Synthesis and Characterization of 3-Hydroxybupivacaine and Deuterated 3-Hydroxybupivacaine for Use in Equine Medication Regulation

Abstract

Bupivacaine is a local anesthetic widely used in equine and human medicine. Use of bupivacaine in performance horses is regulated because its ability to block pain means that it can be misused for advantage in performance horses. In racing regulation, bupivacaine is classified by the Association of Racing Commissioners International (ARCI) as a Class 2 Penalty Class A Foreign substance, the detection of which can lead to significant penalties. In horses, bupivacaine is metabolized by Phase-I hydroxylation to yield 3-hydroxybupivacaine, which is then glucuronidated to yield the Phase-II metabolite bupivacaine-3-hydroxyglucuronide, which is excreted at relatively high concentrations in equine urine. Standard regulatory procedure during urinalysis is to perform an enzymatic hydrolysis, thereby enabling subsequent detection of 3-hydroxybupivacaine, the primary analyte used for bupivacaine regulation in urine samples from competition horses. We now report on the synthesis of 3-hydroxybupivacaine and deuterated 3-hydroxybupivacaine from piperidine-2-carboxylic acid in six successive steps with moderate yield. The compounds were characterized by ¹H and ¹³C NMR and their purity ascertained by HPLC-MS. The deuterated bupivacaine and 3-hydroxybupivacaine were further confirmed by HRMS. The synthesis of these compounds provides certified reference standards and stable isotope-labeled internal standards for drug testing in competitive equine sports including horse racing.