Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B virus

Article abstract of DOI:10.1021/jm010410d

A novel class of 5-substituted acyclic pyrimidine nucleosides, 1-[(2-hydroxyethoxy)methyl]-5-(1-azidovinyl)uracil (9a), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-(1-azidovinyl)uracil (9b), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)

Full text of DOI:10.1021/jm010410d

2032
J . Med. Chem. 2002, 45, 2032-2040
Design a n d Syn th esis of Novel 5-Su bstitu ted Acyclic P yr im id in e Nu cleosid es a s
P oten t a n d Selective In h ibitor s of Hep a titis B Vir u s
Rakesh Kumar,* Mahendra Nath, and D. Lorne J . Tyrrell
Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alberta,
Edmonton, Canada T6G 2H7
Received August 30, 2001
A novel class of 5-substituted acyclic pyrimidine nucleosides, 1-[(2-hydroxyethoxy)methyl]-5-
(1-azidovinyl)uracil (9a ), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-(1-azidovinyl)uracil
(9b), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)uracil (9c), were synthesized
by regiospecific addition of bromine azide to the 5-vinyl substituent of the respective
5-vinyluracils (2a -c) followed by treatment of the obtained 5-(1-azido-2-bromoethyl) compounds
(3a -c) with t-BuOK, to affect the base-catalyzed elimination of HBr. Thermal decomposition
of 9b and 9c at 110 °C in dioxane yielded corresponding 5-[2-(1-azirinyl)]uracil analogues
(10b,c). The 5-(1-azidovinyl)uracil derivatives 9a -c were found to exhibit potent and selective
in vitro anti-HBV activity against duck hepatitis B virus (DHBV) infected primary duck
hepatocytes at low concentrations (EC50 ) 0.01-0.1 µg/mL range). The most active anti-DHBV
agent (9c), possessing a [4-hydroxy-3-(hydroxymethyl)-1-butyl] substituent at N-1, exhibited
an activity (EC50 of 0.01-0.05 µg/ mL) comparable to that of reference compound (-)-â-L-2′,3′-
dideoxy-3′-thiacytidine (3-TC) (EC50 ) 0.01-0.05 µg/mL). In contrast, related 5-[2-(1-azirinyl)]-
uracil analogues (10b,c) were devoid of anti-DHBV activity, indicating that an acyclic side
chain at C-5 position of the pyrimidine ring is essential for anti-HBV activity. The pyrimidine
nucleosides (9a -c, 10b,c) exhibited no cytotoxic activity against a panel of 60 human cancer
cell lines. All of the compounds investigated did not show any detectable toxicity to several
stationary and proliferating host cell lines or to mitogen stimulated proliferating human T
lymphocytes, up to the highest concentration tested.
In tr od u ction
cellular carcinoma. Antiviral therapy can be aimed to
clear the virus load in body fluids of the infected people
and therefore reduce the risk of viral transmission to
others.
Hepatitis B virus (HBV) is a causative agent of acute
and chronic hepatitis. Hepatitis B virus infection is the
world’s ninth leading cause of death.1-3 There are
approximately 400 million people with chronic HBV
infection.4 Chronic HBV infection leads to liver damage,
cirrhosis, and hepatocellular carcinoma.4,5 HBV is prob-
ably the major cause of liver cancer worldwide. Accord-
ing to the World Health Organization report, each day
∼1500 people die of hepatocellular carcinoma and
∼3000 people die of liver cirrhosis, both of which are
secondary to HBV infections. HBV infections are in-
creasingly serious problems among homo- and hetero-
sexual populations, intravenous drug users, organ trans-
plant recipients, patients on renal dialysis, and cancer
patients receiving chemotherapy.6 HBV infections are
present in people in all parts of the world. HBV
infections are also common among human immuno-
deficiency virus (HIV) infected individuals because it
has a similar mode of transmission.
The major therapeutic option for HBV carriers is
R-interferon. However, use of R-interferon is limited,
success rate is low, and serious side effects are ob-
served.8,9 Nucleoside analogues have gained increasing
importance as antiviral agents. The mechanism of
action of nucleoside analogues is suggested to be through
the interaction of their triphosphate derivatives, formed
after cellular metabolic transformation, with HBV DNA
polymerase or reverse transcriptase as substrates and/
or inhibitors. HBV is an incomplete double-stranded
DNA virus. Its DNA replication is unique and includes
a reverse transcriptase catalyzed reverse transcription
step.10 The fact that HBV DNA polymerase is quite
different than human DNA polymerases suggests that
compounds that selectively inhibit HBV replication can
be identified.11
Lamivudine [(-)-â-L-2′,3′-dideoxy-3′-thiacytidine, 3-TC,
1a , Chart 1], a pyrimidine nucleoside analogue, has
been approved for the treatment of HBV infection. The
administration of lamivudine induces significant drop
in the amount of virus in the serum of most HBV
carriers and causes no significant side effects in most
patients. Unfortunately, emergence of drug resistant
HBV may begin after 6-12 months of therapy.12 In
addition, long-term remission after completion of treat-
ment with lamivudine is not commonly observed, and
most patients experience a rebound in viremia once the
New infection with HBV can be prevented by vac-
cination. However, the present vaccination is not effec-
tive for ∼400 million chronic carriers worldwide. An
HBV infected mother can pass the infection to her
nonvaccinated infant at the time of birth.7 It has
been observed that suppression of the replication of
HBV in the liver leads to improved liver pathology and
decreased progression to liver cirrhosis and hepato-
* To whom correspondence should be addressed. Tel: (780) 492-
7545. Fax: (780) 492-7521. E-mail: rakesh.kumar@ualberta.ca.
10.1021/jm010410d CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/09/2002

5-Substituted Acyclic Pyrimidine Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2033
Ch a r t 1
affinity for the cellular DNA polymerase and is a potent
inhibitor of HBV DNA polymerase in vitro.23,24 Thus,
acyclic moieties contribute significantly to potent anti-
HBV activity and selectivity. Despite excellent anti-HBV
activity, all of these purine analogues (ACV, GCV, PCV)
are less water-soluble and are poorly absorbed when
given orally to rodents and humans.25 In contrast,
homologous acyclic derivatives of pyrimidine nucleosides
were found to have excellent water solubility, which
could increase their bioavailabilty as well as facilitate
formulation.26 To increase oral bioavailability, famci-
clovir, 9-[4-acetoxy-3-(acetoxymethyl)-1-butyl]-guanine,
a prodrug of penciclovir has been investigated. Unfor-
tunately, in a placebo-controlled phase III trial, famci-
clovir did not exhibit sufficient efficacy and has been
stopped from further development.27
It has been shown that chronic infection of hepato-
cytes is maintained by the presence of 30-40 copies of
viral covalently closed circular (ccc) DNA in the hepa-
tocyte nucleus. Viral cccDNA is the template for virus
transcription and is dependent upon viral DNA synthe-
sis.28 Therefore, it can be envisioned that continuous and
complete suppression of HBV DNA replication may
deplete cccDNA. It has been shown in in vitro culture
that the combination of 3-TC and PCV act synergisti-
cally as anti-HBV agents and can substantially reduce
cccDNA.29 However, 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.30 Continuous long
term therapy with an array of potent nontoxic individual
anti-HBV agents or combination of anti-HBV agents
may lead to continuous inhibition of viral DNA synthe-
sis resulting eventually in the depletion of the cccDNA
pool. In addition, continuous suppression of viral DNA
synthesis may lead to restoration of T cell immune
responses,31 which in turn could lead to elimination of
HBV infected cells from chronically infected patients.
