Design,Synthesis Of Novel Dual EP2/EP4 Receptor Antagonists,and Their Application In Tumor Immunotherapy

G protein-coupled receptors（GPCRs）as a classic target of life medicine research,play a very important role in the prevention and treatment of various diseases.With the continuous deepening of mechanisms research,its role in tumorigenesis development and immune escape has become more and more clear.Many studies have shown that prostaglandin E₂（PGE₂）is a key mediator in triggering chronic inflammation and acquired immunosuppressive responses.While EP2 and EP4,which belong to the GPCRs family,play an important role in tumor escape,invasion and metastasis.Based on the connection between GPCRs and tumor microenvironment（TME）,this project fully investigated the synergy between the downstream targets EP2 and EP4 of PGE₂,and developed a novel dual EP2/EP4 antagonist for tumor immunotherapy.Based on the structure of EP4 receptor antagonists has been reported,this project comprehensively analyzed structural characteristics of the existing EP2 receptor antagonists,and then summarized the basic pharmacodynamic group mode of dual EP2/EP4 receptor antagonists through comparison and induction.In this paper,we first analyzed the pharmacodynamic groups of the two targets.At the same time,we simulated the interaction between the compound and the binding site by means of molecular docking,and then we used such as bioisostere and other substitution methods to design and synthesize a series of dual-target compounds with 4,7-dihydro-5H-thieno[2,3-c]pyran as the core.Next we combined the corresponding biological activity experiments and successfully screen the hit compound.On this basis,we optimized its structure and evaluated its pharmacokinetic properties,and finally obtained a novel class of dual EP2/EP4 receptor antagonists with high activity,good drug potential and low toxicity.In this project,the existing EP4 receptor antagonists in our laboratory were first analyzed,and it was found that corresponding extension of the hydrophobic side chain is the key to production of EP2 antagonistic activity in the skeleton structure of 4,7-dihydro-5H-thieno[2,3-c]pyran.After several rounds of structure-activity relationship（SAR）summary and chemical structure modification,we determined that the com-pound had the best activity when the 2nd position of 4,7-dihydro-5H-thieno[2,3-c]pyran was biphenyl-related structure.Subsequent c AMP experimental results showed that the inhibitory activity of multiple compounds on both targets was maintained at the nanomol level,and their activities were comparable to those of the positive compounds E7046 and PF-04418948,and they also had good physicochemical properties.After-wards,we selected compound 96 with better in vitro activity and higher druggability as a lead compound for further biological evaluation,the results showed that compound96 can effectively inhibit the expression of immunosuppressive-related genes in macro-phages.In addition,the result of anti-tumor activity of CT26 tumor-bearing mice also showed that the anti-tumor effect of the compound 96 was significantly better than that of the positive drugs E7046 and PF-04418948 alone.It was worth noting that compound96 can further enhance the anti-tumor immune response,inhibit tumor growth and prolong the survival time of mice.In summary,based on PGE₂,a key mediator in the TME,this paper fully inves-tigated EP2 and EP4 which were two major contributors in many physiological and pathological activities,and innovatively combined the two receptors for anti-tumor activity research.Furthermore,we successfully designed and synthesized novel dual EP2/EP4 antagonists with high activity and good druggability through multi-batch chemical structure modification.More in-depth studies have shown that the effect of compound 96 in inhibiting tumor growth was closely related to its ability to promote immune cells to resist tumors.Further blocking the target of tumor immune inflam-matory mediators can effectively improve the anti-tumor immune function in humans.