Design, synthesis, and evaluation of anti-orthoflavivirus Anchimerically HINT1 Activatable (AHA) ProTides.

Abstract

Orthoflaviviruses continue to pose a growing global health burden as climate change, urbanization, and diagnostic challenges drive increasing cases of dengue, Zika, and yellow fever virus infections. Despite this burden, no small-molecule antiviral has been approved for orthoflavivirus infections. Although 2'C-modified nucleoside analogues possess broad-spectrum antiviral potential, their utility is often limited by poor cellular uptake and inefficient intracellular activation. Existing phosphoramidate ProTides depend on esterase-mediated unmasking, which can lead to poor tissue tropism. Here, we expanded our recently reported Anchimeric HINT1-Activatable (AHA) ProTide platform, a non-ester phosphoramidate prodrug strategy designed to bypass carboxylesterase-dependent activation. A series of analogues incorporating diverse purine and pyrimidine nucleobases, 2'C-sugar modifications, and thermolytic thioether masking groups were synthesized and evaluated. Anchimeric activation rates were independent of nucleobase identity but were strongly affected by masking-group chain length, with half-lives of approximately 5 h and 15 h for AHA-Et and AHA-Bt analogues, respectively. Structure-activity relationship studies revealed a pronounced nucleobase dependence, with antiviral activity observed only among purine analogues. In contrast, pyrimidine analogues bearing 2'C-α-fluoro modifications were inactive in Vero cells. Combined with HINT1 kinetic analyses demonstrating poor processing of selected inactive AHA ProTides, these findings indicate that CES1 deficiency alone does not account for the lack of antiviral activity and highlight HINT1 substrate recognition as a critical determinant of successful prodrug activation. Among the compounds evaluated, AHA ProTide 5f showed the strongest activity against DENV-2 (EC50 = 22 ± 1 μM) and ZIKV (EC50 = 3 ± 2 μM) without detectable cytotoxicity up to 100 μM. These findings provide design principles for next-generation HINT1-activated, non-ester phosphoramidate ProTides.

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