Thus, there is a tremendous clinical need to investigate
novel classes of antiviral agents for the chemotherapy
of HBV infections.
use of drug is stopped.13,14 The rebound in viremia has
been suggested to be due to either unaffected virus
infected cells against which nucleoside was ineffective15
and/or intranuclear covalently closed circular DNA
(CCC DNA) species whose replicative intermediates are
nonresponsive to lamivudine.16,17
A promising class of nucleoside analogues for antiviral
chemotherapy belongs to a group with open chain
“acyclic” sugar moieties.18 The effectiveness of acyclic
nucleoside analogues as a substrate or inhibitor of viral
enzymes is likely dependent on the ability of the acyclic
side chain to mimic the interaction of the glycosyl
portion of the natural substrate with the enzyme. The
flexibility of the acyclic chain may allow it to adopt a
conformation favorable for enzyme interaction as sub-
strates or inhibitors.18
Among acyclic purine nucleosides, Acyclovir (ACV,
1b), 9-[(2-hydroxyethoxy)methyl]guanine has been shown
to be a modest inhibitor of HBV DNA polymerase in
triphosphate form, exhibited both in vitro and in vivo
activity in animal models, but demonstrated disappoint-
ing anti-HBV efficacy in human clinical trials.19 Gan-
ciclovir (GCV, 1c), 9-[(2-hydroxy-1-(hydroxymethyl)-
ethoxy)methyl]guanine, structurally similar to ACV but
differs in having the functional equivalent of a 3′-OH
group, is a potent inhibitor of HBV, both in cell culture
and in vivo in animal models.20,21 However, long-term
clinical use of GCV is limited due to its severe dose-
related toxicity.22 Penciclovir (PCV, 1d ), 9-[4-hydroxy-
3-(hydroxymethyl)-1-butyl]guanine, is an acyclic nucleo-
side closely related to GCV with the exception that the
ether oxygen in the acyclic side chain is replaced with
a methylene bridge. PCV has shown anti-HBV activity
in vitro and in vivo.23 The basis of potent and selective
anti-HBV activity of PCV is at the HBV DNA poly-
merase level. The triphosphate derivative of PCV has
a very high affinity for HBV DNA polymerase and a low
Among many pyrimidine nucleosides that have been
studied, C-5 alkyl substituted pyrimidine nucleosides
with 2′-fluoro substituted arabinosyl analogues, FMAU
(1e) [1-(2′-deoxy-2′-fluoro-â-D-arabinofuranosyl)-5-meth-
yluracil] and FEAU (1f) [1-(2′-deoxy-2′-fluoro-â-D-ara-
binofuranosyl)-5-ethyluracil], have been found to exhibit
potent in vitro and in vivo anti-HBV activity.32 The
triphosphate derivatives of these analogues inhibited
viral DNA polymerase of HBV in vitro.33 However, all
of these have been shown to be toxic in vivo.32 In a study
of 5′-triphosphates of 2′-deoxyuridine and 2′-arabino-
uridine analogues of 5-substituted pyrimidine nucleo-
sides, 5-propenyl derivatives (1h ,i) were the most potent
inhibitors of DHBV DNA polymerase with high selectiv-
ity relative to cellular DNA polymerase-R.34 In a group
of 5-substituted 3′-fluoro-2′,3′-dideoxyuridine analogues
in the triphosphate form, structure-activity correlation
studies indicated that substitution at the C-5 position
of the pyrimidine by an alkyl group including a double
bond (1i) increases the inhibitory activity against hu-
man HBV DNA polymerase.35
Therefore, several strategies based on nucleoside ana-
logues have evolved for the treatment of HBV infection

2034 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Kumar et al.
Sch em e 1a
a
Reagents: (i) N-bromosuccinimide, sodium azide, DME, 0 °C; (ii) t-BuOK, THF, 0 °C.
which include (1) substitution of a C-atom in the sugar
ring by a heteroatom,12 (2) â-L-stereochemistry,11 (3)
acyclic purine nucleosides and their derivatives,19-23 and
(4) 5-substituted pyrimidine nucleosides.32 However,
5-substituted acyclic pyrimidine nucleosides have not
been studied for anti-HBV activity.16,17,36,37 Recently, we
reported the synthesis of 5-(1-azidoethyl) analogues (1j-
l) containing acyclic moieties at the N-1 position. The
acyclic pyrimidine nucleosides 1k ,l exhibited appre-
ciable in vitro anti-DHBV activity (EC50 ) 0.1-5 µg/
mL), whereas related homologues (1j) were inactive or
moderately active (EC50 ) >10 µg/mL).38 To expand the
structure-activity relationship and to improve anti-
HBV activity, we now report the synthesis, anti-DHBV
activity, and toxicity of a novel class of 5-substituted
acyclic pyrimidine nucleosides (9a -c, 10b,c). The 5-(1-
azidovinyl) analogues (9a -c) can be considered to be
hybrids of 5-(1-azidoethyl) (1j-l) and 5-vinyl substituent
(1i). 1-[(2-Hydroxyethoxy)methyl]-5-(1-azidovinyl)uracil
(9a ), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-
(1-azidovinyl)uracil (9b), and 1-[4-hydroxy-3-(hydroxy-
methyl)-1-butyl]-5-(1-azidovinyl)uracil (9c) were found
to exhibit highly potent and selective anti-DHBV activ-
ity in vitro. In contrast, related analogues of 9b,c in
which the 5-substituent had undergone intramolecular
cyclization to provide 5-[2-(1-azirinyl) analogues of 1-[(2-
hydroxy-1-(hydroxymethyl)ethoxy]methyl)]uracil (10b)
and 1-(4-hydroxy-3-(hydroxy methyl butyl)uracil (10c),
respectively, were devoid of anti-DHBV activity.
addition of bromine azide across the 5-vinyl substituent
of 2a . For example, the bromine atom in 3a is attached
to a methylene carbon that exhibited resonance at δ
34.0, whereas the azido substituent is attached to a
chiral methine carbon that exhibited resonance at δ
58.2. This regiospecific addition is consistent with
reports that unsymmetrical olefins, capable of halonium
ion formation, were found to favor an unsymmetrical
bridged intermediate of the type illustrated in Scheme
1, even in solvents having a high dipole moment.39
Reaction of 1-[(2-benzoyloxyethoxy)methyl]-5-(1-azido-
2-bromoethyl)uracil (3a ) with t-BuOK in THF yielded
the 5-(1-azidovinyl) analogue 4a (38.7%) and bicyclic
compound 5a (9.8%). A similar reaction employing 1-[(2-
benzoyloxy-1-(benzoyloxymethyl)ethoxy)methyl]-5-(1-
azido-2-bromoethyl)uracil (3b) yielded one major com-
pound, 5-(1-azidovinyl) analogue 4b (66%). In contrast,
reaction of 1-(4-benzoyloxy-3-benzoyloxymethyl-1-butyl)-
5-(1-azido-2-bromoethyl)uracil (3c) with t-BuOK in THF
gave rise to three products. Thus, the direct elimination
of HBr yielded 1-(4-benzoyloxy-3-benzoyloxymethyl-1-
butyl)-5-(1-azidovinyl)uracil (4c, 51.6%, path A), whereas
intramolecular cyclization reactions gave rise to the
bicyclic products 5-(4-benzoyloxy-3-benzoyloxymethyl-
1-butyl)furano-[2,3-d] pyrimidin-6-(5H)-one (5c, 15%,
path B) and 3-azido-2,3-dihydro-5-(4-benzoyloxy-3-ben-
zoyloxymethyl-1-butyl)furano-[2,3-d] pyrimidin-6-(5H)-
one (6, 1.1%, path C), respectively.
