Determination of the dissociation constants (pKa) of basic acetylcholinesterase inhibitors by reversed-phase liquid chromatography

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylamine-diamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18, C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15–7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett σ parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.