.9]
abacavir
72 [3.4-16.9]
a The data are expressed as percent inhibition of viral DNA in the presence of 10 µg/mL of the test compounds as compared to untreated infected controls.
b The drug concentration (µM) required to reduce the viral DNA in infected cells to 50% of untreated infected controls. c The drug concentration required
to reduce the viability of HepG2 cells as determined by MTT assay by 50% of untreated control after 3 days. d The > sign indicates that 50% inhibition was
not reached at the stated (highest) concentration tested. e The drug concentration required to reduce the viability of Vero cells as determined by MTT assay
by 50% of untreated control after 3 days. f Not determined. g (-)-â-L-2′,3′-dideoxy-3′-thiacytidine. h Percent inhibition was calculated by using the formula
(untreated positive control - treated test sample) × 100/untreated positive control. Tests were repeated 2-3 times, and the data for each test compound were
compared with a positive and negative control performed at the same time under identical conditions. For the compounds where the EC50 obtained from
three experiments was within 10% standard deviation, average values are shown, otherwise a range of EC50 values are shown.
Results and Discussion
disubstituted series of compounds, 6-methyl (27), 6-car-
bomethoxy (28), 5-iodo-6-methyl (29), and 5-nitro-6-methyl (30)
derivatives also exhibited anti-DHBV activity, where they were
approximately equiactive (EC50 ) 20.4-40.9 µM). In the
DHBV studies, 5-chloro derivative 18 (EC50 ) 0.4-2.2 µM)
emerged as the most active analogue. The bromo derivative 17
was approximately 9-fold less active (EC50 ) 3.7-18.5 µM)
than 18. In contrast compound 19 with a 5-iodo substituent was
less inhibitory to DHBV replication (EC50 ) 32 µM) than either
17 or 18. These results suggest that halogen substituents at the
5-position are determinants of anti-DHBV activity in this series
of compounds. The anti-DHBV activity exhibited by the most
active compound 18 was 10-times less potent than that of
reference drug 3-TC (EC50 ) 0.04-0.2 µM).
The activity of compounds 17-31 against wild-type HBV
was also examined against human hepatitis B virus in 2.2.15
cells. Most of the compounds active against DHBV retained
anti-HBV activity in 2.2.15 cells. The most active compound
18 exhibited an EC50 value of 4.5-45.4 µM in 2.2.15 cells that
compares favorably to that of reference drug 3-TC (EC50 ) 2.1-
4.4 µM). We note that anti-HBV activity for compounds 17,
18, and 21 in 2.2.15 cells was diminished 2-10-fold and 10-
fold, whereas for compounds 25 and 26, it was improved
approximately by 2-fold as compared to DHBV. The reasons
for the observed differences between the two cell systems may
be related to metabolic peculiarities, genomic organization of
hepadnavirus (i.e., integrated in 2.2.15 cells and nonintegrated
in duck hepatocytes), or inherent differences in the human vs
duck HBV.
To assess the antiviral effect of 5-, 6-, or 5,6-substituted
analogues of 1-[(2-hydroxyethoxy)methyl]uracils (17-31) on
wild-type HBV replication in vitro, primary hepatocytes from
ducks congenitally infected with duck HBV were used. These
cells chronically produce DHBV DNA, and therefore antiviral
activity was determined by analysis of intracellular viral DNA
using dot-blot hybridization. Stable HBV-producing human
hepatoblastoma cell line 2.2.15, which carries HBV DNA stably
integrated into the genome of HepG2 cells, was used to
determine the antiviral activity of compounds 17-31 against
human hepatitis B virus in vitro. To analyze the antiviral effect
of the compounds 17-31 against drug-resistant HBV, human
hepatoma cell line HepG2 transfected with the mutated HBV
genome was used. The cell lines contain single-mutant HBV
(B1 cell line transfected with M204I) or double-mutant HBV
(D88 cell line transfected with L180M/M204V).37 Both resistant
cell lines have mutations that are clinically relevant. The
concentrations required to inhibit 50% of HBV DNA (EC50)
and the 50% cytotoxic concentration (CC50) on HepG2 and Vero
cells are shown in Table 1. Lamivudine (3-TC) and abacavir
were used in these assays as reference antiviral drugs.