The base-catalyzed intramolecular cyclization reac-
tions of 3c to 6, as illustrated in Scheme 2, is analogous
to the reported conversion of 5-[2-[(methylsulfonyl)oxy]-
ethyl]uracil to 2,3-dihydrofurano[2,3-d]pyrimidin-6(5H)-
one using t-BuOK in DMSO.40 The mechanism respon-
sible for the formation of the bicyclic compound 5c
proceeds via an elimination reaction involving expulsion
of HN3 from 6 as shown in Scheme 2, since treatment
of 6 with a saturated solution of ammonia in methanol
gave the bicyclic derivative 7, rather than the azido
compound 8 (Scheme 2). Deprotection of 4a -c with a
saturated solution of NH3 in MeOH yielded target 5-(1-
Ch em istr y
The regiospecific addition of bromine azide to the
5-vinyl substituent of the uracil derivatives (2a -c)
yielded 1-[(2-benzoyloxyethoxy)methyl]-5-(1-azido-2-bro-
moethyl)uracil (3a , 45.5%), 1-[(2-benzoyloxy-1-(benzoyl-
oxymethyl)ethoxy]methyl)]-5-(1-azido-2-bromoethyl)ura-
cil (3b, 84.8%), and 1-(4-benzoyloxy-3-benzoyloxymeth-
yl-but-1-yl)-5-(1-azido-2-bromoethyl)uracil (3c, 81.5%),
respectively. The 13C NMR (J modulation) spectrum of
3a provided conclusive evidence for the regiospecific

5-Substituted Acyclic Pyrimidine Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2035
Sch em e 2a
a
Reagents: (iii) NH3, MeOH, 25 °C.
Sch em e 3a
Resu lts a n d Discu ssion
The anti-DHBV activity for the new class of acyclic
pyrimidine nucleosides, 5-(1-azidovinyl)-(9a -c) and re-
lated 5-[2-(1-azirinyl)-(10b,c) analogues, along with the
reference antiviral drug 3-TC was assessed in confluent
cultures of primary duck hepatocytes obtained from
chronically infected Pekin ducks. These cells chronically
produce HBV DNA, and therefore antiviral activity was
determined by analysis of viral DNA, using dot blot
hybridization. Duck hepatitis B virus (DHBV), a mem-
ber of hepadnaviridae, shares properties of hepatotro-
pism, virion structure, genome organization, replication,
and epidemiology with human HBV.42 DHBV-based in
vitro and in vivo systems have been used extensively
to screen drugs for potential anti-HBV activity.43 It has
been shown that compounds such as 3-TC and PCV,
both potent inhibitors of DHBV, are also potent inhibi-
tors of HBV in chimpanzees as well as humans.44 The
concentrations required to inhibit 50% of DHBV DNA
(EC50), 50% cytotoxic concentration (CC50) on stationary
cells, and 50% inhibitory concentration (IC50) on prolif-
erating cells, are shown in Table 1. Purine acyclic
nucleosides (GCV and PCV) are also included in Table
1 for comparison of their anti-DHBV potency.
Among the new class of acyclic pyrimidine nucleo-
sides, 5-(1-azidovinyl)-(9a -c) analogues exhibited po-
tent in vitro activity against DHBV (EC50 ) 0.01-0.1
µg/mL). The anti-DHBV activity exhibited by analogues
9a -c compares favorably to that of reference drug 3-TC
(EC50 ) 0.01-0.05 µg/mL). Surprisingly, compounds
9a -c were found to be significantly more potent inhibi-
tors of DHBV replication compared to the corresponding
potent acyclic purine nucleosides (1b-d ), as measured
previously in the DHBV infected primary duck hepato-
cyte cultures.45 The 5-(1-azidovinyl)uracil (9b) with 1-[(2-
hydroxy-1-(hydroxymethyl)ethoxy)methyl] substituents
at N-1 possessed higher activity (EC50 ) 0.01-0.1 µg/
mL) than GCV (EC50 ) 1.5 µg/mL). Similarly, 5-(1-azido-
vinyl)uracil (9c) containing 1-[4-hydroxy-3-(hydroxy-
methyl)-1-butyl] moiety demonstrated superior activity
(EC50 ) 0.01-0.05 µg/mL) than PCV (EC50 ) 0.3 µg/
mL). This study reveals an unexpected difference be-
a
Reagents: (iii) NH3, MeOH, 25 °C; (iv) dioxane, 110 °C.
Sch em e 4a
a
Reagents: (v) NaOH, or t-BuOK, DMSO or DME, 25 °C.
azidovinyl) analogues of 1-[(2-hydroxyethoxy)methyl]-
uracil (9a , 60.8%), 1-[(2-hydroxy-1-(hydroxymethyl)-
ethoxy]methyl)]uracil (9b, 58.4%), and 1-[4-hydroxy-3-
(hydroxymethyl)-1-butyl]uracil (9c, 79%) (Scheme 3).
Thermal decomposition, using a procedure similar to
that reported for the conversion of R-azidostyrene to
phenylazirine,41 of the 5-(1-azidovinyl) compounds 9b
and 9c in dry dioxane at 110 °C yielded the correspond-
ing 5-[2-(1-azirinyl)] analogues 10b and 10c in 35.7 and
26% yield, respectively (Scheme 3). In contrast, the
above reaction employing 9a produced the desired
product (10a ) as indicated by the thin-layer chroma-
tography, but could not be eluted out from the silica gel
column due to decomposition.
The synthesis of target compound 1-[4-hydroxy-3-
(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)uracil (9c) was
also attempted by the reaction of unprotected 1-[4-
hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-bromo-
ethyl)uracil (11) with either t-BuOK or NaOH using
DMSO or DME as solvent, which produced bicyclic com-
pound 7 (56%) and a low yield of 9c (11%) (Scheme 4).

2036 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Kumar et al.
Ta ble 1. In Vitro Antiviral Activity against Hepatitis B Virus in Primary Duck Hepatocyte Cultures (DHBV) and Toxicity in
Stationary and Proliferating Cells for 5-Substituted Uracils (9a -c) and Reference Compounds
% inhibition at 10 µg/mLa
[EC50 (µg/mL)]b
toxicity
CC50 (µg/mL)
cell proliferation
IC50 (µg/mL)
no.
