Selective Mcl-1 Inhibitors:Synthesis,Structural Characterization And Antitumor Activity Of 1,9-Disubstituted β-Carboline Derivatives And Their Pt,Ru And Cu Complexes

Cancer is a major disease that seriously threatens human health and social development.Therefore,the prevention and treatment of cancer has become a hot spot and a difficult point for scientists all over the world.Myeloid cell leukemia 1（Mcl-1）is one of the anti-apoptotic members of the Bcl-2 family proteins It is highly expressed in patients with multiple myeloma and is an important factor for the survival of myeloma cells.However,so far,it has been reported that most Mcl-1 inhibitors are still in the early stages of drug development,and without drugs have been approved for clinical application.In order to seek for an ideal and selective small molecule inhibitor of Mcl-1,this thesis uses tryptamine and pyridine-2-carbaldehyde as starting materials to synthesize an intermediate 1-（2-pyridine）-β-carboline（I）,and then,at N-9 position of I,a hydrophilic group（hydroxyalkyl group）and a hydrophobic group（alkyl and alkene group）were introduced,and six 1,9-disubstitutedβ-carboline derivatives L¹～L⁶ were synthesized.Among them,compounds L³～L⁶ are new synthetic,and then using these 6 compounds as active ligands to react with corresponding metal salts,17new metal complexes were synthesized:[Pt（L¹）Cl₂]（1）,[Pt（L²）Cl₂]（2）,[Pt（L³）Cl₂]（3）,[Pt（L⁴）Cl₂]（4）,[Pt（L⁵）Cl₂]（5）,[Pt（L⁶）Cl₂]（6）,[Ru（L¹）Cl₃CH₃OH]（7）,[Ru（L²）Cl₃CH₃OH]（8）,[Ru（L²）Cl₃C₂H₅OH]（9）,[Ru（L⁴）Cl₃CH₃OH]（10）,[Cu（L¹）Br₂（DMSO）]（11）,[Cu（L²）Br₂（DMSO）]（12）,[Cu（L⁵）Br₂（DMSO）]（13）,[Cu（L⁶）Br₂（DMSO）]（14）,[Cu（L¹）Br₂]（15）,[Cu（L³）Cl₂]（16）,[Cu（L⁴）Cl₂]（17）.The structures of these compounds were characterized by electrospray mass spectrometry,nuclear magnetic resonance spectroscopy,single crystal X-ray diffraction analysis and other methods.The stability of 1,9-disubstitutedβ-carboline derivatives and their metal complexes in solution was tested by ultraviolet-visible spectroscopy and HPLC.In order to further study the anti-tumor activity of the 23 compounds synthesized,these compounds were initially tested by MTT method against 8 tumor cell lines（NCI-H460,He La,MGC80-3,Hep G2,SK-OV-3,BEL-7402,SMMC-7721,T-24）and one human normal liver cell HL-7702 proliferation inhibition.For compounds with a tumor cell proliferation inhibition rate greater than 50%,continue to determine its IC₅₀ value.The experimental results show that the ligand introduced into the N-9 position of the hydrophobic group has better activity than the ligand introduced into the N-9 position of the hydrophilic group,and the anti-tumor activity of the corresponding metal complex is also better.Among them,the complexes 1 and 2 had the strongest antitumor activity against cervical cancer cell He La,with IC₅₀ values of 2.87±0.22 and 2.99±0.16μM,respectively.Therefore,we chose complexes 1 and 2 to study the anti-tumor action mechanism.In order to verify whether the complex exerts an anti-tumor effect by inhibiting Mcl-1,the CCK-8 experiment was used to determine IC₅₀ value of the complexes against five Mcl-1dependent cell lines（LP-1,L-363,MOLP-8,NCI-H929,K562）.The experimental results showed that the IC₅₀ values of the complexes on the five Mcl-1 dependent cell lines were all lower than 10μM,while the Pt complexes could more significantly inhibit the proliferation of cells.Ru complexes,cisplatin,ABT-199（Bcl-2 protein inhibitor）,A-1210477（Mcl-1 protein inhibitor）,etc.are generally lower in antitumor activity than Pt complexes.To determine how the compound binds to the Mcl-1 protein,we conducted molecular docking analysis of the compound and the Mcl-1 protein.The results show that the compounds bind to the P2 pocket of Mcl-1,and the complex binds to Mcl-1 stronger than the corresponding ligands.The xenografted mouse model bearing He La was used to study the effect of the complexes 1and 2 on the tumor growth inhibition.Complex 1（10,15 mg/kg）and complex 2（10,15 mg/kg）were intraperitoneally injected into nude mice every day,two weeks later,the relative tumor growth rate（T/C）of complexes 1 and 2 at a dose of 15 mg/kg was 40.28%（P<0.01）and 35.01%（P<0.01）.The inhibition rate of complex 2 was 60.02%（P<0.001）,higher than that of complex1（55.76%,P<0.001）,and slightly lower than that of cisplatin（63.03%,P<0.001）.Flow cytometry,Co-Immunoprecipitation（Co-IP）,Western Blot and other methods were used to further investigate the action mechanism by complexes 1 and 2 induce tumor cell apoptosis.Through Annexin V-FITC/PI double staining,intracellular reactive oxygen species（ROS）level changes,intracellular Ca²⁺level changes,mitochondrial membrane potential（JC-1）changes,etc.the complexes 1 and 2 on tumor cell apoptosis were detected influences.The results show that the complexes 1 and 2 can cause apoptosis of the corresponding tumor cells,increase the level of reactive oxygen species in the cells,release calcium ions,reduce the mitochondrial membrane potential,enhance the membrane permeability,and then induce apoptosis.The results of Co-IP and Western Blot experiments show that complexes 1 and 2 can induce the dissociation of Mcl-1-Bak and Mcl-1-Bax complexes,thereby releasing Bak and Bax,and then mediating the mitochondrial pathway and inducing cell apoptosis.Flow cytometry and Western blotting experiments show that complexes 1 and 2 can induce the release of cytochrome C from tumor cells into the cytoplasm,and cleave PARP,and activate caspase-9 and caspase-3.