N4-hydroxycytosine dioxolane nucleosides and their activity against hepatitis B virus

Article abstract of DOI:10.1081/NCN-200067414

Novel racemic, D- and L-β-dioxolane N⁴-hydroxycytosine nucleosides have been synthesized and evaluated for their activity against hepatitis B virus. None of the synthesized nucleosides demonstrated selective anti-HBV activity. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200067414

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N 4-Hydroxycytosine Dioxolane Nucleosides and Their
Activity Against Hepatitis B Virus
Jinfa Du a , Laurent Hollecker a , Junxing Shi a , Byoung-Kwon Chun a , Kyoichi A. Watanabe
a , Raymond F. Schinazi b c , Tammy Y. Nachman a , Stefania Lostia a , Lieven J. Stuyver a &
Michael J. Otto a
a Pharmasset, Inc. , Tucker, Georgia, USA
b Veterans Affairs Medical Center , Decatur, Georgia, USA
c Emory University , Atlanta, Georgia, USA
Published online: 31 Aug 2006.
To cite this article: Jinfa Du , Laurent Hollecker , Junxing Shi , Byoung-Kwon Chun , Kyoichi A. Watanabe , Raymond F.
Schinazi , Tammy Y. Nachman , Stefania Lostia , Lieven J. Stuyver & Michael J. Otto (2005) N 4-Hydroxycytosine Dioxolane
Nucleosides and Their Activity Against Hepatitis B Virus, Nucleosides, Nucleotides and Nucleic Acids, 24:8, 1209-1214, DOI:
10.1081/NCN-200067414
To link to this article: http://dx.doi.org/10.1081/NCN-200067414
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (8):1209–1214, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200067414
N4-HYDROXYCYTOSINE DIOXOLANE NUCLEOSIDES AND THEIR
ACTIVITY AGAINST HEPATITIS B VIRUS
Jinfa Du, Laurent Hollecker, Junxing Shi, Byoung-Kwon Chun, and
5
Kyoichi A. Watanabe
Pharmasset, Inc., Tucker, Georgia, USA
5
Raymond F. Schinazi
Veterans Affairs Medical Center, Decatur, Georgia, USA and
Emory University, Atlanta, Georgia, USA
Tammy Y. Nachman, Stefania Lostia, Lieven J. Stuyver, and Michael J. Otto
5
Pharmasset, Inc., Tucker, Georgia, USA
Novel racemic, D- and L-b-dioxolane N4-hydroxycytosine nucleosides have been synthesized and
5
evaluated for their activity against hepatitis B virus. None of the synthesized nucleosides demonstrated
selective anti-HBV activity.
Keywords HBV, Dioxolane, Nucleoside
INTRODUCTION
Hepatitis B virus (HBV) is a causative agent of acute and chronic hepatitis.
HBV infection is the world’s ninth leading cause of death. The number of chronic
carriers is estimated to be more than 400 million worldwide, with roughly 4 million
deaths annually from the resulting cirrhosis and hepatocellular carcinoma. Only
three FDA-approved drugs are currently available for the treatment of hepatitis B
infections. The first therapeutic option involves immune stimulation by using
interferon-a. However, IFN-a is only effective in 10–30% of treated patients, making
its usefulness limited, and often serious side effects result in cessation of therapy.[1]
A second therapeutic option involves nucleoside analogues. Two nucleosides
are FDA approved, i.e., lamivudine, (ꢀ)-b-2’,3’-dideoxy-3’-thiacytidine, 3TC and
Received 1 December 2004, accepted 22 March 2005.
This research was supported by NIH SBIR grant 1 R43AI056794-01. R. F. Schinazi is founder of Pharmasset
Ltd., and his particulars have been reviewed by Emory University’s Conflict of Interest Committee. R. F. Schinazi’s
group is supported by the Department of Veterans Affairs and received no funding from Pharmasset to perform
this work.
Address correspondence to Dr. Jinfa Du, Pharmasset, Inc., 1860 Montreal Road, Tucker, GA 30084, USA;
Fax: (678) 395-0030; E-mail: jdu@pharmasset.com
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J. Du et al.
hepsera, 9-{2-[bis(pivaloyloxymethyl)phosphonylmethoxy]ethyl}adenine, adefovir
dipivoxil. Both drugs are now entering widespread clinical use associated with
effective suppression of viral replication, reduced disease activity and improved
liver histology.
Long-term treatment with lamivudine results in the emergence of drug-resistant
HBV, the first signs of which might appear within 6–12 months of therapy. Re-
sistance to 3TC is associated to accumulation of mutations in the viral polymerase,
with rtM204V and rtM204I as the key mutations for resistance. The latter ones
are often accompanied with a compensatory mutation at rtL180M. In addition,
long-term remission after completion of treatment with 3TC is not commonly
SCHEME 1 Reagents and conditions: a) i. uracil, HMDS, reflux, ii. TMSOTf, CH2Cl2; b) Lawesson’s reagent,
ClCH2CH2Cl, reflux; c) NH2OH, EtOH, reflux; NH3, MeOH, RT.

