The Design, Synthesis And In Vitro Anti-tumor Activity Of Betulinic Acid-nitrogen-containing Heterocyclic Derivatives

BackgroundBetulinic acid(BA)is a lupane-triterpenoids of five-membered rings compound,which is mostly distributed in the genus Betula and any other plants.Among them,the highest content is in the bark of the birch.The research of domestic and foreign scholars showed that Betulinic acid has many biological activities,such as anti-tumor,anti-virus,anti-bacterial,anti-inflammatory,et al.Especially,the anti-tumor and inducing tumor cell apoptosis activities of Betulinic acid are significant,which have attracted wide attention in scientific community.In early stage,our experience group had synthetized the derivative of Betulinic acid called TBA(the compound as Betulinic acid combined with ligustrazine)which showed significant antitumor activity.In addition,there were plenty of reports in the literature suggested that Betulinic acid modification is of great significance in the research of finding antitumor drugs.In early stage,our group was guided by the principle of combination,and the active molecule tetramethylpyrazine was introduced into many lupane-triterpenoids of five-membered rings compounds.The anti-tumor activity of these derivatives improved significantly.However,the structure of pyrazine ring is very complex.The study based on piperazine ring is very important to explore the structure-activity relationship of the nitrogenous heterocyclic ring combining with pentacyclic triterpenoid.Many studies have shown that the introduction of nitrogenous heterocycles can significantly enhance the antitumor activity of triterpenoid.Therefore,the introduction of nitrogen containing heterocyclic rings into pentacyclic triterpenoid is expected to find new leading compounds.This study was based on structure-activity relationship analysis,and cyclopentane,cyclohexane,piperidine,pyrrolidine and piperazine five kinds of simple nitrogen heterocyclic were introducted into Betulinic acid to find the new active leading compound.The structure-activity relationship of similar nitrogen containing heterocyclic rings combining with Betulinic acid was discussed systematically,which was expected to be the foundation of this kind of synthesis,and to narrow the discovery cycle of antitumor leading compounds.Research methodsReferring to the chemical transfonnation of medicinal chemistry,the derivatives of Betulinic acid combining with nitrogen heterocycles which linked by amide bonds and linked by carbon-nitrogen bonds were synthesized,and their chemical structures were confirmed by 1H-NMR,13C-NMR and HR-MS respectively.As the biological activity evaluation,HepG2(liver cancer cells),BGC-823(gastric cancer cell),HeLa(cervical cancer cells),SH-SY5Y(neuroblastoma cells)4 tumor cells models the activity of inhibiting the proliferation on tumor cells of derivatives Betulinic acid combining with nitrogen heterocyclic in vitro,and L02(hepatic stellate cells)and H9C2(myocardial cells)2 normal cell models were selected to evaluate cytotoxicity on normal cells by MTT method,DOX(doxorubicin)was used as the positive drug.The preliminary pharmacodynamic analysis was used to investigate the structure-activity relationship.Furthermore,microscopic observation and the DAPI staining were used to observe the effect of Betulinic acid combining with heterocyclic derivative 7e on the morphology of Hela cells.The flow cytometry was also used to explore the mechanism of the effects of 7e in inhibition the proliferation of Hela cells.——Research and Results——There were 26 derivatives of Betulinic acid combining with nitrogen heterocycles linked by amide bonds or linked by carbon-nitrogen bonds were synthesized successively,of which 23 were new compounds unreported,and all compounds were confirmed by 'H-NMR,13C-NMR and HR-MS respectively,and the physicoche parameters related of these derivatives were determined.As their biological activity,MTT method was used to evaluate antitumor activities of derivatives in vitro,the experimental results showed that most of derivatives showed good inhibitory activity on these four kinds of tumor cells,the inhibitory activities of all derivatives are better than that of betulinic acid,in which 7e which was the derivative introducing the structure piperazine via carbon-nitrogen bonds showed the best activity on tumor cells,its IC50 value on the Hela cells was 2.05?M.At the same time,the toxicity of 7e on H9C2 and L02 cells was weaker than that of the positive drug DOX.Combined with cell staining,it was found that 7e could block connections between Hela cells,lead to damage on cells,crack on nuclear,and induce early apoptosis on Hela cells.ConclusionsThere were 26 derivatives Betulinic acid combining with nitrogenous heterocyclic synthesized successively,of which 23 were new compounds unreported,and all compounds were confirmed respectively.Most of derivatives had showed better antitumor activity than their parent nucleu Betulinic acid,and showed less toxicity than positive drug DOX,although the activity was less than that of the positive drug DOX.And in contrast,7e had the best anti-tumor activity.The inhibitory activities of derivatives on tumor cells were enhanced effectively,no matter the nitrogenous heterocyclic rings were introduced through amide bond or carbon-nitrogen bonds.However,the saturated structure was more conducive to the activity.To sum up,in this study,five kinds of simple nitrogenous heterocycles were introduced through the method of chemical synthesis,and the Betulinic acid was the parent nucleus.Then,the structure-activity relationship of these derivatives had been analyzed.The results suggested that the anti-tumor activities of derivatives were significantly different between different kinds of nitrogenous heterocyclic introduced by different kinds of form.Furthermore,this study indicated that the systematic and complete structure-activity relationship analysis for lupane-triterpenoids of five-membered rings compounds combining with nitrogenous—heterocyclic would provide a-basis for the synthesis of similar lupane-triterpenoids of five-membered rings compounds combining with nitrogenous heterocyclic derivatives with antitumor activities,which was expected to narrow the discovery cycle of antitumor leading compounds and had the practical significance in the development of new antitumor drugs.