Butyrylcholinesterase is an enzyme whose levels increase significantly with the progress of the Alzheimer's disease, which makes it a promising drug target in the late stages of the disease. Using High-Performance Liquid Chromatography, we have developed conditions that allowed separation of racemic mixtures of selective butyrylcholinesterase inhibitors into their pure enantiomers. Selected column has a limited number of appropriate mobile phases. Among the selected alkaline mobile phases, a solution of triethylamine and a phosphate buffer did not allow separation of the racemic mixture of inhibitors. Given the fact that separation with borate buffer was successful on analytical coloumn, the method was transferred to semi-preparative column. Milligram amounts of the enantiomers of N-((1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl)methyl)-N-(2-methoxyethyl)-2-naphthamide and N-((1-benzylpiperidin-3-yl)methyl)-N-(2-methoxyethyl)naphthalene-2-sulfonamide were isolated. Further, their optical purity and specific rotation were measured. Using the enzyme assays different level of stereoselectivity of the pure enantiomers was observed. The median inhibitory concentration of pure enantiomers, as well as their racemic mixtures was determined. The most active enantiomer (+)-1 with 13.4 nM IC50 value has more than 10-times stronger affinity for the butyrylcholinesterase active site if compared to distomer (-)-1. In the case of sulfonamide analogue (±)-2, the stereoselectivity of pure enantiomers is markedly reduced, whereby 4.5 nM IC50 value was determined for eutomer (-)-2.
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