Abstract
The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6′ of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.
Footnotes
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Financial support for this study was provided by Università degli Studi del Piemonte Orientale “Amedeo Avogadro”.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.016998.
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ABBREVIATIONS: CA-4, (Z)-combretastatin A-4; HPLC, high-performance liquid chromatography; TBAF, tetrabutylammonium fluoride; TBDM-SCl, t-butyldimethylsilyl chloride; DMF, N,N-dimethylformamide; THF, tetrahydrofuran; M, metabolite; LC-DAD, liquid chromatography-diode array detector; LC-MS, liquid chromatography-mass spectrometry; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; NOE, Nuclear Overhauser effect; AVE8062, 2-amino-3-hydroxy-N-/{methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-vinyl]-phenyl/}-propionamide; P450, cytochrome P450; Oxi4503, (Z)-2′,3′-dihydroxy-3,4,4′,5-tetramethoxy-stilbene-diphosphate.
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↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received June 7, 2007.
- Accepted September 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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