Discovery,Optimization And Bioactivity Of Small Molecule CD73 Inhibitors

In recent years,immunotherapy has become a hot spot in the field of tumor treatment,and new targets have been discovered continuously.The adenosine signaling pathway is a key pathway in tumor immunosuppression.ATP is degraded into AMP under the action of extracellular CD39,and AMP is further metabolized into adenosine under the action of extracellular CD73.Therefore,blocking the adenosine signaling pathway can reduce the immunosuppressive effect of tumors.This thesis mainly focuses on the CD39-CD73-adenosine signaling pathway,and carried out research on the discovery,structure optimization and biological activity evaluation of small molecule CD73 inhibitor lead compounds.The main contents include:1.Discovery of lead compounds for small molecule CD73 inhibitorsUsing the self-built compound library of the research group,using the three-part screening strategy,through the malachite green phosphate reagent method,screening and discovering new framework small molecule CD73 inhibitor lead compounds.When screening the kinase inhibitor library,it was found that the compound MS1013 had a significant inhibitory effect on human CD73 protein at a concentration of 10μM,and its inhibition rate was as high as 92.56%.When screening the natural product library,it was found that the inhibition rate of compound MS1371 was 63.63%at a concentration of 10μM.In addition,this thesis carried out the determination of target selectivity and CD73 inhibitory activity IC₅₀ value.It was found that the compounds MS1013 and MS1371 had no inhibitory activity on CD39,and both showed good inhibitory activity on CD73.Immune activation studies have shown that the natural product MS1371 has an activating effect on IFN-γat 10μM,which is better than the positive control drug AB680.The above results indicate that the natural product MS1371（betulinic acid）can be used as a lead compound for small molecule CD73 inhibitors.Then,a preliminary structure-activity relationship study was conducted on the lead compound betulinic acid using similarity search technology,which provided a direction for subsequent structural optimization.2.Design,synthesis and biological activity of betulinic acid carbamate derivativesAccording to the preliminary structure-activity relationship of lead phenylbutyrate,10kinds of 3-butyric acid carbamate derivatives were designed and synthesized,and their inhibitory activity on CD73 was studied.The results showed that 5 compounds showed certain activity.Among them,the betulinic acid carbamate derivative ZM514 has the most significant inhibitory effect on CD73,and its human-derived CD73 inhibitory activity IC₅₀ value is 1.39±0.01μM,which is 4.24 times higher than that of betulinic acid.Moreover,the IC₅₀ value of the inhibitory activity against mouse CD73 is 14.65±2.50μM,which is 3.70 times higher than that of betulinic acid.The results of in vitro cytotoxicity experiments on tumor cells with high expression of CD73 showed that the compound ZM514 had little toxicity to both MDA-MB-231 and Mia Pa Ca-2 tumor cells,indicating that this type of compound is not a cytotoxic drug.The results of the immune activation study showed that the compound ZM514 has a better IFN-γactivation effect than the positive control drug AB680 at 10μM,and is equivalent to APCP.Therefore,the betulinic acid carbamate derivative ZM514 can be used as a small molecule CD73 inhibitor for further follow-up in-depth research.3.Design,synthesis and biological activity of betulinic acid ester derivativesBased on the structure-activity relationship of betulinic acid,8 betulinic acid ester derivatives were designed and synthesized,and the CD73 inhibitory activity test was carried out.The results showed that the four derivatives showed good inhibitory effects on CD73.Among them,the IC₅₀ values of the inhibitory activity of compounds ZM522 and ZM523 on human CD73 reached the nanomolar level,which was more than 10 times higher than the lead.In addition,the IC₅₀ value of the inhibitory activity of compound ZM522 on murine CD73 was3.06±0.02μM,and its inhibitory effect was significantly enhanced compared to the lead.The results of in vitro cytotoxicity experiments show that the cytotoxic activity of compound ZM522 is equivalent to the lead betulinic acid.The results of immune activation studies show that compound ZM522 has a significant activation effect on IFN-γat 10μM.This study found a new class of small molecule CD73 inhibitor lead compounds with a new framework,which laid a good foundation for the development of small molecule CD73inhibitors with independent intellectual property rights.