PLKs抑制剂ON 01910结构类似物的设计、合成及初步抗肿瘤活性研究

发布时间：2018-09-11 10:02

【摘要】：Plks (Polo-like kinase)的异常表达与肿瘤的发生发展有密切的联系,研究已证实Plks在多种肿瘤细胞中均过量表达。Plks是一种在分裂细胞中表达的丝氨酸/苏氨酸激酶,作用于有丝分裂过程,促进细胞增殖,其含量在细胞周期M期达到峰值。鉴于在细胞增殖过程中的重要作用及其高度保守的结构,Plks成为治疗恶性肿瘤的重要靶标分子,目前已经发现了一系列的Plks抑制剂而且它们的抗肿瘤活性已在大量的体内外活性研究中得以证实。其中,ON01910作为一种有效的Plks抑制剂已经进入临床三期研究阶段,该化合物对多种肿瘤细胞(包括一些抗药性的细胞株)的半数抑制浓度在纳摩尔浓度范围内。ON01910的药物代谢动力学性质表现为血浆蛋白结合率高,消除快,半衰期短,而且低剂量给药时主要在肝脏中消除,高剂量给药时则主要通过尿排泄。更有组织分布研究表明,肝中的药物蓄积最多,其次是肾,不良反应主要表现为轻微的血液毒性和肝损伤,并无严重的骨髓抑制、神经病变或心脏毒性。基于me-too药物研发的一般策略,本文对ON01910进行专利研究并分析总结该类化合物的构效关系,进行局部结构改造,以改变药物的酸碱性、脂水分布系数、体内代谢转化的方向与延长作用时间。同时尝试通过拆分或手性立体合成,将外消旋体类似物两种构型的异构体分别进行研究。另一方面引入羟肟酸基团,使之以前药形式给药,改善ON01910在体内的ADME过程,提高生物利用度,而且可发挥羟肟酸与金属离子的螯合作用,作用于HDAC、APN、MMP等金属蛋白酶,影响多条信号通路而抑制肿瘤的发生发展。以4-甲氧基-3-硝基甲苯为起始原料,经NBS溴代、巯基乙酸(TGA)亲核取代、30%H2O2氧化、与2,4,6-三甲氧基苯甲醛缩合、硝基还原及溴乙酸甲酯(BAM)亲核取代生成(E)2-(2-甲氧基-5-((2,4,6-三甲氧基苯乙烯基磺酰基)甲基)苄胺)乙酸甲酯(L6),L6结构中羧酸甲酯结构被NaOH水解成羧酸(L7)或者与羟胺钾反应引入羟肟酸基团(L8),同时L7分别与氨基乙醇、赖氨酸、精氨酸、胆碱、氢氧化钙、氢氧化镁、氢氧化钾和氢氧化钠生成对应的盐(L9-L16),而且通过相似的制备方法,合成了具有亚磺酰基结构的类似化合物,结构经ESI-MS及1H-NMR验证。除对ON01910类似物进行研究之外,本文在拼合原理的基础上设计合成了4个a-硫辛酸衍生物,其中化合物Ⅰ2、 Ⅰ3由具有光学活性的硫辛酸制得,化合物Ⅱ则通过外消旋α-硫辛酸与抗癌辅助剂替拉扎明(TPZ)缩合制得,目标化合物具有显著的抑制肿瘤细胞增殖活性,为该类化合物的设计与合成提供新思路。本文最后对目标化合物的抗肿瘤活性进行了初步研究。首先,用荧光测定法验证了L8、L21及Ⅰ1、Ⅱ对HDAC酶具有中等抑制活性。同时,采用MTT法测定了目标化合物对ES-2,K562,PC-3,MDA-MB-231、A549及U937细胞株的抑制增殖活性,结果表明这些化合物都具有较好的细胞毒活性,尤其是L7～8、L21～22,对被测定的三株细胞的IC50值均在1μmol·L-1以下。最后以PLC人肝癌荷NOD/SCID鼠模型对化合物L12、L13抗肿瘤活性进行评价,取得了较好的活性结果,对抗肿瘤化合物的研究具有理论指导意义与实际应用意义。
[Abstract]:The abnormal expression of Plks (Polo-like kinase) is closely related to the occurrence and development of tumors. It has been proved that Plks are overexpressed in many kinds of tumor cells. Plks is a serine/threonine kinase expressed in dividing cells, which acts on mitosis and promotes cell proliferation. The content of Plks peaks in M phase of cell cycle. Plks has become an important target molecule for the treatment of malignant tumors due to its important role in cell proliferation and highly conserved structure. A series of Plks inhibitors have been found and their antitumor activities have been confirmed in a large number of studies in vivo and in vitro. ON01910 has been used as an effective Plks inhibitor. The pharmacokinetic properties of ON01910 are high plasma protein binding rate, rapid elimination, short half-life, and mainly in the liver at low doses. The dosage was given mainly through urine excretion. Some studies of tissue distribution showed that the drug accumulation in the liver was the highest, followed by the kidney. The main adverse reactions were mild hematotoxicity and liver injury, without severe myelosuppression, neuropathy or cardiotoxicity.