Among the acyclic nucleosides studied, 5-iodo (19), 5-nitro
(20), 5-hydroxymethyl (25), and 5-carboxyethyl (26) analogues
inhibited the replication of DHBV effectively with EC50 values
of 32-47.6 µM. The corresponding 5-bromo (17), 5-chloro (18),
5-amino (21), and 5-trifluoromethyl (24) analogues were active
and inhibited 50% DHBV replication at 3.7-18.5, 0.4-2.2, 5.0,
and 3.9-39.3 µM, respectively. In the 6-substituted and 5,6-

2052 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Semaine et al.
1-[(2-Hydroxyethoxy)methyl]-5-azauracil (31) possessed sig-
nificantly reduced anti-HBV activity against wild-type duck
HBV and human HBV (25% inhibition at 10 µg/mL), suggesting
that replacement of carbon with a nitrogen atom at the 6-position
of the uracil base is detrimental to antiviral activity.
pyrimidine nucleosides against wild-type and resistant HBV
mutants is important for identification of new and effective anti-
HBV agents with different resistance patterns or mechanisms
of action. Further biological and structure-activity relationship
studies of this new class of anti-HBV agents are ongoing in
our laboratories.
Cell-based DNA replication assays were used to measure the
antiviral activity of compounds 17-31 against lamivudine-
resistant HBV. In the B1 cell line with single-mutant HBV,
compounds 20-23, 26, 27, 29, and 30 showed moderate activity
(20-40%) in HBV replication at 10 µg/mL, whereas 5-chloro
(18), 5-bromo (17), and 5-trifluoromethyl (24) analogues were
notably inhibitory with EC50 values of 4.5-45.4, 18.5-37.7,
and 39.3 µM, respectively. In the D88 cell line with double
mutations (L180M/M204V), compounds 21, 22, 25, 29, and 30
exhibited moderate activity (20-40%) in HBV replication at
the concentration of 10 µg/mL, whereas compound 24 exhibited
an EC50 of 39.3 µM. Encouragingly, acyclic derivatives 17 and
18 showed better response against lamivudine-resistant HBV
with double mutations with EC50 values of 3.7-45.4 µM
suggesting that L180M/M204V changes do not confer signifi-
cant cross-resistance to them. The anti-HBV activity exhibited
by 17 and 18 compares favorably to reference compound
abacavir (EC50 ) 3.4-16.9 µM) used in these assays. Abacavir
has been reported to be active against lamivudine-resistant HBV
where no significant shift in the EC50 was observed for virus
expressing either the M204I mutation or the L180M/L204V
mutations compared to that for the wild-type virus.37 It is
interesting to observe that compounds 17 and 18 were effective
against both single and double mutants, and the EC50 values of
these were approximately similar to that against wild-type HBV
(Table 1). Similar results have been reported for the clinical
drug adefovir where it exhibits EC50 values of 0.58, 0.45-4.9,
and 2.2-9.5 µM, respectively, in the wild-type HepG2 2.2.15
cell line and lamivudine-resistant single- and double-mutant
HBV.38,39 In contrast, the single mutant M204I was >20 times
less susceptible to 3-TC than was wild-type HBV. The introduc-
tion of the double mutations (L180M/M204V) resulted in HBV
that was significantly less sensitive to 3-TC (30% inhibition at
10 µg/mL, and EC50 was not achieved up to 25 µg/mL). The
pattern of activity of lamivudine correlated with clinical in vitro
investigations where the methionine to valine mutation in the
YMDD motif of HBV DNA polymerase resulted in a 45-fold
decrease in lamivudine susceptibility.18
Experimental Section
Melting points were determined with a Buchi capillary apparatus
and are uncorrected. 1H NMR spectra were determined for solutions
in Me2SO-d6 on a Bruker AM 300 spectrometer using Me4Si as an
internal standard. The assignment of all exchangeable protons (OH,
NH) was confirmed by the addition of the D2O. UV spectra were
recorded with a Hewlett-Packard 8453 spectrophotometer. Mi-
croanalyses were within (0.4% of theoretical values for all elements
listed, unless otherwise indicated. Silica gel column chromatography
was carried out using Merck 7734 silica gel (100-200 µM particle
size). Thin-layer chromatography was performed with Machery-
Nagel Alugam SiL G/UV silica gel slides (20 µM thickness). The
5-, 6-, or 5,6-substituted uracils (2-16) were purchased from Sigma-
Aldrich Chemical Co.
