6‑Bromo-8-(4‑[3H]methoxybenzamido)-4-oxo‑4H‑chromene-2-carboxylic Acid: A Powerful Tool for Studying Orphan G Protein-Coupled Receptor GPR35

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 [3H]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 [3H]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. [3H]12 labeled the receptor with high affinity (K D = 5.27 nM). Binding was saturable (B max = 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; K i = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a K i value in the subnanomolar range.