N-α-[(RS)-2-Carboxy-3-phenylpropionyl]-L-leucine

A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase complexed with the phosphonic acid analogue of leucine (LeuP) was used for structure-based design of novel LAP inhibitors and for the analysis of their interactions with the enzyme binding site. The inhibitors were designed by modification of phosphonic group in the LeuP structure toward finding the substituents bound at the S' side of the enzyme. This resulted in two classes of compounds, the phosphonamidate and phosphinate dipeptide analogues, which were synthesized and evaluated as inhibitors of the enzyme. The in vitro kinetic studies for the phosphinate dipeptide analogues revealed that these compounds belong to the group of the most effective LAP inhibitors found so far. Their further modification at the P1 position resulted in more active inhibitors, hPheP[CH(2)]Phe and hPheP[CH(2)]Tyr (K(i) values 66 nM and 67 nM, respectively, for the mixture of four diastereomers). The binding affinities of these inhibitors toward the enzyme are the highest, if considering all compounds containing a phosphorus atom that mimic the transition state of the reaction catalyzed by LAP. To evaluate selectivity of the designed LAP inhibitors, additional tests toward aminopeptidase N (APN) were performed. The key feature, which determines their selectivity, is structure at the P1' position. Aromatic and aliphatic substituents placed at this position strongly interact with the LAP S1' binding pocket, while a significant increase in binding affinity toward APN was observed for compounds containing aromatic versus leucine side chains at the P1' position. The most selective inhibitor, hPheP[CH(2)]Leu, binds to LAP with 15 times higher affinity than to APN. One of the studied compounds, hPheP[CH(2)]Tyr, appeared to be very potent inhibitor of APN (K(i) = 36 nM for the mixture of four diastereomers). The most promising LAP inhibitors designed by computer-aided approach, the phosphonamidate dipeptide analogues, were unstable at pH below 12, because of the P-N bond decomposition, which excluded the possibility of determination of their binding affinities toward LAP.

The synthesis and biological activity studies of the series of structurally different alpha-aminoalkylphosphonates were performed in order to optimise the shape of the side chain of the potential inhibitors in S1 pocket of leucine aminopeptidase [E.C.3.4.11.1]. Analysis of a series of compounds with aromatic, aliphatic and alicyclic P1 side chains enabled to find out the structural features, optimal for that fragment of inhibitors of LAP. The most active among all investigated compounds were the phosphonic analogues of homo-tyrosine (K(i)=120 nM) and homo-phenylalanine (K(i)=140 nM), which even as racemic mixtures were better inhibitors in comparison with the best till now-phosphonic analogue of l-leucine (230 nM). Additional comparison of the inhibitory activity obtained for aminopeptidase N (APN, E.C.3.4.11.2) give insight into structural preferences of both enzymes.

A new diastereoselective synthesis of alpha-aminophosphonates has been developed, based on the reaction, in the presence of trifluoroacetic acid, of trialkyl phosphites with chiral imines derived from (R)- or (S)-1-(alpha-aminobenzyl)-2-naphthol. The reaction proceeds at room temperature in toluene with high diastereoselectivity. The major diastereomer can be separated by crystallization from an appropriate solvent. The relative configuration of both chiral centers of the major diastereomer was determined by single-crystal X-ray structure analysis. The desired alpha-aminophosphonic acids can be obtained in enantiopure form by treatment of the corresponding diastereomers with HCl.