Design,Synthesis And Antitumor Activity Of Thiophene-Triazines As Dual PI3K/mTOR Inhibitors

The PI3K/Akt/m TOR signaling pathway is one of the most important intracellular network pathways regulated by growth factors,which regulates various life activities such as cell survival,metabolism,growth,and proliferation.Previous studies have confirmed that the PI3K-Akt-m TOR signaling pathway is closely associated with tumorigenesis and development.Therefore,the development of drugs targeted on PI3K and m TOR has become a research pot with a lot of attention.In our study,GDC-0941 was selected as the lead compound based on the structural characteristics of PI3K inhibitors.Accumulated studies have demonstrated that the molecular docking between GDC-0941 and PI3Kαprotein as well as the structural analysis of PI3K inhibitors,which indicating that there are many types of cores in the cavity of PI3Kαprotein.The results showed that the space in the ATP binding site is enough for the skeleton transition of thiophene[3,2-d]pyrimidine of GDC-0941.Based on the above research and analysis,the following modifications were made to the lead compounds:（1）According to the principles of the scaffold-hopping and bioisosterism,the parent nucleus of thiophene[3,2-d]pyrimidine of GDC-0941 was skeleton transition,and the triazine ring similar to pyrimidine ring was introduced.The first series of2-aryl-4-morpholine substituted thiophene-triazine compounds were designed and synthesized,with a total of 30 target compounds.（2）Based on the biological activity evaluation and structure-activity relationship of the first series of compounds,the most optimized compound ZBL-23was selected for further optimization.In order to improve the m TOR kinase activity of the target compound,we designed and synthesized the second series of thiophene[3,2-d]pyrimidine compounds containing a small molecule urea structure by splicing the key pharmacophore‘urea fragment’of m TOR inhibitor onto compound ZBL-23 based on combination principles.The second series of substituted alkylaryl urea-4-morpholine substituted thiophene-triazine compounds were designed and synthesized,with a total of 32 target compounds.Three cancer cell lines A549,MCF-7 and Hela were selected for the anti-tumor activities of the 62 target compounds by MTT assay,using GDC-0941 as positive control.The results showed that most compounds of the first series exhibited moderate to excellent antitumor activity.Among them,compound ZBL-23 showed excellent anti-tumor potency for A549,MCF-7 and Hela cell lines with IC50 values of 0.20±0.05μM,1.25±0.11μM and 1.03±0.24μM,respectively.The activity of compound ZBL-23 was better than that of the leading compound GDC-0941.Moreover,most compounds of the second series also exhibited moderate to excellent antitumor activity to A549,MCF-7 and Hela,among which ZBL-39 showed excellent anti-tumor potency for these three cancer cell lines with IC50 values of 0.12±0.07μM,0.39±0.81μM and 2.85±0.71μM,respectively.According to the results of antitumor test,compounds with better antitumor activity were selected for kinase inhibition test of PI3Kαand m TOR and further kinase selectivity test.The data showed that the first series of compounds ZBL-21,ZBL-23 and the second series of compounds ZBL-31 and ZBL-39 were identified as selective PI3Kα/m TOR dual inhibitors,and the IC₅₀ value of inhibition activity among them against PI3Kαkinase was 9.5 n M,7.0 n M,22 n M and 11 n M,respectively.The IC50 values of m TOR kinase inhibitory activities were 69 n M,48n M,29 n M and 32 n M,respectively,which were 7 to 48 times higher than those of GDC-0941.In addition,the first series of compounds ZBL-23 were tested by flow cytometry and AO staining.The results showed that ZBL-23 could promote the induction of A549 cell apoptosis and block the cell cycle of A549 at G0/G1 phase.Western blot experiments were also performed on ZBL-23,which showed that ZBL-23 could effectively inhibit the phosphorylation of p-AKT（S473）and p-GSK3β.Furthermore,the antitumor activity of compound ZBL-39 in nude mice was investigated by MCF-7 cell transplantation model.The data showed that in the MCF-7 cell transplantation model of nude mice,the tumor volume and average tumor weight of high-dose ZBL-39 treatment group were significantly smaller than those of the blank control group,and the tumor inhibition rate was 42.1%.At the same time,the weight of tumor bearing mice in ZBL-39 high-dose treatment group decreased slightly at the beginning of administration,but with the extension of treatment time,the weight of tumor bearing mice gradually recovered.At the end of the experiment,there was no significant difference compared with the blank control group,and there was no significant toxic reaction.Immunohistochemical detection of Ki67,caspase-3,cyt-c,p65 protein and Bcl-2 gene expression in nude mice MCF-7 cell transplanted tumors.Compared with the blank control group,the expression of Ki67 protein in ZBL-39 high-dose treatment group was decreased,while the expression of Bcl-2,caspase-3,cyt-c and p65 were all increased,the data indicating that compound ZBL-39 can induce apoptosis of MCF-7 cell transplanted tumors.Based on the antitumor activity of the compounds and the results of molecular docking,the structure-activity relationship of the compounds can be summarized as follows:（1）The morpholine ring can be well embedded in the hinge region of PI3Kαand m TOR proteins,and form key hydrogen bonds with Val851 and Val2240.One of the key fragments maintain the biological activity of the compound;（2）The secondary amine introduced in the thiophene ring extends to the solvent front of the protein cavity and forms a potential hydrogen bond with Gln859 and Arg2251,among which the introduction of morpholine ring and mesyl piperazine was beneficial to the enhancement of the biological activity of the compounds;（3）The aryl group substituted at the 2 position penetrates into the affinity pocket of the protein and forms hydrogen bonds with multiple amino acid residues.Introduceing the 2-aminopyrimidine group and the methylphenylurea group containing hydrogen bond acceptor/donor atom was the most beneficial to enhance the activity of the compounds.In conclusion,two series of 62 thiophene-triazine compounds as dual PI3Kα/m TOR inhibitors have been obtained by the modification of GDC-0941 as the lead compound,and their structure-activity relationship and mechanism of action have been preliminarily analyzed and discussed,which provided optimization and transformation ideas for the study of dual PI3Kα/m TOR inhibitors,and pointed out the direction for later research.