Synthesis And Anti-tumor Activity Of Indazoles Based On PI3K/AKT/mTOR Signaling Pathway

The PI3K/AKT/mTOR signaling pathway is one of the most common abnormal activation pathways in tumor cells,and is involved in important functions such as tumor cell growth,proliferation,migration,invasion,and tumor angiogenesis.At present,PI3K/AKT/mTOR signaling pathway inhibitors have made positive progress in the treatment of hematological malignancies,renal cancer,liver cancer,lung cancer,etc.As a multi-target tyrosine kinase inhibitor,Sorafenib can used to cure cancers such as liver cancer,kidney cancer and thyroid tumor by inhibiting the PI3K/AKT/mTOR signaling pathway.However,problems such as low oral bioavailability,long half-life and drug resistance have appeared in clinical medicine.In this paper,Sorafenib is used as a template compound to reduce its toxicity and improve its bioavailability through medicinal chemistry.Based on the structure-based drug design strategy,a new class of indazoles was designed.Specifically,6-nitro-1H-indazole was used as the starting material,and compound W1-16 was synthesized through cyclization,Buchwald-Hartwig and suzuki coupling reactions.In order to further improve the lipid solubility and druggability of compound W1-16,the long-chain aryl urea structure was replaced by a cyclopropylcarboxamide structure through structural simplification,and hydrophilicity was introduced at the C-5 or 6 position of the indazole core.In order to replace the benzene ring with a substituted pyrazole group,another series of indazole compounds were designed.Specifically,m-bromoaniline was used as the starting material to synthesize compound W17–25 through two-step substitution and suzuki coupling reactions.The study found that the target compound W1–25 has good antiproliferative effect on HGC-27,Hep G2,HT-29,MCF-7 and A-549 cells in vitro,and compound W24 has the best activity among the four tumor cells with high expression of PIK3CA with IC50 values of 0.43-3.88μM.Edu staining and colony formation experiments showed that compound W24 could inhibit DNA synthesis and proliferation in vitro of HGC-27 cells in a concentration-dependent manner.Western blot experiments proved that compound W24 can reduce the phosphorylation of PI3K,AKT and mTOR,being a PI3K/AKT/mTOR signaling pathway inhibitor.Flow cytometry and Western blot showed that compound W24 could induce endogenous cell apoptosis by reducing the expression of Cyclin B1 protein,arresting the cell cycle in G2/M phase,stimulating the production of ROS and down-regulating Bcl-x L.In addition,compound W24inhibited the migration and invasion of HGC-27 cells by affecting the m RNA expression levels of Snail and Slug,reducing the content of MMP-9.Compound W24also reduced the m RNA expression of HIF1-αand the protein expression of VEGF,which may have certain effects on tumor angiogenesis.The drugability study showed that compound W24 had good pharmacokinetic properties in vitro and in vivo,and had no liver microsome toxicity.The acute toxicity study in mice showed that the LD₅₀ of compound W24 was greater than 2000 mg/kg,and there was no obvious histomorphological changes in mouse organs,indicating that it was safe.In conclusion,25 indazole compounds were designed and synthesized in this project,and the compound W24 was found to have broad-spectrum anti-tumor activity through in vitro anti-proliferation studies.Subsequent mechanism studies showed that compound W24 could inhibit the PI3K/AKT/mTOR signaling pathway,block the HGC-27 cell cycle,induce apoptosis,and inhibit the migration and invasion of HGC-27 cells.Compound W24 also showed good safety and druggability.The results of this paper have accumulated a certain theoretical basis and empirical basis for the subsequent research of PI3K/AKT/mTOR signaling pathway inhibitors in gastric cancer.