Synthesis and evaluation of novel potent HCV NS5A inhibitors.

Judicious modifications to the structure of the previously reported HCV NS5A inhibitor 1, resulted in more potent anti-HCV compounds with similar and in some cases improved toxicity profiles. The synthesis of 19 new NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. For the most potent compounds chemical stability, stability in liver microsomes and inhibition of relevant CYP450 enzymes is also presented.

Synthesis and biological evaluation of new potent and selective HCV NS5A inhibitors.

NS5A inhibitors are a new class of direct-acting antiviral agents which display very potent anti-HCV activity in vitro and in humans. Rationally designed modifications to the central biphenyl linkage of a known NS5A series led to selection of several compounds that were synthesized and evaluated in a HCV genotype 1b replicon. The straight triphenyl linked compound 11a showed similar anti-HCV activity to the clinical compound BMS-790052 and a superior cytotoxicity profile in three different cell lines, with an EC(50) value of 26 pM and a therapeutic index of over four million in an HCV replicon assay. This triphenyl analog warrants further preclinical evaluation as an anti-HCV agent.

Synthesis of purine modified 2'-C-methyl nucleosides as potential anti-HCV agents.

Based on the anti-hepatitis C activity of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine, a series of new modified purine 2'-C-methyl nucleosides was prepared as potential anti-hepatitis C virus agents. Herein, we report the synthesis of both 6-modified and 2-modified purine 2'-C-methyl-nucleosides along with their anti-HCV replication activity and cytotoxicity in different cells.

Antiviral activity and tolerability of amdoxovir with zidovudine in a randomized double-blind placebo-controlled study in HIV-1-infected individuals.

BACKGROUND: Amdoxovir acts synergistically with zidovudine in vitro and the combination prevents or delays the selection of thymidine analogue and K65R mutations. In silico studies have shown that a reduced dose of zidovudine (200 mg) results in decreased zidovudine-monophosphate levels, associated with toxicity, while maintaining zidovudine-triphosphate levels, which are associated with antiviral effects. Here, we aimed to assess the short-term tolerability and antiviral activity of amdoxovir in combination with reduced and standard doses of zidovudine. METHODS: The study was a double-blind, placebo-controlled study in HIV-1-infected patients not receiving antiretroviral therapy and with plasma HIV-1 RNA > or =5,000 copies/ml. Patients were randomized to 10 days of twice-daily treatment with 200 mg zidovudine, 300 mg zidovudine, 500 mg amdoxovir, 500 mg amdoxovir plus 200 mg zidovudine or 500 mg amdoxovir plus 300 mg zidovudine. The mean change in viral load (VL) log(10) and area under the virus depletion curve (AUC(VL)) from baseline to day 10 were determined. Laboratory and clinical safety monitoring were performed. RESULTS: Twenty-four patients were enrolled. The mean VL log(10) change was 0.10 with placebo, -0.69 with zidovudine 200 mg, -0.55 with zidovudine 300 mg, -1.09 with amdoxovir, -2.00 with amdoxovir plus zidovudine (200 mg) and -1.69 with amdoxovir plus zidovudine (300 mg). Amdoxovir plus zidovudine (200 mg) was significantly more potent than amdoxovir monotherapy in AUC(VL) and mean VL decline (P=0.019 and P=0.021, respectively), suggesting synergy. There was markedly decreased VL variability with the combination compared with amdoxovir alone. All adverse events were mild to moderate. CONCLUSION: The combination of amdoxovir plus zidovudine appeared synergistic with reduced VL variability. This combined therapy, including the use of a lower zidovudine dosage, warrants further development for the therapy of HIV infection.

Lack of pharmacokinetic interaction between amdoxovir and reduced- and standard-dose zidovudine in HIV-1-infected individuals.

