【作者】 张鹏飞；

【导师】 毕彩丰；

【作者基本信息】 中国海洋大学 ， 海洋化学工程与技术， 2014， 博士

【摘要】 吲哚-2,3-二酮(2,3-indolinedione)，又名靛红，是广泛存在于海洋生物及人体的具有活性的天然内源性化合物，也是许多药物的重要合成原料之一，吲哚-2,3-二酮可用来合成国产传统抗肿瘤药物——靛玉红，同时也具有抗细菌、抗动脉粥样硬化、降低胆固醇、抗癌、预警帕金森病及调节脑内激素的平衡等重要生物活性。自上世纪以来，癌症逐渐成为严重危害人类身心健康的主要疾患，寻找效果好、副作用低的抗癌药物是生物化学领域的重大研究热点。研究表明许多过渡金属配合物具有一定的生物学活性，将吲哚-2,3-二酮应用于希夫碱配合物的合成与应用研究，对开发更高药效的抗癌新药具有重要意义。由于吲哚-2，3-二酮类衍生物具有其独特的生物活性及希夫碱配合物也有很多的奇特性质，本文选择不同结构的氨基化合物与海洋活性小分子吲哚-2，3-二酮合成了六个系列希夫碱配体，将这些配体与过渡金属离子反应得到了三十二种未见报道的希夫碱配合物，并运用元素分析、红外光谱、紫外光谱、摩尔电导率、热重分析等表征手段对配体及配合物进行了结构表征，推测其可能的配位方式和化学结构；并对配体和部分配合物进行了荧光光谱分析。培养得到了四个化合物的单晶，其中两个为吲哚-2，3-二酮类希夫碱，采用X-射线单晶衍射得到了单晶的精细结构并讨论了其量子化学计算。以蛋白酶体为作用靶点，研究了部分配合物的抗肿瘤活性。具体内容如下：(1)合成了吲哚-2，3-二酮缩2-氨基-4-甲基苯酚配体C15H11N2O2（简写为HL1）及其六种过渡金属配合物。经过表征，金属配合物的组成是[ML1(CH3COO)· H2O](M=Cu、Zn、Ni、Mn、Co、Cd，均为二价金属离子)。(2)合成了吲哚-2.3-二酮缩2-氨基-5-甲基苯酚配体C15H11N2O2(简写为HL2)及其六种过渡金属配合物。经过表征，金属配合物的组成是[ML2(CH3COO)]·2H2O (M=Cu、Zn、Ni、Mn、Co、Cd，均为二价金属离子)。(3)合成了吲哚-2.3-二酮缩2-甲氧基-5-氨基苯酚配体C15H11O3N2(简写为HL3)及其五种过渡金属配合物。经过表征，金属配合物的组成是[ML3(CH3COO)]· H2O (M=Cu、Zn、Ni、Mn、Cd，均为二价金属离子)。(4)合成了吲哚-2.3-二酮缩对氨基水杨酸配体C15H9O4N2(简写为HL4)及其五种过渡金属配合物。经过表征，金属配合物的组成是[ML4(CH3COO)]·3H2O (M=Cu、Zn、Ni、Co、Cd，均为二价金属离子)。(5)5合成了吲哚-2.3-二酮缩L-苯丙氨酸的配体C17H13O3N2（简写为HL）及其五种过渡金属配合物。经过表征，金属配合物的组成是[ML5(CH3COO)]·3H2O (M=Cu、Zn、Co)5；[ML(CH3COO)]·2H2O (M=Ni、Cd，均为二价金属离子)。(6)吲哚-2.3-二酮缩L-6色氨酸配体C19H14O3N3（HL）及其五种过渡金属配合物。经过表征，金属配合物的组成是[ML6(CH3COO)]·2H2O(M=Cu、Co、Cd，均为二价金属离子)；[ML6(CH3COO)]·3H2O (M=Ni，Zn，均为二价金属离子)。(7)利用Achar微分法和Coats-Redfern积分法，对部分配合物进行了非等温热分解动力学处理，得出了配合物某步热分解反应机理、热动力≠≠学方程、相应的动力学参数及活化熵变△S和吉布斯自由能变△G，其结果如下：配合物CuL(CH3COO)· H2O第3步热分解动力学函数为f(α)=1/4(1-α)[-ln(1-α)]-3，热分解动力学方程为：dα/dt=A·e-E/RT·f(α)=A·e-E/RT1/4(1-α)[-ln(1-α)]-3，E=972.04kJ·mol-1, lnA=197.51，r=0.9989≠，△S=54.57J·mol-1·K-1，△G≠=940.84kJ·mol-1；配合物[CdL(CH3COO)]·2H2O第3步热分解动力学函数是：f(α)=1/4(1-α)[-ln(1-α)]-3，热分解动力学方程为：dα/dt=A·e-E/RT·f(α)=A·e-E/RT1/4(1-α)[-ln(1-α)]-3E=255.52kJ/mol，lnA=45.98，r=0.9995≠，△S=12.68J/mol·K≠，△G=248.04kJ/mol；配合物[CuL3(CH3COO)]· H2O第2步热分解动力学函数为：f(α)=(1-α)2，其热分解动力学方程为：dα/dt=A·e-E/RT·f(α)=A·e-E/RT·(1-α)2-1，E=362.30kJ·mol, lnA=69.84≠，r=0.9995，△S=19.28J·mol-1·K-1，△G≠=351.19kJ·mol-1。[ZnL5(CH3COO)]·3H2O、[CoL4(CH3COO)]·3H2O和[CuL6(CH3COO)]·2H2O的热分析数据略。(8)测定了希夫碱配体及其部分金属配合物的荧光光谱，研究了其荧光1性质。结果表明：[NiL(CH3COO)·H2O]1、[CdL(CH3COO)·H2O]、[CuL2(CH3COO)]·2H22O、[ZnL(CH3COO)]·2H2O[NiL2(CH3COO)]·2H2O2、[CdL(CH3COO)]·2H2O、[CuL3(CH3COO)]· H2O、[ZnL3(CH3COO)]· H2O、[CuL4(CH3COO)]·3H2O、[ZnL4(CH3COO)]·3H2O、[CoL4(CH43COO)]·3H2O、[CdL(CH3COO)]·3H2O、[NiL5(CH3COO)]·2H2O、[ZnL6(CH3COO)]·3H2O、[CoL6(CH3COO)]·3H2O的荧光性质较好。与配体相比，部分配合物的荧光强度明显增强，且激发峰和发射峰位置均发生了一定程度的偏移。