【作者】 李乾斌；

【导师】 徐文方；

【作者基本信息】 山东大学 ， 药物化学， 2008， 博士

【摘要】 第一部分研究背景金属蛋白酶是一大类含金属离子的蛋白水解酶。肿瘤细胞生物学研究表明,在金属蛋白酶家族中有两种锌离子依赖性金属蛋白酶:氨肽酶N（Aminopeptidas N,APN/CD13）和基质金属蛋白酶（Matrix Metalloproteinase,MMP）与恶性肿瘤的浸润与转移的发生和发展密切相关。其中APN为锌离子依赖性的金属外肽酶,参与底物N端氨基酸的降解。研究证明,APN在肿瘤发生、机体免疫功能调节以及病毒感染中发挥重要的作用。1、APN在肿瘤细胞表面高水平表达,其在肿瘤细胞的演进过程中的主要作用可能是:（1）降解细胞外基质,促进多种生长因子的释放,加速肿瘤细胞增殖;（2）促进肿瘤细胞侵袭与转移;（3）刺激血管内皮细胞释放肿瘤微血管形成相关因子,促进肿瘤新生血管的形成;（4）增强肿瘤细胞粘附性,而高粘附性的肿瘤细胞又能进一步刺激释放金属蛋白酶,提高降解基底膜的能力。2、APN在粒细胞及淋巴细胞表面大量表达,同时作为细胞表面的分化抗原CD13参与了T淋巴细胞依赖的炎症反应;还能够表达于抗原递呈细胞表面,降解免疫活性物质（如白介素-8）;参与抗原处理和细胞表面的主要组织相容性复合体Ⅱ型（MHC-Ⅱ）粘附抗原决定簇依赖的T细胞对抗原的识别,降低了T细胞的识别能力,同时削弱了巨噬细胞和NK细胞对肿瘤细胞的识别和杀伤能力,使机体免疫力下降。3、APN作为人冠状病毒HCoV-229E和传染性胃肠炎病毒（TGEV）表面的受体,在上呼吸道感染（如:SARS）和急性肠炎中扮演重要角色,且其发挥作用与酶的活性相关。4、APN参与HIV病毒颗粒进入宿主细胞的过程。研究表明,在感染HIV的患者体内APN活性远远高于健康志愿者。在HIV-1入侵宿主细胞时,高表达的APN通过降解能够使HIV-1辅助受体CCR5脱敏的趋化因子fMLP,从而降低细胞的天然免疫功能,并使CCR5增敏,促进病毒进入宿主细胞。5、APN参与内源性镇痛物质内啡肽和脑啡肽的降解,从而引起P物质的过度释放,导致疼痛。6、APN降解血管紧张素,参与机体血压的调节。因此,APN抑制剂有望发展成为一类新型的治疗药物。正因为APN与肿瘤发生发展有着密切关系,以APN为靶点的靶向性抗肿瘤药物研究倍受关注。这方面的研究方向有三:APN单抗和APN配体结合药物研究目前处于起步阶段,小分子APN抑制剂有天然产物和合成抑制剂,研究较为成熟,其中第一个从网状橄榄链霉菌培养液中发现APN抑制剂Bestatin（通用名为Ubenimex）已作为免疫增强剂于1987年在日本上市。近年来又发现了多种和Bestatin结构类似的小分子化合物,如:Probestin,Actinonin,其中Actinonin对APN的抑制活性远高于Bestatin,是一类很有开发应用前景的化合物。第二部分目标化合物的虚拟筛选以及合理设计尽管针对于小分子APN抑制剂的研究比较多,但在2006年之前,由于没有任何关于APN晶体结构的报道,因此研究的焦点主要在于天然产物以及Bestatin类似物的研究,合成抑制剂的研究进展比较缓慢。本研究的特色在于综合利用基于机制（Mechanism-Based）和基于结构（Structure-Based）的药物设计思想进行全新药物设计:基于细胞外基质主要成分为脯氨酸-甘氨酸残基这一推断,构建了一系列小分子杂环化合物库,并充分利用了计算机辅助药物设计软件的优势,对已知活性化合物构建药效团并对所构建的小分子杂环化合物库进行虚拟筛选发现了匹配良好的环酰亚胺骨架。然后借助基于靶点结构的药物设计策略通过深入研究E.Coli APN的三维结构以及酶-抑制剂复合物的结合模式,用柔性对接来模拟目标化合物与靶酶的相互作用,对环酰亚胺骨架的侧链进行合理设计并实现定向合成。本研究所设计的三个系列化合物是逐步优化设计的结果。研究发现,所得的部分量化参数（如FlexX对接结果）与体内外活性试验具有一定相关性,另外,本课题通过深入研究靶点三维结构的差异,通过对侧链进行改造实现了目标化合物对两种金属蛋白酶（APN和MMP）选择性的转变,表明本文设计思想较为合理,对化合物分子设计有指导意义,所构建的各种筛选模型对实验结果具有良好的预测性。此外,本文不仅从整体上考虑化合物结构与酶活性中心的匹配性,而且组成化合物的各结构片段均也选择了具有抗癌活性的片段或组织相容性较好天然氨基酸残基,所设计合成的环酰亚胺类抑制剂在体内经各种酰胺酶、酯酶以及脱甲基化代谢分解后的各级产物能维持较长时间的疗效。另外,在对结构进行优化设计时预先充分考虑到了药物在体内的ADME等动力学特性,将化合物做成盐酸盐,提高化合物的水溶性和生物利用度,从而提高化合物的中标率。第三部分目标化合物的合成在虚拟筛选结合基于靶点结构的合理药物设计的基础上,本着结构多样性的原则,对环酰亚胺的侧链进行结构改造,并通过定向合成得到了94个结构确定的目标化合物。合成方法中充分体现了多肽合成的优势,工艺路线比较新颖,切实可行。本文以两种合成路线对目标化合物进行了合成:1、为了探讨N-乙酸羧基部分的不同取代基对活性的影响,对于3-三甲氧基苯甲酸-2,6-哌啶二酮-N-乙酸系列化合物采用了比较原始的单向合成路线:即先以L-谷氨酸为起始原料,将三甲氧基苯甲酸与其氨基进行缩合,然后再通过脱水环合、甘氨酸胺解得到关键的中间体6,最后充分利用多肽合成优势,以EDCI为缩合剂对羧基进行衍生化得到了一系列没食子酸类三肽化合物。三个甲氧基可以在路易斯酸BBr3存在下脱去甲基保护。2、采用多向合成策略提高合成效率。先合成能够实现结构多样性的中间体,然后再进行衍生化:即先以L-谷胺酰胺或L-天冬酰胺为起始原料经过Boc保护、环合等步骤合成关键的环酰亚胺中间体12和27,再在相转移催化剂TBAI的存在下合成了N-取代的中间体13,18以及28。这些中间体脱去Boc保护基游离出氨基后再以不同的酰氯或混合酸酐进行衍生化。环酰亚胺N-乙酸化合物是由相应的苄酯化合物经催化转移氢化脱去苄酯得到,然后羧基再经混合酸酐方法（与氯甲酸异丁酯形成酸酐）合成异羟肟酸类化合物。对所合成的化合物,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,其纯度经过HPLC分析,大多在96%以上。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。第四部分目标化合物的活性评价及构效关系研究本研究设计合成了3个系列共94个具有环酰亚胺骨架的类肽化合物,并对其活性进行了初步筛选。由于APN和MMP都属于锌离子依赖性金属蛋白酶,并且在肿瘤细胞的侵袭与转移过程中发挥非常重要的作用,体外抑酶筛选时同时针对两种酶进行活性评价。体外细胞测试时采用了特异性表达APN的白血病细胞（HL-60和K562）以及特异性表达MMP-2和MMP-9的人卵巢黏液囊腺上皮癌细胞SKOV3,观察化合物对两种细胞的生长抑制作用以及表达水平的影响。体外抑酶实验显示:SeiresⅠ中的化合物对MMP的选择性都比较高(7j的IC₅₀比值约150),但7I,7o,9a,9b具有较好的APN抑制活性,活性最好的化合物9a的IC₅₀为3.0μM与bestatin的相当(IC₅₀=2.4μM):这说明本研究所引用的构象限制策略确实提高了化合物对靶标的选择性。