【作者】 张久亮；

【导师】 吴继洲；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 随着药物化学、生物化学、结构化学、生理学、分子药理学等学科的发展和交叉渗透，人们逐渐认识并找出了许多药物发挥药理作用所依存的骨架结构，从而也产生了将具有相似生物活性的两类化合物进行结构拼合的设想，以期获得副作用更小、药效更加良好的新化合物。随着实践的不断深入，药物拼合作为一门理论逐渐为人们认识、接受并加以应用。拼合原理主要是指将两种化合物的结构拼合在一个分子内，或者将两个药物的骨架结构兼容在同一分子内，以期减小两种药物的毒副作用，求得二者作用的联合效应。湖北贝母系百合科植物湖北贝母（Fritillaria hupehensis Hsiao et K．C．Hsia）的干燥鳞茎，具有清热化痰、止咳、散结之功效，在中国民间有悠久的用药历史，《中国药典》2000版和2005版均有收载。现代药理实验表明，湖北贝母总生物碱及其单体浙贝乙素均具有较强的镇咳和祛痰活性。蛇胆是一味名贵的中药材，系统的化学和药理实验证实其主要活性成分为牛磺胆汁酸及各种游离的胆汁酸。中药复方蛇胆川贝散是由川贝母和蛇胆两味中药组成，组方相对比较简单，具有清肺止咳、祛痰平喘的良好功效，在我国应用历史悠久，历版《中国药典》均有收载；但由于无法解决作用物质不明确所带来的质量无法控制等弊端，很难走向国际市场；而且由于组方的蛇胆和川贝母同属贵重药材，这类复方制剂还面临着中药材资源严重不足的问题。基于吴继洲教授对湖北贝母长达二十年的研究基础与石朝周教授对蛇胆十几年的研究基础，已经阐明贝母及蛇胆两味药材中的主要活性成分分别为异甾生物碱和各种胆汁酸。为了寻找具有高效低毒的镇咳、祛痰和平喘药物，本课题组以5种胆汁酸和浙贝乙素为原料，借鉴药物化学结构拼合的思路，开展了以下研究。一、胆汁酸浙贝乙素酯合成路线的研究1、酯键拼合的设计和合成以浙贝乙素和5种胆汁酸中的任意一种为原料，利用胆汁酸含有的羧基和浙贝乙素含有的羟基，在脱水剂（DCC）和催化剂（DMAP）条件下进行酯化反应，得到5个新化合物。2、胆汁酸浙贝乙素酯的中试合成经过波谱学及现代分析方法鉴定5个新化合物，确定合成的物质确实是目标化合物。随即大量合成系列胆汁酸浙贝乙素酯（胆酸浙贝乙素酯、鹅去氧胆酸浙贝乙素酯、熊去氧胆酸浙贝乙素酯、猪去氧胆酸浙贝乙素酯、去氧胆酸浙贝乙素酯），为下一步5个新化合物的生物活性筛选打下基础。二、胆汁酸浙贝乙素酯生物活性的筛选在对5个胆汁酸浙贝乙素酯进行活性筛选的研究中，本课题组采用了小鼠氨水引咳、小鼠气管酚红排泌及豚鼠乙酰胆碱-组胺整体引喘三种动物模型，综合评价了5个新化合物的镇咳、祛痰和平喘活性。研究结果表明：在相同剂量下，胆酸浙贝乙素酯（CA-Ver）活性要明显强于其他的胆汁酸浙贝乙素酯，其中尤为值得关注的是，在小鼠氨水镇咳模型的筛选中，CA-Ver显示出了比磷酸可待因和原料药更强的镇咳活性。三、胆汁酸浙贝乙素酯急性毒性的研究对胆汁酸浙贝乙素酯的小鼠急性毒性研究结果表明：5个胆汁酸浙贝乙素酯小鼠灌胃给药的LD₅₀值超过6 g／kg，而且5个胆汁酸浙贝乙素酯小鼠腹腔注射给药的LD₅₀值超过3．5 g／kg，表明两类物质进行结构拼合是可以起到减毒作用的。四、胆酸浙贝乙素酯镇咳机制的初步研究1、胆酸浙贝乙素酯对小鼠大脑单胺类神经递质及其代谢产物的影响采用RP-HPLC-FLD法，以磷酸可待因和氢溴酸右美沙芬为阳性对照药，测定了CA-Ver、浙贝乙素对小鼠脑部五种单胺类神经递质含量的影响。研究结果表明：阳性药右美沙芬的镇咳机制与单胺类神经递质5-HT有关，它能增加动物脑部5-HT的含量以达到镇咳效应；而CA-Ver、浙贝乙素和可待因的镇咳效应与5-羟色胺能机制无关。2、胆酸浙贝乙素酯对中枢阿片受体能镇咳机理的研究采用小鼠辣椒素引咳模型，借助非选择性阿片受体拮抗剂纳诺酮对小鼠进行预处理，探讨CA-Ver可能存在的中枢阿片受体能镇咳机制。研究结果显示阿片受体阻断剂纳诺酮能阻断CA-Ver镇咳效应的发挥，说明CA-Ver的镇咳机理与中枢阿片受体有关。本论文对湖北贝母中的有效成分之一浙贝乙素和蛇胆中的活性成分胆汁酸进行酯键拼合的设计和合成，并对合成的5个新化合物进行了生物活性的筛选，以期寻找到增效减毒的镇咳新药。本课题组对活性最好的CA-Ver进行了较系统的药效学和机制的研究，为将其开发成为镇咳、祛痰新药奠定了坚实的基础。更多还原

【Abstract】 Following the solo and cross-disciplinary development of biochemistry,medicinal chemistry,structural chemistry and physiology,the basic structuresof some drugs had been clearly elucidated,which were the core of theirbioactivities.In order to gain some new compounds with better bioactivity andattenuated toxicity,people attempted to combine two kinds of drugs throughionic bond or ester bond.Many famous drugs in clinic use such assultamicillin and benorilate were discovered through this way.Till now,“combination principle”has been an important idea and method in new drugdiscovery.Hubeibeimu,listed in Pharmacopoeia of the People’s Republic of China（Chp）2000 and Chp2005,is the dried bulb of Fritillaria hupehensis Hsiao etK.C.Hsia.Within the realm of Chinese medicine,Hubeibeimu has beenclaimed to resolve phlegm,stop cough,and clear away heat,and dispelaccumulation;it is widely used in Traditional Chinese Medicine.Recentpharmacological studies demonstrated that the total and single alkaloids （eg,verticinone） of this medicine have antitussive activity.Shedan is a valuableChinese crude drug.Systematic chemical and pharmacological researchstudies have confirmed that its main components are taurocholic acid andvarious kinds of free cholic acid.“Shedan-Chuanbei powder”,consisted ofSnake Bile （Chinese name“Shedan”） and Fritillariae Cirrhosae （Chinese name“Chuanbei”）,is the most popular antitussive,expectorant andantiasthmatic formulation in Chinese communities.It has been used in clinicfor thousand years in China due to the positive potent therapeutic effects,lowtoxicity and minimal side effects.Therefore,it has been officially listed in theChinese Pharmacopoeia （1995,2000,and 2005）.But the clinical applicationof Shedan-Chuanbei powder is now stringently limited because of theshortage of the two crude medicinal