Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists

2-Cyano-1-[4-(1H-imidazol-4-yl)­butyl]-3-[2-(phenylsulfanyl)­ethyl]­guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1–4) subtypes on Sf9 cell membranes (radioligand binding, [35S]­GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)­cyclopentyl]­methyl}-3-[2-(phenylsulfanyl)­ethyl]­guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([35S]­GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.