Study On The Dual-Target Antitumor Drug Design,Synthesis,Biological Evaluation Of Isoxazole-Containing Oxindole Derivatives

Purpose:As a class of natural compounds with high biological activity,indole alkaloids have been extensively developed in anti-tumor,anti-AIDS,antibacterial,and anti-inflammatory drugs in recent years.In particular,the spirooxindole derivatives modified at the C3 position show highly physiological activity in terms of anti-tumor activity.On the other hand,isoxazole as another highly active skeleton,due to its two adjacent electronegative heteroatoms,can enhance the interaction with the receptor and the enzyme target,so that this skeleton exhibits highly physiological activity,which is also common in various antibacterial and antitumor drugs.Meanwhile,due to the special properties of fluorine atoms,more and more fluorine-containing organic compounds are widely used in the field of new drug research and development.Among them,trifluoromethyl is an important fluorine-containing group,which have played a very significant role in the improvement of the lipophilicity of drug molecules,drug metabolism,chemical stability,etc.Therefore,how to introduce this group into drugs to optimize the properties of drugs is also a hot spot in the field of medicinal chemistry.On this basis,we designed a combination of the bioactive oxindole skeleton and isoxazole skeleton,and introduced trifluoromethyl that can improve the properties of the drug,so as to construct a new compound containing three modules.Anti-tumor activity of the novel compounds were tested and evaluated,hoping to provide a new idea and method for the development of new anti-tumor drugs.Methods:1.Construction of isoxazole and trifluoromethyl containing spirooxindole derivatives.First,trifluoroethylamine was condensed with the C3 position of isatin,so that a trifluoromethyl group was introduced at the C3 position of the isatin skeleton,and the corresponding imine isatin 1 was obtained at the same time.At the same time,an electron-deficient olefin 2 was selected as another substrate,and then 1,3-dipole substrate 1 and electron-deficient olefin 2 were used to generate a 1,3dipolar cycloaddition under the catalysis of Br?nsted base.As a result,target structure of the compound was identified by X-ray single crystal diffraction,HRMS,1H-NMR,and 13C-NMR,which laid the foundation for the next step of anti-tumor biological activity determination.2.Measuring the inhibition rate of MDM2 protein by FP-based(fluorescence polarization)MDM2 binding assay,verifying the selective binding inhibition of GPX4 protein by selective cytotoxicity experiment and indirectly measuring the inhibition rate of the compound on GPX4 protein,using antiproliferation assay to test the inhibition rate of MCF-7 breast cancer cells for screening out the most active compound and molecular dynamics simulation tests were carried out to determine the molecular mechanism of compound.Finally,toxicological tests of compounds 3 in normal cells were carried out to confirm their safety.Results:1.A total of 59 racemic compounds were synthesized efficiently through synthetic strategies,and their chemical structures were identified.2.The best active compound 3ag was screened out by FP-based(fluorescence polarization)MDM2 binding assay,selective cytotoxicity and antiproliferation assays.Toxicological assay showed that compounds 3 had no obvious cytotoxicity to human breast epithelial cells even at high concentration.3.The molecular mechanism of binding between compound 3ag and MDM2 protein was preliminarily verified by molecular dynamics simulation tests.Conclusion:Isoxazole-containing isatin derivatives prepared by 1,3-dipolar cycloaddition reaction as MDM2/GPX4 dual-target inhibitors show relatively good anti-tumor activity.