SeiresⅡ以及SeriesⅢ两个系列中的化合物由于游离氨基的引入使得化合物对APN的活性明显增加,其中SeriesⅡ中的17j,22a,22g以及所有含有异羟肟酸基团的化合物24以及SeriesⅢ中的32a,32c,32f,32i,34对APN分别显示了较好的活性和选择性,IC₅₀比值多数在20左右。其中化合物34d的IC₅₀值为2.9μM,24f和34f的IC₅₀分别为1.8μM和1.0μM,均超过了Bestatin。构效关系分析显示,对应于SeriesⅠ的化合物,P1’是影响选择性的关键基团,而且Tyr的存在有利于酶对MMP的抑制活性,在P1’引入脂肪侧链会增加对APN抑制活性,但却损失了对二者的选择性;对于SeriesⅡ和SeriesⅢ化合物而言,游离氨基的存在是体现选择性的关键基团,而且游离氨基是APN抑制剂的必要条件。对于不同取代基活性趋势为异羟肟酸＞羧基＞乙酯,P1为芳香基团时活性较好,但有酚羟基存在时不利于酶的结合。体外白血病细胞生长抑制实验发现对APN抑酶活性较好的几个化合物均体现出对HL-60细胞较强的抑制作用,其中7g,9a,24f,24i,34f的活性均超过了bestatin。其可能的作用机制是环酰亚胺骨架化合物通过与APN结合然后启动下游分子,从而影响DNA的合成,最终诱导细胞凋亡。细胞实验中还通过MTT法测定了目标化合物对白血病细胞K562细胞的生长抑制作用,96孔板中受试化合物浓度低于或达到饱和浓度时,抑制率均低于25%:二倍级梯度升高化合物的含量至15mg/mL,亦没有明显浓度依赖的抑制作用,虽然可能由于化合物在96孔板中析出,影响了试验结果的线性关系,但通过倒置显微镜观察,细胞形态正常,与空白组相比无明显减少,所以推定所合成的化合物基本没有细胞毒作用。由于K562表达APN的水平远远低于HL-60细胞,而K562细胞对药物的敏感性较差,这可能与APN的表达不同有关,同时也间接地说明了化合物对APN具有一定的靶向性。对MMP抑制活性较好的化合物（7i,7r）的体外人卵巢黏液囊腺上皮癌细胞SKOV3生长抑制实验以及明胶酶谱分析实验证明了化合物对MMP的作用有可能不是直接抑制酶活,而是影响MMP-2以及MMP-9的表达,从而发挥其抗肿瘤作用。环酰亚胺类肽抑制剂体内抑瘤试验采用荷H22瘤小鼠抗转移模型,将受试化合物以50mg/kg/d剂量口服给药6天/周,持续给药两周。结果显示:对APN抑制活性较好的化合物（9a,24f,24i,34f,34i）肿瘤转移的抑制率都在50%以上,其肺组织表面的结节数与空白组形成了鲜明的对比。其中化合物24f和9a的抑制肿瘤转移率高达72.91%和81.56%,且没有观测到明显的毒副作用,有望成为一个具有良好开发前景的先导化合物。其可能的作用机制是:1)化合物直接抑制APN的活性,从而抑制APN对血管基底膜的降解;2) APN的活性被化合物抑制后可以激活或抑制某些细胞因子,这些细胞因子可以调控基因的转录,从而影响MMP分子的表达,进而抑制了基质金属蛋白酶对血管基底膜的降解,抑制肿瘤细胞在肺组织的定位。体内活性测试所选用的5个化合物其体外抑酶活性及细胞测试活性也都很高,表明体内外实验结果能够完全吻合。这也说明了以下几点:1)所构建的药效团模型的筛选能力比较强,能够把活性骨架从虚拟的小分子化合物库中筛选出来;2)所建立的体外抑酶实验以及体外细胞测试方法比较可行,可信度比较高;3)本课题的设计思想完全符合最新的药物化学设计思路,而且预先考虑到了化合物的ADME的性质,因此设计比较成功。第五部分结论本课题所设计的环酰亚胺骨架类肽化合物具有很好的金属蛋白酶抑制活性,通过体内外活性筛选发现了有开发潜力的化合物,其中几个有望作为新型氨肽酶N抑制剂先导化合物进行临床前研究。本课题研究中的活性筛选试验定性地显示了化合物结构与活性之间内在关系的趋势,同时建立了该类化合物的构效关系以及定量构效关系模型,为进一步结构优化提供了有益的理论依据,并为发现活性更好的新型抗肿瘤转移先导化合物打下坚实的基础。更多还原

【Abstract】 PartⅠ.Research backgroundMetalloproteinase is a big family of metal-ion containing hydrolases.It is discovered that two zinc-dependent metalloproteinase（Aminopeptidas N,APN/CD13 and Matrix Metalloproteinase,MMP） are involved in the invasion and metastasis of malignant tumors.Among which APN also plays critical roles in tumorgenesis,regulation of immunological function and virus infection as well.a.APN is over-expressed on the surface of tumor cells and the possible function in the process of tumor growth is:1) Degrading extracellular matrix,promoting the release of most growth factor and so speeding up the proliferation of tumor cells;2) Promoting the invasion and metastasis of tumor cells;3) Stimulating the release of cellular factors associated with the neovascularization by vascular endotheilal cells and promoting the angiogenesis of tumor;4) Enhancing the adhesiveness of tumor cells,and in turn the tumor cells with high adhesiveness will stimulate the release of metalloproteinase and enhancing its ability of ECM degradation;b,APN is also over-expressed on the surface of granulocyte and lymphocyte and participate the T-lymphocyte dependent inflammatory reaction as CD13 antigen.APN also has been implicated in the processing and trimming of antigenic