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 (4: 5-heptylpyrazine-2-carboxamide, MIC_M. tuberculosis 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 23 (5-hexyl-N-(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) Mycobacterium tuberculosis 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.

Department of Clinical Microbiology University Hospital Hradec Králové Sokolská 581 Hradec Králové 500 05 Czech Republic

Department of Medical Genetics and Cell Biology School of Basic Medical Sciences Zhengzhou University Zhengzhou Henan 450001 China

Faculty of Pharmacy in Hradec Králové Charles University Ak Heyrovského 1203 Hradec Králové 500 03 Czech Republic

Faculty of Pharmacy University Business Academy Heroja Pinkija 4 Novi Sad 21101 Serbia

Faculty of Science University of Hradec Králové Rokitanského 62 Hradec Králové 500 03 Czech Republic

Medicinal Chemistry Rega Institute for Medical Research KU Leuven Herestraat 49Box 1041 Leuven 3000 Belgium

Pledge Therapeutics Gaston Geenslaan 1 Leuven 3001 Belgium

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