Conformation and Relative Configuration of a Very Potent Glycosylphosphatidylinositol-Anchoring Inhibitor with an Unusual Tricarbocyclic Sesterterpenoidδ-Lactone Skeleton from the FungusPaecilomyces inflatus

Abstract

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 hexadecahydroindeno[5′,6′:4,5]cycloocta[1,2-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.