(9)合成了四个化合物晶体，分别为:吲哚-2，3-二酮缩2-氨基-4-甲基苯1酚（a同HL）、吲哚-2，3-二酮缩2-氨基-5-甲基苯酚（b同HL2）、1-苯基-3-甲基-4-苯甲酰基-5-吡唑啉酮(PMBP)缩2-甲氧基-5-氨基苯酚（c）和PMBP缩2-氨基-5-甲基苯酚（d）。X-射线单晶衍射表征结果表明：a和b的结构类似，为希夫碱晶体；化合物c和d的结构类似，并且c和d发生了质子转移变成了其互变异构体，质子由吡唑酮环上的N原子转移到了希夫碱C=N双键上的N原子上，而不是预想得到的希夫碱。晶体结构测试表明：配体a属单斜晶系，空间点群P2(1)/c.，化学式为：C15H11N2O2，晶胞参数为a=12.6211(11)，b=8.7100(7)，c=11.2835(10)，α=90，β=90.7800(10)，γ=90，V=1240.28(18)3，F(000)=528，Dc=1.351g/cm3。最终偏差因子[对I＞2σ(I)的衍射点] R1=0.0391，wR2=0.0919和R1=0.0699，wR2=0.1135；晶体c属三斜晶系，空间点群P-1.，化学式为：C15H11N2O2，晶胞参数为a=8.8111(9)，b=11.4716(12)，c=11.4947(14)，α=115.376(2)，β=99.5470(10)，γ=97.6320(10)，V=1007.74(19)3，F(000)=420，Dc=1.316g/cm3。最终偏差因子[对I＞2σ(I)的衍射点] R1=0.0643，wR2=0.1479和R1=0.1068，wR2=0.1833；晶体b和晶体d数据略。运用密度泛函方法计算了希夫碱前线轨道的能量和分布、分子静电势（MEP）、自然键轨道NBO电荷分布和稳定化能E2分析，探索分子轨道与活性的关系，对分子可能的活性位点进行了预测。(10)以蛋白酶体为靶点，对合成配合物的抗肿瘤活性进行了研究。对金属配合物采用MTT比色法进行初步筛选，研究其对乳腺癌细胞MDA-MB-231增殖的抑制情况。发现其中三种配合物[CdL3(CH3COO)]·H2O(C1)、[CoL4(CH3COO)]·3H2O(C3)和[ZnL6(CH3COO)]·3H2O(C5)对于人体乳腺癌细胞的增值较好的抑制作用。为了研究化学结构和抗癌活性的关系，本论文选取了与上述三2种配合物结构相近的配合物CdL(CH3COO)·2H2O（C2）、CoL2(CH3COO)·2H2O（C4）和[ZnL5(CH3COO)·3H2O（C6），研究了以上六种配合物对于类糜蛋白酶体（CT-like）的抑制作用；进一步应用蛋白质免疫印迹法(Western blot)研究其对细胞凋亡的相关蛋白的表达和细胞凋亡的形态学变化。实验结果表明配合物C1、C3和C5是通过抑制蛋白酶体的活性来抑制人体乳腺癌细胞的恶性增值。同时本文研究了其抗肿瘤作用的机理，初步探讨了金属配合物化学结构及抗癌活性之间关系，提出了含有苯环与吸电子官能团相连的希夫碱配合物可能具有优异的抑制肿瘤细胞增殖的活性的猜测，对于设计合成新型金属配合物抗癌药物具有一定的指导作用。更多还原

【Abstract】 2,3-indolinedione, also called isatin, is a kind of widespread ocean organisms andendogenous active compounds in the natural body. It is also a very importantpharmaceutical intermediates.2,3–indolinedione can be used to synthesize indirubinwhich has anti-cancer effect. It also has important biological activity in antibacterial,anti atherosclerosis, assimilating cholesterol, anticancer, early warning aboutParkinson’s disease, and regulate the brain hormone balance. Since the20th century,cancer has become one of the main disease for serious harm human health. How tolooking for high efficiency and low side effects anti-cancer drugs has been the scienceresearch focus. In recent years, a large number of transition metal complexes havecertain biological activities.2,3-indolinedione is applied to the synthesis of schiffbase complexes and application research to develop more potential anticancer drugs.Because2,3-indolinedione derivatives has its unique biological activity and theschiff base complexes also has so many features, this paper select amino compoundswhich have different structure and small marine active molecules2,3-indolinedioneto synthesize six series of schiff base ligand and