Development,Hydrolytic Stability And Antitumor Activities In Vitro Of Novel Curcumin Derivatives

Objective:Curcumin(CUR)is an phenol monomer compound extracted from the curcuma rhizome of the curcuma genus of zingiberaceae plants,though CUR has antitumor,antibacterial,antiviral,scavenging free radicals and cardiovascular system aspects of pharmacological activities,etc,and small side effects,its clinical application is limited because of low hydrolytic stability,especially unstable under neutral and alkaline conditions,low bioavailability,quick metabolism in the body,easy oxidation,poor intestinal absorption after oral administration.Therefore,the objective is to modify the chemical structure of CUR,synthesize derivatives CURD 1 and CURD 2,and study the hydrolytic stability,antitumor activities,drug in take,apoptosis and intracellular distribution of synthesized derivatives.Methods:1.The derivatives CURD 1 and CURD 2 were synthesized with triethylamine as catalyst using the idea of "green chemistry ",the structure of derivatives were characterized by UV,NMR,MS and IR.2.The hydrolytic stability of CUR,CURD 1,CURD 2 in 1640 culture medium,1640 medium containing 10%FBS and PBS solution at pH 7.2 were determined by ultraviolet spectrophotometric method.3.The anticancer activities of CUR,CURD 1,CURD 2 for Be17402,SMMC7721 and HepG2 cells and toxicity for L02 cell were measured by MTT method at 48,72,and 96 h.4.The distribution of CUR,CURD 1,CURD 2 in the cancer cells was detected by confocal laser scanning microscope.5.The intake of CUR,CURD 1,CURD 2 in Be17402,SMMC7721 and HepG2 cells were determined by HPLC method.6.The apoptosis of Be17402 cells dosed of CUR,CURD 1 and CURD 2 was respectively inspected with flow cytometry.Results:1.The novel derivatives of CURD 1 and CURD 2 were synthesized successfully,not published in the literature.2.In the aspect of the hydrolytic stability in three different media,the undegraded ratios of CUR,CURD 1,CURD 2 were respectively 8.01～14.75%,19.19-37.54%,38.63～48.05%at 24 h,therefore,the stability of CURD 2 was better than CURD 1 significantly,the stability of CURD 1 was better than CUR.3.The growth inhibiton of CURD 1 and CURD 2 to three cancer cells had a manner of time and dose-dependent;as well as CUR,CURD 1 and CURD 2 were sensitive to Be17402 and SMMC7721 cell lines,insensitive to HepG2 cells,the IC50 of CURD 1 to Be17402 and SMMC7721 were 23.06 and 29.16 μmol/l at 72 h,the IC50 of CUR to Be17402 and SMMC7721 were 21.70 and 28.95 μmol/l at 72 h,the inhibitory activities of CUR and CURD 1 were similar,while the activities of CURD 2 was lower than CURD 1 and CUR;the toxicities of CUR,CURD 1,CURD 2 to normal liver cell L02 were in the order of CUR>CURD 1>CURD 2.4.In the drug group,the nucleus of Be17402,SMMC7721 and HepG2 cells were not only dyed blue by PI,at the same time,the cell nucleus had the green fluorescence of the drugs,but in the blank group,the nucleus of cancer cells only had blue fluorescence of PI.This shows that CURD 1 and CURD 2,as well as CUR,mainly entered into the nucleus of cancer cells for playing an antitumor role.5.In CURD 1 group,the drug content in Be17402,SMMC7721,HepG2 cells were respectively 1.51,0.58,0.61 μmol at 6 h;in CUR group,the drug content were 0.29,0.51,0.51 μmol;in CURD 2 group,the drug content were 0.04,0.33,0.13 μmol.It can be seen that the intake of CUR,CURD 1,CURD 2 for tumor cells were in the order of CURD 1>CUR>CURD2.6.When the concentration of 40 μmol/l,the Be17402 cell apoptosis of CUR,CURD 1 and CURD 2 were 17.6%,18.05%and 16.28%respectively at 72 h.This shows that CURD 1 and CURD 2 both could promote Be17402 cell apoptosis.Conclusion:1.The curcumin phenolic hydroxyl was catalyzed by triethylamine for the first time,this method is simple and feasible with less by-products,and conducive to the separation and purification of product.2.Novel curcumin derivatives CURD 1 and CURD 2 have better hydrolytic stabilities than CUR.3.Derivatives CURD 1 and CURD 2 have good tumor cell proliferation inhibitory activities,and their toxicities to normal liver cells are lower than CUR,they have a higher selectivity in liver cancer cells.4.Derivatives CURD 1 and CURD 2 mainly distributed in the nucleus of tumor cells.5.The intake of CUR,CURD 1,CURD 2 for tumor cells were in the order of CURD 1>CUR>CURD 2.6.CURD 1 and CURD 2 can promote apoptosis of tumor cells,their anticancer mechanism still need further study.