Effect of various pyrimidines possessing the 1-[(2-hydroxy-1- (hydroxymethyl)ethoxy)methyl] moiety, able to mimic natural 2′- deoxyribose, on wild-type and mutant hepatitis B virus replication

Article abstract of DOI:10.1021/jm060102l

Hepatitis B virus (HBV) is the most common cause of chronic liver disease worldwide. Development of drug resistance against clinical anti-HBV drug lamivudine due to long-term use and rebound of viral DNA after cessation of treatment has been a major setba

Full text of DOI:10.1021/jm060102l

J. Med. Chem. 2006, 49, 3693-3700
3693
Effect of Various Pyrimidines Possessing the 1-[(2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl]
Moiety, Able To Mimic Natural 2′-Deoxyribose, on Wild-type and Mutant Hepatitis B Virus
Replication
Rakesh Kumar,*,† Wassila Semaine,†,‡ Monika Johar,† D. Lorne J. Tyrrell,§ and Babita Agrawal*,‡
Department of Laboratory Medicine and Pathology, 1-41 Medical Sciences Building, Department of Surgery, and Department of Medical
Microbiology and Immunology, Faculty of Medicine and Dentistry, UniVersity of Alberta, Edmonton, AB, Canada T6G 2H7
ReceiVed January 31, 2006
Hepatitis B virus (HBV) is the most common cause of chronic liver disease worldwide. Development of
drug resistance against clinical anti-HBV drug lamivudine due to long-term use and rebound of viral DNA
after cessation of treatment has been a major setback of the current therapy. We have synthesized a series
of pyrimidine nucleosides possessing a variety of substituents at the C-5 position, and a 1-[(2-hydroxy-1-
(hydroxymethyl)ethoxy)methyl] flexible acyclic glycosyl moiety at the N-1 position, that have the ability to
mimic the natural 2′-deoxyribosyl moiety. Some of these potential antiviral compounds included variations
at both C-5 and C-6 positions of the uracil base. Other variations of the uracil derivatives were the 6-aza
congeners. 4-Amino and 4-methoxy pyrimidine derivatives were also made. Compounds in which the base
moiety was substituted by 5-chloro- (25), 5-(2-bromovinyl)- (32), or 5-bromo-6-methyl- (37) groups possess
significant activity against duck-HBV, wild-type human HBV (2.2.15 cells), and lamivudine-resistant HBV
containing single and double mutations. No cytotoxicity was seen in host HepG2 and Vero cells, up to the
highest concentration tested. The anti-HBV activity exhibited by compounds 25, 32, and 37 was superior
for human HBV and comparable for DHBV to that of the corresponding purine nucleoside, ganciclovir.
Further, they were only 10-15-fold less inhibitory against human HBV in 2.2.15 cells than the reference
drug, lamivudine. Other compounds in the series were moderately inhibitory against DHBV and wild-type
human HBV. The size of the halogen and the electronegativity of the substituents at the 5- and 6-positions
are important for antiviral activity toward HBV. These compounds were also evaluated for their antiviral
activity for West Nile virus, respiratory syncytial virus, SARS-coronavirus, and hepatitis C virus. They
were generally inactive in these antiviral assay systems (at concentrations up to 100 µg/mL). 1-[(2-Hydroxy-
1-(hydroxymethyl) ethoxy)methyl]-5-fluorocytosine (34) showed some inhibitory activity against hepatitis
C virus. Taken together, these data support our previous observations that the 5-substituted pyrimidine
nucleosides containing acyclic glycosyl moieties have potential to serve as a new generation of potent,
selective, and nontoxic anti-HBV agents for wild-type and lamivudine-resistant mutant HBV.
Introduction
chronic HBV patients are eligible for this treatment. Unfortu-
nately, of those who are treated, R-IFN therapy is effective only
in 30-40% of the cases. It is expensive, requires i.v. admin-
istration, and has serious side effects such as influenza-like
symptoms, depression, insomnia, nausea, abdominal pain, hair
loss, leucopenia, thrombocytopenia, thyroid disorders, and
rashes.2,4,5 Thymosin-R1 (Zadaxin), interleukin-12, and thera-
peutic vaccines have been used in clinical trials as immuno-
therapy regimens, but have not demonstrated sufficient efficacy.6-8
HBV is an incomplete double-stranded DNA virus. Its DNA
replication is unique and includes a reverse transcriptase-
catalyzed reverse transcription step.9 The HBV DNA polymerase
is an essential and multifunctional enzyme, which functions as
a DNA polymerase, reverse transcriptase, RNAse H, coordinates
the assembly of viral nucleocapsids, and catalyzes the generation
of DNA primer. Therefore, different classes of compounds can
interfere at various steps of HBV replication. Furthermore, HBV
DNA polymerase is quite different from human DNA poly-
merases, suggesting that compounds that selectively inhibit HBV
replication can be identified.10
Hepatitis B virus (HBV) infection is a significant problem
affecting more than 5% of the world’s population.1-3 HBV
infections are present in people in all parts of the world. HBV
can cause acute and chronic liver diseases. Chronic HBV
infection often results from exposure in early life, leading to
viral persistence in the absence of a strong antibody or cellular
immune response. There are over 350 million people worldwide
who are chronic carriers of HBV, 6 times the number of people
infected with human immunodeficiency virus (HIV) (50 mil-
lion). Chronic HBV infection leads to liver damage, cirrhosis,
and liver cancer with high mortality. It has been suggested that
suppression of the replication of HBV in the liver leads to
improved liver pathology and decreased progression to liver
cirrhosis and hepatocellular carcinoma, both of which are
secondary to HBV infection and major causes of mortality.