1-[(2-Hydroxyethoxy)methyl]-5-bromouracil (17). To a sus-
pension of 2 (3 g, 15.7 mmol) in 40 mL of dry acetonitrile, 9.0 mL
(44.2 mmol) of bis(trimethylsilyl)acetamide was added, and the
mixture was stirred at room temperature till a clear solution was
obtained. To this solution, 1.2 mL (16.2 mmol) of 1,3-dioxolane,
2.6 g (15.6 mmol) of potassium iodide (KI), and 2.4 mL (22.09
mmol) of chlorotrimethylsilane were added. The reaction mixture
was stirred at room temperature for 16 h at which time TLC
(EtOAc/MeOH, 9.5:0.5, v/v) indicated that the reaction was
completed. The reaction medium was quenched with methanol and
neutralized with 6.0 g of sodium bicarbonate. The solid obtained
was purified by silica gel column chromatography using EtOAc/
MeOH (99:1, v/v) to yield 17 as a solid (3.85 g, 92.5%): mp 150-
152 °C (lit mp 147-148 °C);31 1HNMR (DMSO-d6) δ 3.55 (m,
4H, OCH2CH2O), 4.65 (br s, 1H, OH), 5.14 (s, 2H, NCH2), 8.28
(s, 1H, H-6). Anal. (C7H9BrN2O4) C, H, N.
The above procedure used for the synthesis of 17 was applied
for the preparation of compounds 18-31 with variations as
described in Supporting Information.
In Vitro Antiviral Assay for Duck Hepatitis B Virus (DHBV).
Primary hepatocyte cultures obtained from congenitally infected
ducks were used to determine the anti-DHBV activity of test
compounds, as reported previously.32,34,40,41
In Vitro Antiviral Assay for Human Hepatitis B Virus Using
2.2.15 Cells. The human HBV transfected 2.2.15 cells were obtained
from Dr. M. A. Sells and were used to determine the anti-HBV
activity of test compounds, as reported previously.41,42
In Vitro Antiviral Assay against Lamivudine-Resistant Hu-
man Hepatitis B Virus [B1 (M204I) and D88 (L180M/M204V)]
Cell Lines. The 3TC resistant cell lines B1 and D88 were previously
constructed by Tyrrell et al. and grown out of liquid nitrogen frozen
stocks.37 The D88 cell line contains a double mutation of the HBV
genome (rtL180M/M204V), and the B1 cell line features a single
mutation (rtM204I). The cell culture, treatment with compounds,
and dot-blot hybridization procedures were similar to a procedure
with 2.2.15 cells as described previously.37,41,42
The compounds 17-31 were also evaluated for their antiviral
activities against West Nile virus, respiratory syncytial virus,
SARS coronavirus, and hepatitis C virus. However, none of
these agents exhibited notable inhibition at concentrations up
to 100 µg/mL. 1-[(2-Hydroxyethoxy)methyl]-5-nitrouracil (20)
showed some inhibitory activity against West Nile virus (EC50
> 134 µM). The compounds 17-31 exhibited no in vitro
cytotoxicity against host HepG2 and Vero cells (CC50 >306-
534 µM) (Table 1).
Summary
Cell Cytotoxicity (MTT Assay). Cytotoxicities of test com-
pounds on HepG2 and Vero cells were determined using neutral
red uptake and MTT assays, respectively, as described earlier.32,34
Infection with hepatitis B virus is a medical problem of global
proportions. We describe 5-, 6-, or 5,6-substituted analogues
of 1-[(2-hydroxyethoxy)methyl]uracil as HBV-specific acyclic
pyrimidine nucleosides in vitro. While compounds 17, 18, 21,
22, 24, 29, and 30 showed inhibition against wild-type duck
HBV, human HBV (2.2.15), and lamivudine-resistant single and
double mutants of HBV, the 5-chloro (17) and 5-bromo (18)
analogues containing a modified glycosyl portion are most
effective against all wild-type and mutant HBV tested. The
observed antiviral activities of 5-, 6-, or 5,6-substituted acyclic
Acknowledgment. We are grateful to the Canadian Institutes
of Health Research (CIHR) for an operating grant (No. MOP-
36393) for the financial support of this research. B.A. is thankful
to the Alberta Heritage Foundation for Medical Research
(AHFMR) for a Medical Scholar Award and EG grant. We also
thank the United States National Institutes of Health Antiviral
Research Branch, which provided the antiviral tests for West

Inhibition of HBV Replication by Pyrimidines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2053
(22) Lai, C. L. Genotypic Resistance to Lamivudine in a Prospective,
Placebo Controlled Multicenter Study in Asia of Lamivudine Therapy
for Chronic Hepatitis B Virus Infection: Incidence, Kinetics of
Emergence and Correlation with Disease Parameters. Hepatology
1997, 26, 259A.
