RESUMO
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.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular , Humanos , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos de Purina/química , Nucleosídeos de Purina/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Humanos , Nucleosídeos de Purina/síntese químicaRESUMO
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.
Assuntos
Fármacos Anti-HIV/farmacocinética , Dioxolanos/farmacocinética , Interações Medicamentosas , Infecções por HIV/tratamento farmacológico , Nucleosídeos de Purina/farmacocinética , Inibidores da Transcriptase Reversa/farmacocinética , Zidovudina/farmacocinética , Adulto , Fármacos Anti-HIV/administração & dosagem , Dioxolanos/administração & dosagem , Esquema de Medicação , Feminino , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Nucleosídeos de Purina/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Resultado do Tratamento , Adulto Jovem , Zidovudina/administração & dosagemRESUMO
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.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Amidas , Antivirais/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citidina/análogos & derivados , Humanos , Ácidos Fosfóricos , Pró-Fármacos/uso terapêutico , Nucleosídeos de Pirimidina/uso terapêutico , Uridina/análogos & derivados , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos de Purina/química , Nucleosídeos de Purina/farmacologia , Antivirais/síntese química , Células Cultivadas , Humanos , Nucleosídeos de Purina/síntese química , RNA Viral/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Replicon/genética , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/toxicidade , Linhagem Celular , Desoxicitidina/farmacologia , Desoxicitidina/toxicidade , Hepacivirus/fisiologia , Humanos , Camundongos , RNA Viral/biossínteseRESUMO
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.
Assuntos
Antivirais , Hepacivirus/efeitos dos fármacos , Nucleosídeos de Purina , RNA Viral , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hepacivirus/genética , Estrutura Molecular , Nucleosídeos de Purina/síntese química , Nucleosídeos de Purina/farmacologia , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Replicon/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Flúor/química , Hepacivirus/metabolismo , Ribonucleosídeos/química , Animais , Bovinos , Linhagem Celular , Química Farmacêutica/métodos , Desoxicitidina/síntese química , Desoxicitidina/farmacologia , Vírus da Diarreia Viral Bovina/metabolismo , Desenho de Fármacos , Fluoretos/farmacologia , Humanos , Ácido Fluorídrico/química , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/virologia , Modelos Químicos , Biologia Molecular/métodos , Compostos de Potássio/farmacologia , Nucleosídeos de Pirimidina/química , RNA/química , RNA Ribossômico/química , Ribonucleosídeos/farmacologia , EstereoisomerismoRESUMO
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.
Assuntos
Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Nucleosídeos/síntese química , Antivirais/farmacologia , Carboidratos/química , RNA Polimerases Dirigidas por DNA/química , Desoxiaçúcares/química , Genoma Viral , Hepacivirus/genética , Humanos , Modelos Químicos , Nucleosídeos/química , Ribonucleosídeos/química , Proteínas não Estruturais Virais/químicaRESUMO
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.
Assuntos
Antivirais/farmacologia , Química Farmacêutica/métodos , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Nucleosídeos de Purina/química , Linhagem Celular , Sistema Livre de Células , Desenho de Fármacos , Humanos , Modelos Químicos , Nucleosídeos de Purina/síntese química , Nucleosídeos de Purina/farmacologia , RNA/química , RNA Polimerase Dependente de RNA/química , Ribonucleosídeos , Uridina/químicaRESUMO
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).
Assuntos
Antivirais/síntese química , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hepacivirus/efeitos dos fármacos , Nucleosídeos/síntese química , Antivirais/química , Antivirais/farmacologia , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Hepacivirus/genética , Humanos , Nucleosídeos/química , Nucleosídeos/farmacologia , RNA Viral/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antineoplásicos/síntese química , Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Nucleosídeos de Purina/síntese química , Ribonucleosídeos/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Nucleosídeos de Purina/química , Nucleosídeos de Purina/farmacologia , Ribonucleosídeos/química , Ribonucleosídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antivirais/síntese química , Desoxicitidina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Cristalografia por Raios X , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Desenho de Fármacos , Hepacivirus/fisiologia , Estrutura Molecular , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Desoxirribonucleosídeos/síntese química , Desoxirribonucleosídeos/farmacologia , Genoma Viral , Hepacivirus/efeitos dos fármacos , Replicon , Animais , Bovinos , Hepacivirus/genética , Hepacivirus/fisiologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Nucleosídeos/síntese química , Antivirais/química , Antivirais/farmacologia , Células Cultivadas , Hepacivirus/genética , Humanos , Nucleosídeos/química , Nucleosídeos/farmacologia , RNA Viral/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
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.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , RNA Viral/biossíntese , RNA Viral/genética , Replicon/efeitos dos fármacos , Replicon/genética , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Vírus da Diarreia Viral Bovina/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Fase S/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
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.
Assuntos
Antimetabólitos/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/farmacologia , RNA Viral/análise , Replicon/efeitos dos fármacos , Hepacivirus/genética , Humanos , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.
Assuntos
Antivirais/farmacologia , Zalcitabina/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/síntese química , Antivirais/química , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Células Vero , Zalcitabina/análogos & derivados , Zalcitabina/síntese química , Zalcitabina/químicaRESUMO
A base-modified nucleoside analogue, beta-D-N(4)-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC(90)) of 5.4 microM, an observation that was confirmed by virus yield assays (EC(90) = 2 microM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC(90) of 5 microM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2'-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 micro M) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.