materials,especially for the sake ofanimal protection.In addition,the inherent defects of the most of the complexof traditional Chinese medicine such as the indistinct basal pharmacodynamicmaterials and the difficulties in quality control had blocked them heading intothe international medicinal market.Therefore,it prompts us to search for newbioactive substitutes for Shedan-Chuanbei powder for antitussive drugs.Basedon Prof.Wu Ji-zhou’s nearly 20 years studies on the chemical constituents andthe bioactivities of the Fritillaria hupehensis and Prof.Shi Cao-zhou’s morethan 10 years study on the bioactive constituents of Snake bile,theisosteroidal alkaloids and the bile acids were clearly elucidated as the twokind of major bioactive constituents respectively in bulbs of Fritillaria andSnake bile.In order to gain the new compounds with better bioactivity andattenuated toxicity,we tried to combine two kinds of drugs through ester bond.Enlightened with the“combination principle”in drug discovery,wesynthesized five novel esters of verticinone and bile acids,both of which arethe major bioactive components in Shedan-Chuanbei powder.And then we did a series of studies as follows:ⅠStructure combination and synthetic procedure of bile acids-verticinone estersIn order to gain the new compounds with better bioactivity andattenuated toxicity,we tried to combine two kinds of drugs through ester bond.We synthesized five novel esters of verticinone and bile acids,named ascholic acid-verticinone ester （simplified as CA-Ver in the paper）,chenodeoxycholic acid-verticinone ester （simplified as CDCA-Ver in thepaper）,ursodeoxycholic acid-verticinone ester （simplified as UDCA-Ver inthe paper）,hyodeoxycholic acid-verticinone ester （simplified as HDCA-Ver inthe paper）,deoxycholic acid-verticinone ester （simplified as DCA-Ver in thepaper）.Bile acids-verticinone esters were synthesized from bile acids andverticinone via esterification using DCC as the acid-activating agent,leadingto the linkage of the carboxylic acid group on C24 position of cholic acid withthe hydroxyl group on C3 position of verticinone.CA-Ver,CDCA-Ver,UDCA-Ver,HDCA-Ver and DCA-Ver,five novelesters of verticinone and bile acids,were readily identified by comparison oftheir m.p,[α] _D²⁰,IR,MS,¹H-NMR and ¹³C-NMR data with their componentsteroid monomers.The synthesis of five novel esters are illustrated in Scheme1 （five steps,33% overall yield）.Reaction of cholic acid with absolute MeOHfollowed by esterification resulted in the formation of methyl 3α,7α,12α-trihydroxy-5β-cholan-24-oate 1.Then reaction of 1 with TBDMSiCl via ether bond afforded the intermediate 2.Hydrolysis of the methyl ester groups of 2with 5% sodium hydroxide aqueous solution furnished 3.Then treatment of 3with verticinone in CH₂Cl₂ followed by esterification gave the compound 5α,14α-cevanin-6-O-20β-hydroxy-3β-yl-3α-tert-butyldimethylsilyloxy-7α,12α-dihydroxy-5β-cholan-24-oate 4.In this reaction,DMAP was used as a catalystand DCC as a dehydration agent.Finally,the deprotection reaction of 4 in 5 %HF aqueous solution gave the desired 5α,14α-cevanin-6-O-20β-hydroxy -3β-yl-3α,7α,12α-trihydroxy-5β-cholan-24-oate 5.The chemoselective esterificationreaction of 3 with verticinone in CH₂Cl₂ provided 4 in 58.9% yield.We thenevaluated the antitussive activity and the acute toxicity of the five ester-linkedcompounds.