摘要
肝癌的发生和发展不仅与肝癌的分化异常及增殖过度有关,而且与细胞凋亡的减少有关。因此开发诱导肿瘤细胞凋亡的新药,成为肿瘤治疗的新思路。砷化物(胂化物)作用于肿瘤细胞开始于20世纪70年代,自从三氧化二砷(砒霜的主要成分)成功治疗急性早幼粒白血病(APL)后,人们围绕砷化物(胂化物)开展了一系列的研究,砷剂的抗肿瘤作用越来越受到人们的关注。另外,近年来也有报道显示丹皮酚较高浓度时在体内、外实验中均有抗肿瘤作用,并诱导肿瘤细胞凋亡、影响凋亡相关基因表达。我们从有机胂的角度出发,结合丹皮酚的抗炎、活血和抗肿瘤等特性,合成了一系列的有关丹皮酚和有机胂的衍生物,均为创新化合物。采用正交实验和方差分析对合成化合物的条件进行了探讨。同时对丹皮酚的提取条件进行了优化研究。以创新化合物对人肝癌细胞株HepG2细胞的抗肿瘤活性进行了筛选,获得效果较佳的化合物和最佳的试验浓度,通过HepG2细胞生长抑制实验、诱导HepG2细胞凋亡分析及机制等探讨,探索了一类结构简单、较低浓度下可以诱导体外及体内肿瘤细胞凋亡,而对正常细胞影响较小的新型有机胂药物。
本文的主要研究方法、内容及结论如下:
1. 提取丹皮酚的最佳条件:采用超临界CO2提取法是目前最好的方法。用正交实验手段得到丹皮酚的萃取温度为45℃,萃取压力为100Bar,时间为75min时,萃取效果最佳。提取率为94.1%。
2. 合成了8种关于丹皮酚和有机胂的衍生物,即2-羟基-4-甲氧基苯乙酮缩对氨基苯(3a或S1),2-羟基-4-甲氧基苯乙酮缩对氨基苯胂酸(3b或S6),2-甲氧基-4-羟基-5-乙酰基偶氮苯-4′-胂酸(4a或R2),1-邻羟基-对甲氧基苯甲酰基-2-对氨基苯胂酸基乙烷(5a或M3),1-邻羟基-对甲氧基苯甲酰基-2-苯基-2-对氨基苯胂酸基乙烷(5b或M1),2?-羟基-4?-甲氧基苯甲酰基-溴代甲烷(F1),2?-羟基-4?-甲氧基-5?-溴苯乙酮(F2),2?-羟基-4?-甲氧基-5?-溴苯甲酰基-溴代甲烷(F4-2)。 这8种化合物均为新化合物。通过正交实验,得到了合成它们的最佳条件:
① 化合物3a(S1): 丹皮酚:苯胺 = 1:1时,在正丁醇溶液中回流2h,回流温度118℃,其产率达到最高(25.1%);
② 化合物3b(S6):在正丁醇溶液中,丹皮酚:阿散酸=1:1时,回流3h,产物的产率最高(28.4%);
③ 化合物4a(R2):丹皮酚与阿散酸的摩尔比改变,对产率无影响,反应温度控制在5℃以下,反应时间3h,反应介质pH=8~10时为最佳合成条件,其产率为54.6%;
④ 化合物5a、5b(M3、M1):合成5a化合物的最佳条件为1.5mL浓HCl/EtOH,反应温度为20℃,反应时间35h;合成5b化合物的最佳条件为0.1～0.2 mL浓HCl,20℃,反应35h;
⑤ 丹皮酚的溴化物(F1 F2 F4-2):在Br2/无水醚和0℃下,得F2化合物;在无水甲醇或CHCl3/EtOAc和CuBr2下,得F1化合物;在无水乙醇/ CHCl3/EtOAc和CuBr2下,得F4--2化合物。F1化合物的最佳合成条件为:甲醇为溶剂,丹皮酚: CuBr2=1:2,反应时间控制在5~6h,温度在60~70℃。
3. 用MTT法筛选丹皮酚,阿散酸和8种创新化合物对HepG2细胞生长抑制情况发现;丹皮酚对HepG2细胞在较高浓度(5×10-5 mol/L以上),对细胞活力有影响。浓度越高,抑制HepG2细胞生长越显著。F1、F2、F4-2、S1和阿散酸对HepG2细胞生长抑制无显著影响。胂化物S6、R2、M1、M3在5×10-7 mol/L以上对HepG2细胞的生长有明显的抑制作用,其抑制作用具有时间依赖性。随着时间的增加细胞凋亡比例也在不断增加。随着剂量的增加细胞凋亡的比例也在不断的增加。四种胂化物在1×10-6 mol/L和5×10-7 mol/L时对HepG2细胞毒性较小,对HepG2抑制率大小顺序为:S6>R2>M3 >M1。其初步判断其抑制效果是通过凋亡引起的。
4. 从细胞形态学观察(光学普通显微镜,荧光显微镜,透射电镜)的结果定性地说明,其抑制HepG2细胞生长是由凋亡引起的,且有时间和剂量依赖特性。高浓度时出现坏死,低浓度在时间延长时也有部分坏死现象出现。当四种胂化物在浓度为1×10-6 mol/L、5×10-7 mol/L时,光学显微镜观察只有凋亡现象,荧光显微镜观察到的即有细胞凋亡,又有细胞坏死。电镜观察典型的凋亡现象和少量坏死细胞出现。其结果与MTT法一致。
5. 采用PI单染和Annexin V/PI双染流式细胞仪检测,PI单染时,浓度为5×10-7 mol/L、1×10-6 mol/L四种胂化物对HepG2细胞的凋亡率大小顺序为S6>M3>R2>M1,此定量结果与MTT法一致。胂化物诱导细胞凋亡具有周期特异性。主要特异性抑制细胞G0/G1期(G0/G1期细胞所占百分比明显下降),使S期和G2/M期细胞比例增加。 Annexin V/PI双染时,四种胂化物对HepG2细胞随时间的推移,细胞凋亡率增加,细胞坏死率也相应增加但其增幅较小,即细胞凋亡率远大于细胞坏死率,说明胂化物主要是通过诱导细胞凋亡来实现对肿瘤细胞的杀伤作用的。
