【作者】 陈军；

【导师】 周有骏； 朱驹；

【作者基本信息】 第二军医大学 ， 药物化学， 2008， 博士

【摘要】 恶性肿瘤是当前严重影响人类健康、威胁人类生命的主要疾病之一,世界卫生组织和各国政府卫生部门都把攻克癌症列为一项首要任务。目前,临床上常用的抗癌药物主要是细胞毒类药物,这类药物因其细胞毒的固有性质而具有难以避免的选择性差、毒副作用强、易产生耐药性等缺点。因此,寻找特异性高、毒性小、病人耐受性好的新靶标就成为抗癌药物研究的迫切需要。近年来,随着生命科学研究的飞速发展,众多基于癌细胞发生、发展机制的特异性靶点被鉴定出来,如:参与胞内信号转导的蛋白酪氨酸激酶（PTK）、蛋白激酶C（PKC）、法尼基转移酶（Ftase）,参与细胞周期调节的细胞周期蛋白依赖性激酶（CDK）,诱导肿瘤细胞调亡的Bcl-2蛋白,抑制肿瘤血管生成的血管内皮细胞生长因子（VEGF）,参与胞外基质降解和重建的基质金属蛋白酶（MMP）等。寻找选择性作用于这些特异性靶点的高效、低毒、专属性强的新型抗癌药物已成为当今抗肿瘤药物研究的主要方向。血管生成（angiogenesis）是指从已存在的血管上发育出新的血管系统。正常的血管生成只在某些短期、特定的生理过程中存在,如生殖、伤口愈合等。而异常的血管生成则是肿瘤、风湿性关节炎、糖尿病性视网膜病变等恶性疾病的病理表现之一。自从Folkman（1933-2008）提出血管生成与肿瘤的发生发展密切相关的假说以来,大量的临床实践与实验研究证实了抑制肿瘤介导的血管生成可以有效地抑制肿瘤的生长和转移。今天,已有10个抑制血管生成的抗癌药物上市,全球有超过120万人接受抗血管生成治疗。VEGF受体是抗血管生成的重要靶标。近年来,靶向VEGF受体的小分子抑制剂的研究非常活跃,大量结构各异的抑制剂被报道出来,具代表性的有2-吲哚酮类,4-苯胺基喹唑啉类和2,4-二氮杂萘类等。其中一些化合物已分别进入各期临床评价,显示出良好的应用前景。但目前这些抑制剂仍存在一些问题,例如它们都是ATP的竞争性抑制剂,而细胞内尤其是癌细胞内的ATP浓度能达到5mmol/L以上,因此抑制剂活性应至少达到纳摩尔级水平才能表现出有效的抑制作用。另外,VEGF受体属于酪氨酸激酶超家族,该家族成员广泛参与体内生物信号的传导。由于序列的同源性,它们的ATP结合位点的三维结构是高度保守的。因此如何提高抑制剂在这些家族成员中的选择性也是一个研究重点。所以,寻找高活性,高选择性的VEGF受体抑制剂仍是今后的研究方向。随着计算机辅助药物设计技术的广泛应用,VEGF受体的结构和功能以及与抑制剂的作用机制的逐渐阐明,VEGF受体抑制剂的开发正由随机筛选转为合理药物设计（SBDD）。本课题旨在应用计算机辅助药物设计技术和化学合成手段,在全面探测VEGF受体活性腔的性质、深入探讨受体与抑制剂的作用模式的基础上,设计、合成新型的VEGF受体抑制剂先导化合物,为发现新型高效、低毒的抗癌药物打下坚实的基础。为此,我们主要进行了以下几方面的工作:一、VEGFR-2活性腔性质的研究活性腔的性质以及与配体结合相关的关键残基是基于结构合理药物设计的基础,但目前仍没有对VEGFR-2活性腔进行全面深入分析的报道。为此,我们首先运用活性位点搜寻工具（Binding Site Analysis）确定了VEGFR-2活性腔的形状与大小,然后采用多拷贝同时搜寻法（MCSS）对VEGFR-2活性腔进行分析,计算结果显示活性腔可分为3个疏水性区域和1个极性区域:Ⅰ区为与绞链区毗邻的一个扁平的,由Val846,Ala864,Val897,Val914,Phe916,Leu1033的疏水性侧链组成的疏水腔。Ⅱ区是由Ile886,Leu887,Ile890,Val896,Val897,Leu1017,Ile1042,Phe1047的疏水性侧链组成的1个较大的疏水腔。位于Ⅰ区和Ⅱ区之间的由Lys866,Glu883的极性侧链和Asp1044的骨架羰基氧组成环状极性区域对于配体的结合也很重要。Ⅲ位于活性腔开口向溶剂的位置,是由Leu838和Phe916的侧链组成的小的疏水性区域。Ⅳ区是由Asn921,Leu1033,Cys1043的疏水性残基和Arg1030,Asn1031的亲水性残基组成的一个极性区域。其中Arg1030侧链上的N-H也是关键的氢键供体位点。这些结果与众多文献报道的抑制剂与VEGFR-2的结合模式一致。为了考察VEGFR-2活性腔在与配体结合前和结合后的构象差异,即受体与配体的诱导契合作用,我们对已公布的VEGFR-2游离态晶体结构和8个复合物晶体结构进行了全面的比较。将这9个晶体结构活性腔范围内的肽链的Cα原子进行叠合,计算其RMSD值,比较它们之间的异同。结果发现活性腔内Ⅰ区、Ⅲ区和Ⅳ区内的肽链骨架以及残基侧链的变化均较小,而Ⅱ区内活化环的起始端DFG序列的构象有显著的差异,其中Phe1047侧链的空间取向在不同的复合物中也完全不同。这些构象变化改变了Ⅱ区的形状和大小,这提示我们在模拟VEGFR-2与抑制剂的结合模式时考虑Ⅱ区的柔性变化对获得准确的结果是非常重要的。二、VEGFR-2与抑制剂结合模式的分子模拟研究不同类型抑制剂与靶酶的结合方式对于深刻理解受体-配体相互作用制可以提供丰富的信息,而目前文献对此的报道却是分散的和不全面的。因此我们采用柔性分子对接技术对已报道的各类VEGFR-2抑制剂与VEGFR-2活性腔进行对接研究,考察它们与受体结合特点的异同,确定参与配体结合的关键残基及其空间取向。结果发现:Ⅰ区中有2个关键的氢键作用位点:Glu917骨架上的C=O作为氢键受体,Cys919骨架上的N-H作为氢键供体,绝大部分抑制剂都与这一个或两个氢键位点发生相互作用。Lys868、Glu885和Asp1046形成的极性区域对提高配体亲合力很重要,疏水腔Ⅲ和极性腔Ⅳ是额外增强配体结合力的区域,Ⅳ区的Asn923可提供额外的氢键作用位点。在此基础上,我们采用LIQUID程序建立了VEGFR-2受体的三维药效团模型,它包含与受体结合关键的10个药效点。药效团模型直观地反映了各关键的药效点的空间分布以及彼此之间的关系,可为新抑制剂的设计提供参考和思路。三、全新VEGFR-2抑制剂的设计根据以上研究结果,我们将已报道的抑制剂拆分成小分子片段,用LUDI搜寻各个小片段在活性腔中相应区域内最佳的结合构象和空间取向,再分别连接这些最佳取向的小片段生成配体分子,再用柔性分子对接软件Affinity评价这些新生成的分子与活性腔的亲合力,最后用人工捡视拣选合成可行的目标化合物。