Brefeldin A Purification Using The Cooling Crystallization And Its Structure Modification

Brefeldin A(BFA)is a 13-carbon macrolide lactone antibiotic which possessing antitumor,antiviral,antifungal activities for blocking the of protein secretion process in eukaryotic cells by interfering in the endoplasmic reticulum(ER)to Golgi membrane traffic.However,its therapeutic application is limited by its poor targeting,low bioavailability and solubility.Hence,it is an efficient strategy to synthesize brefeldin A derivatives that remain high anticancer activity and display good pharmacokinetic properties.Firstly,based on the comparison of the yields and the morphology of crystals prepared by uding three different crystallization methods,we chosen cooling crystallization to purify brefeldin A.Via optimization of crystallization process parameters,we prepared brefeldin A sample with purity of 99.6%(w/w)by twice crystallization,in a yield of 42.0%,providing the raw material for the subsequent chemical derivatization.Brefeldin A has two hydroxyl groups at C4 and C7,which can be chemically modified.Due to steric effect,modification with large non-polar groups can cause significant activity loss.Moreover,the C7-OH is more active than C4-OH.Therefore,we decided to modify C7-OH to synthesize brefeldin A derivatives.In ice bath,various amines and triethylamine dissolved in toluene were added slowly to triphosgene in toluene.Reation mixtures were heated to 80℃,and reflux for 3h to synthesize isocyanates.Then the redultant isocynates reacted with the C7-OH of brefeldin A,forming the carbamate derivatives.The carbamate derivatives of brefeldin A were purified by TLC,and their cytotoxicity,antitumor activity against the TE-1 cell line was evaluated.The results showed the derivatives lost the antitumor activity.Further we used trichloroacetimidate ester as glycosyl donors to react with brefeldin A in the presence of BF3·EtO2 and 4A molecular sieve,in nitrogen atmosphere to prepare the corresponding five glycosylated derivatives of brefeldin A at its C7-OH,and then deacetylated by sodium methoxide in methanol.Those noval glycosylated brefeldin A derivatives had GI50 against TE-1 cell in the micromolar range,which is much higher than that of original brefeldin A.Molecular docking indicatied that the conformational change that block the glycosylated brefeldin A access to the binding sites.However,the introduction of polar glycosyl groups retains considerable antitumor activity and raises the aqueous solubility.