• antibiotic resistance (AMR)
• bicyclic nitroimidazoles
• controlled-potential electrolysis
• cyclic voltammetry
• Dimetridazole comparison
• EC mechanism
• electrochemical characterization
• electrochemical sensors
• emerging pollutants
• environmental monitoring
• nitro radical anion
• pH-dependent reactivity
• pharmaceutical contaminants
• Pretomanid
• proton-coupled electron transfer
• redox behavior

This thesis focuses on the electrochemical characterization of Pretomanid, a bicyclic nitroimidazole compound recently approved for the treatment of multidrug-resistant tuberculosis (MDR-TB), with the aim of elucidating its redox behavior, reduction mechanism, and environmental implications. The work stems from the growing global challenges posed by both antimicrobial resistance and the widespread dispersion of pharmaceutical compounds in aquatic ecosystems, issues that have been exacerbated by the misuse and improper disposal of antibiotics. The increasing detection of antibiotics and their metabolites in surface and groundwater, combined with their poor removal by conventional wastewater treatment plants, raises significant concerns about environmental persistence, ecological imbalance, and the contribution of these contaminants to the development and spread of antimicrobial resistance. Pretomanid represents a compound of dual relevance, combining therapeutic importance in the fight against tuberculosis with potential environmental impact as an emerging contaminant, making the investigation of its chemical properties particularly significant.
The primary objective of this research was to investigate Pretomanid’s electrochemical properties under controlled conditions and to provide a mechanistic understanding of its redox processes, while also evaluating its behavior in aqueous systems of environmental relevance. Experimental work was conducted at the J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences using advanced electrochemical techniques such as cyclic voltammetry and controlled-potential electrolysis. Acetonitrile was selected as the primary solvent, using tetrabutylammonium hexafluorophosphate as the supporting electrolyte and Ferrocene as an internal standard to ensure accurate quantification of electron transfer processes. The electrochemical response of Pretomanid was systematically studied by evaluating the influence of multiple parameters, including scan rate, compound concentration, solvent conditions, and pH. To complement these studies, a comparative analysis with Dimetridazole, a structurally simpler and well-characterized nitroimidazole compound, was carried out under identical experimental conditions. This comparison allowed a deeper understanding of the structural and electronic factors governing Pretomanid’s reactivity, stability, and radical intermediate formation.
The initial part of the work involved the accurate determination of the electroactive surface area of the working electrode, calculated using the Ferrocene redox system as a calibration standard. This step was essential for reliable evaluation of the electron transfer kinetics and diffusion-controlled processes. Cyclic voltammetry experiments revealed that Pretomanid undergoes a single-electron reduction in the first step, producing a nitro radical anion as the initial intermediate. At high scan rates, the redox process exhibited quasi-reversible behavior, while at lower scan rates the reversibility decreased markedly, indicating the presence of a rapid follow-up chemical step. This step is likely associated with a proton-coupled reaction of the nitro radical anion, consistent with mechanisms previously hypothesized for similar nitroimidazole compounds but here experimentally confirmed for Pretomanid. The diffusion-controlled nature of the process was supported by the linear dependence between cathodic peak current and the square root of scan rate, as predicted by the Randles–Ševčík equation, and by the absence of peak potential shifts under different conditions. Nicholson’s analysis further confirmed the presence of an EC-type mechanism, consisting of an initial electron transfer followed by a fast and irreversible chemical transformation.
Concentration-dependent voltammetric studies further corroborated these findings, demonstrating a linear relationship between cathodic peak current and Pretomanid concentration, consistent with a diffusion-controlled process. Importantly, no evidence of second-order phenomena such as dimerization or disproportionation was detected, as the peak potential remained stable across the entire range of tested concentrations. Bulk electrolysis experiments were conducted to complement cyclic voltammetry results, enabling quantification of the electrons transferred during reduction. Integration of the current-time curves revealed a one-electron process in the initial step, followed by a four-electron process in subsequent stages, which led to the complete transformation of the nitro group into reduced species.
A comparative study with Dimetridazole (DMZ) provided significant insights into the role of Pretomanid’s bicyclic structure on its reactivity. Under identical experimental conditions in acetonitrile, Dimetridazole displayed a chemically and electrochemically reversible one-electron reduction at approximately −1.65 V vs Fc/Fc⁺, forming a relatively stable radical anion. In contrast, Pretomanid’s radical anion was found to be highly reactive, undergoing rapid proton abstraction from the surrounding medium, eventually leading to hydroxylamine derivatives. This result highlighted the significantly lower stability of Pretomanid’s radical intermediate, attributable to its structural features and electronic distribution, and confirmed the existence of a fast follow-up chemical step absent in the simpler DMZ system.
The influence of pH on Pretomanid’s electrochemical behavior was also investigated in aqueous solutions to simulate environmentally relevant conditions. The experiments demonstrated that Pretomanid’s reduction is strongly pH-dependent, as the reduction peak potential shifted towards more negative values with increasing pH, consistent with proton-coupled electron transfer processes. The highest reduction currents were observed under acidic conditions, particularly at pH 3 and 4, indicating enhanced reducibility in environments where proton availability is high. This finding has important pharmacological and environmental implications, as Pretomanid is designed to act under hypoxic and inflamed tissues, where pH gradients can influence its activation pathway, and its potential persistence in natural waters could be significantly modulated by local pH.
The results obtained in this study provide a detailed mechanistic understanding of Pretomanid’s redox behavior and confirm the nitro group as the primary redox-active site. Unlike previous studies on nitroimidazoles, this thesis experimentally demonstrated the presence of an EC mechanism in Pretomanid, where a reversible one-electron transfer is followed by a rapid, irreversible chemical step. These findings contribute to the comprehension of Pretomanid’s activation pathways in biological systems while also informing future monitoring strategies for its detection in environmental samples.
Furthermore, the comparison between Pretomanid and Dimetridazole revealed critical differences in radical anion stabilization and reactivity, underscoring the importance of structural complexity in governing electrochemical behavior. Such knowledge lays the foundation for the design of improved electrochemical detection methods, exploiting Pretomanid’s distinctive redox properties for sensitive and selective monitoring in pharmaceutical quality control and environmental studies. Importantly, electrochemical techniques demonstrated excellent analytical performance, with detection and quantification limits comparable to, or even better than, conventional methods such as HPLC-MS, highlighting their potential as cost-effective and sustainable alternatives for real-time applications.
Finally, this thesis emphasizes the broader environmental and public health relevance of Pretomanid’s characterization. The increasing prevalence of pharmaceutical contaminants, particularly antibiotics, in aquatic environments, combined with the growing threat of antimicrobial resistance, necessitates the development of robust analytical tools for monitoring and risk assessment. The sensitivity, portability, and low cost of electrochemical sensors make them highly suitable for these purposes, offering a promising approach to track emerging contaminants and evaluate their impact on ecosystems and human health. The work presented here thus provides both fundamental insights into Pretomanid’s chemical reactivity and practical foundations for developing efficient detection technologies, contributing to multidisciplinary efforts aimed at managing pharmaceutical pollution and mitigating the risks associated with antimicrobial resistance.