Therapy of chronic HBV can aim to either inhibit viral
replication or enhance immunological response against the virus,
or both.
R-Interferon (R-IFN, Intron A) has been used for chronic
HBV infections, but its use is limited. Less than one-half of all
The (-) enantiomer of 2′3′-dideoxy-3′-thiacytidine (1a), also
known as lamivudine (3-TC), is a potent inhibitor of HBV
replication.11,12 It is phosphorylated by cellular kinases into the
triphosphate form, which competes with cellular dCTP and is
incorporated into viral DNA by the viral DNA polymerase. This
results in premature chain termination during both the reverse
* Corresponding author. Phone: (780) 492-7545. Fax: (780) 492-7521.
E-mail: rakesh.kumar@ualberta.ca.
† Department of Laboratory Medicine and Pathology.
‡ Department of Surgery.
§ Department of Medical Microbiology and Immunology.
10.1021/jm060102l CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/12/2006

3694 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Kumar et al.
transcription and the DNA synthesis steps of HBV DNA
synthesis.13 Chronically infected individuals treated with lami-
vudine therapy show histological improvement and significant
reduction of viral load.14,15
viral DNA in the hepatocyte nucleus. A drawback of lamivudine
and hepsera therapy is that the template for hepadnavirus
replication, cccDNA, is relatively unaffected.28 Although the
half-life of HBV cccDNA in humans has not been established,
the half-lives of both duck HBV and woodchuck HBV cccDNA
have been shown to be quite long, between 30 and 57 days.29,30
Because of the stability of cccDNA, continuous suppression of
viral replication will likely require long-term anti-HBV therapy.
Prolonged lamivudine monotherapy results in the develop-
ment of lamivudine-resistant HBV variants in approximately
43% of treated patients within a year and 49-58% patients in
2-3 years.31 The appearance of resistant HBV suggests that
lamivudine therapy must be combined with other antiviral drugs
to delay or prevent the emergence of resistance mutants. Thus,
the future treatment of chronic HBV should encompass a
cocktail of antiviral drugs as has been proven effective for anti-
HIV therapy.
Mutations associated with lamivudine resistance occur most
often in the conserved YMDD motif of the nucleotide-binding
site of the viral DNA polymerase.32-34 Single nucleotide changes
at codon 204 of the reverse transcriptase domain of the
polymerase results in the substitution of either valine or
isoleucine for methionine (M204V or M204I). The valine
substitution and occasionally the isoleucine substitution is
accompanied by an additional upstream mutation at codon 180
(L180M).32
Molecular modeling studies suggested that acyclic nucleosides
provide torsional flexibility and a shorter chain connecting the
base and the sugar moiety, as compared to the oxathiolane ring
in lamivudine. The molecular modeling studies also predicted
that the dNTP binding pocket of HBV DNA pol containing
M204V/I+L180M mutations is more constrained and crowded,
allowing acyclic nucleoside analogues to be accommodated
more effectively. This modeling prediction was confirmed with
biochemical studies using HBV polymerase inhibition assays.35
Therefore, nucleoside analogues that have sugar ring systems
that are modified or acyclic may retain activity against lami-
vudine-resistant mutants.35 This discrepancy in the mode of
binding of nucleoside analogues with modified glycosyl moieties
to the dNTP binding pocket of HBV DNA pol permits the
possibility that lamivudine-resistant HBV could be treated with
other nucleoside agents singly or in combination.
Recently another antiviral agent, hepsera, 9-[2-[bis[(pivaloy-
loxy)methoxy]phosphinyl]methoxy]-ethyl]adenine [adefovir dipiv-
oxil] (1b), an oral prodrug of an acyclic nucleotide analogue of
adenosine, has been approved by the FDA for HBV infection.16-19
In this drug, after absorption, the bis-(pivaloyloxymethyl) moiety
is removed from the parental drug, resulting in the active agent
adefovir. Hepsera, being a monophosphate derivative, does not
require initial phosphorylation to the monophosphate for activa-
tion.16 Adefovir diphosphate is an inhibitor of HBV DNA
polymerase (reverse transcriptase) activity and is also a chain
terminator when incorporated into viral DNA. Hepsera was
demonstrated to be well-tolerated, to reduce serum HBV DNA
levels by 4 logs, and lead to seroconversion in 20-27% of the
patients treated for 12 weeks at a daily dose of 30 mg or greater/
day.17,18 However, primary limitations of adefovir therapy are
dose-related side effects such as nephrotoxicity, lactic acidosis,
and severe hepatomegaly with steatosis, and cessation of therapy
may result in serious hepatitis.19
More recently, a cyclopentyl deoxyguanosine analogue,
baraclude, [1S-(1R,3R,4â)]-2-amino-1,9-dihydro-9-[4-hydroxy-
3-3(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6-
one] [entecavir] (1c), in which the oxygen in the furanose ring
is replaced with an exocyclic double bond, has been approved
by the FDA for HBV infection.20 In in vitro studies, it is
significantly more potent than lamivudine in HepG2 2.2.15
hepatoma cells with high therapeutic index and is also an
effective inhibitor of replication of lamivudine-resistant HBV
mutant.21,22 Entecavir was found to be superior to lamivudine
and also efficacious in patients developing resistance to lami-
vudine.23,24 However, this drug also suffers from serious toxicity
problems such as lactic acidosis and hepatotoxicity with
hepatomegaly and steatosis.
