title: 6-Bromo-8-(4-[(3)H]methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic Acid: a powerful tool for studying orphan G protein-coupled receptor GPR35.
abstract: The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [(3)H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [(3)H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [(3)H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn , An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Thimm
authors.forename: Mario
authors.initials: M
authors.lastname: Funke
authors.forename: Anne
authors.initials: A
authors.lastname: Meyer
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D002264:Carboxylic Acids / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D002264:Carboxylic Acids; C111379:GPR35 protein, human; D043562:Receptors, G-Protein-Coupled