【作者】 尚鲁庆；

【导师】 徐文方； 岸岗史郎；

【作者基本信息】 山东大学 ， 药物化学， 2009， 博士

【摘要】 研究目的氨肽酶N（Aminopeptidase N,APN）,亦被称为CD13,是一类锌离子依赖性金属蛋白酶,广泛存在于小肠、肾及中枢神经系统的多种细胞表面。相比于正常细胞,该酶在肿瘤细胞表面高水平表达。研究证明氨肽酶N在肿瘤生长、侵袭和转移过程中发挥着重要作用:（1）降解细胞外基质,促进原发肿瘤的生长侵袭,有利于肿瘤的转移;（2）氨肽酶N是肿瘤新生血管的调节器,可以促进肿瘤新生血管的形成;（3）降解胸腺肽和白介素,降低机体免疫机能。世界卫生组织曾指出70%的晚期癌症患者出现明显的疼痛,癌症引起的疼痛亦是临床治疗癌症的一个难题,往往使用的是阿片类药物。研究报道生物体内具有阿片样物质,脑啡肽即是其中一个。但脑啡肽易被体内的氨肽酶N所降解,令其作为镇痛药具有局限性。正因为氨肽酶N与肿瘤的密切关系,抗氨肽酶N药物的研究是抗肿瘤研究领域的一个热点。目前的研究主要包括氨肽酶N单抗,氨肽酶N配体药物和氨肽酶N抑制剂三方面。其中氨肽酶N单抗仅仅只是用于基础研究;氨肽酶N配体药物研究已有报道APN的特异性受体NGR（asparagine-glycine-arginine）与肿瘤药物偶联后取得令人满意的治疗效果。抗氨肽酶N药物研究较成熟的方面是氨肽酶N抑制剂的开发,Bestatin作为第一个上市的氨肽酶N抑制剂,临床上已经用于治疗急性成年人非淋巴性白血病。近年又发现许多氨肽酶N天然抑制剂,如Probestatin,Amastatin,Curcumin等;另外,人们还合成了许多小分子化合物氨肽酶N抑制剂,如α-氨基磷酸抑制剂,β-氨基硫醇类抑制剂等。本课题组以氨肽酶N为靶点,经十几年的研究亦报道了许多小分子类肽化合物。本研究以氨肽酶N为靶点在充分调研文献的基础上,基于E.Coli氨肽酶N（eAPN）的三维晶体结构及bestatin与eAPN作用模式,通过计算机辅助合理药物设计,合成一系列小分子类肽化合物并对它们进行初步的活性筛选,以期找到具有良好的氨肽酶N抑制活性的先导化合物。实验方法目前为止人氨肽酶N的晶体结构尚未报道,2006年大肠杆菌氨肽酶N的晶体结构以及其与bestatin复合物的晶体结构陆续被报道。eAPN的晶体结构表明氨肽酶N活性区域至少包含有一个阴离子结合位点和三个疏水性口袋,即:位于锌离子左侧的S₁和右侧的S₁’和S₂’口袋。本研究以bestatin作为先导物,充分利用计算机辅助药物设计软件的优势,构建了一系列小分子化合物库,并对所建立的小分子化合物库进行虚拟筛选,寻找到与eAPN活性中心区域匹配良好的1,2-二氨基-3-苯丙烷骨架。利用计算机软件Sybyl将该骨架和bestatin的母核AHPA进行比对,发现二者与eAPN的活性中心作用模式相似,并且二者的溶剂可及化表面也极为相似。在构建的母核1,2-二氨基-3-苯丙烷结构基础上,然后将不同的结构片段与之拼接后再利用柔性对接模拟目标化合物与靶酶的作用模式,依此对1,2-二氨基-3-苯丙烷骨架的侧链进行结构修饰,合理设计目标化合物。本研究以D-苯丙氨酸作为起始原料,经甲酯化、甲磺酰化、叠氮化及还原反应得到关键中间体（2R）-2-叔丁氧羰酰胺基-3-苯基-丙胺,再利用经典的二肽缩合反应,将不同的结构片段与之相拼接得到一系列二肽类化合物。第二系列类三肽化合物是在第一系列类二肽化合物基础上进行结构优化而来,将已制备的不同的二肽与（2R）-2-叔丁氧羰酰胺基-3-苯基-丙胺进行缩合反应,再将保护基团脱去得到一系列类三肽目标化合物。此外,本研究将目标化合物进行了体外抑酶实验、体外HL-60细胞生长测试实验,筛选有氨肽酶N抑制活性的化合物。实验结果本文共设计并合成了79个目标化合物,并对所有化合物通过红外光谱、核磁共振氢谱、电喷雾质谱等方法进行了结构确证。经查阅文献证实,所合成的目标化合物为新型化合物,未见文献报道。本研究第一系列合成了38个以1,2-二氨基-3-苯丙烷为骨架结构的类二肽化合物,体外抑酶实验显示大部分化合物对氨肽酶N具有一定的抑制活性,化合物11j,11k,11p,21显示了良好的酶活性,但抑酶效果和对照物bestatin有一定的差距。分析化合物11j,11k,11p结构特征发现,化合物末端的极性基团（如羟基,巯基,氨基等）可以提高化合物的氨肽酶N抑制活性;另外,化合物21作为本系列中的一个类三肽化合物,显示了不错的抑制活性,推测与其结构有关。在第一系列化合物活性结果基础上,我们以第一系列中11p和21作为先导物,对其进行结构修饰和优化,设计并合成了41个类三肽化合物,体外抑酶实验显示本系列相比于第一系列化合物,活性有明显提高。从结构上来讲,第二系列类三肽化合物与氨肽酶N的活性中心的作用优于类二肽化合物,骨架的苯环可以伸入活性中心的S₁疏水性口袋,而两个侧链R和R₁分别伸向S₁’和S₂’口袋。其中化合物32d,44a,44d,44h的IC₅₀在10μM以下,接近于对照物Bestatin(IC₅₀:3.1±0.6μM)的氨肽酶N抑制活性。体外细胞实验通过MTT法测定了目标化合物对白血病细胞HL-60的生长抑制作用,结果显示对APN酶抑制作用强的化合物11p,21,32d,44a,44d和44h同样有很强的HL-60细胞抑制活性,化合物32d和44h的IC₅₀分别为0.29±0.06mM和0.27±0.08mM,超过了bestatin。此外,基于氨肽酶N在脑啡肽降解过程中发挥着关键作用,本文建立了两套氨肽酶N抑制剂筛选体系用来更好的考察新型氨肽酶N抑制剂的活性。无论是豚鼠回肠平滑肌电针刺激实验体系,还是HPLC检测二肽Tyr-Gly在豚鼠脑蛋白中降解的实验体系,都可以很好地用于评价氨肽酶N新型抑制剂的活性。最后,本文在1,2-二氨基-3-苯丙烷类氨肽酶N抑制剂活性结果基础上,以所合成的化合物为对象,利用计算机软件进行了初步的定量构效关系研究。分子对接研究通过化合物44h的分子对接模式图解析了本课题中氨肽酶N抑制剂与氨肽酶N活性中心的作用位点及作用模式。化合物44h的苯环,赖氨酸侧链及巯基侧链分别占据氨肽酶N活性区域的三个疏水性口袋S₁,S₁’和S₂’,抑制剂与氨肽酶N活性中心区域匹配良好。采用比较分子场分析方法（CoMFA）建立了1,2-二氨基-3-苯丙烷类氨肽酶N抑制剂的定量构效关系（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,建立的CoMFA模型具有较高的交叉验证系数q²和一定的预测能力。研究结论本研究基于氨肽酶N的晶体结构及抑制剂与酶的作用模式,利用计算机软件进行设计并合成的1,2-二氨基-3-苯丙烷类化合物具有很好的氨肽酶N抑制活性。所设计的合成路线科学合理,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化合物。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,为今后新型氨肽酶抑制剂的研究提供了帮助。更多还原

【Abstract】 ObjectiveAminopeptidase N（APN）,as known as CD13,is a member of zinc dependent metalloproteinase.It is widely expressed on the surface of renal and intestinal brush border cells,synaptic membranes in central systems and APN is over-expressed on the surface of tumor cells.APN is demonstrated to play a key role in the process of tumor growth,invasion and metastasis.The functional aspects are briefly summarized:（1）. Degrading extracellular matrix,promoting the growth and invasion of tumor cells;（2）. As a regulator of novel vessels,promoting the angiogenesis of tumor tissues;（3）. Degrading immunoactive substance and bioactive peptides,such as interleukin, thymopentin,enkephalin and so