peptides that protrude out of major histocompatibility complex（MHC） classⅡmolecules on the cell surface.APN is also involved in the down-regulation of signal peptides,such as enkephaline in the brain and can cleave bioactive proteins,including several cytokines and antigen delivering peptides,degrading lots of immunoactive substances,impairing the immunological functions,depressing the recognization of macrophage and NK cells to surface antigen on tumor cells and the ability to kill these cells directly;c.APN was shown to be the major receptor for the transmissible gastroenteritis virus（TGEV）,which causes a severe gastroenteritis in newborn pigs,and for the human coronavirus 229E （HCV229E） which causes upper respiratory infections;d.APN also play a role in the entry of HIV-1 into cells by degrading fMLP,a chemokine associated with the desensitization of CCR5,the co-receptor for HIV-1 entry and up-regulating the expression of CCR5;e.Furthermore,APN is also involved in the down-regulation of signal peptides,such as enkephaline in the brain and will be a promising target for analgesia;f.In recent years,APN has been elucidated to participate in the enzymatic cascade of rennin-angiotensin system in the brain and periphery by cleaving angiotensinⅢto antiotensinⅣand is associated with the regulation of blood pressure.Therefore,APN will be a good target for the design of novel therapeutic agents for the treatment of diseases,such as cancer,leukemia,rheumatoid arthritis, diabetic nephropathy and central nervous system diseases,such as Alzheimer’s disease.The anticancer drug research targeting to APN is closely watched in recent years because of it role in the tumor growth.The research fields include three aspects:APN mono-antibody and APN ligand binding drugs,both of which are in the initial stage; Small molecular APN inhibitors,either natural or synthetic.Bestatin,firstly isolated from a culture filtrate of Streptomyces olivoreticuli,has been used in clinics as an immunoenhancer in Japan in 1987.In recent years,many bestatin analogues were discovered,among which Actinonin is a more active inhibitors.PartⅡ.The Virtual Screen and Rational Design of Target Compounds Although there are lots of small molecular inhibitors reported in recent years,however, there were no any reports of APN crystal structure until 2006.Most researches are focused on natural products and bestatin analogues.And the process of synthetic APN inhibitors is really slow,resulting in few report associated with target structure-based drug design toward APN.The characteristics of this study are the utilization of mechanism-based and structure-based drug design comprehensively.That is,based on the inference that the major components of ECM are Pro-Gly,we build a virtual molecule library with heterocycle as scaffold according to the conformational constraint strategy.And with advantages of computer-aided drug design software,we also build a four-emelment pharmacophore model according to the information of inhibitors with known activity and