(23) Dienstag, J. L.; Schiff, E. R.; Wright, T. L.; Perrillo, R. P.; Hann,
H.-W. L.; Goodman, Z.; Crowther, L. B. S.; Condreay, L. D.;
Woessner, M. B. S.; Rubin, M.; Brown, N. A.; Brown, N. A. The U.
S. Lamivudine Investigator Group. Lamivudine as Initial Treatment
for Chronic Hepatitis B in the United States. N. Engl. J. Med. 1999,
341, 1256-1263.
(24) Lai, C. L.; Chien, R. N.; Leung, N. W.; Chang, T. T.; Guan, R.; Tai,
D. I.; Ng, K. Y.; Wu, P. C.; Dent, J. C.; Barber, J.; Stephenson, S.
L.; Gray, D. F. A One-year Trial of Lamivudine for Chronic Hepatitis
B. Asia Hepatitis Lamivudine Study Group. N. Engl. J. Med. 1998,
339, 64-70.
(25) Leung, N. W. Y.; Lai, C. L.; Chang, T. T.; Guan, R.; Lee, C. M.;
Ng, K. Y.; Lim, S. G.; Wu, P. C.; Dent, J. C.; Edmundson, S.;
Condreay, L. D.; Chien R. N. On behalf of the Asia Hepatitis
Lamivudine Study Group. Extended Lamivudine Treatment in
Patients with Chronic Hepatitis B Enhances Hepatitis B e Antigen
Seroconversion Rates: Results After 3 Years of Therapy. Hepatology
2001, 33, 1527-1532.
Nile virus, respiratory syncytial virus, SARS coronavirus, and
hepatitis C virus through NIAID contract to Dr. R.W. Sidwell.
Supporting Information Available: The details of synthesis
of compounds 18-31 and their spectral and elemental analyses.43
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Malik, A. H.; Lee, W. M. Chronic Hepatitis B Virus Infection:
Treatment Strategies for the Next Millennium. Ann. Intern. Med.
2000, 132, 723-731.
(2) Lok, A. S. Hepatitis B Infection: Pathogenesis and Management. J.
Hepatol. 2000, 32 (Suppl. 1), 89-97.
(3) Kane, M. A. The Global Epidemiology of Hepatitis B. Soz.
PraVentiVmed. 1998, 43 (Suppl. 1), S24-S26.
(4) Medical Microbiology; Mims, C. A., Ed.; Mosby: London, 1993;
Chapter 38.
(5) Perrillo, R. P.; Schiff, E. R.; Davis, G. L.; Bodenheimer, H. C.;
Lindsay, K.; Payne, J.; Dienstag, J. L.; O’Brien, C.; Tamburro, C.;
Jacobson, I. M. A Randomized Control led Trial of Interferon Alfa-
2b Alone and After Prednisone Withdrawal for the Treatment of
Chronic Hepatitis B. The Hepatitis Interventional Therapy Group.
N. Engl. J. Med. 1990, 323, 295-301.
(26) Allen, M. I.; Deslauriers, M.; Andrews, C. W.; Tipples, G. A.;
Walters, K. A.; Tyrrell, D. L.; Brown, N.; Condreay, L. D.;
Lamivudine Clinical Investigation Group. Hepatology 1998, 27,
1670-1677.
(27) Fischer, K. P.; Tyrrell, D. L. Generation of Duck Hepatitis B Virus
Polymerase Mutants through Site-directed Mutagenesis which Dem-
onstrate Resistance to Lamivudine [(-)-Beta-L-2′, 3′-dideoxy-3′-
thiacytidine] in vitro. Antimicrob. Agents Chemother. 1996, 40,
1957-1960.
(6) Wong, D. K. H.; Cheung, A. M.; O’Rourke, K.; Naylor, C. D.;
Detsky, A. S.; Heathcote, J. Effect of Alpha-Interferon Treatment in
Patients with Hepatitis B e Antigen- Positive Chronic Hepatitis B.
A Meta-Analysis. Ann. Intern. Med. 1993, 119, 312-323.
(7) Nevens, F.; Main, J.; Honkoop, P.; Tyrrell, D. L.; Barber, J.; Sullivan,
M. T.; Fevery, J.; DeMan, R. A.; Thomas, H. C. Lamivudine Therapy
for Chronic Hepatitis B: A Six Month Randomized Dose-ranging
Study. Gastroenterology 1997, 113, 1258-1263.