on.Additionally,World Health Organization（WHO） had reported that 70%terminal cancer patients usually suffer the pain.Synthetic analogues of morphine are applied as analgesics but they have undesirable side effects （e.g.addiction）.Numerous researches have shown that one of the most promising approaches appears to potentiate the antinociceptive effected induced by the endogenous opioid substance enkephalin.However,enkephalin is limited in the clinical therapy for easily degradation by aminopeptidase N. More and more researches focus on the field of anti-aminopeptidase N drug as that enzyme has the close relationship with the cancer.The research mainly contained APN mono-antibody,APN ligand binding drug and aminopeptidase N inhibitor.APN mono-antibody is only for research at this time and some reports have showed that APN ligand NGR（asparagine-glycine-arginine） as the receptor of APN linked with anticancer drug can get well therapeutic effect.Most of the research on the anti-aminopeptidase N drug is to find aminopeptidase N inhibitor possessed inhibitory activity.Bestatin,the marketed drug as aminopeptidase N inhibitor,now is clinically used to prolong the survival of patients with acute adult nonlymphcytic leukemia.In recent years,many natural aminopeptidase N inhibitors have been reported,such as Probestatin,Amastatin and Curcumin.Moreover,numerous synthetic APN inhibitors have also been reported,such asα-aminophosphonates APN inhibitors,β-amino-thiols APN inhibitors.In our group,we also had reported numerous synthetic small molecular compounds targeted with aminopeptidase N in the past ten years.Based on the three dimensional crystal structure of E.Coli APN and enzyme-bestatin complex,this study used computer-aided drug design software to design and synthesize a series of compounds.Then we hope to find lead compounds which possess potential aminopeptidase N inhibitory activity.MethodsSo far the three dimensional structure of human APN has not been found and the crystal structure of E.Coli aminopeptidase N was reported in 2006.As the structure of lead compound bestatin,we build a virtual molecule library with the advantages of computer-aided drug design software.The following design strategy is screening the virtual molecule library to obtain the pharmacophore 3-phenylpropane-1,2-diamine, which could satisfy the requirement of the active site of E.Coli APN.We compared the mode of the pharmacophore and AHPA with the active site of APN by the Sybyl 7.0.The result showed that the electrostatic potential surface of 3-phenylpropane-1, 2-diamine and AHPA is very similar.Based on the pharmacophore 3-phenylpropane-1, 2-diamine,various fragments are introduced with the pharmacophore by the FlexX docking aided and virtual screened.As a result,the target compounds are design rationally and synthesized easily.In this research,the D-phenylalanine was used as the starting material.The phenylalanine was suffered esterification,sulfonylation,nitridation and reduction to get the key intermediate（2R）-2-[（tert-butoxycarbonyl）amino]-3-phenyl-propylamine. Then various fragments were coupled with the intermediate to generate a series of dipeptidemimatics.The compounds of seriesⅡare designed based on the optimization of the seriesⅠ.The intermediate was coupled with different prepared dipeptide to generate various analogues of tripeptide.In addition,enzyme assay and HL-60 cell proliferation assay were processed in this research.ResultsIn this research,we obtained 79 target compounds and all of them are novel without any report by now with the structures identified by IR,¹H-NMR and ESI-MS.In series I,we designed and synthesized 38 analogous of dipeptide with the scaffold 3-phenylpropane-1,2-diamine.The enzyme assay showed that most compounds exhibited APN inhibitory activities and the most potential compounds are compound 11p and 21 and compound 11j,11k also exhibited well activity against APN.From the structure of compound 11j,11k and 11p,we know the polar group（e.g.