Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi

Abstract

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12␤-position by the fungus Alternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12␤-hydroxyl cinobufagin, 12␤-hydroxyl desacetylcinobufagin, 3-oxo-12␤-hydroxyl cinobufagin, 3-oxo-12␤-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12␣-hydroxyl cinobufagin. The last five products are new compounds. 12␤-Hydroxylation of cinobufagin by A. alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12␤ also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12␤-hydroxyl cinobufagin and 3-oxo-12␣-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12␤-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.Bufadienolides are steroids with a characteristic ␣-pyrone ring at the C-17 position; they have cardiotonic, blood pressure-stimulating, antiviral, and local anesthetic activities (20). More than 300 bufadienolides have been isolated from natural sources including plants and animals (15,23). These compounds have been reported to have significant antitumor activities (9,19,31,33). Cinobufagin (compound I) is a bufadienolide with a 14␤,15␤-epoxy ring, originally isolated as a major component of the traditional Chinese drug, Chan'Su (also called toad venom or toad poison), which is prepared from the skin secretions of giant toads (10). Cinobufagin can induce apoptosis and elevate intracellular Ca 2ϩ levels (13,14). Against cancer cells it has a 50% inhibitory concentration (IC 50 ) of approximately 10 Ϫ8 mol/liter (30). Unfortunately, it is poorly soluble in water and is toxic to humans; therefore, analogs with improved pharmaceutical properties, such as those obtained from plant cell suspension cultures (27,29), are needed before this compound can be utilized effectively in a clinical setting.Microbial transformation is an important tool for structural modification of organic compounds, especially natural products with complicated structures (17,22). It can be used to synthesize chemical structures that are difficult to obtain by other means (24) and as a model of mammalian metabolism due to the similarity between mammalian and microbial cytochrome P 450 enzyme systems (1,2,4,5,12). The metabolism of cinobufagin in rat liver microsomes produced at least six metabolites (32), resulting primarily from deacetylation at C-16 and epimerization of 3-OH via a 3-keto intermediate. The exact s...

Efficacy and safety of gemcitabine-oxaliplatin combined with huachansu in patients with advanced gallbladder carcinoma

“…Some studies demonstrated that huachansu injection can improve the QOL of patients and has been used as a treatment of cancer in China [26][27][28] . It was reported that digitalis-like cinobufagin can protect prostate cancer cells from proliferation.…”

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes

“…The media were sterilized at 121 8C and 1.06 kg/cm 2 for 30 min. [20] The strains were maintained on potato dextrose agar slants at 4 8C.…”

Biotransformation of Cinobufagin by Cunninghamella elegans

“…4, 5 and 6 were identified as 12b -hydroxy-desacetylcinobufagin, 3-oxo-12a-hydroxybufagin and 12b-hydroxybufagin, respectively, by comparison with the literature [10] and on the basis of various spectroscopic analyses.…”

“…Cunninghamella elegans has been employed for the biotransformation of cinobufagin (1) to afford 5 metabolites. The structures of the transformation products have been characterized as 12a -hydroxybufagin (2), 11a -hydroxybufagin (3), 12b -hydroxy-desacetylcinobufagin (4), 3-oxo-12a -hydroxybufagin (5) and 12b -hydroxybufagin (6) [10] (Fig. 1).…”