Nucleoside Activity Against Hepatitis B Virus
1211
TABLE 1 Anti-HBV Activity and Toxicity of the Selected Compounds
HepAD38 EC90, mM
HepAD79 EC90, mM
6
8
0.43
>10
5.71
>10
10
3TC
0.05
0.04
2.43
>10
ND: not determined; EC90: effective concentration required for reducing
the HBV DNA levels by 90% in 5 days in HepAD38 and 10 days in
HepAD79.
observed, and most patients experience a rebound in viremia once the use of drug
is stopped.[1]
Adefovir dipivoxil is a nucleotide analogue that has been shown to have potent
anti-HBV activity similar to lamivudine. Several clinical studies concluded that
prolonged suppression of HBV replication might be possible without the emer-
gence of resistance. However, at least one case of resistance against adefovir
dipivoxil therapy has now been described and is associated with the selection of a
rtN236T mutation.[2]
Because of the ongoing epidemic and the rise of drug-resistant HBV, it remains
an urgent task to develop additional safe and selective agents for the treatment of
HBV infection. We herein report the synthesis and evaluation of antiviral activity of
racemic and enantiomerically pure D- and L-b-1-[2-(hydroxymethyl)-1,3-dioxolan-4-
yl]-N4-hydroxycytosine against HBV. N4-Hydroxycytosine nucleosides were
selected since we recently determined that N4-hydroxycytosine nucleoside can be
substrate of cytidine deaminase[3–5] and dioxolane cytosine nucleoside showed
potent anti-HBV activity but is toxic.
RESULTS AND DISCUSSION
The synthesis of racemic and enantiomerically pure D- and L-b-dioxolane N4-
hydroxycytosine is shown in Scheme 1. Condensation of ( )-4-acetoxy-2-
(benzoyloxymethyl)-1,3-dioxolane (1) with silylated uracil in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) in methylene chloride afforded
an inseparable mixture of anomers (2). Treatment of the mixture with Lawesson’s
reagent in 1,2-dichloroethane at reflux gave b-4 and a-3y after silica gel
*
1
*
Compound 4: foam, H NMR (CDCl3) d 9.87 (s, 1H, NH, D2O exchangeable), 8.06–7.48 (m, 5H, Bz), 7.45
(d, J = 8.0 Hz, 1H, 6-H), 6.34 (d, J = 4.4 Hz, 1H, 4’-H), 6.13 (dd, J = 1.6, 7.6 Hz, 1H, 5-H), 5.31 (t, J = 2.4 Hz, 1H,
2’-H), 4.70 (m, 2H, 5’-H), 4.25 (m, 2H, 6’-H). Anal Calcd for C15H14N2O5S + 0.1H2O: C, 53.54; H, 4.22; N, 8.33.
Found: C, 53.45; H, 4.19; N, 8.26.
yCompound 3: foam, 1H NMR (CDCl3) d 9.86 (s, 1H, NH, D2O exchangeable), 8.07–7.46 (m, 5H, Bz), 7.22
(d, J = 8 Hz, 1H, 6-H), 6.46 (dd, J = 1.6, 7.6 Hz, 1H, 5-H), 6.28 (dd, J = 2.4, 5.2 Hz, 1H, 4’-H), 5.81 (t, J = 4 Hz, 1H,
2’-H), 4.45 (m, 3H, 5’-HA, 6’-H), 4.16 (m, 1H, 5’-HB). Anal Calcd for C15H14N2O5S + 0.1H2O: C, 53.54; H, 4.22; N,
8.33. Found: C, 53.32; H, 4.18; N, 8.04.

