Dissociation Dynamics of Protonated Nitroimidazoles: The Ortho Effect as the Mechanistic Trigger for Radical Formation

Abstract

Substituted 5-nitroimidazoles are recognized as being significantly more effective in treating hypoxic tumors than their 4-nitroimidazole isomers. While classical Quantitative Structure–Activity Relationship (QSAR) models point to electron affinity (EA) as the critical parameter for this activity, recent gas-phase electron-attachment studies reveal a paradox: isolated radical anions of 1-methyl-5-nitroimidazole (1M5NI) do not spontaneously fragment into essential cytotoxic species such as NO• and OH• radicals. To resolve this dilemma, we investigate the high-energy dissociation pathways triggered instead of activated during radiotherapy. Because these radiosensitizers capture secondary electrons and are immediately protonated in the acidic tumor microenvironment, we utilized Electrospray Ionization (ESI) Orbitrap mass spectrometry to generate the [M + H]+ species. This protonated closed-shell cation serves as a rigorous structural and mechanistic analogue to map the reaction topology of the transient in vivo state. By comparing the protonated 1M5NI ([1M5NI + H]+) and 2-methyl-4-nitroimidazole ([2M4NI + H]+) isomers through high-level computational modeling [DFT and CCSD(T)], we elucidate that while EA serves as the initial trigger, the protonated state is mandatory to unlock the fragmentation pathway. Crucially, the massive energy deposition inherent to radiotherapeutic environments provides the necessary driving force to overcome the high-energy dissociation barriers (>3 eV), channeling the internal energy along a specific normal mode of vibration to eject the cytotoxic radicals. These findings provide a fundamental physical chemistry basis for the clinical efficacy of 5-nitroimidazole drugs.