Amdoxovir (AMDX) inhibits HIV-1 containing the M184V/I mutation and is rapidly absorbed and deaminated to its active metabolite, beta-D-dioxolane guanosine (DXG). DXG is synergistic with zidovudine (ZDV) in HIV-1-infected primary human lymphocytes. A recent in silico pharmacokinetic (PK)/enzyme kinetic study suggested that ZDV at 200 mg twice a day (b.i.d.) may reduce toxicity without compromising efficacy relative to the standard 300-mg b.i.d. dose. Therefore, an intense PK clinical study was conducted using AMDX/placebo, with or without ZDV, in 24 subjects randomized to receive oral AMDX at 500 mg b.i.d., AMDX at 500 mg plus ZDV at 200 or 300 mg b.i.d., or ZDV at 200 or 300 mg b.i.d. for 10 days. Full plasma PK profiles were collected on days 1 and 10, and complete urine sampling was performed on day 9. Plasma and urine concentrations of AMDX, DXG, ZDV, and ZDV-5'-O-glucuronide (GZDV) were measured using a validated liquid chromatography-tandem mass spectrometry method. Data were analyzed using noncompartmental methods, and multiple comparisons were performed on the log-transformed parameters, at steady state. Coadministration of AMDX with ZDV did not significantly change either of the plasma PK parameters or percent recovery in the urine of AMDX, DXG, or ZDV/GZDV. Larger studies with AMDX/ZDV, with a longer duration, are warranted.

Anti-hepatitis C virus activity of novel beta-d-2'-C-methyl-4'-azido pyrimidine nucleoside phosphoramidate prodrugs.

BACKGROUND: 2'-C-methyl and 4'-azido nucleosides have previously demonstrated inhibition of hepatitis C virus (HCV) replication by targeting the RNA-dependent RNA polymerase NS5B. In an effort to discover new and more potent anti-HCV agents, we envisioned synthesizing nucleoside analogues by combining the 2'-C-methyl-moiety with the 4'-azido-moiety into one molecule. METHODS: 2'-C-methyl-4'-azido pyrimidine nucleosides were synthesized by first converting 2'-C-methyl ribonucleosides to the corresponding 4'-exocyclic methylene nucleosides. Treatment with iodine azide, benzoylation of the 2'- and 3'-hydroxy groups, oxidative displacement of the 5'-iodo group with meta-chloroperoxybenzoic acid, and debenzoylation gave the desired 2'-C-methyl-4'-azido uridine and thymidine analogues in good yield. Standard conversion of uridine to cytidine via the 4-triazole yielded 2'-C-methyl-4'-azido cytidine. In addition, 5'-phosphoramidate derivatives of 2'-C-methyl-4'-azido uridine and cytidine were synthesized to bypass the initial phosphorylation step. RESULTS: The prepared nucleosides and their 5'-monophosphate prodrugs were evaluated for their ability to inhibit replication of the hepatitis C virus in a subgenomic replicon cell based assay. Cytotoxicity in Huh7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and ribosomal RNA. Among the newly synthesized compounds, only the 5'-monophosphate nucleoside prodrugs had modest and selective anti-HCV activity. All prepared pyrimidine nucleosides and 5'-monophosphate nucleoside prodrugs displayed no evidence of cytotoxicity at high concentrations. CONCLUSIONS: This work is the first example of both inactive uridine and cytidine analogues of a nucleoside being converted to active anti-HCV nucleosides via 5'-monophosphate prodrugs.

Synthesis and biological activity of 5',9-anhydro-3-purine-isonucleosides as potential anti-hepatitis C virus agents.

In order to study structure-activity relationships among the derivatives and congeners of 5',9-anhydro-3-(beta-D-ribofuranosyl)xanthine for anti-hepatitis C virus activity, a series of 5',9-anhydro-purine-isonucleosides with a substituent (s) at 6- or/and 8-position of the purine moiety were synthesized, and their anti-hepatitis C virus activity and cytotoxicity were evaluated and discussed.

