Insights into Free Drug Release from Efficacious N-Acyl O-Aminophenol Duocarmycin Prodrugs

Acyclic and cyclic N-acyl O-aminophenol prodrugs of duocarmycin analogues were reported as members of a unique class of reductively cleaved prodrugs that map seamlessly onto the duocarmycin family of natural products. Although these prodrugs were explored with the expectations that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles, the remarkable stability of some such prodrugs suggests another mechanism of free drug release is operative. The prototype of such chemically unreactive N-acyl O-aminophenol prodrugs is 1, which proved remarkably efficacious in vivo in vertebrate tumor models; was found to lack the toxicity that is characteristic of traditional chemotherapeutic drugs as well as the free drugs in the class (e.g., myelosuppression); and displayed a preferential site (intracellular), a slow and sustained rate, and a potentially unique mechanism of free drug release. Herein, we detail studies that provide insights into this stereoselective mechanism of free drug release. Combined, the results of the studies are consistent with an exclusive protein-mediated (enantio)selective activation and free drug release from prodrug 1 by N–O bond cleavage preferentially in cancer cell lines versus cultured normal human cell lines effected by a cytosolic cysteine-based enzyme and suggest that the activating protein is one that is selectively expressed, upregulated, or preferentially activated in cancer cell lines, potentially constituting a new oncology targeted precision therapy.