Synthesis and in vitro anti-HCV activity of β-D- and L-2′-deoxy-2′-fluororibonucleosides

Article abstract of DOI:10.1081/NCN-200059224

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and L-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′- deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N⁴-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N⁴-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′- deoxy-2′-fluoro-N⁴-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200059224

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SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D-
and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES
Junxing Shi a , Jinfa Du a , Tianwei Ma b , Krzysztof Pankiewicz a , Steven E. Patterson a
,
Abdalla E. A. Hassan a , Phillip M. Tharnish a , Tamara R. McBrayer a , Stefania Lostia a
,
Lieven J. Stuyver a , Kyoichi A. Watanabe a , Chung K. Chu b , Raymond F. Schinazi c d
Michael J. Otto a
&
a Pharmasset, Inc., Tucker, Georgia, USA
b College of Pharmacy, The University of Georgia, Athens, Georgia, USA
c Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
d Veterans Affairs Medical Center, Decatur, Georgia, USA
Published online: 15 Nov 2011.
To cite this article: Junxing Shi , Jinfa Du , Tianwei Ma , Krzysztof Pankiewicz , Steven E. Patterson , Abdalla E. A. Hassan ,
Phillip M. Tharnish , Tamara R. McBrayer , Stefania Lostia , Lieven J. Stuyver , Kyoichi A. Watanabe , Chung K. Chu ,
Raymond F. Schinazi & Michael J. Otto (2005) SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-
FLUORORIBONUCLEOSIDES, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 875-879, DOI: 10.1081/NCN-200059224
To link to this article: http://dx.doi.org/10.1081/NCN-200059224
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):875–879, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059224
SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF b-D- AND
L-2’-DEOXY-2’-FLUORORIBONUCLEOSIDES
5
Junxing Shi and Jinfa Du
Pharmasset, Inc., Tucker, Georgia, USA
5
Tianwei Ma
College of Pharmacy, The University of Georgia, Athens, Georgia, USA
Krzysztof W. Pankiewicz, Steven E. Patterson, Abdalla E. A. Hassan,
Phillip M. Tharnish, Tamara R. McBrayer, Stefania Lostia, Lieven J. Stuyver, and
5
Kyoichi A. Watanabe
Pharmasset, Inc., Tucker, Georgia, USA
5
Chung K. Chu
College of Pharmacy, The University of Georgia, Athens, Georgia, USA
5
Raymond F. Schinazi
Department of Pediatrics, Emory University School of Medicine,
Atlanta, Georgia, USA and Veterans Affairs Medical Center, Decatur, Georgia, USA
5
Michael J. Otto
Pharmasset, Inc., Tucker, Georgia, USA
5
Based on the discovery of b-D-2’-deoxy-2’-fluorocytidine as a potent anti-hepatitis C virus (HCV)
agent, a series of b-D- and L-2’-deoxy-2’-fluororibonucleosides with modifications at 5 and/or 4
positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral
diarrhea virus (BVDV). The introduction of the 2’-fluoro group was achieved by either fluorination of
2,2’-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of
arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds,
namely b-D-2’-deoxy-2’,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon
cell line, and inhibitory activity against ribosomal RNA. As b-D-N4-hydroxycytidine (NHC) had
previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2’-fluoro
were combined into one molecule, yielding b-D-2’-deoxy-2’-fluoro-N4-hydroxycytidine (12). However,
this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were
devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the
BVDV system cannot reliably predict anti-HCV activity in vitro.
HCV replicon RNA-containing Huh7 cells were provided by Apath, LLC, St. Louis, MO. CKC and RFS are
supported in part by NIH grant RO1-AI-32351. CKC is supported in part by NIH grant 1-RO1-AI-25899. RFS is
supported by NIH grant 2P30-AI-50409, and the Department of Veterans Affairs. RFS and CKC are the founders
and consultants of Pharmasset, Inc., and did not receive any funding from the company for these studies.
Address correspondence to Junxing Shi, Pharmasset, Inc., Tucker, GA, USA.
875
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J. Shi et al.
INTRODUCTION
HCV is an important pathogen affecting nearly 170 million people worldwide.
HCV infections become chronic in about 50% of cases, and about 20% of these
chronically infected patients develop liver cirrhosis that can lead to hepatocellular
carcinoma. The current therapies, based on interferon-alpha (IFN-a) alone or
combination with ribavirin, are only moderately effective. Therefore, there is a need
for more effective anti-HCV agents. Recently, we reported that a sugar-fluorinated
nucleoside, b-D-2’-deoxy-2’-fluorocytidine (1), had potent anti-HCV activity.[1]
Based on the activity of this compound, a series of b-D- and L-analogues were
synthesized and evaluated against HCV in the HCV subgenomic replicon system
and BVDV. In addition, our earlier discovery of a base-modified nucleoside, NHC,
possessing potent anti-HCV activity,[2] prompted us to combine these two features
in one molecule. Herein, we report the synthesis and the biological evaluation of
several b-D- and L-2’-deoxy-2’-fluororibonucleosides.
RESULTS AND DISCUSSION
Two main approaches have been widely used for the synthesis of 2’-deoxy-2’-
fluororibonucleosides: 1) fluorination of anhydronucleosides with hydrogen
fluoride or potassium fluoride, and 2) fluorination of arabinonucleosides with
diethylaminosulfur trifluoride (DAST) or with tetrabutylammonium fluoride
(TBAF) via a sulfonate intermediate.
SCHEME 1 Synthesis of b-D- and L-2’-deoxy-2’-fluororibonucleosides.

