1,8-二乙酰基大黄酸-(2-溴)-乙酯的合成及对骨肉瘤MG-63细胞的作用研究
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摘要
目的设计并合成新的大黄酸衍生物1,8-二乙酰基大黄酸-(2-溴)-乙酯(大黄酸衍生物B),探讨其对骨肉瘤MG-63细胞的作用和机制。方法以大黄酸为原料合成了1,8-二乙酰基大黄酸-(2-溴)-乙酯,经UV、IR、NMR确定合成产物的结构,并利用HPLC测定其纯度。采用MTT法测定大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的体外生长抑制作用;流式细胞仪检测细胞凋亡和细胞周期分布。结果经UV、IR、~1H NMR、~(13)C NMR进行分子结构表征,确证合成的目标化合物为1,8-二乙酰基大黄酸-(2-溴)-乙酯,纯度>98%。MTT结果表明,大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的IC_(50)值分别为110.60、25.78μmol·L~(-1)。流式细胞仪检测细胞凋亡和周期结果表明,80μmol·L~(-1)的大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的凋亡率分别为(6.87±0.53)%、(48.84±2.20)%,且主要将细胞周期阻滞在S期。结论合成化合物1,8-二乙酰基大黄酸-(2-溴)-乙酯的体外抗肿瘤活性明显优于大黄酸,且具有阻滞骨肉瘤MG-63细胞周期进程和促进其凋亡的作用。
AimTo design and synthesize of a new rhein derivative 1,8-diacetyl rhein(2-bromo)-ethyl ester(rhein derivatives B)and explore its effect on osteosarcoma MG-63 cells and the related mechanism.Methods1,8-diacetyl rhein(2-bromo)-ethyl ester was synthesized from rhein and its structure was identified by UV,IR and NMR spectra.The purity of the synthetic product was determined by HPLC.The in vitro anti-proliferative activity of rhein and the synthetic product on osteosarcoma MG-63 cells were determined by MTT assay.Apoptosis and cell cycle distribution were detected by flow cytometry.Results The molecular structure of 1,8-diacetyl rhein(2-bromo)-ethyl ester was confirmed by UV,IR,~1H NMR and~(13)C NMR,and the purity was higher than 98%.The IC_(50)values of rhein and rhein derivatives B on osteosarcoma MG-63 cells were 110.60μmol·L~(-1)and 25.78μmol·L~(-1),respectively.The Resultsof flow cytometry showed that the apoptotic rates of rhein and rhein derivatives B at the concentration of 80μmol·L~(-1)were(6.87±0.53)%and(48.84±2.20)%,respectively,and the cell cycle was mainly arrested in S phase on MG-63 cells.Conclusions The anti-tumor activity of 1,8-diacetyl rhein-(2-bromo)-ethyl ester is superior to that of rhein in vitro,and the mechanisms may be associated with blocking the process of cell cycle of osteosarcoma MG-63 cells and initiating apoptosis.
AimTo design and synthesize of a new rhein derivative 1,8-diacetyl rhein(2-bromo)-ethyl ester(rhein derivatives B)and explore its effect on osteosarcoma MG-63 cells and the related mechanism.Methods1,8-diacetyl rhein(2-bromo)-ethyl ester was synthesized from rhein and its structure was identified by UV,IR and NMR spectra.The purity of the synthetic product was determined by HPLC.The in vitro anti-proliferative activity of rhein and the synthetic product on osteosarcoma MG-63 cells were determined by MTT assay.Apoptosis and cell cycle distribution were detected by flow cytometry.Results The molecular structure of 1,8-diacetyl rhein(2-bromo)-ethyl ester was confirmed by UV,IR,~1H NMR and~(13)C NMR,and the purity was higher than 98%.The IC_(50)values of rhein and rhein derivatives B on osteosarcoma MG-63 cells were 110.60μmol·L~(-1)and 25.78μmol·L~(-1),respectively.The Resultsof flow cytometry showed that the apoptotic rates of rhein and rhein derivatives B at the concentration of 80μmol·L~(-1)were(6.87±0.53)%and(48.84±2.20)%,respectively,and the cell cycle was mainly arrested in S phase on MG-63 cells.Conclusions The anti-tumor activity of 1,8-diacetyl rhein-(2-bromo)-ethyl ester is superior to that of rhein in vitro,and the mechanisms may be associated with blocking the process of cell cycle of osteosarcoma MG-63 cells and initiating apoptosis.
