Discovery and Optimisation of wt-RET/KDR-selective Inhibitors of RETV804M Kinase

Discovery and Optimisation of wt-RET/KDR-selective Inhibitors of RETV804M Kinase Rebecca Newton,1,* Bohdan Waszkowycz,1 Chitra Seewooruthun,2 Daniel Burschowsky,2 Mark Richards,3 Samantha Hitchin,1 Habiba Begum,1 Amanda Watson1, Eleanor French,1 Niall Hamilton,1 Stuart Jones,1 Li-Ying Lin,4 Ian Waddell,1 Aude Echalier,2 Richard Bayliss,3 Allan M. Jordan1 and Donald Ogilvie.1 1Drug Discovery Unit, Cancer Research UK Manchester Institute University of Manchester, Alderley Park, Macclesfield, SK10 4TG. U.K. 2Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester, LE1 7RH, UK. 3Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. 4Leicester Drug Discovery & Diagnostics Centre (LD3), Maurice Shock Building, University of Leicester, University Road, Leicester, LE1 7RH, UK. *Telephone: +44 (0)161-306-6272. Email: rebecca.newton@cruk.manchester.ac.uk.

Preparative HPLC Some compounds were purified by preparative HPLC on a Waters FractionLynx MS autopurification system, with a Waters XBridge 5 µm C18, 100 mm × 19 mm i.d. column, running at a flow rate of 20 mL/min with UV diode array detection (210-400 nm) and mass-directed collection using both positive and negative mass ion detection.
Purifications were performed using buffered acidic or basic solvent systems as appropriate. Compound retention times on the system were routinely assessed using a 30-50 µL test injection and a standard gradient, and then purified using an appropriately chosen focussed gradient as detailed below, based upon observed retention time.

4-(1,3-Benzodioxol-5-yl)-1H-pyrazol-3-amine (24)
Prepared an a manner similar to that previously described. 1 To a stirred suspension of sodium methoxide (1. To this solid was added methanol (25 mL) and water (5 mL). The mixture was cooled to 0 °C before addition of semicarbazide (2.1 g, 27.92 mmol). The mixture was stirred at 0 °C for 10 min, then for a further hour at room temperature keeping the pH at 9-10 with 5N sodium hydroxide. The reaction mixture was then diluted with water (200 mL), cooled in an ice-bath and allowed to stir overnight. The resulting beige precipitate was isolated by filtration. To this solid was added methanol (150 mL) and 5N sodium hydroxide solution (15 mL) and the mixture was heated at reflux for 30 minutes. After cooling, water (300 mL) was added and the mixture was cooled in an ice-bath and allowed to stir overnight. The resulting precipitate was filtered and dried in vacuo to return the title compound
The protein was eluted with 20 mM Tris-Cl pH 8.0, 200 mM NaCl, 1 mM DTT, and 3 mM d-Desthiobiotin. The protein was treated with Precission protease overnight at 4 °C and further purified by gel filtration using a Superdex 75 16/60 column into 20 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM DTT. The purified protein was concentrated to 3 mg/mL using centrifugal filter units with a molecular weight cut-off of 10 kDa.

Crystallisation, data collection and structure determination
Crystals of RET V804M were grown at 18 °C by sitting-drop vapour diffusion against 0.1 M sodium citrate pH 4.5-5.5, 2.0 M sodium formate (mother liquor). For drop sizes of 0.5 µL concentrated protein + 0.5 µL mother liquor the crystals grew readily within one week. The crystals were soaked by adding 1 µl mother liquor containing an additional 30% ethylene glycol and 2 mM optimised lead compound 13 or 34 and incubating at 18 °C overnight. Crystals were cryo-cooled in liquid nitrogen and data collection was performed at Diamond Light Source beamline I04. For crystals soaked in compound 13 two datasets were collected from two separate crystals (1200 and 2980 images with 0.15° and 0.05° oscillation range, respectively) and merged for increased completeness and resolution of the data. For compound 34 1800 images with 0.1° oscillation range were collected. The data were processed with XDS 6 and merged using AIMLESS. 7 The structure was solved by molecular replacement in PHASER 8 using a non-phosphorylated RET tyrosine kinase domain (PDB ID: 2IVS) 9 as a search model. Refinement was performed by alternatingly using REFMAC5 10 and COOT 11 , with B factors being refined isotropically. Both final models show well-defined electron density for the respective compounds, which were modelled with full occupancy.