6. 通过加入外源性SOD和对胞液SOD的测量,发现胂化物引起细胞凋亡的过程,有活性氧的参与,并且胂化物还导致细胞内SOD值的减少,正是由于这种降低,才导致了细胞内特别是线粒体中活性氧大量聚集,启动了细胞凋亡过程,通过对SOD值的分析,证明了活性氧在细胞凋亡中的作用。
7. 通过对线粒体膜电位的测定发现,R2作用后HepG2细胞线粒体跨膜电位下降,且呈时间、剂量依赖性,说明线粒体膜电位的降低可能是R2诱导HepG2细胞
凋亡的机制之一。
8.
The generation and evolution of hepatoma are related not only to abnormal differentiation and over-proliferation of hepatoma cells but also to apoptosis decrease. To develop the new drugs with inducing apoptosis of tumor cells will be a new strategy for curing cancer. Since arsenic trioxide has successfully used to cure acute promyelocytic leukemia(APL), arsenic compounds have then used to cure cancer since beginning of 1970s. From that, a series of arsenic compound are studied. Effect of arsenic compounds on cancer has paid increasing attention. Beyond that, paeonol is reported to be an effective against cancer in vivo and in vitro and be able to induce the apoptosis of tumor cells and affect the express of apoptotic genes. From these point of views, combining arsenic compounds with paeonol might be new drugs for anti-inflammatory, and its promotion of blood flow and anti-tumor. In this present, a series of derivatives of paeonol with arsenic compounds are synthesized, all of which are innovative compounds. The synthetic conditions of these compounds have been studied by orthogonal experiment and variance analysis. The optimization on extractive conditions of paeonol has been also studied. Anti-tumor activity of the innovative compounds on HepG2 cell line is screened, and the most effective and the optimal testing concentration are determined. Attempting to find out a kind of new organic arsenic drugs, simple structure and apoptosis induced to tumor cell in vivo and in vitro at a lower concentration, some methods are used by men of the growth inhibition assay, apoptotic analysis, and effective mechanism with HepG2 cell line.
The main results and conclusions are as follows:
1. The optimal extractive conditions of paeonol
The supercritical carbon dioxide is used to extraction of paeonol from Dabpi(a Chinese herb). The optimal extractive condition is obtained by orthogonal experiment, and the extractive effect, that is, extractive temperature is 45℃, the extractive pressure is 100Bar, and the extractive time is 75 minutes. The extraction rate is 94.1%.