Among acyclic purine nucleosides, acyclovir 9-[(2-hydroxy-
ethoxy)methyl]guanine (1d) has been shown to be a modest
inhibitor of HBV DNA polymerase in triphosphate form.
Acyclovir exhibited both in vitro and in vivo activity in animal
models, but demonstrated disappointing anti-HBV efficacy in
human clinical trials.36 Ganciclovir, 9-[(2-hydroxy-1-(hydroxy-
methyl)ethoxy)methyl]guanine (1e), structurally similar to acy-
clovir but differing in the functional equivalent of a 3′-OH
group, is also a potent inhibitor of HBV, both in cell culture
against duck-HBV (DHBV) and in in vivo animal models.37,38
However, long-term clinical use of ganciclovir is limited due
to its severe dose-related toxicity.39 In contrast, pyrimidine
nucleosides with a 1-[(2-hydroxyethoxy)methyl] group have
been explored as anti-herpes agent with no significant activity.40-44
Recent work in our laboratory has led to the identification
of acyclic pyrimidine nucleosides as a new family of potent
and selective inhibitors of DHBV replication.45,46 More recently,
various 5-substituted acyclic pyrimidines containing a 1-[(2-
hydroxyethoxy)methyl] glycosyl moiety at the N-1 position
showed potent in vitro activity in both chronically infected
primary duck hepatocytes and a human HBV DNA transfected
human hepatoblastoma cell line (HepG2 2.2.15).47 Intriguingly,
Treatment with lamivudine, hepsera, or baraclude in HBV-
infected patients leads to a quantitative decrease in HBV
replication. However, after cessation of treatment, virus DNA
levels rebounded to baseline pretreatment (or even higher)
levels.25-27 This problem of virus reappearance can be in part
explained by the mechanism by which HBV maintains the
infection at the level of individual hepatocytes. It has been
shown that chronic infection of hepatocytes is maintained by
the presence of 30-40 copies of covalently closed circular (ccc)

Effect of Various Pyrimidines on HBV Replication
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3695
Scheme 1a
a Reagents: (i) Hexamethyldisilazane, trimethylchlorosilane, potassium nonaflate, dry acetonitrile, reflux; (ii) 40% aqueous methylamine, 25 °C.
these compounds also exhibited in vitro activity against lami-
vudine-resistant HBV containing single and double mutations.47
In our studies, we noted that, among acyclic pyrimidines,
substituents at the C-5 position of the pyrimidine base are
important determinants of anti-HBV activity against duck HBV,
wild-type human HBV, and lamivudine-resistant HBV in vitro
at noncytotoxic concentrations. It was therefore of interest to
examine the anti-HBV effect of various 5-substituted acyclic
pyrimidine nucleosides containing a 1-[(2-hydroxy-1-(hydroxy-
methyl)ethoxy)methyl] moiety with a 3′-OH like group, to
determine if they have increased affinity for viral DNA
polymerase, thus providing improved anti-HBV activity and
selectivity.
In an effort to further explore the structure activity relation-
ships (SARs), we now report the synthesis and antiviral activities
of several 5-substituted pyrimidines with a 1-[(2-hydroxy-1-
(hydroxymethyl)ethoxy)methyl] acyclic chain. We herein also
present modifications of the 4-, 5-, and 6- positions in this new
series of promising antivirals and report their anti-HBV activity.
The results presented here extend our previous observations and
suggest the usefulness of the acyclic pyrimidine nucleosides as
HBV inhibitors.
Chemistry
Acyclonucleosides 22-24, 26-29, 32-35, and 39-41 were
synthesized by the reaction of respective silylated uracils with
bromomethyl ether as described earlier.40-44 Similar synthetic
routes for the preparation of unknown acyclic derivatives (25,
30, 31, and 36-38) were adopted (Scheme 1). Thus, 5-substi-
tuted uracils (5, 10, 11) were refluxed with hexamethyldisilazane
(HMDS) and a catalytic amount of chlorotrimethylsilane for
about 3 h. The resulting clear solutions were evaporated in vacuo
to remove excess HMDS, and the silylated intermediates
obtained were coupled with 2-(bromomethoxy)-1,3-propenediyl
dibenzoate43 in dry acetonitrile at reflux temperature for 6-12
h to produce the precursor ester derivatives. These derivatives
were subsequently deprotected with 40% aqueous methylamine
at 25 °C to yield the target 1-[(2-hydroxy-1-(hydroxymethyl)-

3696 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Kumar et al.
