Synthesis and the Stereoselective Enzymatic Hydrolysis of Flurbiprofen-Basic Amino Acid Ethyl Esters

Abstract

Ethyl esters of flurbiprofen L-arginine (FP-Arg-OH), flurbiprofen L-lysine (FP-Lys-OH) and flurbiprofen p-guanidino-L-phenylalanine (FP-GPA-OH) were synthesized and then the release of flurbiprofen enantiomers from these derivatives in the presence of trypsin (Tp), carboxypeptidase B (CPB) and car-boxypeptidase Y (CPY) were examined in order to evaluate their availability as prodrugs for flurbiprofen (FP). The ester bonds of the three racemic FP derivatives were hydrolyzed by Tp at about 3 to 20 times the rates of N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), a specific substrate for Tp. (R)- FP was released faster than (S)-FP by either CPB or CPY from both FP-Arg-OH and FP-Lys-OH. On the other hand, FP-GPA-OH was not hydrolyzed at all by CPB and the hydrolysis rate of this compound by CPY was very slow. (S)-Flurbiprofen L-arginine ethyl ester ((S)-FP-Arg-OEt) was separated from (R)-FP-Arg-OEt by high-performance liquid chromatography. A comparison of the kinetic parameters for the tryptic hydrolysis of the two optically active FP-Arg-OEt diastereomers and those of Bz-Arg-OEt suggested that the orientation of the scissile bond in each diastereomer to the catalytic center of Tp is more favorable than that of Bz-Arg-OEt. However, no significant difference was found between the kinetic parameters for the two diastereomers, suggesting that the orientational difference between (S)-FP and (R)-FP in the diastereomers does not have any effect on the tryptic hydrolysis of the ester bond. On the other hand, for the hydrolysis of the diastereomers by CPB and CPY, the kcat values for (R)-FP-Arg-OH were larger than those for (S)-FP-Arg-OH, while the Km values for the former were almost the same as those for the latter. These results indicate that the orientation of the scissile bond in (R)-FP-Arg-OH to the catalytic sites of CPB and CPY was more favorable than that in (S)-FP-Arg-OH. (S)-FP-Arg-OEt is thus considered to be a promising optically active water-soluble prodrug for the sustained release of FP.