The Evolution of Phosphoramidates from Small-Molecule Inhibitors to Tunable Cleavable Linkers
Ley, Corinne Ley
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During the development of a series of phosphoramidate-based inhibitors to prostate-specific membrane antigen, we observed a trend in increasing acid stability as the distance between the phosphorus center and α-carboxylate of the P1 residue is increased. While the mechanism of this influence is not fully understood, we designed a new generation of phosphoramidate inhibitors based on trans-4-hydroxyproline as the P1 residue to restrict the interaction of the α-carboxylate to the phosphoramidate core. These hydroxyproline inhibitors demonstrated comparable IC50 values to earlier generations as well as enhanced thermal and acid stability, which is desired for use with imaging or therapeutic radionuclides such as 68Ga or 177Lu. Further capitalizing on this trend in stability of the P-N bond of phosphoramidates, we have developed a second-generation of tunable pH-sensitive linkers to release amine-containing drugs for controlled-release applications. Key to the pH-triggered amine release from these linker is a proximal carboxylic acid to promote the hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general-acid type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine. However, the proximity of the neighboring carboxylic acid attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. While the tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies in which pH changes can trigger the release of turn-on dyes, antibody drug conjugates (ADC), small-molecule drug conjugates (SMDC) and drug eluting stents (DES), the promise of oral delivery of drug-conjugates is expected to have broad impact in applications for clinically relevant targeted drug delivery.