Studies On The Synthesis, Pharmacological Action And Antitussive Mechanism Of Bile Acids-Verticinone Esters

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:â… Structure 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.â…¡Screening 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?â…¢The 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.â…£The 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.