Table 1. In Vitro Activity Against Wild-type (Duck HBV and Human HBV) and Drug-Resistant Hepatitis B Virus, and Toxicity of Various Acyclic
Analogues of 2′-Deoxy Pyrimidine Nucleosides
% inhibition @10 µg/mLa
[EC50 (µM)]b
toxicity CC50 (µM)
DHBV
primary duck
hepatocytes
2.2.15
wild-type
HBV
B1 cell line
M204I
mutant HBV
D88 cell line
L180M/M204V
mutant HBV
HepG2
cellsc
Vero
cellse
no.
R1
R2
X
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
H
F
Br
Cl
NO2
NH2
CH3
C2H5
CH2OH
CH2CH2OH
CHdCHBr
H
F
Br
H
Br
I
H
CH3
CH3
H
O
O
O
O
O
O
O
O
O
O
O
0
0
NDf
ND
ND
56 [20-40]
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
40 [>40]
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
30 [>32.4]
ND
ND
ND
ND
30
72 [3.4-16.9]
ND
>462
>427
>338
>400
>383
>432
>434
>410
>406
>384
>311
>465
>429
>323
>434
>324
>280
>460
>432
>404
>462
>427
>338
>400
>383
>432
>434
>410
>406
>384
>311
>465
>429
>323
>434
>324
>280
>460
>432
>404
H
H
H
H
H
H
H
H
H
H
24 [>42.7]
48 [>33.8]
69 [4-20]
45 [>38.3]
37 [>43.2]
45 [>43.4]
35 [>41.0]
0
26 [>42.7]
40 [>33.8]
60 [20-40]
40 [>38.3]
30 [>43.2]
37 [>43.4]
30 [>41.0]
0
28 [>38.4]
53 [30]
0
0
25 [>38.4]
75-90 [3-15]
0
20 [>42.9]
45 [>32.3]
27 [>43.4]
73 [16.2-32.4]
44 [>28.0]
41 [>46.0]
45 [>43.2]
25 [>40.4]
96 [0.04-0.21]
35 [>32.3]
0
CH3
CH3
CH3
O
O
O
O
O
S
55 [32.4]
38 [>28.0]
32 [>46.0]
37 [>43.2]
0
51 [32.4]
ND
ND
ND
ND
3-TCg
88 [2.1-4.4]
45 [>44.0]
80-92 [3.4-16.9]
ND
Abacavir
GCVh
[4.0]i
20% @ 100 µMj
ND
ND
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 (>) 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,
g
by 50% of untreated control after 3 days. f Not determined. (-)-3-TC (lamivudine). h Ganciclovir. i Data taken from Shaw et al. j Data taken from Kruining
et al.
ethoxy)methyl]uracils (25, 30, 31) as shown in Scheme 1. The
5,6-disubstituted acyclic pyrimidine nucleosides 37 and 38 were
synthesized similarly by direct alkylation of the silylated
6-methyl (16), 5-bromo-6-methyl (17), and 5-iodo-6-methyl (18)
bases, respectively (Scheme 1). The site of alkylation of the
5,6-disubstituted acyclonucleosides (36-38) 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).
HepG2 cells. To analyze the antiviral effects of the compounds
22-41 against the drug-resistant HBV, human hepatoma cell
line HepG2 transfected with 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).50 Both resistant cell lines have
mutations that are clinically relevant. In all of the cell lines used,
the antiviral activity was determined by analysis of intracellular
viral DNA, using dot blot hybridization. The concentrations
required to inhibit 50% of HBV DNA (EC50), and 50% cytotoxic
concentration (CC50) on HepG2 and Vero cells, are shown in
Table 1. Lamivudine and abacavir were used in these assays as
reference antiviral drugs. Purine acyclic nucleoside ganciclovir
(GCV) is also included in Table 1 for comparison of their anti-
HBV potency.
Results and Discussion
The anti-HBV activities of the synthesized acyclic pyrimidine
nucleosides (22-41) were evaluated against DHBV in confluent
cultures of primary duck hepatocytes obtained from chronically
infected Pekin ducks.48,49 DHBV is a member of the family
hepadnaviridae and has a virion structure and genome organiza-
tion similar to that of human HBV. Thus, DHBV has been used
extensively to screen potential drugs to control chronic HBV
infection. The anti-HBV activity of the acyclic pyrimidines was
also assessed in confluent cultures of the human hepatoma cell
line 2.2.15 that chronically produces infectious HBV. 2.2.15 is
a stable human HBV-producing human hepatoblastoma cell line,
which carries HBV DNA stably integrated into the genome of
Among the acyclic pyrimidine nucleosides investigated,
5-chloro (25), 5-(2-bromovinyl) (32), and 5-bromo-6-methyl
(37) analogues exhibited the most promising in vitro activity
against DHBV (EC50 ) 4-20, 3-15, 16.2-32.4 µM, respec-
tively). Interestingly, the anti-DHBV activity exhibited by
compounds 25 and 32 was found to be similar to that of
corresponding potent acyclic purine nucleoside ganciclovir (EC50
) 4 µM), as measured previously in the DHBV infected primary

Effect of Various Pyrimidines on HBV Replication
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3697
duck hepatocyte cultures.51 At a concentration of 10 µg/mL,
test compounds 22, 23, 24, 26-31, 33-36, and 38-41 exhibited
modest or no inhibition of DHBV replication, and the percent
inhibition of viral replication was as follows: 22 (0%), 23
(24%), 24 (48%), 26 (45%), 27 (37%), 28 (45%), 29 (35%), 30
(0%), 31 (25%), 33 (0%), 34 (20%), 35 (45%), 36 (27%), 38
(44%), 39 (41%), 40 (45%), and 41 (25%).
