Original article a-Aminoalkylphosphonates as a tool in experimental optimisation of P1 side chain shape of potential inhibitors in S1 pocket of leucine- and neutral aminopeptidases Marcin DrTg a, *, Jolanta Grembecka a , Malgorzata Pawelczak b , Pawel Kafarski a, * a Institute of Organic Chemistry, Biochemistry and Biotechnology, University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland b Faculty of Chemistry, University of Opole, Opole, Oleska 48, Poland Received 26 October 2004; received in revised form 14 February 2005; accepted 24 February 2005 Available online 20 April 2005 Abstract The synthesis and biological activity studies of the series of structurally different a-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. © 2005 Elsevier SAS. All rights reserved. Keywords: Aminophosphonates; Inhibitor; Leucine aminopeptidase; Aminopeptidase N 1. Introduction Aminopeptidases are enzymes that catalyse the removal of amino acids from the N-terminus of a peptide or protein. These enzymes have been found in bacteria, yeast, plants, animal and human tissues, what is considerably ascribed to their broad substrate specificity [1]. They play such a biologi- cal functions as protein maturation, activation and degrada- tion of bioactive peptides, hormone level regulation and con- trol of cell proliferation [2,3]. It is well known, that elevated concentration of these enzymes was observed in such patho- logical disorders as cancer and cataracts [1,3]. In the case of the carcinogenesis, the methionine aminopeptidases has been shown to be a promising target for development of anti- angiogenesis agents [4]. Additionally, the importance of these enzymes arises from the observation, that some of them might play the role in the apoptosis of cancer cells, what makes them interesting targets in oncological research (with methion- ine and neutral aminopeptidases being the most promising targets) [5]. Aminopeptidases can be subdivided into two major groups, with the first one being responsible for the hydrolysis of amino acids with hydrophobic side chains from N-terminus of the peptide or protein, and the second, which specifically remove other amino acid residues [3]. Within the first group, leucine aminopeptidase (LAP, E.C.3.4.11.1) is one of the most detailed characterised enzyme with respect to its sequence, structure and mechanism of action. Human LAP plays an important role in the early stages of HIV infection, where the elevated concentration of this enzyme was observed [6]. The unusual activity of this enzyme has also been observed in such pathological disorders as can- cer, inflammation of liver or eye cataract [7–9]. Because of this leucine aminopeptidase is often called the stress enzyme. Substantial insight into the structure of the LAP derives from X-ray structures of the enzyme from bovine lens deter- mined in its native form and complexed with transition state inhibitors such as: bestatin, amastatin, phosphonic analogue of L-leucine and L-leucinal coordinated to the active site. * Corresponding author. Tel.: +48 71 320 4027; fax: +48 71 328 4064. E-mail address: marcin.drag@pwr.wroc.pl (M. DrTg). European Journal of Medicinal Chemistry 40 (2005) 764–771 www.elsevier.com/locate/ejmech 0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.ejmech.2005.02.011