Design,Synthesis And Antitumor Activity Of Thiophene Triazine PI3K/mTOR Dual Target Inhibitors Containing Arylurea

Cancer has become the leading cause of death in my country,and it is urgent to develop cancer therapeutics.The PI3K/AKT/mTOR pathway is prone to dysregulation in human cancers,making it an important target to develop anticancer drugs by blocking PI3K and mTOR.In recent years,a variety of inhibitors targeting PI3K and mTOR have shown significant efficacy in clinical treatment.Among them,GDC-0941,an orally effective PI3K inhibitor,has good pharmacokinetic properties.However,the drug resistance and side effects generated by GDC-0941 as a single inhibitor cannot be ignored.Therefore,in this paper,GDC-0941 was used as the prototype to split its thienopyrimidine skeleton and introduced a symmetrical triazine structure as the core by using the skeleton transition method.The urea fragment that enhanced the inhibitory power of mTOR was then introduced,and nitrogen atoms was connected to enhance the binding power to PI3K and mTOR protein.Finally,compounds XIN-1～XIN-29 were designed and synthesized from morpholine and2,4,6-trichloro-1,3,5-triazine through nucleophilic substitution,Suzuki coupling and reductive amination,then obtained by ¹H NMR,¹³C NMR and TOF-MS.The MTT method was used to screen the toxicity of XIN-1～XIN-29 to MCF-7,Hela,Hela-MDR and A549 cells.Among them,compounds XIN-2 and XIN-10showed excellent anti-proliferative activity against 9 cancer cells,and they had the strongest inhibitory ability on breast cancer cell MCF-7,with IC₅₀ values of 0.30±0.05μM and 0.21±0.01μM,respectively.Secondly,XIN-2 and XIN-10 had significant inhibitory activities on PI3K and mTOR kinases,with IC₅₀ values of171.4 n M and 50.8 n M for PI3K kinase;IC₅₀ values for mTOR kinase were 10.1 n M and 21.4 n M,which were stronger than GDC-0941.Further in-depth pharmacological experiments were conducted on the preferred compounds XIN-2 and XIN-10 to explore their mechanism of action.AO staining and FITC/PI double staining apoptosis assay showed that XIN-2 and XIN-10induced apoptosis of MCF-7,A549 and Hela cells.Cycling experiments showed that they blocked MCF-7 cell proliferation in S phase and A549 and Hela cells in G0/G1phase.Reactive oxygen species assay and mitochondrial membrane potential detection demonstrated that they could increase the level of ROS in MCF-7 cells and reduce the mitochondrial membrane potential to induce apoptosis.Cloning and migration experiments showed that XIN-2 and XIN-10 effectively inhibited the proliferation and migration of MCF-7 cells.Real-time quantitative PCR experiments showed that they significantly inhibited the expression of PI3K and mTOR genes in MCF-7 cells.In addition,Western blot results showed that they dose-dependently inhibited the phosphorylation of AKT-S473 and blocked the phosphorylation of the downstream pathway mTOR.The anticancer activity of compound XIN-2 in nude mice was investigated by MCF-7 cell xenograft model.The tumor inhibition rate of the XIN-2 high-dose treatment group（75 mg/kg）was 40.14%There was no pathological reaction in the organs of nude mice by organ HE staining.The tunel staining and immunohistochemical detection of xenografts showed that XIN-2 induced the apoptosis of MCF-7 cell xenografts.PCR results showed that it significantly inhibited the expression of PI3K and mTOR in MCF-7 xenografts.In conclusion,compound XIN-2 can effectively inhibit the proliferation of MCF-7 xenografts.In conclusion,in this paper,GDC-0941 was modified as the lead compound to obtain 29 novel thiophene-triazine PI3K/mTOR dual inhibitors containing aromatic urea,and their structure-activity relationship and anticancer activity were evaluated.Preliminary analysis and discussion were carried out,which laid the foundation for the further study of PI3K/mTOR dual inhibitors in the future.