Use of chiral liquid chromatography for the evaluation of stereospecificity in the carbonyl reduction of potential benzo[ c]fluorene antineoplastics benfluron and dimefluron in various species

Abstract

Benfluron (B) [5-(2-dimethylaminoethoxy)-7 H-benzo[ c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C 7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway ( O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm × 4.6 mm ID, 5 μm) using a mobile phase acetonitrile–1 M NaClO 4 (40:60, v/v). Another mobile phase, methanol–1 M NaClO 4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min −1 in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.