H ₂ O ₂ –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 <i>in vivo</i> activation remains poorly defined. We report a novel H₂O₂-responsive phenylboronic nitrogen mustard prodrug (<b>10a</b>) and its precisely deuterated analogue (<b>10b</b>), designed to elucidate the activation pathway of ROS-responsive agents. These isotopologues differ only in ethyl substituents─hydrogen (<b>10a</b>) versus deuterium (<b>10b</b>)─enabling isotope-resolved tracking of metabolic transformations <i>in vivo</i>. Co-administration of <b>10a</b> and <b>10b</b> in triple-negative breast cancer xenograft mice identified two metabolites, providing the first definitive <i>in vivo</i> evidence of oxidative deboronation as the primary activation mechanism. Prodrug <b>10a</b> 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.