Conformation and Relative Configuration of a Very Potent Glycosylphosphatidylinositol-Anchoring Inhibitor with an Unusual Tricarbocyclic Sesterterpenoid δ-Lactone Skeleton from the Fungus Paecilomyces inflatus

In the course of our screening for glycosylphosphatidylinositol (GPI) inhibitors, we found a fungal strain, Paecilomyces inflatus, which inhibited GPI anchoring in yeast. Bioassay-guided fractionation with gel filtration, MPLC on normal phase and prep. HPLC on reversed-phase yielded a minor secondary metabolite, the substituted hexadecahydroindeno5′,6′:4,5]cycloocta1,2-c]pyranyl heptanoate 1 , that inhibited GPI synthesis in vitro by yeast microsomes with a MIC of 3.4 nm. Ester 1 specifically inhibited GPI synthesis in eukaryotic, including mammalian cells, but had no significant activity in protozoa. Based on spectroscopic evidence, including UV, FT-IR, FAB-MS, ESI-HR-MS, ¹H-NMR, ¹³C-NMR, DQ-COSY, ROESY, HSQC, and HMBC data, the metabolite 1 was shown to have an unusual tricarbocyclic sesterterpenoid δ-lactone skeleton. Its solution conformation and relative configuration was elucidated with ¹H,¹H coupling constants and detailed analysis of its ROESY data, coupled with inspection of Dreiding models. In the preliminary investigations on structure-activity relationships, the three derivatives 2 – 4 of ester 1 were prepared by acetylation, catalytic hydrogenation, and intramolecular alkene addition of a hydroxy group, respectively. The test results revealed that the modifications of substituents at the δ-lactone ring or of the C=C bonds of 1 caused a loss of activity by a factor of ca. 500 to 5000. Therefore, it seems that the δ-lactone ring and the C=C bonds in 1 are essential for the potent GPI-inhibitory activity.