Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.     

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.     

Synthesis and anti-HCV activity of 3′,4′-oxetane nucleosides

Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials. As part of our continuing efforts to discover novel anti-HCV agents, 3′,4′-oxetane cytidine and adenosine nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 2′-mono/diflouro analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro. Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be converted to their corresponding diphosphates.     

Cyclic monophosphate prodrugs of base-modified 2'-C-methyl ribonucleosides as potent inhibitors of hepatitis C virus RNA replication

A new series of heterobase-modified 2'-C-methyl ribonucleosides was synthesized and tested as inhibitors of hepatitis C virus (HCV) RNA replication. The nucleosides showed a weak inhibitory activity in a HCV replicon system (EC(50)=92 microM) and did not exhibit any cytotoxicity (CC(50)>300 microM). Cyclic monophosphate (cMP) prodrugs of the same nucleosides were synthesized and also tested in the HCV replicon system. Prodrugs exhibited strong potency (EC(50)=0.008 microM) without significant cytotoxicity (CC(50)>50 microM).     

Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl purine nucleosides as inhibitors of hepatitis C virus RNA replication

A series of purine nucleosides containing the 2'-deoxy-2'-fluoro-2'-C-methylribofuranosyl moiety were synthesized and evaluated as potential inhibitors of the hepatitis C virus in vitro. Of the nucleosides that were synthesized, only those possessing a 2-amino group on the purine base reduced the levels of HCV RNA in a subgenomic replicon assay.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

Benzimidazole inhibitors of hepatitis C virus NS5B polymerase: identification of 2-[(4-diarylmethoxy)phenyl]-benzimidazole

A series of 1-cycloalkyl-2-phenyl-1H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp). A SAR study was performed and led to identify the 2-[(4-diarylmethoxy)phenyl]-benzimidazoles as potent inhibitors. They inhibit subgenomic HCV RNA replication in the replicon cells at low micromolar concentrations (EC(50) as low as 1.1microM). They are selective against DNA polymerases (IC(50)>10microM) and exhibit low cytotoxicity.     

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).     

Synthesis of Novel 4′α-Trifluoromethyl-2′β-C-Methyl-Carbodine Analogs as Anti-Hepatitis C Virus Agents

Novel 4?′α-trifluoromethyl-2?′β-methyl carbocyclic nucleoside analogs have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell cultures. Construction of cyclopentene intermediate 10a was achieved via sequential Johnson–Claisen orthoester rearrangement and ring-closing metathesis starting from the α-trifluoromethyl-α,β-unsaturated ester 5. Stereoselective dihydroxylation and desilylation yielded the target carbodine analogs. The synthesized nucleoside analogs mentioned above (18 and 19) were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides showed neither significant antiviral activity nor toxicity up to 50 μM.     

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.     

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).     

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Synthesis and in vitro anti-HCV activity of beta-D- and 1-2'-deoxy-2'-fluororibonucleosides

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides 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 beta-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 beta-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 beta-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.     

6-Hydrazinopurine 2'-methyl ribonucleosides and their 5'-monophosphate prodrugs as potent hepatitis C virus inhibitors

A series of 6-hydrazinopurine 2'-methyl ribonucleosides was synthesized and tested for its inhibitory activity against the hepatitis C virus (HCV). The lack of antiviral activity of these nucleosides was associated with a poor affinity for adenosine kinase, which prompted us to synthesize several of their 5'-monophosphate prodrugs. Some of these prodrugs exhibited more than 1000-fold improvement in anti-HCV activity when compared to their parent nucleosides (EC(50) of 24 nM vs 92 microM for the parent).     

New furano- and pyrrolo[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidine nucleosides and their 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphates: Synthesis and biological properties

Bicyclic furano[2,3-d]pyrimidine ribonucleosides were synthesized by Pd(0)-and CuI-catalyzed coupling of 5-iodouridine with terminal alkynes. The treatment of the resulting nucleosides with ammonia or methylamine solution in aqueous alcohol resulted in pyrrolo-and N ⁷-methylpyrrolo[2,3-d]pyrimidine nucleosides. 5′-O-Triphosphates of bicyclic nucleosides were obtained by the treatment of the nucleosides with POCl₃ in the presence of a “proton sponge.” The 5′-O-triphosphates are not substrates for HCV RNA-dependent RNA polymerase, but are effective substrates for HCV RNA helicase/NTPase and did not inhibit ATP hydrolysis. Only 3-(β-D-ribofuranosyl)-6-decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one showed a moderate anti-HCV activity in the HCV replicon system and efficiently inhibited replication of bovine viral diarrhea virus (BVDV) in KCT-cells, other compounds being inactive. None of the compounds were cytotoxic within the tested range of concentrations.     

Identification of [(naphthalene-1-carbonyl)-amino]-acetic acid derivatives as nonnucleoside inhibitors of HCV NS5B RNA dependent RNA polymerase

A novel series of HCV NS5B RNA dependent RNA polymerase inhibitors containing a naphthalene carboxamide scaffold were identified by high throughput screening. Optimization of substituents by parallel synthesis and the iterative design towards understanding structure-activity relationship to improve potency are described. Tetra substituted naphthalene 31 displayed potent activity with IC(50) of 120 nM against HCV NS5B enzyme and was selective over a panel of polymerases.     

Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) and identification of a novel active 5'-triphosphate species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.     

I. Novel HCV NS5B polymerase inhibitors: discovery of indole 2-carboxylic acids with C3-heterocycles

SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 μM, replicon EC(50)>100 μM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 μM, replicon EC(50)=1.4 μM) and 7r (NS5B IC(50)=0.017 μM, replicon EC(50)=0.3 μM) with improved enzyme and replicon activity.