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J. Du et al.
FIGURE 1 Effect of selected compounds on HepG2 cell proliferation. , cell proliferation in the absence of
.
drug; 5, cell proliferation in the presence of 1 mM of ( )-b-1-[2-(hydroxymethyl)-1,3-dioxolan-4-y1]-N4-hydroxy-
cytosine (6); &, cell proliferation in the presence of 1 mM of D-b-1-[2-(hydroxymethyl)-1,3-dioxolan-4-y1]-N4-
hydroxycytosine (8); , cell proliferation in the presence of 1 mM of L-b-1-[2-(hydroxymethyl)-1,3-dioxolan-4-y1]-
ꢁ
N4-hydroxycytosine (10).
*
chromatographic separation in excellent yields. The final nucleoside 6 was
obtained by treatment of b-4-thiocytosine analog 4 with aqueous hydroxylamine in
ethanol at reflux followed by deprotection with methanolic ammonia in good yield.
Similarly, coupling of enantiomerically pure (2R)- and (2S)-4-acetoxy-2-(benzoyloxy-
methyl)-1,3-dioxolane (7 and 9) with silylated uracil followed by reaction with
Lawesson’s reagent, a/b-isomers separation, replacement with hydroxylamine and
deprotection afforded the corresponding (2R,4R or D)- and (2S,4S or L)-b-1-[2-
(hydroxymethyl)-1,3-dioxolan-4-yl]-N4-hydroxycytosine (8 and 10).y The enantio-
merically pure 7 and 9 were prepared by the reported method.[6]
The assignment of the anomeric configuration of these nucleosides was
established on the basis of the characteristics of the proton NMR spectra. The 2’-
proton of the a-anomer 3 appeared at a lower field (5.81 ppm) than that (5.31 ppm)
of the b-anomer 4. However, the 5’-proton of the a-anomer 3 appeared at a higher
field (4.30 ppm) than that (4.70 ppm) of the b-anomer 4. Chemical shifts of specific
protons were affected by their electronic environment. The proton signals shifted to
lower field in NMR spectra when they were located on the same side as the
Compound 6: crystals, mp. 159–161°C. 1H NMR (DMSO-d6) d 10.00, 9.55 (ss, 2H, NH, NOH, D20
*
exchangeable), 6.94 (d, J = 8.4 Hz, 1H, 6-H), 6.20 (d, J = 4.8 Hz, 1H, 4’-H), 5.58 (d, J = 7.2 Hz, 1H, 5-H), 5.13 (t,
J = 6.0 Hz, 1H, OH, D2O exchangeable), 4.86 (t, J = 3.2 Hz, 1H, 2’-H), 4.05 m 2H, 5’-H), 3.58 (m, 2H, 6’-H). Anal
Calcd for C8H11N3O5: C, 41.92; H, 4.84; N, 18.33. Found: C, 42.13; H, 4.84; N, 18.40. HRMS (FAB) obsd, m/z
230.0784, calcd for C8H11N3O5+ H, m/z 230.0777, (M + H)+.
y1H-NMR is identical to that of ( )-nucleoside.

Nucleoside Activity Against Hepatitis B Virus
1213
pyrimidine base due to its deshielding effect. The assignment for these nucleosides
is consistent with reports by others with similar pyrimidine nucleosides.[7]
The synthesized nucleosides 6, 8, and 10 were evaluated in vitro for their
biological activity against HBV. These compounds were tested in an HBV-
inducible system under control of tetracycline. In the presence of tetracycline, the
HBV production is suppressed, but upon removal of tetracycline from the culture
media, these cells produce HBV particles. The compounds were evaluated in two
different cells lines; e.g., HepAD38 expressing the wild type virus, and HepAD79,
expressing the lamivudine resistant virus.[8,9] The methodology for testing was
essentially as described previously.[10]
The results of the antiviral testing are shown in Table 1. The racemic mixture 6
and the enantiomerically pure L-10 showed potent antiviral activity against wild-
type virus and a modest activity against the 3TC resistant virus. Compound D-8 did
not show any antiviral activity.
L-Dioxolane cytidine is a notoriously toxic compound.[11] Description of the
antiviral effect as given in Table 1 is calculated from the amount of viral DNA in
cell supernatant. These values do not take into account the possible toxic side
effects that these compounds might have on cell proliferation. Therefore, a separate
toxicity experiment was conducted. HepG2 cells were exposed for 14 days to 1 mM
of the test compound, with the medium being renewed every 4 days. The results are
shown in Figure 1. While the racemic mixture 6 and the L-enantiomer 10 show
significant inhibition of cell proliferation, the D-enantiomer 8 is rather indistin-
guishable from the untreated control.
It is concluded from these toxicity results that the observed antiviral effect of 6
and 10 might be secondary to the inhibitory effect on cell proliferation. A favorable
therapeutic window (antiviral efficacy/cellular toxicity) could not be established.
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