Synthesis and evaluation of Aziridine-containing compounds as inhibitors of porcine kidney diamine oxidase

Abstract

Diamine oxidase is an enzyme that catalyzes the oxidative deamination of diamines. Putrescine and cadaverine are among the best substrates of porcine kidney diamine oxidase. In this study, a variety of diamines with structures that are related to putrescine and cadaverine, namely N-(3-aminopropyl)aziridine, N-(4-aminobutyl)aziridine, N-(5-aminopentyl)aziridine, N-(6-aminohexyl)aziridine, N-(7-aminoheptyl)aziridine and the rigid analogue, N-(p-aminomethylbenzyl)aziridine have been synthesized and their interactions with diamine oxidase evaluated. These aziridinyl-containing compounds were synthesized in three steps from the condensation of aziridine with bromonitriles to yield aziridinylnitriles. Reduction of the nitriles with LiAlH{dollar}\sb4{dollar} provided the aziridinyl amines. These aziridinyl derivatives were relatively stable in aqueous buffer solutions at 37{dollar}\sp\circ{dollar}C. All of the compounds contain an aziridine ring as an alkylating group to inactivate the enzyme. Calculated IC{dollar}\sb{lcub}50{rcub}{dollar} values ranged from 0.01 to 1.7 mM. The activity of these inhibitors correlated with the separation length of the two nitrogen atoms of the diamine analogues. Four-carbon and five-carbon chain analogues were more potent than the other analogues tested. 3-Aziridinylpropylhydrazine, which contains an aziridine ring as well as a hydrazine group, was synthesized by reduction of the hydrazone formed between 3-aziridinylpropanal and hydrazine. This bifunctional inhibitor showed an IC{dollar}\sb{lcub}50{rcub}{dollar} value of 0.0085 {dollar}\mu{dollar}M which classifies it as one of the most potent inhibitors of diamine oxidase reported. All of the inhibitors showed time-dependent irreversible inhibition of diamine oxidase, which suggests that the formation of an enzyme-inhibitor complex is a reversible step, and a subsequent irreversible step leads to the inactivation of the enzyme. The inhibition pattern depends on the length of the inter-nitrogen distance in the analogues. Evaluation of the kinetics of inhibition of putrescine deamination indicated that the analogues of up to 5 or 6 carbons were competitive inhibitors. The 7-carbon analogue, N-(7-aminoheptyl)aziridine, showed mixed-competitive inhibition and a high IC{dollar}\sb{lcub}50{rcub}{dollar} value. The observed switching of the inhibitory mechanism is consistent with an additional binding region in the active site of diamine oxidase which can be accessed by longer-chained, lower-affinity inhibitors.