Studies On Design,Synthesis,Structural Characterization And Antitumor Activity Of Mcl-1 Targeted Copper(?) Complexes With ?-Carbolines

Mcl-1(myeloid cell leukemia 1)is an anti-apoptotic Bcl-2 family member of proteins which plays an important role in the survival of various cancer cells.However,the development of inhibitors of Mcl-1 has been challenging.Up to now,no metal-based Mcl-1inhibitor has been reported.Indole scaffold was used as a starting point to develop metal-based Mcl-1inhibitors.The combination of indole scaffold and 2,2-Bipyridine forms 1-(2-pyridyl)-?-carboline.In order to improve the binding affinity to the hydrophobic pocket of Mcl-1,a variety of hydrophobic groups were introduced into the N-9 position of 1-(2-pyridyl)-?-carboline.Herein,a series of9-substituted?-carboline derivatives(2-24)and their copper complexes(25-56)were designed and synthesized.Their structures were characterized by NMR,ESI-MS,elemental analysis and X-ray diffraction analysis.At the cellular level,the antitumor activities of these compounds have been tested by MTT method against five cell lines.At the molecular level,their interactional properties with Mcl-1(myeloid cell leukemia 1)were tested by fluorescence polarization-based binding assay,enzyme-linked immunosorbent assay,docking study and SPR.The mechanism of action of copper complexes wasfurther investigated by co-immunoprecipation(co-IP),Si RNA transfection,flow cytometry and western blot.In vivo antitumor activity,toxicity,and preliminary pharmacokineticstudyof these compounds were evaluated.The main contents are as following:1.Design,synthesis,structural characterization and stability in solution of copper complexes.3D-QSAR was used to evaluate the structure-activity relationship of?-carboline derivatives.The result suggests that antitumor activity of?-carboline derivatives can be improved by introduced a hydrophobic group or a bulky group at N-9 site.Basedon the 3D-QSAR results,a series of 9-substituted?-carboline derivatives(2-24)were designed and synthesized.The structures of these compoundswere characterized by NMR spectrometry and ESI-MS.Thirty two copper(?)complexes(25-56)with?-carboline ligands were synthesized.The structures of these complexes were characterized by ESI-MS,elemental analyses and single crystal X-ray diffraction analysis.All these copper(?)complexes are mononuclear structure.In each case the metal center Cu(?)is tetra-coordinated and surrounded by one bidentate?-carboline ligand and two chloridion ions or two nitrate ions.The stability of complexes wasinvestigated by UV-Vis spectroscopy and ESI-MS.2.In vitro antitumor activity.The cytotoxicity of compounds 2-24 and their copper(?)complexes(25-56)against four tumor cell lines(NCI-H460,MGC80-3,Hep G2,T-24)and the normal human liver HL-7702 cells was tested by MTT assay.The cytotoxicity of compounds2-7,13-16 and 18are higher than that of 1-(2-pyridyl)-?-carboline.Moreover,all the 9-substitude?-carboline derivatives(2-24)were low toxiticy toward the normal human liver HL-7702 cell line.The copper(?)complexes(25-56)showed enhanced cytotoxicity than copper salt and their ligands,suggesting a synergistic effect upon the combination of copper(?)and the ligands.The structure and activity relationship analysis of thesecopper complexes suggested that the hydrophobic groups at N-9 position of?-carbolineligands contributed to the increased cytotoxicity toward cancer cells.The complex 38,39,48,49 were selected for further study.3.The mechanism of action of copper complexes.The interactions of these copper complexesand Mcl-1 were furtherstudiedusing various methods.In addition,the action mechanism of these copper complexes was further investigated at the cellular level.(1)The result of fluorescence polarization-based binding assay,enzyme-linked immunosorbent assay and SPR assaysuggest that the binding affinity of copper complexes 25-56to Mcl-1 are much higher than that of their ligands or copper salts.The structure activity relationship analysis of these copper complexes showed that the hydrophobic groups at N-9position of?