(8) Liaw, Y. F.; Leung, N. W.; Chang, T. T.; Guan, R.; Tai, D. I.; Ng,
K. Y.; Chien, R. N.; Dent, J.; Roman, L.; Edmundson, S.; Lai, C. L.
Effects of Extended Lamivudine Therapy in Asian Patients with
Chronic Hepatitis B. Asia Hepatitis Lamivudine Study Group.
Gastroenterology 2000, 119, 172-180.
(9) Gilson, R. J. C.; Chopra, K.; Murray, L. I. Placebo-controlled Phase
I/II study of Adefovir Dipivoxil in Patients with Chronic Hepatitis
B Infection. Hepatology 1996, 24 (Suppl. 1), 281A.
(10) Hepsera Full Prescribing Information. Gilead Sciences. 09/2002, pp
1-18.
(11) Torresi, J.; Locarnini, S. Antiviral Chemotherapy for the Treatment
of Hepatitis B Virus Infections. Gastroenterology 2000, 118 (Suppl.
2), 83-103.
(12) Delaney, W. E., IV.; Edwards, R.; Colledge, D.; Shaw, T.; Torresi,
J.; Miller, T. G.; Isom, H. C.; Bock, C. T.; Manns, M. P.; Trautwein,
C.; Locarnini, S. Cross-resistance Testing of Antihepadnaviral
Compounds Using Novel Recombinant Baculoviruses Which Encode
Drug-Resistant Strains of Hepatitis B Virus. Antimicrob. Agents
Chemother. 2001, 45, 1705-1713.
(13) Zoulim, F. Detection of Hepatitis B Virus Resistance to Antivirals.
J. Clin. Virol. 2001, 21, 243-253.
(14) Leung, N. Treatment of Chronic Hepatitis B: Case Selection and
Duration of Therapy. J. Gastroenterol. Hepatol. 2002, 17, 409-414.
(15) Colledge, D.; Locarnini, S.; Shaw, T. Synergistic Inhibition of
Hepadnaviral Replication by Lamivudine in Combination with
Penciclovir in vitro. Hepatology 1997, 26, 216-225.
(16) Leung, N. Clinical Experience with Lamivudine. Semin. LiVer Dis.
2002, 22 (Suppl. 1), 15-22.
(17) Tipples, G. A.; Ma, M. M.; Fischer, K. P.; Bain, V. G.; Kneteman,
N, M.; Tyrrell, D. L. Mutation in HBV RNA-dependent DNA
Polymerase Confers Resistance to Lamivudine in vivo. Hepatology
1996, 24, 714-717.
(18) Bartholomew, M. M.; Jansen, R. W.; Jeffers, L. J.; Reddy, K. R.;
Johnson, L. C.; Bunzendahl, H.; Condreay, L. D.; Tzakis, A. G.;
Schiff, E. R.; Brown, N. A. Hepatitis-B-Virus Resistance to
Lamivudine Given for Recurrent Infection after Orthotopic Lver
Transplantation. Lancet 1997, 349, 20-22.
(19) Liaw, Y.-F. Therapy of Chronic Hepatitis B: Current Challenges
and Opportunities. J. Viral Hepatol. 2002, 9, 393-399.
(20) Ling, R.; Mutimer, D.; Ahmed, M.; Boxall, E H.; Elias, E.; Dusheiko,
G. M.; Harrison, T. J. Selection of Mutations in the Hepatitis B Virus
Polymerase During Therapy of Transplant Recipients with Lamivu-
dine. Hepatology 1996, 24, 711-713.
(28) Melegari, M.; Scaglioni, P. P.; Wands, J. R. Hepatitis B Virus Mutants
Associated with 3TC and Famciclovir Administration are Replication
Defective. Hepatology 1998, 27, 628-633.
(29) Das, K.; Xiong, X.; Yang, H.; Westland, C. E.; Gibbs, C. S.;
Sarafianos, S. G.; Arnold, E. Molecular Modeling and Biochemical
Characterization Reveal the Mechanism of Hepatitis B Virus Poly-
merase Resistance to Lamivudine (3TC) and Emtricitabine (FTC). J
Virol. 2001, 75, 4771-4779.
(30) Seigne`res, B.; Pichoud, C.; Germon, S.; Furman, P.; Tre´po, C.;
Zoulim, F. Inhibitory Activity of New Nucleoside Analogues on
Wild-type and YMDD Mutants of the Hepadnavirus Reverse Tran-
scriptase. AntiViral Res. 2000, 46, A56.
