【作者】 杨康辉；

【导师】 徐文方；

【作者基本信息】 山东大学 ， 药物化学， 2010， 博士

【摘要】 研究背景：氨肽酶N(APN/CD13; EC 3.4.11.2),是一类锌离子依赖性金属蛋白酶,其广泛存在于人的多种组织中,在不同细胞(上皮细胞、内皮细胞、成纤维细胞和白细胞等)表面均有表达。相比于正常细胞,该酶在肿瘤细胞表面高水平表达,其在恶性肿瘤生长、侵袭、转移以及新血管的形成过程中扮演重要角色,成为研究抗癌药物的一个重要靶点。目前,已有报道的天然抑制剂如：Bestatin, Probestatin, Amastatin, Curcumin等在预防和治疗肿瘤方面取得一定的积极效果。Bestatin作为第一个上市的APN抑制剂,临床上已经用于治疗急性成年人非淋巴性白血病。另外,人们还合成了许多小分子APN抑制剂,如α-氨基磷酸类抑制剂,β-氨基硫醇类抑制剂,以及本课题组报道的环酰亚胺类抑制剂,L-赖氨酸衍生物抑制剂等。本研究以APN为靶点,设计合成了一系列小分子类肽化合物,并对其进行初步的生物活性筛选,以期发现具有良好APN抑制活性的抗癌先导化合物。研究方法：在本实验室前期研究工作中发现的活性先导物1,2-二氨基-3-苯丙烷结构基础上,结合文献调研,解析APN的三维晶体结构及其与抑制剂的作用模式和特点,选择了与该活性结构相类似的氯霉胺为基本骨架,并在此基础上选择能与酶活性中心的疏水性口袋相互作用的不同侧链与之拼合进行衍生化。结构修饰主要考虑以下三个因素：1)引入不同体积和长度的疏水侧链,考察立体效应对酶的作用。2)侧链中选择不同的吸电子基和斥电子基,考察不同电性效应对酶的作用。3)侧链中选择可能与酶相互作用的氢键基团,以增强化合物与酶的亲和力。本研究综合利用计算机辅助药物设计软件的优势,对设计的化合物与酶进行柔性对接打分,选择分值较高的化合物进行化学合成。同时,对其分子量、脂水分配系数、氢键受体和氢键供体数进行了预测,采用Lipinski规则进一步筛选,最终设计了三个系列115个全新结构的目标化合物。目标化合物的合成以氯霉胺为起始原料,采用了氨基保护,选择性氧化,溴代,叠氮化,还原,缩合等多个反应步骤完成,采用红外光谱、核磁共振氢谱、电喷雾质谱、X-ray单晶衍射等方法确证其化学结构。生物活性研究选用体外抑酶(APN, MMP-2)活性试验、肿瘤细胞(HL-60,ES-2,K549等)生长抑制试验和小鼠体内抗肿瘤转移试验对目标化合物进行了初步活性评价,从中筛选出具有较好活性的氯霉胺类APN抑制剂作为抗癌先导化合物。研究结果：本研究最终合成了115个目标化合物,其中大部分化合物产率较高,纯度良好,所有化合物进行了化学结构确证。经文献检索,所合成的目标化合物为新型结构,未见相关报道。本研究合成的78个A系列目标化合物中,大部分化合物对APN具有抑制活性,其中化合物9e(9e’),9m(9m’),9r(9r’)和9s(9s’)活性较好。B系列化合物相比于A系列化合物,整体上活性有明显提高。其中,化合物12w,12x和12y的IC5035μM以下,接近于阳性对照药Bestatin的APN抑制活性。然而,B系列化合物同样也显示出较强的MMP-2抑制活性。在C系列中,化合物16k,161与阳性对照药Bestatin的活性相当,且对MMP-2的抑制活性较低,显示了良好的抑酶选择性。本研究在对上述目标化合物进行抑酶活性试验的基础上,利用计算机软件进行了初步定量构效关系研究。采用比较分子力场分析方法(CoMFA)建立了具有较高的交叉验证系数q2和一定预测能力的定量构效关系(QSAR)模型。通过对CoMFA模型立体场等势线图和静电场等势线图的分析,提出了化合物下一轮结构优化的方案,为氯霉胺类APN抑制剂的进一步研究奠定了基础。体外细胞抑瘤实验结果表明,多数化合物对高表达APN的肿瘤细胞显示出良好的抑制效果。其中,化合物161、12w表现出与阳性对照药相当的肿瘤细胞抑制活性。体内小鼠抗肿瘤转移试验结果证明,化合物12w和161均有一定的抑制荷肝癌H22小鼠肿瘤细胞转移的作用。其中,化合物12w的抑制率为48.51%,小于阳性对照药Bestatin(抑制率为58.04%),161的抑制率达到了56.49%,接近阳性对照药Bestatin。研究结论：本研究基于APN的晶体结构及抑制剂与酶的作用模式,借助计算机软件进行合理药物设计并合成的氯霉胺类化合物具有较好的APN抑制活性。所设计的合成路线科学合理,起始原料经济易得。通过初步的活性测试发现了具有进一步研究价值的活性化合物。其中,化合物12w,161可以作为抗癌先导化合物用于下一轮的结构优化。同时,基于化合物结构和活性数据建立了有一定预测能力的定量构效关系模型,为今后新型APN抑制剂的研究奠定了基础。更多还原