2. Eight synthesized derivatives of paeonol with arsenic compounds
2-hydroxy-4-methoxy-acetophenonesemi-p-aminobenzene(3a), 2-hydroxy-4-methoxy-acetophenonesemi-p-aminobenzenearsonic acid(3b),
2- methoxy-4-hydroxy-5- acetylazobenzene-4’-arsonic acid(4a),
1-o-hydroxy-p-methoxybenzoyl-2-p-aminobenzenearsonicacidethane(5a),
1-o-hydroxy-p- methoxybenzoyl-2-phenyl-2-p-aminobenzenearsonic acid-ethane(5b),
2’-hydroxy-4’-methoxybenzoyl-bromomethane(F1), 2’-hydroxy-4’-methoxy-5’-bromoacetophenone(F2), 2’-hydroxy-4’-methoxy-5’-bromobenzoyl- bromo-methane(F4-2), respectively.
All of the eight compounds are innovative compounds, and the optimal synthetic conditions were as the following:
● The yield of compound 3a (S1) reaches a maximum of 25.1% in such conditions as: (1) n (paeonol) :n (aniline)= 1:1; (2)The solution is normal butyl alcohol;(3) The refluxing time is 2 hours; (4)The refluxing temperature is 118℃.
● The yield of compound 3b (S6) reaches a maximum of 28.4% in such conditions as:(1)The mol ratio between paeonol and arsanilic acid is 1:1 in normal butyl alcohol solution; (2)The refluxing time is 3 hours.
● Changes of the mol ratio between paeonol and arsanilic acid have no effect on the yield of compound 4a (R2). The yield is 54.6% in these conditions: (1)The reaction temperature is controlled under 5℃; (2)The reaction time is 3 hours; (3)The pH value of reaction medium range from 8 to 10.
● The optimal synthetic conditions of compound 5a (M3) are 1.5mL concentrated HCl/EtOH, the reaction temperature of 20℃, and the reaction time of 35 hours. The optimal synthetic conditions of compound 5b (M1) are 0.1-0.2mL concentrated HCl, the reaction temperature of 20℃, and the reaction time of 35 hours.
● Paeonol bromide (F1, F2, F4-2): (1)compound F2 is synthesized in the condition of Br2/ absolute ether and a temperature of below zero;(2)Compound F1 is synthesized in the condition of absolute methanol or CHCl3/EtOAc and CuBr2; (3)Comp
引文
[1] 章灵华,肖培根,黄艺等. 丹皮酚的药理与临床研究进展. 中国中西医结合杂志,1996,16(3):187-190
[2] 孙国平,沈玉先,张玲玲,周爱武,魏 伟,徐叔云. 丹皮酚对HepA荷瘤小鼠免疫调节和抑瘤作用研究. 中国药理学通报,2003,19(2):160-162
[3] 孙国平,沈玉先,张玲玲等. 丹皮酚的体内外抗肿瘤作用. 安徽医科大学学报,2002,37(3):183-185
[4] Lin Q S., Fu F Y. Component Chemistry of Chinese Herbal Medicine(Zhong Cao Yao Cheng Fen Hua Xue). People’s Medical Press, China, 1980
[5] Y. L. Kwong, D. Todd. Delicious Poison: Arsenic Trioxide for the Treatment of Leukemia. Blood, 1997, 89(9): 3487
[6] 张鹏,王树叶,胡龙虎等. 三氧化二砷注射液治疗急性早幼粒细胞白血病72例. 中华血液学杂志, 1996, 17(2):58-60
[7] Mer J. Ancient Remedy Performes New Tricks. Science, 1996, 273(2): 578-580
[8] Look AT. Arsenic and apoptosis in the treatment of acute promyelocytic leukemia. Natl Cancer Inst, 1998, 90(3): 86-88
[9] Gollagher RE. Arsenic:new life for an old potion. N Engl J Med, 1998, 339(19): 1389-1391
[10] Tamm I, Paternostro G, Zapata J M. Treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med, 1999, 340(16): 1043-1045
[11] Kroemer G,de The H. Arsenic trioxide,a novel mitochondriotoxic antichacer agent. J Natl Cancer Inst, 1999, 91(9): 743-745
[12] Vincent PC. Arsenic: a new place. Pathology, 1999, 31(2): 81-82
[13] Ming D S., Qi X M., Xie Y., et al. Dimensionality and lock-in transition in Tl2Ba2CuO6 single crystal J. of Shanxi Medical Univ.(Shanxi Yixueyuan Xuebao),1994,25(30):291
[14] Duan X X. Handbook of Practicality Fine Organic Synthesis,Chemical Industry Press. China. 2000
[15] Tang D S. Chinese Traditional Medicine Chemistry,People’s Medical Press. China. 1986
[16] 孟庆华,冀亚飞,张燕秋 . 丹皮酚的提取及其衍生物的合成 ,徐州师范大学1998,16(2):41
[17] 方岩雄,黄剑明,张昆.用超临界CO2从丹皮提取丹皮酚. 精细化工,2001,18(4):201
[18] 赵凤岭, 陆明旦. 2-羟基-4-甲氧基苯乙酮的制取及其结构测定.山东轻工业学院学报,1996,10(2):48
[19] Zhang P.,Wang S Y.,Hu L H., et al. Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging, Chinese J. of Hematology (Zhonghua Xueyexue Zazhi), 1996, 17(2):12
[20] State Pharmacopeia Commission of Ministry of Health P.R. China. Pharmacopeia of Ministry of Health P.R. China.(1),People’s Medical Press. China. 1979
[21] Afzal M etal . A modified method or the synthesis of O-hydroxy aromatic aldehydes and ketones. Bull Col Sci., 1973, 14:41
[22] Wang X Y. Evidence of a role for neuropeptide Y and monoamines in mediating the appetite suppressive effect of growth hormone Journal of Mathematical Medicine, 2000, 13(4): 344
[23] Chen G Q et al . Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia(APL). Blood ,1997,89(9): 3345
[24] 章灵华,肖培根,黄艺.丹皮酚的药理与临床研究进展,中国中西医结合杂志, 1996,16(3):187-190.
