Application of benzamide compound with fluorophores
A technology of benzamide and fluorescent group, applied in the application field of benzamide compounds with fluorescent group
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Embodiment 1
[0020] Example 1 Compound I inhibits the activity of nicotinamide phosphoribosyltransferase
[0021] The following enzyme refers to nicotinamide phosphoribosyltransferase, which can be purchased from Kangtai Biotechnology Co., Ltd. (003-82) or American ChemDiv Company (M049-0244), and can also be prepared by yourself.
[0022] 1. Enzyme preparation: inoculate BL21(DE3) plysS cells transformed with recombinant plasmid (Nampt-pET 28a+) in 2×YT medium (37 μg / ml chloramphenicol and 75 μg / ml kanamycin) Shake overnight at 37°C, collect the cells and resuspend them with 20 times the original volume of fresh medium, culture at 37°C until OD 600 About 0.6, induced by 0.5 mM IPTG, 28 ℃ for 5 hours. The cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH=7.5), and the cells were lysed by ultrasonication at 200 W, 1 second and 9 seconds apart, for a total of 30 minutes. The lysate was centrifuged at 12500 rpm, 4°C for 50 minutes, an...
Embodiment 2
[0029] Example 2 Compound I inhibits the proliferation of human liver cancer cells in vitro
[0030] We investigated the inhibitory effect of compound I on the proliferation ability of human liver cancer cells in vitro. Compound I with gradient concentration was used to act on the cell lines in the logarithmic growth phase for 2 days, and the cell viability method was used to detect:
[0031] 1. Digest the HepG 2 cells in the logarithmic growth phase, pipette into a single cell suspension, inoculate in a 96-well culture plate, 1×10 4 Cells / well, 100 μl medium per well, 37°C, 5% CO 2 Incubate overnight in the incubator.
[0032] 2. After the cells adhered to the wall, a gradient concentration of the test compound I was added and cultured in an incubator for another 2 days.
[0033] 3. Determination of cell viability: Add 10 μl of cell viability reagent (CCK-8 reagent) to a 96-well cell culture plate in turn, mix well and react at 37 °C for 30 minutes, and detect the OD val...
Embodiment 3
[0035] Example 3 Fluorescence imaging of compound I in human liver cancer cells
[0036] We observed the fluorescence imaging of compound I in human hepatoma cell HepG 2 by confocal laser microscopy. The specific method is:
[0037] 1. Digest the cells in the logarithmic growth phase, pipette into a single cell suspension, inoculate in a culture dish, and store at 37 °C, 5% CO 2 Incubate overnight in the incubator.
[0038]2. After the cells adhered to the wall, add compound I with a final concentration of 3 μM, and for the solvent control, add the same volume of solvent DMSO, namely dimethyl sulfoxide.
[0039] 3. After 30 minutes, observe the fluorescence of the compound under a confocal laser microscope.
[0040] 4. After the observation, the culture solution containing the compound was quickly removed, washed with phosphate buffer saline for 3 times, and then the fluorescence in the cells was observed again under the laser confocal microscope.
[0041] 5. Immediately...
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