The application of phosphoramidate ProTide technology to the potent anti-HCV compound 4'-azidocytidine (R1479).

We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the potent anti-HCV compound 4'-azidocytidine (R1479), with variation on the ester, amino acid and aryl moiety of the ProTide. Sub-muM inhibitors of HCV emerge. The compounds are all non-cytotoxic in the replicon assay. We herein report detailed SARs for each of the regions of the ProTide.

Application of the phosphoramidate ProTide approach to 4'-azidouridine confers sub-micromolar potency versus hepatitis C virus on an inactive nucleoside.

We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4'-azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4'-Azidouridine did not inhibit HCV, although 4'-azidocytidine was a potent inhibitor of HCV replication under similar assay conditions. However 4'-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively deliver 4'-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy.

Bicyclic nucleoside inhibitors of varicella-zoster virus modified on the sugar moiety: 3' and 5' derivatives.

Bicyclic furanopyrimidine nucleoside analogues (BCNAs) have been previously reported as potent and selective anti-varicella-zoster virus (VZV) agents. Few modifications on the sugar moiety have been considered so far but some of them have shown interesting activity against human cytomegalovirus (HCMV) while losing activity against VZV. In addition, recent work has led to an entirely new series of anti-HCMV bicyclic furopyrimidine agents, acting through a non-nucleoside mechanism. In order to further investigate structure-activity relationship studies on the sugar moiety, some 3'- and 5'-chloro derivatives and 5'-deoxygenated derivatives have been synthesized. The lack of anti-VZV activity of the 5'-modified derivatives is further proof of a mechanism of action involving VZV thymidine kinase (TK)-mediated phosphorylation. Similarly, the replacement of the 3'-OH with chlorine showed a decrease of antiviral activity, which can be correlated to the lack of interaction with VZV TK as demonstrated by enzyme assays. These results confirm free 5'-OH and 3'-OH as necessary requirements for efficient recognition by VZV TK and for potent anti-VZV activity in cell culture.

Preclinical development of bicyclic nucleoside analogues as potent and selective inhibitors of varicella zoster virus.

OBJECTIVES: To progress the anti-varicella-zoster-virus (VZV) aryl bicyclic nucleoside analogues (BCNAs) to the point of Phase 1 clinical trial for herpes zoster. METHODS: A new chromatography-free synthetic access to the lead anti-VZV aryl BCNAs is reported. The anti-VZV activity of lead Cf1743 was evaluated in monolayer cell cultures and organotypic epithelial raft cultures of primary human keratinocytes. Oral dosing in rodents and preliminary pharmacokinetics assessment was made, followed by an exploration of alternative formulations and the preparation of pro-drugs. We also studied uptake into cells of both parent drug and pro-drug using fluorescent microscopy and biological assays. RESULTS: Cf1743 proved to be significantly more potent than all reference anti-VZV compounds as measured either by inhibition of infectious virus particles and/or by viral DNA load. However, the very low water solubility of this compound gave poor oral bioavailability (approximately 14%). A Captisol admixture and the 5'-monophosphate pro-drug of Cf1743 greatly boosted water solubility but did not significantly improve oral bioavailability. The most promising pro-drug to emerge was the HCl salt of the 5'-valyl ester, designated as FV-100. Its uptake into cells studied using fluorescent microscopy and biological assays indicated that the compound is taken up by the cells after a short period of incubation and limited exposure to drug in vivo may have beneficial effects. CONCLUSIONS: On the basis of its favourable antiviral and pharmacokinetic properties, FV-100 is now being pursued as the clinical BCNA candidate for the treatment of VZV shingles.

Bicyclic nucleoside inhibitors of Varicella-Zoster virus: the effect of branching in the p-alkylphenyl side chain.

Further to the discovery of bicyclic furanopyrimidine nucleoside analogues (BCNAs) as potent anti-VZV agents, a branched series of this family of compounds was synthesised. The aim was to study the impact of the geometry and steric hindrance in the side chain as well as lipophilic role of this moiety on biological activity. The results showed a detrimental effect of branching on antiviral activity, with a different magnitude depending on the position of branching in the side chain. This study again showed the importance of this moiety for biological activity, as well as the limited efficacy of the ClogP value as a tool for predicting the potency of BCNAs, while suggesting an alternative rationale behind the design of future series.