A Series of Oxyimine-Based Macrocyclic Dinuclear Zinc(II) Complexes Enhances Phosphate Ester Hydrolysis, DNA Binding, DNA Hydrolysis, and Lactate Dehydrogenase Inhibition and Induces Apoptosis

A symmetrical macrocyclic dizinc­(II) complex (1) has been synthesized by using the ligand (L1) [μ-11,24-dimethyl-4,7,16,19-tetraoxa-3,8,15,20-tetraazatricyclo-[20.3.1.110,13] heptacosa-1(25),2,7,9,11,13(27),14,20,22(26),23-decaene-26,27-diol]. A series of unsymmetrical macrocyclic dizinc­(II) complexes (2–6) has been synthesized by Schiff base condensation of bicompartmental mononuclear complex [ZnL] [μ-3,16-dimethyl-8,11-dioxa-7,12-diazadicyclo-[1.114,18] heptacosa-1,3,5(20),6,12,14,16,18(19)-octacaene-19,20-diolato)­zinc­(II)] with various diamines like 1,2-diamino ethane (L2), 1,3-diamino propane (L3), 1,4-diamino butane (L4), 1,2-diamino benzene (L5), and 1,8-diamino naphthalene (L6). The ligand L1 and all the zinc­(II) complexes were structurally characterized. To corroborate the consequence of the aromatic moiety in comparison to the aliphatic moiety present in the macrocyclic ring on the phosphate ester hydrolysis, DNA binding and cleavage properties have been studied. The observed first order rate constant values for the hydrolysis of 4-nitrophenyl phosphate ester reaction are in the range from 2.73 × 10–2 to 9.86 × 10–2 s–1.The interactions of complexes 1–6 with calf thymus DNA were studied by spectroscopic techniques, including absorption, fluorescence, and circular dichroism spectroscopy. The DNA binding constant values of the complexes were found in the range from 1.80 × 105 to 9.50 × 105 M–1, and the binding affinities are in the following order: 6 > 5 > 1 > 2 > 3 > 4. All the dizinc­(II) complexes 1–6 effectively promoted the hydrolytic cleavage of plasmid pBR322 DNA under anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 6 under physiological conditions give the observed rate constant (kobs) of 4.42 ± 0.2 h–1, which shows a 108-fold rate acceleration over the uncatalyzed reaction of ds-DNA. The comparison of the dizinc­(II) complexes 1–6 with the monozinc­(II) complex [ZnL] indicates that the DNA cleavage acceleration promoted by 1–6 are due to the efficient cooperative catalysis of the two close proximate zinc­(II) cation centers. The ligand L1, dizinc­(II) complexes 1, 3, and 6 showed cytotoxicity in human hepatoma HepG2 cancer cells, giving IC50 values of 117, 37.1, 16.5, and 8.32 μM, respectively. The results demonstrated that 6, a dizinc­(II) complex with potent antiproliferative activity, is able to induce caspase-dependent apoptosis in human cancer cells. Cytotoxicity of the complexes was further confirmed by the lactate dehydrogenase enzyme level in HepG2 cell lysate and content media.

本研究采用配体L1 [μ-11,24-二甲基-4,7,16,19-四氧杂-3,8,15,20-四氮杂三环[20.3.1.1^10,13]二十七碳-1(25),2,7,9,11,13(27),14,20,22(26),23-癸烯-26,27-二醇] 合成了一种对称大环双核锌(II)配合物（macrocyclic dizinc(II) complex）1。通过双室单核配合物[ZnL] [μ-3,16-二甲基-8,11-二氧杂-7,12-二氮杂二环[1.1^11,18]二十七碳-1,3,5(20),6,12,14,16,18(19)-八碳烯-19,20-二醇合锌(II)] 与多种二胺（包括1,2-乙二胺（L2）、1,3-丙二胺（L3）、1,4-丁二胺（L4）、1,2-苯二胺（L5）及1,8-萘二胺（L6））发生希夫碱缩合反应，合成了一系列不对称大环双核锌(II)配合物（2~6）。已对配体L1及所有锌(II)配合物进行了结构表征。为验证大环环内芳香基团相较于脂肪基团对磷酸酯水解、DNA结合及切割活性的影响，本研究开展了相关实验。对硝基苯基磷酸酯水解反应的表观一级速率常数范围为2.73×10^–2 ~ 9.86×10^–2 s^–1。采用紫外-可见吸收光谱、荧光光谱及圆二色谱（circular dichroism spectroscopy）等光谱技术，研究了配合物1~6与小牛胸腺DNA（calf thymus DNA）的相互作用。配合物的DNA结合常数（DNA binding constant）范围为1.80×10^5 ~ 9.50×10^5 M^–1，结合亲和力排序为：6 > 5 > 1 > 2 > 3 > 4。所有双核锌(II)配合物1~6均可在厌氧及有氧条件下有效促进质粒pBR322 DNA（plasmid pBR322 DNA）的水解切割。在生理条件下，配合物6介导的DNA水解动力学数据显示其表观速率常数（observed rate constant, kobs）为4.42 ± 0.2 h^–1，相较于未催化的双链DNA（ds-DNA）反应，速率提升了108倍。将双核锌(II)配合物1~6与单核锌(II)配合物[ZnL]对比可知，1~6介导的DNA切割加速效应源于两个紧密邻近的锌(II)阳离子中心的高效协同催化（cooperative catalysis）。配体L1、双核锌(II)配合物1、3及6对人类肝癌HepG2癌细胞（human hepatoma HepG2 cancer cells）具有细胞毒性（cytotoxicity），其半数抑制浓度（IC50）分别为117、37.1、16.5及8.32 μM。研究结果表明，具备强效抗增殖活性（antiproliferative activity）的配合物6可诱导人类癌细胞发生半胱天冬酶依赖的细胞凋亡（caspase-dependent apoptosis）。通过检测HepG2细胞裂解液及细胞培养液中的乳酸脱氢酶（lactate dehydrogenase, LDH）水平，进一步验证了配合物的细胞毒性。