3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

Charles University Faculty of Pharmacy in Hradec Králové Akademika Heyrovského 1203 50005 Hradec Králové Czech Republic

Faculty of Natural Sciences Department of Biochemistry Comenius University in Bratislava Ilkovičova 6 Mlynská dolina 842 15 Bratislava Slovakia

Global Health Institute École Polytechnique Fédérale de Lausanne 1015 Lausanne Switzerland

Regional Institute of Public Health Department of Bacteriology and Mycology Partyzánské náměstí 7 70200 Ostrava Czech Republic

Univ Lille CNRS Inserm CHU Lille Institut Pasteur de Lille U1019 UMR 9017 CIIL Center for Infection and Immunity of Lille F 59000 Lille France

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