[25] XIU Yu-Bin(秀宇彬).Anti-cancer Chinese Herbal Medicine Pharmacology and Application(抗癌中药药理与应用),Haerbin(哈尔滨):Heilongjiang science technique Press(黑龙江科学技术出版社),1999:175
[26] SUN Guo-Ping(孙国平),SHEN Yu-Xian(沈玉先),ZHANG Ling-Ling(张玲玲).丹皮酚的体内外抗肿瘤作用. J. Anhui Medi. Univ.(安徽医科大学学报),2002,37(3):183
[27] House H O Auth.(著),HUA Wen-Ting(花文廷)transl.(译).The Modern Synthesis Reaction(现代合成反应), Beijing(北京):Beijing University Press(北京大学出版社), 1985:360
[28] 陈俊杰,李裕林.多羟基苯乙酮的α-溴化反应研究. 厦门大学学报(自然科学报), 1993,32(2):249-251
[29] 章灵华,肖培根,黄艺.丹皮酚的药理与临床研究进展,中国中西医结合杂志, 1996,16(3):187-190.
[30] 秀宇彬.抗癌中药药理与应用,哈尔滨:黑龙江科学技术出版社,1999. 175-680.
[31] 孙国平,沈玉先,张玲玲.丹皮酚的体内外抗肿瘤作用,安徽医科大学学报 ,2002,37(3):183-185
[32] 韩广甸,赵树伟,李述文,等编译.有机制备化学手册(中卷),北京:石油化学工业出版社,1977.304.
[33] L. Carroll King and G.Kenneth Ostrum. Seletive Bromination with Copper(II) Bromide J. Org. Chem.,1964,29:3459-3462.
[34] 陈俊杰,李裕林. 多羟基苯乙酮的α-溴化反应研究厦门大学学报(自然科学版),1993,32(2):249-251.
[35] K. B. Doifode and M. G. Marathey. Reactions of Cupric Bromide in Dioxane Formation of
Bromo-ohydroxyacetophenone J. Org. Chem., 1964,29:2025-2026.
