Antiviral activity of 2,3′-anhydro and related pyrimidine nucleosides against hepatitis B virus

Article abstract of DOI:10.1016/j.bmcl.2010.08.120

Various 2,3′-anhydro analogs of 5-substituted 1-(2-deoxy-β-d- lyxofuranosyl)uracils (10-15) and a related 1-(3-O-mesyl-2-deoxy-β-d- lyxofuranosyl) pyrimidine nucleoside analog (18) have been synthesized for evaluation as a new class of potential anti-HBV

Full text of DOI:10.1016/j.bmcl.2010.08.120

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6790–6793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiviral activity of 2,30-anhydro and related pyrimidine nucleosides
against hepatitis B virus
Naveen C. Srivastav a, Michelle Mak a, Babita Agrawal b, D. Lorne J. Tyrrell c, Rakesh Kumar a,
⇑
a Department of Laboratory Medicine and Pathology, 1-71 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7
b Department of Surgery, University of Alberta, Edmonton, AB, Canada T6G 2H7
c Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
a r t i c l e i n f o
a b s t r a c t
Various 2,30-anhydro analogs of 5-substituted 1-(2-deoxy-b-
D-lyxofuranosyl)uracils (10–15) and a
related 1-(3-O-mesyl-2-deoxy-b-D-lyxofuranosyl) pyrimidine nucleoside analog (18) have been synthe-
sized for evaluation as a new class of potential anti-HBV agents. The compounds 10, 12, and 15 demon-
strated most potent anti-HBV activities against duck HBV (DHBV) and human HBV with EC50 values in the
Article history:
Received 4 July 2010
Revised 21 August 2010
Accepted 24 August 2010
Available online 20 September 2010
range of 2.5–10 and 5–10
nucleoside 18 also demonstrated significant anti-HBV activity against DHBV with an EC50 value of
2.5 g/mL, however, it was less active against HBV in 2.2.15 cells (EC50 = >10 g/mL).
lg/mL, respectively, at non-toxic concentrations (CC50 = >200 lg/mL). The
Keywords:
Hepatitis B virus
Pyrimidine nucleosides
l
l
Ó 2010 Elsevier Ltd. All rights reserved.
Hepatitis B virus (HBV) is a major cause of acute and chronic
hepatitis in humans. According to the World Health Organization
(WHO), HBV infection is one of the top 10 leading causes of death
due to infectious diseases.1 There are ꢀ400 million chronic carriers
of HBV worldwide and 20–30% of them will die due to exacerba-
tion of chronic liver diseases such as cirrhosis, hepatocellular car-
cinoma (HCC), and liver failure.2 HCC is fifth among most
common cancer in humans. The probability of developing chronic
HBV infection is reciprocal to the age of acquiring the infection.
HBV has been known to be transmitted perinatally, sexually, and
parenterally.2 At the present time, only six agents are available
for the treatment of chronic HBV infection; immunomodulatory
episomal covalently closed circular (ccc) viral DNA in the hepato-
cyte nucleus.8 Viral cccDNA has a long half-life, is the template
for virus transcription and is replenished by viral DNA synthesis.
However, it can be expected that continuous and complete sup-
pression of HBV DNA replication may deplete cccDNA.9,10 Short-
term therapy with HBV DNA synthesis inhibitors cannot deplete
the pool of cccDNA, and this is the reason for rapid rebound of viral
replication after cessation of therapy.7,9,10
The two major problems of current therapy; development of
resistance and viral rebound may be addressed by continuous
long-term therapy with potent non-toxic, non-cross-resistant
anti-HBV agents or combination of anti-HBV agents. Substantive
and continued suppression of viral DNA polymerase/RT may lead
to continuous inhibition of viral DNA synthesis resulting eventu-
ally in the depletion of the cccDNA pool.10 Thus, there is an urgent
need to investigate additional new classes of antiviral agents for
HBV infections. Although anti-HBV nucleos(t)ides target HBV poly-
merase, their specific modes of action can differ and may involve
anti-priming, anti-reverse transcriptase, anti-DNA dependent
DNA polymerase and/or combination of these mechanisms.3 Also,
cross-resistance between nucleos(t)ides does not necessarily oc-
cur.3,4 These two attributes reinforce the notion that the search
for novel anti-HBV agents must be continued and intensified.
