【作者】 郭瑞霞；

【导师】 史清文； 李力更；

【作者基本信息】 河北医科大学 ， 药理学， 2014， 博士

【摘要】 第一部分槲皮素的结构修饰及活性评价槲皮素是一种分布最广泛的黄酮类化合物，约有100多种植物中含有槲皮素。研究发现槲皮素具有广泛的药理作用和生物活性，如抗氧化、抗癌、抗菌、抗病毒及镇痛作用等，因此对槲皮素及其衍生物的生物活性研究越来越受到关注。然而槲皮素脂溶性较差、生物利用度较低，使其开发利用受到了限制。槲皮素-O-甲基化衍生物是在体内代谢的中间体并被认为是最有生物活性的结构，所以对槲皮素进行甲基化修饰及活性研究具有相当重要的意义。目的：以槲皮素为研究对象，通过直接甲基化和先用保护基对槲皮素的羟基进行保护，然后再甲基化两种途径，获取槲皮素甲基化衍生物，用1H NMR、13C NMR、HSQC和HMBC等鉴定化合物结构，对结构修饰的产物进行抗肝癌活性筛选，为发现新的药物先导化合物做出一些基础研究工作。方法：将精制后的槲皮素和适量的DMF置于三口烧瓶中，加入适量的无水碳酸钾，室温搅拌10min后加入甲基化试剂碘甲烷，在室温或0℃反应，TLC监测至反应结束，用稀盐酸将反应液调至中性，乙酸乙酯萃取3次，合并萃取液，水洗，无水硫酸钠干燥过夜，旋蒸除去溶剂，残液用硅胶柱色谱进行分离，根据反应温度和反应物投料比的不同，可得单甲基槲皮素至四甲基槲皮素。将槲皮素溶于DMF中，加入适量的无水碳酸钾，室温搅拌10min后加入苄基化试剂氯化苄，60℃反应，TLC监测至反应结束，用稀盐酸将反应液调至中性，乙酸乙酯萃取3次，合并萃取液，水洗，无水硫酸钠干燥过夜，旋蒸除去溶剂，残液用硅胶柱色谱进行分离，根据反应温度和氯化苄用量的不同，可主要得三苄基槲皮素和四苄基槲皮素，然后再用甲基化试剂碘甲烷对苄基槲皮素分别进行甲基化。用二氯二苯甲烷对槲皮素的邻二酚羟基进行保护，然后再用甲基化试剂碘甲烷对其进行甲基化。产物应用ESI-MS、1H NMR鉴定化合物的结构、部分化合物经13C NMR、HSQC和HMBC等鉴定结构。结果：不用保护基，直接用碘甲烷对槲皮素进行甲基化，室温条件下反应主要得3,7,3′,4′-O-四甲基槲皮素（2a）和3,7,4′-O-三甲基槲皮素（2b），0℃反应，主要得7,4′-O-二甲基槲皮素（2c）、3,7-O-二甲基槲皮素（2d）和3-O-甲基槲皮素（2e）。用氯化苄对槲皮素的羟基进行保护，在70℃、氯化苄是槲皮素的5倍（摩尔数）时，主要得四苄基槲皮素（3b）；在60℃、氯化苄是槲皮素的3倍（摩尔数）时，主要得三苄基槲皮素（3a）；用碘甲烷分别对三苄基槲皮素和四苄基槲皮素进行甲基化反应，得到了三苄基甲基槲皮素（4a），而未能得到四苄基甲基槲皮素（4b）。用二氯二苯甲烷对槲皮素的邻二酚羟基进行保护得化合物5，由于5的收率太低，而未对其进行甲基化反应。结论：本研究对槲皮素进行了结构修饰，直接用碘甲烷和槲皮素反应，在不同的温度及投料比条件下，共得到5个槲皮素甲基化衍生物；先将槲皮素的羟基保护起来，然后再进行甲基反应的实验方案和直接甲基化的实验方案相比，反应步骤多，分离提纯困难、收率低。抗肝癌活性实验结果表明，槲皮素的甲基化程度越高，抗肝癌细胞（人肝癌细胞株Bel-7402、人肝癌细胞株Hep G2、大鼠肝癌细胞株SMMC-7721）增殖能力越弱。第二部分土木香的提取分离目的：以中药材土木香根为研究对象，利用硅胶柱色谱、硝酸银硅胶柱色谱等分离技术对其进行分离，获得异土木香内酯和土木香内酯单体化合物，用1H NMR鉴定化合物结构，为下一步的结构修饰奠定物质基础。方法：取粉碎后的药材约5Kg，用99%乙醇加热回流2小时提取3次，合并滤液，除去溶剂得浸膏，即土木香根的粗提物,约600g。将得到的土木香浸膏分散于氯化钠饱和水溶液中，依次用石油醚、二氯甲烷进行萃取，得石油醚提取物浸膏约135g、二氯甲烷提取物浸膏约70g。采用硅胶柱色谱、硝酸银硅胶柱色谱对以上部两种浸膏进行分离可得异土木香内酯和土木香内酯两个化合物，用1H NMR对化合物的结构进行鉴定。结果：通过乙醇提取、石油醚和二氯甲烷萃取和柱色谱等步骤从土木香根中分离得到异土木香内酯和土木香内酯两个化合物，为下一步的结构修饰奠定了物质基础。结论：本研究主要是从土木香根中提取其主要活性成分异土木香内酯和土木香内酯，经过乙醇提取、石油醚和二氯甲烷萃取、柱色谱分离等步步骤可以得到异土木香内酯和土木香内酯。第三部分异土木香内酯的结构修饰—Heck反应目的：以异土木香内酯为研究对象，通过Heck反应对其进行结构修饰，通过1H NMR数据对化合物的结构进行鉴定。对结构修饰的产物进行抗肝癌活性筛选，为发现新的药物先导化合物做出一些基础研究工作。方法：在醋酸钯和三（邻甲苯基）膦的催化下和三乙胺的存在下，在DMF溶液中加入碘代芳烃对异土木香内酯进行结构修饰，120°C加热反应10-12h，反应完毕后用乙酸乙酯萃取，最后通过硅胶柱色谱进行分离纯化，洗脱剂为石油醚-乙酸乙酯（15:1）。用MTT法测定异土木香内酯衍生物3a-3d对三种肝癌细胞的抑制活性。结果：优化了Heck反应的合成条件：醋酸钯和三（邻甲苯基）膦是最佳的催化体系，温度120℃反应10-12h。反应物萃取之后用硅胶柱层析法进行分离纯化，得对氯苯异土木香内酯（9a）、对苯异土木香内酯（9b）、对甲苯异土木香内酯（9c）和对甲氧基苯异土木香内酯（9d）4个化合物，收率60-91%。结论：本研究建立了用碘代芳烃对异土木香内酯进行结构修饰的方法，优化了反应条件。用MTT法测定异土木香内酯衍生物9a-9d，对氯苯异土木香内酯（9a）和对甲氧基苯异土木香内酯（9d）对Hep G2肿瘤细胞的增殖抑制活性和紫杉醇基本相同，优于顺铂。第四部分异土木香内酯的结构修饰—Michael加成反应目的：以异土木香内酯为研究对象，用活性亚甲基化合物通过Michael加成反应对其进行结构修饰，产物通过1H NMR、13C NMR、HRMS进行结构鉴定。对结构修饰的产物进行抗肝癌活性筛选，为发现新的药物先导化合物做出一些基础研究工作。方法：将活性亚甲基化合物溶于乙醇中，加入适量的碱催化剂后室温搅拌，然后再加异土木香内酯，室温搅拌反应，TLC监测反应进程，反应混合物调为中性用后乙酸乙酯萃取、然后通过硅胶柱色谱进行纯化，洗脱剂为石油醚-乙酸乙酯（15:1）。用MTT法测定异土木香内酯衍生物11a-11g对三种肝癌细胞（人肝癌细胞株Bel-7402、人肝癌细胞株Hep G2、大鼠肝癌细胞株SMMC-7721）的抑制活性。结果：以异土木香内酯为底物，用7种活性亚甲基化合物通过Michael加成反应对其进行结构修饰，共得到了7个异土木香内酯衍生物，即氰乙酸甲酯异土木香内酯（11a）、氰乙酸乙酯异土木香内酯（11b）、丙二酸二甲酯异土木香内酯（11c）、丙二酸二乙酯异土木香内酯（11d），硝基甲烷异土木香内酯（11e）、硝基乙烷异土木香内酯（11f）、乙酰乙酸乙酯异土木香内酯（11g）所有化合物经核磁氢谱、碳谱和高分辨质谱进行结构鉴定。以化合物11a和11c为例优化了反应条件，目标化合物收率可达71~94%。结论：本研究建立了用活性亚甲基化合物对异土木香内酯进行结构修饰的方法，优化了反应条件，合成了7个未见文献报道的化合物11a-11g。用MTT法测定合成的7个异土木香内酯衍生物的抗肝癌活性，氰乙酸甲酯（11a）异土木香内酯和氰乙酸乙酯异土木香内酯（11b）对Hep G2肿瘤细胞增殖有一定的抑制作用，其抑制活性大于顺铂，小于紫杉醇。第五部分土木香内酯的结构修饰—Michael加成反应目的：以土木香内酯为研究对象，用活性亚甲基化合物通过Michael加成反应对其进行结构修饰，所合成的化合物通过1H NMR、13C NMR对化合物的结构进行鉴定。方法：将活性亚甲基化合物溶于乙醇中，加入适量的碱催化剂，室温搅拌，然后再加土木香内酯，室温搅拌反应，TLC监测反应进程，反应混合物调为中性用后乙酸乙酯萃取、然后通过硅胶柱色谱进行纯化，洗脱剂为石油醚-乙酸乙酯（15:1）。结果：以土木香内酯为底物，用活性亚甲基化合物通过Michael加成反应对其进行结构修饰，共得到了6个土木香内酯衍生物，即氰乙酸甲酯土木香内酯（13a）、丙二酸二甲酯异土木香内酯（13b）、丙二酸二乙酯异土木香内酯（13c），硝基甲烷异土木香内酯（13d）、硝基乙烷异土木香内酯（13e）、乙酰乙酸乙酯异土木香内酯（13f），所有化合物经核磁氢谱、碳谱进行结构鉴定。结论：本研究建立了用活性亚甲基化合物对土木香内酯进行结构修饰的方法，优化了反应条件，合成了6个未见文献报道的化合物13a-13f。更多还原

