Synthesis of N3,5′-cyclo-4-(β-D-ribofuranosyl)-vic- triazolo[4,5-b]pyridin-5-one and its 3′-deoxysugar analogue as potential anti-hepatitis C virus agents

Article abstract of DOI:10.1081/NCN-200059314

We recently discovered a novel compound, identified as N ³,5′-cyclo-4-(β-D-ribofuranosyl)-vic-triazolo[4,5-b] pyridinin-5-one, with anti-hepatitis C virus (HCV) activity in vitro. The structure was confirmed by chemical synthesis from 2-hydroxy

Full text of DOI:10.1081/NCN-200059314

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SYNTHESIS OF N3,5′-CYCLO-4-(β-D-RIBOFURANOSYL)-
VIC-TRIAZOLO[4,5-b]PYRIDIN-5-ONE AND ITS 3′-
DEOXYSUGAR ANALOGUE AS POTENTIAL ANTI-HEPATITIS
C VIRUS AGENTS
Peiyuan Wang a , Laurent Hollecker a , Krzysztof W. Pankiewicz a , Steven E. Patterson
a , Tony Whitaker b , Tamara R. McBrayer b , Phillip M. Tharnish b , Lieven J. Stuyver b ,
Raymond F. Schinazi c , Michael J. Otto b & Kyoichi A. Watanabe a
a Department of Chemistry, Pharmasset, Inc., Tucker, Georgia, USA
b Department of Biology, Pharmasset, Inc., Tucker, Georgia, USA
c Department of Pediatrics, Emory University, School of Medicine/Veterans Affairs Medical
Center, Decatur, Georgia, USA
Published online: 15 Nov 2011.
To cite this article: Peiyuan Wang , Laurent Hollecker , Krzysztof W. Pankiewicz , Steven E. Patterson , Tony Whitaker ,
Tamara R. McBrayer , Phillip M. Tharnish , Lieven J. Stuyver , Raymond F. Schinazi , Michael J. Otto & Kyoichi A. Watanabe
(2005): SYNTHESIS OF N3,5′-CYCLO-4-(β-D-RIBOFURANOSYL)-VIC-TRIAZOLO[4,5-b]PYRIDIN-5-ONE AND ITS 3′-DEOXYSUGAR
ANALOGUE AS POTENTIAL ANTI-HEPATITIS C VIRUS AGENTS, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 957-960
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):957–960, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059314
SYNTHESIS OF N3,5’-CYCLO-4-(b-D-RIBOFURANOSYL)-vic-
TRIAZOLO[4,5-b]PYRIDIN-5-ONE AND ITS 3’-DEOXYSUGAR
ANALOGUE AS POTENTIAL ANTI-HEPATITIS C VIRUS AGENTS
Peiyuan Wang, Laurent Hollecker, Krzysztof W. Pankiewicz, and
5
Steven E. Patterson
Department of Chemistry, Pharmasset, Inc., Tucker, Georgia, USA
Tony Whitaker, Tamara R. McBrayer, Phillip M. Tharnish, and
5
Lieven J. Stuyver
Department of Biology, Pharmasset, Inc., Tucker, Georgia, USA
5
Raymond F. Schinazi
Department of Pediatrics, Emory University, School of Medicine/
Veterans Affairs Medical Center, Decatur, Georgia, USA
5
Michael J. Otto
Department of Biology, Pharmasset, Inc., Tucker, Georgia, USA
5
Kyoichi A. Watanabe
Department of Chemistry, Pharmasset, Inc., Tucker, Georgia,
USA
We recently discovered a novel compound, identified as N3,5’-cyclo-4-(b-D-ribofuranosyl)-vic-
triazolo[4,5-b]pyridinin-5-one, with anti-hepatitis C virus (HCV) activity in vitro. The structure was
confirmed by chemical synthesis from 2-hydroxy-5-nitropyridine. It showed anti-HCV activity with
EC50 =19.7 mM in replicon cells. Its 3’-deoxy sugar analogue was also synthesized, but was inactive
against HCV in vitro.
5
INTRODUCTION
Since hepatitis C virus (HCV) is responsible for the second most common
cause of viral hepatitis, much attention has been given toward the discovery of
clinically useful anti-HCV agents. HCV is an RNA virus that replicates without the
Supported in parts by NIH grants 1R43 AI-52868 (biology) and 1 R43 AI-056720 (chemistry). We would like
to acknowledge the contribution of D. L. Barnard, J. D. Morrey, and D. F. Smee at the Institute for Antiviral
Research at Utah State University for their testing of RNA viruses under NIH Contract No. N01-A1-85348. Dr.
Schinazi is a founder and consultant of Pharmasset, Inc. His laboratory did not receive any funding from
Pharmasset, Inc.
Parts of this work were presented at the International Society for Nucleosides, Nucleotides & Nucleic Acids,
XVI International Roundtable, Minneapolis, Minnesota, USA, September 12–16, 2004.
Address correspondence to Peiyuan Wang, Department of Chemistry, Pharmasset, Inc., Tucker, GA 30084,
USA.
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P. Wang et al.
involvement of DNA. It is one of the most important Flaviridae infections in
humans and an estimated 170 million people worldwide are HCV carriers.[1]
Currently, there is no effective cure for this disease and the only medicines available
are alpha interferon, either alone or in combination with ribavirin.[2] However, the
therapeutic value of these treatments has been compromised largely due to adverse
effects,[2,3] which highlights the need for the development of additional options
for treatment.
RESULTS AND DISCUSSION
Chemistry
Recently, a novel HCV agent was identified by our group as N3,5’-cyclo-4-(b-D-
ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one[4] from Pharmasset’s compound li-
brary (6, Scheme 1). Compound 6 was synthesized from the known 1-(2,3,5-tri-O-
benzoyl-b-D-ribofuranosyl)-5-nitropyridin-2-one (2a),[5] which was obtained in 70%
yield by condensation of the trimethylsilylated pyridine with 1-O-acetyl-2,3,5-tri-O-
benzoyl-D-ribofuranose (1) under Vorbru¨ggen’s conditions. The 1H NMR spectrum
of the product was consistent with those previously reported.[5] The benzoyl
protecting groups were removed and 1-(b-D-ribofuranosyl)-5-nitropyridin-2-one (2b)
was obtained in 81% yield. Treatment of 2b with sodium azide in DMF at 110–
120°C for 12 hr afforded the vic-triazolopyridine nucleoside 3 in 60% yield. Reaction
of 2a with NaN3 in DMF at 80–95°C afforded only 4-(2,3,5-tri-O-benzoyl-b-D-
ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one (4). However, 5 was produced when
the reaction temperature was increased to 110°C or higher with the concomitant
SCHEME 1 Reagent: (a) silylated 5-nitropyrimidine-2-one/SnCl4/DCE; (b) NaN3, DMF, (at 110–120°C, 12 h,
from 2b to 3; at 80–95°C, 24 h NaN3, DMF, from 2a to 4; at 110–120°C, 48 h, from 2a to 5); (c) NH3/MeOH, rt;
(d) 0.5 M NaOMe in MeOH, rt.