[36] CALLIGARIS M R. Comprehensive Coordination Chemistry Oxford Pregamon . Press. 1987
[37] RENCH F A, BLANZ F J. The carcinostatic activity of alpha-(N) heterocyclic carboxaldehyde thiosemicarbazones. I. Isoquinoline-1-carboxaldehyde thiosemicarbazone Cancer Research,1965, 25(9):1454
[38] Scovil J P, KLAYAN D L , FRANCHINO C F. 2-Acetylpyridine thiosemicarbazones. 4. Complexes with transition metals as antimalarial and antileukemic agents . J. Med. Chem., 1982,25(10 ): 1261
[39] 乐芝凤,祝心德,陈金池等.含硫席夫碱的合成、表征和抗菌活性. 华中师范大学学报,1989,23: 1
[40] 田君濂. 毕思玮. 高恩庆.等. 甲酰基甲酸氨基硫脲席夫碱二价金属离子配合物的研究, 应用化学, 1994, (1): 5
[41] 陈新斌,杨艳,桂明德. Schiff碱配合物拟酶催化反应的取代基效应研究, 高等学学校化学学报, 2000, 21(11): 1684-1687
[42] 王红 李淑兰,刘德信.邻香兰素与邻氨基苯甲酸Schiff碱铜(Ⅱ)配合物的合成、晶体结构和热分解动力学 高等学学校化学学报, 1994,15(4):485-489
[43] 李淑兰,刘德信,崔学桂. 邻香兰素乙二胺合镍(II)配合物的合成研究. 山东大学学报, 1993,28(4):422-451
[44] Ferraro J R, Basile L J, Garcia-Ineguez,et al. Concerning the thermochromic mechanism of copper(II) and nickel(II) complexes of N,N-diethylethylenediamine Inorg. Chem., 1976,15(10):2342
[45] Hadjoudis E, Vitiorakis M, Moustakali-Marridis L. Photochromism and thermochromism of schiff bases in the solid state and in rigid glasses Tetrahedron, 1987,43(7):1345
[46] Joshi G B ,Patel G S,Tthakor V M. J .Indian Chen Soc,1975, 52(1):47
[47] Joshi G B ,Patel G S, Thakor V M. J. Indian Chem. Soc.,1974,51(5):573
[48] Guerrio p, Casellato U , Tamburini S , et al. Lanthanide complexes with compartmental schiff bases Inorg. Chem. Acta , 1987 129:127
[49] 姚克敏,李冬成,沈联芳. 镧系与邻氨基苯甲酸型Schiff碱配合物的合成、表征及催化活性. 化学学报,1993,51(7):677-682
[50] 黄世伟,刘萍,芳氧乙酸的快速合成. 化学试剂,1992,14(5):313
[51] 谢晶曦,常俊标,王绪明编著. 红外光谱在有机化学和药物化学中的应用(修订版),北京:科学出版社,2001,12
[52] 张汉辉, 郑戚编著. 波谱学,厦门:厦门大学出版,1998.6
[53] 邵建本, 刘益, 杨婷蓉. 新的氟哌酸衍生物合成及其体外抑菌试验. 西南国防医药, 1994, 4
(4):193-195
[54] 刘波, 高惠强. 苯并噻唑偶氮吡唑和苯并噻唑偶氮嘧啶的合成. 有机化学 1994, 14(2):206-210
[55] 邢其毅, 徐瑞秋, 周政编.《基础有机化学(下册)》. 北京: 高等教育出版社 1983. 9 p719-720
[56] W. Carruthers. Some Modern Methods of Organic Synthesis.(Third Edition) Cambridge University Press 1986
[57] R.T. Morrison, R.N. Boyd. ORGANIC CHEMISTRY (4rd ed)Allyn and Bacon 1983
[58] A. H. 勃拉特主编, 南京大学化学系有机化学教研组译. 有机合成(第二集). 北京: 科学出版社, 1964. 4
[59] 万道正. 曼尼希反应和曼尼希碱化学. 北京: 科学出版社, 1986
[60] Tramontini M, Angiolini L, Mannich Bases: Chemistry and Uses, CRC Press Inc, Boca Raton, FL1994
[61] Tramontini M, Angiolini L. Further advances of the chemistry of Mannich bases . Tetrahedron, 1990 , 46(6): 1791
[62] Michael A, Bernhard W, Nikolaus R. Angew Chem Int Ed ,1998 , 37: 1045
[63] Yi Lin, Zou Junhua , Lei Huangshu, et al. The Mannich Reaction of Cyclic Ketones, Aromatic Aldehydes and Aromatic Amines. Org. Prep Proced Int, 1991, 23(5): 673-676
[64] 邹君华. 间硝基苯甲酮基或间氯苯甲醛与间氯苯甲醛与芳香胺和芳香酮的Mannich反应. 有机化学. 1996, 16: 218-222
[65] 易林, 邹君华, 徐秀娟. 芳香酮与芳香醛及芳香胺的Mannich反应. 化学通报, 1991,(5): 20-21
[66] 范莉, 杨大成. 对氨基苯甲酸乙脂与4-苯基-2-丁酮和芳香醛的Mannich反应. 西南师范大学学报(自然科学版), 1998, 23(6): 726-729
[67] 雷皇书, 林晓梅, 王宁, 邹君华. 芳香胺的Mannich反应研究. 西南师范大学学报(自然科学版), 1995, 20(1): 44-48
[68] 杨大成, 顾雪元, 奉萍. 对氨基苯甲酸乙脂与苯乙酮和芳香醛的Mannich反应. 西南师范大学学报(自然科学版), 1997, 22(1): 54-57
[69] 杨大成, 章国林, 杨宁等. 对-甲苯乙酮与芳香醛和芳香胺的Mannich反应. 高等学校化学学报, 2000, 21(11): 1694-1696
[70] Yi Lin, Zou Junhua, Xu Xiujuan, et al. The Mannich reaction between aromatic ketones, aromatic aldehydes and aromatic amines. Synhesis(Gr), 1991, 9: 717
