Synthesis And Anti-cancer Effect Of Oleanolic Acid Covalent Inhibitors

The mechanism of tumor is complex,and as a natural product,oleanolic acid（OA）has the characteristics of multi-target action,which has unique advantages in the field of anti-tumor.In addition,covalent inhibitors containing electrophilic active groups have become a hot spot in the field of antitumor drugs in the past decade.The introduction of electrophilic active groups may further improve the antitumor activity of OA derivatives.In this paper,the covalent inhibitor design strategy was applied to the structural modification of OA.By adjusting the reactivity and modification site of covalent reactive groups,an electrophilic warhead was introduced into the A ring of OA to design and synthesize a series ofα,β-unsaturated amides,non-Michael receptors andα,β-unsaturated ketone OA derivatives,a total of 36,and their in vitro anti-tumor effects were discussed.（1）The in vitro cell proliferation inhibitory activity of these compounds on tumor cell lines MCF-7,Hep G2,He La,MDA-MB-231,MDA-MB-468 and JIMT-1 was tested by MTT method.Most of the derivatives have good antiproliferative activities,especially compounds Y02(MDA-MB-468 cells,IC₅₀=0.58μM),27a(MCF-7 cells,IC₅₀=0.83μM;JIMT-1cells,IC₅₀=0.86μM),Y03(MCF-7 cells,IC₅₀=0.68μM;JIMT-1 cells,IC₅₀=0.77μM),and Y04(MCF-7 cells,IC₅₀=0.65μM;JIMT-1 cells,IC₅₀=0.78μM),could selectively inhibit breast cancer cells.Cell scratch experiments showed that compounds 27a,Y03,and Y04 could significantly inhibit the migration of MDA-MB-231 and MCF-7 cells at a concentration of 4μM.In addition,compounds 27a,Y03,and Y04 can dose-dependently induce apoptosis and increase of reactive oxygen species（ROS）in MCF-7 cells.（2）OA derivatives can induce the production of reactive oxygen species in tumor cells,leading to tumor cell death,which may be related to cell ferroptosis.The viability of MCF-7cells increased after treated with ferroptosis inhibitors Fer-1 and DFO,and decreased after treated with ferroptosis inducers Fe³⁺and Erastin,while the viability did not significantly decreased by the addition of other metal ions,indicating that OA derivatives can induce ferroptosis.In addition,most of the tested OA derivatives induced a greater than 50%decrease in glutathione peroxidase 4（GPX4）activity,suggesting that these OA derivatives may induce ferroptosis through the GPX4 pathway.（3）The non-specific reaction of electrophilic warheads with glutathione（GSH）may lead to toxicity in vivo,so we analyzed the reactivity of OA derivatives with GSH in vitro.When the molar ratio of OA derivative and GSH was 1:10,after reacting at 37^oC for 2 hours,the percentage of unreacted compounds was analyzed,and it was found that more than 79%of the compounds were unreacted,and the reactivity was lower than that of the positive control drug CDDO-Me（75.69%）,suggesting that such OA derivatives may have lower GSH reaction toxicity in vivo.