Application of substituted piperazine-1,4-diamide compounds in pharmacy
A technology of diamides and compounds, which can be used in medical preparations containing active ingredients, antipyretics, metabolic diseases, etc., and can solve the problem of decreased expression of liver fatty acid β-oxidation genes, etc.
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Embodiment 1
[0038] Example 1. Determination of the activation activity of substituted piperazine-1,4-diamides on SIRT1
[0039] 1) Protein expression
[0040] First, from the plasmid pcDNA3.1-SIRT1 (a gift from the research group of Professor Liu Depei, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), the catalytic domain of human SIRT1 and the cDNA including the N-terminal and C-terminal partial sequences were amplified, and the cloning vector pBlunt-Simple After the Vector connection sequence is correct, it is connected with the Escherichia coli expression vector pET-30a(+), the constructed recombinant expression plasmid is named pET-SIRT1, transformed into the Escherichia coli expression host BL21(DE3), single clones are picked, and sequenced for identification. Shake flasks with correct sequenced single colonies were cultured at OD 600 When the value is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM, culture at 15°...
Embodiment 2
[0059] Example 2. Culture of cells
[0060] HepG2, ABCA1-LUC HepG2, U2OS and HEK293 cells are all adherent cells, which are passaged every 48 hours. After the cells are full, discard the old medium, rinse the cells with PBS and discard, add an appropriate amount of trypsin, digest the cells at room temperature for about 2 minutes, discard the digestion solution, and immediately add the medium containing 10% FBS to inhibit trypsin Vitality, gently blow and tap the cells in the culture flask with an elbow pipette to make the cells completely detach from the bottom of the bottle and disperse them into a single cell suspension by blowing and blowing. Then inoculate the cell suspension in a new cell flask at a ratio of 1:3, or discard an appropriate amount of cell suspension, then add an appropriate amount of complete medium, and put it in an incubator to continue culturing. Culture conditions: 37°C, 5% CO 2 .
[0061] Mouse mononuclear macrophage RAW264.7 is a semi-adherent cel...
Embodiment 3
[0063] Example 3. The effect of compound (I) on the expression level of acetylated p53 protein in human osteosarcoma U2OS cells induced by doxorubicin.
[0064] 1) Cells treated with compounds: Human osteosarcoma cells U2OS were pre-treated with McCoy's5a medium containing 10% FBS at a rate of 3×10 per well. 5 Cells were seeded in 6-well plates at 37°C, 5% CO 2cultured to logarithmic phase. After the cells were completely adhered to the wall, the cell solution was aspirated, replaced with blank McCoy's5a medium, and added with a final concentration of 5 μM doxorubicin hydrochloride (DOX), 10 μM SIRT1-specific inhibitor EX-527 and a final concentration of 0.1, 1, 10 μM positive compound, 37°C, 5% CO 2 Incubate under conditions for 6h.
[0065] 2) Protein preparation: After 6 hours, discard the medium, rinse the cells twice with pre-cooled PBS, trypsinize and collect the cells with the medium, centrifuge at 800rpm for 3min, suspend the cells with PBS, and centrifuge at 800rpm...
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