【Abstract】 Objective:Aminopeptidase N (APN/CD13; EC 3.4.11.2), a zinc-dependent membrane metalloproteinase, is present in a wide variety of human tissues and the surface of cell types (endothelial, epithelial, fibroblast, leukocyte). Compared with normal cells, APN is over-expressed on the surface of some tumor cells. It is demonstrated that this enzyme plays a crucial role in the process of tumor growth, invasion, metastasis and promoting the angiogenesis. Therefore, APN is considered as an important target in anti-cancer drug discovery and development.So far, many natural inhibitors such as Bestatin, Phebestin, Probestatin, Amastatin, Curcumin have been reported and proved to be clinically efficacious for the treatment of tumor. Bestatin, the first marketed drug as natural aminopeptidase N inhibitor in Japan in 1987, now is clinically used to prolong the survival of patients with acute adult nonlymphcytic leukemia. In recent years, numerous synthetic APN inhibitors(APNI) have been reported, such asα-aminophosphonates APNI, a-aminoboronic acid APNI,β-thiols APNI,2,6-piperidinedione-N-acetamide derivatives APNI, L-lysine derivatives APNI, AHPA (β-Amino-α-Hydroxyl-phenylbutanoic acid) derivatives APNI and so on.In this study, we designe and synthesize a series of small molecule peptido-mimetics based on the APN as target. Their preliminary biological activity screening are subsequently conducted in order to find out effective APN inhibitors as anti-cancer lead compounds.Methods:According to the binding characteristics learned from the three dimensional crystal structure of APN and the mode of action of APN-inhibitor complex reported in literatures, we choose the chloramphenicol amine as the novel scaffold based on the active structure of 3-phenylpropane-1,2-diamine scaffold which was previous reported by our group. To improve the interaction with the hydrophobic pockets in the active site of APN, several fragments were introduced to the scaffold. This scaffold is optimized with following chemical modification:i) The different space volume and length of side chains were chosen to investigate the interaction of stereoscopic effect with APN. ii) Various electron-attracting groups and electron-pushing groups were introduced so as to investigate the interaction of electric effect with APN. iii) The groups which can form hydrogen bond with the enzyme were taken to enhance affinity. All the target compounds were rationally designed by CADD, FlexX docking and virtual screening. Meantime, the properties of all these compounds like molecular weight, lipid-water partition, the number of hydrogen bond receptor and hydrogen bond donor were predicted and screened by Lipinski rules before synthesis.115 novel target compounds were designed and synthesized by using chloramphenicol amine as starting material, through amino protection, selective oxidation, bromination, azide, reduction, condensation and other reaction steps. The structure were confirmed by IR,1H NMR, ESI-MS, X-ray crystal diffraction methods.In addition, enzyme assay (APN, MMP-2), cancer cell (HL-60, ES-2, K562, A549, H7402,2PLC) proliferation assay and the mice experiment in vivo were processed in this research.Results:In this study 115 target compounds were synthesized and most of the compounds were obtained in high yield and good purity. All of the compounds were novel without any relevant reports in literature.In series A, the preliminary activity evaluation against APN showed that some compounds possessed moderate inhibitory activities. Among 78 target compounds, 9e(9e’),9m(9m’),9r(9r’) and 9s(9s’) were the most potential. Compared with series A,25 compounds in series B exhibited better inhibitory activities. The IC50 of compounds 12w,12x and 12y were below 35μM, which were close to that of the positive control Bestatin. However, the high inhibition of MMP-2 was also observed. In series C, compound 161 was found to be the most active. The inhibitory activity was similar to that of Bestatin. Generally speaking, compounds in series C showed low activities towards MMP-2. It is worth mentioning that compounds 16k and 161 not only had a high inhibition against APN, but also represented low inhibition towards MMP-2, which suggested that both the compound have good selectivity against enzymes.The structure activity relationship (SAR) was summarized based on the test results of the target compounds with the scaffold chloramphenicol amine in the previous enzyme assay. Comparative Molecular Field Analysis (CoFAR) was utilized to establish the QASR model of target compounds, which showed good cross-validated coefficient q2 and predictive potency. According to the the steric contour map and the electrostatic contour map of the CoMFA model, a program on the further structural optimization was proposed.The test results of growth inhibition against six tumor cells in vitro indicated that most potent APN inhibitors displayed good inhibitory effect against the growth of these tumor cells. Compound 161 exhibited very similar potency against the proliferation of tumor cells compared with that of Bestatin. Compound 12w showed better inhibitory effect towards all the tumor cells than Bestatin.In vivo experiments, compound 12w and 161 showed high potency against tumor cell H22 metastasis with the inhibition rates 48.51% and 58.04% respectively in mice. While the inhibition rate for the positive control Bestatin was 56.49%.Conclusions:In conclusion, based on CADD and the virtual screening with APN as target chloramphenicol amine derivatives were designed and synthesized as APN inhibitors. We reported a convenient and economical method of the synthesis of APN inhibitors. Preliminary activity assays showed that most compounds displayed good APN inhibitory effects. Some compound such as 12w,161 showed better activities than the positive control Bestatin and could be used as lead compounds in the future. We also established a QASR model of target compounds, which is beneficial for the design of novel APN inhibitor in the further study.更多还原