bufadienolide and bufogenin

Resibufogenin (RB), a major bioactive bufadienolide, has the potential anticancer activity. In the present work, biotransformation of RB by Actinomucor elegans AS 3.2778 yielded five products, namely 3-oxo-resibufogenin (1), 3-epi-resibufogenin (2), 3-epi-12-oxo-hydroxylresibufogenin (3), 3α-acetoxy-15α-hydroxylbufalin (4), and 3-epi-12α-hydroxylresibufogenin (5), respectively. Among them, metabolites 3 and 4 are previously unreported. The chemical structures of metabolites 1-5 were fully elucidated on the basis of 2D NMR and HR-MS. The highly stereo- and regio-specific isomerization, hydroxylation, and esterification reactions were observed in the biotransformation process of RB by A. elegans. Their cytotoxicities against A549 and H1299 cells were evaluated.

Topics: Antineoplastic Agents; Biotransformation; Bufanolides; Drug Screening Assays, Antitumor; Humans; Hydroxylation; Molecular Structure; Mucorales; Nuclear Magnetic Resonance, Biomolecular

Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan'su. Biotransformation of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl 15beta-hydroxyl bufotalin, via an unprecedented 14beta,15beta-epoxy ring cleavage and a regio-selective acetoxylation. This product showed significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure-activity relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR research.

Topics: Acetylation; Antineoplastic Agents; Biotransformation; Bufanolides; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Nocardia; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Structure-Activity Relationship

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.

Topics: Alternaria; Biotransformation; Bufanolides; Cholenes; Chromatography, High Pressure Liquid; Hydroxylation; Kinetics; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity