Study On The Synthesis Of Arctigenin Derivatives And Their Anti-Tumor Activities And Mechanisms In Vitro

With the rapid growth and aging of the world’s population,cancer has gradually become an important cause of morbidity and mortality in the world,so the research on tumor cells has gradually become a hot direction.Because many anti-tumor drugs have great side effects,the development of high-efficiency and low-toxicity natural products and the synthesis of derivatives with natural producs in the research of anti-tumor active drugs have become hotspots.At the same time,it wass also more and more important to explore their anti-tumor mechanism.Lignan compounds were widely found in Chinese herbal medicines and had abundant pharmacological effects.Recent studies have shown that many lignan compounds have potential anti-tumor activities.Arctigenin（ARG）is an important lignan compound,which has been reported to have tumor effects.In this study,a series of arctigenin derivatives were synthesized based on the structure of arctigenin skeleton,and their anti-tumor activities and mechanism were explored,in order to select out the anti-tumor lead compounds with better activity and provide an important theoretical basis for the subsequent development of the arctigenin derivatives anti-tumor drugs.The main research contents and results were as follows:1.Synthesis and structural characterization of arctigenin derivativesBased on the preliminary exploration of the research group,arctigenin was used as parent nucleus,and synthesized with BOC-amino acid,and acetylamino acid and carboxylic acid respectively,and 31 arctigenin derivatives were obtained.Among them,the structure of arctigenin BOC-amino acid ester and arctigenin ester hydrochloride derivatives 1-15 and arctigenin carboxylic acid ester derivatives24-31 have been analyzed and characterized in the early stage of our research group.Therefore,in this article,the structure identification of arctigenin acetyl amino acid ester derivatives 16-23 were carried out by spectral and other methods.At the same time,the melting point was detected,and the 8 arctigenin acetyl amino acid ester derivatives were all new compounds after identification,and the deviation range of melting point of each compound was not more than 2℃.2.Study on the anti-tumor activity of arctigenin and its derivativesFive types of cancer cells,including human prostate cancer cell PC-3M,human hemangioendothelial cell Hem ECs,human non-small cell lung cancer cell A549,human liver cancer cell Hep G2,and human breast cancer cell MCF-7 were selected for anti-tumor activity screening of arctigenin and its 31 derivatives by CCK-8method（Cell Counting Kit-8）in vitro.The results showed that different compounds had different anti-tumor activities against different tumor cells.All compounds including arctigenin showed strong anti-tumor activity against PC-3M cells,compounds 1,12,13,14,16 and 17 showed significant inhibitory effects.Among them,compound 1 had strong anti-tumor activity on PC-3M cells,and the cell proliferation inhibition was the best and stronger than the positive control5-fluorouracil（5-FU）,its IC₅₀=7.08±0.32μmol/L.For Hem ECs cells,most of the compounds showed great anti-tumor activity,and the activity was second only to PC-3M cells,especially compounds 1,2,3,6,15,16,22 and 27.Compounds 1,2,6,8,9,14 and 26 showed strong antitumor activities on A549 cells,among which compound 1 showed the strongest antitumor activity and was stronger than 5-FU.In addition,from the IC₅₀ values of the compounds on Hep G2 cells and MCF-7 cells,the anti-tumor activity of arctigenin and its derivatives was weaker than other tumor cells.According to the screening resuls of anti-tumor activity,compound 1 with strong activity and human prostate cancer cell PC-3M were selected as the research objects for in-depth follow-up studies.3.Study on the effect and mechanism of preferred compound on PC-3M cellsThe effect and mechanism of compound 1 on PC-3M cells were further investigated by plate cloning formation assay,wounding healing assay,PI staining,DAPI staining,Annexin V-FITC/PI staining and acridine orange staining.The number of colonies in the plate cloning formation experiment was observed,compared with the control group and the ARG group,the number of colonies in compound 1 group was less,indicatied that compound 1 had a strong anti-proliferation effect PC-3M cells.In the wounding healing assay,the scratch healing degree of compound 1 group was significantly lower than control group and ARG group,and with a concentration-dependent manner,indicated that compound 1 had a significant inhibitory effect on the migration of PC-3M cells.In cell cycle detection,the cycle distribution data showed that compound 1 could block PC-3M cells in G0/G1 phase.Subsequently,the apoptotic effects of compound 1 on PC-3M cells were evaluated from three aspects:cell morphology,flow cytometry detection and Western blot detection of apoptosis-related proteins.DAPI staining fluorescence results showed that the fluorescence intensity of compound 1 group was relatively stronger,and with the increase of drug concentration,the fluorescence intensity gradually strengthened,and the number of cells gradually reduced.The results of flow cytometry showed that the number of apoptotic cells in the compound 1 group was relatively more,and the expression levels of apoptosis-related protein Bax and Cleaved caspase-3 gradually increased with the increase of concentration,while the expression level of Bcl2decreased gradually with the increase of concentration,indicated that compound 1could induce PC-3M cell apoptosis.It can be seen that the number of cell lysosomes in the compound 1 group increased,and the orange-red fluorescence gradually increased with the increase of concentration from the fluorescence results of acridine orange staining.At the same time,the expression levels of autophagy-related protein Beclin-1 and LC3B-II were gradually increased with the increase of concentration,and the expression level of P62 gradually decreased with the increase of concentration,indicated that compound 1 could induce PC-3M cell autophagy.4.The effect of preferred compound on PI3K/Akt/m TOR signaling pathwayThe PI3K/Akt/m TOR signaling pathway is closely related to cell growth and survival,etc,and it is the upstream pathway that regulated cell apoptosis and autophagy.Therefore,in order to further explored the mechanism of compound 1inducing PC-3M cell death,we selected m TOR（PDB:4JT6）,a key protein in the PI3K/Akt/m TOR signaling pathway that regulated apoptosis and autophagy,as a target protein for molecular simulation docking.The results of molecular docking showed that compound 1 could produce higher binding energy with the domain,formed a more stable molecular conformation,and thus more stably binded to m TOR.Therefore,it was preliminarily proved that compound 1 could induce apoptosis and autophagy of human prostate cancer cell PC-3M through the PI3K/Akt/m TOR signaling pathway.In addition,the PI3K/Akt/m TOR signaling pathway and related proteins were further detected,and the PI3K inhibitor LY294002 was co-administered at the same time,which further proved that compound 1 could induce apoptosis and autophagy of human prostate cancer cell PC-3M through the PI3K/Akt/m TOR signaling pathway.In summary,this artical used arctigenin as the core to design and synthesize a series of arctigenin derivatives and screened them for anti-tumor activity in vitro,and then the compound of strongest antitumor activity was further explored antitumor function and mechanism,this artical provided a theoretical basis and a new perspective on the structural modification of arctigenin and its anti-tumor effect and mechanism in vitro.