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In this study, we synthesized and evaluated a new spirocyclic piperidine derivative 3, containing a 4-fluorobutyl side chain, as a PET radioligand for neuroimaging of σ₁ receptors. In vitro, compound 3 displayed high affinity for σ₁ receptors (K(i) =1.2 nM) as well as high selectivity. [¹⁸F]3 radiosynthesis was performed from the corresponding tosylate precursor, with high radiochemical yield (45-51 %), purity (>98 %), and specific activity (>201 GBq μmol⁻¹). Metabolic stability of [¹⁸F]3 in the brain of CD-1 mice was verified, and no penetration of peripheral radiometabolites into the cerebral tissue was observed. Results of ex vivo autoradiography revealed that the distribution of [¹⁸F]3 in the brain corresponded to regions with high σ₁ receptor density. The highest region-specific total-to-nonspecific ratio was determined in the facial nucleus (4.00). Biodistribution studies indicated rapid and high levels in brain uptake of [¹⁸F]3 (2.2 % ID per gram at 5 min p.i.). Pre-administration of haloperidol significantly inhibited [¹⁸F]3 uptake into the brain and σ₁ receptor-expressing organs, further confirming in vivo target specificity.

Objectives: The vesicular acetylcholine transporter (VAChT) is a transmembrane protein located in presynaptic vesicles of cholinergic neurons and is discussed to be an interesting target for PET imaging of neurodegenerative disorders. Previous developments of VAChT radiotracers are based on vesamicol (trans-2-(4-phenylpiperidino)cyclohexanol) and are focused on structural modifications in order to keep the high affinity of vesamicol to VAChT and to lower the affinity to σ1 and σ2 receptors, two off-targets which are also addressed by vesamicol. The moderate selectivity of several VAChT-ligands is one of the limiting factors for a successful application as radiotracer. Based on numerous literature data and our own intensive synthetic work in this field, we observed that structural modifications based on single-target structure affinity considerations are rarely successful. Therefore, we synthesized different series of systematically modified vesamicol analogs as basis for the development of a 3D-QSAR model, which will consider for the first time the affinity to VAChT as well as to the σ1 and σ2 receptors. Methods: Nucleophilic ring opening of an epoxide precursor with a secondary amine was used to synthesize a large number of vesamicol derivatives. By varying the epoxide precursor, structural modifications in ring A of the vesamicol skeleton were realized (left part of the figure). Due to the selection of different amines, the rings B and C were sterically or electronically modified (right part of the figure). The binding affinities to VAChT were determined with competitive binding experiments using preparations of PC12 cells stably transfected with ratVAChT cDNA and (-)-[3H]vesamicol. To determine the binding affinity to the sigma receptors, rat cortex membranes and (+)-[3H]pentazocin for σ1 and rat liver membranes and [3H]DTG for σ2 receptors were used. Results: So far we synthesized 55 derivatives belonging to the four classes of vesamicols, F-benzoylvesamicols, benzovesamicols and azavesamicols (Fig.) and determined their binding affinity to VAChT, σ1 and σ2 receptors. A few of them such as benzovesamicol or FBT are already known and were used as reference compounds. Interestingly, the VAChT binding data obtained by our test system differ to some extent from the values reported in literature. Therefore, VAChT binding data of selected vesamicol derivatives obtained either with PC12 cells or membranes of pig and/or rat brain are compared and differences of Ki values are discussed. The use of preparations obtained by freezing and thawing of PC12 cells stably transfected with ratVAChT cDNA and (-)-[3H]vesamicol was found to be the most practicable and reliable method. As a preliminary trend we could observe that modifications in ring B and C led to a loss of VAChT affinity, whereas modifications in ring A partly increased VAChT affinity, which was however often accompanied by an increase in σ1/σ2 receptor affinity. Figure. Vesamicol as chemical lead and derived new analogs Conclusion: Based on our previous results, we have to conclude that VAChT affinity suitable for PET imaging correlates often with significant off-target affinity to the σ1 and σ2 receptors. Acknowledgment: Supported by DFG (WE 2927/4-1).

Phosphodiesterases (PDEs) are enzymes that play a major role in cell signalling by hydrolysing the second messengers cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) throughout the body and brain. Altered cyclic nucleotide-mediated signalling has been associated with a wide array of disorders, including neurodegenerative disorders. Recently, PDE5 has been shown to be involved in neurodegenerative disorders such as Alzheimer's disease but its precise role has not been elucidated yet. To visualize and quantify the expression of this enzyme in brain, we developed a radiotracer for specific PET imaging of PDE5. A quinoline based lead compound has been structurally modified resulting in the fluoroethoxymethyl derivative ICF24027 with high inhibitory activity towards PDE5 (IC50 = 1.89 nM). Radiolabelling with fluorine-18 was performed by a one-step nucleophilic substitution reaction using a tosylate precursor (RCY(EOB) = 12.9 ± 1.8%; RCP >99%; SA(EOS) = 70-126 GBq/µmol). In vitro autoradiographic studies of [18F]ICF24027 on different mouse tissue as well as on porcine brain slices demonstrated a moderate specific binding to PDE5. In vivo studies in mice revealed that [18F]ICF24027 was metabolized under formation of brain penetrable radiometabolites making the radiotracer not suitable for PET imaging of PDE5 in brain.