CC趋化因子受体4拮抗剂的设计、合成与活性评价

发布时间：2018-08-05 14:48

【摘要】：CC趋化因子受体4（CC chemokine receptor4，CCR4）属于G蛋白偶联受体家族，广泛表达于免疫系统的细胞表面，特别是Treg细胞和Th2细胞。CCR4对T细胞迁移到胸腺和T细胞发育成熟起到重要作用。CCR4有三个天然特异性配体：胸腺活化调节趋化因子（TARC）、巨噬细胞衍生的趋化因子（MDC）和趋化素样因子1（CKLF1）。其中TARC和MDC是CCR4的高亲和性配体。细胞表面的CCR4与配体结合，可激活偶联的G蛋白，进而发生级联细胞激活效应，包括激活磷脂酶C和蛋白激酶C的活化，同时也激活了Ras和Rho。通过这一系列的信息传递实现趋化靶细胞向特定位置迁移，发挥生物学效应。CCR4可以调节T细胞迁移到身体炎症部位，包括皮肤和肺。研究证实，CCR4与哮喘、过敏性鼻炎、异位性皮炎、系统性红斑狼疮和类风湿性关节炎等免疫相关性疾病的发生发展密切相关。同时，CCR4在肿瘤微环境中起到调控作用，诱导肿瘤的免疫逃逸和促进肿瘤细胞的转移等。因此，CCR4已经成为治疗免疫相关性疾病和肿瘤免疫治疗的重要的药物靶标，研发选择性拮抗CCR4的小分子药物将为上述疾病的治疗提供新的选择，尤其是在治疗哮喘和CCR4阳性的恶性肿瘤等疾病方面具有广阔的应用前景。目前为止，只有拮抗CCR4的单克隆抗体Mogamulizumab于2012年被批准上市，用于治疗复发或难治性CCR4阳性的成人T细胞性白血病淋巴瘤，还没有小分子CCR4拮抗剂应用于临床，大部分小分子CCR4拮抗剂都处于生物活性测试或临床前研究阶段。相信随着研究的深入，会有很多的小分子CCR4拮抗剂应用于临床，为哮喘和CCR4阳性的恶性肿瘤等疾病患者提供新的治疗方案。目前，没有CCR4晶体结构的报道，CCR4与其他GPCR受体同源性较低，现有的CCR4拮抗剂与受体作用位点不是很明确，这些不利因素限制了利用计算机辅助药物设计的方法进行目标化合物的设计。本文从hit to lead的思路出发，采用基于配体的Me-too策略，在总结目前已报道的活性较高的氨基嘧啶类CCR4拮抗剂的结构特征和规避现有化合物专利保护的基础上，设计了两种三取代嘧啶胺类新化合物。已公开报道的小分子CCR4拮抗剂依据化合物结构母核的不同可分为：三取代噻唑烷酮衍生物、三取代内酰胺衍生物、2-氨基噻唑衍生物、氨基嘧啶衍生物、芳基磺酰胺衍生物、3-哌嗪基香豆素衍生物、2-苯基取代吗啉衍生物和双哌啶羧酸胺衍生物等。基于2-氨基噻唑和芳基磺酰胺两类CCR4拮抗剂的结构特点，根据化合物类药原理和生物电子等排原理，采用活性片段拼接和逆合成分析的方法，设计了萘磺酰胺噻唑烷酮类新化合物。共设计合成了两大类65个新结构目标化合物，对关键中间体和目标化合物的合成方法进行了研究和优化。所有化合物的结构经1H-NMR、ESI-MS和HRMS等确证。采用细胞趋化抑制实验和非标记全细胞检测实验对目标化合物进行了活性评价，并对优选化合物进行了CCR4受体选择性实验研究。采用MTT法评价了目标化合物的细胞毒作用。细胞趋化抑制实验结果表明，大部分目标化合物在1μM浓度下对TARC和MDC介导的CCR4转染的L1.2细胞的趋化运动都表现出了不同程度的趋化抑制作用，其中部分化合物表现出了高于阳性化合物的抑制活性，且化合物对二者的抑制作用效果基本相同。对活性较好的优选目标化合物测试了IC50值，发现化合物N-14、N-36和N-39表现出了与阳性化合物1相近的抑制活性，IC50值为别为64nM、69nM、77nM和78nM。三个化合物还表现出了对CCR4拮抗作用的选择性。非标记全细胞检测实验结果表明，化合物N-39与阳性化合物BMS-397具有相当的抑制活性，IC50值分别为1.913μM和1.062μM。MTT实验结果表明化合物（N-51除外）在10μM浓度下对细胞无毒性。化合物N-39表现出了高活性和安全性以及较好的受体选择性，值得进一步的研究。根据活性评价结果，本文对目标化合物进行了初步的构效关系分析：嘧啶母核2位哌嗪基连接的羰基是保持活性的必需基团。与羰基相连的取代基以位含氨基的饱和取代基活性高，取代基不含杂原子或为芳香取代基活性明显下降。嘧啶母核4位的取代基团对活性影响较大，2,4-卤素双取代的苄氨基有利于活性的保持，，单取代的活性明显下降。嘧啶母核6位取代基的空间结构和极性大小对活性有很大的影响，空间结构和极性都很小的氯或甲基取代活性最好，其中取代基为甲基时，化合物对TARC和MDC介导的细胞趋化运动都有稳定的抑制活性。这对进一步开发高效、低毒、选择性强的CCR4拮抗剂具有一定的指导意义。
[Abstract]:CC chemokine receptor 4 (CC chemokine receptor4, CCR4) belongs to the G protein coupled receptor family, which is widely expressed on the cell surface of the immune system, especially Treg and Th2 cells..CCR4 plays an important role in the migration of T cells to the thymus and T cells..CCR4 has three day specific ligands: thymic activation regulates chemokine. Macrophage derived chemokine (MDC) and chemokine like factor 1 (CKLF1). TARC and MDC are highly affinity ligands for CCR4.
The binding of CCR4 to the ligand on the cell surface activates the coupling of G protein, and then the activation effect of cascade cells, including activation of phospholipase C and protein kinase C, and activates Ras and Rho. through this series of information transfer to realize localization and migration of chemotaxis target cells, and the biological effect.CCR4 can regulate the migration of T cells. CCR4 is closely related to the development of immune related diseases such as asthma, allergic rhinitis, atopic dermatitis, systemic lupus erythematosus and rheumatoid arthritis, and CCR4 plays a regulatory role in the tumor microenvironment, inducing immune escape and promoting tumor cells. Transfer, etc.
Therefore, CCR4 has become an important drug target for the treatment of immune related diseases and tumor immunotherapy. The development of small molecular drugs that selectively antagonize CCR4 will provide a new choice for the treatment of these diseases, especially in the treatment of asthma and CCR4 positive cancer masks. The monoclonal antibody Mogamulizumab against CCR4 was approved in 2012 for the treatment of recurrent or refractory CCR4 positive adult T cell leukemia lymphoma. There is no small molecule CCR4 antagonist applied to the clinic. Most of the small molecule CCR4 antagonists are in bioactivity test or pre clinical stage. In addition, there will be many small molecule CCR4 antagonists used in clinical, for asthma and CR4 positive malignant tumors and other diseases to provide new treatment.