[71] B. shivarama Holla, B. Veerendra , M. K. Shivananda, et al. Synthesis Characterization and anticancer activity studies on some Mannich bases derived from 1,2,4-triazoles. European
Jouenal of Medicinal Chemistry, 2003, 38: 759-767
[72] 董金华, 徐莉英 , 计志忠. 2-取代环戊(烯)酮芳胺Mannich碱的合成及抗炎活性. 中国药物化学杂志, 1998, 8(1): 5-9
[73] Gul, H. Inci; Vepsalainen, Jouko ; Gue, Mustafa; et al. Cytotoxic activities of mono and bis Mannich bases derived from acetophenone against Renca and Jurkat cells. pharmaceutica Acta Helvetiae, 2000 , 74(4) :393-398
[74] Dimmock, J R; Erciyas, E; Kumar, P; et al. Mannich bases of phenolic azobenzenes possessing cytotoxic activity. European Journal of Medicinal Chemistry , 1997 , 32(7-8): 583-594
[75] Adam Fedorwicz, Janez Mavri, Piotr Bala, et al. Molecular dynamics study of the tautomeric equilibrium in the Mannich dase. Chemical Physics Letters , 1998, 289: 457-462
[76] Alexander, E. R., Underhill , E. J . Amer. Chem. Soc., 1949, 71: 4014
[77] Jhomas, F. Cummings, J. Reid Shelton. J. Org. Chem., 1960, 25: 419
[78] Samoilenko, T. G. et al. Kauch. Rezina , 1976, (1): 22-23
[79] Dryanska Veneta, Jasheva. Mikhova Bozhanka . Phas-transfer-catalyzed additions, part13. A convenient synthesis of α、β-diary-β-arylamino ketones by reaction of aiky l benzylketones and azomethines. J. Chem. Synop., 1995, 10: 418-419
[80] Nguyen Minh Khoi, Tran Manh Hung Giang Thi Son, Chu Thi Loc, et al. Synthesizing β-amino ketones derivatives of benzyliden-aryl-amine. Tap Chi Duoc Hoc., 1995 , (5) : 1、 3(Vietnameses)
[81] Zou Junhua . The Mannich reaction of diakyl ketones, aromatic aldehydes and aromatic amines. Org Prep Proceolint , 1996 , 28(5): 554-558
[82] 邹君华, 易林. 苯乙酮、 对甲氧基苯甲醛和芳香胺的Mannich反应. 西南师范大学学报(自然科学版), 1991, 16(1): 66-70
[83] 邹君华, 易林. 芳香胺参加的Mannich反应-3-芳胺基-3-苯基-1-(4-溴苯基)丙酮的合成. 化学研究与应用,1991, 3(2): 87-88
[84] 邹君华, 易林. 苄叉丙酮与芳香醛和芳香胺的Mannich反应. 西南师范大学学报(自然科学版), 1998, 23(3): 298-302
[85] 陈胡兰, 邹君华. 对甲氧基苯乙酮与苯甲醛(对甲氧基苯甲醛)、芳香胺的Mannich反应. 西南师范大学学报(自然科学版), 1999, 24(2): 190-195
[86] Gautier, J. A., Moicque, M., Quan, D. Q. The Mannich reaction of aromatic aldehydes and aromatic amines . Acad Sci., 1964 , 258 :3731
[87] Brands, R. , Roth , H. J. The Mannich reaction of aromatic amines . Arch. Pharm., 1967, 300 : 1005
[88] 陈光旭 , 徐秀娟. 芳香胺的Mannich反应研究 化学学报, 1982, 40 :463
[89] Bedoya , L.M ; Sanchez-Palomino S ; Abad M.J. Anti-HIV activity of medicinal plant extracts. Joural of Ethnopharmacology ,2001, 77(1): 133—11
[90] Mosmann T. Rapid Colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983, 65(1) :55
[91] Carmichael J, Degraff W G, Gazdar A F, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing . Cancer Res, 1987, 47(4): 936
[92] 郑耘,杨善民,颜江华,陈瑞川. MTT比色法在测定细胞活性中的影响因素. 福建医学院学报,1995,29(6):195—198
[93] Sargent JM , Talyor CG. Appraisal of the MTT assay as a rapid test of chemosensitiviy in acute myeloid leukemia . Br .J . Cancer, 1989, 60 : 206
[94] Twentyman PR , Fox NE , Rees JKH . Chemosensitivity testing of fresh leukmia cell using the MTT colorimetric assay. Br J Haematol , 1989, 71: 19
[95] Denizot F. Rapid colorimetric assay for cell growth and survial modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Method , 1986, 89:271