inhibition at 100 µM)38 and only 10-15-fold less than the
reference drug lamivudine. This contrasts to their anti-DHBV
activity. The differential activity between DHBV and HBV
could be attributed to metabolic peculiarities, genomic organiza-
tion of hepadnavirus (i.e., integrated in 2.2.15 cells and
nonintegrated in duck hepatocytes), and/or inherent differences
in the human versus duck HBV. Interestingly, like anti-DHBV
activity, 5-unsubstituted parent molecules (22, 33) did not show
any activity against human HBV in 2.2.15 cells, and similar
SARs were observed among various 5-substituents. We further
noted that among the compounds 22-41 possessing the 1-[(2-
hydroxy-1-(hydroxymethyl) ethoxy)methyl] moiety, the antiviral
activity against human HBV in 2.2.15 cells was reduced as
compared to their 1-[(2-hydroxyethoxy)methyl] counterparts.47
These results are similar to the observations made for anti-
DHBV activity.
Cell-based DNA replication assays were used to measure the
antiviral potency of selected compounds 25 and 37 against
lamivudine-resistant HBV in a B1 cell line with a single mutant
HBV (M204I), and in a D88 cell line with a double mutation
(L180M/M204V) (Table 1). The 5-chloro (25) and 5-bromo-
6-methyl (37) derivatives retained activity against single mutant
HBV as compared to the wild-type HBV. The anti-HBV activity
of 37 was slightly diminished against double-mutant HBV.
These results suggest that compounds 25 and 37 are still
sensitive to the lamivudine-resistant single and double mutant
HBV strains.
It is noteworthy that compounds 25 and 37 were inhibitory
against both single and double mutants, and the % inhibition
of HBV DNA replication was comparable to that against wild-
type HBV (Table 1). Similar results have been reported for the
clinical drug adefovir where it exhibited EC50 values of 0.58,
0.45-4.9, and 2.2-9.5 µM, respectively, in a wild-type 2.2.15
cell line, and in lamivudine-resistant single and double mutant
HBV.54,55 In contrast, single mutant M204I was >20 times less
susceptible to lamivudine than was wild-type HBV. The
introduction of the double mutants (L180M/M204V) resulted
in HBV that was significantly less sensitive to lamivudine (30%
inhibition @10 µg/mL, and EC50 was not achieved up to 25
µg/mL). The pattern of activity of lamivudine correlated with
clinical investigations where M to V mutation in the YMDD
motif of HBV DNA polymerase resulted in a 45-fold decrease
in lamivudine susceptibility.56
Compounds 22-41 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. Compound 1-[(2-hydroxy-1-(hydroxymethyl)-
ethoxy)methyl]-5-fluorocytosine (34) showed some inhibitory
activity against hepatitis C virus (EC50 ) 132 µM).
Surprisingly, the 5-unsubstituted parent uracil and cytosine
derivatives (22, 33) were completely devoid of anti-HBV
activity, suggesting that in this series of compounds function-
alized substituents at the 5-position of pyrimidine contribute to
the antiviral activity. Concerning substitutions by halogens, it
appears that the size of the substituent plays an important role,
because 5-bromo (24) and 5-chloro (25) derivatives have higher
inhibitory activity than the corresponding 5-fluoro (23) analogue.
In the case of the 5-bromo compound (24), alteration of the
4-position by replacement of the oxygen with methoxy (35) did
not influence antiviral activity. Similarly, variation of the
thymine derivative (28) with 6-azathymine (40) did not affect
activity. At a concentration of 10 µg/mL, compound 28 (45%
inhibition) bearing a 5-methyl group had DHBV inhibition
similar to that of the 5-bromo (24) compound (48% inhibition).
Substitution at the 5-position of the uracil base by a longer
saturated unsubstituted alkyl group (C2H5) or substituted alkyls
(CH2OH, CH2CH2OH) reduced the inhibitory activity in contrast
to 5-methyl (28), 5-bromo (24), or 5-chloro (25) analogues.
Introduction of a double bond at the 5-position of the base
increased the inhibitory activity as in compound 32. These
results are in agreement with our previous observations where
5-substituents possessing a double bond provided the most
potent anti-HBV activity.41,52 Similar conclusions were also
made by Reimer et al.53 and Tao et al.10 who showed that
substitution at the 5-position of a pyrimidine nucleoside by an
alkyl group containing a double bond increased the inhibitory
activity of their triphosphate derivatives against HBV DNA
polymerase.
The differences in biological activities for acyclic analogues
of pyrimidine nucleosides are likely related to the nature of the
substituents at the 5-position of the pyrimidine ring because
the conformations with respect to the glycosidic linkage, the
1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] chain, are simi-
lar in our compounds 22-41.
