Studies On Synthesis,Structural Characterization And Antitumor Activity Of Cu(Ⅱ) Complexes With Enhanced Chemodynamic Therapy And Rh(Ⅲ) Complex With Nitroxide Radical

Although platinum drugs are widely used in the treatment of various cancers,they lack selectivity and have serious toxic side effects,such as nephrotoxicity and neurotoxicity.Thus,it is urgent to develop non-platinum-based drugs and new treatment strategies to overcome these toxic side effects.When there are too many free radicals in the organisms,they will react directly with DNA,proteins and lipids,resulting in cell dysfunction.Therefore,free radicals can be used in cancer treatment.In the existing free radical therapeutic modalities,improving intracellular reactive oxygen species（ROS）is the most common treatment strategy.The production of ROS is highly oxygen dependent,but tumor tissues are usually in a hypoxic environment,which will greatly limit the effectiveness of cancer treatment.Basing on tumor microenvironment characteristics,chemodynamic therapy（CDT）proposed by professor Bu et al.has attracted extensive attention.CDT doesn’t require oxygen,which uses metal ions（iron,copper,manganese,cobalt ions,etc.）mediated Fenton/Fenton-like reaction to convert excess H₂O₂ in tumor tissues into highly cytotoxic hydroxyl radical（·OH）to induce cancer cell apoptosis.Nonetheless,cancer cells usually deplete·OH produced by CDT by producing excessive glutathione（GSH）.As a consequence,it is necessary to reduce the GSH level in tumor cells to enhance the efficiency of CDT.Apart from CDT,killing cancer cells with compounds containing free radicals is also an effective antitumor treatment strategy under the hypoxia condition of tumor.Based on the above background,this dissertation mainly covered two aspects:1)Cu（II）complexes of quinoline derivatives with enhanced CDT by depleting GSH were synthesized and their antitumor mechanisms were studied.2)Rh（III）complex with oxoglaucine containing stable nitroxide radical targeting G-actin and activator protein（AP-1）was synthesized and its antitumor mechanism was studied.The main contents are as follows:1.Cu（Ⅱ）complexes（Cu1,Cu2 and Cu3）with 3-substituted 2-fluoroquinoline derivatives as ligands were synthesized to enhance CDT by depleting GSH.The results of nuclear magnetic resonance（NMR）spectroscopy,high-resolution mass spectrometry（HRMS）,UV-Vis spectrometry and electron paramagnetic resonance（EPR）spectroscopy suggested that Cu1,Cu2 and Cu3 oxidized GSH to oxidized glutathione（GSSG）,Cu（II）was reduced to Cu（I）,and the generated Cu（I）catalyzed H₂O₂ to·OH.MTT assay results demonstrated that the toxicity of the copper（II）complexes to all test cell lines was Cu1>Cu2>Cu3,except that Cu1 and Cu2 had similar cytotoxicity to MGC80-3 cells.Among the test cells,Cu1,Cu2 and Cu3 had overall good activity against SK-OV-3 cells,with IC₅₀ of 5.27±0.59,9.40±0.83 and 16.06±1.94μM,respectively.Therefore,SK-OV-3 cells were selected to investigate the antitumor mechanism.Inductively coupled plasma-mass spectrometry（ICP-MS）experiment results indicated that the order of copper uptake by SK-OV-3 cells was Cu1>Cu2>Cu3 after Cu1,Cu2 and Cu3 acted on SK-OV-3cells,which was consistent with the antitumor activity.Using the GSH and GSSG detection kit,it was found that the content of GSH decreased and the content of GSSG increased after Cu1 and Cu2 acted on SK-OV-3 cells.By flow cytometry,it was found that pre-incubation of L-buthionine sulfoximine（L-BSO）inhibited the synthesis of intracellular GSH and weakened the ability of Cu1 and Cu2 to induce early apoptosis.At the cellular level,it was determined that Cu1 and Cu2 exerted antitumor activity by depleting intracellular GSH.The results of fluorescence imaging showed that intracellular·OH level increased.These results suggested that Cu1 and Cu2 released Cu（I）by depleting GSH,resulting in Fenton-like reaction in SK-OV-3 cells.By fluorescence imaging,Western blot and flow cytometry,it was confirmed that the production of·OH induced mitochondrial damage,caspase cascade activation and endoplasmic reticulum stress,which eventually led to cell cycle arrest in sub-G1 phase and apoptosis.Importantly,Cu1 and Cu2 also had significant antitumor effect in the SK-OV-3 xenograft model without obvious systemic toxicity.The antitumor rates of Cu1（20 mg/kg）and Cu2（50mg/kg）were 48.43%and 58.06%respectively.In conclusion,Cu1 and Cu2 have excellent antitumor activity in vivo and in vitro through enhanced CDT by