P. Wang et al.
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SCHEME 2 Reagent: (a) silylated 5-nitropyrimidine-2-one/SnCl4; (b) NaOMe/MeOH; (c) BzCl/Py; (d) NaN3,
DMF, 115°C, 4 d; (e) NaOMe/MeOH.
decrease of 4. Compound 5 was obtained in 44% yield after prolonged heating.
Saponification of 5 with MeONa/MeOH furnished 6 in 62% yield.
The 3’-deoxy analogue 12 was synthesized by the condensation of 1,2-O-acetyl-
5-O-methoxycarbonyl-3-deoxy-D-glyceropentofuranose (7, Scheme 2) with 5-nitro-
pyridine-2-one to afford 8 in 79.5% yield as the first step. The methoxycarbonyloxy
group at C-5’ was not a good leaving group, and underwent saponification upon
treatment with two equivalent of NaOMe/MeOH to produce the free nucleoside 9.
Without purification, 9 was benzoylated to 10 in 97% yield from 8. Treatment of
10 with 1.5 equivalent of NaN3 in DMF at 115°C for 4 days gave 11.
Saponification of 11 with 1.5 equivalent of NaOMe/MeOH at room temperature
for 1 hr afforded 12 in 62% yield as colorless solid after silica gel chromatographic
purification with a stepwise gradient of MeOH (0 to 4%) in CH2Cl2.
Antiviral Assay
Compound 6 was evaluated in the HCV subgenomic RNA provided by
Apath LLC (St. Louis, MO).[6] Compound 6 had an anti-HCV effect with an
EC50=19.7 mM and EC90=79.8 mM. In addition to the antiviral effect, ribosomal
RNA was also reduced. Huh7 replicon cells were kept in culture for 7 days, either
in presence (100 mM) or in absence of the compound. These experiments showed
that 6 caused a cytostatic effect at high concentrations (CC50 = 30.6 mM). Con-
comitantly with the slower cell proliferation, a significant decrease in intracellular
HCV RNA was observed. Compound 6 does not inhibit purified HCV RNA-
dependent RNA polymerase (NS5B) in vitro when tested up to 100 mM.[7] In
addition, 6 was tested against a range of other RNA viruses including Influenza
viruses A and B, respiratory syncytial virus, rhinovirus, parainfluenza virus,
Pinchinde virus, Venezuelian equine encephalitis virus, yellow fever, West Nile,

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P. Wang et al.
adenovirus type 1, Punta Toro A, hepatitis B virus, and bovine viral diarrhea virus.
The compound was inactive and generally nontoxic,[8–10] with the exception of
weak activity against influenza B virus with an EC50 of 28 mM. Compound 12
showed no activity in the HCV replicon system. As expected, 6 was inactive against
DNA viruses, such as HIV-1 in primary human lymphocytes and HSV type 1 in
Vero cells.[9]
CONCLUSIONS
A novel, potential anti-HCV agent was discovered from the Pharmasset
compound library. The structure was identified as N3,5’-cyclo-4-(b-D-ribofuranosyl)-
vic-triazolo[4,5-b]pyridin-5-one (6) and confirmed by chemical synthesis. Compound
6 inhibited production of HCV-RNA, in the HCV-subgenomic replicon cell line
(Huh7 cells) with EC50 = 19.7 mM and CC50 = 30.6 mM. Compound 6 did not
inhibit HCV-RNA polymerase in vitro, suggesting that this nucleoside does not
interact with this viral enzyme. The 3’-deoxy analogue 12 was synthesized and it
showed no antiviral activity in the replicon system, suggesting that the presence of a
3’-OH group was important for anti-HCV activity.
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