Based on the general strategy of me-too drug development, ON01910 was patented, and its structure-activity relationship was analyzed and summarized. Local structural modification was carried out to change the acidity and basicity of the drug, the distribution coefficient of lipids and water, the direction of metabolic transformation in vivo and prolong the action time. On the other hand, the introduction of hydroxamic acid groups into the formerly administered drugs can improve the ADME process of ON01910 in vivo and enhance its bioavailability. Moreover, the chelation of hydroxamic acid with metal ions can also be exerted, acting on HDAC, APN, MMP and other metalloproteinases, affecting multiple signaling pathways. Inhibition of tumor development. 4-methoxy-3-nitrotoluene was used as the starting material, NBS bromination, mercaptoacetic acid (TGA) nucleophilic substitution, 30% H2O2 oxidation, condensation with 2,4,6-trimethoxybenzaldehyde, nitroreduction and methyl bromoacetate (BAM) nucleophilic substitution to form (E) 2-(2-methoxy-5-((2,4,6-trimethoxystyrenesulfonyl) methyl) benzylamine) ethyl Methyl ester of acid (L6), methyl ester of carboxylic acid in L6 structure is hydrolyzed by NaOH to carboxylic acid (L7) or reacted with potassium hydroxylamine to introduce hydroxamic acid group (L8), at the same time L7 reacts with aminoethanol, lysine, arginine, choline, calcium hydroxide, magnesium hydroxide, potassium hydroxide and sodium hydroxide to form corresponding salts (L9-L16). Similar compounds with sulfonyl structure were identified by ESI-MS and 1H-NMR.
In addition to the study of ON01910 analogues, four a-lipoic acid derivatives were designed and synthesized on the basis of the principle of conjugation. Compounds I 2 and I 3 were synthesized from lipoic acid with optical activity. Compound II was synthesized by condensation of racemic alpha-lipoic acid with antitumor adjuvant tirazamine (TPZ). Inhibition of tumor cell proliferation activity provides new ideas for the design and synthesis of such compounds.
In the end, the antitumor activity of the target compounds was preliminarily studied. Firstly, L8, L21 and I 1, II showed moderate inhibitory activity to HDAC enzyme by fluorescence assay. Meanwhile, the antiproliferative activity of the target compounds to ES-2, K562, PC-3, MDA-MB-231, A549 and U937 cell lines was determined by MTT method. All of them had good cytotoxic activity, especially L7-8 and L21-22. The IC50 values of the three cells were all below 1 micromol.L-1. Finally, the anti-tumor activities of L12 and L13 were evaluated by the NOD/SCID mouse model of PLC human liver cancer, and the results were satisfactory. The study of anti-tumor compounds has theoretical significance and practical significance. Inter application significance.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914

【共引文献】