Coumarin-stavudine (d4T) novel hybrid ProTides with dual-functionality and enhanced anti-HIV activity

Innovative anti-HIV strategies are urgently needed to address challenges in vaccine development and multidrug resistance. ProTides are a clinically validated prodrug strategy that improves nucleoside monophosphate delivery by bypassing the first phosphorylation step. Conventional ProTides employ phenol or 1-naphthol aryl groups, which release potentially toxic byproducts upon activation. We report the first use of coumarin-based fluorophores (4MU or 4TFMU) as aryl masking groups in stavudine (d4T) ProTides, creating hybrid profluorophores with dual antiviral and fluorescent tracking capabilities. Eight hybrid ProTides were synthesised and evaluated against HIV-1 (III_B) and HIV-2 (ROD) in MT-4 cells. Five ProTides retained activity in thymidine kinase deficient C8166-TK^- cells, confirming bypass of the first phosphorylation step. ProTide 21 showed potent activity (IC₅₀: 80 nM for HIV-1, 140 nM for HIV-2) and high selectivity indices (1549 and 923), outperforming d4T. Enzymatic activation was verified by ³¹P NMR. Surprisingly, two phosphorodiamidate derivatives were isolated, revealing a new class of phosphorodiamidating reagents enabling efficient synthesis of diamidate prodrugs. This multifunctional ProTide platform combined enhanced potency, reduced toxicity, and built-in fluorescence, offering promising avenues for next generation nucleoside and non-nucleoside ProTide and diamidate based therapeutics.