【Abstract】 Part one Structural modification of quercetin and their activityevaluationQuercetin is one of the most widely distributed of flavonoids, and about100types of plants contain quercetin. It was reported that quercetin hasextensive pharmacological effects and biological activities, such asantioxidant, anticancer, antibacterial, antiviral, analgesic action and so on.Therefore, the research on biological activities of quercetin and its derivativeshas attached more and more attention. However, the development andutilization of quercetin is limited due to its poor fat-solubility and lowbioavailability. Methylated derivatives of quercetin are proposed to be themost biologically active intermediates in the metabolism of the body, so it isof great importance to develop the methylation routes of quercetin and thecorresponding biological activities.Objective: The objective of this project is to synthesize methylatedderivatives of quercetin via direct or indirect two methods, followed by thebioactivity screening against tumor cells to provide basic information forfurther drug discovery. The structures of the synthesized compounds wereelucidated by means of1H NMR,13C NMR, HSQC and HMBC. To exploidthe activities of synthesized quercetin derivatives for tumor cells by bissayscreen. We do some base research works for discovering new drug leadcompounds.Methods: The refined quercetin in DMF was added anhydrous K2CO3.After stirring at room temperature for10min, methyl iodide was added, andthe reaction was kept at room temperature or0°C until the TLC showed a fullconversion. The mixture was neutralized with HCl, extracted with EtOAc,washed with H2O and dried over anhydrous Na2SO4. After concentration and column chromatography, mono-, di-, tri-, or tetra-methylated quercetins wereobtained based on the ratio of reactants. The refined quercetin in DMF wasadded anhydrous K2CO3. After stirring at room temperature for10min,benzyl iodide was added, and the reaction was kept at room60°C until theTLC showed a full conversion. The mixture was neutralized with HCl,extracted with EtOAc, washed with H2O and dried over anhydrous Na2SO4.After concentration and column chromatography, tri-or tetra-methylatedquercetins were obtained based on the ratio of reactants. The obtainedcompounds were methylated with methyl iodide. The two ortho OH groupsof quercetin were protected with dichlorodiphenylmethane, followed bymethylation with methyl iodide. The products were characterized withESI-MS and NMR.Results: The direct methylation of quercetin without protection mainlyafforded3,7,3′,4′-O-tertmethylquercetin and3,7,4′-O-trimethylquercetin whenthe reaction was carried out at room temperature, while when it was done at0℃, the products were mainly7,4′-O-dimethylquercetin,3,7,-O-dimethylquercetin and3-O-methylquercetin. Benzylation of quercetinaffored3,7,3′,4′-O-tertbenzylquercetin when5equiv. benzyl chloride