Discovery of 3,3′-(2,4-Diaminopteridine-6,7-diyl)diphenol as an Isozyme-Selective Inhibitor of PI3K for the Treatment of Ischemia Reperfusion Injury Associated with Myocardial Infarction Moorthy S. S. Palanki,* ,² Elena Dneprovskaia, ² John Doukas, ² Richard M. Fine, ‡ John Hood, ² Xinshan Kang, ‡ , Dan Lohse, ² Michael Martin, ² Glenn Noronha, ² Richard M. Soll, ² Wolfgang Wrasidlo, ² Shiyin Yee, ² and Hong Zhu ² TargeGen Inc., 9380 Judicial DriVe, San Diego California 92121, and BioPredict, Inc., Suite 201, 660 Kinderkamack Road, Oradell, New Jersey 07649 ReceiVed October 19, 2005 In studies aimed toward identifying effective and safe inhibitors of kinase signaling cascades that underlie ischemia/reperfusion (I/R) injury, we synthesized a series of pteridines and pyridopyrazines. The design strategy was inspired by the examination of naturally occurring PI3K inhibitors such as wortmannin and quercetin, and building a pharmacophore-based model used for optimization. Structural modifications led to hybrid molecules which incorporated aminopyrimidine and aminopyridine moieties with ATP mimetic characteristics into the pharmacophore motifs to modulate kinase affinity and selectivity. Elaborations involving substitutions of the 2 and 4 positions of the pyrimidine or pyridine ring and the 6 and 7 positions of the central pyrazine ring resulted in in ViVo activity profiles which identified potent inhibitors of vascular endothelial growth factor (VEGF) induced vascular leakage. Pathway analysis identified a diaminopteridine- diphenol as a potent and selective phosphatidylinositol-3-kinase (PI3K) inhibitor. The structure-activity relationship studies of various analogues of diaminopteridine-diphenol-based on biochemical assays resulted in potent inhibitors of PI3K. Introduction The vascular endothelium plays a key role in physiological homeostasis by regulating functions such as blood plasma- interstitial fluid balance via vascular permeability control. Ischemic tissue damage results in part from the disruption of this homeostatic balance. As first shown by Dvorak et al., 1 an ischemic environment triggers upregulation of vascular endot- helial growth factor (VEGF) expression, followed by VEGF- mediated vascular leakage. This process is regulated through complex interactions between VEGF and structural proteins in the endothelium that form the intercellular junctions between neighboring endothelial cells, VE-cadherin and -catenin. Phosphorylation of these junctional proteins induces confor- mational changes in these molecules, with subsequent losses in cell-cell adhesion. 2 This in turn leads to a reduction in the vascular barrier that controls fluid movement from the blood compartment to the interstitium. The importance of kinases in regulating major cellular signaling events such as cell survival, 3-6 cell migration, 7-10 and transformation and apoptosis 11-14 has long been recognized, and perturbation in these pathways has been implicated in a number of diseases including cancer and inflammation. 15,16 The critical roles that kinases play in regulat- ing the vascular barrier have been extensively researched. 17-19 These studies suggest that the inhibition of the implicated signaling pathways might prove therapeutically effective for the treatment of diseases and injuries associated with microvascular barrier function such as ischemic conditions where edema accumulation is believed to contribute to tissue damage. In the regulation of microvascular processes, two major signaling pathways, the Src family and the PI3K signaling cascade in endothelial cells (Figure 1), are of pivotal importance. A frequent target for intervention downstream of receptor tyrosine kinases is Src, 20-23 but investigations on PI3K inhibitors for a while was confined to natural products such as wortmannin, analogues of wortmannin, 24 semisynthetic viridine analogues, quercetin, LY294002 (a quercetin analogue), and staurosporin. 25-27 Even though wortmannin is very potent inhibitor of PI3K, its development was hampered by its toxicity, insolubility, and aqueous instability. Several analogues of wortmannin were developed to circumvent these challenges. One such analogue is a pegylated wortmannin derivative, PWT-458, currently being developed by Wyeth Research. 28 This pegylation of wortmannin resulted in a compound with improved plasma stability and reduced toxicity. Studies with synthetic, small molecules have focused primarily on the catalytic domain and specifically the ATP binding site of these proteins. Several inhibitors of PI3K were reported recently including 1-benzopyran-4-ones, 29 ben- zoxazines, 30 benzothiophenes, 31 tetrazole benzofurancarboxa- mides, 32 pyrimidine carboxamides, 33 and fused azolepyrim- idines. 34 * Author to whom correspondence should be addressed. Telephone: (858)-678-0760. Fax: (858)-678-0762. E-mail: Palanki@targegen.com. ² TargeGen Inc. ‡ BioPredict. Figure 1. Kinase pathways regulating endothelial cell junction integrity. The two major signal transduction pathways in the regulation of intercellular integrity shown in this figure are Src and PI3K. PI3K can be activated via multiple growth factors or through G protein coupled PAFs. Signaling through Src can also affect gene transcription via ERK activation. 4279 J. Med. Chem. 2007, 50, 4279-4294 10.1021/jm051056c CCC: $37.00 © 2007 American Chemical Society Published on Web 08/09/2007