Comparison of anti-DHBV activity of 5-bromo (24) and
5-chloro (25) derivatives with related acyclic analogues 1-[(2-
hydroxyethoxy)methyl]-5-bromo(or chloro)uracil47 revealed that
chloro and bromo substituents at C-5 positions are important
determinants of potent anti-DHBV activity, where the chloro
analogue is more potent than the bromo analogue.47 However,
the activity of compounds 24 and 25 containing 1-[(2-hydroxy-
1-(hydroxymethyl)ethoxy)methyl] moiety was reduced as com-
pared to their 1-[(2-hydroxyethoxy)methyl] counterparts.47
Similar observations were also made with compounds containing
5-amino (27), 5-hydroxymethyl (30), 6-methyl (36), or 5-iodo-
6-methyl (38) substituents.
The in vitro cytotoxicities of compounds 22-41 were
assessed in host HepG2 cells as well as Vero cells. No toxicities
were observed for these analogues (CC50 ) >280 to >465 µM
(Table 1)).
The antiviral activity of compounds 22-41 was also exam-
ined against wild-type human hepatitis B virus in 2.2.15 cells.
Most of the 5-substituted-[(2-hydroxy-1-(hydroxymethyl)ethoxy)-
methyl]uracils active against DHBV retained anti-HBV activity
in 2.2.15 cells. The most active compounds 25, 32, and 37
exhibited EC50 values in the 20-40 µM range that compare
favorably to their activity against DHBV. However, it was
noteworthy that the anti-HBV activity of compounds 25, 32,
and 37 in 2.2.15 cells was found to be superior to the
corresponding acyclic purine nucleoside ganciclovir (20%
The fact that compounds 25, 32, and 37 show selective anti-
HBV activity and low toxicity to host cells suggests that they
have higher affinity for the virus-specific DNA polymerase than
the host cell enzymes. It is well known that HBV replicates by
a multistep mechanism involving the reverse transcription of a
pregenomic RNA intermediate, and known anti-HBV agents
such as lamivudine and adefovir may act as chain terminators.
The distinctive structure of nucleosides 25, 32, and 37 may
imply that they may not act as chain terminators and their mode
of action may be different from that of clinically used agents.

3698 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Kumar et al.
thickness). 5- and/or 6-substituted uracils (2-21) were purchased
from Sigma-Aldrich Chemical Co.
The precise mechanism of action of these compounds remains
unclear. However, by analogy with other antiviral nucleosides,
the potent anti-HBV activity of these compounds could likely
be attributed to their phosphorylation by cellular kinases
followed by selective inhibition of HBV DNA polymerase by
their triphosphate derivatives acting as substrates and/or inhibi-
tors of HBV DNA polymerase. There is no known HBV-
encoded enzyme such as thymidine kinase of the herpes-viruses
that could initiate the phosphorylation of anti-HBV nucleosides.
In the case of acyclic purine nucleoside, penciclovir, it has been
shown that in HBV infected or uninfected host cells, the
phosphorylations of penciclovir to its triphosphate form are
similar and brought about by cellular enzymes.57 However, the
precise cellular enzymes that may be responsible for phospho-
rylate acyclic nucleosides are not clear.
5-Chloro-1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-
uracil (25). A mixture of 5 (250 mg, 1.71 mmol), 0.74 g (1.8 mmol)
of 2-(bromomethoxy)-1,3-propanediyl-dibenzoate, 0.29 mL (1.79
mmol) of hexamethyldisilazane, 1.2 mL (11.05 mmol) of trimeth-
ylchlorosilane, and 1.5 g (4.44 mmol) of potassium nonaflate in
100 mL of dry acetonitrile was refluxed for 5 h at 85 °C. The
reaction was brought to room temperature. The solid material was
filtered off and washed with dichloromethane (20 mL). The mother
liquor and washings were combined and concentrated on rota-vacuo.
The residue obtained was redissolved in dichloromethane (300 mL)
and washed with water. The organic phase was dried over anhydrous
sodium sulfate, filtered, concentrated, and purified with silica gel
column chromatography to yield the intermediate 1-[(2-benzoyloxy-
(1-benzoyloxymethyl)ethoxy)methyl]uracil that was deprotected
using 40% aqueous methylamine (20 mL) at room temperature for
2 h. The product was purified by silica gel column chromatography
using MeOH:CHCl3 (1.2:8.8, v/v) as eluent to yield 25 as a solid
Summary
1
(200 mg, 46.72%): mp 142-145 °C. H NMR (DMSO-d6): δ
In conclusion, our work demonstrates anti-HBV properties
of a new subclass of acyclic pyrimidine nucleoside analogues
supporting our strategy for the identification of new anti-HBV
agents based on modifications in the sugar moiety of pyrimidine
nucleosides. The results obtained here are consistent with our
previous observations that 5-substituted acyclic pyrimidine
nucleosides are endowed with selective inhibitory activity in
vitro not only against wild-type but also for resistant mutant
HBV. In this study, compounds possessing 5-chloro, 5-(2-
bromovinyl), and 5-bromo-6-methyl substituents on the hetero-
cyclic ring were found to be good inhibitors of HBV replication
against DHBV, human HBV, and lamivudine-resistant HBV
with a single mutation. The most active compounds exhibit only
10 times lower in vitro anti-HBV activity in 2.2.15 cells than
lamivudine, but activity similar to that of the corresponding
acyclic guanine derivative ganciclovir against DHBV. Percent
inhibition of viral replication by compounds 25 and 37 for
resistant mutant HBV was close to that against wild-type HBV
in contrast to lamivudine where single and double mutants of
HBV were significantly less sensitive than wild-type HBV.
