Design and Multistep Synthesis of Ligands for the GPR18 and GPR183: Related Orphan G Protein-Coupled Receptors with Immunoregulatory Functions

Abstract

GPR18 and GPR183 are closely related G protein-coupled receptors (GPCRs) with significant structural similarity. Despite differences in ligand-binding profiles, both respond to lipophilic molecules and share overlapping expression patterns, particularly in immune-related tissues. These similarities suggest that they may have related biological functions and play crucial roles in immunomodulation. For GPR18, starting from the recently published indolylethylaminoxanthine PSB-KK1415, which acts as an agonist (EC₅₀ = 0.0191 µM), the lead optimization strategy focused on enhancing potency to enable radiolabeling for radioligand binding assays and on identifying sites suitable for fluorophore attachment to create fluorescent probes for use in BRET (Bioluminescence Resonance Energy Transfer) assays. Parallel efforts were made to refine physicochemical properties to support both approaches. Although modifications also targeted the indolylethylamino portion, the primary focus was on optimizing the N7-position of the xanthine core. Five distinct multi-step synthetic strategies were implemented to modify position N7 of the xanthine scaffold, yielding promising candidates. Among these, FPE-173 (EC₅₀ = 0.207 µM), featuring a benzyl substituent at N7 with a para-positioned diamino linker, emerged as a strong candidate for fluorescence labeling, while FPE-211 (EC₅₀ = 0.0179 µM), containing a 7-chloroquinoline at N7, demonstrated excellent potential for a radiolabeled ligand. These tool compounds are expected to facilitate further exploration of GPR18's physiological and pathological roles, aiding in its validation as a therapeutic target. For GPR183, the indolylethylaminobenzamide FPE-48B was identified as a promising antagonist, with an IC₅₀ of 4.06 µM. This compound shares structural features with potent known antagonists such as ML401 and NIBR189. These insights guided the design and synthesis of three series: benzoic acid amides, piperazine derivatives, and cinnamic acid amides. A consistent structure-activity relationship (SAR) trend was observed, with p-halogen substitution significantly enhancing potency. Starting from FPE-48B, a 4-fold increase in potency was achieved within the benzoic acid amides class, with FPE-156 emerging as the most potent compound (IC₅₀ = 1.03 µM). Development of the other two classes resulted in compounds that exhibit low micromolar potency, including FPE-ML33 (IC₅₀ = 1.65 µM) from the cinnamic acid amide series, and FPE-ML54 (IC₅₀ = 1.35 µM) from the piperazine series. While further optimization is required, these scaffolds provide a foundation for developing GPR183 antagonists and exploring broader applications. Overall, this work enhances our understanding of GPR18 and GPR183, providing valuable leads for future drug discovery efforts. The novel ligands developed herein not only advance the tools available for investigating these receptors but also pave the way for new therapeutic strategies targeting immune and inflammatory diseases.