最终,我们拣选出两类分子3,4-二氢异喹啉类和1-异喹啉酮类进行有机合成。四、新型VEGFR-2抑制剂的合成与活性研究在本节中,我们共合成了24个异喹啉类化合物,除化合物22,23外,其它目标化合物均为首次报道,所有目标化合物均经¹HNMR、质谱、红外结构确定。3,4-二氢异喹啉类的通用合成方法是:3,4,5-三甲氧基苯甲醛与硝酸甲烷缩合成硝基苯乙烯,然后用LiAlH4还原成3,4,5-三甲氧基苯乙胺,再跟各种取代的苯甲酰氯制成酰氨,再经Bischler-Napieralski环合制得目标产物,总收率33%。产物还可进一步经钯碳脱氢制成芳构的异喹啉或用NaBH₄还原成1,2,3,4-四氢异喹啉。1-异喹啉酮类的通用合成方法是:高邻苯二甲酸与氨水制成铵盐,高温脱水制得高邻苯二酰亚胺,然后与各种取代的苯甲醛经Knoevenagel缩合、NaBH₄还原、稀盐酸水解制得目标产物,总收率9%。采用了人脐静脉内皮细胞抑制实验和鸡胚尿囊膜实验考察了目标化合物的抗血管生成活性。人脐静脉内皮细胞抑制实验结果表明:本类异喹啉类化合物具有一定的的抑制血管内皮细胞增殖的活性。其中活性最强的化合物6的IC₅₀达到了3.14μmol/L。鸡胚尿囊膜实验表明目标化合物具有不同程度的抑制血管生成的作用,其中,化合物10的活性最强,浓度为1μg/ml时即表现出明显的血管生成抑制作用。更多还原

【Abstract】 Malignant tumor is currently the major disease threatening human’s health and life. So anti-cancer is then taken as an important task by WHO and Health Department of countries. Most of the current anti-cancer drugs in clinic are cytotoxic which bears properties of nonselective, severer side-effect and drug resistance. Therefore, new targets with high efficiency and low toxicity is the urgent requirement for developing anti-cancer drugs. Recently, many targets based on mechanics of cancer developing are identified, such as: protein tyrosine kinase （PTK） involving cellular signal transduction, protein kinase C （PKC）, farnesyl transferase （Ftase）, cyclin-dependent protein kinases （CDK）, Bcl-2 protein, vascular endothelial growth factor （VEGF）, matrix metallo-proteinase （MMP）, etc. Discovering novel potent anti-cancer drugs based on these new targets are main task in medicinal chemistry.Angiogenesis refers to the formation of new capillaries sprouting or splitting from pre-existing vessels. physiological angiogenesis in the adult is associated with the maintenance of the vasculature, female reproductive cycling, and wound healing. Pathological angiogenesis is upregulated in a wide range of diseases such as retinopathies, arthritis, endometriosis and cancer. Starting from Folkman’s studies in the early 70s, it has been demonstrated that the growth of primary tumors and their subsequent metastasis are angiogenesis-dependent processes. Today, there are 10 anti-angiogenesis agents as anti-cancer drugs approved by US FDA and more than 1.2 million people taking anti-angiogenesis therapy.VEGF receptors are critical targets of anti-angiogenesis. In the recent years, various kind of small molecule inhibitors targeting VEGFR have been reported such as 2-indolinones, Quinazolines and Phthalazines. Some inhibitors are under clinical trials value and show promising prospect. Otherwise, some shortages still remain. For example, as a ATP competitor, it’s IC50 should be below nanomolar level to show efficacy. On the other hand, VEGFR belongs to receptor tyrosine kinase super family, of which members play broadly roles in signal transduction. Their ATP