We recently identified lyxofuranosyl pyrimidine nucleosides as
treatment Interferon-a, nucleoside/nucleotide antivirals lamivu-
dine (LMV), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine
(LdT), and tenofovir (TDF).3 It has been observed that suppression
of the replication of HBV in the liver by antiviral drugs leads to im-
proved liver pathology and decreased progression to liver cirrhosis
and hepatocellular carcinoma, resulting in reduced mortality and
morbidity.4 However, the currently available therapy is inadequate
due to development of adverse effects. Continued treatment with
monotherapy leads to development of resistance.3,5 In addition,
the most common limitation with the current therapy is viral
rebound to baseline or even higher viral levels following cessation
of treatment.6,7 This problem of virus re-appearance can be in part
explained by the mechanism by which HBV maintains the infection
at the level of hepatocytes. It has been shown that chronic infection
of hepatocytes is maintained by the presence of 30–40 copies of
a new class of potential anti-HBV agents where 1-(20-deoxy-b-
lyxofuranosyl)thymine (1) and 1-(20-deoxy-b-
-lyxofuranosyl)-5-
trifluoromethyluracil (2) exhibited appreciable in vitro inhibition
of HBV in 2.2.15 cells (EC50 = 10 g/mL) at non-toxic concentra-
tions (CC50 = >200 -lyxofurano-
g/mL). In contrast 1-(20-deoxy-b-
syl)-4-thiothymine (3) was inactive.11 As part of our ongoing
D-
D
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l
D
⇑
Corresponding author. Tel.: +1 (780) 492 7545; fax: +1 (780) 492 7521.
E-mail address: rakesh.kumar@ualberta.ca (R. Kumar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.120

N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6790–6793
6791
investigation of novel anti-HBV agents, it was of interest to
discover more potent anti-HBV agents with low or no cytotoxicity.
anosyl)-5-ethyluracil (14),15 and 2,30-anhydro-1-(2-deoxy-2-
fluoro-b- -lyxofuranosyl)-5-ethyluracil (15) were synthesized by
D
the reaction of nucleosides 4–9 with triphenylphosphine and
diisopropyl azodicarboxylate in dry CH3CN using the procedure
reported by Balagopala et al.17 (Scheme 1). The products were
purified on silica gel column chromatography and structural
assignments of the synthesized derivatives were made based on
1H NMR and elemental analysis studies. In addition to anhydro
compounds 10–15, we also synthesized 1-(3-O-mesyl-2-deoxy-b-
X
R
HN
1, R = CH3 X=O
2, R = CF3 X=O
3, R = CH3 X=S
O
N
HO
O
OH
D
-lyxofuranosyl)-4-thiothymine (18) as illustrated in Scheme 2 to
investigate its potential activity against HBV. 1-(5-O-Trityl-2-
deoxy-b-
-lyxofuranosyl)-4-thiothymine (16)18 upon treatment
with mesyl chloride in dry pyridine gave 1-(3-O-mesyl-5-O-tri-
D
Pyrimidine nucleosides with an additional linkage between ura-
cil base and carbohydrate portion, termed as anhydro or cyclo
nucleosides have been studied in earlier investigations in an
attempt to find more potent antiviral and antitumor agents,12,13
tyl-2-deoxy-b-D-lyxofuranosyl)-4-thiothymine (17), which was
detritylated to yield 1819 in 52% yield (Scheme 2).
for example, 2,20-anhydro-1-b-
D-arabinofuranosylcytosine (cycloC)
The anti-HBV activity of nucleoside analogs 10–15 and 18, along
withthe reference antiviral compound (ꢁ) 3-TC was assessed in con-
fluent cultures of primary duck hepatocytes obtained from chroni-
cally infected Pekin ducks according to the procedure previously
reported by us.20,21 Duck hepatitis B virus (DHBV), a member of hep-
adnaviridae, shares properties of hepatotropism, virion structure,
genome organization and replication with human HBV.22 DHBV-
based in vitro and in vivo systems have been used extensively to
screen drugs for potential anti-HBV activity.23 It has been shown
that compounds like 3-TC and penciclovir, both potent inhibitors
of DHBV, are also potent inhibitors of HBV in chimpanzees as well
as humans.23,24 The anti-HBV activity of the compounds 10–15
and 18 was also assessed in confluent cultures of the human hepa-
toma cell line 2.2.15 that chronically produces infectious HBV. The
anti-HBV activity data of compounds 4–9 in 2.2.15 cells is also
included for comparisons. 2.2.15 is a stable human HBV-producing
human hepatoblastoma cell line, which carries HBV DNA stably
integrated into the genome of HepG2 cells.25,26 Table 1 shows the
concentrations required to inhibit 50% of HBV DNA (EC50) and 50%
cytotoxic concentration (CC50) on Huh-7 cells.
