非平面特异性CDK4抑制剂的设计、合成及抗肿瘤活性研究

发布时间：2018-02-24 13:32

本文关键词： CDK4抑制剂抗肿瘤 DNA嵌入作用　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：CDK是细胞分裂周期各个阶段检查点的重要组成部分,是健康细胞生长和增殖所需的。CDK4在细胞分裂周期的G0/G1期具有非常特殊的功能,CDK4抑制剂可以导致细胞周期的早期G1阻滞,从而防止不受控制的细胞生长。最近的研究表明Cdk4是介入癌症治疗的重要靶标,因为Cdk4活性的失调可导致癌症。Fascaplysin是一种五环季铵盐,最初从斐济海绵Fascaplysinopsis Bergquist sp中分离,具有广泛的活性,它也是一种有潜力的细胞周期蛋白依赖性激酶4(CDK4)抑制剂,可以引起正常和肿瘤细胞系的细胞周期停滞G1期。然而,Fascaplysin由于其本身毒性副作用,严重限制了其作为抗癌药物的潜力。Fascaplysin在细胞水平显示出特殊的毒性,进一步的研究表明,fascaplysin的平面结构可以嵌入双链DNA分子,类似于DNA插入剂的属性。本研究的主要目标是开发有潜力非平面具有CDK4特异性的fascaplysin类似物,不嵌入或不与DNA相互作用,从而达到避免在细胞水平的细胞毒性。本课题通过断键开环思路设计了两类基于fascaplysin结构非平面特异性CDK4抑制剂,采用经典的Pictet-Spengler反应,通过环合,酰化合成了四氢β-咔啉类目标化合物,采用异氰酸酯法合成了吲哚类目标化合物。随后,通过1H-NMR、13C-NMR以及ESI-MS对20个化合物进行结构确证。为探索20目标化合物的抗肿瘤活性,分别进行了细胞增殖抑制实验,细胞周期、凋亡实验。MTT法检测肿瘤细胞增殖抑制,实验结果显示目标化合物对肿瘤细胞增殖抑制活性最好为b5(5μMIC5030μM),其中作用于A-549的IC50为8.53±1.65μM。吲哚类化合物肿瘤细胞抑制活性最好为a9(IC5090μM),正常细胞毒性IC50为119.54±38.57μM。四氢β-咔啉类化合物中苯环上硝基取代较甲氧基和卤素取代活性要差,苯环硝基取代抑制活性IC50值大于200μM,而苯环甲氧基和卤素取代IC50小于100μM。两类化合物细胞抑制活性弱于对照品fascaplysin(0.2μMIC501.1μM),但对正常细胞毒性有一定程度降低。PI染色法检测细胞周期实验表明fascaplysin及目标化合物b5较空白组G0/G1期比例降低,S期比例显著提高,呈现S期阻滞;目标化合物a9、b3使A549细胞G0/G1期增加,均呈现G1期阻滞。AnnexinV/PI双染凋亡细胞检测表明化合物a9、b3及b5对A-549均产生凋亡诱导,b5诱导能力最强,a9次之,最弱为b3。
[Abstract]:CDK is an important part of the checkpoint in all stages of cell division cycle. CDK4, which is necessary for healthy cell growth and proliferation, has a very special function in the G _ 0 / G _ 1 phase of cell division cycle. CDK4 inhibitor can lead to early G1 arrest of cell cycle. This prevents uncontrolled cell growth. Recent studies have shown that Cdk4 is an important target for interventional cancer therapy because the imbalance in the activity of Cdk4 causes cancer. Fascaplysin, a pentacyclic quaternary ammonium salt, was originally isolated from the Fijian sponge Fascaplysinopsis Bergquist sp. Has a wide range of activities and is also a potential cyclin dependent kinase 4 CDK4 inhibitor, which can cause cell cycle arrest in normal and tumor cell lines in G1 phase. However, Fascaplysin is due to its own toxic side effects. The potential of Fascaplysin as an anticancer drug is severely restricted. Fascaplysin exhibits special toxicity at the cell level. Further studies have shown that the planar structure of fascaplysin can be embedded in double-stranded DNA molecules. Properties similar to DNA inserts. The main objective of this study is to develop fascaplysin analogues with potential nonplanar CDK4 specificity, without embedding or interacting with DNA. In order to avoid cytotoxicity at the cell level, two kinds of nonplanar specific CDK4 inhibitors based on fascaplysin structure were designed by using the idea of breaking the bond and opening the loop. The classical Pictet-Spengler reaction was used, and the cyclization was carried out through cyclization. Tetrahydrogen 尾 -carboline compounds were synthesized by acylation and indole target compounds were synthesized by isocyanate method. The structures of 20 compounds were confirmed by 1H-NMR-13C-NMR and ESI-MS. Cell proliferation inhibition assay, cell cycle, apoptosis assay and MTT assay were used to detect tumor cell proliferation inhibition. The results showed that the best inhibitory activity of the target compound on tumor cell proliferation was b5N5 渭 MIC5030 渭 M-1, among which the IC50 acting on A-549 was 8.53 卤1.65 渭 M.The inhibitory activity of indoles on tumor cells was best at a9IC5090 渭 M-1, and the normal cytotoxic IC50 was 119.54 卤38.57 渭 M.tetrahydro尾 -carbolinized. The nitro-substitution activity of benzene ring is worse than that of methoxy and halogen. The inhibitory activity of benzene-nitro-substituted IC50 was more than 200 渭 m, while the inhibitory activity of benzyl methoxy and halogen substituted IC50 was less than 100 渭 M. the inhibitory activity of the two compounds was weaker than that of the reference substance fascaplysin(0.2 渭 MIC501.1 渭 M, but the cytotoxicity of the two compounds to normal cells was decreased to a certain extent. Pi staining was used to detect the cell. The periodic experiments showed that the ratio of fascaplysin and target compound b5 was significantly lower than that of the blank group in G _ 0 / G _ 1 phase, and the ratio of S phase was significantly higher than that of control group. The results showed that the G _ 0 / G _ 1 phase of A549 cells was increased by A9B _ (3), and the apoptotic cells of A549 cells were detected by G _ 1 phase arrest and Annexin V _ (P) Pi double staining. The results showed that A9B _ (3) and b _ (5) had the strongest apoptosis-inducing ability to A-549, followed by B _ (3) and B _ (9).
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

【参考文献】