【作者】 李敏；

【导师】 马立英；

【作者基本信息】 郑州大学 ， 药学（专业学位）， 2023， 硕士

【摘要】 蛋白酶体（Proteasome）是一种蛋白质复合物,主要通过破坏肽键来降解细胞不需要的或受到损伤的蛋白质,其对蛋白的选择性降解在生命过程中发挥着重要作用。自2003年第一个蛋白酶体抑制剂硼替佐米获FDA批准上市以来,泛素-蛋白酶体系统逐渐受到更多的关注,同时也成为多种疾病特别是癌症治疗的重要靶点。S期激酶相关蛋白2（S-phase kinase-associated protein 2,Skp2）是泛素-蛋白酶体系统中SCF^Skp2E3泛素连接酶的底物识别单元,Skp2作为细胞周期中重要的调节因子,可以特异性识别磷酸化底物细胞周期调控因子p27,p21等,并介导其泛素化降解,从而促进肿瘤的发生和发展。相关研究发现,Skp2在多种肿瘤细胞中异常高表达,并常与肿瘤治疗的不良预后有关。因此研究开发Skp2小分子抑制剂对于肿瘤相关研究具有重要意义。本论文在课题组前期工作的基础上,设计合成了一系列结构新颖的吡嗪类Skp2抑制剂。具体研究工作如下:1.为了发现新的Skp2抑制剂,课题组前期利用高通量筛选（HTS）从内部化合物库中发现了具有2,3-二苯基-5-取代吡嗪结构的苗头化合物779(IC₅₀=42.77μM),并借助计算机辅助药物设计,综合应用生物电子等排及骨架跃迁等药物设计策略对化合物779进行结构优化后发现了先导化合物L-1(IC₅₀=45.44μM)。本论文对化合物L-1进行结构修饰及构效探索,主要集中在C-5位取代基的优化上,以及对C-2位苯环替换或在苯环上引入不同取代基团,总共设计合成了78个吡嗪类Skp2抑制剂,并通过¹H NMR、¹³C NMR以及HR-MS对化合物结构进行了确证。2.本论文通过HTRF法（均相时间分辨荧光法）对所合成化合物进行了Skp2抑制活性评价,结果显示大多数化合物表现出优于先导化合物的Skp2抑制作用。其中抑制活性在10μM以下的化合物共计27个,6个目标化合物Skp2抑制活性达到了1μM以下。通过构效分析发现吡嗪环C-5位单取代时引入苯甲腈基团活性最优,环合取代时引入氨甲基哌啶活性最优,吡嗪环C-2位苯环上引入吸电子取代基活性更优。通过对化合物体外抗肿瘤细胞活性评价后发现,化合物13r对食管癌TE-1细胞有一定的抑制作用(IC₅₀=15.46μM),因此本论文继续对化合物13r在相关生物作用机制方面进行探究。结果表明,化合物13r可以抑制食管癌TE-1细胞的增殖与迁移,诱导肿瘤细胞凋亡及导致细胞周期阻滞。综上所述,本论文总共设计合成了78个全新的吡嗪类Skp2抑制剂,其中化合物13r可以有效地抑制食管癌TE-1细胞的生长。新型吡嗪类Skp2抑制剂的设计开发不仅丰富了Skp2抑制剂的结构类型,同时也可作为潜在抗癌药物的先导化合物用于进一步优化与研发。更多还原

【Abstract】 Proteasomes are protein complexes that degrade unwanted or damaged proteins by cells primarily by breaking peptide bonds,and their selective degradation of proteins plays an important role in the life process.Since the first proteasome inhibitor bortezomib was approved by the FDA in 2003,the ubiquitin-proteasome system has gradually attracted more attention and become an important target for the treatment of various diseases,especially cancer.S-phase kinase-related protein 2（Skp2）is the substrate recognition unit of SCF^Skp2 E3 ubiquitin ligase in ubiquitin-proteasome system,Skp2 as an important regulator in the cell cycle,it can specifically identify the phosphorylation substrate cell cycle regulators p27,p21,and mediate its ubiquitination degradation,so as to promote the occurrence and development of tumor.Related studies have found that Skp2 is abnormally highly expressed in a variety of tumor cells,and is often associated with the poor prognosis of tumor therapy.Therefore,the development of small-molecule Skp2 inhibitors is of great significance for tumor-related research.Based on the previous work of the research group,a series of structural novel pyrazine Skp2 inhibitors were designed and synthesized.The specific research work is conducted as follows:1.In order to discover new Skp2 inhibitors,our research group used high-throughput screening（HTS）to discover the seedling compound 779(IC₅₀=42.77μM)with structure of 2,3-Diphenyl-5-substituted pyrazine.Then the lead compound L-1(IC₅₀=45.44μM)was found after optimizing the structure of compound 779 with the help of computer-aided drug design and comprehensive application of drug design strategies such as bioelectronic isoplatoon and skeleton transition.In this paper,the structural modification and structure-activity exploration of compound L-1 mainly focused on the optimization of C-5 substituents and the replacement of C-2 benzene rings or the introduction of different substituents on the benzene ring.A total of 78pyrazine Skp2 inhibitors were designed and synthesized,and the compound structure was confirmed by ¹H NMR,¹³C NMR and HR-MS.2.In this paper,the Skp2 inhibitory activity of the synthesized compounds was evaluated by HTRF method（homogeneous time-resolved fluorescence method）,and the results showed that most of the compounds showed better Skp2 inhibition than the lead compounds.Among them,a total of 27 compounds had inhibitory activity below10μM,and 6 target compounds Skp2 inhibition activity reached less than 1μM.Through structure-activity analysis,it was found that the introduction of benzonitrile group in the C-5 position of the pyrazine ring had the best activity,the introduction of aminomethylpiperidine in cyclic substitution was the best,and the electron-absorbing substituent introduced on the C-2 benzene ring of the pyrazine ring was more active.After evaluating the anti-tumor cell activity of compounds in vitro,it was found that compound 13r had a certain inhibitory effect on esophageal cancer TE-1 cells(IC₅₀=15.46μM).Therefore,this paper continues to explore the relevant biological mechanism of compound 13r.The results showed that compound 13r could inhibit the proliferation and migration of esophageal cancer TE-1 cells,induce apoptosis of tumor cells and lead to cell cycle arrest.In summary,a total of 78 novel pyrazine Skp2 inhibitors were designed and synthesized,among which compound 13r can effectively inhibit the growth of esophageal cancer TE-1 cells.The design and development of novel pyrazine Skp2inhibitors not only enriches the structural types of Skp2 inhibitors,but also serves as a lead compound for potential anticancer drugs for further optimization and development.更多还原