9a
X
R
DHBV primary duck hepatocytes
HFFc
Veroe
HFF f
Daudig
human T cellsh
O
O
C
-
H
84 [0.01-0.1]
86 [0.01-0.1]
93 [0.01-0.05]
96 [0.01-0.05]
[1.5]
>100d
>100
>100
ND
>100
>200
>200
>100
>100
100
>100
ND
>50
>50
>50
ND
>50
>50
>50
>50
9b
9c
CH2OH
CH2OH
-
3-TCi
GCVj,k
PCVj,l
[0.3]
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
b
infected controls. The drug concentration (µg/mL) required to reduce the viral DNA in infected cells to 50% of untreated infected controls.
c The drug concentration (µg/mL) required to reduce the uptake of neutral red stain by uninfected cell monolayers to 50% of untreated
d
uninfected human foreskin fibroblast (HFF) cell controls after 7 days. “>” 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 The drug concentration (µg/mL) required to reduce the proliferation of HFF cells to 50% of
g
untreated controls. The drug concentration (µg/mL) required to reduce the proliferation of Daudi cells to 50% of untreated controls.
h
The drug concentration (µg/mL) required to reduce the proliferation of PHA stimulated human peripheral blood T lymphocytes to 50%
of untreated PHA stimulated controls. (-)-â-L-2′,3′-dideoxy-3′-thiacytidine. Data taken from Shaw et al.45 Ganciclovir. Penciclovir.
i
j
k
l
tween novel N-alkoxy derivatives of pyrimidine and
purine in their ability to inhibit DHBV replication and
emphasizes the influential effect of the novel 5-substi-
tuted pyrimidine base.
The observation that 1-[4-hydroxy-3-(hydroxymethyl)-
1-butyl]-5-(1-azidovinyl)uracil (9c) showed better activ-
ity than 1-[(2-hydroxyethoxy)methyl]-5-(1-azidovinyl)-
uracil (9a ) and 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)-
methyl]-5-(1-azidovinyl)uracil (9b) is consistent with
previous observations reported in the case of purine
acyclic nucleosides that 9-[4-hydroxy-3-(hydroxymethyl)-
1-butyl]guanine (PCV) exhibited higher anti-DHBV
activity than 9-[(2-hydroxyethoxy)methyl]guanine (ACV)
and 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-
guanine (GCV).45 It has been suggested that greater
antiviral potency of PCV relative to that of GCV is
probably partly due to the increased intracellular stabil-
ity of its nucleotides, as well as other factors such as
phosphorylation efficiency or recognition by the viral
polymerase.45
Interestingly, modification of the 5-(1-azidovinyl) sub-
stituent in the novel pyrimidine acyclic nucleosides
(9b,c) to the corresponding 5-[2-(1-azirinyl)] analogues
(10b,c) provided compounds that were devoid of any
anti-DHBV activity in the in vitro assay at 10 µg/mL.
These results suggested that an acyclic substituent at
the C-5 position of the pyrimidine ring is essential for
anti-HBV activity.
Comparison of anti-DHBV activity of 9a -c with their
related 5-(1-azidoethyl) analogues (1j-l) revealed that
anti-DHBV activity of 1j (EC50 ) >10 µg/mL)38 was
remarkably improved by alteration of the 5-(1-azido-
ethyl) substituent to the 5-(1-azidovinyl) moiety (9a ,
EC50 ) 0.01-0.1 µg/mL). Similar observations were also
obtained with novel compounds 9b (EC50 ) 0.01-0.1
µg/mL) and 9c (EC50 ) 0.01-0.05 µg/mL), when com-
pared with their respective 5-(1-azidoethyl) analogues
(1k , EC50 ) 1-10 µg/mL; 1l, EC50 ) 0.1-10 µg/mL).38
These results suggest that the 1-azidovinyl moiety at
the 5-position of the pyrimidine ring of 9a -c is an
important determinant for potent anti-HBV activity.
Compounds 9a -c inhibited DHBV replication at con-
centrations that were significantly lower than the
highest concentration range tested to determine the
toxicity to several host cells (Table 1). Up to a concen-
tration of 50 µg/mL, compounds 9a -c did not have
any visual effect on morphology and viability of duck
hepatocytes.
The precise mechanism of action of these compounds
is currently unknown. However, by analogy with other
antiviral nucleosides, the potent and selective anti-
DHBV activity of compounds 9a -c is likely due to their
phosphorylation by cellular kinases followed by selective
inhibition of HBV DNA synthesis by acting as substrate
and/or as inhibitor of HBV DNA polymerase by their
triphosphate derivatives.36,37 The derivative 9c, with
1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] moiety demon-
strated remarkable anti-DHBV activity with a 50%
effective concentration (EC50) of 0.01-0.05 µg/mL, while
compounds 9a and 9b with 1-[(2-hydroxyethoxy)methyl]
and 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]
moieties, respectively, were slightly less inhibitory to
DHBV replication (EC50 ) 0.01-0.1 µg/mL) (Table 1).
The cytotoxic activities of compounds 9a -c and 10b,c
were also determined by the National Cancer Institute
(NCI, USA) using an in vitro assay46 in which com-
pounds were evaluated against approximately 60 hu-
man tumor cell lines (e.g., melanoma, leukemia, non-
small cell lung, small cell lung, colon, central nervous
system, ovarian, prostate, and renal cancers). None of
the compounds showed significant activity or selectivity
in these assays at the highest concentration tested (30
µg/mL) (data not shown), indicating that novel com-
pounds 9a -c and 10b,c do not have cytotoxicity against
various human cell lines nor any anticancer activity.
The potent anti-HBV compounds 9a -c were tested
in vitro for their toxicity against several other cell lines
(Table 1). None of these compounds exhibited in vitro

5-Substituted Acyclic Pyrimidine Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2037
triethylamine (1.5 mL) (dried over calcium hydroxide) in dry
DMF (50 mL) was maintained at 70 °C with stirring until an
intense red color appeared. 1-[(2-Benzoyloxy-1-(benzoyloxy-
methyl)ethoxy)methyl]-5-iodouracil47 (1.0 g, 1.81 mmol) and
vinyl acetate (10 mL, 110 mmol) were then added, and the
reaction was allowed to proceed at 70 °C for 6 h with stirring.
The solvent was removed in vacuo, and the residue obtained
was purified by elution from a silica gel column starting with
chloroform and then changing to chloroform-methanol (98:2,
v/v). Removal of the solvent from the combined fractions
containing the desired products yielded 2b as a viscous oil (480
toxicity against stationary phase cells [Vero cells (CC50
> 100 or > 200 µg/mL), HFF cells (CC50 > 100 µg/mL)]
and proliferating cell lines [HFF (IC50 g 100 µg/mL),
Daudi (IC50 > 50 µg/mL)]. In rapidly proliferating fresh
human T lymphocyte cell culture, cellular DNA synthe-
sis, as monitored by the incorporation of [methyl-3H]-
thymidine into DNA, was also not affected by com-
pounds 9a -c in concentrations up to 50 µg/mL (highest
concentration tested).
1
mg, 59%). H NMR (CDCl3) δ: 4.4-4.65 (m, 5H, CH, OCH2),
Su m m a r y
5.20 (dd, J ) 11 Hz, J ) 2.1 Hz, 1H, -CHdCH H′), 5.38 (m,
2H, NCH2), 5.86 (dd, J ) 18.0 Hz, J ) 2.1 Hz, 1H, -CHd
CHH′), 6.28 (dd, J ) 18 Hz, J ) 11.0 Hz, 1H, -CHdCHH′),
7.30-8.05 (m, 11H, H-6, benzoyl hydrogens), 8.26 (s, 1H, NH).