wasadded at70℃, and3,7,4′-O-tritbenzylquercetin when3equiv. benzylchloride was added at60℃. Methylation of Compound3a with methyl iodideafforded Compound4a. However, methylation of Compound3b under thesame conditions failed to produce Compound4b. Protection of the ortho OHgroups of quercetin with dichlorodiphenylmethane to obtain Compound5withreally low yield, so further methylation was not carried out.Conclusion: The structural modification of quercetin was carried out.Five methylated derivatives are synthesized from quercetin and methyl iodideunder different conditions. The indirect methylation route via protection led toseparation difficulty and low yield. The liver cancer efficacy tests showed thatmethylated quercetins are less active than the parent compound. Higher degreeof methylation led to lower bioactivity. Part Two The Extraction and Separation of Inula heleium RootsObjective: The objective of this project is to obtain isoalantolactone andalantolactone from the root of I. helenium, we used the technologies of Silicagel column chromatography, Silver nitrate silica gel column chromatography,and HPLC, etc. The structures of the pure compounds were elucidated bymeans of1H NMR. Material basis were provided for the further structuralmodification of isoalantolactone and alantolactone.Methods: The crushed roots of Inula helenium (5.0Kg) in99%ethanolwas reflux for2hr. After filtered, the alcohol extract was concentrated to yield600g crude product, which was suspended in brine and extracted withpetroleum ether, dichloromethane in turn. Two fractions were obtained:petroleum ether fraction135g and dichloromethane fraction70g. Thefractions were subjected to silica gel column chromatography and silvernitrate silica gel column chromatography to get pure compounds. Thecompounds were1H NMR, and13C NMR.Results: Isoalantolactone and alantolactone were isolated from the rootsof I.helenium through extraction and silica gel column chromatographyfurtherstructural modification.Conclusion: The main active ingredients isoalantolactone andalantolactone were isolated from the roots of I. helenium through extractionand silica gel column chromatography.Part Three Structural Modification of Isoalantolactone–Heck ReactionObjective: Structural modification of isoalantolactone was proceededthrough Heck Reaction and the synthesized compounds were elucidated bymeans of1H NMR. These compounds were evaluated toxic activities againstthree different hepatoma cell lines, Bel-7402, SMMC-7721and Hep G2so asto provide information for further drug discovery.Methods: Aromatic iodide, Pd(OAc)2, were added to a solution ofisoalantolactone in DMF. Then, add a small amount of triethylamine. Thereaction mixture was heated for10-12h at120°C. The mixture was cooledroom temperature and poured into water and extracted with ethyl acetate. After evaporation of the solvent, the residue was purified by silica gel columnchromatography. These compounds were evaluated for toxic activities againstthree different hepatoma cell lines by MTT method.Results: Optimizing the Heck reaction conditions: Pd(OAc)2, andtris(o-tolyl)phosphine was the best catalytic systems, reacted10-12hours at120℃. Compounds9a-9d were purified by silica gel column chromatography,the yields