At present, without the report of CCR4 crystal structure, the homology of CCR4 and other GPCR receptors is low. The existing CCR4 antagonist and receptor site are not very clear. These disadvantages restrict the design of target compounds using the method of computer-aided drug design. Based on the idea of hit to lead, this paper uses ligand based Me-to. O strategy, on the basis of summarizing the structural characteristics of the present reported active amino pyrimidine CCR4 antagonists and avoiding the patent protection of existing compounds, two new compounds of three substituted pyrimidine amines were designed. The publicly reported small molecular CCR4 antagonists can be divided into three substituted thiazoles based on the difference of the compound structure parent nucleus. Ketone derivatives, three substituted lactam derivatives, 2- amino thiazole derivatives, amino pyrimidine derivatives, aryl sulfonamide derivatives, 3- piperazine coumarin derivatives, 2- phenyl substituted morpholine derivatives and dipiperidine carboxylic amines derivatives and so on. Structure characteristics based on 2- amino thiazole and aryl sulfonamide two CCR4 antagonists, according to compound drugs A new compound of naphthalamidone thiazolidone was designed by means of active fragment splicing and inverse synthesis. A total of two kinds of 65 new structural target compounds were designed and synthesized. The synthesis methods of key intermediates and target compounds were studied and optimized. The structure of all the compounds was 1H-N MR, ESI-MS and HRMS are confirmed.
The activity of the target compounds was evaluated by cell chemotaxis inhibition test and unlabeled whole cell test. The selective experimental study of CCR4 receptor was carried out on the preferred compound. The cytotoxicity of the target compound was evaluated by MTT method. The results of cell chemotaxis inhibition experiment showed that most of the target compounds were at 1 micron M concentration. The chemotaxis of CCR4 transfected L1.2 cells mediated by TARC and MDC showed different degrees of chemotactic inhibition. Some of the compounds showed higher inhibitory activity than the positive compounds, and the inhibitory effect of the compounds on the two were basically the same. The IC50 value of the preferred target compounds with better activity was tested and discovered. The compound N-14, N-36 and N-39 showed the inhibitory activity similar to the positive compound 1. The IC50 value was not 64nM, 69nM, 77nM and 78nM. also showed the selectivity to the CCR4 antagonism. The unlabeled whole cell detection experimental results showed that the compound N-39 and the positive compound BMS-397 had the considerable inhibitory activity, the IC50 values were respectively. The experimental results of 1.913 M and 1.062 micron M.MTT showed that the compounds (except N-51) were nontoxic to the cells at the concentration of 10 mu M. Compound N-39 showed high activity and safety and better receptor selectivity. It was worth further study.
According to the results of the activity evaluation, the preliminary structure-activity relationship of the target compounds was analyzed. The carbonyl group connected by 2 piperazines in the pyrimidine nucleus is a necessary group to maintain the activity. The substituent connected to the carbonyl group is highly active with the saturated substituent containing the amino group, and the substituent without the heteroprecursor or the aromatic substituent activity obviously decreases. The substituent groups of the 4 sites of the parent nucleus have great influence on the activity. The double substituted benzyl amino group of 2,4- halogen is beneficial to the retention of the activity, and the activity of the mono substituent is obviously decreased. The space structure and polarity of the 6 substituents of the pyrimidine parent nucleus have great influence on the activity, and the space structure and the polarity of the substituent are the best, in which the substituents are taken as the substituents When methylated, the compounds have stable inhibitory activity on cellular chemotaxis mediated by TARC and MDC, which has certain guiding significance for the further development of high efficiency, low toxicity, and selective CCR4 antagonists.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914

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