Enzyme-assisted synthesis and structural characterization of nitrocatecholglucuronides

Enzyme-assisted synthesis and characterization are described for 3-O-beta-n -glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N,N-diethy1-3-(3,4-dih ydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-( 3,4- dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (nitecapone) 3a and 4'- methyl-3,4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-g lucuronides 5b and 6b of the aglycon 1,2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucur onidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1 -o-beta-D- and 2-O-beta-D-glucuronides. Purification of the crude products by C-18 solid-phase extraction and/or flash chromatography gave compounds 1 b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were c haracterized on the basis of UV and IR spectra and confirmed with FAB-MS an d NMR spectroscopy.