title: Entwicklung von 18F-markierten subtyp-selektiven Orexin- und Muskarin- Rezeptorliganden für die Bildgebung mit Hilfe der Positron-Emissions-Tomographie
title: Development of 18F-labeled subtype selective orexin and muscarinic receptor ligands for imaging by positron emission tomography
Creators: name: Bolik, Kim-Viktoria
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2024
Subjects: subject: Orexin receptor
subject: Muscarinic receptor
subject: GPCR
subject: PET ligand
subject: 18F-labeling
subject: Boroxine precursors
subject: Copper-mediated radiofluorination
subject: Neuroimaging
subject: Cancer imaging
subject: Subtype-selectivity
subject: Boronic acid anhydride
subject: Apparent molar activity
subject: Boronic acid anhydride precursor

description: Non-invasive positron emission tomography (PET) has proven an extremely useful tool for neuroimaging and cancer imaging. PET radioligands that subtype selectively address G-protein coupled receptors (GPCRs) are still needed, e.g. for the muscarinic acetylcholine 3 receptor (M3R) and the orexin 1 receptor (OX1R). Subtype selective 18F-labeled PET ligands for these GPCRs may lead to profound understanding of their role in neurological disorders and distribution in the brain, as well as to receptor-specific tumor imaging in the periphery. The aim of this work was to develop subtype selective 18F-labeled M3R and OX1R ligands, preferably by applying and optimizing the direct late-stage copper-mediated radiofluorination. As lead structures in the development of the novel radioligands served OFH243 (Ki value of 69 pM for the M3R1) and JH257 (Ki value of 1.2 nM for the OX1R2). Optimization of the 18F-labeling of boronic acid pinacol ester (Bpin) precursor 1 allowed a minimization of the reaction scale from 500 µL3 to 200 µL. Thereby over 60% of the precursor could be saved and the radiochemical yield (RCY) increased from 55%3 to 88%. A series of novel boroxine precursors were synthesized by two step reactions from Bpin derivatives in overall yields of 99%. These precursors were then investigated in 18F labeling studies, systematically optimizing the reaction conditions. A direct comparison of Bpin and trifunctional boroxine precursors showed that boroxines were at least as amenable to radiofluorination, providing high RCYs of up to 78% after 5 min and being less prone to byproduct formation. Radioligand [18F]21 (as 18F-labeled analog of OFH243) was obtained with a 2 fold increased apparent molar activity (AMA) of more than 60 MBq/nmol by addressing boroxine 19 (compared to Bpin precursor 18) and an activity yield (AY, referred to starting activity, non-decay corrected) of 35% after 69 min applying 1 GBq [18F]fluoride as starting activity. Specific binding of [18F]21 to the muscarinic acetylcholine receptors (mAChRs) was confirmed by in vitro autoradiography with rat brain slices. Furthermore, the data indicated an additional binding site for the [18F]21 within the striatum as both pirenzepine (40 µM, as M1R selective ligand) and 4 DAMP (1 µM, as non-selective mAChR ligand) at high concentrations were not able to completely displace [18F]21. In a first in vivo PET imaging study in a rat, a maximum brain uptake of 0.2 %ID/g and a high signal-to-noise ratio was observed and ascribed to the high target affinity and rapid excretion of unbound [18F]21 by the P glycoprotein 1 (P gp). Metabolite studies proved a high in vivo radiochemical stability of 98% of [18F]21 over 120 min in rats. Even though [18F]21 showed promising properties as PET ligand for neuroimaging, further experiments are required to evaluate the M3R over M1R selectivity. Radiosynthesis of the benzo[d]oxazole-substituted dual orexin receptor ligand [18F]47 resulted in a low RCY of 12% after 20 min. Thus, structural modifications through exchange of the heteroarene to quinoxaline and to different benzo[d]thiazoles offered interesting structural analogs of JH257 and culminated in the preparation of 18F-labeled OX1R ligand [18F]44 (Ki value of 44 for OX1R: 760 pM with 76 fold selectivity for the OX1R over OX2R) for neuroimaging. Radioligand [18F]44 was obtained in an AY of 22% after 76 min with an AMA of 2.1 MBq/nmol from boroxine precursor 46. In vivo PET imaging studies in rats showed that both, [18F]44 and its dual orexin receptor analog [18F]29 demonstrated increased maximum brain uptake of up to 0.37 %ID/g and 0.18 %ID/g, respectively, in the presence of cyclosporine A or suvorexant. After multistep-synthesis of the benzo[d]thiazole-substituted alkyne 55, 55 was used as starting material for the preparation of 48 and [18F]48. The 18F glycoconjugate [18F]48 was obtained with an AY of 7% after 126 min. Both ligands may offer new guidance for the further development of novel ligands for OX1R-specific tumor imaging.