Inhibition of hepatitis C replicon RNA synthesis by beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine: a specific inhibitor of hepatitis C virus replication.

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.

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.

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.

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

We recently discovered a novel compound, identified as N3, 5-cyclo-4-(beta-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 microM in replicon cells. Its 3'-deoxy sugar analogue was also synthesized, but was inactive against HCV in vitro.

Synthesis and anti-hepatitis C virus activity of nucleoside derivatives of N3, 5'-anhydro-4-(beta-D-ribofuranosyl)-8-aza-purin-2-ones.

A series of N3, 5-Anhydro-4-(beta-D-ribofuranosyl)-8-azapurin-2-ones were prepared in multistep reactions from uridine as potential anti-hepatitis C virus (HCV) agents. The synthetic details as well as biological evaluations are discussed.

Synthesis and structure-activity relationships of novel anti-hepatitis C agents: N3,5'-cyclo-4-(beta-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one derivatives.

Several 6- and 7-monosubstituted N3,5'-cyclo-4-(beta-d-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one derivatives as well as the 5-thiono analogue were synthesized, providing structure-anti-hepatitis C virus (HCV) activity relationships for the series. Among the compounds synthesized, the 6-bromo, 7-methylamino, and 5-thiono analogues exhibited more potent anti-HCV activity in an HCV subgenomic replicon cell based assay (EC90 = 1.9, 7.4, and 10.0 microM, respectively) than the lead compound N3,5'-cyclo-4-(beta-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one (EC90 = 79.8 microM).

Cytostatic 6-arylpurine nucleosides. 6. SAR in anti-HCV and cytostatic activity of extended series of 6-hetarylpurine ribonucleosides.

Significant anti-HCV activity of 6-hetarylpurine ribonucleosides has been discovered and is reported here for the first time and compared with cytostatic effect. An extended series of 6-hetarylpurine nucleosides has been prepared by heterocyclizations in position 6 of purine nucleosides or by cross-couplings of 6-chloropurine nucleosides with hetarylboronic acids, -stannanes, or -zinc halides. The most anti-HCV active were purine ribonucleosides bearing pyrrol-3-yl or 2-furyl groups exerting EC(90) = 0.14 and 0.4 microM, respectively.

Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication.

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.

Synthesis and anti-viral activity of a series of d- and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic HCV replicon system.

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of 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 key step in the synthesis, the introduction of 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 compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). 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 always predict anti-HCV activity.

Synthesis of N3,5'-cyclo-4-(beta-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridin-5-one, a novel compound with anti-hepatitis C virus activity.

A novel anti-hepatitis C virus (HCV) agent, N(3),5'-cyclo-4-(beta-D-ribofuranosyl)-vic-triazolo[4,5-b]pyridinin-5-one, was identified, and the structure was confirmed by chemical synthesis from 2-hydroxy-5-nitropyridine.

Inhibition of the subgenomic hepatitis C virus replicon in huh-7 cells by 2'-deoxy-2'-fluorocytidine.

2'-Deoxy-2'-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5'-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.

Manadomanzamines A and B: a novel alkaloid ring system with potent activity against mycobacteria and HIV-1.

Two novel alkaloids, named manadomanzamines A (1) and B (2), were isolated from an Indonesian sponge Acanthostrongylophora sp. (Haplosclerida: Petrosiidae). Their structures were elucidated and shown to be a novel organic skeleton related to the manzamine type alkaloids. Their absolute configuration and conformation were determined by CD, NOESY, and molecular modeling analysis. The microbial community analysis for the sponge that produces these unprecedented alkaloids has also been completed. Manadomanzamines A (1) and B (2) exhibited strong activity against Mycobacterium tuberculosis (Mtb) with MIC values of 1.9 and 1.5 mug/mL, respectively. Manadomanzamines A and B also exhibit activities against human immunodeficiency virus (HIV-1) and AIDS opportunistic fungal infections.

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites.

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.