【作者】 徐利保；

【导师】 李松；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2014， 博士

【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。更多还原

【Abstract】 The CC chemokine receptor4(CCR4) belongs to the superfamily of Gprotein-coupled receptors, is broadly expressed on the cells of immunesystem,specificallyTreg cells and T helper2cells. It plays a key role inT cells migrateto the thymus andT cell maturation and education. Three natural chemokine ligandsfor CCR4have been identified as thymus and activation-regulated chemokine(TARC), macrophage-derived chemokine (MDC) and chemokine-like factor1(CKLF1). TARC and MDC are highly specific ligands with strong affinities forCCR4.Chemokine ligands binding to CCR4on the cell surface can activate coupledG-protein and a number of intracellular signaling pathways, including activation ofphospholipase C, protein kinase C,proteins Ras and Rho. CCR4plays a biologicaleffect to lead to the target cell migration to a specific location by the series ofinformation transmission. CCR4can regulate T cell migration to the sites ofinflammation in the body, including the skin and lungs.The studies have demonstratedthat CCR4and its ligands are closely related to the development of asthma, allergicrhinitis and atopic dermatitis, systemic lupus erythematosus and rheumatoid arthritisand other diseases. Meanwhile, CCR4is expressed in the tumor microenvironmentand play a role in regulating the tumor growth. CCR4can induce tumor immuneescape and promote metastasis of tumor cells.Therefore, CCR4has become an important potentialtherapeutic target forallergicinflammatory diseases and cancer immunotherapy.The selectively small molecularCCR4antagonists as drug will provide a new option for treatment of these diseases, inparticular treatment of asthma and CCR4-positive cancer so on.To date, only CCR4monoclonal antibody Mogamulizumab has been approved forlisting for the treatment of relapsed or refractory CCR4-positive adult T-cell leukemialymphoma in2012.The majority of small molecule CCR4antagonists are in bioassay or preclinical research stage.With in-depth research,there will be many smallmolecular CCR4antagonists used in clinical and provide new treatment options forpatients with asthma and CCR4-positivecancer in the future.To date, crystal structure of CCR4hasn’t been reported. CCR4does not have highhomology with other GPCRs. Binding sites of CCR4antagonists is not very clear.These disadvantage factors limit the use of computer-aided drug design in designingaimed compounds. Based on hits to leaddesign ideas and me-too strategy, wedesigned two types of novel trisubstituted pyrimidine amine derivatives as CCR4antagonists through summarizing structural features of many high activity pyrimidineamines CCR4antagonists and avoiding the existing compound patents. The publiclyreported CCR4antagonists can be subdivided into eight categories depending on thegeneral structure of the compounds. These categories are trisubstituted thiazolidinonederivatives, trisubstituted lactam derivatives,2-aminothiazole derivatives, aminopyrimidine derivatives, aryl sulfonamide derivatives,3-piperazinyl coumarinderivatives,2-phenyl morpholine derivatives and bipiperidinyl carboxylic acid amidederivatives so on. We take advantage of active fragment splicing and inverse syntheticanalysis to design novel naphthalene sulfonamide thiazolidinedione derivatives asCCR4antagonists on the basis of structural features of2-aminothiazole and arylsulfonamide derivatives. We have synthesized sixty five novel compounds, andoptimized methods of the synthesis of target compounds and key intermediates.Themolecular structures have been confirmed by1H-NMR, ESI-MS and HRMS.The activities of these compounds were evaluated by the chemotaxis inhibitionassay and label-free cellular assay. And the preferred compounds were studiedthrough CCR4receptor selective experiment. The cell cytotoxicity of compounds hasbeen evaluated by MTT experiment. Chemotaxis experiments indicated that most ofthe compounds at1μM showed ability to inhibit. Some of the compounds exhibithigher inhibitory activity than positive compounds.And inhibitory effects of TARCand MDC mediatedcells chemotaxis are the same. We tested IC50values of preferredcompounds. Compounds N-14, N-36and N-39(IC50values of64,77and69nM,respectively) showed similar activity with the positive control compound1(IC50value of78nM). Meanwhile the compounds show high selectivity for CCR4.Thelabel-freecellular assay experimental results show that the compound N-39(IC50value of1.913μM) has considerable inhibitory activity with the compound BMS-397(IC50valueof1.062μM).The MTT experiment results show that the compounds (excludeN-51)are non-cytotoxic at10μM.Compound N-39has shown higher activity, better safetyand receptor selectivity, andbeen worth further study.The preliminary structure-activity relationships of these trisubstituted pyrimidineamide derivatives were discussed based on the obtained experimental data.The resultsfrom the SAR study indicated that for maintaining the activity it was critical that thesubstituents which attached to piperazinyl group located in the2-position of thepyrimidine nucleus contain carbonyl groups. The substituents contain a saturatedcarbonyl group with the-position nitrogen atom, which attached to carbonyl group,and have high activity. The activity of the compounds which contain the substituentsthat don’t contain hetero atom or the aromatic substituents decreased significantly.The substituents which located in the4-position of the pyrimidine nucleus play animportant role in maintaining the activity. A benzyl amide group substituted withhalogen at the2-and4-positions is beneficial for maintaining the activity. The size ofthe spatial structure and the polar of the substituents which located in the6-position ofthe pyrimidine nucleus play an important role in maintaining the activity. Small andless polar substituents, Cl and methyl group, are more potent. Wherein the substituentis methyl, the compounds have stabilized activity of inhibiting TARC and MDCmediated cells chemotaxis.The discussion of SARs should be helpful for furtherdevelopment of the new CCR4antagonists which are more potent, lower toxicity andmore specificity.更多还原