Design, Synthesis, and Enzymatic Evaluation of N 1 -Acyloxyalkyl- and N 1 -Oxazolidin-2,4-dion-5-yl-Substituted -lactams as Novel Inhibitors of Human Leukocyte Elastase Rui Moreira,* ,† Ana Bela Santana, † Jim Iley,* ,‡ Joa ˜ o Neres, § Kenneth T. Douglas, § Peter N. Horton, | and Michael B. Hursthouse | CECF, Faculdade de Farma ´ cia, Universidade de Lisboa, Av. Forc ¸ as Armadas, 1600-083 Lisboa, Portugal, Department of Chemistry, The Open University, Milton Keynes MK7 6AA, U.K., Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics, School of Pharmacy & Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, U.K., and EPSRC National Crystallography Service, School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, U.K. Received February 11, 2005 Human leukocyte elastase (HLE) is a serine protease that very efficiently degrades various tissue matrix proteins such as elastin. The imbalance between HLE and its endogenous inhibitors leads to excessive elastin proteolysis and is considered to be responsible for the onset of chronic obstructive pulmonary disease (COPD). A novel series of C-3-, C-4-, and N-1- substituted azetidin-2-ones were prepared as potential mechanism-based inhibitors of HLE to restore the protease/antiprotease imbalance. N-Acyloxyalkylazetidin-2-ones, 4, and their carbamate counterparts, 5, are weak HLE inhibitors, being 5 times less active than their bicyclic oxazolidin-2,4-dione-substituted analogues, 6, containing an electron-withdrawing substituent at C-4. Compounds 6 containing a C-4 substituent exist as two diastereomeric pairs of enantiomers, each pair presenting similar inhibitory activity against HLE. Comparative docking experiments with the C-4-substituted oxazolidin-2,4-dione inhibitors 6 suggest that only the 4R,5′S and 4S,5′S diastereomers consistently interact with the -lactam carbonyl carbon atom accessible to the serine hydroxyl oxygen. Introduction Human leukocyte (or neutrophil) elastase (HLE, EC 3.4.21.37) is a member of the chymotrypsin superfamily of serine proteases that very efficiently degrades various tissue matrix proteins such as elastin, when released from the azurophilic granules of polymorphonuclear leukocytes (neutrophils) 1 due to inflammatory stimuli and mediators. 2,3 In healthy individuals, the proteolytic activity of HLE is regulated by potent antiproteases such as R 1 -antitrypsin and secretory leukocyte protein- ase inhibitor. The imbalance between HLE and its endogenous inhibitors leads to excessive elastin pro- teolysis and destruction of connective tissues and is considered to be responsible for the onset of chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic obstructive bronchitis. 4,5 Selec- tive inhibitors of neutrophil elastase can restore the protease/antiprotease imbalance and thus are important candidates for the treatment of COPD and other inflam- matory disorders such as rheumatoid arthritis and cystic fibrosis. 7-10 -Lactams are well-known serine protease inhibitors that acylate the nucleophilic serine residue of a wide range of enzymes, 11 including HLE. 8 Cephalosporin sulfones, for example, 1, have been reported as one of the first -lactam irreversible inhibitors of HLE. 12-15 Compound 1 and other cephalosporin sulfone analogues promote the acylation of the catalytic serine and alky- lation of the histidine residue, probably via a mecha- nism-based inhibition pathway. 16 The need for improv- ing oral bioavailability prompted the design of monocyclic -lactams. Merck’s pioneering work led to the develop- ment of several -lactam inhibitors, for example, 2, that contain a phenol leaving group at C-4, 17-19 two of them reaching clinical trials. 5 Recently, we reported that -lactams 3 (LG ) OCOR or OCONHR) (see Scheme 1) are time-dependent inhibitors of HLE. 20 These -lac- tams were designed as potential mechanism-based inhibitors because the leaving group (LG) in 3 can be expelled following the initial Ser-195 attack at the -lactam carbonyl atom to generate an electrophilic imine within the enzyme active site (Scheme 1). This reactive functionality has the potential of reacting with a second amino acid residue within the active site (e.g., His-57), leading to an inactivated enzyme through a double hit. In addition to a good leaving group, LG, compounds 3 contain two ethyl groups at C-3 required for molecular recognition by the enzyme and an electron- withdrawing substituent, EWG, at C-4 required for * To whom correspondence should be addressed: Tel +351 217946477; fax +351 217946470; e-mail rmoreira@ff.ul.pt or j.n.iley@open.ac.uk. † Universidade de Lisboa. ‡ The Open University. § University of Manchester. | University of Southampton. 4861 J. Med. Chem. 2005, 48, 4861-4870 10.1021/jm0501331 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/06/2005