Further biochemical studies to elucidate the antiviral mechanism
of these acyclic pyrimidine nucleosides and synthesis of
additional derivatives are warranted.
The problems of HBV resistance and rebound of viral DNA
after drug removal underline the urgent need to design and
discover new antiviral strategies and agents. Short-term treat-
ment with lamivudine is usually insufficient to clear the virus.
It is possible that resistant mutants may also develop against
the investigated new class of compounds. However, new agents
are required to be used alone, sequentially, and/or in combination
with other drugs to bring more complete virus suppression and
prevent or delay drug-resistance problems. There are potential
benefits of combination therapy to suppress virus maximally
for a longer period and stop the emergence of lamivudine-
resistant HBV isolates in patients undergoing monotherapy.
3.40-3.50 (m, 4H, OCH2CH2O), 3.55 (m, 1H, CH), 4.62 (m, 2H,
OH), 5.20 (s, 2H, NCH2), 8.18 (s, 1H, H-6), 11.80 (s, 1H, NH).
5-Hydroxymethyl-1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)-
methyl]uracil (30). The reaction of 10 (200 mg, 1.41 mmol) with
2-(bromomethoxy)-1,3-propanediyldibenzoate (0.610 g, 1.55 mmol),
hexamethyldisilazane (0.23 mL, 1.43 mmol), trimethylchlorosilane
(0.95 mL, 8.74 mmol), and potassium nonaflate (1.24 g, 3.66 mmol)
in 100 mL of dry acetonitrile at 80 °C for 7 h was followed by
debenzolyation using 50 mL of 40% aqueous methylamine at room
temperature, using the procedure outlined for the preparation of
22. Purification of the product by silica gel column chromatography
using MeOH:EtoAc (1.5:8.5, v/v) as the eluent yielded 30 as a syrup
(80 mg, 23.1%). 1H NMR (DMSO-d6): δ 3.41-3.58 (m, 5H, OCH2-
CH2OCH), 4.15 (s, 2H, CH2OH), 4.60-5.0 (m, 3H, OH), 5.20 (s,
2H, NCH2), 7.58 (br, 1H, H-6), 11.65 (bs, 1H, NH).
5-(2-Hydroxyethyl)-1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)-
methyl]uracil (31). The reaction of 11 (200 mg, 1.28 mmol) with
2-(bromomethoxy)-1,3-propanediyldibenzoate (0.553 g, 1.41 mmol),
hexamethyldisilazane (0.26 mL, 1.61 mmol), trimethylchlorosilane
(0.96 mL, 8.84 mmol), and potassium nonaflate (1.12 g, 3.31 mmol)
in 100 mL of dry acetonitrile at 80 °C for 10 h was followed by
debenzolyation using 45 mL of 40% aqueous methylamine at room
temperature, using the procedure outlined for the preparation of
22. Purification of the product by silica gel column chromatography
using MeOH:EtOAc (1.5:8.5, v/v) as the eluent yielded 31 as a
1
solid (190 mg, 57.02%): mp 115-118 °C. H NMR (DMSO-d6):
δ 2.36 (m, 2H, CH2), 3.35-3.60 (m, 7H, OCH2CH2O, CH, CH2-
OH), 4.55-4.62 (m, 3H, OH), 5.18 (s, 2H, NCH2), 7.52 (s, 1H,
H-6), 11.25 (s, 1H, NH).
1-[(2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl]-6-methyl-
uracil (36). The reaction of 16 (200 mg, 1.59 mmol) with
2-(bromomethoxy)-1,3-propanediyldibenzoate (0.69 g, 1.76 mmol),
hexamethyldisilazane (0.26 mL, 1.61 mmol), trimethylchlorosilane
(1.1 mL, 10.13 mmol), and potassium nonaflate (1.39 g, 4.11 mmol)
in 100 mL of dry acetonitrile at 80 °C for 10 h was followed by
debenzolyation using 30 mL of 40% aqueous methylamine at room
temperature, using the procedure outlined for the preparation of
22. Purification of the product by silica gel column chromatography
using MeOH:EtOAc (1:9, v/v) as the eluent yielded 36 as a white
solid (140 mg, 38.35%): mp 140-142 °C. UV λmax (H2O): 206
nm shoulder (ꢀ ) 10 200); (0.01 N NaOH) 207 nm (ꢀ ) 16 600).
1H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3), 3.30-3.58 (m, 5H,
OCH2CH2O, CH), 4.60 (m, 2H, OH), 5.32 (s, 2H, NCH2), 5.46 (s,
1H, H-5), 11.15 (br s, 1H, NH).
