Compound (SMILES): COc1ncc(N2CCOc3ccc(O[C@H]4CCN(C(=O)c5cnco5)C4)cc32)cc1C#N
InChIKey: AAZKJQXQGRGOED-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay: Determination of Enzymatic PI3K Alpha and PI3K Delta Isoform Inhibition1.1 Test of Lipid Kinase ActivityThe efficacy of the compounds of examples 1-117 as PI3 kinase inhibitors can be demonstrated as follows:The kinase reaction is performed in a final volume of 50 μl per well of a half area COSTAR, 96 well plate. The final concentrations of ATP and phosphatidyl inositol in the assay are 5 μM and 6 μg/mL, respectively. The reaction is started by the addition of PI3 kinase, e.g. PI3 kinase 6.p110δ.The components of the assay are added per well as follows 10 μl test compound in 5% DMSO per well in columns 2-1.    Total activity is determined by addition 10 μl of 5% vol/vol DMSO in the first 4 wells of column 1 and the last 4 wells of column 12.    The background is determined by addition of 10 μM control compound to the last 4 wells of column 1 and the first 4 wells of column 12.    2 mL Assay mix are prepared per plate   1.912 mL of HEPES assay buffer      8.33 μl of 3 mM stock of ATP giving a final concentration of 5 μM per well      1 μl of [33P]ATP on the activity date giving 0.05 μCi per well      30 μl of 1 mg/mL PI stock giving a final concentration of 6 μg/mL per well      5 μl of 1 M stock MgCl2 giving a final concentration of 1 mM per well    20 μl of the assay mix are added per well.    2 mL Enzyme mix are prepared per plate (x* μl PI3 kinase p110(3 in 2 mL of kinase buffer). The Enzyme mix is kept on ice during addition to the assay plates.    20 μl ‘Enzyme mix’ are added/well to start the reaction.    The plate is then incubated at room temperature for 90 minutes.    The reaction is terminated by the addition of 50 μl WGA-SPA bead (wheat germ agglutinin-coated Scintillation Proximity Assay beads) suspension per well.    The assay plate is sealed using TopSeal-S (heat seal for polystyrene microplates, PerkinElmer LAS [Deutschland] GmbH, Rodgau, Germany) and incubated at room temperature for at least 60 minutes.    The assay plate is then centrifuged at 1500 rpm for 2 minutes using the Jouan bench top centrifuge (Jouan Inc., Nantes, France).    The assay plate is counted using a Packard TopCount, each well being counted for 20 seconds.      The volume of enzyme is dependent on the enzymatic activity of the batch in use.In a more preferred assay, the kinase reaction is performed in a final volume of 10 μl per well of a low volume non-binding CORNING, 384 well black plate (Cat. No. #3676). The final concentrations of ATP and phosphatidyl inositol (PI) in the assay are 1 μM and 10 μg/mL, respectively. The reaction is started by the addition of ATP.The components of the assay are added per well as follows:50 nl test compounds in 90% DMSO per well, in columns 1-20, 8 concentrations (⅓ and 1/3.33 serial dilution step) in single.    Low control: 50 nl of 90% DMSO in half the wells of columns 23-24 (0.45% in final).    High control: 50 nl of reference compound (e.g. compound of Example 7 in WO 2006/122806) in the other half of columns 23-24 (2.5 μM in final).    Standard: 50 nl of reference compound as just mentioned diluted as the test compounds in columns 21-22.    20 mL buffer are prepared per assay   200 μl of 1M TRIS HCl pH7.5 (10 mM in final)      60 μl of 1M MgCl2 (3 mM in final)      500 μl of 2M NaCl (50 mM in final)      100 μl of 10% CHAPS (0.05% in final)      200 μl of 100 mM DTT (1 mM in final)      18.94 mL of nanopure water    10 mL PI are prepared per assay   200 μl of 1 mg/mL 1-alpha-Phosphatidylinositol (Liver Bovine, Avanti Polar Lipids Cat. No. 840042C MW=909.12) prepared in 3% OctylGlucoside (10 μg/mL in final)      9.8 mL of buffer    10 mL ATP are prepared per assay.
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 447.45, ALogP 2.77, HBA 9, HBD 0, PSA 113.95, QED 0.58