Query String: DEFEROXAMINE
DEFEROXAMINE BDBM50393308
BDBM47715 MLS000028713 SMR000058548 N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-1,4-dioxobutyl]-hydroxyamino]pentyl]-N''-(5-aminopentyl)-N''-hydroxybutanediamide;methanesulfonic acid DEFEROXAMINE DEFEROXAMINE MESYLATE N''-(5-azanylpentyl)-N-[5-[[4-[5-[ethanoyl(oxidanyl)amino]pentylamino]-4-oxidanylidene-butanoyl]-oxidanyl-amino]pentyl]-N''-oxidanyl-butanediamide;methanesulfonic acid N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N''-(5-aminopentyl)-N''-hydroxybutanediamide;methanesulfonic acid cid_62881 N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-keto-butanoyl]-hydroxy-amino]pentyl]-N'-(5-aminopentyl)-N'-hydroxy-succinamide;mesylic acid
- HIF-1alpha transactivation assays NIH3T3-EPO-luc cells were maintained at 37° C. in a humidified atmosphere containing 5% CO2 in DMEM supplemented with 10% fetal calf serum (FBS), and 1% (v/v) penicillin/streptomycin. Deferoxamine (DFX) was purchased from Sigma-Aldrich (USA). Cells (1×104/well in 96-well plates) were seeded the day before the assay. The next day, the cells were stimulated with increasing concentrations of either Cannabidiol (CBD), VCE-004 or compounds II to X. After six hours of stimulation the cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shaker. Luciferase activity was measured using a microplate luminometer (Berthold) following the instructions of the luciferase assay kit (Promega, Madison. Wis., USA).
- Electrochemiluminescence (ECL) Assay A homogeneous end point electrochemiluminescence (ECL) assay is performed using a biotinylated BACE substrate. The Km of the substrate is approximated at 50 uM. A typical reaction contains approximately 0.6 nM enzyme, 0.25 uM of the substrate, and buffer (50 mM Pipes, pH 6.5, 0.1 mg/ml BSA, 0.2% CHAPS, 15 mM EDTA and 1 mM deferoxamine) in a total reaction volume of 100 ul. The reaction proceeds for 1-2 hrs and is then stopped by the addition of 150 uL of a quench cocktail solution (25 ul M Tris-HCl, pH 8.0, 50 ul INC buffer (2% BSA, 0.2% Tween-20 and 0.05% sodium azide diluted in Phosphate buffered saline (PBS) plus 75 uL PBS), containing Streptavidin coated magnetic beads and ruthenylated antibody which specifically recognizes the C-terminal residue of the product. The samples are subjected to M-384 (Igen Inc., Gaithersburg, MD) analysis. Under these conditions, less than 10% of substrate is processed by BACE 1.
- HIF-1alpha transactivation assays HaCaT-EPO-Luc cells were maintained at 37° C. in a humidified atmosphere containing 5% CO2 in DMEM supplemented with 10% fetal calf serum (FBS), and 1% (v/v) penicillin/streptomycin. The cells (1×105/well in 24-well plates) were seeded the day before the assay and then stimulated with increasing concentrations of either Cannabidiol (CBD), VCE-004 or compounds II to X. After six hours of stimulation the cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCi2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shake. Luciferase activity was measured in the cell lysates as indicated for NIH3T3-EPO-Luc cells. The RLUs are calculated and the EC50 and IRA (Intrinsic relative activity) values in both cell lines were determined relative to 150 μM deferoxamine (DFX) using the following equation: IRA coefficient=(EC50-DFX×Emax)/(EC50×Emax-DFX), where EC50 and Emax denote EC50 and Emax of the agonist, and EC50-DFX and Emax-DFX denote EC50 and Emax values of the standard agonist DFX.
- End Point Electrochemiluminescence (ECL) Assay A homogeneous end point electrochemiluminescence (ECL) assay is performed using a biotinylated BACE substrate. The Km of the substrate is approximated at 50 uM. A typical reaction contains approximately 0.6 nM enzyme, 0.25 uM of the substrate, and buffer (50 mM Pipes, pH 6.5, 0.1 mg/ml BSA, 0.2% CHAPS, 15 mM EDTA and 1 mM deferoxamine) in a total reaction volume of 100 ul. The reaction proceeds for 1-2 hrs and is then stopped by the addition of 150 uL of a quench cocktail solution (25 ul M Tris-HCl, pH 8.0, 50 ul INC buffer (2% BSA, 0.2% Tween-20 and 0.05% sodium azide diluted in Phosphate buffered saline (PBS) plus 75 uL PBS), containing Streptavidin coated magnetic beads and ruthenylated antibody which specifically recognizes the C-terminal residue of the product. The samples are subjected to M-384 (Igen Inc., Gaithersburg, Md.) analysis. Under these conditions, less than 10% of substrate is processed by BACE 1. The enzyme used in these studies is soluble (transmembrane domain and cytoplasmic extension excluded) human protein produced in a baculovirus expression system. To measure the inhibitory potency for compounds, 10 concentrations of inhibitors are prepared starting from 200 uM with three fold series dilution. Solutions of the inhibitor in DMSO are included in the reaction mixture (final DMSO concentration is 10%). All experiments are conducted at rt using the standard reaction conditions described above. To determine the IC50 of the compound, a four parameter equation is used for curve fitting. The errors in reproducing the dissociation constants are typically less than two-fold. In particular, the compounds of the following examples had activity in inhibiting the beta-secretase enzyme in the aforementioned assay, generally with an IC50 from about 1 nM to 200 uM. Such a result is indicative of the intrinsic activity of the compounds in use as inhibitors of beta-secretase enzyme activity.