(31) Kelley, J. L.; Kelsey, J. E.; Hall, W. R.; Krochmal, M. P.; Schaeffer,
H. J. Pyrimidine Acyclic Nucleosides. 1-[(2-Hydroxyethoxy)methyl]-
pyrimidines as Candidate Antivirals. J. Med. Chem. 1981, 24, 753-
756.
(32) Kumar, R.; Sharma, N.; Nath, M.; Saffran, H. A.; Tyrrell, D. J.
Synthesis and Antiviral Activity of Novel Acyclic Nucleoside
Analogues of 5-(1-Azido-2-haloethyl)uracils. J. Med. Chem. 2001,
44, 4225-4229.
(33) Kumar, R.; Tyrrell, D. J. Novel 5-Vinyl Pyrimidine Nucleosides with
Potent anti-Hepatitis B Virus Activity. Bioorg. Med. Chem. Lett.
2001, 11, 2917-2920.
(34) Kumar, R.; Nath, M.; Tyrrell, D. J. Design and Synthesis of Novel
5-Substituted Acyclic Pyrimidine Nucleosides as Potent and Selective
Inhibitors of Hepatitis B Virus. J Med. Chem. 2002, 45, 2032-2040.
(35) Robins, M. J.; Hatfield, P. W.; Balzarini, J.; De Clercq, E. Nucleic
Acid Related Compounds. 47. Synthesis and Biological Activities
of Pyrimidine and Purine “Acyclic” Nucleoside Analogs. J. Med.
Chem. 1984, 27, 1486-1492.
(36) Rosowsky, A.; Kim, S.-H.; Wick, M. Synthesis and Antitumor
Activity of an Acyclonucleoside Derivative of 5-Fluorouracil. J. Med.
Chem. 1981, 24, 1177-1181.
(37) Walters, K. A.; Tipples, G. A.; Allen, M. I.; Condreay, L. D.;
Addison, W. R.; Tyrrell, L. Generation of Stable Cell Lines
Expressing Lamivudine-Resistant Hepatitis B Virus for Antiviral-
Compound Screening. Antimicrob. Agents Chemother. 2003, 47,
1936-1942.
(38) Ono, S. K.; Kato, N.; Shiratori, Y.; Kato, J.; Goto, T.; Schinazi, R.
F.; Carrilho, F. J.; Omata, M. The polymerase L528M mutation
cooperates with nucleotide binding-site mutations, increasing hepatitis
B virus replication and drug resistance. J. Clin. InVest. 2001, 107,
449-455.
(39) Perrillo, R.; Schiff, E.; Yoshida, E.; Statler, A.; Hirsch, K.; Wright,
T.; Gutfreund, K.; Lamy, P.; Murray, A. Adefovir dipivoxil for the
Treatment of Lamivudine-resistant Hepatitis B Mutants. Hepatology
2000, 32, 129-134.
(21) Chan, T. M.; Fang, G. X.; Tang, C. S. O.; Cheng, I. K. P.; Lai, K.
N.; Ho, S. K. N. Preemptive Lamivudine Therapy Based on HBV
DNA Level in HBsAg-positive Kidney Allograft Recipients. Hepa-
tology 2002, 36, 1246-1252.
(40) Lee, B.; Luo, W.; Suzuki, S.; Robins, M. J.; Tyrrell, D. L. J. In vitro
and in ViVo Comparison of the Abilities of Purine and Pyrimidine
2′,3′-Dideoxynucleosides to Inhibit Duck Hepadnavirus. Antimicrob.
Agents Chemother. 1989, 33, 336-339.

2054 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Semaine et al.
(41) Tuttleman, J. S.; Pugh, J. C.; Summers, J. W. In vitro Experimental
Infection of Primary Duck Hepatocyte Cultures with Duck Hepatitis
B Virus. J. Virol. 1986, 58, 17-25.
(42) Sells, M. A.; Chen, M. L.; Acs, M. L. Production of Hepatitis B
Virus Particles in Hep G2 Cells Transfected with Cloned Hepatitis
B Virus DNA. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1005-1009.
(43) Robins, M. J.; Hatfield, P. W. Nucleic Acid Related Compounds.
37. Convenient and High-yield Synthesis of N-[(2-hydroxyethoxy)-
methyl] Heterocycles as “Acyclic Nucleoside” Analogues. Can. J.
Chem. 1982, 60, 547-553.
JM058271D