Preliminary Study On The Design, Synthesis And Anti-tumor Activity Of Glycyrrhetinic Acid Derivatives

Background:Cancer is a major disease which seriously affect all over the world,with a high morbidity and mortality.Especially as a huge country like our China,with a largest population in the world,the number of deaths because of cancer each year are millions,which seriously threatens people's lives and health.Therefore,it is a vital mission for modern researchers and scientists to find corresponding effective drugs for malignant tumors.In recent years,finding and developing new anti-tumor drugs from natural products has been the research focus.Glycyrrhetinic acid,as an active ingredient of pentacyclic triterpenes in traditional Chinese medicine licorice,large number of researches around the world have shown that it has a promising application prospect as a lead compound to optimize the antitumor effect.Our research group have synthesized more than 100 derivatives of glycyrrhetic acid in the previous researches.This research is the third part in the synthesis and activity of glycyrrhetic acid derivatives.Combining the previous studies on glycyrrhetic acid and related literature,the introduction of an lipophilic group at the C-30 position can significantly enhance the anti-tumor activity of glycyrrhetic acid,and previous research by our research group showed that the combination of amino acids and triterpenoids can increase its inhibitory effect on tumors and can effectively improve the solubility and selectivity of drugs.Research purpose:This project was based on researches in the early stage of the our laboratory of glycyrrhetic acid and related literature:1)C-3 hydroxyl group was changed to amino group;2)By introducing esterophilic group benzyl at the C-30 position of glycyrrhetic acid,and the small molecule amino acid was introduced at the C-3 position.Two series of glycyrrhetinic acid derivatives with different bonding methods were obtained,combined with antitumor activity screening,the effect of bonding methods on the activities of different derivatives was going to be explored and lead compounds with significantly improved activity were expected to obtain.At the same time,during the research process,the problem of epimerism was further explored,which would provide an important reference for future scholars' researches on the derivatization of glycyrrhetic acid.Research methods:In this experiment,36 glycyrrhetic acid derivatives were synthesized by organic synthesis,mainly divided into two series:1)C-3 position ester bond derivatives,using glycyrrhetinic acid as the nucleus to introduce a benzyl group at the C-30 position and at the same time different kinds of amino acids were introduced at the C-3 position,which were GA-OH series(GO,C-3 position was an ester bond);2)C-3-position amide bond derivative,the glycyrrhetinic acid C-3 hydroxyl group was converted to amino to obtain 3-aminoglycyrrhetinic acid GA-NH2,using this as the nucleus to introduce benzyl and amino acids,which were GA-NH2 series(GN,C-3 position was an amide bond).In addition,the intermediate product GN-BN epimer was separated,the structure was confirmed,and the activity was evaluated during the research.All glycyrrhetic acid derivatives were confirmed by 1H-NMR,13C-NMR,HRMS and other methods,and physical parameters such as melting point and optical rotation were measured.Four kinds of tumor cells(human lung cancer cells A549,human cervical cancer cells Hela,human liver cancer cells HepG2 and human breast cancer cells MCF-7)and one normal cell(canine kidney epithelial cells MDCK)were selected to measure the activity and toxicity of all the derivatives.MTT experiments were performed for evaluation.The effects of drugs on the nucleus morphology of tumor cells were evaluated by cell staining,and the antitumor mechanism of dominant compounds was preliminarily explored by flow cytometry.All the researches above will provide important reference for follow-up scholars on the structural modification of glycyrrhetic acid and pentacyclic triterpenes.Research results:A total of 36 glycyrrhetic acid derivatives were synthesized by organic synthesis in this paper,including 32 new compounds,and all compounds were screened for their antitumor activity by MTT experiments.The structure-activity relationship analysis results showed that:1.The activity of de-Boc glycyrrhetinic acid derivatives was better than that of non-de-Boc derivatives.At the same time,the activity of glycyrrhetinic acid derivatives connected by amide bonds was better than the derivatives of ester bonds;2.The exposure of amino groups when connecting amino acids played a key role in the activity of the compound.Most of the activities of different tumor cells can reach less than 5?M,which was tens times higher than the activity of glycyrrhetinic acid.3.Epimer 3?-GN-BN and 3?-GN-BN were separated and confirmed in structure,and no significant difference in antitumor activity was found between them.According to the MTT evaluation,compound 29 was the dominant compound(IC50A549=2.109±0.11?M).Cell staining and molecular biology preliminary exploration of this compound revealed that the cytotoxic mechanism of this compound was:promote the nuclear fragmentation of A549 cells,induce early apoptosis of the cells,and block cell division staying in the S phase,which could be defined as multiple pathways to induce tumor cell apoptosis.Conclusion:This subject was to carry out continuous research on the design,synthesis and activity characterization of glycyrrhetinic acid derivatives,further structural modifications were carried to explore improvement of the activity of glycyrrhetic acid by changes in the bonding mode and the introduction of small molecular amino acids.It was found that all amide-linked compounds had better activity than ester-linked compounds,and all the de-protected derivatives had stronger activity.It could be inferred that exposure of the amino group played a decisive role on whether the glycyrrhetic acid derivative had good activity.Among them,the best compound 29 could induce tumor cell apoptosis,and it was proved that 3?-GN-BN and 3?-GN-BN,which are easily overlooked intermediates,were produced during the synthesis process,and the difference in antitumor activity was not significant.From the above studies,we can conclude that the bonding modes in C-3 position has a certain improvement on the antitumor activity of glycyrrhetic acid,and all of the compounds has significantly enhanced the activity through such structural modification,without changing the apoptosis-inducing effect of glycyrrhetic acid.The related research results have reached the research objective of the subject,and have important reference value for the follow-up scholars' researches on glycyrrhetic acid and pentacyclic triterpenes.