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 novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A.

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.

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 0.1 mM. However, a submicromolar active agent could be derived from 1 by the application of the ProTide technology. All the phosphoramidates prepared were L-alanine derivatives with variations in the aryl moiety and in the ester part of the amino acid. The benzyl ester and the l-naphthyl phosphate (18) had the best activity in replicon assay. Phosphoramidates (18-21) achieved a significant improvement in antiviral potency over the parent nucleoside (1) with no increase in cytotoxicity.

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.

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. The compounds were evaluated in tissue culture versus three different tumour cell lines, using thymectacin as the control. It is notable that minor structural modification of the parent phenyl methoxyalaninyl structure of thymectacin leads to significant enhancements in potency. In particular, replacement of the methyl ester moiety in the lead by a benzyl ester gave a 175-fold boost in potency versus colon cancer HT115. This derivative emerges as a low micromolar inhibitor of HT115 cells and a new lead for further optimisation.

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. Modifications in the lipophilicity of dideoxyadenosine and its direct intracellular delivery as a mononucleotide may thus enhance the ability to inhibit adenylyl cyclase and depress electrotonic signalling via myoendothelial gap junctions.