Anal. (C24H22N2O7) C, H, N.
In conclusion, we present the synthesis and in vitro
biological evaluation of a novel class of hitherto un-
known 5-substituted acyclic pyrimidine nucleosides
(9a-c, 10b-c). Compounds possessing a 5-(1-azidovinyl)
substituent were found to be strong and selective
inhibitors of DHBV replication in vitro at concentrations
several orders of magnitude lower than their toxicity
thresholds in a variety of cell lines. The fact that
the compounds 9a -c show potent and selective anti-
DHBV activity suggests that they have higher affinity
for the virus specific DNA polymerase than the host
cell enzymes. Among the compounds investigated, 9c
emerged as the most potent and selective anti-DHBV
agent. The spectrum of anti-DHBV activity and cyto-
toxicity of compounds 9a -c in culture is similar or
better to that of established anti-HBV agents that are
currently used clinically for HBV infections. Thus,
compounds 9a -c have emerged as a new series of
compounds with potent anti-DHBV activity and merit
further studies to develop as potential chemotherapeutic
agents for anti-HBV therapy. Additional in vitro and
in vivo tests as well as studies of structure-activity
relationships and mechanism of action of this new
family of compounds are ongoing in our laboratories.
1-[4-Ben zoyloxy-3-(ben zoyloxym eth yl)-1-bu tyl]-5-vin yl-
u r a cil (2c). Benzoyl chloride (600 mg, 4.2 mmol) was added
to a solution of 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-
vinyluracil38 (450 mg, 1.87 mmol) in dry pyridine (50 mL), and
the reaction was allowed to proceed at 25 °C with stirring for
48 h. The reaction mixture was poured onto ice water (50 mL)
and extracted with dichloromethane (3 × 50 mL). The di-
chloromethane extract was dried over sodium sulfate, the
solvent was removed in vacuo, and the residue obtained was
purified by elution from a silica gel column using chloroform-
methanol (97:3, v/v) as eluent to yield 2c as a viscous oil (550
mg, 65.4%), which was used directly in the next reaction step.
1-[(2-Ben zoyloxyet h oxy)m et h yl]-5-(1-a zid o-2-b r om o-
eth yl)u r a cil (3a ). N-Bromosuccinimide (NBS, 350 mg, 1.96
mmol) was added in aliquots to a precooled (-5 °C) suspension
prepared by mixing a solution of 2a (61 mg, 1.95 mmol) in
1,2-dimethoxyethane (25 mL) with a solution of sodium azide
(510 mg, 7.84 mmol) in water (1.2 mL). The initial yellow color
faded away after each addition. When all the NBS was
consumed, the reaction mixture was allowed to stand at 0 °C
for 30 min, prior to pouring onto ice-water (25 mL) and
extraction with dichloromethane (3 × 50 mL). The dichloro-
methane extract was washed with cold water (25 mL) and
dried (Na2SO4), and the solvent was removed in vacuo to yield
a residue which was purified by elution from a silica gel
column using chloroform:methanol (98:2, v/v) as eluent to yield
Exp er im en ta l Section
Melting points were determined with a Buchi capillary
apparatus and are uncorrected. 1H NMR and 13C NMR spectra
were determined for solutions in CDCl3, DMSO-d6, or CD3OD
on a Bruker AM 300 spectrometer using Me4Si as an internal
standard (1H NMR). The assignment of all exchangeable
protons (OH, NH) was confirmed by the addition of the D2O.
13C NMR spectra were acquired using the J modulated spin-
echo technique where methyl and methine carbon resonances
appear as positive peaks, and methylene and quaternary
carbons appear as negative peaks. Microanalyses were within
(0.4% of theoretical values for all elements listed, unless
otherwise indicated. Preparative thin-layer chromatography
(PTLC) was performed using Whatman PLK 5F plates, 1.0 mm
in thickness, and silica gel column chromatography was
carried out using Merck 7734 silica gel (100-200 µM particle
size). Wa r n in g: Halogenated solvents such as dichloromethane
must not be used in certain reactions, such as those described
for the preparation of products 3a -c, since its reaction with
sodium azide may produce potentially explosive polyazido-
methane.
1-[(2-Ben zoyloxyeth oxy)m eth yl]-5-vin ylu r acil (2a). Ben-
zoyl chloride (410 mg, 2.9 mmol) was added to a solution of
1-[(2-hydroxyethoxy)methyl]-5-vinyluracil38 (614 mg, 2.89 mmol)
in dry pyridine (30 mL), and the reaction was allowed to
proceed at 25 °C with stirring for 6 h. Removal of the solvent
in vacuo and purification of the product by elution from a silica
gel column using chloroform-methanol (99:1, v/v) as eluent
gave 2a as a viscous oil (615 mg, 67.3%) which was used
directly in the next reaction step.
1
3a (388 mg, 45.5%) as a viscous oil. H NMR (CDCl3) δ: 3.58
(m, 1H, CHH′Br), 3.78 (m, 1H, CHH′Br), 3.97 (m, 2H, OCH2-
CH2OBz), 4.48 (m, 2H, OCH2CH2OBz), 4.83 (m, 1H, CHN3),
5.28 (s, 2H, NCH2), 7.42-8.06 (m, 6H, H-6, benzoyl hydrogens),
9.90 (s, 1H, NH, exchanges with deuterium oxide). 13C NMR
(CDCl3) δ: 34.0 (CH2Br), 58.2(CHN3), 63.3 (OCH2CH2), 68.0
(NCH2), 111.5 (C-5), 128.4, 129.6, 129.7, 133.2 (Ar-C), 141.8
(C-6), 150.6 (C-2 CdO), 162.0 (C-4 CdO), 166.3 (Ar CdO).
Anal. (C16H16N5O5Br) C, H, N.
1-[(2-Ben zoyloxy-1-(ben zoyloxym eth yl)eth oxy)m eth yl]-
5-(1-a zid o-2-br om oeth yl)u r a cil (3b). Reaction of 2b (30 mg,
0.066 mmol) with N-bromosuccinimide (13 mg, 0.073 mmol),
using the procedure described for the preparation of 3a , and
purification of the product by silica gel column chromatography
using dichloromethane-methanol (97:3, v/v) as eluent afforded
1
3b as a viscous oil (32 mg, 84.8%). H NMR (CDCl3) δ: 3.45
(m, 1H, CHH′Br), 3.65 (m, 1H, CHH′Br), 4.40-4.65 (complex
m, 6H, CH, OCH2, CHN3), 5.35 (m, 2H, NCH2), 7.38-8.04 (m,
11H, H-6, benzoyl hydrogens), 8.30 (s, 1H, NH). Anal.
(C24H22N5O7Br) C, H, N.
1-[4-Ben zoyloxy-3-(b en zoyloxym et h yl)-1-b u t yl]-5-(1-
a zid o-2-br om oeth yl)u r a cil (3c). A mixture of 2c (510 mg,
1.14 mmol), saturated solution of sodium azide in water (300
mg, 4.6 mmol), N-bromosuccinimide (223 mg, 1.25 mmol), and
1,2-dimethoxyethane (50 mL) was stirred at 0 °C for 30 min.
