Synthesis Of Oleanolic Acid Derivative And Its Antitumor Effects On Non-Small Cell Lung Cancer Cell Line A549

Lung cancer is a fateful disease with the highest incidence rate and mortality rate in all kinds of cancer worldwide,for which there is no effective treatment so far.It is still a pressing matter of the moment to screen for safer,more effective,stable and controllable lung cancer drugs.In this study,two oleanolic acid derivatives,QDGS-6 and QDGS-7 were synthesised and identified.Furthermore,their antiproliferation effect on non-small cell lung cancer cell line A549 were studied.1.Screening the optimal synthesis of two new derivatives from oleanolic acid.A new oleanolic acid derivative named QDGS-6 was synthesized from oleanolic acid by oxidation,Borch reduction amination,and condensation.Meanwhile,another oleanolic acid derivative,QDGS-7 was synthesized through esterification.Through single factor experiment,the best synthesis condition of QDGS-6 is as follows:the ratio of the two materials,3-amino-12-ene-28-oleanolic acid and?3,5,6-trimethylpyrazine-2-yl?-formic acid is 1:2;reaction the temperature for the reaction is 25?;the reaction time is 24 hours.The yield of the reaction is 72.7%.The best synthesis condition of QDGS-7 is:oleanolic acid:norcantharidin=1:4,reaction temperature is 90?,reaction time is 8 hours.The yield was 77.9%2.Completing the structure elucidation of two new derivatives from oleanolic acid.Compound QDGS-6:MS of[C₃₈H₅₇N₃O₃]⁺:theoretical m/z of[M+H]⁺=605.44,measured m/z of[M+H]⁺=604.90;ES-MS of[C₃₈H₅₇N₃O₃]^-:theoretical m/z of[M-H]^-=603.44,measured m/z of[M-H]^-=602.84.¹H NMR?600 MHz,CDCl₃??8.03?d,J=10.1 Hz,1H?,5.31?s,1H?,3.81?td,J=10.3,5.6 Hz,1H?,2.93?s,3H?,2.85?dd,J=13.6,3.6 Hz,1H?,2.55?d,J=22.3 Hz,6H?,2.01?dd,J=13.4,10.2 Hz,1H?,1.96¹.87?m,2H?,1.84¹.71?m,2H?,1.72¹.56?m,8H?,1.52?t,J=12.7 Hz,1H?,1.39?ddd,J=34.5,28.1,12.5 Hz,3H?,1.30¹.02?m,8H?,1.01⁰.90?m,15H?,0.80?s,3H??¹³C NMR?151 MHz,CDCl3??183.60,164.48,153.83,151.36,147.47,143.70,139.24,122.52,56.60,56.06,47.64,46.59,45.93,41.66,41.01,39.28,38.97,37.96,37.04,33.86,33.08,32.62,32.47,30.68,28.68,27.70,25.91,25.42,23.61,23.40,23.00,22.86,21.90,21.56,18.63,17.15,16.61,15.31.Therefore,QDGS-6 is identified as 3-N-?3,5,6-trimethylpyrazine-2-formyl?-oleanolic acid.Compound QDGS-7:MS[C₃₈H₅₆O₇]^-:theoretical m/z of[M-H]^-=623.4,measured m/z of[M-H]^-=623.64.Therefore,QDGS-6 is preliminarily identified as 3-norcantharidin-oleanolic acid.3.Discovering QDGS-6 and QDGS-7 inhibition of NSCLC A549 cell proliferation,much better than oleanolic acid in vitro.The effects of QDGS-6 and QDGS-7 on the proliferation and apoptosis of A549were studied by CCK-8,EdU and flow cytometry test,while 5-fluorouracil was served as thepositive control.It was found that QDGS-6 and QDGS-7 could inhibit proliferation and induce apoptosis of A549 in a dose-dependent way:As the concentration of QDGS-6 and QDGS-7 increased,A549 cells became round and vacuolated gradually.Furthermore,the activity of Caspase 3 in A549 cells was enhanced after treatment of QDGS-6 and QDGS-7,which may be one of the mechanisms of the proliferation inhibition on A549.Besides,compared to oleanolic acid and 5-fluorouracil,it was also found that QDGS-6 and QDGS-7 had lower IC50in A549 proliferation test,and higher selection index?SI?in the 16HBE proliferation test,which means that QDGS-6 and QDGS-7 were safer than oleanolic acid and5-fluorouracil in vitro.Moreover,QDGS-7 has better inhibitory activity on A549 cells than QDGS-6.