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

Eron Saxon1, Dana Stambekova2, Thilini Nimasha Fernando Ponnamperumage1

  • 1Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University of Wisconsin-Milwaukee, 2000 E. Kenwood Boulevard, Milwaukee, Wisconsin 53211, United States.

Journal of Medicinal Chemistry
|December 2, 2025
PubMed

Insights

This study introduces a novel boron-based prodrug activated by hydrogen peroxide (H2O2) for targeted cancer therapy. Precision deuteration confirmed oxidative deboronation as the key in vivo activation mechanism, leading to selective cancer cell killing with minimal toxicity.

Area of Science:

  • Medicinal Chemistry
  • Oncology
  • Pharmacology

Background:

  • Boron-based prodrugs show promise for selective cancer treatment by responding to reactive oxygen species (ROS).
  • The in vivo activation mechanisms of ROS-responsive prodrugs are not fully understood, limiting their therapeutic optimization.

Purpose of the Study:

  • To design and synthesize a novel hydrogen peroxide (H2O2)-responsive phenylboronic nitrogen mustard prodrug and its deuterated analogue.
  • To elucidate the in vivo activation pathway of ROS-responsive boron-based anticancer prodrugs using isotope-resolved tracking.
  • To evaluate the therapeutic efficacy and safety of the developed prodrug in preclinical cancer models.

Main Methods:

  • Synthesis of a novel H2O2-responsive phenylboronic nitrogen mustard prodrug (10a) and its precisely deuterated analogue (10b).
  • Co-administration of isotopologues 10a and 10b in triple-negative breast cancer xenograft mouse models.
  • Isotope-resolved metabolic tracking to identify in vivo metabolites and elucidate the activation pathway.
  • Assessment of H2O2-inducible DNA-alkylating activity, cancer cell proliferation inhibition, and tumor growth suppression in vivo.

Main Results:

  • Identification of two key metabolites, providing the first definitive in vivo evidence for oxidative deboronation as the primary activation mechanism.
  • Prodrug 10a demonstrated H2O2-inducible DNA-alkylating activity.
  • Selective inhibition of proliferation in high ROS-expressing MDA-MB-468 cancer cells compared to nonmalignant MCF-10A cells.
  • Significant suppression of tumor growth in vivo with no observable toxicity.

Conclusions:

  • Precision deuteration serves as a powerful mechanistic probe for investigating drug activation pathways in vivo.
  • Oxidative deboronation is confirmed as the principal in vivo activation mechanism for this class of ROS-responsive boron-based prodrugs.
  • The developed boron-based prodrug platform offers a rational approach for designing and optimizing targeted cancer therapeutics with enhanced selectivity and safety.

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