95(P38) Structure Determination and Bioactivity of a New Steroidal Saponin, Furcreastatin

Tumor suppressor gene p53 is mutated in about 50% of human neoplasias. The wild-type p53 protein induces G1 block in the cell cycle and enhances apoptosis. We searched for microbial and plant secondary metabolites that induce selective cell death in mutated p53-expressing fibroblasts. As a result we isolated a novel steroidal saponin, furcreastatin, from the plant Furcraea foetida. Extraction of the dried leaves (15.64g) of Furcraea foetida with 80% aqueous EtOH, followed by concentration and extraction with BuOH, gave a crude material that was purified by column chromatography on silica gel, and then by gel filtration to yield furcreastatin (1) (211mg) as colorless needles. The IR spectrum of 1 exhibited typical absorption bands of alcoholic C-O at 1000-1200cm^<-1>, suggesting the presence of saccharide components. HRFABMS (neg.) showed m/z 1385.6266 (M-H) (C_<63>H_<101>O_<33> requires 1385.6225). Furthermore, fragment ion peaks at m/z 1239 and m/z 1223 in FABMS (neg.) indicated the presence of two terminal deoxyhexosyl and hexosyl units in the branched saccharide chain. By 2D NMR studies, the structure of 1 was proposed to be a steroidal saponin having a new hexasaccharide chain consisting of 4 moles of glucopyranose, and 1 mole of each of galactopyranose and rhamnopyranose. The new hexasaccharide sequence was determined by HMBC and NOESY. The β anomeric configurations for the glucose and galactose were assigned based on their large coupling constants of anomeric protons. The anomeric configuration of the rhamnose was determined to be α, by comparison of NMR chemical shift data of 1-H (a low-field shift in α anomer), and C-3 and C-5 (high-field shifts in α anomers). The gross structure of the aglycon with 27 carbons was elucidated to be 3-hydroxyspirostan-12-one based on the results of ^1H-^1H COSY, HMBC, and NOESY experiments. Acid hydrolysis of 1 gave an aglycon and three monosaccharides. The aglycon was identical to the authentic hecogenin in all respects. The D and L configurations of the three monosaccharides were determined to be D-glucose, D-galactose, and L-rhamnose, respectively, by measurements of optical rotation values. Therefore, the structure of 1 was decided to be (3β,5α,25R)-3-hydroxyspirostan-12-one 3-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-{β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)}-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside]. Furcreastatin showed selective cytotoxicity on mutant p53-overexpressing Balb/c 3T3 10 (1)-mp53 cells at 4〜8μg/mL.