Acid epimerization of 20-keto pregnane glycosides is determined by 2D-NMR spectroscopy.

Carbohydrates influence many essential biological events such as apoptosis, differentiation, tumor metastasis, cancer, neurobiology, immunology, development, host-pathogen interactions, diabetes, signal transduction, protein folding, and many other contexts. We now report on the structure determination of pregnane glycosides isolated from the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae). The observation of cicatrizant, vulnerary and cytostatic activities in some humans and animals of Ceropegia fusca Bolle, a species endemic to the Canary Islands, encouraged us to begin a pharmacological study to determine their exact therapeutic properties. High resolution (1)H-NMR spectra of pregnane glycosides very often display well-resolved signals that can be used as starting points in several selective NMR experiments to study scalar (J coupling), and dipolar (NOE) interactions. ROESY is especially suited for molecules such that ωτ(c) ~ 1, where τ(c) are the motional correlation times and ω is the angular frequency. In these cases the NOE is nearly zero, while the rotating-frame Overhauser effect spectroscopy (ROESY) is always positive and increases monotonically for increasing values of τ(c). The ROESY shows dipolar interactions cross peaks even in medium-sized molecules which are helpful in unambiguous assignment of all the interglycosidic linkages. Selective excitation was carried out using a double pulsed-field gradient spin-echo sequence (DPFGSE) in which 180° Gaussian pulses are sandwiched between sine shaped z-gradients. Scalar interactions were studied by homonuclear DPFGSE-COSY and DPFGSE-TOCSY experiments, while DPFGSE-ROESY was used to monitor the spatial environment of the selectively excited proton. Dipolar interactions between nuclei close in space can be detected by the 1D GROESY experiment, which is a one-dimensional counterpart of the 2D ROESY method. The C-12 and C-17 configurations were determined by ROESY experiments.

Pregnane steroidal glycosides and their cytostatic activities.

Four new steroidal glycosides such as 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-tigloyl-14-ß-hydroxy-17-ß-pregnane (1), 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-(2'-amino)-benzoyl-14-ß-hydroxy-17-ß-pregnane (2), 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-14-ß-dihydroxy-17-α-pregnane (3) and 3-O-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside-12-ß-14-ß-dihydroxy-17-ß-pregnane (4) were isolated from the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae), a crassulacean acid metabolism plant, an endemic species to the Canary Islands that has been used in traditional medicine as a cicatrizant, vulnerary and disinfectant. The dichloromethane extract exhibited significant cytostatic activity against HL-60, A-431 and SK-MEL-1 cells, human leukemic, epidermoid carcinoma and melanoma cells, respectively. As shown in Table I, compounds 1 and 2 showed very similar IC(50) values. The acetylation of 1 to give the diacetate 5 increases 5-fold the cytotoxicity against HL-60 cells. Compounds 3 and 4 did not show cytotoxicity at the assayed concentrations. With respect to the compounds containing only the steroid ring (6-8), the presence of a charged O-amino-benzoyl but not a tigloyl group improved the cytotoxicity.

Biosynthesis of antitumoral and bactericidal sanguinarine.

A simple, rapid, and reliable TLC method for the separation and determination of sanguinarine has been established. This intensively studied biologically active alkaloid has a wide range of potentially useful medicinal properties, such as antimicrobial, antiinflammatory, and antitumoral activities. Sanguinarine has also been incorporated into expectorant mixtures and has a strong bactericidal effect upon gram-positive bacteria, particularly Bacillus anthracis and staphylococci. These medicinal properties are due to the interaction of sanguinarine with DNA. A fibre-optic-based fluorescence instrument for in situ scanning was used for quantitative measurements. The sanguinarine was determined over the range 5-40 ng and a detection limit of 1.60 ng. The method was applied to the quantification of sanguinarine in tissue culture extracts of Chelidonium majus L.