Identification of pyrazolo[1,5-a]pyrimidine-3-carboxylates as B-Raf kinase inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 10, 2009, Pages 2735-2738

  Gopalsamy, Ariamala ^a   Ciszewski, Greg ^a   Hu, Yongbo ^a   Lee, Frederick ^a   Feldberg, Larry ^a   Frommer, Eileen ^a   Kim, Steven ^a   Collins, Karen ^a   Wojciechowicz, Donald ^a   Mallon, Robert ^a  

^a WYETH RESEARCH   (United States)

Author keywords

B Raf; B Raf Inhibitor; Pyrazolo 1,5 a pyrimidine 3 carboxylate

Indexed keywords

B RAF KINASE; BRUTON TYROSINE KINASE; CARBOXYLIC ACID DERIVATIVE; CYCLIN DEPENDENT KINASE 4; MAMMALIAN TARGET OF RAPAMYCIN; PHOSPHATIDYLINOSITOL 3 KINASE; PHOSPHATIDYLINOSITOL 3 KINASE ALPHA; PHOSPHOINOSITIDE DEPENDENT PROTEIN KINASE 1; PHOSPHOTRANSFERASE; PROTEIN KINASE B; PROTEIN KINASE LCK; PROTEIN KINASE LYN; PROTEIN TPL2; PROTEIN TYROSINE KINASE; PYRAZOLE DERIVATIVE; PYRAZOLO[1,5 A]PYRIMIDINE 3 CARBOXYLIC ACID; PYRIMIDINE DERIVATIVE; SOMATOMEDIN C RECEPTOR; UNCLASSIFIED DRUG; VASCULOTROPIN RECEPTOR 2;

ANTINEOPLASTIC ACTIVITY; ARTICLE; CANCER CELL CULTURE; CRYSTAL STRUCTURE; DRUG SELECTIVITY; DRUG SYNTHESIS; HUMAN; HUMAN CELL; IC 50; MOLECULAR DOCKING; SIGNAL TRANSDUCTION; STRUCTURE ACTIVITY RELATION;

CELL LINE, TUMOR; COMPUTER SIMULATION; HUMANS; PROTEIN KINASE INHIBITORS; PROTO-ONCOGENE PROTEINS B-RAF; PYRIMIDINES; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 65349179117     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.03.129     Document Type: Article

References (11)

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8. B-Raf kinase assay: Flag/6his-tagged recombinant human B-Raf (from Sf9 insect cells; specific activity ∼200 U/ml) was used to phosphorylate human non-active Mek1 (GST tagged from E. coli); resultant phospho-MEK1 was measured by a phospho-specific polyclonal Ab from Cell Signaling Technology (#9154). B-Raf assay stock solutions were: assay dilution buffer (ADB) containing 20 mM MOPS, pH 7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol; Magnesium/ATP Cocktail (0.5 mM ATP, 75 mM MgCl2) in ADB; active B-Raf in ADB; non-active GST-MEK1 in ADB; and TBST-Tris (50 mM, pH 7.5), NaCl (150 mM), Tween-20 (0.05%). Kinase assays were carried out in a shaking incubator. The protocol was: in 96 well plate format B-Raf (0.4U/assay) and non-active MEK1 (0.4 μg/assay) were incubated with 0.1 mM ATP/ 15 mM MgCl2 for 60 min at 30 °C. This mixture was transferred to anti-GST Ab (Pharmacia) coated 96 well plates (Nunc), and then incubated for 60 min at 30 °C. Plates were then washed 3× with TBST, and then anti-phospho MEK1 Ab was added with incubation for 60 min at 30 °C. Plates were then washed 3× with TBST, and anti-rabbit Ab/Europium conjugate (Wallac) was added with incubation for 60 min (30 °C). A Wallac (Victor) Plate Reader was used to collect data that was analyzed in excel for single point and IC50 determinations.
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11. 50: 4.6 μM).