binding site is highly conserved, so selectivity of inhibitors among those RTKs are also important. With development of computer-aided drug design technology, elucidating of structure and function of VEGFR and binding mode of receptor and inhibitor, discovering new VEGFR inhibitors is turning to rational drug design from random screen.Our aim is to design and synthesis a novel VEGFR inhibitor lead compound based on exploring properties of VEGFR active site and identifying interaction model between VEGFR and inhibitors by using CADD methods. For this sake, we have achieved following works:1. Study of properties of VEGFR active siteThe properties of active site and critical residues involving ligands binding is the base of rational drug design. Unfortunately, there is no reports about comprehensive study of active site of VEGFR-2 yet. So, we identified the shape and size of the active site of VEGFR-2 using Binding Site Analysis method. Then Multiple Copy Simultaneous Search （MCSS） method was applied to study the properties of the active site. The results show that the active site can be divided into three hydrophobic sections and one polar section by the characters bound with ligand: SectionⅠis a hydrophobic pocket formed by the hydrophobic side chain of Val846, Ala864, Val897, Val914, Phe916, Leu1033; Section II is a relatively larger hydrophobic lumen formed by the hydrophobic side chain of Ile886, Leu887, Ile890, Val896, Val897, Leu1017, Ile1042 and Phe1047. A ring-like polar section formed by the side chain of Lys866, Glu883 and the carbonyl oxygen on the skeleton of Asp1044 between SectionⅠandⅡ, is very important to ligand binding; SectionⅢlies at the active site open to solvent, and is a small hydrophobic area formed by the side chains of Leu838 and Phe916; SectionⅣis a polar section formed by the hydrophobic residues of Asn921, Leul033, Cys1043 and hydrophilic residues of Arg1030, Asn1031.In order to study the differences of conformation of active site between before and after ligand’s binding, we compared free state crystal structure with 8 published complex crystal structures of VEGFR-2. These 9 structures were superimposed by Cαof peptide around the active site while RMSD values were calculated and the differences of conformation were examined. The results show that the skeleton of peptide and side chains of residues in sectionⅠ,ⅢandⅣare comparatively similar but the conformation of DFG motif at the beginning of the activation loop in section II is changed greatly. These changes result in different shape and size of section II. It implicates that the flexibility of section II must be considered when carrying out molecular docking.2. Molding of binding mode of VEGFR-2 and inhibitorsStudying of various binding