The Synthesis,Antitumor Mechanism Studies Of Pyrazolo[4,3-d]pyrimidine Derivatives

Pyrazolo[4,3-d]pyrimidine,as an isomer of pyrazolopyrimidine,is a fused heterocycle consisting of a pyrazole ring and a pyrimidine ring,and has gained a significant attention in the field of biology and medicinal chemistry.Pyrazolo[4,3-d]pyrimidine derivatives have been proven to have a number of pharmacological activities,including anti-tumor,anti-infection,phosphodiesterase inhibitors,adenosine antagonists and cytokinin antagonists.Therefore,the structural modification and modification of pyrazolo[4,3-d]pyrimidine derivatives and the screening of novel compounds with significant anti-tumor activity are very significant issues in the treatment of tumors.This experiment was based on the structure-activity relationship of pyrazolo[4,3-d]pyrimidines and the results of previous research of the research group.The pyrazolo[4,3-d]pyrimidine substituted with 3,4,5-trimethoxyphenyl at the C-5position was used as the starting material.Novel pyrazolo[4,3-d]pyrimidine derivatives4a-4o was designed and synthesized through the structural modification of its C-7position.All the compounds'structures were confirmed by ¹H NMR,¹³C NMR and HR-MS.In this study,the MTT assay was used to evaluate the in vitro anti-tumor activity of target compounds on four tumor cells?human hepatoma SMMC-7721 cells,human gastric cancer SGC-7901 cells,human glioma U87-MG cells,and human gastric cancer MGC-803 cells?.The tumor activity showed that compounds 4i,4j,4m,and 4n had significant inhibitory effects on four tumor cell lines,and compound 4n had the strongest inhibitory effect on the proliferation of glioma U87-MG cells with an IC₅₀ of2.22?M.The IC₅₀0 value of compound 4n was less than the glioma positive control drug temozolomide?TMZ?.Therefore,we chose compound 4n to investigate the proliferation,apoptosis and autophagy of U87-MG cells by using MTT assay,Western blot technique and flow cytometry on glioma U87-MG cells.After the autophagy inhibitor 3-MA was used to inhibit the autophagy induced by compound 4n,the proliferation and apoptosis of U87-MG cells were detected by MTT method,flow cytometry and Western blot respectively,and the role of autophagy in proliferation and apoptosis on glioma U87-MG cells was explored.The results showed that compound 4n could significantly inhibit the proliferation of glioma U87-MG cells in a time-and dose-dependent manner,although it could induce apoptosis,the overall apoptosis level was not high;Compound 4n could induces autophagy by inhibiting PI3K/Akt/mTOR pathway in U87-MG cells;The autophagy induced by compound 4n in glioma U87-MG cells is a protective mechanism that promotes cell proliferation and inhibits cell apoptosis;therefore,we can inhibit autophagy induced by compound 4n in U87-MG cells to increase the rate of apoptosis,thereby further enhancing the anti-glioma effect of compound 4n.It provides more new theoretical basis for the anti-tumor studies of pyrazolo[4,3-d]pyrimidine derivatives.