The search for bioactive constituents from Annona bullata Rich. (Annonaceae)

This project involves the isolation and characterization of biologically active compounds, especially antitumor and pesticidal compounds, from the bark of Annona bullata Rich. (Annonaceae) by bioactivity-directed fractionation. Partitions and consecutive chromatographic separations of the EtOH extract, monitoring each step with brine shrimp lethality tests, resulted in the isolation of eleven bioactive acetogenins, two lesser active known diterpenes, two known alkaloids, and one known plant steroid. Seven of the acetogenins are new to the literature. The carbon skeletons of the new acetogenins (bullatacin, bullatacinone, bullatalicin, bullatalicinone, 4-deoxyasimicin, uvariamicin IV, and bullatencin) were determined by chemical and spectral methods including MS, UV, IR, $\sp1$H-NMR, $\sp{13}$C-NMR and 2D COSY. The absolute stereochemistries, except for the chiral center at position 36, of several of these compounds were analyzed by making their Mosher esters and examining relevant $\sp1$H-NMR and $\sp{19}$F-NMR spectra. The known acetogenins (squamocin and uvariamicins I, II, and III), diterpenes (16$\alpha$-hydroxy-($-$)-kauranoic acid and ($-$)-kaur-16-en-19-oic acid), alkaloids (liriodenine and atherospemidine), and the plant steroid ($\beta$-sitosterol-$\beta$-D-glucopyranoside) were identified. The isolated acetogenins showed highly potent cytotoxicities in human solid tumor cell lines, e.g., bullatacin is over a billion times more cytotoxic than adriamycin, an anticancer antibiotic. The ED$\sb{50}$ values are at the level of killing cancer cells at one molecule per cell. Results in a panel of 45-57 human tumor cell lines at NCI for bullatacin, bullatacinone, bullatalicin, bullatalicinone, squamocin, and 4-deoxyasimicin showed selective actions. Some SAR\u27s are apparent. A number of mechanism-based assays have been performed and have ruled out several possible mechanisms of action for these potent acetogenins. Experiments at the Purdue Cell Culture Laboratories have indicated acetogenins, like adriamycin, are pumped out of the cell in multi-drug-resistant cell lines and must be acting inside the cells. Bullatacin is a superb inhibitor of (a) the mitochondrial electron transport system (rat liver mitochondria) and (b) NADH-ubiquinone reductase (complex I of the transport chain purified from beef heart mitochondria). The I$\sb{50}$ values are certainly at the low nanomolar level and could be in the subnanomolar range. (Abstract shortened with permission of author.