br GPR a G protein coupled receptor GPCR

GPR35, a G protein-coupled receptor (GPCR), was discovered and classified as an orphan GPCR in 1998 and deorphanized in 2006 by the discovery of kynurenic imipramine hcl as the endogenous agonist. Since its discovery, limited references on the GPR35 receptor have appeared, due in part to a scarcity of endogenous or exogenous ligands that regulate GPR35 with sufficient potency and selectivity. The deorphanization of GPR35 receptor has even been debated due to the poor potency of kynurenic acid (IC of 39μM) and it has recently been proposed that GPR35 should be renamed to CXCR8 due to a reported binding to CXCL17. GPR35 has gained special consideration as a result of its association with many diseases including type-2 diabetes, nociceptive pain, inflammation, mild mental retardation syndrome, metabolic disorders, and gastric cancer. Based on the association of GPR35 with these major diseases and disorders, there is a current and urgent need for compounds to regulate the GPR35 receptor and serve as research tools in molecular characterization of GPR35 receptor. Herein, we report a structure-activity relationship (SAR) study of benzothiazoles as antagonists of GPR35. This research sets the foundation for the design of future potent and selective ligands. An image-based high-throughput, high-content primary screen using the Molecular Libraries Probe Production Initiative evaluated ∼300,000 compounds. This screen identified a few relatively potent and selective GPR35 antagonists: benzothiazole (CID1231538), rhodanine (CID2286812), and pyrazole (CID2745684; ). Compound displayed high antagonism activity at GPR35 (IC=20.1nM), however, this compound has a rhodanine ring. Compounds with a rhodanine ring have been recommended to be excluded from library screening results due to the potentially misleading results, as described as Pan Assay Interference Compounds (PAINS). Compound is also a potent GPR35 antagonist (IC=160nM), but the thiosemicarbazone functionality could also react in a nonselective manner, similar to the rhodanine ring. Although benzothiazole was not as potent as the other potential leads (IC=0.55μM), it was selected because it could be synthetically modified and had a greater opportunity for selectivity since it was inherently less reactive for covalent attachment. The route to synthesize compounds – and – initiated with ring-opening of phthalic anhydride using 6-amino-2-mercaptobenzothiazole to form acid in 96% yield or with 5-amino-2-mercaptobenzoimidazole to form compound , also in good yield (95%) (). Alkylation of thiol with 4-chlorobenzyl chloride, benzyl bromide, or 4-(chloromethyl) pyridine hydrochloride successfully produced thioethers , , and , respectively. These different aryl groups would test the potential and type of aromatic stacking. Formation of the phthalisoimide ring was accomplished using acetic anhydride and triethylamine. Phthalisoimide intermediates , , and were produced in 91%, 96%, and 87% yields, respectively, and were opened using morpholine, piperidine, or pyrrolidine to produce final analogues , , , , and . The morpholine surrogates were chosen to evaluate if there is a requirement for hydrogen bonding in that region of the receptor. Analogue was synthesized following the same protocol, except using 5-amino-2-mercaptobenzoimidazole as starting substrate. The benzimidazole group was selected for synthesis to test the role of the endocyclic sulfur. Benzimidazole may also provide a positive potential surface instead of negative potential surface of the sulfur. Oxidation of the exocyclic sulfur to a sulfoxide or sulfone could potentially provide strong and directional hydrogen bonds. Sulfoxide and sulfone were synthesized using different amounts of -chloroperoxybenzoic acid (CPBA) to improve this interaction (). Due to the chiral nature of the sulfoxide, enantiomers and were individually synthesized following procedures similar to those used to synthesize Nexium (esomeprazole). The individual sulfoxide enantiomers ( and ) were synthesized using titanium tetraisopropoxide and the respective enantiomers of diethyl tartrate and cumene hydroperoxide as the oxidant. The absolute configuration of sulfoxides and were inferred based on the close analogy to the process for esomeprazole. It was decided that the confirmation of the absolute configurations would be made if either of the two enantiomers were significantly potent.