Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like the reference antagonist ciproxifan (hH3R pKi value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues.

A piperidine-based lead structure for the human histamine H 3 receptor (hH 3 R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH 3 R ligand with affinity in the nanomolar concentration range (K i hH 3 R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH 3 R preference. Bodilisant was used for visualization of hH 3 R in hH 3 R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH 3 R.KEYWORDS: histamine, H 3 receptor, nonimidazole derivative, GPCR, BODIPY, HEK-293 cells, tissue labeling, fluorescence confocal laser scanning microscopy, displacement, pharmacological tool T he human histamine H 3 receptor is one of the four human histamine receptor subtypes (hH 1−4 R). It is a membranebound class A family G-protein-coupled receptor (GPCR) (G i/o coupled) mainly expressed in the central nervous system (CNS) acting as an auto-as well as a heteroreceptor.1 The human histamine H 3 receptor (hH 3 R) modulates the release of several neuronal neurotransmitters.2,3 Because of different central effects of several hH 3 R antagonists in preclinical and clinical trials, we have seen a need for labeled hH 3 R ligands to be taken as diagnostic tools to further investigate neurological disorders in the CNS based on receptor distribution, occupation, and regulation. In histochemistry, fluorescentlabeled antibodies are of common use. 4 For the manufacturing process of antibodies, mostly animal experiments are required. 5,6 These antibodies have to be labeled in a followup work step with a second labeled antibody for immunofluorescence; merely a few primary antibodies are directly labeled.7 Most antibodies are sensitive to temperature and have to be stored in freezers. The main disadvantage as compared to small fluorescent GPCR ligands is their undisplacement properties without target destruction. Small fluorescent GPCR ligands can be displaced by other ligands and consequently enable the development of fluorescence displacement assays. There are many efforts to design fluorescent GPCR ligands on the strength of their prominence as the largest and most versatile group of cell surface receptors, therefore responsible for various pharmacological functions. 8,9 Recent research in our working group resulted in chalconebased fluorescent hH 3 R ligands able to label hH 3 R in cells and human tissue 10 based on earlier results (Mirisant-405, Mirisant-470, and Benz-Mirisant-405).11−13 Because these early ligands were not usable under all conditions, we wanted to design fluorescent hH 3 R ligands with more properties in common in one molecule, that is, higher hH 3 R affinity, versatile fluorescent wavelength, optimized fluorescence intensity, and thus a better signal-to-noise ratio. The decision has been made for 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye as a...

Exchange of the lipophilc part of ortho-substituted cinnamic acid lead structures with different small molecule fluorophoric moieties via a dimethylene spacer resulted in hEP3R ligands with affinities in the nanomolar concentration range. Synthesized compounds emit fluorescence in the blue, green, and red range of light and have been tested concerning their potential as a pharmacological tool. hEP3Rs were visualized by confocal laser scanning microscopy on HT-29 cells, on murine kidney tissues, and on human brain tissues and functionally were characterized as antagonists on human platelets. Inhibition of PGE2 and collagen-induced platelet aggregation was measured after preincubation with novel hEP3R ligands. The pyryllium-labeled ligand 8 has been shown as one of the most promising structures, displaying a useful fluorescence and highly affine hEP3R antagonists.