【作者】 祁俊生；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 生物医学工程， 2004， 博士

【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%; <WP=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<WP=7>凋亡的机制之一。8.更多还原

【Abstract】 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 paeonolThe 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), <WP=9>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更多还原