Studies On Structural Modification Of Quercetin, Isoalantolactone And Alantolactone

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.