depleting GSH.2.The structures of Cu1 and Cu2 were optimized.Twelve new enhanced CDT agents Cu（L¹）₂–Cu(L¹²)₂ were obtained by halogen substitution of the OCH₃ group in Cu1 or the H atom in Cu2.ICP-MS results demonstrated that the lipophilicity of most of the complexes was improved after halogenation of the ligands.It was confirmed that Cu（L¹）₂–Cu(L¹²)₂ could produce·OH through depleting GSH by NMR and EPR.The results of MTT assay demonstrated that Cu（L¹）₂–Cu(L¹²)₂ had strong cytotoxicity to T24 cells,and the most toxic complexes were Cu（L⁴）₂（R²=F）and Cu(L¹⁰)₂（R⁴=F）.After treatment with Cu（L⁴）₂ and Cu(L¹⁰)₂,the intracellular GSH/GSSG ratio decreased and·OH level increased,indicating that Cu（L⁴）₂ and Cu(L¹⁰)₂ enhanced CDT by depleting intracellular GSH.The results of Western blot and autophagy double labeled virus transfection showed that Cu（L⁴）₂ and Cu(L¹⁰)₂ increased the expression of LC3B-II and SQSTM1/p62 in T24cells,promoted the formation of autophagosome,but hindered the fusion of autophagosome and lysosome,thus blocking the autophagy flow.Pre-incubation with rapamycin attenuated the ability of Cu（L⁴）₂ and Cu(L¹⁰)₂ to induce intracellular ROS production and early apoptosis,indicating that Cu（L⁴）₂ and Cu(L¹⁰)₂ promote apoptosis by inhibiting autophagy flow and further enhance the effect of CDT.Besides,the pharmacokinetic properties of Cu(L¹⁰)₂were significantly improved compared with that of Cu1 and Cu2,with a longer half-life(t_1/2=4.9 h),larger area under the plasma concentration-time curve(AUC_0-24h=10.9 mg·h·L^-1),and higher maximal plasma concentration(C_max=2.6 mg/L).For the complex Cu1,t_1/2=3.5 h,AUC_0-24h=7.3 mg·h·L^-1,C_max=1.8 mg/L.For the complex Cu2,t_1/2=3.5 h,AUC_0-24h=6.2 mg·h·L^-1,C_max=1.3mg/L.Moreover,the tumor inhibition rate of Cu(L¹⁰)₂（58.8%）was also higher than that of Cu1（53.4%）and Cu2（39.6%）in the T24 xenograft model.The inhibition rate of Cu（L⁴）₂（51.2%）was equivalent to that of Cu1（53.4%）and higher than that of Cu2（39.6%）.The results showed that the introduction of F,especially R⁴,into the ligand could significantly improve the lipophilicity of the complex,the pharmacokinetic properties of the complex,and the tumor inhibition rate of the complex in nude mouse implanted with T24 cells.Cu（L⁴）₂ and Cu(L¹⁰)₂ are metal complexes that enhance the effect of CDT by depleting GSH and inhibiting autophagy flow.This provides a new idea for the design of metal antitumor drugs.3.Rh complexes[Rh（OG）Cl₃CH₃CH₂OH]（1）and[Rh（OG=O）Cl₂（DMSO）₂]（2）were successfully synthesized by the coordination of oxoglaucine（OG）ligand with Rh Cl₃.The structure of complex 2 was further analyzed by HRMS,NMR,EPR and X-ray photoelectron spectroscopy（XPS）analysis.It was demonstrated that complex 2 was Rh（Ⅲ）complex with nitroxide radical.The results of HRMS,MTT assay and structural analysis suggested that the cytotoxicity of complex 2 to BEL-7402 was significantly stronger than that of complex 1,possibly due to the existence of nitroxide radical in complex 2.The results of fluorescence photography,actin polymerization experiments and flow cytometry suggested that complex 2 inhibited the polymerization of G-actin by ROS overproduction,resulting to arrest the cell cycle of BEL-7402 cells in G2/M phase.By reverse transcription polymerase chain reaction（RT-PCR）and Western blot,it was found that complex 2induced the expression of c-Fos,c-Jun and c-Jun phosphorylation,but the inhibition of c-Fos,c-Jun and c-Jun phosphorylation by adding si RNA and MAPK inhibitors enhanced the ability of complex 2 to induce apoptosis.Co-immunoprecipitation（Co-IP）test and luciferase reporter assay（AP-1 transcriptional activity）results showed that complex 2promoted the interaction between SIRT 1 and c-Fos,therefore c-Fos and c-Jun could not combine to inhibit the transcriptional activity of AP-1,to play an antitumor role.It was discovered that complex 2 induced the decrease of mitochondrial membrane potential,activated caspase-3/9 and induced cell apoptosis through flow cytometry.More importantly,in the BEL-7402 xenograft mouse model,complex 2 significantly inhibited the growth of BEL-7402 tumor,and the tumor inhibition rate was 60.9%at 16 mg/kg.It was found through pathological sections that complex 2 would not cause obvious damage of the heart,liver,spleen,lung and kidney of mice.Complex 2 is a Rh（Ⅲ）complex with nitroxide radical that induced mitochondrial mediated apoptosis by inhibiting G-actin polymerization and AP-1 transcriptional activity.