Discovery and Total Synthesis of Crossiguanipyrazines with Potent Activity Against <i>Mycobacterium tuberculosis</i>

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains one of the most devastating infectious diseases, with rising antimicrobial resistance exacerbating the urgent need for new therapeutic agents. Here, we report the discovery of crossiguanipyrazines A-I, a rare family of potent anti-TB alkylpyrazines from the underexplored actinobacterium Crossiella cryophila DSM 44230. Crossiguanipyrazines (CGPs) feature an unusual 3-methoxyl-2,5,6-trialkyl-pyrazine scaffold with variable hydroxylation and N-prenylation. The stereochemistry of CGPs A-G was determined by Mosher's analysis and electronic circular dichroism spectroscopy. Stable isotope-labeling revealed a unique biosynthetic origin from arginine and acetate. Inhibition with a cytochrome P450 inhibitor suppressed hydroxylation, while feeding experiments with stable isotope-labeled CGP I demonstrated its role as a precursor for other CGPs, implicating P450 enzymes in CGP diversification. CGPs C and H exhibited potent anti-TB activity in the low micromolar range without mammalian cell cytotoxicity, unlike previously reported cytotoxic trialkylpyrazine-type natural products. Structure-activity relationship (SAR) studies highlighted the critical roles of hydroxylation and N-prenylation in modulating biological activity. Furthermore, we achieved the first total synthesis of CGP I, which validated the chemical structure and provided a foundation for further optimization of this compound class. These findings suggest that CGPs can serve as useful starting points for anti-TB drug development.