SYSTEMS NEUROSCIENCE ORIGINAL RESEARCH ARTICLE published: 26 March 2012 doi: 10.3389/fnsys.2012.00014 Novel chalcone-based fluorescent human histamine H 3 receptor ligands as pharmacological tools MiriamTomasch, J. Stephan Schwed, Lilia Weizel and Holger Stark* ZAFES/CMP/ICNF, Biocenter, Institute of Pharmaceutical ChemistryJohannWolfgang Goethe University, Frankfurt am Main, Germany Edited by: Maria Beatrice Passani, Universita’ di Firenze, Italy Reviewed by: Marco De Amici, Università degli Studi di Milano, Italy Marlon Cowart, Abbott Laboratories, USA *Correspondence: Holger Stark, Biocenter, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany. e-mail: h.stark@pharmchem. uni-frankfurt.de Novel fluorescent chalcone-based ligands at human histamine H 3 receptors (hH 3 R) 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 hH 3 R in the same concentration range like the reference antagonist ciproxifan (hH 3 RpK i value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluores- cence could be excluded. All synthesized chalcone compounds could be used to visualize hH 3 R proteins in stably transfected HEK-293 cells using confocal laser scanning fluores- cence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH 3 R visualization in different tissues. Keywords: human histamine H 3 receptor, fluorescent ligand, fluorescence confocal laser scanning microscopy, pharmacological tool INTRODUCTION Histaminergic receptors belong to class A of membrane bound G-protein-coupled receptors (GPCRs). They consist of four sub- types, the histamine H 1 ,H 2 ,H 3 , and H 4 receptors (Walter and Stark, 2012). The H 3 receptor has a neurotransmitter function. High receptor densities could be found in different areas of the central nervous system (Martinez-Mir et al., 1990; Sander et al., 2008). Possible indications of H 3 receptor antagonists/inverse ago- nists could be the treatment of cognitive and sleep disorders as well as schizophrenia, epilepsy, adipositas, and neuropathic pain (Girard et al., 2004). To get information on etiopathol- ogy and accumulation or depletion of human histamine H 3 receptors (hH 3 R) and to accelerate the clinical development of pharmaceuticals in the screening of drugs it is interesting to design labeled H 3 receptor ligands. To date several different tech- niques have been used to measure receptor occurrence in tissues. Radioactive competition is often used for instance in brain slices with [ 3 H](R)-alpha-methylhistamine (Martinez-Mir et al., 1990), [ 3 H]N α -methylhistamine (Le et al., 2009), [ 3 H]-A-349821 (Miller et al., 2009), [ 125 I]iodoproxyfan (Ligneau et al., 1994; Stark et al., 1996), and [ 125 I]iodophenpropit (Jansen et al., 2000). Adversely, analysis of ex vivo autoradioactivity often takes longer time (Le et al., 2009) and synthesis and storage of radioligands causes high costs and special equipment/rooms. Radioactive exposure as generally known is very harmful. Fluorescent ligands are pre- ferred over radioligands in terms of safety precautions and often applicability. Fluorescent ligands and their use for the localiza- tion and detection of GPCRs is still a topical area of investigation (Kuder and Kiec-Kononowicz, 2008). Attempts to design fluores- cent human hH 3 R were established with motif structure elements of Sangers reagent, dansyl, NBD, cyanoisoindol, and tetramethyl- rhodamine groups (Amon et al., 2006, 2007; Cowart et al., 2006; Kuder et al., 2010). Most of these compounds showed high affinity at histamine H 3 receptors (hH 3 R K i : 0.1–10 nM), but their fluores- cence absorption and emission wavelengths were mainly between 300 and 500 nm. In this wavelength range interactions with cel- lular autofluorescence occur and in addition to this problem, most of these ligands possess low fluorescence intensities. A PhD thesis from the working group of Prof. Buschauer (University Regensburg, Erdmann, 2010) presented further fluorescent human histamine H 3 ligands. Applied fluorophores possess good fluo- rescent features but are expensive or difficult to synthesize. The aim of this study was the optimization of the fluorescent proper- ties taking advantage of already published fluorescent ligands as lead structures. A literature survey indicated bio-active fluorescent benzylidine tetralones (Kamakshi et al., 2010) possessing different antibacterial activity and useful physicochemical data (Al-Ansari, 1998). Tomecková et al. (2004) reported on related cyclic chalcone analogs demonstrating biological effects on mitochondrial outer membrane via fluorescence microscopy. These results motivated us to use chalcones as fluorescent element for labeling of histamine H 3 receptor ligands to generate novel fluorescent pharmaceutical tools (Figure 1). MATERIALS AND METHODS CHEMISTRY All reagents and solvents were purchased from VWR (Darm- stadt, Germany), Sigma-Aldrich (Steinheim, Germany), Alfa Aesar (Ward Hill, MA, USA), Perkin Elmer Life and Analytical Sciences (Rodgau, Germany), and Acros Organics (Geel, Belgium), and were used without further purification (unless otherwise stated). 1 H and 13 C NMR spectra were recorded on a AV 250 Spektrom- eter (5.9 T; 1 H: 250 MHz; 13 C: 63MHz), AV 300 Spektrometer (7.1 T; 1 H: 300 MHz; 13 C: 75 MHz),or AV 400 Spektrometer (0.4 T; 1 H: 400 MHz, 13 C: 100 MHz): Bruker (Rheinstetten, Germany). Electro-spray-ionization MS (ESI MS) was performed on a: VG Frontiers in Systems Neuroscience www.frontiersin.org March 2012 |Volume 6 | Article 14 | 1