Removal of the solvent in vacuo gave a viscous oil, which was
purified by silica gel column chromatography. Elution with
chloroform-methanol (96:4, v/v) yielded 3c (530 mg, 81.5%)
1
1-[(2-Ben zoyloxy-1-(ben zoyloxym eth yl)eth oxy)m eth yl]-
5-vin ylu r a cil (2b). A mixture of palladium(II) acetate (26 mg,
0.11 mmol), triphenylphosphine (57 mg, 0.22 mmol), and
as a syrup. H NMR (CDCl3) δ: 1.92 (m, 2H, NCH2CH2), 2.36
(m, 1H, CH), 3.52-3.80 (m, 2H, NCH2), 4.0 (m, 2H, CH2Br),
4.48 (m, 4H, OCH2), 4.82 (m, 1H, CHN3), 7.36-8.0 (m, 11H,

2038 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Kumar et al.
H-6, benzoyl hydrogens), 9.38 (s, 1H, NH). Anal. (C25H24N5O6-
Br) C, H, N.
(m, 4H, OCH2CH2), 4.72 (m, 1H, OH), 5.03 [d, J ) 2 Hz, 1H,
C(N3)dCHH′], 5.18 (s, 2H, NCH2), 5.95 [d, J ) 2 Hz, 1H,
C(N3)dCHH′], 7.98 (s, 1H, H-6), 11.68 (br, 1H, NH). 13C NMR
(CD3OD) δ: 61.9 (OCH2CH2), 72.1 (OCH2CH2), 78.6 (NCH2),
101.8 [C(N3)dCH2], 109.6 (C-5), 138.6 [C(N3)], 144.0 (C-6),
152.0 (C-2 CdO), 163.2 (C-4 CdO). Anal. (C9H11N5O4) C, H, N.
1-[(2-Hyd r oxy-1-(h yd r oxym eth yl)eth oxy)m eth yl]-5-(1-
a zid ovin yl)u r a cil (9b). A solution of 4b (350 mg, 0.71 mmol)
in a saturated solution of ammonia in methanol (25 mL) was
stirred at 25 °C for 48 h. Removal of solvent in vacuo yielded
a residue which was purified by silica gel column chromatog-
raphy using chloroform-methanol (90:10, v/v) as eluent to
yield 9b (120 mg, 58.4%) as a solid: mp 129-130 °C. 1H NMR
(DMSO-d6) δ: 3.30-3.50 (m, 4H, OCH2), 3.55 (m, 1H, CH),
4.65 (m, 2H, OH), 5.02 [d, J ) 2 Hz, 1H, C(N3)dCHH′], 5.25
(s, 2H, NCH2), 5.92 [d, J ) 2 Hz, 1H, C(N3)dCHH′], 7.98 (s,
1H, H-6), 11.60 (s, 1H, NH);13C NMR (CD3OD) δ: 62.7 (OCH2),
78.1 (NCH2), 82.2 (CH), 101.7 [C(N3)dCH2], 109.5 (C-5), 138.7
[C(N3)], 144.4 (C-6). Anal. (C10H13N5O5) C, H, N.
1-[4-Hyd r oxy-3-(h yd r oxym eth yl)-1-bu tyl]-5-(1-a zid ovi-
n yl)u r a cil (9c). A solution of 4c (220 mg, 0.45 mmol) in a
saturated solution of ammonia and methanol (30 mL) was
stirred at 25 °C for 5 days. Removal of the solvent in vacuo
yielded a residue that was purified by silica gel column
chromatography using chloroform-methanol (92:8, v/v) as
eluent to yield 9c (100 mg, 79%) as a solid: mp 126-127 °C.
1H NMR (CD3OD) δ: 1.6-1.8 (m, 3H, CH, NCH2CH2), 3.6 (m,
4H, OCH2), 3.90 (t, J ) 7.2 Hz, 2H, NCH2), 5.02 (d, J ) 2 Hz,
1H, C(N3)dCHH′), 6.10 (d, J ) 2 Hz, 1H, C(N3)dCHH′), 7.90
(s, 1H, H-6);13C NMR (CD3OD) δ: 29.0 (NCH2CH2), 41.9 (CH),
48.2 (NCH2), 63.4 (OCH2), 101.3 [C(N3)dCH2], 109.0 (C-5),
138.6 [C(N3)], 145.0 (C-6), 151.9 (C-2, CdO), 163.4 (C-4, Cd
O). Anal. (C11H15N5O4) C, H, N.
1-[(2-Hyd r oxy-1-(h yd r oxym eth yl)eth oxy)m eth yl]-5-[2-
(1-a zir in yl)]u r a cil (10b). A solution of 9b (82 mg, 0.28 mmol)
in dry dioxane (45 mL) was heated at 110 °C for 3 h. Removal
of the solvent in vacuo gave a viscous oil, which was purified
by silica gel column chromatography. Elution with chloroform-
methanol (82:18, v/v) gave 10b as a dark brown oil (26 mg,
35.7%). 1H NMR (DMSO-d6) δ: 1.30 (d, 2H, azirinyl hydro-
gens), 3.30-3.55 (m, 4H, OCH2), 3.62 (m, 1H, CH), 4.70 (br,
2H, OH), 5.30 (s, 2H, NCH2), 8.45 (s, 1H, H-6). 13C NMR (CD3-
OD) δ: 16.35 (azirinyl CH2), 62.4 (OCH2), 78.0 (NCH2), 82.5
(CH), 102.7 (C-5), 154.0 (C-6), 161.6 (azirinyl C-2). Anal.
(C10H13N3O5‚1/2H2O) C, H, N.
[(2-Ben zoyloxyet h oxy)m et h yl]-5-(1-a zid ovin yl)u r a cil
(4a ) a n d 5-[(2-Ben zoyloxyeth oxy)m eth yl]fu r a n o-[2,3-d ]-
p yr im id in -6-(5H)-on e (5a ). Potassium tert-butoxide (425 mg,
1.87 mmol) was added to a suspension of 3a (832 mg, 2.58
mmol) in dry THF (100 mL) at -5 °C with stirring. The cooling
bath was removed, and the reaction mixture was stirred at 0
°C for 3 h. Removal of the solvent in vacuo gave a residue
which was dissolved in dichloromethane (50 mL) and washed
with cold water (25 mL), the dichloromethane fraction was
dried (Na2SO4), and the solvent was removed in vacuo. The
reaction mixture was separated by silica gel column chroma-
tography using chloroform-methanol (95:5, v/v) as eluent to
give two products, which eluted in the following order:
Fraction 1 (4a ): 358 mg, 38.7%; mp 129 °C. 1H NMR (CDCl3)
δ: 3.98 (m, 2H, OCH2CH2), 4.50 (m, 2H, OCH2CH2), 5.08 [d,
J ) 2 Hz, 1H, C(N3)dCHH′], 5.30 (s, 2H, NCH2), 6.33 [d, J )
2 Hz, 1H, C(N3)dCHH′], 7.40-8.10 (m, 6H, H-6, benzoyl
hydrogens), 8.65 (s, 1H, NH). Anal. (C16H15N5O5) C, H, N.
