Synthesis of Triphosphate Nucleoside Prodrugs: γ‐ProTriPs

Abstract Although monophosphate nucleoside prodrug approaches have been extensively investigated, leading to the development of several key antiviral and anticancer drugs, less attention has been given to the design of triphosphate prodrugs for the delivery of triphosphorylated nucleotide analogues. Expanding on this strategy, we report here an efficient synthetic methodology for preparing nucleoside triphosphate prodrugs, in which the γ‐phosphate of a nucleotide is masked with an amino acid ester and an aryloxy group (γ‐ProTriP). This approach aims to achieve the direct intracellular release of the triphosphate nucleotide active species, circumventing metabolic bottlenecks and potential toxicity that are often associated with the accumulation of nucleoside analogues and/or their mono‐ and diphosphate species. This article outlines the synthetic strategy for preparing γ‐ProTriP derivatives using either microwave‐accelerated synthesis or conventional heating methods. The approach is exemplified by the preparation of a clofarabine γ‐ProTriP, which emerges as a promising alternative to traditional monophosphate prodrug strategies. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol : Preparation of triphosphate aryloxy phosphoramidate of adenosine, uridine, and clofarabine with microwave heating Alternate Protocol : Preparation of triphosphate aryloxy phosphoramidate of adenosine with conventional heating Support Protocol 1 : Cation exchange of UDP disodium salt to UDP di(triethylammonium) salt Support Protocol 2 : Synthesis of di(triethylammonium) salt of clofarabine 5′‐diphosphate Support Protocol 3 : Synthesis of pentafluorophenyl phosphorylating reagents