The authors thank Dr J Aoki University of

The authors thank Dr. J. Aoki, University of Tokyo, for kindly providing 5E5-ATX antibody. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A.E.K. (KR4391/1-1), research grants “Primary sclerosing cholangitis” from the Deutsche Crohn-Colitis-Vereinigung and the Norwegian Primary Sclerosing Cholangitis Foundation to U.B., the Wellcome Trust and Biomedical Research Centre at Imperial College London (UK) to C.W. and a ZonMW TOP (The Netherlands) grant to R.O.E. (#TOP 40-00812-98-10054).
Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (ENPP2), is a secreted lysophospholipase D (lysoPLD) that cleaves choline from phosphatidylcholine (LPC) forming phosphatidic Mevastatin australia (LPA), a mitogen and motility factor that has been implicated in the pathophysiology of cancer, , and many other biological processes such as vascular development, lymphocyte homing and inflammation., , LPA consists of a single fatty acyl chain, a glycerol backbone and a free phosphate group. Several structurally diverse forms of LPA exist with different acyl-chain lengths and saturations. LPA acts through a number of LPA receptors and the great variety of cellular and biological actions of LPA is explained by the fact that the six known LPA receptors show broad tissue expression and can couple to at least three distinct G proteins, which, in turn, feed into multiple effector systems (). The ATX–LPA axis has been implicated in several disease states including inflammation, pulmonary fibrosis, and tumour progression. ATX is processed along the classical export pathway and secreted as a catalytically active glycoprotein. ATX’s major lipid substrate, LPC, is secreted by the liver and is abundantly present in plasma and interstitial fluids. Potent and selective inhibitors of ATX would clearly be of value to elucidate the biology of this target and an overview of the patent and primary literature describing the development of novel ATX inhibitors has recently been published. Clinical trials have recently been initiated with GLPG1690 which is the first autotaxin inhibitor to enter the clinic and is under evaluation for the treatment of idiopathic pulmonary fibrosis. Herein we report the discovery of a new class of small molecule inhibitors of ATX which have been optimized to deliver compounds suitable for in vivo studies. A high throughput biochemical screen using the FS-3 assay was run using the CRT compound collection of 87,865 compounds. The screen was run at 30μM and the confirmed hit rate, defined as giving >50% inhibition at 30μM, was 1.2%. After IC determination in the FS-3 assay, a number of distinct chemical series were identified. Compounds from each series were then screened in the enzyme coupled (EC) biochemical assay which uses the more physiologically relevant substrate LPC C16:0 and measures the release of choline. The absolute potencies of the compounds in the enzyme coupled biochemical assay were generally lower and this helped prioritise between the chemical series so that chemistry effort could be focused on the most promising scaffolds. Based on the potency, ligand efficiency (LE) and chemical tractability, the imidazo[4,5-]pyridine () was selected as an attractive starting point for further chemistry. Compounds, prepared to explore the structure activity relationships of the hit , were synthesized by the routes described in , , , . The route used to modify the amide group (R in compound ) and the -benzyl group on the core (R in compound ) is shown in . Thus, 2-chloro-3-nitro-pyridine was reacted with CsCO and the appropriate amine in methanol at reflux to give via a SAr reaction. The nitro group in was hydrogenated over 10% Pd/C in ethanol to afford pyridine-2,3-diamine . This diamine could alternatively be obtained through a reduction of the nitro group with iron in HCl. Compound was reacted with succinic anhydride in dioxane at reflux and then treated with sulfuric acid in ethanol under reflux to give the ester . Reaction of with KOBu and the appropriate amine in the microwave at 150°C afforded the final compounds of general formula . Alternatively the carboxylic acid obtained from the reaction of with succinic anhydride, was treated with HBTU, triethylamine and the required amine at room temperature to afford . Alternative coupling agents can also be used to perform this amide formation.