ⅡScreening on the bioactivities of bile acids-verticinone estersIn the screening study of the bile acids-verticinone esters,we employedthree animal modes such as cough induced by ammonia water in mice,phenolred secretes in the trachea of mice and acetylcholine-histamine inducedasthma in guinea pigs to systematically evaluate the antitussive,expectorantand antiasthmatic effects of five bile acids-verticinone esters.The resultsshowed that CA-Ver had much more potent bioactivities than the other fouresters.Moreover,the much more potent antitussive effect of CA-Ver thancodeine phosphate also deserved advanced study.Based on the studies ofpharmacology,whether do the five ester-linked compounds have synergicpharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids?ⅢThe acute toxicity of bile acids-verticinone estersA further acute toxicity study showed that the LD₅₀ values of the fiveester-linked compounds exceeded 3.5 g/kg by intraperitoneal injection in mice.There were no deaths or any signs of toxicity observed after oraladministration of single doses of the five ester-linked compounds at any doselevel up to the highest dose tested （6 g/kg）,which was the no-observed-adserved-effect level （NOAEL）.Uniformly,there were no deaths after theintraperitoneal injection at any dose level up to highest dose tested （3.5 g/kg）.No mortalities had occurred during the study and whole observations did notindicate evidences of substance-related toxicity.So the acute toxicity of CA-Ver,CDCA-Ver,UDCA-Ver,HDCA-Ver and DCA-Ver were considered asunclassified,since a dose of 6 g/kg （by gavage） or 3.5 g/kg （by intraperitonealinjection） did not induce deaths or toxic symptoms.ⅣThe antitussive mechanism of cholic acid-verticinone ester1 The influences that CA-Ver made to the five kinds of monoamineneurotransmitters in mice’s brainBy the reverse-phase HPLC method with fluorescent light detector,usingcodeine and dextromethorphan as positive drugs,we assayed the influencesthat CA-Ver and verticinone made to the five kinds of monanineneurotransmitters in mice’s brain.The results indicated that the antitussivemechanism of dextromethorphan concered with monanine neurotransmitters 5-HT and it could increase the amount of 5-HT in animal’s brain to producethe antitussive effects;while the antitussice effects of CA-Ver,verticinone andcodeine had no relationship with serotonergic mechanisms.2 The central mechanism （modulated by opioid receptor） of CA-VerThe test on capsaicin-induced cough model of mice pretreatment withnaloxone,a non-selective opioid receptor antagonist,was taken forobservation of CA-Ver’s central antitussive mechanism.The studydemonstrated that p.o.administration of CA-Ver produced a dose-dependentantitussive effect in mice.The antitussive effect of CA-Ver was significantlyreduced by pretreatment naloxone.In a parallel study,codeine phosphate,oneof the most commonly used potent antitussive agents,was used as the positivecontrol.The antitussive effect of codeine phosphate,a centrally-acting,narcotic antitussive drug was also significantly reduced by pretreatmentnaloxone.These results indicate that the naloxone-sensitive opioid receptorplays an important role in mediating the antitussive effect of CA-Ver.All theresults indicated that the antitussive effects of CA-Ver had no relationshipwith serotonergic mechanisms but central opioid mechanism just as codeine.Our present study not only synthesized the five novel esters of verticinoneand bile acids,which will be used as antitussive and expectorant agents infuture,it could also be an attempt at application structure combination idea tothe research and development of TCM,which may exploit a novel field ofnew drug design from TCM.Cumulatively,this study highlights a conjugated extension of esterification with verticinone and single bile acid and illustratesa potential benefit to structure combination of natural products.Furthermore,our current project could build the foundation to further study structure-activity relationship of verticinone and to develop a new antitussive drug innext step.更多还原