Screening Of Antitumor Activity And Study Of Mechanism Of Action Of Benzo-nitrogen-containing Heterocyclic Small Molecule Compound

Secondary resistance mutations in cancer cells are a response to certain small-molecule inhibitors.Drug-resistance mutations can arise in various forms,and these mutations inevitably lead to cancer recurrence.Therefore,the design and development of novel antitumor small molecule inhibitors are of great significance for cancer therapy.Through literature research,it is found that many anti-tumor small molecule inhibitors have benzo nitrogen-containing heterocyclic structures,such as gefitinib and osimertinib.Therefore,based on the compounds with benzoazepine heterocyclic structure in the research group,this subject evaluated their inhibitory activity on tumor cell lines with high epidermal growth factor receptor（EGFR）expression and P53 mutation,and demonstrated their inhibitory activity in cell lines.To explore the anti-migration,pro-apoptosis and cycle arrest effects of highly active small molecules on tumor cells,and the possible mechanism of active compounds on the important targets of tumor therapy EGFR and P53 at the protein and transcription levels.The development and design of molecular inhibitors provide ideas and directions.In this paper,the compounds with benzo-nitrogen-containing heterocyclic structures in the research group are used as the research objects,and the MTT method is used,p-quinazolinone derivatives,3-pyrazole indole derivatives,indole 3-position fused ring substituted derivatives,etc.Three series,totaling 110 compounds,were assayed for in vitro antiproliferative activity.The selected cell lines are:EGFR high expression cell line-lung cancer cell A549,prostate cancer cell PC-3 and MDM2 high expression cell line-liver cancer cell Hep-G2,human myeloid leukemia cell K562.The research contents are as follows:1)Scratch test was used to evaluate the anti-migration effect of highly active compounds on tumor cells;2)DAPI staining test and Annerxin V-FITC/PI double staining method were used to analyze their ability to induce apoptosis;3)PI staining was used to evaluate its cycle arrest effect on tumor cell division;4)Western blotting（Western Blot,）was used to evaluate its effect on the expression levels of EGFR and P53-related important proteins in tumor cells;5)Real-time Fluorescence quantitative PCR was used to evaluate its effects on EGFR and P53-related important genes at the gene level of tumor cells.The research results showed that 1)among the three series of benzonitrogen-containing heterocyclic small molecule compounds,the 3-pyrazolindole derivatives series had the best anti-proliferative activity against the four tumor cell lines;2)The IC₅₀values of compounds S-4and O-6 against the four tumor cell lines are all at the sub-micromolar level;3)Compounds S-4and O-6 had strong inhibitory activities on the proliferation and migration of the four tumor cells;4)Compounds Both S-4 and O-6 may inhibit tumor cell migration and cell cycle by inhibiting the phosphorylation and activation process of EGFR and inhibit the transcription of MDM2 gene,thereby increasing the level of P53 protein and inducing cell apoptosis.