Abstract
Cytochrome P-450 (P-450)-catalyzed oxidation of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester gives rise to 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester and to 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester, identified in this work. A pyridine hydroxymethyl diester of the sort of the latter compound is novel; under acidic or dehydrating conditions the diester is readily converted to a cyclic lactone (2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid 5-ethyl ester lactone). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester was not hydroxylated to form this hydroxymethyl compound or lactone, but 1,4-dihydro-2-hydroxymethyl-4-phenyl-6-methyl-3,5-pyridinedicarboxyli c acid diethyl ester was enzymatically oxidized to give both products. The rates of oxidative carboxylic ester cleavage and methyl hydroxylation varied among individual forms of P-450 tested. Experiments with 2H and 3H labels were used to estimate an intrinsic kinetic deuterium isotope effect of 15 for ethyl ester cleavage by rat liver P-450PB-B in a reconstituted system. Rat liver microsomal systems showed kinetic deuterium and tritium isotope effects of 8 and 11, respectively, and this deuterium isotope effect was not attenuated in either intra- or intermolecular competitive experiments. When deuterium was present in the ethyl (ester) groups, increases in the rate of 2-methyl hydroxylation were observed in rat liver microsomes and with purified P-450 beta NF-B (but not with P-450PB-B). Deuteration of the methyl groups gave rise to kinetic isotope effects of 7-11, but no increases were seen in the rates of ester cleavage. These studies and those on rates of substrate disappearance indicate that isotopically sensitive branching (metabolic switching) observed in these systems is not necessarily bidirectional.
Highlights
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
2,6-dimethyl-4-phenyl-3,6-pyridinedicarboxyliaccid sively as calcium channel blockers; these compounds are diethyl ester gives rise to 2,6-dimethyl-4-pheny1-3,5- inactivated by oxidation to their pyridine derivatives pyridinedicarboxylic acid monoethyl ester and to 2- (Raemsch and Sommer, 1983)
When deuterium was presentin with large kinetic hydrogen isotope effects, consistent with the ethyl groups, increases in the rate of 2- the view that hydrogen atom abstractionoccurs, and thishigh methyl hydroxylation were observed in rat liver microsomes and with purified P - ~ ~ O ~ P(b-uBt not with P-450pB-B)
Summary
The lactone prepared by this procedure was identical with that isolated from the preparative microsomal oxidation of 2,6-dimethyl-4-pheny1-3,5pyridinedicarboxylic acid diethyl ester as judged by m.p., comethyl-4-phenyl-3,5-pyridinedicarboxyliaccid monoethyl ester (O), chromatography on HPLC, NMR, andEIMS(datanot 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylicacid shown). Formation of Different Products of 2,6-Dimethy1-4-phenyl3,5-pyridinedicarboxylicAcid Diethyl Ester by Individual P450s-Varying amounts of the monoacid and the hydroxymethyl compound and itsderived lactone were formed by the five different P-450s examined (Table I). These transformations were first identified in liver microsomes isolated from untreated rats, the most active P-450s (of those examined) were twothat areknown to be highly inducible by xenobiotic chemicals. Kinetic tritium isotope effects forOxidative ester cleavage of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylaiccid diethyl ester byP-450s
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