The inhibitory action against MEK1 was examined by quantitative evaluation of the phosphorylation of a peptide by a ERK2 protein in the presence of CH5424802. The inhibitory activity against Raf 1 was assessed by analyzing the capacity of the CTEP GluR Chemical kinases to phosphorylate MEK1 in the clear presence of CH5424802. Cells were lysed in Cell Lysis Buffer containing 1mMPMSF,1% phosphate inhibitor cocktail 1,1% phosphate inhibitor cocktail 2, and Complete Mini, EDTA Free 1. Cell lysates were put through sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically used in Immobilon P membranes. After blocking in Blocking One, the membranes were incubated individually in the principal antibodies diluted with anti ALK, anti STAT3, antiPhospho STAT3, anti AKT, anti Phospho AKT, anti p44/ 42 MAP Kinase, anti Phospho ERK1/2, anti ALK, anti Phospho ALK, and anti actin. For the detection of phosphorylated ALK in NCIH2228 cells, cell lysates were immunoprecipitated Metastasis with anti phosphotyrosine antibody. The immunoprecipitants were then gathered with ProteinG Sepharose and put through immunoblot analysis utilizing an anti ALK antibody. The membranes were incubated by having an anti rabbit or anti mouse IgG, HRP related antibody. The bands were found with ECL Plus accompanied by LAS 4000. Cells were incubated with various levels of substance and cultured in 96 well plates over night for the suggested time. For spheroid cell growth inhibition assay, cells were incubated over night, seeded on spheroid plates, and then treated with compound for the indicated times. The viable cells were measured by the CellTiter Glo_ Luminescent CX-4945 solubility Cell Viability Assay. Caspase 3/7 assay was examined using the Caspase Glo 3/7 Assay Kit. Cell lines were used to judge the antitumor activity of CH5424802 in vivo. They were produced as s. D. tumors in SCID or nude mice. Beneficial studies were started when the cyst reached _250 or _350 mm3. Mice were randomized to treatment groups to receive vehicle or CH5424802 for the indicated duration. Final concentration of car was 0. 02 N HCl, 10% DMSO, 10% Cremophor EL, 15% PEG400, and 15% HPCD. The length and width of the tumor mass were measured, and the tumor volume was determined as: TV page1=39 /2. Tumor growth inhibition was determined using the following formula: tumor growth inhibition #3 100, where T and T0 are the mean tumor volumes on a specific experimental day and on the first day of treatment, respectively, for the experimental groups and similarly, where C and C0 are the mean tumor volumes for the control group. The effective dose for 50% inhibition was determined from the values of tumor growth inhibition on the last experimental time using XLfit type.