CRM1抑制剂的设计、合成及抗肿瘤活性评价

发布时间：2018-11-24 18:46

【摘要】：CRM1(Chromosome region maintenance 1),也叫XPO1(Exportin 1),它是一种真核蛋白,调控核蛋白,r RNA,sn RNA和m RNA等蛋白和RNA出入细胞核。CRM1能够转运大部分肿瘤抑制蛋白和原癌蛋白,其表达异常与肿瘤的发生发展以及耐药性等密切相关。因此,CRM1已经成为了肿瘤药物研究的靶点之一。目前已经报道的大多数CRM1抑制剂都具有α,β不饱和δ内酯环,包括天然来源的哥纳香甲素、Leptomycin B等化合物。本课题对α,β不饱和δ内酯环的初步研究发现,内酯环不饱和双键的取代会使目标化合物的抗肿瘤活性消失,而不同的侧链对活性影响较大。因此,在本课题组前期研究的基础上,以发现的先导化合物对照2和哥纳香甲素的基本结构为骨架,开展先导化合物的结构优化和构效关系研究,发现新型的CRM1抑制剂。具体研究工作如下:一、6-氧/氮/硫甲基-5,6-二氢-吡喃-2-酮的合成及活性研究以对照2为先导化合物,保留不饱和内酯环,对侧链进行修饰,在6位引入不同的杂原子(氧、氮、硫),通过取代、开环、酯化、氧化、关环等反应合成14个氧甲基化合物,4个氮甲基化合物,1个硫甲基化合物,1个亚砜甲基化合物,并评价了它们的抗肿瘤活性,它们的IC50值都大于或等于2.31μM。苯环上存在吸电子基团的氧甲基化合物、氮甲基化合物、硫甲基化合物抗肿瘤活性都减弱或者消失,而其它供电子基团的氧甲基化合物、亚砜基化合物都有良好的抗肿瘤活性。说明苯环上的吸电子基团和6-杂原子对化合物的抗肿瘤活性影响很大。二、6-氮乙基-5,6-二氢-吡喃-2-酮的合成及活性研究基于有较好抗肿瘤活性的6-亚砜基化合物,通过对氨基进行酰化,研究酰胺类化合物对生物活性的影响。利用酰化、成环、硫代等反应得到17个6-氮乙基化合物,并评价其抗肿瘤活性,发现它们大部分化合物抗肿瘤活性消失,说明侧链的酰胺键对抗肿瘤活性影响可能很大。三、候选化合物对Hela细胞周期、凋亡和CRM1蛋白表达量的影响CRM1在Hela细胞中高表达,因此,基于前期研究结果,选择活性最好的化合物I11,评价其对Hela细胞凋亡率和周期分布的影响。结果表明I11在20μM浓度时可以将Hela细胞阻滞在G1期,诱导22.7%细胞凋亡。并且通过免疫蛋白印迹技术,发现化合物I11能够明显的降低CRM1蛋白的表达量。综上,基于哥纳香甲素的基本骨架,对前期发现潜在的CRM1抑制剂,进行了结构优化和构效关系研究,共合成目标化合物37个,结构经过1H NMR,13C NMR和HRMS确证,均为未见文献报道的新化合物。其中化合物I11对MGC-803、Hela、H1299的IC50值分别达到了2μM、4μM、6μM。化合物I8对MGC-803、Hela的IC50值都达到了8μM。化合物I11能够促进Hela细胞凋亡和阻滞细胞周期停滞在G1期。并且发现化合物I11能够明显的降低CRM1蛋白的表达量。上述研究结果,为深入研究新型的CRM1抑制剂奠定了良好的基础。
[Abstract]:CRM1 (Chromosome region maintenance 1, also known as XPO1 (Exportin 1), is a eukaryotic protein that regulates nuclear proteins, such as r RNA,sn RNA and m RNA, and RNA access nuclei. CRM1 can transport most tumor suppressor proteins and proto-oncoproteins. Abnormal expression is closely related to tumor development and drug resistance. Therefore, CRM1 has become one of the targets of cancer drug research. At present, most of the CRM1 inhibitors reported have 伪, 尾 unsaturated 未 lactone ring, including natural sources of gonadin, Leptomycin B and other compounds. The preliminary study of 伪, 尾 unsaturated 未 lactone rings shows that the substitution of unsaturated double bonds of lactone ring will make the antitumor activity of the target compounds disappear, but different side chains have great influence on the activity. Therefore, on the basis of the previous studies in our group, the structure optimization and structure-activity relationship of the leading compounds were studied based on the basic structure of the pilot compound control 2 and gonadin, and a new type of CRM1 inhibitor was found. The specific research works are as follows: 1. Synthesis and activity of 6-oxy / nitrogen / thiomethyl-6-dihydro-pyran-2-ketone. The unsaturated lactone ring was retained and the opposite chain was modified with control 2 as the lead compound. By introducing different heteratomic atoms (oxygen, nitrogen, sulfur) at 6 sites, 14 oxomethyl compounds, 4 nitromethyl compounds, 1 thiomethyl compound, 1 sulfoxide methyl compound were synthesized by substitution, ring opening, esterification, oxidation, ring closing and so on. Their antitumor activities were evaluated, and their IC50 values were higher than or equal to 2.31 渭 M. The antitumor activity of oxomethyl compounds, azomethyl compounds and thiomethyl compounds in benzene ring were all decreased or disappeared, while the other oxygen methyl compounds, sulfoxide group, which supplied electron groups, all had good antitumor activity. The results show that the electron-absorbing groups and 6-heteroatoms on the benzene ring have great influence on the anti-tumor activity of the compounds. Synthesis and activity of 2,6-azoethyl-5-dihydro-6-dihydro-pyran-2-one based on 6- sulfoxide compounds with good antitumor activity, the effects of amides on biological activity were studied by acylation of amino groups. Seventeen 6-azoethyl compounds were obtained by acylation, cyclization and thioacetylation, and their antitumor activities were evaluated. It was found that the antitumor activity of most of the compounds disappeared, indicating that the side chain amide-bond might have a great effect on the anti-tumor activity. Third, the effect of candidate compounds on the cell cycle, apoptosis and CRM1 protein expression of Hela cells. Therefore, based on the results of previous studies, the most active compound I11 was selected. To evaluate its effect on apoptosis rate and cell cycle distribution of Hela cells. The results showed that I11 could block Hela cells in G1 phase at 20 渭 M concentration and induce 22. 7% apoptosis. Western blot analysis showed that compound I _ 11 could significantly reduce the expression of CRM1 protein. In conclusion, based on the basic skeleton of gonadin, the potential CRM1 inhibitors were studied. 37 target compounds were synthesized, and their structures were confirmed by 1H NMR,13C NMR and HRMS. They are new compounds which have not been reported in the literature. The IC50 values of compound I11 to MGC-803,Hela,H1299 reached 2 渭 MU 4 渭 MU 6 渭 M. respectively. The IC50 values of compound I8 to MGC-803,Hela were all up to 8 渭 M. Compound I 11 can promote Hela cell apoptosis and arrest cell cycle arrest in G 1 phase. It was also found that compound I 11 could significantly reduce the expression of CRM1 protein. These results lay a good foundation for the further study of new CRM1 inhibitors.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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