Preparation Of BTK Inhibitor Of Pyrimidine Derivatives And Its Preliminary Pharmacological Activity Of The Anti-chronic B Lymphocyte Leukemia

Objective:Design and synthesis a series of bruton's tyrosine protein kinase?BTK?inhibitors which possess 2,4-diaryl pyrimidine structure,to screen out the better activity and lower toxicity better compounds by the preliminary evaluating their inhibitory activity on chronic lymphocytic leukemia B.Methods:In the first part of this paper,a novel series of 2,4-diaryl pyrimidines was prepared by introducing the amino fragment into the C-2 position of the diphenylaminopyrimidine skeleton based on the molecular heterozygous strategy using the BTK inhibitor Spebrutinib as a lead compound.Also the target molecules were identified by ¹HNMR,¹³CNMR,HRMS and MS.In the second part,Spebrutinib and Ibrutinib were used as positive control drugs.At the kinase level,the half-maximal inhibitory concentration of BTK tyrosine kinase(IC₅₀)was determined by quantitative analysis of the remaining ATP content after the kinase reaction.In vitro screening of tumor cells,using CCK-8 colorimetric method,selected human B lymphoma cells with high BTK expression Ramos and Raji,determined the IC50 value of the target molecule inhibition rate on tumor cells in 48 h;AO/EB staining was performed to examine the inhibitory effect of the optimal compound on tumor cells Ramos;normal peripheral blood mononuclear cells?PBMCs?were extracted and the toxic effects of optimal compounds on normal immune cells were detected;flow cytometry To investigate the effect of optimal compounds on the apoptosis of B lymphoma cells and the mechanism of action on the cell cycle.Results:In the first part,12 target molecules were synthesized and purified,and the NMR data was consistent with the structure of the target molecule.The characteristics of high-resolution mass spectra were consistent with the molecular mass of the target molecule.The second part of the compounds preliminary activity test results show that the synthesized target molecules mostly have good anti-tumor activity,of which D12 has excellent inhibition of BTK kinase activity,and the IC₅₀ is 0.23nM.At the same time,compound D10 has very good for Ramos cells and Raji cells,and the activity of IC₅₀ was 10.5?M and 19.1?M,respectively.The results of AO/EB fluorescence staining showed that compared with the blank group,the number of tumor cells was significantly reduced after treatment with compound D10,and apoptosis and nuclear condensation occurred.The optimal compound D10 had almost no toxic effects on normal peripheral blood mononuclear cells?PBMCs?.The flow cytometric study of D10,an optimal compound,significantly increased the apoptosis of Ramos in B-lymphoma cells and inhibited the cell cycle at G0/G1 stage.Conclusions:A series of diphenylamine pyrimidine derivatives were prepared successfully.And preliminary studies on the anti-tumor pharmacological activity revealed that most of the synthetic compounds had strong activity in inhibiting the proliferation of tumor cells.Some compounds such as D10 inhibited the activity of BTK kinase and inhibited tumor cell activity better than positive compound Spebrutinib.It can be further developed as a candidate compound.The structural modifications in this study indicate that the structural optimization of the left amino side chain of the diphenylaminopyrimidine skeleton can effectively improve the antitumor activity and toxicity of the drug,and provide an effective structure-activity relationship theoretical basis for the structural optimization of such molecules.