[96] 艾立平,任中原 . 培养HepG2细胞的几点体会. 天津医学院学报, 1994,18(13):27
[97] 孙国平,沈玉先,张玲玲等. 丹皮酚的体内外抗肿瘤作用. 安徽医科大学学报,2002,37(3):183-184
[98] Styblo M , Yamauchi H , Thomas D J . Comparative in vitromethylation of trivalent and pentavalent arsenical. Toxicol Appl Pharmacol, 1995, 135(2) :172-178
[99] 赵卫红、秦好长、闫福岭主编. 细胞凋亡. 河南省:河南医科大学出版社,1997
[100] 杨广烈. 荧光与荧光显微镜. 光学仪器,2001,23(4):18-29
[101] 黄守固,孔北华. 三氧二砷对人卵巢癌细胞株3AO细胞增殖和凋亡影响的研究. 山西医科大学学报,2001, 32(5):403-405
[102] 邓友平,林晨,陈洁平. 三氧化二砷诱导人宫颈癌雪白凋亡及Bcl-2高表达对其影响. 中国药物学与毒理学杂志,1999,13(4):288
[103] 聂林,张洹. 三氧化二砷对恶性淋巴瘤细胞株凋亡研究. 肿瘤,2001,21(2):79
[104] 林晨,邓友平,郑杰. 三氧化砷诱导人肿瘤细胞凋亡和G2+M其阻滞但引起人永生化 宫颈上皮细胞G1其阻滞. 中国医学科学院学报,200,22(2):124-129
[105] 陈洪,秦叔逵,陈惠英. 三氧化二砷诱导人肝癌细胞株SMMC-7721凋亡的试验研究. 肿瘤防治研究,1998,25(5):336
[106] 闻勤生,罗俊卿,焦成松. 三氧化二砷诱导肝癌细胞HepG2凋亡作用。第三军医大学学报,2001,23:913-914
[107] Watson NC,Di YM,Orr MS. Influence of ioinzing radiation on proliferation,c-myc expression and the indiation of apoptotic cell death in two breast tumor cell lines differing in
p53 status. Int. J Radiat Biol,1997,72(5):547-559
[108] Skouteris GG, Schroder C H. C-myc is required for the G0/G1-stransition of primary hepatocytes stimulated with a deleted form of hepatocytes growth factor. Biochem,1996,316:879-886
[109] 周玫、陈瑗、脂质过氧化作用及其对细胞的损伤. 自由基医学. 北京:人民军医出版社,1992:30-48
[110] Loun JW. Molecular mechanism of action of anticancer agents involving free radical intermediates Adv Free Radical Biol Med .1985,1(4):225
[111] Rubin R ,Farber JL. Mechanism of the killing of cultured hepatocyyes by hydrogen peroxides .Arch Biochen Biophys 1984, 228 (6) : 450
[112] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mam malian organs . Physiological Reviews , 1979, 59(3) : 527-605
[113] Xu J X,Li X,Zhang Y X,et al.In: Lester P.eds.Mitochondrial respiratory chain:A Salf defense system against oxygen toxicity Proceedings of the international symposium on natural antioxidants: molecular mechanis and health effects . Champaign , Illinois : AOCS Press 1996 , 530-539
[114] Allen R G, Balin A K . Oxidative on development and differentiation: An overview of a free radical theory of deveiopment. Free Radical Biology & Medicine , 1989 , 6(6) : 631-661
[115] Skulachev V P . Role of uncoupied and non coupled oxidations in maintenance of safely low levels of oxygen and its one electron reductants. Quart Rev Biophys , 1996 , 29(1) :169-202
[116] Lass A, Sohal R S. Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species Free Radic Biol Med , 1999 , 27(1/2) :220-226
[117] Skulachev V P. Why are mitochondria involved in apoptosis. FEBS Lett , 1996 , 397 : 7-10
[118] Zoratti M, Szabo I.The mitochondria permeability transition. Biochim Biophys Acta , 1995 , 1241(2) : 139-176
[119] Skulachev V P.Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett,1998, 423(2) :275-280
[120] Orrenius S. Mechanisms of oxidative cell damage. In : Poli G eds. Free Radicals;From Basel , 1993 : 47-64
[121] Ma Y X, Ogino T, Kawabata T et al. Cupric nitrlotriacetate induced apoptosis in HL-60 cellsassociation with lipid peroxidation, release of cytochrome c from mitochondria, and activation of caspase-3. Free Radic Biol Med , 1999, 27 (1/2) : 227-233
