A kind of indolinone compound and its preparation method and application

[0038] (1) Screening of hTERT expression inhibitors

[0039]①Cell transfection: HeLa cells were inoculated in two 3cm sterile cell dishes, and the cells were spread to 60-70% and transfected with plasmid pGL2 or pGL2-hTERT promoter respectively, and inoculated into 96-well plates after 6 hours, 8000 per well cells.

[0040] ② Cells treated with small molecule compounds: five concentrations (1, 2, 4, 8, 16 μM) of each compound were used to treat the cells, 3 secondary wells for each concentration, and cultured in an incubator for 36 hours.

[0041] ③Cell lysis: Add 55 μL Extraction buffer [according to the final concentration of 1% TritonX-100, 15mM MgCl2, 4mM EGTA, 1mM DTT, 25mM glycyglycine, make up 10mL with deionized water, store in the dark at 4°C] in the well, and mix well. Lyse on ice for 30 min.

[0042] ④Take 5μL Assay cocktail buffer [add deionized water in order, the final concentration is 30mM ATP (pH 5.0), 0.1M KH2PO4 (pH 7.8), 0.1M MgCl2, mix well, prepare and u...