5-Bromo-6-methyl-1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)-
methyl]uracil (37). The reaction of 17 (200 mg, 0.98 mmol) with
2-(bromomethoxy)-1,3-propanediyldibenzoate (0.57 g, 1.46 mmol),
hexamethyldisilazane (0.2 mL, 1.24 mmol), trimethylchlorosilane
(0.73 mL, 6.72 mmol), and potassium nonaflate (0.86 g, 2.53 mmol)
in 100 mL of dry acetonitrile at 80 °C for 9 h was followed by
debenzolyation using 40 mL of 40% aqueous methylamine at room
Experimental Section
Melting points were determined with a Buchi capillary apparatus
and are uncorrected. 1H NMR spectra were determined for solutions
in DMSO-d6 or CD3OD on a Bruker AM 300 spectrometer using
TMS as an internal standard. The assignment of all exchangeable
protons (OH, NH) was confirmed by the addition of D2O. UV
spectra were recorded with a Hewlett-Packard 8453 spectropho-
tometer. Microanalyses 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

Effect of Various Pyrimidines on HBV Replication
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3699
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temperature, using the procedure outlined for the preparation of
22. Purification of the product by silica gel column chromatography
using MeOH:EtOAc (0.8:9.2, v/v) as the eluent yielded 37 as a
white solid (80 mg, 26.54%): mp 145-147 °C. UV λmax (H2O):
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N. Engl. J. Med. 1990, 323, 295-301.
1
209 nm (ꢀ ) 7800); (0.01 N NaOH) 213 nm (ꢀ ) 10 700). H
NMR (DMSO-d6): δ 2.58 (s, 3H, CH3), 3.30-3.46 (m, 4H, OCH2-
CH2O), 3.56 (m, 1H, CH), 4.38 (m, 1H, OH), 4.60 (m, 1H, OH),
5.42 (s, 2H, NCH2), 11.75 (s, 1H, NH).
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1-[(2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-iodo-6-
methyluracil (38). The reaction of 18 (200 mg, 0.79 mmol) with
2-(bromomethoxy)-1,3-propanediyldibenzoate (0.47 g, 1.2 mmol),
hexamethyldisilazane (0.16 mL, 1.0 mmol), trimethylchlorosilane
(0.53 mL, 4.88 mmol), and potassium nonaflate (0.7 g, 2.07 mmol)
in 100 mL of dry acetonitrile at 80 °C for 9 h was followed by
debenzolyation using 35 mL of 40% aqueous methylamine at room
temperature, using the procedure outlined for the preparation of
22. Purification of the product by silica gel column chromatography
using MeOH:EtOAc (1.5:8.5, v/v) as the eluent yielded 38 as a
solid (80 mg, 28.31%): mp 142-143 °C. UV λmax (H2O): 215
1
nm (ꢀ ) 8500); (0.01 N NaOH) 214 nm (ꢀ ) 14 800). H NMR
(DMSO-d6): δ 2.68 (s, 3H, CH3), 3.25-3.42 (m, 4H, OCH2CH2O),
3.50 (m, 1H, CH), 4.38 (m, 1H, OH), 4.60 (m, 1H, OH), 5.40 (s,
2H, NCH2), 11.60 (s, 1H, NH).
Biological Evaluations. In Vitro Antiviral Assay (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.45-49 The
details of the assay are provided in the Supporting Information.
In Vitro Antiviral Assay (Human Hepatitis B Virus, 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.47,49,58 The details of the
assay are provided in the Supporting Information.
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 from liquid
nitrogen frozen stocks.50 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
identical to those as described previously for 2.2.15 cells.47,49,50,58
The details of the assay are provided in the Supporting Information.
Cell Cytotoxicity (MTT Assay). Cytotoxicities of test com-
pounds on human hepatoblastoma cell line (HepG2) and Vero cells
were determined using neutral red uptake and MTT assays,
respectively, as described earlier.45-47 The details of the assay are
provided in the Supporting Information.
Acknowledgment. We are grateful to the Canadian Institutes
of Health Research (CIHR) for an operating grant (MOP-36393)
for the financial support of this research. B.A. is thankful to
the Alberta Heritage Foundation for Medical Research (AH-
FMR) 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
Nile virus, respiratory syncytial virus, and SARS CoV through
NIAID contract to Dr. R. W. Sidwell.
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I/II Study of Adefovir Dipivoxil in Patients with Chronic Hepatitis
B Infection. Hepatology 1996, 24 (Suppl. 1), 281A.
(18) Malik, A. H.; Lee, W. M. Chronic Hepatitis B Virus Infection:
Treatment Strategies for the Next Millennium. Ann. Intern. Med.
2000, 132, 723-731.
(19) Hepsera Full Prescribing Information. Gilead Sciences. 09/2002,
1-18.
(20) Zoulim, F. Entecavir: A New Treatment Option for Chronic Hepatitis
B. J. Clin. Virol., in press.
(21) De Clercq, E. Perspectives for the Treatment of Hepatitis B Virus
Infections. Int. J. Antimicrob. Agents 1999, 12, 81-95.
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Innaimo, S.; Colonno, R. J.; Standring, D. N.; Clark, J. M. Efficacy
of the Carbocyclic 2′-Deoxyguanosine Nucleoside BMS-200475 in
the Woodchuck Model of Hepatitis B Virus Infection. Antimicrob.
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R. J. Efficacies of Entecavir Against Lamivudine-resistant Hepatitis
B Virus Replication and Recombinant Polymerases In Vitro. Anti-
microb. Agents Chemother. 2002, 46, 2525-2532.
Supporting Information Available: The details of the synthesis
and in vitro antiviral assays against duck hepatitis B virus, human
hepatitis B virus, and lamivudine-resistant human hepatitis B virus;
microanalytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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