from60%to91%. Inhibitory activity of9a and9d on theproliferation of tumor cells are basically same to taxinol, supor to cisplatin andMTX.Conclusion: This study established a method to modify the structure ofisoalantolactone with aryl iodides and optimized the reaction conditions.Compounds9a-9d were evaluated for toxic activities against three differenthepatoma cell lines by MTT method. The results showed that compounds9aand9d had good inhibitory activity against hepatoma cell lines.Part Four Structural Modification of Isoalantolactone–MichaelReactionObjective: The objective of this project is to modify isoalantolactonethrough Michael reaction and the synthesized compounds were elucidated bymeans of1H NMR,13C NMR and HRMS. These compounds were evaluatedtoxic activities against three different hepatoma cell lines, Bel-7402,SMMC-7721and Hep G2so as to provide information for further drugdiscovery.Methods: Active methylene compounds and proper base were added to asolution of isoalantolactone in anhydrous EtOH. The mixture was stirred atroom temperature for2h. The reaction mixture was adjusted to neutral withdilute hydrochloric acid solution. After evaporation of the solvent, the residuewas purified by silica gel column chromatography. The synthesizedcompounds were evaluated for toxic activities against three different hepatomacell lines by MTT method.Results: Seven isoalantolactone derivatives11a-11g were synthesizedthrough Michael reaction. These compounds were elucidated by means of1H NMR,13C NMR and HRMS. To exploid the active constituents for tumor cellsby activity screen. The reaction conditions were optimized, the yields from71%to94%.Conclusion: This study established a method to modify the structure ofisoalantolactone with active methylene compounds and optimized the reactionconditions. Six new compounds11a-11g were synthesized. These compoundswere evaluated for toxic activities against three different hepatoma cell lines.The results showed that compounds11a and11b have inhibitory activity onthe proliferation of tumor cells. Inhibitory activity on tumor cells was greaterthan less than cisplatin a, less than taxinol.Part Five Structural Modification of Alantolactone–Michael ReactionObjective: Structural modification of alantolactone was proceededthrough Michael reaction and the synthesized compounds were elucidated bymeans of1H NMR and13C NMR. These compounds were evaluated for toxicactivities against three different hepatoma cell lines, Bel-7402, SMMC-7721and Hep G2. To laid the groundwork for discoverying lead compounds in drugstudy.Methods: Active methylene compounds and proper base were added to asolution of isoalantolactone in anhydrous EtOH. The mixture was stirred atroom temperature. The reaction mixture was adjusted to neutral with dilutehydrochloric acid solution. After evaporation of the solvent, the residue waspurified by silica gel column chromatography.Results: Six alantolactone derivatives13a-13f were synthesized throughMichael reaction. These compounds were elucidated by means of1H NMRand13C NMR.Conclusions: This study established a method to modify the structure ofalantolactone with active methylene compounds and optimized the reactionconditions. Six new compounds13a-13f were synthesized.更多还原