Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 16, 2010, Pages 4795-4799

  Blackburn, Christopher ^a   Duffey, Matthew O ^a   Gould, Alexandra E ^a   Kulkarni, Bheemashankar ^a   Liu, Jane X ^a   Menon, Saurabh ^a   Nagayoshi, Masayuki ^a   Vos, Tricia J ^a   Williams, Juliet ^a  

^a MILLENNIUM PHARMACEUTICALS INC   (United States)

Author keywords

B Raf inhibitor; Parallel synthesis; Pyrazoline

Indexed keywords

B RAF KINASE; B RAF KINASE INHIBITOR; PYRAZOLINE DERIVATIVE; PROTEIN KINASE INHIBITOR; PYRAZOLE DERIVATIVE; PYRIDINE DERIVATIVE; RECOMBINANT PROTEIN;

ARTICLE; BINDING SITE; DRUG STRUCTURE; DRUG SYNTHESIS; ENZYME ACTIVITY; ENZYME ASSAY; ENZYME INHIBITION; ENZYME PHOSPHORYLATION; HIGH THROUGHPUT SCREENING; HUMAN; HUMAN CELL; HYDROGEN BOND; IC 50; SIGNAL TRANSDUCTION; AMINO ACID SUBSTITUTION; ANTAGONISTS AND INHIBITORS; CHEMISTRY; DRUG SCREENING; GENETICS; METABOLISM; STRUCTURE ACTIVITY RELATION; SYNTHESIS;

AMINO ACID SUBSTITUTION; DRUG EVALUATION, PRECLINICAL; HIGH-THROUGHPUT SCREENING ASSAYS; PROTEIN KINASE INHIBITORS; PROTO-ONCOGENE PROTEINS B-RAF; PYRAZOLES; PYRIDINES; RECOMBINANT PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 77955416938     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.06.110     Document Type: Article

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9. 50 values >10 μM for a panel of kinases that included CAMKII, CDK1, CDK2E, CHK1, CHK2, CKII, KDR2, FLT3, CSF1R, KIT2, LCK2, PKA2, and FGFR1.
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13. 1H NMR spectroscopy and mass spectrometry.
14. Inhibition of Raf kinase activity in whole cells was assessed by determining the decrease in phosphorylation of the Raf kinase substrate pERK. This whole cell ELISA assay utilized a human melanoma cell line (A375) possessing the mutation B-Raf V600E. A375 cells seeded overnight were incubated with Raf inhibitors for 3 h at 37 °C. At the end of the incubation, the cells were fixed, permeabilized, blocking buffer added and the plates were incubated overnight. After the blocking buffer was discarded, the plates were incubated with anti-phospho-ERK antibody for 1 h followed by treatment with anti-horse radish peroxidase. Optical density was read at 650 nm for the substrate tetramethybenzidine.
15. 50 values of 17 nM and 18 nM, respectively, for inhibition of the WT enzyme.
16. 50 values >10 μM for a panel of kinases that included KDR2, FLT3 and CSF1R, KIT2, AKT2, and FGFR1.
17. 50 values of 1.2 and 14 μM in the enzymatic and cellular assays, respectively. The later eluting isomer was considerably more active (15 nM in the enzymatic assay and 280 nM in the cellular assay).