-OH,-SH, -NH₂） of the compound can increase the activity of the compound.In addition, compound 21 is an analogue of tripeptide and shows the best APN inhibitory activity, maybe owing to the structure difference of the compound 21.Base on the results of the seriesⅠ,we optimized the structure of compounds with the compound 11p and 21 as lead compound.In this series,we obtained 41 target compounds and preliminary result showed that most of target compounds displayed better APN inhibitory activities than the seriesⅠ.The compounds in seriesⅡstructurally matched the active of the enzyme are better than these of compound in seriesⅠ.When the compounds in seriesⅡwere matched with the enzyme,the phenyl group of the pharmacophore could inserted to the S₁ pocket,and two side chains R,R₁ inserted to the S₁’ and S₂’, respectively.The IC₅₀ of compound 32d,44a,44d,44h are less than 10μM,which are similar to that of bestatin.The results of in vitro growth inhibition against HL-60 cells indicated that most potent APN inhibitor displayed good inhibitory effect against the growth of HL-60 cells.Compound 11p,21,32d,44a,44d and 44h,which showed well enzyme inhibitory activities against APN,also exhibited good potency against the proliferation of HL-60 cell.Moreover,compound 32d,44h exhibited the inhibitory effect of cell proliferation with IC₅₀ values of 0.29±0.06 mM and 0.27±0.08 mM,respectively, which showed better potency than that of bestatin.Additionally,based on the key role of APN in the process of degradation of enkephalin,we established two useful systems for the in vitro evaluation of the novel APN inhibitor.Potentiating effect of APN inhibitors on the twitch inhibition of enkephalin in guinea pig ileum experiment and effects of APN inhibitors on the hydrolysis of Tyr-Gly during the incubation with the striatal membrane of guinea pig experiment,both of them could be used to evaluate the activity of novel APN inhibitor.Finally,the structure activity relationship（SAR） was showed based on the structure and the activity data of the target compounds with the scaffold 3-phenylpropane-1,2-diamine.In order to investigate the interaction of our target compounds with APN, flexible docking of the compound 44h was built and docked into the active site of APN（PDB code:2DQM） using Sybyl7.0.The binding studies showed the phenyl group inserted to S₁ pocket,the thiol group orientated to S₁’ pocket and the lysine side chain plunged into pocket S₂’ Comparative Molecular Field Analysis（CoFAR） was utilized to establish the quantitative structure activity relationship（QSAR） of the target compounds.The steric contour map and the electrostatic contour map of the CoMFA model showed the model had good cross-validated coefficient q² and predictive potency.ConclusionsIn conclusion,based on the crystal structure of eAPN and the mode of bestatin with the enzyme,we utilize the computer-aided drug design software to design and synthesize the target compounds with the scaffold 3-phenylpropane-1,2-diamine.We reported a convenient and economical method of the synthesis of APN inhibitors. Preliminary activity assays showed most compounds possess APN inhibitory activity and the IC₅₀ of compound 32d,44a,44d,44h is less than 10μM which can be used in the further research.We also established a QASR model of target compounds which is beneficial for the design APN inhibitor in the future.更多还原