We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.

New substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble intracellular prodrugs for the free bioactive phosphate to establish relationship(s) between compound structure and in vitro antiviral activity. The majority of compounds demonstrated an elevation of in vitro potency relative to that of the parent nucleoside, and unlike d4T, all retained full activity in thymidine kinase-deficient cells. The compound bearing a p-chloro aryl group (8e) expressed nanomolar activity in vitro, a 14-fold increase in activity relative to that of the unsubstituted phosphoramidate (100-fold compared to d4T). An assay using pig liver esterase was used to establish the stability of the compounds to enzymatic degradation. While there was no apparent correlation between in vitro activity and half-life of enzymatic degradation, there was a close correlation between compound lipophilicity, determined by octanol/water partition coefficient, and in vitro potency. We suggest that substitutions made to the aryl moiety of the aryl phosphoramidate of d4T that result in enhancing lipophilicity may serve to increase the cellular uptake of the prodrug by passive diffusion, leading to the expression of antiviral potency at reduced prodrug concentrations.