Supplementary MaterialsIENZ_1376666_Supplementary_Materials. with an Isolera Primary program with 254?nm detector (Biotage, Charlotte, NC, USA) utilizing 230C400 mesh silica gel BAY 80-6946 supplier snap cartridges. All chemical substances and solvents had been bought from Aldrich, VWR or USA Scientific, USA and had been utilized as received. 0.53 (CH2Cl2/CH3OH, 10:1), 1H NMR (DMSO-d6) 6.76C6.77 (m, 0.58 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 6.79C6.80 (m, 0.59 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 6.76C6.77 (m, 0.59 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 6.78 (d, 0.52 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 3.73 (s, 3?H), 6.64 (d, 0.59 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 6.82 (d, 0.50 (CH2Cl2/CH3OH, 10:1), 1H NMR (400?MHz DMSO-d6) 5.78 BAY 80-6946 supplier (s, 2?H), 6.54C6.55 (m, 0.57 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.86 (d, 0.60 (CH2Cl2/CH3OH, 10:1); 1H KCNRG NMR (400?MHz, DMSO-d6) 7.04 (d, 0.68 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.80 (d, 0.54 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 2.30 (s, 3?H), 6.80C6.82 (m, 2?H), 7.19C7.21 (m, 2?H), 7.68C7.22 (m, 2?H), 8.27 (s, 1?H), 9.19 (s, 1?H); 13C NMR (400?MHz DMSO-d6) 154.05, 151.30, 151.25, 140.79, 137.97, 128.75, 123.20, 122.48, 121.18, 117.95, 104.11, 99.25, 21.74; HRMS (ESI) (M?+?H)+: Calcd for C13H13N40.70 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.81 (d, 0.61 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.49 (d, 0.57 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 2.19 (s, 3?H), 6.27 (d, 0.65 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.84 (d, 0.66 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 6.75 (d, 0.63 (CH2Cl2/CH3OH, 10:1); 1H NMR (400?MHz, DMSO-d6) 3.97 (s, 2?H), 6.92 (d, microplate audience. Kinase activity assays had been performed in triplicate at each focus. The luminescence data were analysed using the computer software, Graphpad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Binding affinities for EGFR, AURKA and AURKB The assay was performed externally at DiscoverX Corporation using a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilised, active-site directed ligand24. The assay is performed by combining three components: DNA-tagged kinase, immobilised ligand and a test compound. The ability of the test compound to compete with the immobilised ligand was measured via quantitative PCR of the DNA tag. An 11-point 3-fold serial dilution of each test compound was prepared in 100% of DMSO at 100 final test concentration and subsequently diluted BAY 80-6946 supplier to 1 1 in the assay (final DMSO concentration?=?1%). Compound BAY 80-6946 supplier Kd was determined using a compound top concentration?=?30,000?nM. If the initial Kd determined was 0.5?nM (the lowest concentration tested), the measurement was repeated with a serial dilution starting at a lower top concentration. Binding constants (Kd) were calculated with a standard dose-response curve using the Hill equation. Proliferation and cell killing assays in SCCHN cells FADU, BHY, SAS and CAL cell lines were obtained from ATCC-LGC and were cultured in DMEM (Invitrogen, Germany) supplemented with 10% of heat activated bovine serum (FBS, PAA, Germany), 1% of glutamine, 1% of penicillin-streptomycin (Invitrogen, Germany). To measure proliferation, SCCHN cells were split, reseeded (5??105 in 25?cm2 flasks) and counted at the indicated time points. Cells were then replated at the initial density. The fold increase in cell number was calculated, all given results are based on triplicate experiments. To BAY 80-6946 supplier assess cell death 5??105 cells were stained with propidium iodide (PI, Sigma, Germany). Following incubation, cells were cleaned, resuspended in PBS, and analysed by.