In vitro activity: AZM475271 is a potent, selective and oral inhibitor of Src kinase with IC50 of 5 nM and with potential anticancer activities; it has no inhibitory activity on Flt3, KDR, Tie-2. It demonstrated strong dose-dependent inhibition of Src tyrosine kinase activity in the L3.6pl human pancreatic carcinoma cell line. Deregulated activity of the nonreceptor tyrosine kinase c-Src is believed to result in signal transduction, cytoskeletal and adhesion changes, ultimately promoting a tumor-invasive phenotype. inhibitors with high affinity and specificity for the tyrosine kinase domain of the c-Src enzyme. Maximum reduction of Src kinase activity was observed after incubation for 4 hours with ≥5 μmol/L. The IC50 concentration of AZM475271 to inhibit the phosphorylation of c-src, lck, and c-yes was 0.01, 0.03, and 0.08 μmol/L, respectively, in comparison with an IC50 of 0.7 μmol/L AZM475271 to inhibit KDR.

Kinase Assay: L3.6pl cells were plated into 10-mm dishes. After overnight incubation, the cells were treated for 4 hours with AZM475271 (1–10 μmol/L). After washing cells with ice-cold PBS, lysates were collected using lysis buffer [50 mmol/L HEPES (pH 7.2), 150 mmol/L NaCl, 1 mmol/L EGTA, 20 mmol/L NAF, 1% Triton X-100, 10% glycerol, 1 mmol/L β-glycerophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L Na3VO4] complemented with protease inhibitor mixture tablets (Roche Diagnostics, Mannheim, Germany). After centrifugation for 10 minutes at 9,000 × g, the supernatant was incubated at 4°C for 4 hours with 15 μg of v-src (Ab-1) monoclonal antibody (Oncogene Research Products, San Diego, CA) preadsorbed to protein A- and protein G-Sepharose (Sigma, Munich, Germany). The immune complex was washed three times with lysis buffer and two times with kinase buffer A [30 mmol/L HEPES (pH 7.5), 5 mmol/L MnCl2, 2 mmol/L dithiothreitol, and 0.1 mmol/L Na3VO4]. Beads were finally resuspended in 4 μL of 5× kinase buffer containing 5 μg of enolase (Sigma) and 10 μCi of [γ-33P]ATP (Perkin-Elmer, Wellesley, MA). After incubation at 30°C for 10 minutes, assays were terminated by the addition of 20 μL of 2× Laemmli sample buffer. The samples were heated at 95°C for 5 minutes and analyzed by SDS-12% PAGE. The gels were dried and subjected to autoradiography. Densitometry of the gels was performed by Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD).

Cell Assay: AZM475271 demonstrated strong dose-dependent inhibition of Src tyrosine kinase activity in the L3.6pl human pancreatic carcinoma cell line. Maximum reduction of Src kinase activity was observed after incubation for 4 hours with ≥5 μmol/L. The IC50 concentration of AZM475271 to inhibit the phosphorylation of c-src, lck, and c-yes was 0.01, 0.03, and 0.08 μmol/L, respectively, in comparison with an IC50 of 0.7 μmol/L AZM475271 to inhibit KDR