structure.The cyclic-imide scaffold was obtained by virtual screening of the heterocycle library with this pharmacophore.The following design strategy is structure-based drug design.That is,to design the side chain of cyclic-imide according to the information of the requirement of the active site of E.Coli APN and enzyme-inhibitor complex.Flexsible docking was used for modeling the interaction between target compounds and enzyme.The side chain of cyclic-imide can be designed rationally and synthesized easily.The compounds in three series are a result of optimization step by step.It is discovered that there is some relevance between part of the quantitative parameters （such as docking score） and the in vitro or in vivo activity of compounds.Furthermore, based on the analysis of the difference between the active site of target enzymes,we found the change of the selectivity from MMP to APN,indicating that the design strategy of this study is rational and is significant for the design of novel target compound.In addition,the experimental results can be well predicted by all kinds of screen model constructed.Additionally,the matching of the structure of compounds with the active site of enzyme is not the only consideration of this study.The fragments used in the side chain of the target compounds also apply the fragments with anticancer activity and natural amino acid side chain,which can be compatible with tissues. In addition,the ADME and pharmacokinetics properties of the target compounds have also been considered in advance.The compounds exist in the form of hydrochloride salt with better water solubility and bioavailability and so increasing the hit rate.PartⅢ.The Synthesis of Newly Designed Target CompoundsOn the basis of virtual screening and target structure-based drug design.We obtained 94 structural confirmed compounds with varieties in the side chain of cyclic-imide. The polypeptide synthesis advantages are thoroughly reflected in the synthesis scheme,most of which are novel and feasible.Two strategy are applied for the synthesis of target compounds:Firstly,the 3-trimethoxybenzoic-2,6-piperidinedione-N-acetic acid derivatives are synthesized with L-Glu as starting material and the important intermediate 6 was obtained after acylation,cyclization by dehydration and aminolysis by Gly.The target compounds are obtained with EDCI as condensing agent for polypeptide synthesis from 6 with different amino acid derivatives.The methyl group can also be deprotected by Lewis acid BBr₃.Secondly,the intermediates that can be used for the variety of structure were synthesized in advance.12 and 27 can be obtained by the protection of amino group of L-Gln or L-Asn by Boc and then cyclization of amide withα-carboxyl group. The NH of cyclic-imide can be alkalization by halogenated hydrocarbon in the existence of phase transfer catalyst TBAI to form N-substituted intermediates of 13, 18 and 28.The Boc group of these three compounds can be deprotected by TFA and then the free amino group was acylated by different