引文
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[2]Mamo T,Mladek A C,Shogren K L,et al.Inhibiting DNA-PK CS radiosensitizes human osteosarcoma cells[J].Biochem Bioph Res Co,2017,486(2):307-13.
[3]Meyers P A,Schwartz C L,Krailo M,et al.Osteosarcoma:a randomized,prospective trial of the addition of ifosfamide and or muramyl tripeptide to cisplatin,doxorubicin,and high-dose methotrexate[J].J Clin Oncol,2005,23(9):2004-11.
[4]唐敏,李鸿,周国梅,等.大黄酸调控Rac1/LIMK1/cofilin信号通路抑制卵巢癌细胞运动与侵袭[J].中国药理学通报,2016,32(3):366-72.[4]Tang M,Li H,Zhou G M,et al.Rhein inhibits the movement and invasion of human ovarian carcinoma cells through Rac1/LIMK1/cofilin signaling pathway[J].Chin Pharmacol Bull,2016,32(3):366-72.
[5]Sun H,Luo G,Chen D.A comprehensive and system review for the pharmacological mechanism of action of rhein,an active anthraquinone ingredient[J].Front Pharmacol,2016,7:1-16
[6]Gonnot V,Tisserand S,Nicolas M,et al.Total synthesis of rhein and diacerhein via a directed ortho metalation of an aromatic substrate[J].Tetrahedron Lett,2007,48(40):7117-9.
[7]Xu X,Qi X,Yan Y,et al.Synthesis and biological evaluation of rhein amides as inhibitors of osteoclast differentiation and bone resorption[J].Eur J Med Chem,2016,123:769-76.
[8]Cai J,Duan Y,Yu J,et al.Bone-targeting glycol and NSAIDS ester prodrugs of rhein:synthesis,hydroxyapatite affinity,stability,anti-inflammatory,ulcerogenicity index and pharmacokinetics studies[J].Eur J Med Chem,2012,55:409-19.
[9]Legendre F,Heuze A,Boukerrouche K,et al.Rhein,the metabolite of diacerhein,reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis[J].Scand J Rheumatol,2009,38(2):104-11.
[10]潘智育,李竟,陈云龙,等.8-溴乙氧基大黄酸的合成及其对肝癌Hep G2.2.15细胞乙肝病毒抑制作用的研究[J].中国药理学通报,2016,32(8):1175-80.[10]Pan Z Y,Li J,Chen Y L,et al.Synthesis of 8-bromo-ethoxy Rhein and evaluation of its inhibition effect on hepatitis B virus in human hepatoma cells Hep G2.2.15[J].Chin Pharmacol Bull,2016,32(8):1175-80.
[11]Yao G Y,Ye M Y,Huang R Z,et al.Synthesis and antitumor activities of novel rheinα-aminophosphonates conjugates[J].Bioorg Med Chem Lett,2014,24(2):501-7.
[12]Liang Y K,Yue Z Z,Li J X,et al.Natural product-based design,synthesis and biological evaluation of anthra[2,1-d]thiazole-6,11-dione derivatives from rhein as novel antitumour agents[J].Eur J Med Chem,2014,84:505-15.
[13]Chen Y Y,Chiang S Y,Lin J G,et al.Emodin,aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 human tongue cancer cells[J].Anticancer Res,2010,30(3):945-51.
[14]Shi P,Huang Z,Chen G.Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells[J].Am J Chin Med,2008,36(4):805-13.
[15]Wang J,Liu S,Yin Y,et al.FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis[J].Apoptosis,2014,20(3):399-409.