more than30kinds of complexes.four kinds of single crystals were cultivated and determined through X-ray singlecrystal diffraction. The possible chemstrical structure were determined throughelemental analysis, IR, UV spectra, molar conductivity, TG-DTG analysis and soon..Fluorescence properties of ligands and their complexes have been studied and thecomplexes which have good fluorescrnce properties have been chosen. The inhibitionand inducing apoptosis on breast cancer cells of complexes have been also studied bytargeting the cellular proteasome. The details of work are as follow:(1) The ligand and6transition metal complexes derived from2,3-indolinedioneand2-amino-4-methyl phenol were synthetized. The compositions of the complexes are determind to be [ML1(CH3COO)· H2O](M=Cu、Zn、Ni、Mn、Co、Cd，all of them are divalent metal ions; HL1: C15H11N2O2).(2) The ligand and6transition metal complexes derived from2,3-indolinedioneand2-amino-5-methyl phenol were synthetized. The compositions of thecomplexes are determind to be [ML2(CH3COO)]·2H2O (M=Cu、Zn、Ni、Mn、Co、Cd，all of them are divalent metal ions; HL2：C15H11N2O2).(3) The ligand and5transition metal complexes derived from2,3-indolinedioneand2-methoxy-5-amino phenol were synthetized. The compositions of thecomplexes are determind to be [ML3(CH3COO)]· H2O (M=Cu、Zn、Ni、Mn、Cd，all of them are divalent metal ions; HL3：C15H11O3N2).(4) The ligand and5transition metal complexes derived from2,3-indolinedioneand p-aminosalicylic acid were synthetized. The compositions of thecomplexes are determind to be [ML4(CH3COO)]·3H2O (M=Cu、Zn、Ni、Co、Cd，all of them are divalent metal ions; HL4：C15H9O4N2).(5) The ligand and5transition metal complexes derived from2,3-indolinedioneand L-phenylalanine were synthetized. The compositions of the complexes aredetermind to be [ML5(CH3COO)]·3H2O (M=Cu、Zn、Co，all of them aredivalent metal ions; HL5：C17H13O3N2)；[ML5(CH3COO)]·2H2O (M=Ni、Cd，both of them are divalent metal ions).(6) The ligand and5transition metal complexes derived from2,3-indolinedioneand L-tryptophan synthetized. The compositions of the complexes aredetermind to be [ML6(CH3COO)]·2H2O (M=Cu、Co、Cd，all of them aredivalent metal ions; HL6：C19H14O3N3)；[ML6(CH3COO)]·3H2O (M=Ni，Zn，both of them are divalent metal ions)(7) Both Achar differential and Coats-Redfern integral method were used to dealtwith non isothermal kinetics of thermal decomposition for part of thecomplexes.The pyrolysis reaction mechanism, kinetics of thermaldecomposition equation, the corresponding kinetic