acyl chloride or mixture anhydride.The cyclic-imide-N-acetic acid derivatives can be obtained from its benzyl ester derivatives by catalytic transfer hydrogenation to deprotect the benzyl group. And the hydroxamate acids derivatives can be obtained according to the methods of mixture anhydride from its acid with isobutyl chloroformate.All the targeted compounds are novel without any report by now with the structures identified by IR, ¹H-NMR and ESI-MS.The purity of part compounds are analyzed by HPLC,with the area%of more than 96%. PartⅣ.The Biological Evaluation and SAR & QSAR StudyThe in vitro and in vivo preliminary activity evaluations were performed as for the newly synthesized 94 target compounds.The in vitro enzyme inhibitory activity were determined against APN and MMP,both of which are zinc-dependent metalloproteinase and closely associated with the invasion and metastasis of tumor cells,The cells assay is performed toward leukemia cells（HL-60 and K562） which express APN and SKOV3（which can express MMP-2 and MMP-9）.The in vitro enzyme assay displayed that the compounds in SeiresⅠare all selective to MMP(the IC₅₀ ratio of 7j is 150).However,the inhibitory activity of 71,7o,9a,9b against APN is pretty high,and the IC₅₀ value of most active compound 9a（3.0μM） is equal to that of bestatin(IC₅₀=2.4μM),indicating that the introduction of conformational constraint can indeed improved the selectivity toward target enzyme. The free group in SeiresⅡand SeriesⅢis a decisive factor for the inhibition of APN, as the activity increased by an order of magnitude.Among which 17j,22a,22g in SeriesⅡand 32a,32c,32f,32i in SeriesⅢand all hydroxamate acid compounds 24/34 in both series displayed potent activity and high selectivity toward APN with the IC₅₀ ratio about 20.The IC₅₀ of 34d was 2.9μM.24f and 34f is more potent that bestatin against APN with IC₅₀ value of 1.8μM and 1.0μM,respectively. The SAR and QSAR analysis displayed that the substituents in P1’ site in SeriesⅠis important for the selectivity of MMP and the Try residue is more favorable.The introduction of aliphatic residue can improve the activity against APN with a loss of selectivity.As for the compounds in SeriesⅡand SeriesⅢ,free amino group is a necessary condition for the inhibitory activity of APN and the activity tendency of target compounds with different substituents in P1 is CONHOH＞COOH＞COOEt,and aromatic ring without a hydroxyl group is more favorable.The results of in vitro growth inhibition against leukemia cells indicated that most potent APN inhibitor displayed good inhibitory effect against the growth of HL-60 cells, among which 7g,9a,24f,24i,34f is more potent than bestatin.The possible mechanism may be that the downstream factor is influenced when cyclic-imide derivatives interact with APN,and then the DNA synthesis is inhibited followed by the promotion of apoptosis of tumor cells.The cells assay was also performed to leukemia cell K562.The inhibitory rate is still lower than 25%when the concentration of compounds is nearly