-carbolineligands resulted in the increase in binding affinity,which is similar with the SAR in MTT assay.Complexes 38,39,49 competed with the BH3 peptide for binding with Mcl-1 in a dose-dependent manner,with the Kivalues of 2.1,1.2 and 1.4n M,respectively.(2)Docking study showed that,complexes 38,39,48,and49binds to the P2 binding pocket of Mcl-1.Complex38 can form two aromatic H-?interactions with the Ala227 and Arg263 of the Mcl-1 protein.Complex 39can form two H-?interactions with Arg263 and a strong hydrogen bond with His224 of Mcl-1.Complex 49can formcation-?interaction with Arg263 of Mcl-1.In addition,copper complexes have better docking score than their ligands.(3)Complexes 38,39,49showed higher binding affinity to Mcl-1 than other Bcl-2 family proteins.The most potent complex 39showed 356-795 foldselectively for Mcl-1 over other Bcl-2 family proteins.(4)The results of CCK-8 assay showed that complexes 38,39,48,49selectively inhibite the growth of Mcl-1-dependent cell lines.To the contrast,the cytotoxic agent cisplatin showed no selectivity for these cell lines.(5)Si RNA transfectionand flow cytometry analyses suggest that complexes39,49 kill tumor cells by Bax/Bak mediated apoptosis.(6)Co-immunoprecipation(co-IP)and immunoblot suggest that complexes39,49disrupted binding of Bax and Bak to Mcl-1 in cells.(7)Further study showed that complexes39,49 can release ofcytochrome c into cytosol,activation of caspase-3/9 and induce PARP cleavage in cells.(8)Complex 39,49 induce a caspase dependent apoptosis in cells.4.The toxicity,preliminary pharmacokinetic study and in vivo antitumor activity of copper complexes.The toxicity of copper(?)complexes were evaluated.(1)Complexes38,39,48,49(LD₅₀=32.5,33.6,34.6 and 29.1mg/kg,respectively)showed reduced acutetoxicity compared with cisplatin(LD₅₀=11.1mg/kg).(2)The complexes38,39,48,49 treated animals showed no body weight loss or other side effects in experimental period as compared to cisplatin.(3)Serological analysis and histopathological evaluation of toxicity against the heart,liver,and kidney in mice indicated no drug-related side effect of complexes38,39,48,49 at the dose of 10mg/kg.On the other hand,treatment of cisplatin causes renal tubular lesions.The pharmacokinetics of complexes 38,39,48,49 was also investigated.(1)Complex 39found to have a peak concentration of 46.89?g/L at 20 min.The C_max,MRT and AUC_0-t of complex 39 is higher than complex 38.Complex 49found to have a peak concentration of64.9 ng/ml at 20 min.The C_max,AUC_0-t and AUC_0-?of complex 48 were lower than complex 49.(2)The plasma was also analyzed by ESI-MS,the result suggest that complexes38,39existed in the blood in the form of[Cu LCl]⁺and the complexes48,49existed in the blood in the form of[Cu L(NO₃)]⁺,suggesting that the?-carboline ligand remained chelating to copper center.The in vivo antitumor activity of copper complexes was evaluated by NCI-H460 xenograft model.(1)The mice treated with 38,39,48,49(10mg/kg)showed the relative tumor increment rates(T/C)of 60.9%(P<0.01),40.8%(P<0.001),62.6%(P<0.01),46.2%(P<0.001)on day 7,respectively.The inhibitory rate of complex 39(56.6%,P<0.001)is higher than cisplatin(54.3%,P<0.001).The inhibitory rates of complexes38(34.6%,P<0.01),48(32.9%,P<0.01)and49(53.5%,P<0.001)are lower than that of complex 39.(2)The pathological study showed that complexes38,39 and 49 were more effective in inducing tumor necrosis compared with cisplatin.(3)Moreover,a dose-dependent increased mean survival timecould be observed in mice treated with complex 39(49 days)or49(48days)compared with control group(36 days).On the basis of these positiveresults,complex 39may have thepotential to be further developed as an antitumor agent with high efficiency and low toxicity.