Enhanced Plasma Stability and Potency of Aryl/Acyloxy Prodrugs of a BTN3A1 Ligand.

While ester-based phosphonate prodrugs excel at delivering payloads into cells, their instability in plasma is a hurdle for their advancement. Here, we synthesized new aryl/acyloxy prodrugs of a phosphonate BTN3A1 ligand. We evaluated their phosphoantigen potency by flow cytometry and ELISA and their plasma and cellular metabolism by LC-MS. These compounds displayed low nanomolar to high picomolar potency. Addition of a -isopropyl group to the phenyl substituent and use of cyclohexyl or -methoxybenzyl groups as the acyloxy substituent significantly increased human, but not mouse or rat, plasma stability without negatively impacting potency. Combinations of these prodrug moieties further improved stability, with the best combination achieving a half-life of over 12 h in human plasma, a marked improvement on prior compounds. In contrast, oxane analogs improved water solubility and cellular payload delivery but remained unstable in human plasma. The studies suggest that certain ester-based phosphonate prodrugs quickly deliver active payloads inside cells and show substantial stability in human plasma.