Design,Synthesis And SAR Analysis Of Prostanoid EP4 Receptor Antagonists,and Their Application In Tumor Immunotherapy

EP4 receptor is a downstream prostaglandin receptor for prostaglandin E₂(PGE₂),belongs to G-protein coupled receptor(GPCR)superfamily.An increasing number of studies have indicated that PGE₂/EP4 signaling pathway is continuously activated in most solid tumors,inhibiting innate and acquired anti-tumor immune responses in the tumor microenvironment by triggering cancer-promoting chronic inflammation,it plays a key role in influencing of tumor development,immune evasion and resistance to cancer immunotherapy.Pharmacological blockade of EP4 signaling has been proved to be able to largely reverse PGE₂-induced immunosuppression and enhance anti-tumor immune response.The present EP4 inhibitors can be broadly grouped into two categories:the first-generation ligands bearing sulfonyl urea/acylsulfonamide moieties,and the second-generation EP4 antagonists containing carboxamido-benzoic acid and related scaffolds.In order to improve medicinal properties of the designed compounds,we turned our attention to the second-generation EP4 antagonists with better drug-like properties.In the initial of our work,we comprehensively investigated the structure types of the second-generation EP4 antagonists and summarized their basic template including three essential structural parts:a core skeleton,a benzoic acid-containing branch chain and a hydrophobic side chain.In this paper,we reasonably designed some core skeletons by employing the bioisosteric and skeleton transition strategies,and successfully screened two kinds of hit compounds bearing 1H-1,2,3-triazole and 4,7-dihydro-5H-thieno[2,3-c]pyran.After several rounds of substituent modification and dominant fragment integration,we identified two class of novel EP4 receptor antagonists with high activity,high selectivity,and good drug-like properties.Through analyzing the structure-activity relationship of triazole compounds,we found some important rules as follows:firstly,the integration of hydrophobic groups to N-1 of 1H-1,2,3-triazole was beneficial to improve the inhibitory activity of compounds;secondly,after configuration identification by X-ray crystallography or 2D NMR spectroscopy,we determined 1,5-regioisomer was preferred configuration for the 1,2,3-triazole analogs;more importantly,we found that the R²substituents in the triazole ring had an important impact on the EP4 antagonism activity,a conjugated system composed of an unsaturated bond linked to the triazole ring was generally beneficial for EP4activity.After in vitro EP4 antagonistic activity evalution in calcium flux experiment and c AMP Glo Sensor experiment,we found that compound 10m exhibited very high inhibitory activities against EP4 receptor(6.1 n M and 18.7 n M,respectively).Besides,10m had no significant interaction with human EP1-3 receptors at concentrations up to10?M,and showed good bioavailability and reasonable half-time(F=48.0%,t_1/2=4.7h).Then we further evaluated the anti-tumor effect of 10m and found that 10m exhibited better antitumor potency than the active comparator E7046 in a mouse CT26colon cancer model.Further results indicated that the ability of 10m to suppress tumor growth was correlated with its effects on CD8⁺T cell-mediated anti-tumor immunity.The design idea of 4,7-dihydro-5H-thieno[2,3-c]pyran analogs was derived from structural research of the early second-generation EP4 antagonists.MK-2894,bearing thiophene as core skeleton,is a highly potent and selective second-generation EP4antagonist.Owing to poor physical and chemical properties and low drug-like properties,this compound has no clinical progress since 2010.According to the predicted data of MK-2894,it is preliminarily judged that this compound has poor drug-like properties,and may not have the potential for further development.Therefore,we next focused on the structural optimization of MK-2894 and tactfully designed bicyclic heteroaryl by employing skeleton transition strategy.After several rounds of structural modifications,we identified 4,7-dihydro-thieno[2,3-c]pyran as the preferred core skeleton,and the inhibitory activity of the compounds was further improved during subsequent modifications of R² on thieno[2,3-c]pyran ring.Calcium flux assay results showed that multiple compounds exhibited favorable antagonistic activity on EP4receptors(IC₅₀<1 n M).Among them,the antagonistic activity of compound 30d reached to 54 p M,which was approximately 157-fold more potent than active comparator E7046(EP4 Ca ²⁺IC₅₀=8.5 n M).Compared with MK-2894,thieno[2,3-c]pyran analogs have better water solubility and metabolic stability.In the process of structural modifications,compound 31g,a superior compound in terms of in vitro biological activity and drug-like properties,was choosed as the lead compound for evaluating antitumor activity of CT26 tumor-bearing mice and found that 31g exhibited more significantly antitumor potency than E7046.Furthermore,we evaluated the antitumor effect of compound 31g combined with PD-1 antibody.The results showed that compound 31g combined with PD-1 antibody could more significantly inhibit tumor growth than monotherapy and prolong the median survival time of CT26 tumor-bearing mice.In conclusion,EP4 receptor,one of promising tumor immunotherapy targets,was selected to study in the paper,after comprehensive investigation of the present EP4antagonists,we have successfully designed and synthesized two classes of novel EP4receptor antagonist with high activity,high selectivity,and good drug-like properties.Further in vivo antitumor activity evaluation showed that lead compounds 10m and 31g exhibited more significantly antitumor potency than E7046 in mouse CT26 colon cancer model,and the mechanism of its anti-tumor activity was proved to be correlated to the improvement of antitumor immune function.