J. Shi et al.
877
We utilized the direct fluorination approach for the preparation of b-D-2’-deoxy-
2’-fluorouridine (2) and D-2’-deoxy-2’-fluorocytidine (1) from their anhydronucleo-
sides by HF-pyridine or KF, as the literature reported.[3,4] The halogenation of 1
gave 5-halogenated analogues 2--4. For the preparation of 5-fluoro and 5-methyl
substituted analogues, DAST fluorination was adopted. Thus, 5-substituted 3’,5-
THP-protected arabinonucleosides were treated with DAST, resulting in the
protected 2’-deoxy-2’-fluoronucleosides. After deprotection and/or amination, 5-
substituted D-2’-deoxy-2’-fluoronucleosides 5 6 were obtained (Scheme 1).
b-D-2’-Deoxy-2’-fluoro-N4-hydroxycytidine (12) was synthesized from b-D-2’-
deoxy-2’-fluorouridine (7) by acetylation, sulfonation, hydroxyamination, and
deprotection (Scheme 1). 4-Thio analogue 10 and 4-methylthio analogue 11 were
synthesized by thioation and methylation, as described in the literature.[5] Similarly,
b-D-2’-deoxy-2’-fluoroadenosine (28) was prepared using DAST fluorination,
following a literature procedure.[6]
All the L-series 2’-deoxy-2’-fluororibonucleosides were synthesized by DAST
fluorination. The corresponding arabinonucleosides were prepared either by Holy’s
method,[7] or Vorbru¨ggen sugar-base condensation. After fluorination, the resulting
nucleosides were aminated and/or deprotected to give compounds 13--15 and
19--21, while the 5-halogenated (Cl, Br, I) nucleosides 16--18 and 22--24 were
prepared by halogenation of the corresponding nucleosides. 5-Ethynyl nucleoside
25 was synthesized from 5-iodouridine nucleosides via palladium-mediated
reaction, and hydrogenation of 25 resulted in 5-ethyl analogue 26 (Scheme 1).
b-L-5-Bromovinyl-2’-deoxy-2’-fluorouridine (27) was prepared by a published
procedure.[8]
The synthesized 2’-deoxy-2’-fluororibonucleosides were evaluated in BVDV
and HCV subgenomic replicon RNA-containing Huh7 cells, as described
previously.[2,9] Briefly, for the BVDV assay, Madin-Darby bovine kidney (MDBK)
cells were infected with cpBVDV (NADL strain) in DMEM/F12 media in the
presence or absence of test compounds. After a 3-day incubation, viral RNA was
extracted and analyzed using quantitative real-time RT-PCR (Q-RT-PCR). For the
HCV replicon assay, HCV subgenomic replicon RNA-containing Huh7 cells were
incubated in the presence or absence of tested compounds for 4 days. After
TABLE 1 Anti-BVDV and Anti-HCV Activity of 2’-Deoxy-2’-Fluororibonucleosides In Vitro
EC90
(mM)
BVDV
EC90
(mM)
HCV
CC50
(mM)
rRNA
Compound
Configuration
Base
4-Substitution
5-Substitution
F
1
5
11
12
D
D
D
D
C
C
U
C
>100
ND
>100
ND
5.4
5.6
9.35
>100
>100
5.0
ꢀ100
>100
<1
SCH3
NHOH
8.7
>100
>100
14.4
NHC
ribavirin
1.5
ND: not determined; EC90: effective concentration required for reducing the HCV RNA or BVDV levels by 90%
in 96 or 72 h; CC50: cytotoxic concentration required for reducing the rRNA levels by 50% in 96 h.

