【摘要】：CC趨化因子受體4（CC chemokine receptor4，CCR4）屬于G蛋白偶聯(lián)受體家族，廣泛表達(dá)于免疫系統(tǒng)的細(xì)胞表面，特別是Treg細(xì)胞和Th2細(xì)胞。CCR4對T細(xì)胞遷移到胸腺和T細(xì)胞發(fā)育成熟起到重要作用。CCR4有三個天然特異性配體：胸腺活化調(diào)節(jié)趨化因子（TARC）、巨噬細(xì)胞衍生的趨化因子（MDC）和趨化素樣因子1（CKLF1）。其中TARC和MDC是CCR4的高親和性配體。 細(xì)胞表面的CCR4與配體結(jié)合，可激活偶聯(lián)的G蛋白，進而發(fā)生級聯(lián)細(xì)胞激活效應(yīng)，包括激活磷脂酶C和蛋白激酶C的活化，同時也激活了Ras和Rho。通過這一系列的信息傳遞實現(xiàn)趨化靶細(xì)胞向特定位置遷移，發(fā)揮生物學(xué)效應(yīng)。CCR4可以調(diào)節(jié)T細(xì)胞遷移到身體炎癥部位，包括皮膚和肺。研究證實，CCR4與哮喘、過敏性鼻炎、異位性皮炎、系統(tǒng)性紅斑狼瘡和類風(fēng)濕性關(guān)節(jié)炎等免疫相關(guān)性疾病的發(fā)生發(fā)展密切相關(guān)。同時，CCR4在腫瘤微環(huán)境中起到調(diào)控作用，誘導(dǎo)腫瘤的免疫逃逸和促進腫瘤細(xì)胞的轉(zhuǎn)移等。 因此，CCR4已經(jīng)成為治療免疫相關(guān)性疾病和腫瘤免疫治療的重要的藥物靶標(biāo)，研發(fā)選擇性拮抗CCR4的小分子藥物將為上述疾病的治療提供新的選擇，尤其是在治療哮喘和CCR4陽性的惡性腫瘤等疾病方面具有廣闊的應(yīng)用前景。目前為止，只有拮抗CCR4的單克隆抗體Mogamulizumab于2012年被批準(zhǔn)上市，用于治療復(fù)發(fā)或難治性CCR4陽性的成人T細(xì)胞性白血病淋巴瘤，還沒有小分子CCR4拮抗劑應(yīng)用于臨床，大部分小分子CCR4拮抗劑都處于生物活性測試或臨床前研究階段。相信隨著研究的深入，會有很多的小分子CCR4拮抗劑應(yīng)用于臨床，為哮喘和CCR4陽性的惡性腫瘤等疾病患者提供新的治療方案。 目前，沒有CCR4晶體結(jié)構(gòu)的報道，CCR4與其他GPCR受體同源性較低，現(xiàn)有的CCR4拮抗劑與受體作用位點不是很明確，這些不利因素限制了利用計算機輔助藥物設(shè)計的方法進行目標(biāo)化合物的設(shè)計。本文從hit to lead的思路出發(fā)，采用基于配體的Me-too策略，在總結(jié)目前已報道的活性較高的氨基嘧啶類CCR4拮抗劑的結(jié)構(gòu)特征和規(guī)避現(xiàn)有化合物專利保護的基礎(chǔ)上，設(shè)計了兩種三取代嘧啶胺類新化合物。已公開報道的小分子CCR4拮抗劑依據(jù)化合物結(jié)構(gòu)母核的不同可分為：三取代噻唑烷酮衍生物、三取代內(nèi)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-苯基取代嗎啉衍生物和雙哌啶羧酸胺衍生物等。基于2-氨基噻唑和芳基磺酰胺兩類CCR4拮抗劑的結(jié)構(gòu)特點，根據(jù)化合物類藥原理和生物電子等排原理，采用活性片段拼接和逆合成分析的方法，設(shè)計了萘磺酰胺噻唑烷酮類新化合物。共設(shè)計合成了兩大類65個新結(jié)構(gòu)目標(biāo)化合物，對關(guān)鍵中間體和目標(biāo)化合物的合成方法進行了研究和優(yōu)化。所有化合物的結(jié)構(gòu)經(jīng)1H-NMR、ESI-MS和HRMS等確證。 采用細(xì)胞趨化抑制實驗和非標(biāo)記全細(xì)胞檢測實驗對目標(biāo)化合物進行了活性評價，并對優(yōu)選化合物進行了CCR4受體選擇性實驗研究。采用MTT法評價了目標(biāo)化合物的細(xì)胞毒作用。細(xì)胞趨化抑制實驗結(jié)果表明，大部分目標(biāo)化合物在1μM濃度下對TARC和MDC介導(dǎo)的CCR4轉(zhuǎn)染的L1.2細(xì)胞的趨化運動都表現(xiàn)出了不同程度的趨化抑制作用，其中部分化合物表現(xiàn)出了高于陽性化合物的抑制活性，且化合物對二者的抑制作用效果基本相同。對活性較好的優(yōu)選目標(biāo)化合物測試了IC50值，發(fā)現(xiàn)化合物N-14、N-36和N-39表現(xiàn)出了與陽性化合物1相近的抑制活性，IC50值為別為64nM、69nM、77nM和78nM。三個化合物還表現(xiàn)出了對CCR4拮抗作用的選擇性。非標(biāo)記全細(xì)胞檢測實驗結(jié)果表明，化合物N-39與陽性化合物BMS-397具有相當(dāng)?shù)囊种苹钚�，IC50值分別為1.913μM和1.062μM。MTT實驗結(jié)果表明化合物（N-51除外）在10μM濃度下對細(xì)胞無毒性�；衔颪-39表現(xiàn)出了高活性和安全性以及較好的受體選擇性，值得進一步的研究。 根據(jù)活性評價結(jié)果，本文對目標(biāo)化合物進行了初步的構(gòu)效關(guān)系分析：嘧啶母核2位哌嗪基連接的羰基是保持活性的必需基團。與羰基相連的取代基以位含氨基的飽和取代基活性高，取代基不含雜原子或為芳香取代基活性明顯下降。嘧啶母核4位的取代基團對活性影響較大，2,4-鹵素雙取代的芐氨基有利于活性的保持，，單取代的活性明顯下降。嘧啶母核6位取代基的空間結(jié)構(gòu)和極性大小對活性有很大的影響，空間結(jié)構(gòu)和極性都很小的氯或甲基取代活性最好，其中取代基為甲基時，化合物對TARC和MDC介導(dǎo)的細(xì)胞趨化運動都有穩(wěn)定的抑制活性。這對進一步開發(fā)高效、低毒、選擇性強的CCR4拮抗劑具有一定的指導(dǎo)意義。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R914