[122] Karbowski M, Kurono C, Wozniak M, et al. Free radical-induced megamitochondria formation and apoptosis. Free Radic Biol Med, 1999 ,26(1/2) : 396-409
[123] Yoshida H, Kong Y Y, Yoshida R, et al. Apafl is required for mitochondria pathways of apoptosis and brain development. Cell , 1998 , 94(6) : 739-750
[124] Marzo I, Brenner C, Zamzami N, et al. Bax and adenine nucleotide translocator cooperate in the mitochondria control of apoptosis. Science, 1998, 281(5385) : 2077-2031
[125] Finucane D M, Wetzel E B, Waterhouse N J, et al. Bax-induced caspase activation and apoptosis via cyyochrome c release from mitochondria is inhibitable by Bcl-Xl. J Biol Chem, 1999, 274(4) :2225-2233
[126] Ptonka A. Biochem Biophys Res Commun, 1978, 81:1344
[127] Stevez E, Alvarog G, Sampson J, Acinda B ey al. Liposome-deivered Superoxide dismutase prevents nitric oxide dependent motor neuron death induced by trophi factor withdrawal. Free Radical Biology ang Medicine, 2060, 3(28): 437-446
[128] Barbara A, Elke B, Martina P et al. Retinonc acid reduces apoptosis and oxidative stress by preservation of SOD Protein level. Free Radical Biology and Medicine. 2001, 10(30): 1067-1077
[129] 张宝. 锰超氧化物歧化酶过表达对H2O2诱导PC12细胞凋亡的影响.第一军医大学学报,2000,20(6): 510-512
[130] 王展翔、廖军鲜、许勇钢等. 抗氧化剂对三氧化二砷诱导HL-60细胞凋亡作用的影响.白血病,2000,3(9):153-155
[131] Yabuki Munehisa, Kariya Shinji, Ishisaka Rumi, ey al. Resistance to nitric oxide—mediated apoptosis in HL-60 Variant cell is associated with increased activities of Cu, Zn-superoxide dismutase and catalase. Free Radical Biology and Medicine, 1999, 3-4(26): 325-332
[132] 聂崇兴、赵双星. MTT法测定人红细胞超氧化物歧化酶总酶活力.湖南医科大学学报,1995,20(5): 485-487
[133] Kroemer G, Zamzami N, Susin S. Mitochondrial control of appoptosis. Immumol Today, 1997, 18 (1) :44-51.
[134] Green D R, Read J C. Mitochondria and apoptosis. Science, 1998, 281(5381): 1309-1312.
[135] Marchetti P, Castedo M, Susin SA, et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med, 1996, 184: 1155-1162
[136] Zamami N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med, 1995, 181: 1661-1672.
[137] Zamzami N, Marchetti P, Castedo M, et al. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell
death. J Exp Med, 1995, 182(2): 367-377
[138] Nguyen N, Branton PE, Walton PA, et al. Role of membrane anchor dormain of Bcl-2 in suppression of apoptosis caused by EIB-defective adenovirus. J Biol Chem, 1994, 269(24): 16521-16524
[139] Osborne BA, Smith SW, Liu ZG, et al. Identification of genes induced during apoptosis in T cells. Immunol Rev, 1994, 142(12): 301-320
[140] Wolvetang EJ, Johnson KL, Krauer K, et al. Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett, 1994, 339(1): 40-44.
[141] Pu YS, Hour TC, Chen J, et al. Arsenic trioxide as a novel anti-cancer agent against human transitional carcinoma-characterizing its apoptotic pathway. Anticancer Drugs, 2002, 13(3): 293-300
[142] Nakagawa Y, Akao Y, Morikawa H, et al. Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines. Life Sci, 2002, 70(19): 2253-2269
[143] 袁伯俊,王治乔主编. 新药临床前安全性评价与实践. 北京 军事医学科学出版社,1997 . 11
[144] 陈奇. 中药药理研究方法学. 北京:人民卫生出版社 ,1994 .
[145] 徐叔云. 药理实验方法学. 北京: 高等教育出版社, 1995:204
[146] 纪云晶. 实用毒理学手册. 北京:中国环境科学出版社 ,1991 . 4
[147] Acute oral Toxicity-fixed dose procedure. OECD guideline for testing of chemicals. 2001