2-(3-苯[b]噻吩)-6,7-亞甲二氧基喹啉-4-酮類緣物的設計、合成與抗癌活性、4-苯基-1H-苯[b][1,4]二氮雜環-2(3H)-酮衍生物的設計、合成與生物活性; I. Design, synthesis and anticancer activity of 2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one analogs. II. Design, synthesis and biological activity of 4-phenyl-1H-benzo[b][1,4]diazepin-2(3H)-one derivatives.

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题名:	2-(3-苯[b]噻吩)-6,7-亞甲二氧基喹啉-4-酮類緣物的設計、合成與抗癌活性、4-苯基-1H-苯[b][1,4]二氮雜環-2(3H)-酮衍生物的設計、合成與生物活性;I. Design, synthesis and anticancer activity of 2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one analogs. II. Design, synthesis and biological activity of 4-phenyl-1H-benzo[b][1,4]diazepin-2(3H)-one derivatives.
作者:	張昱勛;Yu-Hsun Chang
贡献者:	中國醫藥大學：藥物化學研究所博士班
关键词:	喹;啉;-4-酮;凋亡;微管;2-phenyl-4-quinolone;combretastatin A-4;apoptosis;microtubule
日期:	2009-07-09
上传时间:	2009-08-13 09:37:11 (UTC+8)
摘要:	Part I. 由於azo-flavonoids 的優異抗癌活性，本實驗室參考之前的實驗成果，整合2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-2133)的6,7-methylenedioxy 基團結構與CoMFA 計算模型中的2位雜環概念，設計、合成出一系列新的2-aryl-6,7-methylenedioxyquinolin-4-one類緣物，之後以數種人類腫瘤細胞作為細胞毒性測試對象，建立出各個化合物結構和藥理活性間的關係。結果顯示，衍生物1、37及39展現出強效的抗癌能力，其中化合物1對Hep 3B 表現出選擇活性，而化合物37對HL-60、HCT-116、Hep 3B及KB-VIN等細胞呈現優異的抑制效果，至於化合物39則是對各個細胞株皆顯現出廣泛的毒殺作用，且IC50濃度介於0.07−0.19 μM間。而從微管的作用測試也證實，該系列新quinolone衍生物仍保有抗微管聚合的良好能力。接續藥物結構和活性關係間的研究，CWC-8 (化合物39)被選為進一步探討抗癌活性機轉的藥物，過程中以U-2 OS骨癌細胞作為測試對象。根據MTT的結果顯示，CWC-8對U-2 OS的抑制有劑量及時間依存性的關係，其IC50濃度為4.97±0.24 μM，而在流式細胞儀的檢驗中，CWC-8也表現出它對細胞週期G2/M arrest與apoptosis的現象，之後於CDK1 kinase和western blotting試驗裡，則呈現CDK1酵素活性提昇、CDK1、Fas/CD95、FADD、cytosolic cytochrome c、caspase-8/-9/-3 active form、Apaf-1、AIF、Bax等蛋白質含量增加但Bcl-2下降之凋亡效果，此外，在caspase-8/-9/-3和其對應酵素抑制劑的混合試驗中，pan-caspase及caspase-8/-9/3 inhibitors皆有反轉CWC-8細胞毒性的能力。總合相關試驗的結果顯示，CWC-8擁有發展為抗癌藥物的潛在條件。 Part II. 蛋白質本身的mobility特性，長久以來在藥物設計領域中一直被忽略，構形誘導是ligand與目標蛋白相互作用時所形成的一種能量平衡過程，它在期間所產生的構形變化並不會自發性地出現在未和ligand結合時的平常狀態，由於蛋白質的這種彈性特性，普遍限制了現今合理藥物設計模式的發展。因此，我們在研究中提出，增加ligand結構彈性可以迎合目標蛋白變動性質的策略觀點，之後以本實驗室先前的抗腫瘤化合物2-phenyl-4-quinolones為基礎骨架，合成出相對具有flexibility特性之4-phenyl-1H-benzo[b][1,4]diazepin-2(3H)-one系列闊環衍生物。出乎預期的，該類化合物在體外細胞毒性篩選試驗結果中，並無明顯的抗癌活性，僅有化合物7-4、7-5、7-9、7-15、7-16、7-20及7-27等對HL-60細胞株展現程度上的抑制效果。雖說在抗癌測試上不盡理想，不過化合物7-4卻在random screening測試中另外發現其抑制細胞發炎的能力，它主要是是透過PKA-dependent pathway來達到抑制經fMLP誘導生成的superoxide anion，同時它還能活化protein phosphatase 2A酵素而使得phosphodiesterase 4之活性被抑制，進而提昇cAMP濃度的作用。 Part I. As part of our continuing investigation of azo-flavonoid derivatives as potential anticancer drug candidates, a series of 2-aryl-6,7-methylenedioxyquinolin-4-one analogs was designed and synthesized. The design combined structural features of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-2133), a previously discovered compound with potent in vivo antitumor activity, and 2-arylquinolin-4-ones identified by CoMFA models. The newly synthesized analogs were evaluated for cytotoxicity against several human cancer cell lines, and structure-activity relationship (SAR) correlations