Studies On The Synthesis,Antitumor Efficacy And Anti-Fibrotic Activity Of Novel β-carboline Derivatives

The Carboline alkaloid has shown significant anti-inflammatory,anti-tumor,anti-virus,and other broad pharmacological activities.In this paper,21 newβ-carboline alkaloid derivatives were synthesized using the synthesis technology method of our research group.We focused on their anti-tumor and anti-fibrotic activities in vitro and then determined their mechanism of action by using theories and methods of oncology and modern molecular biology.It mainly includes the following contents:（1）A brief overview of the risks of tumor and kidney diseases and their treatment.Discovery and research progress of the biological activities of alkaloid derivatives represented byβ-Carboline alkaloid.Based on these above,the significance and research content of this paper will be explained.（2）21β-carboline alkaloid derivatives were synthesized.The chemical structure was characterized by using analytical techniques such as IR,HR-ESI-MS,and NMR.The UV spectroscopy and high-performance liquid chromatography results confirmed that Compound 3 and Compound 7 were stable under physiological conditions.（3）The in vitro antitumor activities of the 21β-carboline alkaloid derivatives determined by using MTT assay against several tumor cell lines consisting of T-24、HCC-827、MGC-803、NCI-H-460、SK-OV-3、A549/CDDP、Hep-G2、He La、4T1、A549、MCF-7、BEL-7404 and human normal liver cell line LO-2with human embryonic lung fibroblast WI-38 indicating that Compound 3 showed a high inhibitory effect on almost all tumor cell lines,while low proliferative inhibition of normal cell line.The cell cycle assay results showed that Compound 3 blocked the cell cycle of T24,SK-OV-3,A549,and Hela tumors at S phase in a dose-dependent manner,while the MGC-803,Hep-G2,NCI-H-460 tumor cells were arrested at the G2/M phase.Western blot results demonstrated that Compound 3 may cause DNA damage in T24 cells by activating the ATM-p53-p21-CDK2 pathway.Furthermore,cell morphology detection and fluorescence detection showed that Compound 3 was able to enter the nucleus of T24 tumor cells,thereby disturbing the normal DNA replication of the cells,inhibiting the cell cycle,and ultimately hindering the division of tumor cells.The results of flow cytometry also showed that the compound 3 can also cause early apoptosis in T24,MGC-803,He La and NCI-H-460 tumor cells.And Compound 3 caused the influx of extracellular Ca²⁺and the release of endoplasmic reticulum Ca²⁺by increasing the level of active oxygen in T24 and MGC-803 cells,which led to an increase in total Ca²⁺content in the cells,resulting in mitochondrial transmembrane potential.Decreased,thereby activating Caspase-3/8/9 in T24 and MGC-803 cells,ultimately inducing apoptosis.The WB results showed that Compound 3 significantly reduced the ratio of anti-apoptotic proteins（Bcl-2,Bcl-xl）/pro-apoptotic proteins（Bak,Bax）.The expression levels of apoptosis-inducing factors Bim and Apaf-1 were increased,and the protein expression level of Caspase-12 was also increased.In summary,compound 3 not only induces apoptosis in T24 tumor cells through the mitochondrial-mediated intrinsic pathway,but it may also be related to endoplasmic reticulum and death receptor pathway.（4）TGF-β1-induced NRK-49F cells were screened for viability using 20β-carboline derivatives by MTT method.The results showed that the compound 7 had the most obvious cell viability inhibition effect on TGF-β1-induced NRK-49F cells,and showed a concentration-dependent relationship.Therefore,Compound7 was used as a research object for anti-fibrosis in vitro.Immunofluorescence experiments showed that typical fibroblast-myofibroblastic transdifferentiation occurred in NRK-49F cells induced by TGF-β1,while Compound 7 significantly inhibited this morphological change.Flow cytometry results showed that compound 7 could block TGF-β1-induced NRK-49F cells at G2 phase,but TGF-β1-induced NRK-49F cells may not induce early apoptosis.Western blot analysis showed that Compound 7 can inhibit the activation of NRK-49F cells and reduce the production and accumulation of ECM（Collagen I,COL3A1,Fibronectin,α-SMA）induced by TGF-β1-induced myofibroblasts.Western blot analysis showed that compound 7 can inhibit the activation of NRK-49F cells induced by TGF-β1 and reduce the ECM（Collagen I,COL3A1,Fibronectin,α-SMA）induced by TGF-β1 induced myofibroblasts,production and accumulation.In summary,it is shown that Compound 7 exerts a role in alleviating the progression of renal fibrosis by inhibiting the deposition of extracellular matrix,thereby exhibiting potential anti-renal fibrosis activity.