Identification of a Potent, Selective, and Orally Active Leukotriene A 4 Hydrolase Inhibitor with Anti-Inflammatory Activity Cheryl A. Grice,* Kevin L. Tays, Brad M. Savall, Jianmei Wei, Christopher R. Butler, Frank U. Axe, Scott D. Bembenek, Anne M. Fourie, Paul J. Dunford, Katherine Lundeen, Fawn Coles, Xiaohua Xue, Jason P. Riley, Kacy N. Williams, Lars Karlsson, and James P. Edwards Johnson & Johnson Pharmaceutical Research & DeVelopment, L.L.C., 3210 Merryfield Row, San Diego, California 92121 ReceiVed December 14, 2007 LTA 4 H is a ubiquitously distributed 69 kDa zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity. As a hydrolase, LTA 4 H stereospecifically catalyzes the transformation of the unstable epoxide LTA 4 to the diol LTB 4 , a potent chemoattractant and activator of neutrophils and a chemoattractant of eosinophils, macrophages, mast cells, and T cells. Inhibiting the formation of LTB 4 is expected to be beneficial in the treatment of inflammatory diseases such as inflammatory bowel disease (IBD), asthma, and atherosclerosis. We developed a pharmacophore model using a known inhibitor manually docked into the active site of LTA 4 H to identify a subset of compounds for screening. From this work we identified a series of benzoxazole, benzthiazole, and benzimidazole inhibitors. SAR studies resulted in the identification of several potent inhibitors with an appropriate cross-reactivity profile and excellent PK/PD properties. Our efforts focused on further profiling JNJ 27265732, which showed encouraging efficacy in a disease model relevant to IBD. Introduction The proinflammatory mediator leukotriene B 4 (LTB 4 a ) is a potent chemoattractant and activator of neutrophils and a chemoattractant of eosinophils, macrophages, mast cells, and T cells. It is implicated in disorders such as inflammatory bowel disease (IBD), 1,2 rheumatoid arthritis (RA), 3–5 chronic obstruc- tive pulmonary disease (COPD), 6,7 asthma, 8–12 cancer, 13,14 and atherosclerosis. 15–20 The generation of LTB 4 in vivo is regulated by the action of leukotriene A 4 hydrolase (LTA 4 H). LTA 4 H is a ubiquitously distributed 69 kDa zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity, the crystal structure of which was published in 2001. 21 As a hydrolase, it stereospecifically catalyzes the transformation of the unstable epoxide LTA 4 to the diol LTB 4 and is a key enzyme in the arachidonic acid cascade downstream of the initial action of 5-lipoxygenase (5-LO) and FLAP (5-lipoxygenase-activating protein) as shown in Figure 1. LTA 4 H also functions as an anion-dependent aminopeptidase, processing arginyl di- and tripeptides with high efficiency. The biological relevance of this activity is unknown. 22,23 It is important to note that LTA 4 H- deficient mice have been generated; these mice develop normally and are healthy. 24 Studies of LTA 4 H deficient mice in several models of inflammation clearly establish the contribution of LTB 4 in the inflammatory process 24 and strongly support the pursuit of LTA 4 H inhibitors as potential treatments for inflam- matory diseases. A 1996 review details work on inhibitors of 5-LO and FLAP, as well as receptor antagonists of LTD 4 and LTB 4 . 25 Inhibitors of 5-LO and antagonists of LTD 4 are currently marketed drugs used in the treatment of asthma. A large number of reports suggesting the use of LTB 4 receptor antagonists as potential therapeutics have also appeared, and although several com- pounds have been evaluated in human clinical trials, no drugs have been marketed to date. Additionally in 2000, four groups simultaneously identified a second GPCR for LTB 4 that shares a 45.2% homology with the first receptor. 26–29 These two receptors, currently termed BLT1 (high affinity, cloned in 1997, primarily expressed on neutrophils) 30 and BLT2 (low affinity, expressed in the liver, spleen, and peripheral leukocytes), 28 are both anticipated to play a role in the inflammatory process. Recent reports established the presence of BLT1 and BLT2 receptors on human and murine mast cells and demonstrated chemotaxis of mast cells to LTB 4 . 31 In order to achieve efficacy in vivo, an antagonist of both receptors may be needed; inhibition of LTA 4 H would obviate the need for a dual antagonist. 32 We also speculate that inhibitors of LTA 4 H may have advantages over inhibitors of 5-LO. For example, blockade of 5-LO prevents the formation of LTA 4 , which in turn is one source of the lipoxins, LXA 4 and LXB 4 (lipoxygenase interaction products), as shown in Figure 2. 33–36 In contrast, the inhibition of LTA 4 H would not hinder the production of the lipoxins because it acts downstream of this branch point in the cascade. Additionally, researchers at Johnson & Johnson PRD in La Jolla have shown that 33r, vide infra, when tested in murine whole blood and when dosed orally at 30 mpk in a zymosan-induced peritonitis model, selectively inhibited LTB 4 production without affecting cysteinyl leukotriene production and maintained the production of the anti-inflammatory mediator LXA 4 . 37 The significance of these findings is related to the role of lipoxins, which are known endogenous anti-inflammatory agents that participate in the resolution process 38 and are postulated to recruit monocytes. In the presence of lipoxins monocytes undergo maturation to macrophages, which clear inflamed areas of necrotic and apoptotic neutrophils. Importantly, the resulting macrophages lack the capacity to cause additional tissue damage, as they cannot generate superoxide ions. 39–41 * To whom correspondence should be addressed. Phone: (858) 784-3054. Fax: (858) 450-2089. E-mail: cgrice@prdus.jnj.com. a Abbreviations: LTA 4 H, leukotriene A 4 hydrolase; LTA 4 , leukotriene A 4 ; LTB 4 , leukotriene B 4 ; IBD, inflammatory bowel disease; PK/PD, pharmacokinetic/pharmacodynamic; RA, rheumatoid arthritis; COPD, chronic obstructive pulmonary disease; 5-LO, 5-lipoxygenase; FLAP, 5-lipoxyge- nase-activating protein; LTD 4 , leukotriene D 4 ; GPCR, G-protein-coupled receptor; BLT1, B leukotriene 1 receptor; BLT2, B leukotriene 2 receptor; LXA 4 , lipoxin A 4 ; LXB 4 , lipoxin B 4 ; CADD, computer-assisted drug design; TBAF, tetrabutylammonium fluoride; MPO, myeloperoxidase activity; hERG, human ether-a-go-go related gene; SEM-Cl, 2-(trimethylsilyl)ethoxym- ethyl chloride; CDI, carbonyl diimidazole. J. Med. Chem. 2008, 51, 4150–4169 4150 10.1021/jm701575k CCC: $40.75  2008 American Chemical Society Published on Web 06/28/2008