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84862781933
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For the reaction between 2-benzoimidazole-2-ylacetate and 2-aminobenzaldehyde U.S. Patent 6479512 B1
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Fraley, M. E.; Hambaugh, S. R.; Hungate, R. W.
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30
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84862822971
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50 using Prism version 5.01 (GraphPad)
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50 using Prism version 5.01 (GraphPad).
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31
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84862822976
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50 values were estimated using Prism Software
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50 values were estimated using Prism Software.
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32
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84862778602
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Microsomal stability test: NADPH dependent oxidative metabolism of 21 in human and rat liver microsomes was determined on single-time-point 30 min. The reaction mixtures consisted of human or rat liver microsomes (0.5 mg/ml; BD Gentest) in 100 mM potassium phosphate buffer (pH 7.4) and final concentration of 21 was 2 μM. After pre-incubation of reaction mixture at 37 °C for 5 min, the reaction was initiated by addition NADPH regeneration solution (BD Biosciences) and the reaction was terminated by adding three times volume of ice-cold acetonitrile with imipramine (80 ng/ml) as internal standard at single-time-point 30 min. After pretreatment of biological samples with vortex and centrifuging, the samples were analyzed by LC/MS/MS system (AB Sciex 2000 Qtrap)
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Microsomal stability test: NADPH dependent oxidative metabolism of 21 in human and rat liver microsomes was determined on single-time-point 30 min. The reaction mixtures consisted of human or rat liver microsomes (0.5 mg/ml; BD Gentest) in 100 mM potassium phosphate buffer (pH 7.4) and final concentration of 21 was 2 μM. After pre-incubation of reaction mixture at 37 °C for 5 min, the reaction was initiated by addition NADPH regeneration solution (BD Biosciences) and the reaction was terminated by adding three times volume of ice-cold acetonitrile with imipramine (80 ng/ml) as internal standard at single-time-point 30 min. After pretreatment of biological samples with vortex and centrifuging, the samples were analyzed by LC/MS/MS system (AB Sciex 2000 Qtrap).
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84862822975
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In vitro VEGF-induced HUVEC proliferation assay: Pooled HUVECs (Lonza) were grown in a 1:1 mixture of EBM-2 medium with EGM-2 supplements and used at passage eight or lower. HUVECs were placed at a density of 10,000 cells/well on a collagen type I precoated 96-well plate. After 24 h, the cells were incubated for 24 h in the presence of rhVEGF (20 ng/mL) in the presence of the compound under examination. Cells were assayed for proliferation by the Premix WST-1 proliferation assay system (Takara)
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In vitro VEGF-induced HUVEC proliferation assay: Pooled HUVECs (Lonza) were grown in a 1:1 mixture of EBM-2 medium with EGM-2 supplements and used at passage eight or lower. HUVECs were placed at a density of 10,000 cells/well on a collagen type I precoated 96-well plate. After 24 h, the cells were incubated for 24 h in the presence of rhVEGF (20 ng/mL) in the presence of the compound under examination. Cells were assayed for proliferation by the Premix WST-1 proliferation assay system (Takara).
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35
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84862822979
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In vitro angiogenesis assay (tube formation): 24-well plates were coated with 200 mL of a 1:1 mixture of Matrigel (10 mg/mL) and EBM-2 medium with EGM-2 supplements and incubated at 37 °C for 1 h. HUVECs (Lonza) were placed at a density of 40,000/well and incubated for 16-18 h. After incubation, the plates were fixed and images were captured using a Nikon camera (50×) and analyzed using Image-Pro Plus software (Media Cybernetics)
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In vitro angiogenesis assay (tube formation): 24-well plates were coated with 200 mL of a 1:1 mixture of Matrigel (10 mg/mL) and EBM-2 medium with EGM-2 supplements and incubated at 37 °C for 1 h. HUVECs (Lonza) were placed at a density of 40,000/well and incubated for 16-18 h. After incubation, the plates were fixed and images were captured using a Nikon camera (50×) and analyzed using Image-Pro Plus software (Media Cybernetics).
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12144289984
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