1
Fraction 2 (5a ): 80 mg, 9.8%; mp 180 °C. H NMR (CDCl3)
δ: 4.08 and 4.50 (2m, 4H total, OCH2CH2), 5.52 (s, 2H, NCH2),
6.40 (d, J ) 2.5 Hz, 1H, OCH dCH), 7.32 (d, J ) 2.5 Hz, 1H,
OCHdCH), 7.40-8.02 (m, 5H, benzoyl hydrogens), 8.10 (s, 1H,
H6). Anal. (C16H14N2O5) C, H, N.
1-[(2-Ben zoyloxy-1-(ben zoyloxym eth yl)eth oxy)m eth yl]-
5-(1-a zid ovin yl)u r a cil (4b). Potassium tert-butoxide (243 mg,
2.16 mmol) was added to a suspension of 3b (620 mg, 1.08
mmol) in dry THF (250 mL) at -5 °C with stirring. The cooling
bath was removed, and the reaction mixture was stirred at 5
°C for 3 h. Removal of the solvent in vacuo gave a residue,
which was dissolved in dichloromethane and washed with cold
water (25 mL). The dichloromethane fraction was dried (Na2-
SO4), and the solvent was removed in vacuo. The purification
of the residue by elution from a silica gel column using
dichloromethane-methanol (96:4, v/v) as eluent yielded 4b as
a viscous oil. 1H NMR (CDCl3) δ: 4.40-4.65 (m, 5H, CH,
OCH2), 5.0 [d, J ) 2 Hz, 1H, C(N3)dCHH′], 5.38 (s, 2H, NCH2),
6.25 [d, J ) 2 Hz, 1H, C(N3)dCHH′], 7.40-8.08 (m, 11H, H-6,
benzoyl hydrogens), 8.18 (s, 1H, NH). Anal. (C24H21N5O7) C,
H, N.
1-[4-Ben zoyloxy-3-(b en zoyloxym et h yl)-1-b u t yl]-5-(1-
a zid ovin yl)u r a cil (4c), 5-[4-Ben zoyloxy-3-(b en zoyloxy-
m eth yl)-1-bu tyl]fu r a n o-[2,3-d ]p yr im id in -6-(5H)-on e (5c),
a n d 3-Azid o-2,3-d ih yd r o-5-[4-ben zoyloxy-3-(ben zoyloxy-
m eth yl)-1-bu tyl]fu r a n o-[2,3-d ]p yr im id in -6-(5H)-on e (6).
Reaction of potassium tert-butoxide (208 mg, 1.84 mmol) with
3c (530 mg, 0.93 mmol), using the procedure described for the
preparation of 4a and purification of the product using
chloroform-methanol (97:3, v/v) as eluent yielded three prod-
ucts which eluted in the following order:
1-[4-Hyd r oxy-3-(h yd r oxym eth yl)-1-bu tyl]-5-[2-(1-a zir i-
n yl)]u r a cil (10c). A solution of 9c (26 mg, 0.09 mmol) in dry
dioxane (10 mL) was stirred at 110 °C for 4 h. Removal of the
solvent in vacuo gave a dark brown residue, which was purified
by PTLC using chloroform-methanol (80:20, v/v) as develop-
1
ment solvent to yield 10c (6 mg, 26%) as a dark yellow oil. H
Fraction 1 (4c): 235 mg, 51.6%; mp 111 °C dec. 1H NMR
(CDCl3) δ: 1.95 (m, 2H, NCH2CH2), 2.40 (m, 1H, CH), 4.0 (t,
J ) 7.2 Hz, 2H, NCH2), 4.50 (m, 4H, OCH2), 5.05 [d, J ) 2.0
Hz, 1H, C(N3)dCHH′], 6.32 [d, J ) 2.0 Hz, 1H, C(N3)dCHH′],
7.40-8.08 (m, 11H, H-6, benzoyl hydrogens), 8.26 (s, 1H, NH).
Anal. (C25H23N5O6) C, H, N.
NMR (CD3OD) δ: 1.30-1.50 (m, 2H, azirinyl hydrogens),
1.65-1.85 (m, 3H, CH, NCH2CH2), 3.60 (m, 4H, OCH2), 4.0 (t,
J ) 7.2 Hz, 2H, NCH2), 8.42 (s, 1H, H-6). 13C NMR (CD3OD)
δ: 16.15 (azirinyl CH2), 29.0 (NCH2CH2), 42.2 (CH), 48.0
(NCH2), 63.0 (OCH2), 102.2 (C-5), 154.5 (C-6), 161.5 (azirinyl
C-2). Anal. (C11H15N3O5‚1/2H2O) C,H,N.
1
Fraction 2 (5c): 63 mg, 15%. H NMR (CDCl3) δ: 2.08 (m,
Rea ction of 1-[4-Hyd r oxy-3-(h yd r oxym eth yl)-1-bu tyl]-
5-(1-a zid o-2-br om oeth yl)u r a cil (11) w ith Sod iu m Hy-
d r oxid e. A solution of sodium hydroxide (5 mg, 0.12 mmol)
in water 12 µL) was added to a solution of 1138 (32.6. mg, 0.09
mmol) in DMSO (5 mL) at ice-bath temperature with stirring.
Removal of the solvent under high vacuum gave a residue that
was purified by silica gel column chromatography using
chloroform-methanol (88:12, v/v) as eluent to yield a mixture
2H, NCH2CH2), 2.38 (m, 1H, CH), 4.26 (t, J ) 7.2 Hz, 2H,
NCH2), 4.48 (m, 4H, OCH2), 6.50 [d, J ) 2.6 Hz, 1H, OCHd
CH], 7.32 [d, J ) 2.5 Hz, 1H, OCHdCH], 7.38-8.08 (m, 11H,
H-6, benzoyl hydrogens). Anal. (C25H22N2O6) C, H, N.
Fraction 3 (6): 5 mg, 1.1%. 1H NMR (CDCl3) δ: 1.92 (m,
2H, NCH2CH2), 2.38 (m, 1H, CH), 4.26 (m, 2H, NCH2), 4.40
(m, 4H, OCH2), 4.62-4.80 [m, 2H, furanyl CH2], 4.90 [m, 1H,
furanyl CHN3], 7.38-8.08 (m, 11H, H-6, benzoyl hydrogens),
Anal. (C25H22N2O6‚1/4H2O) C, H, N.
1
of 9c (3 mg, 11%) and 7 (12 mg, 56%). The H NMR spectrum
for this mixture of 9c and 7 was identical to that of 9c and 7
prepared by other methods described in this study.