【參考文獻】

相關(guān)期刊論文 前8條

1 楊娉婷,沈輝,趙麗娟,魯靜,肖衛(wèi)國,姜紅;趨化因子受體CCR4在活動期類風(fēng)濕關(guān)節(jié)炎患者外周血CD4~+T細(xì)胞表達(dá)的研究[J];中華風(fēng)濕病學(xué)雜志;2005年07期

2 鞏宏偉;綦輝;孫薇;姜丹;肖軍海;楊曉虹;王應(yīng);李松;;哌嗪嘧啶類CCR4拮抗劑的設(shè)計、合成及生物活性[J];高等學(xué)�；瘜W(xué)學(xué)報;2013年09期

3 胡艾希,陳平,楊鄭,伍小云,陳科;溴化銅對芳基烷基酮的選擇性溴化反應(yīng)研究[J];中國藥物化學(xué)雜志;2002年06期

4 趙芳;肖軍海;王應(yīng);李松;;芳酰胺基噻唑類衍生物的合成及其CCR4趨化抑制活性研究[J];中國藥物化學(xué)雜志;2009年01期

5 高源;趙臨襄;;Plerixafor(Mozobil)[J];中國藥物化學(xué)雜志;2009年04期

6 王應(yīng);馬大龍;;新細(xì)胞因子趨化素樣因子(CKLF1)的發(fā)現(xiàn)與新藥開發(fā)[J];中國藥學(xué)雜志;2011年04期

7 吉海杰;胡金鳳;陳乃宏;;以趨化因子受體為靶點的抗哮喘小分子藥物研究進展[J];藥學(xué)學(xué)報;2011年11期

8 陳蘭妹;胡春;;Maraviroc(Selzentry)[J];中國藥物化學(xué)雜志;2008年01期

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