The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile-Part 2.

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability.

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.

The application of the phosphoramidate ProTide approach confers micromolar potency against hepatitis C virus on inactive agent 4'-azidoinosine: kinase bypass on a dual base/sugar modified nucleoside.

Novel phosphoramidate ProTides derived from 4'-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low microM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.

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.

We report on the synthesis of the anti hepatitis C virus (HCV) agent 4'-azidoadenosine (1) and the application of the phosphoramidate ProTide technology to this nucleoside. The synthesis of 1 was achieved through an epoxide intermediate followed by regio- and stereoselective ring opening by azidotrimethylsilane in the presence of a Lewis acid. Compound 1 did not inhibit HCV replication in cell culture at concentrations up to 0.

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.

Anti-cancer ProTides: tuning the activity of BVDU phosphoramidates related to thymectacin.

Based on our wide ranging knowledge of phosphoramidate ProTides as anti-viral agents we have tuned the lead anti-cancer agent thymectacin in the ester and amino acid regions and revealed a substantial enhancement in in vitro potency versus colon and prostate cancer cell lines. Twelve analogues have been reported, with yields of 29-78%. The compounds are fully characterised and data clearly reveal the presence of two phosphate diastereoisomers, as expected, in roughly equi-molar proportions.

Enhanced inhibition of the EDHF phenomenon by a phenyl methoxyalaninyl phosphoramidate derivative of dideoxyadenosine.

In rabbit arteries endogenous production of cAMP facilitates electrotonic signalling via gap junctions, thus explaining the ability of P-site inhibitors of adenylyl cyclase to attenuate EDHF-type responses. In the present study, we show that a lipophilic phosphoramidate pronucleotide derivative of dideoxyadenosine, 2',3'-ddA-PMAPh, exhibits enhanced activity as an inhibitor of EDHF-type smooth muscle hyperpolarizations induced by acetylcholine (ACh) compared to the parent nucleoside 2',3'-ddA, and that the effects of both compounds can be reversed by the cAMP phosphodiesterase inhibitor IBMX. Neither 2',3'-ddA nor 2',3'-ddA-PMAPh depress ACh-evoked endothelial hyperpolarization directly.