Targeting <i>Mycobacterium tuberculosis</i> : The Role of Alkyl Substitution in Pyrazinamide Derivatives

Tuberculosis (TB) remains a significant global health challenge due to the rapid emergence of drug resistance. Despite substantial progress in anti-TB drug development, effective treatment options are limited. In this study, we report the synthesis and biological evaluation of pyrazinamide (PZA) derivatives with 5-alkyl and 5-alkanamido modifications, designed to enhance antimycobacterial activity by increasing lipophilicity and improving penetration of the lipid-rich mycobacterial cell wall. A positive correlation between the length of the 5-alkyl chain and antimycobacterial activity was observed, with maximal potency achieved with the heptyl substituent (<b>4</b>: 5-heptylpyrazine-2-carboxamide, MIC_<i>M. tuberculosis</i> H37Rv = 3.13 μg/mL). In series C with phenyl substitution on the C-2 carboxamide, different simple substituents were tolerated on the benzene ring (both electron-donating and electron-withdrawing, both lipophilic and hydrophilic), and the length of the alkyl chain was the main determinant of the antimycobacterial activity. Compound <b>23</b> (5-hexyl-<i>N</i>-(3-trifluoromethylphenyl)-pyrazine-2-carboxamide) exerted MIC = 3.13 μg/mL and selectivity index (SI, compared to HepG2 cells) >25. Notably, the tested compounds exhibited significant activity against multidrug-resistant (MDR) <i>Mycobacterium tuberculosis</i> strains while maintaining favorable selectivity profiles and low cytotoxicity. In contrast, 5-alkanamido derivatives (series B and D) were devoid of antimycobacterial activity. Mechanistic investigations revealed that unlike PZA, the 5-alkyl pyrazinamide derivatives are not hydrolyzed by mycobacterial pyrazinamidase (PncA), indicating a distinct mode of action. While molecular modeling initially suggested enoyl-ACP reductase (InhA) as a potential target of series C, subsequent experimental validation disproved this hypothesis; thus, the precise mechanism of action remains to be elucidated.