is a highly effective antitumor agent in animal models12 and humans
with toxicity less than that of its parent compound (araC).13 CycloC
acts as a depot form and slowly hydrolyzes to araC under physiolog-
ical conditions.12 2,50-Anhydro analogs of pyrimidine nucleosides
have also been investigated in an effort to increase the antiviral
activity and decrease the cytotoxicity.14 However, to our knowledge,
2,30-anhydro pyrimidine nucleosides have not been explored for
biological evaluation as potential anti-HBV agents. In this Letter,
we have investigated anti-HBV activities of various 5-substituted
2,30-anhydro pyrimidine nucleosides (10–15) and a related analog
(18) as potential antiviral agents. In analogy with cycloC, it is
postulated that they may possess improved cellular penetration,
physiological distribution and absorption, and undergo enzymatic
hydrolysis in cell culture or in vivo to parent nucleosides.
The test compounds 2,30-anhydro-1-(2-deoxy-b-
syl)uracil (10),15 2,30-anhydro-1-(2-deoxy-b-
-lyxofuranosyl)-5-
fluorouracil (11),15 2,30-anhydro-1-(2-deoxy-b-
-lyxofuranosyl)-
-lyxofuranosyl)-5-tri-
D-lyxofurano-
D
D
thymine (12),15 2,30-anhydro-1-(2-deoxy-b-
D
fluoromethyluracil (13),16 2,30-anhydro-1-(2-deoxy-b-
D
-lyxofur-
O
O
R
HN
R
(i)
N
O
N
O
R1
O
N
HO
4, R = H,
5, R = F,
6, R = CH3, R1 = H
7, R = CF3 ,R1 = H
8, R = C2H5 , R1 = H
9, R = C2H5, R1 = F
R1 = H
R1 = H
10, R = H,
11, R = F,
R1 = H
R1 = H
O
R1
HO
12, R = CH3, R1 = H
13, R = CF3 ,R1 = H
14, R = C2H5, R1 = H
15, R = C2H5 R1 = F
OH
Scheme 1. Reagents and conditions: (i) triphenylphosphine, diisopropyl azodicarboxylate, dry CH3CN, ꢁ15 °C to 0 °C, 2–5 h.
S
S
S
CH3
(i)
CH3
(ii)
CH3
HN
HN
HN
N
N
N
O
O
O
TrO
TrO
HO
O
O
O
OH
OMs
OMs
16
17
18
Scheme 2. Reagents and conditions: (i) mesyl chloride, dry pyridine, 0–5 °C, 40 h; (ii) 80% AcOH, 90 °C, 0.5 h.

6792
N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6790–6793
Table 1
In vitro antiviral activities of pyrimidine nucleosides against duck HBV and human HBV replication
O
O
S
CH3
R
R
HN
N
HN
O
O
N
N
N
O
HO
HO
HO
O
O
O
R1
R1
OMs
HO
4-9
10-15
18
R1
DHBV primary duck
hepatocytes % inhibition
EC50
2.2.15 HBV %
inhibition @
10
EC50
g/mL)
Cytotoxicity CC50
g/mL)
b
b
e
Compd
R
(l
g/mL)
(
l
(l
@ 10
l
g/mLa
l
g/mLa
4
5
6
7
8
9
H
F
H
H
H
H
H
F
H
H
H
H
H
F
NDf
ND
ND
ND
ND
ND
67
30
56
35
32
—
—
—
—
—
—
0
ND
0
—
—
—
—
>10
<10
5
—
10
—
>200
ND
CH3
CF3
C2H5
C2H5
H
>200
>100
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
0
40
90
65
0
54
25
51
53
45
10
11
12
13
14
15
18
3-TCd
2.5–5
—
F
CH3
CF3
C2H5
C2H5
—
10
>10c
—
10
10
>10
0.2
65
70
2.5–5
2.5
0.05
—
—
—
96 (0.5–1.0)
88 (0.5–1)
a
b
c
d
e
f
The data is expressed as percent inhibition of viral DNA in the presence of 10
The drug concentration ( g/mL) required to reduce the viral DNA in infected cells to 50% of untreated infected controls.
(>) sign indicates that 50% inhibition was not reached at the stated (highest) concentration tested.
-20,30-dideoxy-30-thiacytidine (Lamivudine, 3-TC).
Concentration required to reduce Huh-7 cell viability by 50%.
