本文關(guān)鍵詞： CDK4抑制劑 抗腫瘤 DNA嵌入作用　出處：《蘭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：CDK是細(xì)胞分裂周期各個(gè)階段檢查點(diǎn)的重要組成部分,是健康細(xì)胞生長和增殖所需的。CDK4在細(xì)胞分裂周期的G0/G1期具有非常特殊的功能,CDK4抑制劑可以導(dǎo)致細(xì)胞周期的早期G1阻滯,從而防止不受控制的細(xì)胞生長。最近的研究表明Cdk4是介入癌癥治療的重要靶標(biāo),因?yàn)镃dk4活性的失調(diào)可導(dǎo)致癌癥。Fascaplysin是一種五環(huán)季銨鹽,最初從斐濟(jì)海綿Fascaplysinopsis Bergquist sp中分離,具有廣泛的活性,它也是一種有潛力的細(xì)胞周期蛋白依賴性激酶4(CDK4)抑制劑,可以引起正常和腫瘤細(xì)胞系的細(xì)胞周期停滯G1期。然而,Fascaplysin由于其本身毒性副作用,嚴(yán)重限制了其作為抗癌藥物的潛力。Fascaplysin在細(xì)胞水平顯示出特殊的毒性,進(jìn)一步的研究表明,fascaplysin的平面結(jié)構(gòu)可以嵌入雙鏈DNA分子,類似于DNA插入劑的屬性。本研究的主要目標(biāo)是開發(fā)有潛力非平面具有CDK4特異性的fascaplysin類似物,不嵌入或不與DNA相互作用,從而達(dá)到避免在細(xì)胞水平的細(xì)胞毒性。本課題通過斷鍵開環(huán)思路設(shè)計(jì)了兩類基于fascaplysin結(jié)構(gòu)非平面特異性CDK4抑制劑,采用經(jīng)典的Pictet-Spengler反應(yīng),通過環(huán)合,�；铣闪怂臍洇�-咔啉類目標(biāo)化合物,采用異氰酸酯法合成了吲哚類目標(biāo)化合物。隨后,通過1H-NMR、13C-NMR以及ESI-MS對(duì)20個(gè)化合物進(jìn)行結(jié)構(gòu)確證。為探索20目標(biāo)化合物的抗腫瘤活性,分別進(jìn)行了細(xì)胞增殖抑制實(shí)驗(yàn),細(xì)胞周期、凋亡實(shí)驗(yàn)。MTT法檢測(cè)腫瘤細(xì)胞增殖抑制,實(shí)驗(yàn)結(jié)果顯示目標(biāo)化合物對(duì)腫瘤細(xì)胞增殖抑制活性最好為b5(5μMIC5030μM),其中作用于A-549的IC50為8.53±1.65μM。吲哚類化合物腫瘤細(xì)胞抑制活性最好為a9(IC5090μM),正常細(xì)胞毒性IC50為119.54±38.57μM。四氫β-咔啉類化合物中苯環(huán)上硝基取代較甲氧基和鹵素取代活性要差,苯環(huán)硝基取代抑制活性IC50值大于200μM,而苯環(huán)甲氧基和鹵素取代IC50小于100μM。兩類化合物細(xì)胞抑制活性弱于對(duì)照品fascaplysin(0.2μMIC501.1μM),但對(duì)正常細(xì)胞毒性有一定程度降低。PI染色法檢測(cè)細(xì)胞周期實(shí)驗(yàn)表明fascaplysin及目標(biāo)化合物b5較空白組G0/G1期比例降低,S期比例顯著提高,呈現(xiàn)S期阻滯;目標(biāo)化合物a9、b3使A549細(xì)胞G0/G1期增加,均呈現(xiàn)G1期阻滯。AnnexinV/PI雙染凋亡細(xì)胞檢測(cè)表明化合物a9、b3及b5對(duì)A-549均產(chǎn)生凋亡誘導(dǎo),b5誘導(dǎo)能力最強(qiáng),a9次之,最弱為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 鹵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 鹵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).
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96

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