Highly potent and selective acylguanidine-type histamine H2 receptor agonists: synthesis and structure-activity relationships of mono- and bivalent ligands

Potent and selective histamine H2 receptor (H2R) agonists, including brain-penetrating compounds, are required as pharmacological tools to evaluate the (patho)physiological role of H2Rs. Moreover, H2R agonists might be of therapeutic value as drugs, for example, in the treatment of acute myelogenous leukemia. Previously, acylguanidine-type H2R agonists with reduced basicity were synthesized in our laboratory, resulting in improved bioavailability and CNS penetration compared to the corresponding guanidines. Based on the preceding work, this thesis aimed at the design, the synthesis and the pharmacological characterization of novel NG-acylated hetarylpropylguanidines to elaborate the structure-activity relationships (SAR) in more detail. A central aspect of this project was the development of bivalent acylguanidine-type H2R agonists. The prepared compounds were investigated for H2R agonism in GTPase and [35S]GTPγS binding assays at guinea pig (gp) and human (h) H2R-GsαS fusion proteins including various H2R mutants, at the isolated gp right atrium (in cooperation with Prof. Elz, University of Regensburg), and, with respect to H2R selectivity, in GTPase assays for activity on recombinant human H1, H3 and H4 receptors. In addition, representative compounds were investigated regarding their hemolytic and cytotoxic properties as well as their potential to bind to plasma proteins. NG-Acylated 3-(2-aminothiazol-5-yl)propylguanidines proved to be H2R partial to full agonists. Within this series, highest potencies resided in compounds having a two- or three-membered carbon chain between carbonyl group and phenyl or cyclohexyl ring, respectively. Notably, the introduction of a free amino group at an appropriate distance to the pharmacophoric moiety was beneficial with respect to H2R agonistic potency. In contrast to their imidazole analogs, the aminothiazoles were highly selective for the H2R vs. other HR subtypes. Thus, this study substantiates previous results, confirming that the 2-aminothiazole and the imidazole moiety are bioisosteric groups at the H2R but not at the H3R and H4R. Moreover, in contrast to amthamine, the 4-methyl group at the thiazole ring did not significantly contribute to the H2R agonism of NG-acylated 3-(2-amino-4-methylthiazol-5-yl)propylguanidines. Bivalent H2R agonists were synthesized by connecting the guanidine groups of two molecules by NG-acylation with dicarboxylic acids of different structure and length (spacer lengths ≈ 6 – 27 Å). The bivalent ligands proved to be up to two orders of magnitude more potent than monovalent acylguanidines and up to 4000 times more potent than histamine at the gpH2R (compounds with octanedioyl to decanedioyl spacers). These are the most potent histamine H2R agonists known to date. However, due to insufficient spacer lengths of the most active compounds, the tremendous gain in potency compared to monovalent analogs cannot be explained by simultaneous occupation of the orthosteric recognition sites of a H2R dimer. The high potency rather results from interaction with an accessory (allosteric?) binding site at the same receptor protomer. Replacing the second hetarylpropylguanidine moiety with simple alkyl guanidine groups afforded rather high H2R agonistic activities (EC50 values in the low nanomolar range), whereas all other variations in this part of the molecule led to drastically decreased potencies. A further decrease in potency resulted from the elimination of the second guanidino group, corroborating the importance of a basic centre at an appropriate distance to the pharmacophore to obtain highly potent bivalent H2R agonists. These results are consistent with the concept of interaction with the orthosteric and an accessory binding site of one H2R protomer, i. e. the accessory binding site can accommodate the second acylguanidine portion. All investigated compounds were significantly more potent and efficacious at the gpH2R relative to the hH2R. These differences might help to verify the suggested model of bivalent ligand - receptor interactions via identification of species-dependent molecular determinants of the orthosteric and the accessory binding site in hH2R and gpH2R, respectively. Investigations on gpH2R and hH2R mutants/chimera confirmed the key role of non-conserved Tyr-17 and Asp-271 in TM1 and TM7 in the gpH2R for species-selective H2R activation and suggested that the e2 loop does not participate in direct ligand - receptor interaction. To explore the topology of this putative accessory binding site in more detail, further studies on H2R mutants are necessary. In conclusion, bioisosteric and bivalent approaches applied in this thesis led to highly potent and selective pharmacological tools for more detailed investigations of the H2R. However, in view of cell based in vitro investigations or future in vivo experiments, the drug-like properties of these H2R agonists should be further improved

In vitro and in vivo studies on the binding and permeation of ketotifen and norketotifen atropisomers in the central nervous system

Ketotifen (K) is a first-generation antihistamine with antiinflammatory potency. It penetrates the blood-brain barrier (BBB) and causes a sedative side effect. Norketotifen (N) is an active metabolite of K. The S-atropisomer of N (SN), however, has antihistaminic and antiinflammatory properties but less sedating side effect than RN and K. This may be due to: (1) higher concentrations of K and RN than SN in the central nervous system (CNS) and/or (2) higher affinity of K and RN than SN for rat brain H1 receptors. The aim of this thesis was to investigate the mechanism of why SN lacks a sedative side effect. To determine concentrations of racemic K and N and atropisomers of K and N in buffer solutions and bio-matrices, nonchiral and chiral high-performance liquid chromatography (HPLC) assays were developed and validated. Log P and log D of K and N (both octanol/buffer and liposomes/buffer) were determined to aid in interpretation of in vitro cell studies. Rat brain endothelial (RBE4) and colorectal adenocarcinoma (Caco-2) cell monolayers were used as in vitro models of the BBB to study the stereoselective uptake and permeability of K and N atropisomers. To investigate the distribution of K and N atropisomers between brain tissue and plasma, the total and free brain-to-plasma (B/P) ratios of K and N atropisomers were measured 5 min post-administration of racemic K and N through the rat tail vein. The affinity of K and N atropisomers to brain H1 receptors was investigated by determining the extent of inhibition of [3H] mepyramine binding to H1 receptors in rat brain cell membranes. The in vitro studies indicated active mechanisms for transporting K and N in both RBE4 and Caco-2 cell lines; however, none of these mechanisms were stereoselective. Interestingly, for both cell lines, more N was found binding non- specifically to cell membranes than that of K, though in a non-stereoselective manner. Liposomes/buffer distribution studies aided in interpretation of these results. Similarly, the total and free B/P ratios of K and N atropisomers suggested a predominant influx mechanism involved in transporting of K and N through the rat BBB. However, this mechanism was not stereoselective for either K or N atropisomers. In addition, K and N non-specifically bound to rat plasma protein and brain tissues in different degree but the non-specific binding was not stereoselective either. In contrast, H1 receptor affinity results suggested a stereoselective binding of SN for the H1 receptors in rat brain, in that SN had a lowest affinity compared with RN, SK and RK. Significant differences in the affinity for the H1 receptors were found between SN and SK, SN and RK, moreover, between SN and RN. Although the difference was significant but not substantial compared to some published stereoselective affinity for the H1 receptors, a similar degree of difference was observed and published by other research groups. Thus the lowest affinity of SN for the H1 receptors could participate in the observed less sedative effect caused by SN. In conclusion, there was no stereoselective transport of SN through the BBB either in vitro or in vivo, and there was no stereoselective non-specific binding of SN to rat plasma proteins or brain tissues. The lower sedative effect of SN is due to a lower uptake of N than K into the brain and reduced binding of SN to CNS H1 receptors