Stereoselective inhibition of human butyrylcholinesterase by phosphonothiolate analogs of (+)- and (-)-cocaine

The hydrolysis of cocaine (benzoylecgonine methyl ester) to ecgonine methyl ester by human butyrylcholinesterase (BuChE; EC 3.1.1.8) has been shown previously to constitute an important means to detoxicate this material to pharmacologically inactive metabolites. The naturally occurring (-)-cocaine is hydrolyzed to ecgonine methyl ester approximately 2000 times slower than the unnatural (+)-cocaine isomer. In good agreement with previous studies, (-)-cocaine bound to human BuChE with relatively good affinity and competitively inhibited the hydrolysis of the spectrophotometric substrate butyrylthiocholine with a Ki value of 8.0 microM. Similarly, (+)-cocaine also showed relatively high affinity for the human BuChE and competitively inhibited butyrylthiocholine hydrolysis with a Ki value of 5.4 microM. The phosphonothiolates corresponding to the transition state analogs for both (-)- and (+)-cocaine hydrolysis were synthesized and tested as inhibitors of human BuChE-catalyzed hydrolysis of butyrylthiocholine. The phosphonothiolate corresponding to the transition state for (-)-cocaine hydrolysis was a competitive inhibitor with a Ki value of 55.8 microM. The phosphonothiolate corresponding to the transition state for (+)-cocaine hydrolysis gave a Ki value of 25.9 microM, but, in addition, it also showed irreversible inhibition with a ki of inactivation of 68.8 min-1 M-1. It is likely that the mechanism-based inhibitor described herein may find use as a mechanistic probe of butyrylcholinesterase action and also possibly aid in the purification of this class of esterases.