Not determined.
lg/mL of the test compounds as compared to untreated infected controls.
l
(ꢁ)-b-
L
Among the synthesized compounds, 10, 12, 15, and 18 elicited
[as in 18, EC50 = 2.5 lg/mL (DHBV) and 45% inhibition @ 10 lg/mL
significant activity against DHBV with EC50 values of 2.5–5, 10,
(2.2.15 cells)] also influenced the antiviral activity dramatically
since its non-mesylated compound (3) had no anti-HBV activity
in our earlier studies.11 Reasons for the increase and decrease in
activities of the compounds compared to the parent analogs are
not clear at this point. However, our results indicate that the
modifications studied in this Letter can lead to the modulation of
the anti-HBV activity in in vitro, possibly due to changes in proper-
ties such as cell delivery, slow release, and/or resistance against
enzymatic hydrolysis.
Hydrolytic stability of compounds 10–15 was determined in sal-
ine and PBS buffer at physiological pH at 25 °C and 37 °C up to 48 h,
and in fetal bovine serum at 37 °C for 24 h. No conversion to parent
nucleosides (4–9), lyxo (30-OH up) derivatives or degradation prod-
ucts was obtained under these conditions. The potent anti-HBV
activity of compounds 10, 12, and 14 as compared to their parent
nucleosides 4, 6, and 8 and decreased inhibition of HBV by 15 as
compared to its parent analog 9 suggests that 2,30-anhydro nucleo-
sides (10–15) possess anti-HBV activity on their own.
2.5–5, and 2.5
found to provide only 30–35% inhibition at 10
l
g/mL, respectively. Compounds 11, 13, and 14 were
g/mL concentra-
l
tion. In these studies, we noted that incorporation of a fluorine
atom at the 20-arabino position of 14 led to an increased DHBV
inhibition (as compared to compound 15). When the test com-
pounds 10–15 and 18 were evaluated against HBV in 2.2.15 cells,
only nucleosides 10, 12, and 13 retained their activity, 15 and 18
had reduced inhibition of viral replication, and 11 lost the inhibi-
tory action. In this system, on the other hand, compound 14
showed increased activity (EC50 = 10
lg/mL) as compared to its
anti-DHBV activity (32% inhibition @ 10
lg/mL). The marked dif-
ferences obtained between DHBV and HBV activities could be
attributed to inherent differences in human versus duck HBV, met-
abolic differences between the two cells and/or genome organiza-
tion of hepadnavirus (i.e., integrated in 2.2.15 cells and non-
integrated in duck hepatocytes).
It was interesting to note that in 2.2.15 cells, 2,30-anhydro ana-
log (12) was almost as active as its lyxo (non-anhydro) derivative
The fact that nucleosides 10, 12, 14, 15, and 18 exhibiting anti-
DHBV or anti-HBV activity in a human hepatoma cell line carrying
the HBV (2.2.15 cells) and/or against both viruses show no host cell
(1, EC50 = 10
much less active (25% inhibition @ 10
(2, EC50 = 10
g/mL).11 In contrast, 2,30-anhydro-1-b-
syluracil (10) (EC50 = 5
g/mL) and 2,30-anhydro-1-b-
syl-5-ethyluracil (14) (EC50 = 10
l
g/mL),11 and the 2,30-anydro derivative (13) was
lg/mL) than the lyxo analog
l
D
-lyxofurano-
-lyxofurano-
cytotoxicity (CC50 = >200 lg/mL), suggests that they can selectively
l
D
inhibit HBV DNA polymerases after phosphorylation by cellular
kinases. Similar mechanisms of action have been suggested for
other antiviral nucleosides.24
lg/mL) gained significant activity
compared to their 30-OH (lyxo) derivatives that proved to be
completely inactive in our previous studies.11 In line with the
observations made with 2,30-anhydro analogs 10 and 14, we were
interested to examine if blocking the 30-OH lyxo group in com-
pound 3 by other means could enhance anti-HBV activity. It was
noteworthy that incorporation of a mesyl group at the 30-position
Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) for an operating Grant (MOP-36393) for the financial

N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6790–6793
6793
obtained was purified by elution from a silica gel column using MeOH/CHCl3
(15:85, v/v) as eluent to give 13 (0.63 g, 67%) as a white solid; mp 190–192 °C.
1H NMR (DMSO-d6) d 2.54 and 2.68 (2 m, 2H, H-20), 3.54 (t, J = 5.80 Hz, 2H, H-
50), 4.24 (m, 1H, H-40), 5.07 (t, J = 5.50 Hz, 1H, 50-OH), 5.36 (br s, 1H, H-30), 6.04
(d, J = 3.66 Hz, 1H, H-10), 8.46 (s, 1H, H-6). Anal. (C10H9F3N2O4) C, H, N.
