Applications of 1-benzyl-4-(2,4-dichloro phenethylamino)piperidine in preparation of antitumor medicines
A kind of dichlorophenethylamino and benzyl technology, which is applied in the application field of 1-benzyl-4-(2,4-dichlorophenethylamino) piperidine in the preparation of antitumor drugs
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Embodiment 1
[0031] Example 1: The inhibitory activity of M22 on NEDD8 activating enzyme was detected by western blot.
[0032] Add 2 μL NEDD8 activating enzyme (2.5 μM), 2 μL NEDD8 (250 μM), 2 μL Abc12 (50 μM) and 10 μL M22 aqueous solution (0-90 μM) in a series of concentration ranges into a 100 μL tube. After mixing the reaction solution evenly, incubate at 37°C for 10 min. Then 2 μL of Mg-ATP (1.25 μM) solution was added. After the reaction solution was mixed evenly, it was incubated at 37°C for 1 h. After the reaction was completed, 2 μL of EDTA (1 mM) aqueous solution was added to terminate the reaction. Protein samples were collected, and the protein level of Ubc12-NEDD8 in the reaction system was detected by western blot. The results showed that M22 dose-dependently inhibited the production of Ubc12-NEDD8 and effectively inhibited the activity of NEDD8 activating enzyme, see figure 1 .
Embodiment 2
[0033] Example 2: ATP competition experiment.
[0034]Add 2 μL NEDD8 activating enzyme (2.5 μM), 2 μL NEDD8 (250 μM), 2 μL Abc12 (50 μM) and 10 μL M22 aqueous solution (33.3 μM) into a 100 μL tube. After mixing the reaction solution evenly, incubate at 37°C for 10 min. Then add 2 μL of Mg-ATP solution of different concentrations (1.25-10000 μM). After the reaction solution was mixed evenly, it was incubated at 37°C for 1 h. After the reaction was completed, 2 μL of EDTA (1 mM) aqueous solution was added to terminate the reaction. Protein samples were collected, and the protein level of Ubc12-NEDD8 in the reaction system was detected by western blot. The results showed that with the increase of ATP concentration, the content of Ubc12-NEDD8 gradually increased, and the activity of NEDD8 activating enzyme was restored. To illustrate that M22 is an ATP-competitive inhibitor of NEDD8 activating enzyme, see figure 2 .
Embodiment 3
[0035] Example 3: Western Blot detection of the effect of M22 on the activity of NEDD8 activating enzyme in cells.
[0036] Western Blot was used to detect the effects of different concentrations of M22 (0-90 μM) on the contents of intracellular related proteins Uba3-NEDD8 (the complex of NEDD8 activating enzyme β subunit and NEDD8) and Ubc12-NEDD8 after the A549 cells were treated for 24 hours. The results show that M22 has a dose-dependent inhibitory effect on the generation of Uba3-NEDD8 and Ubc12-NEDD8 on lung cancer A549, see image 3 . The compound can inhibit NEDD8 activating enzyme activity at the cellular level.
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