Discovery of Further Pyrrolidine trans-Lactams as Inhibitors of Human Neutrophil Elastase (HNE) with Potential as Development Candidates and the Crystal Structure of HNE Complexed with an Inhibitor (GW475151) Simon J. F. Macdonald,* ,† Michael D. Dowle,* ,† Lee A. Harrison, † Geoffrey D. E. Clarke, † Graham G. A. Inglis, † Martin R. Johnson, † Pritom Shah, † Robin A. Smith, ‡ Augustin Amour, § Gill Fleetwood, | Davina C. Humphreys, ‡ Christopher R. Molloy, ‡ Mary Dixon, ⊥ Rosalind E. Godward, ⊥ Alan J. Wonacott, # Onkar M. P. Singh, # Simon T. Hodgson, † and George W. Hardy † Medicinal Chemistry 1, In Vitro Pharmacology, Systems Research, Respiratory Systems, Stevenage CEDD DMPK, and Computational and Structural Sciences, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom Received March 18, 2002 Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development. Described here is the discovery of three further derivatives of pyrrolidine trans-lactams, which fulfill the criteria required for back-up candidates 28, 29, and 32. These include increased activity in inhibiting HNE in human whole blood (HWB) and comparable pharmacokinetic properties, in particular clearance, in two species. To provide a rapid assessment of clearance, cassette dosing in dog was used. Modern array techniques, including the synthesis of mixtures, were used to synthesize compounds rapidly. Having selected three potential compounds as back-up candidates, they were prepared as single enantiomers and profiled in in vitro and in vivo assays and evaluated pharmacokinetically in rat and dog. These compounds are highly potent and selective HNE inhibitors, with a prolonged pharmacodynamic action. Pharmacokinetically, these compounds are comparable with 1 while they are more potent in HWB. Compound 28, however, has a higher clearance. One of these compounds, 32, was cocrystallized with HNE, and features of this structure are described and compared with the cocrystal structure of 1 in porcine pancreatic elastase. Introduction 1. Background. A considerable body of work now describes inhibitors of human neutrophil elastase (HNE), a serine protease from the trypsin class, for the treat- ment of respiratory diseases such as chronic bronchitis, cystic fibrosis, and emphysema. 1 We have introduced pyrrolidine trans-lactams as inhibitors of HNE 2 and described some of their properties. In particular, we have described their intracellular mechanism and the medicinal chemistry program 3 that led to the discovery 4 of a drug candidate GW311616A 1 suitable for develop- ment. 5 The final phase of our program was to generate potential back-up development compounds in case 1 failed in the drug development phase. We describe here the goals for the back-up compounds and the medicinal chemistry strategy and its application using a library, pools, and array techniques. We report our development and use of cassette dosing in dog for the rapid evaluation of clearance. A summary of the biological data of the three potential back-up compounds and the crystal structure of one of these compounds in HNE is de- scribed. We particularly highlight the value of the cassette approach and the judicial use of modern medicinal chemistry techniques in aiding the rapid discovery of these back-up compounds. We also highlight the HNE crystal structure, one of very few published. Although GW311616A was a suitable compound for development, it did contain features that were regarded as potential liabilities. From a structural viewpoint, we were keen to remove the acrylamide functionality since it was perceived as a potential toxicological risk because it is a Michael acceptor. There were also concerns over the long-term photostability of the double bond. 6 Con- sidering the biological profile of 1, the only available measure of its potential efficacy in humans is its potency, ex vivo in human whole blood (HWB) (0.67 µM). This is less potent than the Merck compound 7 L-694,458 (0.45 µM). We also noted that despite the excellent bioavailability of GW311616A in rat (60%) and dog (100%), its clearance in rat (79 mL/min/kg) and dog (28 mL/min/kg) approximated to liver blood flow (about 66 and 38 mL/min/kg, respectively). These concerns directed our goals for the back-up compounds. Thus, our first two goals were to improve potency in HWB and maintain, if not reduce, the clearance in rat and dog. To address these goals, we could replace the piperidinocrotyl side chain and/or the * To whom correspondence should be addressed. Fax: +44 1438 76 36 15. E-mail: sjfm5947@gsk.com and mdd3463@gsk.com. † Medicinal Chemistry 1. ‡ In Vitro Pharmacology. § Systems Research. | Respiratory Systems. ⊥ Stevenage CEDD DMPK. # Computational and Structural Sciences. 3878 J. Med. Chem. 2002, 45, 3878-3890 10.1021/jm020881f CCC: $22.00 © 2002 American Chemical Society Published on Web 08/03/2002