17. Balagopala, M. I.; Ollapally, A. P.; Lee, H. J. Nucleosides Nucleotides Nucleic Acids
1996, 15, 899.
support of this research. B.A. is thankful to the Alberta Innovates-
Health Solutions (formerly Alberta Heritage Foundation for Medi-
cal Research, AHFMR) for a Senior Scholar Award.
References and notes
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19. Experimental synthesis of 1-(3-O-mesyl-2-deoxy-b-
D-lyxofuranosyl)-4-
thiothymine (18). To an ice cooled solution of 1-(5-O-trityl-2-deoxy-b-
D-
lyxofuranosyl)-4-thiothymine (16, 0.5 g, 0.99 mmol) in dry pyridine (20 mL)
was added mesyl chloride (0.13 mL, 1.68 mmol) drop wise with stirring. Then,
the reaction mixture was kept in the refrigerator for 40 h. After the addition of
water (1 mL), the solvent was evaporated and the resulting residue was
dissolved in CHCl3 (50 mL), washed with water (2 ꢂ 25 mL) and dried over
anhydrous Na2SO4. The solvent was removed in vacuo and the residue thus
obtained was purified on a silica gel column using MeOH/CHCl3 (2:98, v/v) as
eluent to give 17 (0.22 g, 38%) as a sirup. 1H NMR (CDCl3) d 1.98 (s, 3H, CH3),
2.49–2.58 (m, 1H, H-20), 2.74–2.88 (m, 1H, H-200), 2.77 (s, 3H, CH3), 3.34–3.42
(m, 1H, H-50), 3.63–3.71 (m, 1H, H-500), 4.27 (m, 1H, H-40), 5.29 (m, 1H, H-30),
6.23 (dd, J = 7.94 Hz, 2.75 Hz, 1H, H-10), 7.24–7.52 (m, 16H, 50-O-trityl and H-6),
10.05 (br s, 1H, NH). A solution of 17 (0.20 g, 0.34 mmol) in 80% aqueous AcOH
(20 mL) was heated at 90 °C for 0.5 h. The solvent was removed in vacuo and
the crude product thus obtained was purified on a silica gel column using
EtOAc/hexane (70:30, v/v) as eluent to give 18 (0.06 g, 52%) as a solid; mp 136–
138 °C. 1H NMR (DMSO-d6) d 1.98 (s, 3H, CH3), 2.35 (dd, J = 15.56 Hz, 2.13 Hz,
1H, H-20), 2.81–2.91 (m, 1H, H-200), 3.23 (s, 3H, CH3), 3.73 (t, J = 5.49 Hz, 2H, H-
50), 4.10–4.15 (m, 1H, H-40), 5.06 (t, J = 5.49 Hz, 1H, 50-OH), 5.27 (t, J = 4.28 Hz,
1H, H-30), 6.09 (dd, J = 7.93 Hz, 2.75 Hz, 1H, H-10), 7.56 (s, 1H, H-6), 12.77 (s, 1H,
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13
NH). C NMR (CD3OD) d 17.43 (CH3), 38.31 (CH3), 40.63 (C-20), 60.52 (C-50),
80.54 (C-30), 84.66 (C-40), 86.04 (C-10), 120.19 (C-5), 133.14 (C-6), 149.82 (C-2),
192.51 (C-4). Anal. (C11H16N2O6S2) C, H, N, S.
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16. Experimental synthesis of 2,30-anhydro-1-(2-deoxy-b-
D
-lyxofuranosyl)-5-
trifluoromethyl uracil (13).
A
dried mixture of 5-trifluoromethyl-20-
deoxyuridine (7; 1.0 g, 3.38 mmol) and triphenylphosphine (1.78 g,
6.79 mmol) was suspended in acetonitrile (50 mL) and the reaction mixture
was cooled to ꢁ15 °C in ice-methanol bath. Diisopropylazodicarboxylate
(1.32 mL, 6.73 mmol) was slowly added with vigorous stirring in 10 min,
maintaining the reaction temperature below ꢁ5 °C. The reaction mixture was
then stirred at 0 °C for 3 h. The solvent was removed in vacuo and the residue
25. Sells, M. A.; Chen, M. L.; Acs, M. L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1005.
26. Korba, B. E.; Boyd, M. R. Antimicrob. Agents Chemother. 1996, 40, 1282.