saturated.The inhibitory effect can not be discovered even in the higher concentration and there was no phenomenon of abnormality about the shape of cells,indicating that the compounds are not cytotoxtic.The sensitivity of K562 to the inhibitors is less than that of HL-60 because of its lower expression of APN,in some degree suggesting that the newly synthesized compounds can target APN well.The cells assay against the growth of SKOV3 display that the compounds（7i,7r） with moderate activity against MMP can strongly inhibit the expression of MMP-2, MMP-9,and this may be the major reason for their anticancer effect. We used the anti-metastasis model of mice bearing H22 tumor cell to evaluate the inhibiting activities of cyclic-imide derivatives in vivo.All tested compounds were orally administered（p.o.） at a dose of 50 mg/kg,6 days/week for two weeks.The test results demonstrated that most of potent APN inhibitors（9a,24f,24i,34f,34i）showed significant anticancer activities（inhibitory rate＞50%） and were devoid of toxicological effects,among which the best compounds were 24f and 9a with the inhibitory rate as high as 72.91%and 81.56%,respectively,showing that they might be a promising lead compound and is worth of further study.The possible mechanism of anti-metastasis effect may be:1) to inhibit the activity of APN directly,and then inhibit the degradation of ECM;2) Some cellular factor that can regulate the transcription of gene was activated or inhibited following the inhibition of APN,and the expression of MMP or other molecules was influenced and in turn inhibiting the ECM degradation and the location of tumor cells in lung tissues. The five compounds used in in vivo experiment assay possess potent inhibitory effect in in vitro enzyme and cells assay and both of the experimental results can be fully consistent.All of these discoveries prove the following points:1) The screening ability of the pharmacophore constructed in advance is strong enough to screen out the potent cyclic-imide scaffold from the virtual library;2) The methods used for in vitro and in vivo activity assay are stable and can be reproduced with high degree of confidence;3) The design strategies applied in this study is identical to the novel ideas in modem medicinal chemistry fields,and the pre-consideration of ADME properties result in successful achievements.PartⅤ.ConclusionThe cyclic-imide peptidomimetics derivatives designed in this study displayed potent metalloproteinase inhibitory activity.And some potent compounds can be screened in the in vitro and in vivo experiments and is promising and valuable of further study and used in pre-clinical study.The results of activity assay experiments can manifest quantitatively the tendency of the relationship between the structure and activity and meanwhile build the SAR and QSAR model.All of these discoveries provide a beneficial theoretical basis for the optimization of lead structure and lay a solid foundation for the discovery of novel more potent anti-metastasis agents in future.更多还原