Appl Environ Microbiol 70(6): 3521-3527

Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi

The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, People's Republic of China

^{Corresponding author. Mailing address: The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road #38, Beijing 100083, People's Republic of China. Phone: 86-10-8280-1516. Fax: 86-10-8280-2700. E-mail:}nc.ude.umjb@adg.

Received 2003 May 30; Accepted 2004 Mar 1.

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 12β-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 12β-hydroxyl cinobufagin, 12β-hydroxyl desacetylcinobufagin, 3-oxo-12β-hydroxyl cinobufagin, 3-oxo-12β-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12α-hydroxyl cinobufagin. The last five products are new compounds. 12β-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-12β also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12β-hydroxyl cinobufagin and 3-oxo-12α-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12β-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.

Abstract

Bufadienolides are steroids with a characteristic α-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 14β,15β-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^{levels (}13, 14). Against cancer cells it has a 50% inhibitory concentration (IC₅₀) of approximately 10^{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 P₄₅₀ 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 structures of these metabolites are still unknown since only small amounts of samples were recovered.

The objective of this study was to utilize Alternaria alternata as an in vitro model to prepare cinobufagin derivatives that are potential mammalian metabolites. Five of the six metabolites obtained are new compounds, and two significantly inhibit the growth of cultured human cancer cells.

Acknowledgments

We thank Guangzhong Tu, Beijing Institute of Microchemistry, for recording NMR spectra. M. Ye thanks Yuxin Sheng and Peng Han for their technical assistance.

Acknowledgments

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