mode of different inhibitors and the receptor can provide much useful information for understanding the mechanism of the interaction between ligand and receptor. So various kind of inhibitors were docked into the active site of VEGFR-2 by flexible docking method and the critical residues involving ligand’s binding were identified. In section I, there are two critical action sites for hydrogen bonding: C=O on Glu917 skeleton as hydrogen bond receptor; N-H of Cys919 skeleton as hydrogen bond donor which are used by most inhibitors. The side chain of Lys868, Glu885 and the carbonyl oxygen on the skeleton of Asp1046 between SectionⅠandⅡcan provide additional action site for ligand binding to improve affinity. Hydrophobic pocketⅢand polar areaⅣcan also contribute to improve affinity. N-H on the side chain of Asn923 in sectionⅣis the critical hydrogen bond donor site, too.3. de novo design of VEGFR-2 inhibitorBase on above work, small molecular segments from reported inhibitors were put into the active site valued their affinity by MCSS. The favorable segments in each section of the active site were linked by LUDI program to construct new ligand molecues. Several scoring functions such as empirical binding free energy function, CScore, were applying to value the affinity of these constructed molecules. Human’s knowledge of organic synthesis was also introduced in to value the possibility of synthesis. At last, we picked out two kinds of molecules 3,4-dihydroisoquinolines and 1-isoquinolinones as our target molecules to synthesis.4. Synthesis and inhibition test of novel VEGFR-2 inhibitorsThe general synthesis method of 3,4-dihydroisoquinolines is: 3,4,5-trimethoxy-benzaldehyde and nitromethane condensed to give 1,2,3-trimethoxy- 5-（2-nitrovinyl） benzene, then treated by LiAlH4 to give 3,4,5-trimethoxy- phenylethanamine, then treated by various substituted benzoyl chloride to give amide, then cyclized via Bischler-Napieralski reaction to give target productions. The productions can further dehydroed by Pd/C or reduced by NaBH₄ to give corresponding isoquinoline or tetrahydroisoquinoline.The general synthesis method of 1-isoquinoliones is: Homophthalic acid and aqua ammonia mixed to give ammonium, then dehydrolysised in high temperature to give homophtalimide, then condensed with various Benaldehyde via Knoevenagel reaction, then reduced by NaBH₄, hydrolysised by dilute HC1 solution to give target productions . 24 products have been synthesized and 22 among them are not reported in literature. The target products show anit-angiogenesis activity by HUVEC assay and CAM method. Compound 6 shows IC50 of inhibition to HUVEC up to 3.14μmol/L and compound 10 inhibits angiogenesis of CAM greatly at the concentration of 1μg/ml.更多还原