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 M. tuberculosis (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 againstM. abscessus, 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 B2 with potent activity against MTB (MIC: B2 also has good safety and oral pharmacokinetic (PK) profiles. Further chiral resolution revealed its R-enantiomer (B2-1) as a more active form. Mechanistic investigations showed that B2-1 establishes a stable electrostatic interaction with Asp326 ofM. abscessusDprE1. 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.