Phosphonic Acid Analogs of Fluorophenylalanines as Inhibitors of Human and Porcine Aminopeptidases N: Validation of the Importance of the Substitution of the Aromatic Ring

Abstract

A library of phosphonic acid analogs of phenylalanine substituted with fluorine, chlorine and trifluoromethyl moieties on the aromatic ring was synthesized and evaluated for inhibitory activity against human (hAPN) and porcine (pAPN) aminopeptidases. Fluorogenic screening indicated that these analogs are micromolar or submicromolar inhibitors, both enzymes being more active against hAPN. In order to better understand the mode of the action of the most active compounds, molecular modeling was used. It confirmed that aminophosphonic portion of the enzyme is bound nearly identically in the case of all the studied compounds, whereas the difference in activity results from the placement of aromatic side chain of an inhibitor. Interestingly, both enantiomers of the individual compounds are usually bound quite similarly.

Synthesis and Inhibitory Studies of Phosphonic Acid Analogues of Homophenylalanine and Phenylalanine towards Alanyl Aminopeptidases

“…Novel library of the phosphonic acid analogues of homophenylalanine and phenylalanine have been synthesized by multistep reaction using commercially available fluorinated 3-phenylpropionic acids and fluorinated and brominated 2-phenylacetic acids, as starting materials. Preselection of starting substrates was done basing on previous study [ 10 ] taking into consideration their availability. Because of limitation on the usefulness of the previously described multistep reaction (hydrogenolytic removal of bromine from phenyl ring during reduction of oxime to amine group) and lack of products upon synthesis of homophenylalanine analogues by this procedure, we decided to convert carboxylic acid precursors to corresponding aldehydes.…”

“…The results of the enzymatic studies, presented in Table 1 , indicate quite significant potency of these compounds towards both enzymes. The data in Table 1 are supplemented with literature inhibitory constants obtained for compounds 17t , 17o and 17s [ 10 ] , which are analogues of compounds 17c , 17d and 17e differing in that they contain chlorine in place of bromine in aromatic rings.…”

“…Substitution of their phenyl moieties with fluorine atoms at any position is beneficial. Position of those substitutions seems to be less pronounced than found for analogues of phenylalanine [ 10 ]. However, it does not significantly change their affinities to both aminopeptidases, with compound 15c being only 5 times better inhibitor than 15a .…”

“…Supplementation of the previously described set of phosphonic acid analogues of phenylalanine [ 10 ] had shown that they display lowest affinities towards the studied aminopeptidases. Albeit, the replacement of chlorine by bromine is favorable, especially in the case of porcine enzyme.…”

“…Since the sequence identity of the two enzymes is 79.3% and sequence similarity 89.4%, the porcine enzyme is often used as a model one in studies on aminopeptidase N inhibitors development. On the other hand, our previous studies [ 9 , 10 ] indicated that low-molecular inhibitors of these enzymes could be bound substantially differently. Therefore, we undertook studies, which are a continuation of previous works and complements the library of the phosphonic acid analogues of phenylalanine, which phenyl group is substituted with halogens.…”