parameters, activation entropy change△S≠and gibbs free energy change≠△Gof the complexes insome step were obtained. The part results are as follows:The thermal decomposition kinetic function of complexCuL1(CH3COO)· H2O in step3may be definded as f(α)=1/4(1-α)[-ln(1-α)]-3，and the kinetic equation of thermal decomposition may be definded as dα/dt=A·e-E/RT·f(α)=A·e-E/RT1/4(1-α)[-ln(1-α)]-3，E=972.04kJ·mol-1, lnA=197.51，r=0.9989，△S≠=54.57J·mol-1·K-1≠，△G=940.84kJ·mol-1.The thermal decomposition kinetic function of complex[CdL2(CH3COO)]·2H2O in step3may be definded asf(α)=1/4(1-α)[-ln(1-α)]-3，and the kinetic equation of thermal decompositionmay be definded as dα/dt=A·e-E/RT·f(α)=A·e-E/RT1/4(1-α)[-ln(1-α)]-3，E=972.04kJ·mol-1, lnA=197.51≠，r=0.9989，△S≠=54.57J·mol-1·K-1，△G=940.84kJ·mol-1.The thermal decomposition kinetic function of complex[CuL3(CH3COO)]· H2O in step2may be definded as f(α)=(1-α)2，and thekinetic equation of thermal decomposition may be definded as：dα/dt=A·e-E/RT·f(α)=A·e-E/RT·(1-α)2，E=972.04kJ·mol-1, lnA=197.51，r=0.9989，△S≠=54.57J·mol-1·K-1，△G≠=940.84kJ·mol-1.The TG-DTG analysis data of [ZnL5(CH3COO)]·3H2O、[CoL4(CH3COO)]·3H2O和[CuL6(CH3COO)]·2H2O the complexes wereomitted.(8) The fluorescence spectra of ligands and part of their metal based complexeswere measured. Meanwhile, the related fluorescence properties were studied.The results show that the complexes such as [NiL1(CH3COO)·H2O],[CdL1(CH3COO)·H2O],[CuL2(CH3COO)]·2H2O,[ZnL2(CH3COO)]·2H2O,[NiL2(CH3COO)]·2H2O,[CdL2(CH3COO)]·2H2O,[CuL3(CH3COO)]· H2O,[ZnL3(CH3COO)]· H2O,[CuL4(CH3COO)]·3H2O,[ZnL4(CH3COO)]·3H2O,[CoL4(CH3COO)]·3H2O,[CdL4(CH3COO)]·3H2O, [NiL5(CH3COO)]·2H2O,[ZnL6(CH3COO)]·3H2O,[CoL6(CH3COO)]·3H2O have good fluorescence properties. Comparing withthe with the corresponding ligands which almost have little fluorescenceintensity, their fluorescence intensity of these complexes enhancedsignificantly. Besides, excitation and emission peaks have shifted to somerange.(9) Four single crystals were cultivated. They are2,3–indolinedione–2-amino-4-methyl phenol (a, the same as L1),2,3–indolinedione–2-amino-5-methyl phenol (b, the same as L2), PMBP–2-methoxy-5-aminophenol(c), PMBP–2-methoxy-5-amino phenol (d), respectively. The resultsof characterization show that the structure of compound a is similar to b andthe structure of c is similar to d. The proton transferation have taken place inthe compounds c and d to become their tautomer. Proton transferred from N ofpyrazole ketones ring to N atom of Schiff base C=N double bond. The