Preparation,Structure Modification And Activity Evaluation Of Germacrone

The morbidity and mortality of cancer is high and the situation of our country is particularly serious.The treatment of cancer mainly includes surgery and chemotherapy.Ho wever,because of the high recurrence and drug resistance,it is a research hotspot to find a new anti-cancer drug with high efficiency and low toxicity.Zedoary as an important Chinese herbal medicine is widely used and it has anti-inflammatory,antiviral,anti-tumor effect.Volatile oil as the main active component of Zedoary,also has the same pharmacological activity.Germacrone is a kind of sesquiterpene compounds isolated from zedoary oil,which was reported to have significant anticancer activity.However,there are some defect such as excessive target,low bioavailability and poor stability,so the researchers are trying to find a variety of constitutive property,clear targets and high efficiency and low toxicity of germacrone derivatives.So far,there are rarely reports about the method of drug synthesis to modify the structure of germacrone.The discovery and optimization of lead compounds from natural products is one of the important approaches for the research and development of new drugs.This research isolated and purified germacrone,synthesized its derivatives and evaluated their activities.The results showed that the derivatives had strong and broad-spectrum anticancer activities.The main contents and results are as follows:Extraction process of germacrone was optimized by using Central Composite Design Response Surface methodology,and determination of its content was tested by high performance liquid chromatography（HPLC）,the average yield of extraction was 8.13%,which was in accordance with the predicted values.The zedoary oil containing germacrone crude was separated repeatedly by silica gel column chromatography,and then further isolated by semi preparative liquid phase to obtain germacrone.The results showed that the purity was 99.0% by HPLC and its structure was confirmed by 13C-NMR,1H-NMR.Based on the structure of germacrone,through the structural modification of C-8 carbonyl,the reduction product was synthesized and modified the C-8 hydroxyl,we designed and synthesized two series of germacrone derivatives including（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-acetate,（3E,7E）-3,7-di methyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-methylbenzoate,（3E,7E）-3,7-dimet hyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-3-methylbenzoate,（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-（trifluoromethyl）benzoate,（Z）-（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-3-phenylacrylate,（3E,7E）-3,7-dime thyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-ylmethanesulfonate,（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yltrifluoromethanesulfonate,（3E,7E）-3,7-dimet hyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-ethanesulfonate,（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-ylbenzenesulfonate,（3E,7E）-3,7-dimethyl-10-（pro pan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-methylbenzenesulfonate,（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-chlorobenzenesulfonate,the mechanism of synthesis route was studied and optimized.The structure of all compounds was confirmed by 13C-NMR,1H-NMR,MS and elemental analysis.The synthetic route was simple,the material was easy to obtain and the yield was high.The anticancer activity of germacrone and its derivatives on human hepatocellular carcinoma cell line Bel-7402,HepG2 cell line,human non-small cell lung cancer A549 cell line and Hela cell line of cervical cancer were determined by MTT method,The results showed that germacrone derivatives had good antitumor activity and most of them were stronger than germacrone,Among them,the formic acid ester derivative（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-methylbenzoate had the strongest activity and the IC₅₀ value of the HepG2 cell line is 68.23 μM;the sulfonic ester derivative had the best activity for p-methyl benzene sulfonate,and the IC₅₀ value of the HepG2 cell line was 56.22 μM.At the same time,the c-Met kinase activity of germacrone derivatives were tested,the results showed that the activity of（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-methylbenzoate and（3E,7E）-3,7-dimethyl-10-（propan-2-ylidene）cyclodeca-3,7-dien-1-yl-4-methylbenzenesulfo-nate were the strongest,which coincided with the MTT experiment.According to the MTT test and c-Met kinase activity experiments,the two kinds of germacrone derivatives were investigated by molecular docking studies,docking results showed that most compounds had interaction force with c-Met sites.There were strong hydrophobic interaction between the（E,E）-1,5-decadiene ring system of germacrone and the target protein crystal,other forces including π-π stacking interaction and σ-π conjugative effect.In order to further verify the results of molecular docking,molecular dynamics simulations were performed on the best docking compounds,in the simulation process of 8 ns,combination of derivatives and protein pocket were stable and reliable,ΔGpred result was-28.36 kcal/mol and less than-25 kcal/mol.In summary,this work mainly extracted and purified high purity germacrone from zedoary oil and optimized the extraction process and purification method.Furthermore,we designed and synthesized a total of 11 germacrone derivatives which were less reported and all reactions were carried out on the mechanism research and condition optimization.Germacrone derivatives had good anti-tumor activities for four different cancer cells and c-Met kinase,the preliminary mechanism of these derivatives were confirmed and explained by molecular docking and molecular dynamics simulations.