Discovery, Design, and Synthesis of Novel 2-Benzyl-2,7-diazaspiro[3.5]nonane Benzothiazinones with Broad-Spectrum Antimycobacterial Activity

Despite their nanomolar potency against <i>M. tuberculosis</i> (MTB), benzothiazinone (BTZs) DprE1 inhibitors like PBTZ169 are ineffective against nontuberculous mycobacteria (NTM) due to a Cys387Ala mutation in the NTM DprE1 target. Here, 529 BTZs in our lab were screened against<i>M. abscessus</i>, and two hits with the 2-benzyl-2,7-diazaspiro[3.5]nonane scaffold showed enhanced activity. Subsequent optimization led to the discovery of lead compound <b>B2</b> with potent activity against MTB (MIC: < 0.01 μg/mL) and NTM strains (MIC: < 0.03125-2.5 μg/mL). Notably, <b>B2</b> also has good safety and oral pharmacokinetic (PK) profiles. Further chiral resolution revealed its R-enantiomer (<b>B2-1</b>) as a more active form. Mechanistic investigations showed that <b>B2-1</b> establishes a stable electrostatic interaction with Asp326 of<i>M. abscessus</i>DprE1. As this residue is highly conserved among mycobacterial species, such an interaction likely underpins its broad-spectrum activity. Therefore, our findings provide a blueprint for developing next-generation BTZs with broad-spectrum activity against mycobacteria.