H2O2–Responsive Anticancer Prodrug: Synthesis, Precision Deuteration in Search of In Vivo Metabolites, and Activation Pathway

Boron-based reactive oxygen species (ROS)-activated prodrugs offer a promising strategy for enhancing cancer selectivity, yet their in vivo activation remains poorly defined. We report a novel H2O2-responsive phenylboronic nitrogen mustard prodrug (10a) and its precisely deuterated analogue (10b), designed to elucidate the activation pathway of ROS-responsive agents. These isotopologues differ only in ethyl substituentshydrogen (10a) versus deuterium (10b)enabling isotope-resolved tracking of metabolic transformations in vivo. Co-administration of 10a and 10b in triple-negative breast cancer xenograft mice identified two metabolites, providing the first definitive in vivo evidence of oxidative deboronation as the primary activation mechanism. Prodrug 10a exhibited H2O2-inducible DNA-alkylating activity, selectively inhibited the proliferation of high ROS-expressing MDA-MB-468 cancer cells over nonmalignant MCF-10A cells, markedly suppressed tumor growth without observable toxicity. This study highlights precision deuteration as a mechanistic probe and establishes a platform for rational design and optimization of boron-based anticancer prodrugs.