Design, Synthesis And In Vitro Bioactivity Evaluation Of Betulin-amino Acid Derivative

Research ObjectivesBetulin is lupane-triterpenoids of five-membered rings and can be obtained from a variety of traditional Chinese medicine medicinal plants,such as seed of wild jujube.Besides,in birch betulin accounts for a relatively greater part in all kinds of chemical composition and is to obtain easily.And in the aspects of pharmacologic,it has broad prospects including antitumor,antiviral,anti-inflammatory and other biological activities.However,as a natural compound,betulin still has some disadvantages,such as poor solubility,low bioavailability,relatively weak of biological activity and these problems seriously restrict its further development and application in clinical therapy.With the application of combination principle and pair-herb compatibility principle,our research group had selected ligustrazine(TMP),active ingredient in Rhizoma Chuanxiong joint,to combine with some active compounds.By this means,it was helpful to improve bioavailability and reduce adverse reactions.Therefore,by chemical method,this study had introduced TMP and kinds of amino acids into betulin,with the expectation to obtain novel betulin derivatives with better bioactivity and less toxicity.Research MethodsBased on the combination principle in medicinal chemistry and clinical application of tradition Chinese medicine,this study selected betulin and ligustrazine,active ingredients of Chinese herbal medicine,as the basic core.Then,by means of chemical method,including esterification reaction and amidation reaction as well as the protection and deprotection reaction,a series of Ligustrazine-betulin-amino acids derivatives and acetyl peptide derivatives had been obtained.Regarding to biological activity,we tested those compounds for inhibiting tumor cells proliferation by MTT assay and cell toxicity to normal cells on various of tumor cells in vitro including HepG2(liver cancer cells),BGC-823(stric cancer cell),HeLa(cervical cancer),HT-29(colon cancer cell)and MDCK(canine kidney epithelial cells).Besides,combining Giemsa,DAPI,AO/EB staining method,this study explored effects of betulin and TB-01 on morphology change for HepG2 cells.Further,this study had explored the mechanism of the effects of TB-01 on inhibition the proliferation of HepG2 cells by flow cytometry.Research ResultsIn the aspect of chemistry,we had designed and syntheses nineteen(Ligustrazine-betulin)-amino acid derivatives,and the structure of the compounds had been confirmed by means of 13C-NMR,1H-NMR and HR-MS,which determined the relevant physical parameters.And regarding to biological activity,the experimental results showed that most TB-amino acid derivatives of four kinds of tumor cells showed good inhibitory activity,and the inhibitory effect was better than that of betulin and TB,among which TB-01 showed that the promising activity of anti-tumor cells and the relatively weaker inhibitory effect on MDCK than DDP.In addition,the result of cell staining and flow cytometry had demonstrated that TB-01 could cause HepG2 cells nuclear damage and lysis and induce early apoptosis,and blocked the cell cycle of HepG2 cells in G1 phase.ConclusionsIn this study,nineteen new compounds had been designed and synthesized,and the pharmacological activities of the derivatives had been evaluated.The experimental results demonstrated that it was effective to introduce ligustrazine and amino acids into the C-3 hydroxyl and carboxyl group on the C-28 of betulin that improve the solubility,and enhance its inhibitory effect on tumor cells in vitro.Among them,glycine derivatives,TB-01,showed a quite potential antitumor activity.The structure-activity relationship suggested that anti-tumor activity of aliphatic amino acid derivatives was better than that of aromatic amino acid derivatives and heterocyclic amino ones of in vitro.Besides,among aliphatic amino acid derivatives,small molecule amino acid derivatives showed better bioactivity than long chain amino acid derivatives,and the introduction of acetyl amino acid failed to further enhance the antitumor activity of amino acid derivatives of betulin.This research completed the structural modification of betulin,which improved its physicochemical properties,and increased its biological activity and to some degree verified that it was a useful strategy that the introduction of amino acid into active ingredients of Chinese herbal medicine.It provided reference for the development of effective ingredients of Chinese herbal medicine and traditional Chinese medicine.