Design, Synthesis And Activity Evaluation Of Liver-targeted Drug Glycyrrhetinic Acid Amino Acid Derivatives

Background:Glycyrrhetinic acid(GA),an effective anti-tumor ingredient,was isolated from traditional Chinese medicine Glycyrrhiza.It was reported that there were abundant binding sites in liver cells for GA,so it was a hot spot to discover liver cancer-targeted drugs based on GA at home and abroad.Compounds TOGA and TNGA were synthesized in our laboratory previously,which were formed by ester and amide bonds to the carboxyl position of GA,respectively,with poor anti-tumor activities.According to relevant reports,the introduction of amino acid residues was helpful for improving activity and reducing toxicity,which offered significant direction for the discovery of novel anti-cancer drugs.Research purpose:This project was intended to improve the anti-tumor activity of the GA derivatives TOGA and TNGA.Based on the targeting property of GA itself,amino acid residues were introduced into the structures of TOGA and TNGA through the principle of chemical combination.Moreover,with the study on the mechanism and drug properties,the efficient and low-toxic compounds were expected to be identified finally.Research methods:TOGA and TNGA were adopted as structure-modification precursors,with the introduction of different amino acid residues,the amino acid derivatives of GA were obtained.And the final structures were characterized by NMR and MS spectrometries.These compounds were evaluated by five cancer cell lines(HepG2 human hepatoma cell model,Hela human cervical cancer model,A549 human lung cancer model,BGC-823 human gastric cancer model,and MDCK canine kidney epithelial cells)to pick up the best one with high-efficiency and low-toxicity.Furthermore,the structure-activity relationship was study through the preliminary activity assay to confirm the functional groups,deduce and verify the possible mechanisms of the activity.And finally,necessary druggability evaluations in vitro and in vivo would be carried out for the selected lead compound for further development of new anti-cancer drugs.Research results:34 GA amino acid derivatives were synthesized and their anti-tumor activities were screened by MTT assays.Most of the compounds were improved in activity compared to TOGA and TNGA.Among these compounds,TOGA-X1,TOGA-X4,TOGA-X5 and TOGA-X17 showed even more potent activity than the positive drug DDP.Combined with the results of cyto-toxicity test in normal cell line,TOGA-X4 was finally selected for its high-efficiency and low-toxicity.Interestingly,TOGA-X4 was demonstrated to induce early apoptosis in HepG2 cells through cell staining and streaming results.In addition,the druggable properties of TOGA-X4 were conducted for the development of new drugs.Through the in vitro plasma stability studies,we found that after in vitro plasma incubation for 2 hours,the remaining compound content was 62.7%;and after in vitro incubation of liver microsomes for 1 hour,the remaining compound content was 71.3%.So,the compound could be considered stable in vitro.TOGA-X4 was prepared into microsphere formulation to increase its solubility and then in vivo plasma stability was also explored.It was found that the compound gradually decreased in vivo but the prototype could be still detected after 72 hours.Through animal in vivo distribution experiments,we found that in the five organs of the heart,liver,spleen,lung and kidney,the distribution of the compound in the liver gradually accumulated with time and eventually stabilized,which could be considered with obvious liver targeting.In nude HepG2-xenograft mice model,TOGA-X4 had the tumor inhibition rate of 49.5%at the dose level of 80 mg/kg,which was comparable to the positive 5-Fu;however,TOGA-X4 had much better tolerability and safety in liver,kidney,and heart than the positive.Conclusion:After the introduction of amino acid residues,the activity of the final compound was improved,and the anti-cancer activity of the TOGA derivatives was generally stronger than that of the TNGA derivatives.The TOGA-X4,with high-efficiency and low-toxicity,was selected as the candidate compound,which could promote early apoptosis of liver cancer cells.It was stable in plasma and liver microsomes in vitro,and the in vivo distribution experiments showed that TOGA-X4 had good liver targeting effect.In addition,the nude xenograft mice results showed that the compound was potent with obvious anti-tumor effect in vivo.In summary,the purpose of the project was achieved and it offered the foundation for the research and development of new drugs.