Design and synthesis of a series of novel pyrazolopyridines as HIF 1-a prolyl hydroxylase inhibitors Namal C. Warshakoon, * Shengde Wu, Angelique Boyer, Richard Kawamoto, Sean Renock, Kevin Xu, Matthew Pokross, Artem G. Evdokimov, Songtao Zhou, Carol Winter, Richard Walter and Marlene Mekel Procter & Gamble Pharmaceuticals Inc, 8700 Mason-Montgomery road, Mason, OH 45040, USA Received 12 June 2006; revised 26 July 2006; accepted 1 August 2006 Abstract—Recently resolved X-ray crystal structure of HIF-1a prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1a prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC 50 of 11 lM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1a. Ó 2006 Elsevier Ltd. All rights reserved. Hypoxia inducible factor (HIF) is a transcriptional com- plex that plays a key role in mammalian oxygen homeo- stasis and regulates a host of hypoxic response genes that regulate angiogenic, glycolytic, and erythropoietic processes. 1–3 The subunit components, HIF-1a and HIF-1b (ARNT) are constitutively expressed and regulation is achieved by the selective destruction of HIF-1a. HIF-1a is a major regulatory point of cellular response to hypoxia (decreasing partial pressure of oxygen). In the presence of oxygen, a family of non-heme iron containing prolyl hydroxylases (EGLN-1, EGLN-2, and EGLN-3) effects post-translational modification of HIF-1a by hydroxylation of proline 564 and proline 402 in the oxygen dependent degradation domain (ODD) via a reaction that requires 2-oxoglutarate (2OG) and ascorbate. 4–6 Proline hydroxylation then targets HIF- 1a subunits for proteasomal degradation via binding to the VHL (Von Hippel Lindau tumor suppressor pro- tein), elongin C/B, CuI2, Rbx1 ubiquitin ligase complex. Since the post-translational hydroxylation of HIF-1a is controlled by oxygen concentration, under hypoxia the hydroxylation of HIF-1a is inhibited, and HIF-1a binds to ARNT to form a functional transcriptional activator that turns on genes with hypoxic response elements (e.g., VEGF, EPO, glycolytic enzymes). 7 Therefore, inhibition of HIF-1a prolyl hydroxylases, resulting in HIF-1a sta- bilization and has a potential to be a viable therapeutic approach for ischemic diseases including myocardial infarction, stroke, peripheral arterial disease, heart fail- ure, diabetes, and anemia. Neither crystal structure of the procollagen nor that of HIF prolyl hydroxylases has been reported in the litera- ture. However, atomic resolution structures have been obtained for other 2-oxoglutarate dioxygenases including bacterial proline 3-hydroxylase, deacetoxy- cephalosporin C synthase, clavaminate synthase, and asparaginyl hydroxylase. 8 The similarity of cofactor requirements for the catalytic step suggests that these structural data should provide significant information concerning the active site of HIF prolyl hydroxylase to aid synthesis of small molecule inhibitors. We sought to identify suitable small molecule HIF-1a prolyl hydroxylase inhibitors based on structure-based design approach utilizing the recently solved crystal structure of EGLN-1 9 in complex with the isoquinoline inhibitor 1 10 (Fig. 1). 0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2006.08.017 Keywords: Prolyl hydroxylase inhibitors; HIF1-a; Hypoxia; Ischemia; Peripheral arterial disease (PAD); Anemia; Pyrazolopyridines. * Corresponding author. Tel.: +1 513 622 4467; fax: +1 513 622 0523; e-mail: Warshakoon.nc@pg.com Bioorganic & Medicinal Chemistry Letters 16 (2006) 5687–5690