NO-donor Ursolic Acid Derivatives:Design,Synthesis,and Antitumor Evaluation

ObjectiveUrsolic acid（UA）is a nutural pentacyclic triterpenoid carboxylic acid extracted from traditional Chinese herbs,and studies have shown that it unleashes a wide range of biological activities,including anti-HIV,anti-diabetic,anti-malaria,antibacterial,antiviral,hepatoprotective,anti-inflammatory and anti-tumor.And ursolic acid is a widely used raw material for medicine and cosmetics.However,UA possesses considerable disadvantages,including of strong hydrophobicity,low bioavailability and Gastrointestinal tract irritation.Accordingly,its clinical application was limited.In this study,a series of NO-donor UA prodrugs based on conjugates of UA and nitric oxide donor hybrid have been synthesized with the aim to improve the partition coefficient and the bioavailability of UA.To obtain new antitumor potential drugs,the antitumor activities of these compounds were also evaluated in this study.MethodsUA was modified on the positons of C-2,C-3 and/or C-28.Molecular docking of all the compounds with four protein kinases（CDK2,Bcl-2,EGFR and VEGF）was conducted by using SYBYL X 1.1.2 software.NO-donor ursolic acid derivatives were synthesized by oxidation,nucleophilic substitution,nucleophilic addition and condensation reaction.The derivatives were purified by column chromatography,and their structures were identified by nuclear magnetic resonance spectroscopy,IR and liquid chromatography-mass spectrometry.The antitumor activity of various derivatives on human hepatoma Hep G-2cells,human breast cancer MCF-7 cells,human colon cancer HT-29 cells,and human lung cancer A549 cells was detected by MTT assay.And the oil distribution coefficient（log P）of the active compound（2-4’-chlorobenzylidene-3-oxo-ursolic acid-2’-nitrooxyl ethyl ester,UANO 11）was determined by high performance liquid chromatography.The effects of UANO 11 on the apoptosis of HT-29 cells were investigated by using flow cytometry and immunofluorescence.UANO 11 was also subjected to docking studies with CDK2 and Bcl-2 proteins and their interaction modes were analyzed.ResultsIn this study,21 NO donor ursolic acid derivatives were synthesized successfully.All compounds were checked for greater than 95%purity with HPLC.The molecular docking study showed that all the ursolic acid derivatives could be combined with 4 proteins（CDK2,Bcl-2,EGFR,VEGF）well.The results of in vitro antitumor activity indicated that 2-4’-chlorobenzylidene-3-oxo-ursolic acid-2’-nitrooxyl ethyl ester displayed the best inhibitory activity on human colon cancer HT-29 cells.The IC₅₀value is 4.28（?）M;The log P of UANO 11 is between-1 and 2,which indicates that the bioavailability of UANO 11 is good.Moreover,UANO 11 is able to induce apoptosis of HT-29 cells in a dose dependent manner.The results of cell cycle test showed that UANO 11 can play an anti-tumor role by blocking cell cycle and inducing apoptosis of HT-29 cells.ConclusionBy comparing with ursolic acid,UANO 11 has higer distribution coefficient In the and the bioavailability.Hence,UANO 11 exhibited greater potential oil/water antiproliferative effects than ursolic acid.UANO11 is therefore worthy of further research as a new antitumor agent for the treatment of colon cancer cells.