| Physena madagascariensis Noronha ex Thouars is a 3--8 meter tall woody dicotyledon tree endemic to Madagascar. The bark of this plant is rubbed on clothing as an effective repellent of terrestrial leeches which are abundant in the region of the island where the tree is found. Anticancer screens on the extracts of the leaves of P. madagascariensis indicated significant activity against two human breast cancer cell lines, which prompted us to study this folk medicine. Using activity against a human breast cancer cell line, MDA-MB-435 (human mammary adenocarcinoma), to guide the isolation of the active constituents, four new dinoroleanane derivatives, remangilone A--D (1--4), were obtained following repeated flash column chromatography and HPLC. Both remangilone A (1) and C (3), the only two natural products purified in sufficient quantities for the bioassay, were found to be cytotoxic against two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435), and induced apoptosis at concentrations of 2.3 muM. The rather unusual 2-hydroxy-24-nor-1,4-dien-3-one functionality in the A-ring was believed to be the pharmacophore. Antibacterial and antifungal screens indicated that antimicrobial activity was also prevalent in the 30% aqueous methanol-soluble fraction of the crude methanolic extracts. This activity was traced to two new prenylated flavanones, remangiflavanone A (5) and B (6). A new dimeric flavonoid, remangiflavanone C (7), featuring a methylene linkage between the two flavonoid monomeric subunits, was also isolated, along with small amounts of the monomeric flavonoid, remangiflavanone D (8), corresponding to the dehydrogenated product of 5. Later assays revealed that 5 and 6 also showed significant activity against certain cancer cell lines. In an effort to improve upon the anticancer activity of remangilone A and C, a new synthetic route to 24-nortriterpene derivatives incorporating the 2-hydroxy-24-nor-1,4-dien-3-one pharmacophore was undertaken. Betulinic acid has recently been shown to exhibit significant cytotoxicity against a variety of cultured human tumor cells both in vitro and in vivo, particularly against human melanoma. The free carboxylic acid group of betulinic acid is essential for cytotoxicity against melanoma. Thus, a hybrid of the remangilones and betulinic acid that contained the 2-hydroxy-24-nor-1,4-dien-3-one pharmacophore was synthesized for bioactivity evaluation. |