1-[(2-H yd r oxyet h oxy)m et h yl]-5-(1-a zid ovin yl)u r a cil
(9a ). A solution of 4a (357 mg, 1.0 mmol) in a saturated
solution of ammonia in methanol (50 mL) was stirred at 25
°C for 84 h. Removal of solvent in vacuo yielded a residue
which was purified by silica gel column chromatography using
chloroform-methanol (92:8, v/v) as eluent to yield 9a (154 mg,
60.8%) as a solid: mp 141-142 °C. 1H NMR (DMSO-d6) δ: 3.50
R ea ct ion of 3-Azid o-2,3-d ih yd r o-5-[4-b en zoyloxy-3-
(b en zoyloxym et h yl)-1-b u t yl]fu r a n o-[2,3-d ]p yr im id in -6-
(5H)-on e (6) w ith Meth a n olic Am m on ia . A solution of 6
(15 mg, 0.3 mmol) in a saturated solution of ammonia in
methanol (1 mL) was stirred at 25 °C for 5 days. Removal of
the solvent in vacuo and purification of the residue obtained

5-Substituted Acyclic Pyrimidine Nucleosides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2039
on PTLC using chloroform-methanol (90:10 v/v) as a develop-
ment solvent afforded 7 (4.3 mg, 60%) as a syrup. 1H NMR
(CD3OD) δ: 1.68-1.84 (m, 3H, CH, NCH2CH2), 3.58-3.64 (m,
4H, OCH2), 2.75 (t, J ) 7.2 Hz, 2H, NCH2), 6.75 (d, J ) 2.5
Hz, 1H, OCHdCH), 7.58 (d, J ) 2.5 Hz, 1H, OCHdCH), 8.58
(s, 1H, H-6). 13C NMR (CD3OD) δ: 29.3 (NCH2CH2), 42.13
(CH), 51.6 (NCH2), 63.5 (OCH2), 105.8 (C-3), 107.9 (C-3a), 145.0
(C-4), 146.3 (C-2), 157.5 (C-6), 173.1 (C-7a). Anal. (C11H14N2O4),
C, H, N.
In Vit r o An t ivir a l Assa y (Du ck Hep a t it is B Vir u s,
DHBV). Pekin duck eggs were obtained from a duck colony
maintained at the University of Alberta farm and were stored
in a 37 °C egg incubator until hatching occurred. Newly
hatched ducklings were infected with a 50 µL intravenous
injection of duck serum containing DHBV. Persistently in-
fected ducks were identified by detection of DHBV DNA in
sera by dot hybridization.48
Primary cultures of duck hepatocytes were prepared from
9-14 day old DHBV infected ducklings using a modified
method.43 Cells were cultured in 60 mm cell culture dishes in
4 mL of L-15 medium containing 5% fetal bovine serum,
penicillin G sodium (10 IU/mL), streptomycin sulfate (10 µg/
mL), and nystatin (25 U/mL). The test compounds were always
added in triplicate to the hepatocyte cultures on day 2 and
were maintained in culture with media changed every second
day until day 12. Cells were harvested at day 14. Initially,
with media containing drugs at concentrations of 200, 100, 50,
25, 12.5, 6.3, and 1.5 µg/mL. DMSO was also included as
control. Plates were incubated for 3 days at 37 °C. The color
reaction involved adding 10 µL of MTT reagent per well,
incubating 4 h at 37 °C, and then adding 100 µL of solubili-
zation reagent. Plates were read on an ELISA plate reader
(Abs 560-650 nm) following an overnight incubation at 37 °C.
Cell P r olifer a tion Assa y: HF F a n d Da u d i Cells. The
effect of test compounds on proliferation of HFF cells was
determined according to previously reported procedure.49 The
procedure for determining the effect of test compounds on
proliferating Daudi cells was essentially the same as that for
HFF cells except that the trypsin treatment to detach the cells
from the wells was not required because Daudi wells grow in
cell suspensions.
Hu m a n T Cells. Enriched T cell populations were purified
from buffy coats obtained from normal Canadian Blood Service
donors, by using nylon wool columns according to published
procedures.50 Test compounds dissolved in DMSO at 10 mg/
mL were prepared as stock solutions. In 96-well flat bottom
plates, test compounds were plated in triplicate wells starting
from 50 µg/mL concentrations. Serial dilutions of compounds
were prepared at 1:2, 1:5, or 1:10 dilutions for up to eight
dilutions. All of the dilutions were made with serum free
AIM V media for lymphocytes (Life Technologies, ON). Control
wells with the same DMSO concentration as those with test
compounds, were also prepared in triplicate wells. A total of
2 × 105 T cells in AIM V media were added to each of these
prepared wells. This was followed by the addition of phyto-
hemagglutinin (PHA, Sigma Chemical Company) to make up
a final concentration of 1 µg/mL of PHA. Positive control wells
with PHA and T cells and negative control wells with T cells
in media alone, were also prepared on each plate. The cultures
were incubated for 3-4 days in 5% CO2 and 95% humidity at
37 °C. After 3-4 days, [3H]thymidine (1 µCi/well) was added
to each well. Eighteen hours after pulsing with radioactive
thymidine, the cells were harvested onto nylon filters, and [3H]-
thymidine incorporation into the DNA of proliferating cells was
measured by liquid scintillation counting. CPM of wells
containing test compounds were always compared with CPM
of the wells containing stimulated T cells and corresponding
DMSO concentration.
the test compounds were screened at
a 10 µg/mL final
concentration. Inhibition in DHBV replication at 10 µg/mL was
calculated as the average of triplicate wells. Standard devia-
tions were within 10% of the average values. After initial
testing, the compounds were serially diluted to determine more
precise anti-DHBV EC50 values. The hepatocytes were lysed
with 1.0 mL of lysis buffer containing 10 mM Tris-HCl and
1% SDS. The lysate was digested with 0.5 mg/mL proteinase
K and extracted with an equal volume of phenol saturated with
Tris-HCl EDTA and 0.1% 8-hydroxyquinoline, followed by
extraction with chloroform. Concentrated NaCl (5 M) was
added to the aqueous phase to yield a final concentration of
0.5 M NaCl, and the DNA was precipitated with two volumes
of 95% ethanol. The DNA pellet was washed with 70% ethanol
and dried. The dried DNA was dissolved in 100 µL of a solution
containing Tris-HCl EDTA.
DNA samples were applied to a nylon filter (Hybond-N,
Amersham) using a Bio-Dot (Bio-Rad Laboratories) microfil-
tration apparatus. DNA on the filter was denatured with
NaOH/NaCl at room temperature for 30 min and neutralized
in Tris-HCl/NaCl. The filters were exposed to ultraviolet
irradiation for 3 min. DNA hybridization was initiated by
adding a recently prepared DHBV (32P) DNA probe at 106
CPM/mL and incubating overnight. Filters were washed twice
in 1xSSC (20xSSC in 3 M NaCl plus 0.3 M sodium citrate, pH
7.0)-0.1% SDS at 65 °C for 2 h and 1xSSC at room temper-
ature for 30 min. After an autoradiographic image was
obtained, the filters were exposed in a phosphoimaging screen
for 1-2 h, samples were quantitated by a Fujix BAS1000, and
the percentage density of phosphoimaging units were calcu-
lated.43 3-TC was used as the reference compound. 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. Percent inhibition was
calculated by using the following formula: (untreated positive
control - treated test sample) × 100/untreated positive control.
Ack n ow led gm en t. We are grateful to the Alberta
Heritage Foundation for Medical Research (AHFMR,
Canada) for an establishment grant (R.K.) and the
Medical Research Council of Canada/Canadian Insti-
tutes of Health Research for an operating grant (MOP-
36393) (R.K. and D.L.J .T.) for financial support of this
research. R.K. is a recipient of an AHFMR Medical
Scholar Award. We thank Ms. J yy-Shiang Huang for
providing excellent technical assistance.
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