878
J. Shi et al.
incubation, total cellular RNA was extracted. Replicon RNA and an internal
control were amplified in a single-step multiplex RT-PCR protocol, as described
previously.[9] Recombinant interferon alfa-2a (for HCV) and ribavirin (for BVDV)
were used as positive controls in these experiments. The results using some
nucleoside analogues are summarized in Table 1 (the rest of the nucleosides
showed EC50 and CC50 values over 100 mM).
All the synthesized nucleosides showed no inhibitory activity against BVDV,
and all the L-series nucleosides showed no anti-HCV activity. In the D-series, among
5-substituted cytidine analogues, only b-D-2’-deoxy-2’,5-difluorocytidine ( 5)
exhibited anti-HCV activity and inhibitory activity against ribosomal RNA. The
5-chloro, 5-bromo, 5-iodo, and 5-methyl substituted 2’-deoxy-2’-fluorocytidine
analogues showed no anti-HCV activity. Uridine analogues b-D-2’-deoxy-2’-
fluorouridine, b-D-2’-deoxy-2,5’-difluorouridine, and b-D-2’-fluorothymidine were
not active against HCV. Replacement of the amino with a thiol group at the
4-position also resulted in an inactive compound 10. However, the 4-methylthio
analogue 11 demonstrated inhibition of ribosomal RNA. Surprisingly, the N4-
hydroxylamino analogue (12) showed neither activity against HCV nor inhibitory
activity to ribosomal RNA.
b-D-2’-Deoxy-2’-fluororibonucleoside analogues are known to be active against
some RNA viruses. Several analogues of this class showed high activity against
influenza A and B, and parainfluenza 1.[10] Also, b-D-2’-deoxy-2’-fluorocytidine has
demonstrated inhibitory activity against herpes simplex virus type 1 and 2,
pseudorabies and equine abortion virus.[11] It is surprising that our findings indicate
that b-D-2’-deoxy-2’-fluororibonucleosides possess anti-HCV activity, as some earlier
works on HCV polymerase concluded that in order to be recognized by HCV
RNA dependent RNA polymerase, a ribonucleoside was needed.[12] The discovery
that b-D-2’-deoxy-2’-fluorocytidine possesses potent anti-HCV activity suggests that
the 2’-fluoro instead of the 2’-hydroxyl group is recognized by the HCV RNA
polymerase. In terms of Van der Vaal radii, the fluorine atom is closer to a
hydrogen atom than a hydroxyl group. However, the fluorine atom may mimic the
hydroxyl group in terms of electronegativity and the ability to form a hydrogen
bond. The conformational study of 2’-fluorinated nucleosides also suggests that
2’-deoxy-2’-fluororibonucleosides are more like ribonucleosides than 2’-deoxyribo-
nucleosides, since it has been confirmed that both 2’-deoxy-2’-fluororibonucleosides
and ribonucleosides adopt a 3’-endo conformation.[13,14] However, the fact that the
anti-HCV activity of b-D-2’-deoxy-2’-fluorocytidine can be abolished by the addition
of 2’-deoxycytidine, not by cytidine[1] demonstrates that 2’-deoxy-2’-fluororibonu-
cleosides are recognized as 2’-deoxynucleosides in at least one step in the metabolic
pathway in HCV replicon cells.
It seems that the anti-HCV activity resides with the b-D-nucleosides. To the best
of our knowledge, no L-enantiomer has been reported to possess any specific anti-
HCV activity. As more L-nucleosides are evaluated against HCV in vitro, this
hypothesis will be further tested.

J. Shi et al.
879
In summary, a series of b-D- and L-2’-deoxy-2’-fluororibonucleosides were
synthesized and evaluated for in vitro anti-HCV and anti-BVDV activity, as well as
their inhibition of ribosomal RNA. The study revealed that b-D-2’-deoxy-
2’,5-difluorocytidine showed lower anti-HCV potency and higher toxicity against
ribosomal RNA than b-D-2’-deoxy-2’-fluorocytidine. All of the other 5-modified b-D-
nucleosides were not active against HCV, and all the L-series compounds were
devoid of anti-HCV activity. The 4-methylthio analogue exhibited inhibitory activity
against ribosomal RNA. None of the tested compounds demonstrated anti-BVDV
activity. Surprisingly, b-D-2’-deoxy-2’-fluoro-N4-hydroxylcytidine was neither active
nor toxic to liver cells.
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