Design and Synthesis of Hydrazinopeptides and Their Evaluation as Human Leukocyte Elastase Inhibitors Laure Guy, Joe ¨lle Vidal,* and Andre ´ Collet* Ste ´ re ´ ochimie et Interactions Mole ´ culaires (UMR CNRS 117), E Ä cole Normale Supe ´ rieure de Lyon, 46 alle ´ e d’Italie, 69364 Lyon Cedex 07, France Augustin Amour and Miche `le Reboud-Ravaux* De ´ partement de Biologie Supramole ´ culaire et Cellulaire, Laboratoire d’Enzymologie Mole ´ culaire Fonctionnelle, Institut Jacques Monod (UMR CNRS 7592/Universite ´ s Paris VI and VII), 2 place Jussieu, Tour 43, 75251 Paris Cedex 05, France Received July 15, 1998 The name hydrazinopeptide designates peptidic structures in which one of the native CONH links is replaced by a CONHNH (hydrazido) fragment. In this paper, we report the synthesis of such hydrazinohexapeptides (3-5) analogous to Z-Ala-Ala-Pro-Val-Ala-Ala-NH i Pr (6), a substrate of human leukocyte elastase (HLE; EC 3.4.21.37), cleaved by this serine protease between the Val4 and Ala5 residues. In hydrazinopeptides 3-5, the Ala5, Val4, or Pro3 residue, respectively, of the model peptide, has been replaced by the corresponding R-L-hydrazino acid. In 3, the bond likely to be cleaved by HLE is the one involving the CONHNH link, while in 4 and 5, this link is normally shifted away by one or two amino acid units from the catalytic serine. On incubation with HLE, hydrazinopeptide 3 proved to be a substrate and was cleaved between Val4 and NHAla5, like peptide 6. In contrast, 4 and 5 proved to bind to HLE without being cleaved, featuring properties consistent with reversible competitive inhibition. General guidelines for the synthesis of hydrazinopeptides are also reported in this paper. These guidelines take into account the chemical specificity of hydrazino acids, while being fully compatible with the conventional peptide coupling techniques. The utilization of orthogonally bisprotected hydrazino acids 1 where the N  and N R atoms bear a Boc and a Bzl group, respectively, is recommended for the easy construction of such hydrazinopeptides. Introduction Among the variety of pseudopeptide structures cur- rently investigated in organic and medicinal chemistry, 1 little attention has been devoted to hydrazinopeptides, in which one of the native amino acids has been replaced by the corresponding R-hydrazino acid, to form a -CO- NH-NH-C* (hydrazide) link (Chart 1). This modifica- tion of the peptide bond was first introduced by Niedrich 2 in the mid-1960s for a series of eledoisin analogues, to increase the resistance toward proteolysis while keeping the physiological activity of the natural peptide unaf- fected. This goal has been met, at least partially, in these eledoisin mimics. 3 The subsequent lack of interest for this class of pseudopeptides is probably due to the difficulty of the synthesis of L- or D-R-hydrazino acids and to the absence of efficient methodologies for their insertion in peptide chains. During the past decade, several methods allowing the preparation of L- or D-R-hydrazino acids 1 either free, 4 N  -protected, 5,6 or N  ,N R -orthogonally bisprotected 7 have been reported. This circumstance has renewed the interest for hydrazinopeptides in making these com- pounds more easily accessible. Short hydrazinopeptide models incorporating one hydrazino acid coupled with one or two amino acids have been synthesized. Struc- tural studies (X-rays, NMR and IR spectroscopies, molecular modeling) 8-10 have revealed in these com- pounds a common conformational bias, called a hy- drazino turn, 11 which folds the peptide backbone locally by way of a well-defined intramolecular bifurcated H-bonding pattern (see Chart 1). We address here the problem of the development of hydrazinopeptidic inhibitors for serine proteases. Hu- man leukocyte elastase (HLE; EC 3.4.21.37) which is involved in the pathogenesis of chronic inflammatory diseases such as pulmonary emphysema was chosen as Chart 1. Hydrazinopeptides and Related Pseudopeptides 4833 J. Med. Chem. 1998, 41, 4833-4843 10.1021/jm980419o CCC: $15.00 © 1998 American Chemical Society Published on Web 10/30/1998