First Example of Phosphoramidate Approach Applied to a 4‘-Substituted Purine Nucleoside (4‘-Azidoadenosine):  Conversion of an Inactive Nucleoside to a Submicromolar Compound versus Hepatitis C Virus

Perrone, Plinio; Daverio, Felice; Valente, Rocco; Rajyaguru, Sonal; Martin, Joseph A.; Lévêque, Vincent; Pogam, Sophie Le; Nájera, Isabel; Klumpp, Klaus; Smith, David B.; McGuigan, Christopher

doi:10.1021/jm070362i

Cited by 67 publications

(49 citation statements)

References 24 publications

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“…For nucleosides that could not be efficiently converted to their monophosphate forms, one could synthesize the monophosphate or phosphonate prodrugs. The phosphoramidate approach was successfully used to convert inactive nucleoside analogs to potent inhibitors of HCV (23,24). Other examples of nucleotide phosphonates are adefovir disoproxil and tenofovir disoproxil, which are in clinical use for treatment of hepatitis B virus and HIV, respectively.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

Chen

Yin

Duraiswamy

et al. 2010

Antimicrob Agents Chemother

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We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxymethyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC 50 ) and cytotoxic concentration (CC 50 ) values of 0.7 M and >100 M, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14 C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.The family Flaviviridae consists of three genera, Flavivirus, Pestivirus, and Hepacivirus. Many viruses from the genus Flavivirus are arthropod-borne and cause significant human diseases. The four serotypes of dengue virus (DENV) alone pose a health threat to 2.5 billion people worldwide, leading to 50 to 100 million human infections and 500,000 cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) each year (11). Besides DENV, other flaviviruses, such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), also cause frequent outbreaks throughout the world. No clinically approved antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are available only for YFV, JEV, and TBEV. The development of a successful DENV vaccine has been challenging due to the facts that (i) the vaccine should simultaneously induce a long-lasting protection against all four DENV serotypes and (ii) an incompletely immunized individual may be sensitized to the lifethreatening DHF or DSS. The challenge associated with the development of a vaccine underlines the importance of antiviral development for DENV and other flaviviruses.The flavivirus genome is a single-strand RNA of plus-sense polarity. The single open reading frame from the 11-kb viral genome encodes three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Nonstructural proteins are responsible for viral replication. Of those, NS3 and NS5 have enzymatic activities. The N-terminal domain of NS3 (with NS2B as a cofactor) functions as a serine protease, and the C-terminal domain acts as an RNA helicase, an RNA triphosphatase, and an NTPase (10,34,35). The N-...

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Section: Discussionmentioning

confidence: 99%

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

Chen

Yin

Duraiswamy

et al. 2010

Antimicrob Agents Chemother

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“…Options to further increase the antiviral potency of nucleoside analogs as inhibitors of HCV replication include optimization of nucleotide analog incorporation efficiency by HCV NS5B, or optimization of phosphorylation efficiency to increase the intracellular concentration of the nucleoside triphosphate analog. In particular, the first step in the pathway to the triphosphate, the formation of nucleoside monophosphate, appears to be a limiting step for a majority of nucleoside analogs (9,26,27).…”

Section: Discussionmentioning

confidence: 99%

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Klumpp

Kalayanov

et al. 2008

Journal of Biological Chemistry

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RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2-␣-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of ␣-hydroxy moieties. 2-Deoxy-2-␤-fluoro-4-azidocytidine (RO-0622) and 2-deoxy-2-␤-hydroxy-4-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC 50 ‫؍‬ 171 ؎ 12 nM and 24 ؎ 3 nM for RO-9187 and RO-0622, respectively; CC 50 >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4-azidocytidine) or 2-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC 50 values 8 -150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2-␣-deoxy-4-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection. Hepatitis C virus (HCV)3 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation. Current treatment options available to HCV-infected persons have limitations with regard to efficacy and tolerability. Only about 50% of individuals infected with HCV genotype 1 achieve sustained virological response when treated with a combination of pegylated interferon ␣ and ribavirin (1, 2). In addition, high viral load, age, body weight, co-infection with human immunodeficiency virus, and cirrhosis negatively affect the probability of achieving sustained virological response (3, 4). Therefore, there is an urgent need to develop new and more effective therapies for the treatment of HCV infection. A number of new antiviral candidates are currently being evaluated in clinical studies, the majority targeting either the HCV protease or HCV polymerase enzymes, which are essential for viral replication (5). The HCV RNA-dependent RNA polymerase, NS5B, contains the active site responsible for viral RNA synthesis and functions as part of a membrane-associated replicase complex. Nucleoside and non-nucleoside inhibitors of HCV polymerase h...

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“…On the other hand, the method employing t -BuMgCl as a reagent has been successfully employed to prepare d4U and ddU, 170 ddA and d4A, 171 d - and l -carbocyclic d4A and ddA, 172 l -2′-deoxythreofuranosyl 3′-aryloxyphosphoramidate prodrugs, 173 3TC, 174 l -carbocyclic 2′,3′-dideoxy-2′,3′-didehydro-7-deazaadenosine, 175 2′,5′-dideoxyadenosine, 176 2′,3′-dideoxy-3′-fluoroadenosine, 177 2′-fluoro-6′-methylene-carbocyclic adenosine, 178 4′-azidouridine, 179 cytidine 180 and adenosine, 181 and 2′-methyl-4′-azidouridine and -cytidine 182 prodrugs (Figure 38). …”

Section: Nucleoside Monophosphate Prodrugsmentioning

confidence: 99%