【作者】 陈颖；

【导师】 黄惠明； 谢宝刚；

【作者基本信息】 南昌大学 ， 药物分析， 2012， 硕士

【摘要】 氨肽酶N (Aminopeptidase N, APN),又名CD13,是一类膜型锌离子依赖型的外肽酶,广泛分布于人体各个组织,其中在小肠、肾、肝以及脑组织中含量较高。研究表明,APN能降解细胞外基质(extracellular matrixc,ECM),参与肿瘤细胞对正常细胞基底膜和细胞间质、基质的侵入以及对血管壁的穿透,最终促进肿瘤细胞的侵袭和远行转移,因此APN在肿瘤的侵袭转移机制中发挥着重要的作用。此外APN作为肿瘤新血管的调节器能够促进肿瘤新血管的生成。研究证实,APN还是传染性胃肠炎病毒(TGEV)、呼吸道感染的人冠状病毒229E(HCoV-229)和人类巨细胞病毒(HCMV)的受体。鉴于此,抑制APN的活性,能有效抑制肿瘤的增殖和新血管的生成,并能调节机体的免疫系统。APN类抑制剂已成为抗肿瘤抗病毒药物的研发热点。本文在分析总结APN抑制剂(主要是天然产物Bestatin)类型及其活性部位结构特征的基础上,运用计算机辅助药物设计技术(Computer-Aided Drug Design,CADD)分析和优化,设计并合成了一个系列共11个结构全新的琥珀酰胺类APN抑制剂。值得一提的是该系列产物中,苯并噻唑是重要母环。据文献报道,苯并噻唑及其衍生物具有较好的抗肿瘤生物活性,特别是苯并噻唑的2位取代活性更高。所以本文以2位苯并噻唑为母核,经过酰化、水解,再酰化得到目标化合物。利用红外光谱(IR)、核磁共振氢谱(’H-NMR)、电喷雾离子化质谱(ESI-MS)等技术对目标化学结构进行表征。确证均为新型化合物,未见文献报道。本文最后对上述目标化合物进行了初步体外活性研究,在抑制肿瘤细胞K562、ES-2细胞增殖活性实验中,运用MTT法以不同浓度梯度的重复实验得到各目标化合物的抑制率,并通过SPSS-13.0软件计算得到化合物的生物活性值IC50。其中化合物5F(2,3-dihydroxy-N1-(6-methoxybenzo[d]thiazol-2-yl)-N4-(p-tolyl) succinamide)对ES-2细胞的增殖有较好的抑制作用,其IC50值为1.12μM((Bestatin0.98μM);目标化合物对K562的活性抑制作用不明显。另外,本文还对部分目标化合物进行了体外抑制APN活性实验,实验以L亮氨酰-对硝基苯胺作为底物,测定目标化合物对APN的抑制活性。结果表明,目标化合5D(2,3-dihydroxy-N1-(6-methoxybenzo[d]thiazol-2-yl)-N4-(4-methoxyphenyl)succinamide)、5F对APN具有较好的生物活性作用。这两种化合物均在苯并噻唑的2位上有-CHO3取代。结合上述体外实验结果表明,苯并噻唑的2位为-CHO3取代时的抗肿瘤活性最好。综上所述,本文初步设计合成和筛选出一个抗肿瘤活性较好的目标化合物,研究结果可为合成结构新颖、高效低毒的氨肽酶N抑制剂提供参考。更多还原

【Abstract】 Aminopeptidase N (APN), also known as CD13, is a membrane-bound zinc-dependent exopeptidase. It’s widely distributed in various tissues with high levels, such as intestine, kidney, liver and central nervous system. APN plays an important role in tumor progression by regulating processes such as tumor invasion and metastasis. As APN can degrade extracellular matrix (ECM), which would help to tumor cells invading the basement membrane and matrix, penetrating the vessel wall, tumor cells can proliferate and metastasize quickly. In addition, APN can promote tumor angiogenesis as a regulator of new blood vessels of the tumor.It was reported that APN is a viral receptor for human coronavirus229E (HCoV-229E), porcine transmissible gastroenteritis virus (TGEV), human cytomegalovirus (HCMV). Inhibition of APN can effectively decrease the growth of tumor cells and regulate the body’s immune system. Therefore, APN has been implicated as a novel therapeutic target for Anti-virus and cancer treatment.In this paper, based on the summary of structural features of APN inhibitors (Natural product:Bestatin) and three-dimensional structural of APN active site, we designed some novel structural APN inhibitors by computer-aided drug design technology and then11compounds were synthesized.It is worthy of note that the Benzothiazole is key to the parent ring in the series of products. It has been reported that the Benzothiazole and its derivatives have high anti-tumor activity, especially2-benzothiazole. The target compounds were synthesized by the process of cyclization, acidylation, hydrolyzation and acidylation followed by2-benzothiazole. Eleven novel Amber lactam derivatives were synthesized and their structures were confirmed by IR、1H-NMR、ESI-MS.Finally, the inhibitory activities of these compounds to K562and. ES-2cells were measured by MTT assay in vitro. Then the values of IC50for these compounds were obtained. The results suggested that the compound (5f)(2,3-dihydroxy-N1-(6-methoxybenzo[d]thiazol-2-yl)-N4-(p-tolyl) succinamide) had highest activity against ES-2(IC50=1.12μM), which was closed to that of Bestatin (IC50=0.98μM). Whereas, all compounds were shown low activities against K562. Furthermore, inhibitory activities against APN by these compounds were determined using L-leucyl-p-nitroaniline as the substrate. Our results indicated that the compound5D (2,3-dihydroxy-N1-(6-methoxybenzo[d]thiazol-2-yl)-N4-(4-methoxyphenyl) succinamide) and5F had higher inhibitory activities against APN. The structure of two compounds have-OCH3substituent group instead of-H in para-position of2-benzothiazole. Combined with the results of inhibition of cells in vitro, the2-Benzothiazole with-OCH3substituent group in para-position showed highest anti-tumor activity.In summary, a potentially good anti-tumor activity compound were designed, sysnthesized and screened preliminarily in vitro in this paper, which would provide new insight and approach for the synthesis of novel inhibitor of aminopeptidase N.更多还原