X-raycrystallography shows that: The legend a is in monoclinic system, P-1spacegroup, molecular formula: C24H21N3O3, Unit cell parameter: a=12.6211(11)，b=8.7100(7)，c=11.2835(10)，α=90，β=90.7800(10)，γ=90，V=1240.28(18)3，F(000)=528，Dc=1.351g/cm3, R1=0.0391，wR2=0.0919for I＞2σ(I).The legend c is in triclinic system, P2(1)/c space group, molecular formula:C15H11N2O2, Unit cell parameter: a=8.8111(9)，b=11.4716(12)，c=11.4947(14)，α=115.376(2)，β=99.5470(10)，γ=97.6320(10)，V=1007.74(19)3，F(000)=420，Dc=1.316g/cm3, R1=0.0643，wR2=0.1479forI＞2σ(I).The crystal data for compound b and d are omitted. The study of legends arecalculated by using the density functional method the frontier orbital energyand distribution, molecular electrostatic potential (MEP), NBO chargedistribution and stabilization energy E2analysis, to explore the molecularorbitals and the relationship between activity and active site of the molecules. (10) The anticancer activity of metal complexes were studied using protease fortargets in this paper. The complexes were sifted from32complexes using3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT method)in human breast cancer, to research proliferation inhibition of the MDA-MB-231breast cancer cell. The result showed that there are three complexes [CdL3(CH3COO)]· H2O (C1),[CoL4(CH3COO)]·3H2O (C3) and [ZnL6(CH3COO)]·3H2O (C5) which can inhibit the vicious proliferation of humanbreast cancer cells. In order to study the relationship between chemicalstructure and antitumor activity, three complexes CdL2(CH3COO)·2H2O（C2）、CoL2(CH3COO)·2H2O（C4）and [ZnL5(CH3COO)·3H2O（C6）which have similar chemical structure with complexes C1, C3and C5to studied on chymotrypsin-like (CT-like) activity of six complexes. Furtherstudy is carried on the application of protein immunoblot technique (Westernblot) to study the expression of apoptosis related proteins and morphology ofcell apoptosis. The results show that the above three complexes inhibit thevicious proliferation of human breast cancer cells through the inhibition of theproteasome activity. Forthermore, this paper preliminary discusses therelationship between the chemical structure and antitumor activity of metalcomplexes. The conclusion that the schiff base complexes with the specialstructure which is the benzene ring connected with electron-withdrawingfunctional groups maybe have excellent proliferation inhibiting activity fortumor cell is presented. It plays a guiding role for designing and synthetisingnew metal complexes for anti-cancer drugs.更多还原