The Design,Synthesis And Antitumor Activity Of Oleanolic Acid Derivatives

Oleanolic acid（OA）is a common natural product.It belongs to the pentacyclic triterpene compounds,and widely found in many plants and exists in the form of free and glycosides.It has a wide range of biological activities.Such as hepatoprotection,hypoglycemic,lipid-lowering,anti-tumor,antihypertensive,anti-inflammatory and anti-HIV infection.In recent years,the main active site of OA has been found in its A ring,C-3 and C-28.However,it has poor water solubility,low bioavailability,so it clinical application had limited.Survivin,a member of the apoptosis-inhibitory protein family,plays a key role in cell division and in inhibiting apoptosis.Survivin inhibitors research is the forefront of the international field,only a small number of clinical trials into the current phase.This paper suggests that the antitumor activity of pentacyclic triterpenoids is related to the target of Survivin.In this paper,Using the techniques of computer-aided drug design,through the use of the natural product,Oleanolic Acid,the docking of Survivin and known active small molecules was simulated and then the key amino acid residue fragment of the target protein was analyzed.It led to the discovery of active groups capable of binding to the critical sites,the introduction of the active groups onto the A-ring of OA,and the modification of the carboxyl group at the C-28 position using amidation,based on the inverse synthesis analysis and Lipinski’s five rules,we eliminated the molecules which had difficulty in synthesis,low drug-induced toxicity and toxicity and optimized the synthesis of 15 oleanolic acid analogues.The compounds were screened for their antitumor activity.Further tests of the apoptotic rate were performed on well-active compound I₃ and Western blot was used to detect the expression of Caspase3 and Caspase7.In this paper,oleanolic acid as the raw material.Fifteen compounds were designed and synthesized.At first the hydroxyl group at the 3-position of oleanolic acid is oxidized and then the carbonyl group is introduced tert-butanol at A ring to give 2,3-dioxo oleanolic acid while amide or ester is formed at the 28-position,the intermediates reacted with ethylenediamine and potassium hydroxide to introduce pyrazine and imidazole into A ring,and 15 compounds,5’-methyl-olean-2-ene-[2,3-b]-pyrazine-12-ene-28-oic acid ester compounds（I₁^I₅）,5’-methyl-olean-2-ene-[2,3-b]-pyrazine-12-ene-28-carboxylic acid amide compounds（II₁^II₅）and olean-2-ene-[2,3-b]-imidazole-12-ene-28-oic acid ester（III₁^III₅）were obtained.The reaction was monitored by TLC.The crude product was separated and purified by column chromatography.The structures of these compounds have been confirmed by MS,NMR and IR.In the present study,the cytotoxic activity of the compounds was determined by MTT assay.The tumor cells were selected as human liver cancer（HepG2）and human gastric cancer cell（SGC7901）.Gefitinib and Adriamycin as positive control.The results showed that compounds I₃,Ⅱ₅ andⅢ₄ exhibited more potent cytotoxicity on HepG2 and SGC7901 than positive drugs,and the inhibitory rates on HepG2 were 77.7%,61.7%and78.6%,IC₅₀0 values were 3.35μmol,7.75μmol and 3.18μmol,respectively.The inhibitory rates on SGC7901 were were 60.6%,65.1%and 57.6%,IC₅₀ values were 6.13μmol,3.32μmol and 7.96μmol,respectively.The apoptosis ratio induced by compound I₃ on HepG2 cells were quantitatively determined by flow cytometric analysis.The results further demonstrated that compound I₃ suppressed HepG2 cell proliferation by inducing apoptosis.Western blotting（WB）was used to detect the expression of Caspase-3,-7protein in the cells,the results showed that cleaved Caspase-3,-7 protein expression increased significantly with the compound concentration,indicating that I₃ promote HepG2 cell apoptosis.This paper has help for further study on oleanolic acid compounds and pentacyclic triterpenes to provide some practical value.