Enantiomeric resolution, stereochemical assignment and toxicity evaluation of TPA enantiomers.

Abstract

Tetrahydro-α-(1-methylethyl)-2-oxo-1(2H)-pyrimidineacetic acid (TPA) is a critical intermediate in the synthesis of HIV protease inhibitors. A simple and efficient method for the separation and determination of TPA enantiomers was developed. The TPA was separated into its enantiomers with an enantiomeric purity of 99% using an HPLC system equipped with a Chiralpak OD-H column. Semi-preparative HPLC enantioseparations were carried out for further enrichment of the enantiomers. The validity of this method was evaluated on the basis of its precision, accuracy, linearity and recovery. The method was observed to be suitable for the rapid separation and semi-preparation of TPA isomers. The separated enantiomers were identified by optical rotation and high-resolution electrospray ionization mass spectrometry. Furthermore, the stereochemical structures of the TPA enantiomers were definitively confirmed using a combination of experimental and calculated electronic circular dichroism spectra. The toxicity of the separated pure enantiomers against Oryzias melastigma was evaluated using the median lethal concentration (LC50 ) values. The results indicated that (S)-(-)-TPA is ~2.5 times more toxic than its enantiomorphism.