Research On Chemical Synthesis Of Phenanthroline Derivatives And Their Mechanism Of Inhibiting Colorectal Cancer Via PI3K/AKT/mTOR Pathway

Colorectal cancer（CRC）is a common malignant tumor,and it is one of the biggest killers in human health.It is estimated that there were 1.8 million new cases of CRC worldwide in 2019,and that more than 880,000 people died from the disease.Chemotherapy is a necessary and effective means for the treatment of advanced CRC.Chemical drugs have been widely used in the clinical treatment of various cancers including CRC,prolonging the life of patients.However,the current chemotherapeutic drugs still have disadvantages such as toxicity to the digestive system and damage to the liver function.Therefore,the medical community has been developing novel chemical structures to search for new chemotherapeutic drug candidate structures,or studying the anticancer activity of known drugs and chemical molecules to increase the choice of chemotherapeutic drugs.Among many chemical structures,phenanthroline derivatives have promising development value.As an excellentπ-electron acceptor,phenanthroline can stably bind to G-quadruplex DNA to inhibit the proliferation of cancer cells.Many derivatives and metal complexes of phenanthroline have been reported to exert exciting toxicity against a variety of cancer cells.We found that 1H-imidazo[4,5-f][1,10]phenanthroline（IPM713）derivatives own best development potential among many phenanthroline derivatives.They have strong toxicity to a variety of cancer cells with IC₅₀ value usually about 1μM,and can induce apoptosis and block cell cycle.Therefore,the development of novel IPM713 derivatives is of great significance for the treatment of CRC.Our previous study developed two novel IPM713 derivatives with great anti-CRC activity in vitro.But these two derivatives possessing poor selectivity,cause certain toxicity to normal cells.Based on the previous experimental exploration and literature review of these derivatives,present research proposed to develop a novel anticancer 1H-imidazole[4,5-f][1,10]phenanthroline derivative（IPM714）with great selective role.According to the literature statistics,the anticancer activity of IPM713 was studied only rarely,and there is no complete evaluation of its anticancer activity.This study will systematically evaluate the anticancer effects of IPM713 and IPM714 in vitro,and demonstrate their anticancer mechanisms on CRC cell line（HCT116）from cellular and protein levels.Data support for the development and evaluation of 1H-imidazole[4,5-f][1,10]phenanthroline derivatives and finding a new candidate compound for the treatment of CRC are also included.The results demonstrated that IPM713 and IPM714 showed strong toxicity to a variety of cancer cells,and the inhibition of HCT116 was strongest among eight cancer cell lines.The selective inhibition to HCT116 cells was found between HCT116 and HIEC cells,and IPM714 played higher selective role to cancer cells.Both drugs were more toxic to HCT116 cells than 5-Fluorouracil（5-Fu）at the same concentration.The plate colony formation assay experiment further confirmed that the two compounds inhibited the proliferation of HCT116 cells.Through staining and flow cytometry analysis,it was found that IPM713 could block HCT116 cells in G0/G1 phase,while IPM714 could block HCT116 cells in S phase,both of which could inhibit the proliferation of cancer cells by blocking cell cycle.IPM713 and IPM714 could induce HCT116 cell apoptosis in a dose-dependent manner.Consistent with these results,Hoechst experiments showed that IPM714 treatment resulted in nuclear pyysis in HCT116 cells.Additionally,IPM714 reduced the migration ability of HCT116 cells.Molecular docking indicated that mammalian target of rapamycin（mTOR）might be a potential binding protein of IPM713 and IPM714.IPM713 and IPM714 could down-regulate the expression of phosphatidylinositol 3-kinase（PI3K）,phospho-protein kinase B（p-AKT）and mTOR protein.IPM713 significantly increased the expression of caspase-3,which further confirmed its induction of apoptosis.IPM714 could also down regulate the expression of p-mTOR.Studies at the mRNA level showed that IPM714 induced the increased expression of PI3KCA and AKT genes,but the expression of PI3K and AKT protein was not significantly increased.In conclusion,a novel 1H-imidazole[4,5-f][1,10]phenanthroline derivative,IPM714,was synthesized in this study.Together with IPM713,the in vitro anticancer activity and anticancer mechanism of IPM714 were evaluated.The results showed that IPM713and IPM714 had broad-spectrum anticancer activity in vitro,and IPM714 had more obvious selective inhibition on CRC cells.The anticancer mechanism of IPM713 and IPM714 may be through inhibiting the abnormally activated PI3K/AKT/mTOR signaling pathway in cancer cells,affecting the downstream signal transmission of apoptosis,cell proliferation and cell cycle,thus blocking cell cycle,inducing apoptosis and leading to the death of cancer cells.This study reported a novel potential compound for anti-CRC therapy in vitro and evaluated anticancer activity of 1H-imidazole[4,5-f][1,10]phenanthroline in vitro.