Undescribed ansatrienin analogs from Streptomyces flaveolus including two N-acetylcysteine conjugates

Ansatrienins, triene-containing C17-benzene ansamycins, have garnered attention owing to their potent cytotoxic properties. In this study, we identified five undescribed ansatrienin analogs from Streptomyces flaveolus: trienomycins M - O (1–3), N-AcCys-ansatrienin 3a (4), and N-AcCys-ansatrienin B (5). Their structures and relative configurations were elucidated using HR-ESI-MS and NMR spectroscopic analyses, whereas their absolute configurations were determined through biosynthetic considerations and time-dependent density functional theory/electronic circular dichroism calculations. Notably, trienomycin M (3) is the first reported ansatrienin with a C-25 hydroxyl group. Compounds 4 and 5 featured an N-acetylcysteine (N-AcCys) moiety attached to C-21 via a thioether linkage. Antiproliferative activity assays revealed that trienomycin N (2) exhibited significant activity against HeLa, HuCCT1, and PANC-1 cell lines, with IC₅₀ values of 0.81, 1.76, and 1.84 μM, respectively. In contrast, compounds 1, 4, and 5 were completely inactive. These findings suggest that C-25 oxidation and C-21 N-AcCys modification negatively affected cytotoxicity. The discovery of these undescribed ansatrienins provides valuable insights into structure-activity relationships and enhances our understanding of the role of host microorganisms in shaping natural products.