were established. Analogs 1, 37, and 39 showed potent cytotoxicity against different cancer cell lines. Compound 1 demonstrated selective cytotoxicity against Hep 3B (hepatoma) cells. Compound 37 was cytotoxic against HL-60 (leukemia), HCT-116 (colon cancer), Hep 3B (hepatoma), and SK-MEL-5 (melanoma) cells. Compound 39 exhibited broad cytotoxicity against all seven cancer cell lines, with IC50 values between 0.07−0.19 μM. Results from mechanism of action studies revealed that these new quinolone derivatives function as anti-tubulin agents. Continuing with the screening result, CWC-8 (compound 39) was chosen to detect the mechanism of anticancer activity in the experiment. We have defined the viability inhibition and apoptotic mechanisms of CWC-8 on human osteogenic sarcoma U-2 OS cells. According to the MTT assay, the cell viability was inhibited by CWC-8 in a dose- and time-dependent manner, with an IC50 of 4.97±0.24 μM. CWC-8 treatment induced G2/M arrest and apoptosis in U-2 OS cells by cell cycle and flow cytometry analysis. Western blotting and CDK1 kinase assay showed that CWC-8 treatment caused a time-dependent increase of Cyclin B and CDK1 protein levels and activity during G2/M arrest. CWC-8 treatment also caused a time-dependent increase in Fas/CD95, FADD, cytosolic cytochrome c, caspase-8/-9/-3 active form, Apaf-1, AIF, Bax protein levels, and decrease in Bcl-2 protein level. CWC-8 also promoted caspase-8/-9 and -3 activities; however, pretreatment of cells with pan-caspase, caspase-8/-9 and -3 inhibitors led to reduced cell growth inhibition action. Taken together, these findings show CWC-8 could be a potential candidate for cancer therapy. Part II. The intrinsic mobility of proteins has often been ignored in drug design field. Conformational induction is the energy balance process in which ligand converts protein into a conformation that would not spontaneously adopt in its unligated state. This flexible property of protein limits the development of rational drug design model nowadays. Therefore, we proposed a concept that describes raise of ligand flexibility is the strategy to accommodate protein mobility. In this study, we synthesized a series of 4-phenyl-1H-benzo[b][1,4]diazepin-2(3H)-one derivatives which are more flexible than our previous antitumor compound− 2-phenyl-4-quinolones. Unexpectedly, the cytotoxicity screening result shows little activity of these derivatives. Only 7-4, 7-5, 7-9, 7-15, 7-16, 7-20 and 7-27 compounds exhibt the potency to inhibt HL-60 cell line. Despite the unideal outcome in anti-cancer test, we acquired the anti-inflammation activity of compound 7-4 via random screening insteadly. It inhibited fMLP-induced superoxide anion production through a PKA-dependent pathway and increased cAMP by activating protein phosphatase 2A, which subsequently inhibited phosphodiesterase 4.
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