Synthesis and biological evaluation of 5-arylamino-6-chloro-1H-indazole-4,7-diones as inhibitors of protein kinase B/Akt

Bioorganic and Medicinal Chemistry Letters

Volumn 16, Issue 23, 2006, Pages 6001-6005

  Ko, Jong Hee ^a,b   Yeon, Seung Woo ^a,b   Ryu, Jung Su ^a,b   Kim, Tae Yong ^a,b   Song, Eun Ha ^a,b   You, Hea Jung ^a,b   Park, Rae Eun ^a,b   Ryu, Chung Kyu ^a,b  

^a Ewha Womans University   (South Korea)

^b ILDONG Pharmaceutical Co   (South Korea)

Author keywords

1H indazole 4,7 dione; Akt; Antitumor; Inhibitor

Indexed keywords

ACETYL COENZYME A CARBOXYLASE 2 INHIBITOR; ENZYME INHIBITOR; THIAZOLYLPHENYL ETHER DERIVATIVE; UNCLASSIFIED DRUG;

ARTICLE; DRUG POTENCY; ENZYME ACTIVITY; ENZYME INHIBITION; HYDROPHOBICITY; STRUCTURE ACTIVITY RELATION;

AMINATION; ANIMALS; ANTINEOPLASTIC AGENTS; CATALYSIS; CELL LINE, TUMOR; CHLORINE; GLYCOGEN SYNTHASE KINASE 3; HUMANS; INDAZOLES; MICE; MICE, NUDE; MOLECULAR STRUCTURE; NEOPLASMS; PHOSPHORYLATION; PROTEIN KINASE INHIBITORS; PROTO-ONCOGENE PROTEINS C-AKT; STRUCTURE-ACTIVITY RELATIONSHIP; XENOGRAFT MODEL ANTITUMOR ASSAYS;

EID: 33750510883     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.08.120     Document Type: Article

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8. 2 and 95% humidity in a 37 °C chamber. Akt kinase assay in vitro: Akt1/PKB protein kinase (10 U/μL) was incubated at 37 °C for 10 min in reaction buffer (20 mM HEPES, 14.3 mM NaCl, 1 mM EDTA, and 2 mM DTT) containing the test compounds dissolved in DMSO. This mixture was added 2 mM ATP 3 μL and GSK3 fusion protein (1 mg/mL) 1 μL and incubated at 37 °C for 30 min. The reaction was stopped by incubation for 5 min at 70 °C. This sample was separated by 12% SDS-PAGE and transferred to PVDF membranes by Western blotting or Dot blotting. After blocking by incubation in TBST (10 mM Tris-HCl, pH 7.5, 170 mM NaCl, and 0.5% Tween 20) containing 5% (w/v) skim milk for 10 min, membrane was incubated overnight at 4 °C with primary antibody (1:1000). Subsequent to washing with TBST, membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) and then washed. Membrane was incubated with ECL (Amersham Bio., UK) substrate reagent and exposed to X-ray film. Phosphorylated GSK3 fusion protein was quantified by a densitometric analysis using ImageMaster 2D platinum v5.0 (Amersham Bio., UK). For the investigation of inhibitor type, the initial velocity v was obtained as the intensities of phosphorylated GSK3 substrates (arbitrary unit/30 min). Data points were collected in triplicate, plotted on Lineweaver-Burk double reciprocal plots, and fitted to linear regression.
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