N-Benzoylpyrazoles Are Novel Small-Molecule Inhibitors of Human Neutrophil Elastase Igor A. Schepetkin, ² Andrei I. Khlebnikov, ‡ and Mark T. Quinn* ,² Department of Veterinary Molecular Biology, Montana State UniVersity, Bozeman, Montana 59717, and Department of Chemistry, Altai State Technical UniVersity, Barnaul 656038, Russia ReceiVed May 22, 2007 Human neutrophil elastase (NE) plays an important role in the pathogenesis of pulmonary disease. Using high-throughput chemolibrary screening, we identified 10 N-benzoylpyrazole derivatives that were potent NE inhibitors. Nine additional NE inhibitors were identified through further screening of N-benzoylpyrazole analogues. Evaluation of inhibitory activity against a range of proteases showed high specificity for NE, although several derivatives were also potent inhibitors of chymotrypsin. Analysis of reaction kinetics and inhibitor stability revealed that N-benzoylpyrazoles were pseudoirreversible competitive inhibitors of NE. Structure-activity relationship (SAR) analysis demonstrated that modification of N-benzoylpyrazole ring substituents modulated enzyme selectivity and potency. Furthermore, molecular modeling of the binding of selected active and inactive compounds to the NE active site revealed that active compounds fit well into the catalytic site, whereas inactive derivatives contained substituents or conformations that hindered binding or accessibility to the catalytic residues. Thus, N-benzoylpyrazole derivatives represent novel structural templates that can be utilized for further development of efficacious NE inhibitors. Introduction Acute respiratory distress syndrome (ARDS a ), chronic ob- structive pulmonary disease (COPD), and cystic fibrosis (CF) are progressive diseases that are frequently fatal. 1-3 Unfortu- nately, there are currently few effective therapeutic treatments for these syndromes. Inflammation associated with these pulmonary diseases is predominantly due to neutrophils and is associated with excessive release of neutrophil granule proteases, such as neutrophil elastase (NE, EC 3.4.21.37). 4,5 NE is a serine protease that is synthesized in neutrophils and stored in azurophilic granules. 6 While the primary role of NE appears to be in microbial killing in the phagosome, excessive NE release into extracellular fluids can cause major tissue damage. 7 For example, NE is released in large amounts during pulmonary inflammation, resulting in a protease/antiprotease imbalance, and this imbalance appears to be a major pathogenic determinant in COPD and ARDS. 5,8-10 NE is a member of the chymotrypsin family of serine proteases and is expressed primarily in neutrophils but is also present in monocytes and mast cells. It can degrade a variety of extracellular matrix proteins, including elastin, fibronectin, laminin, collagen, and proteoglycans (reviewed in refs 11 and 12). NE also can activate several matrix metalloproteinases (MMP-2, -3, and -9) 13 and seems to play an important physiologic role in tissue repair through its ability to regulate growth factors and modulate cytokine expression at epithelial and endothelial surfaces. 14,15 However, excessive NE activity can lead to severe pathology through the degradation of elastin and collagen in the airways, resulting in microvascular injury and interstitial edema. 16 Given the destructive potential of unregulated NE, it is not surprising that inhibition of NE activity in pulmonary tissues has been considered a promising strategy to improve the outcome of pulmonary diseases. 17,18 A number of therapeutic strategies have focused on the use of recombinant or purified preparations of two endogenous NE inhibitors described above, R 1 -antitrypsin and secretory leukocyte protease inhibitor; how- ever, use of these inhibitors has been problematic. 19 Many types of peptide and nonpeptide inhibitors, employing both reversible and irreversible mechanisms of action, have also been reported (reviewed in refs 15, 19, and 20). Among the most potent NE inhibitors are -lactams, 21 tert-butyloxadiazoles, 22 and peptidyl trifluoromethyl ketones. 23 Nevertheless, the primary chemical scaffolds of the most potent NE inhibitors were discovered 15- 20 years ago, 24,25 and modification of these scaffolds is still the current focus of most NE inhibitor development because moving away from these core scaffolds would be difficult and time-consuming. 26 Conversely, we propose that new NE inhibi- tors with different structural and/or physicochemical properties from those described so far could lead to novel and useful leads for the development of anti-inflammatory drugs. Although some novel approaches have been developed for high-throughput screening (HTS) to identify NE inhibitors on a large scale, there are still only a few reports on HTS of elastase inhibitors. 27-29 Thus, we utilized HTS of a chemical diversity library containing 10 000 druglike molecules to identify novel inhibitors of NE that have core structures distinct from currently known leads. Notably, the hits obtained from our screen included 10 N- benzoylpyrazole derivatives, which were potent NE inhibitors. Furthermore, analysis of 43 additional N-benzoylpyrazole derivatives resulted in the identification of nine more potent NE inhibitors with K i e 1 µM. Evaluation of target specificity showed that most of NE inhibitors were selective for NE and chymotrypsin but not other proteases tested. Finally, molecular modeling approaches demonstrated that active N-benzoylpyra- zole derivatives were able to effectively dock within the NE catalytic site so that Michaelis complex formation and synchro- nous proton transfer were favored, while binding of inactive derivatives into the pocket was sterically hindered or catalyti- cally unfavorable. * To whom correspondence should be addressed. Phone: 406-994-5721. Fax: 406-994-4303. E-mail: mquinn@montana.edu. ² Montana State University. ‡ Altai State Technical University. a Abbreviations: ARDS, adult respiratory distress syndrome; COPD, chronic obstructive pulmonary disease; HTS, high-throughput screening; NE, neutrophil elastase; RP-HPLC, reverse-phase high-performance liquid chromatography. 4928 J. Med. Chem. 2007, 50, 4928-4938 10.1021/jm070600+ CCC: $37.00 © 2007 American Chemical Society Published on Web 09/12/2007