Molecular tools for G-protein coupled receptors: Synthesis, pharmacological characterization and [3H]-labeling of subtype-selective ligands for histamine H4 and NPY Y2 receptors

G-protein coupled receptors (GPCRs) are the most important class of biological targets of approved pharmacotherapeutics and hold great potential for the development of future drugs as well. Histamine and neuropeptide Y (NPY) receptors are representative examples of aminergic and peptidergic GPCRs, respectively. Even though the histamine H4 receptor (H4R) has been suggested as a potential therapeutic target for the modulation of various inflammatory and immunological disorders, its (patho)physiological role is far from being fully understood. Therefore, additional potent and receptor subtype selective H4R ligands are required as molecular tools. In search for agonists for the human (h) and murine (m) H4R, a series of 2-arylbenzimidazoles was synthesized, inspired by the structure of a recently published H4R agonist (N-[2-(1H-imidazol-4-yl)ethyl]-3-[4-(5-fluoro-4-methyl-1H-benzo[d]imidazol-2-yl)-3-methyl¬phenoxy]¬propan-1-amine). The compounds were pharmacologically characterized in vitro in radioligand and [35S]GTPgammaS binding assays using membrane preparations of Sf9 insect cells or HEK293 cells stably expressing the hH4R or the mH4R. Extension of the carbon chain between the imidazole and the amino group by two methylene groups was tolerated, revealing higher affinities and potencies (Ki = 0.58 nM, EC50 = 1.2 nM). In addition to the introduction of a 5-methyl substituent at the imidazole ring, rigidization to a spinaceamine derivative was most effective regarding H4R selectivity. Structural changes aiming at replacing the imidazole moiety, e. g. by guanidines, amides and aromatic residues, resulted in a decrease in affinity at the hH4R. Although affinities and activities at the mH4R were lower compared to the data for the hH4R, surprisingly, the 5-methylimbutamine derivative revealed an EC50 value of 7.0 nM at the mH4R (alpha = 0.8). Aiming at H4R agonists with increased selectivity for the H4R over the other HxR subtypes, the combination of VUF 8430 (S-(2-guanidinylethyl)isothiourea, a molecule devoid of an imidazole; Ki (hH4R) = 17.7 nM) with the putatively bioisosteric acylguanidine moiety of the NG-acylated imidazolylalkylguanidines was applied. This approach resulted in compounds with reduced hH4R affinities compared to the reference substance. Highest hH4R affinity (Ki ~100 nM) were found for small alkanoyl residues (propanoyl/isobutyryl) at the guanidine group. Due to a shortage of high affinity radioligands for the investigation of the histamine H2 receptor, the squaramide [3H]UR-DE257 was synthesized and pharmacologically characterized. The Kd‐value (20 nM) determined by kinetic experiments was in agreement with the dissociation equilibrium constant (31 nM) from saturation analysis. [3H]UR-DE257 proved to be a useful tool to determine the H2R binding affinities of unlabeled ligands in competition binding experiments. The tritium-labeled version of the widely used potent H4R antagonist JNJ7777120 ((5-chloro-1H-indol-2-yl)(4-methylpiperazine-1-yl)methanone) was synthesized and pharmacologically characterized. The investigation of [3H]JNJ7777120 revealed that this compound is far from being an ideal standard ligand or, not to mention, an optimal pharmacological tool. Due to high non-specific binding, the purported benefit of [3H]JNJ7777120, a selective radiolabeled H4R antagonist, compared to the non-selective HxR agonist [3H]histamine was not confirmed. Nevertheless, the results of competition binding experiments were in accordance to reported data. The exceptionally high affinity of JNJ7777120 for the murine H4R, reported in literature, was not reproduced. The guanidine-acylguanidine bioisosteric approach was applied to the preparation of the tritiated argininamide-type Y2R-selective radioligand [3H]UR-PLN208. Pharmacological investigation revealed differences between peptidic agonists and nonpeptide antagonists regarding displacement of the radioligand, compatible with the concept of insurmountable antagonism and pseudo-irreversible binding.. Time-dependent depression of the NPY-induced Ca2+-response was demonstrated in the fura-2 assay on hY2R expressing CHO cells The data suggest different or partially overlapping binding sites of BIIE 0246-like antagonists and NPY, supporting the idea of ligand-specific receptor conformations. Taken together, several of the synthesized 2-arylbenzimidazoles harbor the potential of molecular tools and will hopefully contribute to a better understanding of the structure-activity- and structure-selectivity relationships of H4R ligands on human and murine receptor orthologs. The synthesized and pharmacologically characterized tritium-labeled radioligands [3H]UR-DE257, [3H]JNJ7777120 and [3H]UR-PLN208 for GPCRs are proven pharmacological tools for the detection of the respective receptor in vitro and for investigations on the antagonistic binding mode, respectively, as well as for the identification and characterization of new ligands at the respective target