Molecular imaging of σ receptors: synthesis and evaluation of the potent σ1 selective radioligand [18F]fluspidine.

PURPOSE: Neuroimaging of σ(1) receptors in the human brain has been proposed for the investigation of the pathophysiology of neurodegenerative and psychiatric diseases. However, there is a lack of suitable (18)F-labelled PET radioligands for that purpose. METHODS: The selective σ(1) receptor ligand [(18)F]fluspidine (1'-benzyl-3-(2-[(18)F]fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was synthesized by nucleophilic (18)F(-) substitution of the tosyl precursor. In vitro receptor binding affinity and selectivity were assessed by radioligand competition in tissue homogenate and autoradiographic approaches. In female CD-1 mice, in vivo properties of [(18)F]fluspidine were evaluated by ex vivo brain section imaging and organ distribution of intravenously administered radiotracer. Target specificity was validated by organ distribution of [(18)F]fluspidine after treatment with 1 mg/kg i.p. of the σ receptor antagonist haloperidol or the emopamil binding protein (EBP) inhibitor tamoxifen. In vitro metabolic stability and in vivo metabolism were investigated by LC-MS(n) and radio-HPLC analysis. RESULTS: [(18)F]Fluspidine was obtained with a radiochemical yield of 35-45%, a radiochemical purity of ≥ 99.6% and a specific activity of 150-350 GBq/µmol (n = 6) within a total synthesis time of 90-120 min. In vitro, fluspidine bound specifically and with high affinity to σ(1) receptors (K (i) = 0.59 nM). In mice, [(18)F]fluspidine rapidly accumulated in brain with uptake values of 3.9 and 4.7%ID/g and brain to blood ratios of 7 and 13 at 5 and 30 min after intravenous application of the radiotracer, respectively. By ex vivo autoradiography of brain slices, resemblance between binding site occupancy of [(18)F]fluspidine and the expression of σ(1) receptors was shown. The radiotracer uptake in the brain as well as in peripheral σ(1) receptor expressing organs was significantly inhibited by haloperidol but not by tamoxifen. Incubation with rat liver microsomes led to a fast biotransformation of fluspidine. After an incubation period of 30 min only 13% of the parent compound was left. Seven metabolites were identified by HPLC-UV and LC-MS(n) techniques. However, [(18)F]fluspidine showed a higher metabolic stability in vivo. In plasma samples ∼ 94% of parent compound remained at 30 min and ∼ 67% at 60 min post-injection. Only one major radiometabolite was detected. None of the radiometabolites crossed the blood-brain barrier. CONCLUSION: [(18)F]Fluspidine demonstrated favourable target affinity and specificity as well as metabolic stability both in vitro and in animal experiments. The in vivo properties of [(18)F]fluspidine offer a high potential of this radiotracer for neuroimaging and quantitation of σ(1) receptors in vivo.

Synthesis, pharmacological activity and structure affinity relationships of spirocyclic σ(1) receptor ligands with a (2-fluoroethyl) residue in 3-position.

In order to develop a fluorinated radiotracer for imaging of σ(1) receptors in the central nervous system a series of (2-fluoroethyl) substituted spirocyclic piperidines 3 has been prepared. In the key step of the synthesis 2-bromocinnamaldehyde acetal 5 was added to piperidones 6 with various substituents at the N-atom. Unexpectedly, this reaction led to 2-benzoxepines 8, which were contracted with acid to afford the spirocyclic 2-benzofuranacetaldehydes 9. The best yields were obtained, when the transformations up to the alcohols 10 were performed without isolation of intermediates. Generally the (2-fluoroethyl) derivatives 3 have higher σ(1) affinity and σ(1)/σ(2) selectivity than the corresponding (3-fluoropropyl) derivatives 2. The most promising candidate for the development as radiotracer is the (2-fluoroethyl) derivative 3a (WMS-1828, fluspidine, 1'-benzyl-3-(2-fluoroethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]), which shows subnanomolar σ(1) affinity (K(i)=0.59nM) and excellent selectivity over the σ(2) subtype (1331-fold) as well as some other receptor systems. The novel synthetic strategy also allows the systematic pharmacological evaluation of intermediate alcohols 10. Despite their high σ(1) affinity (K(i)=6-32nM) and selectivity the alcohols 10 are 10-30-fold less potent than the bioisosteric fluoro derivatives 3.

Evaluation of spirocyclic 3-(3-fluoropropyl)-2-benzofurans as sigma1 receptor ligands for neuroimaging with positron emission tomography.

A series of various N-substituted 3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,4'-piperidines] (7) has been synthesized. In receptor binding studies, the N-benzyl derivative 7a (WMS-1813) revealed extraordinarily high sigma(1) receptor affinity (K(i) = 1.4 nM) and excellent sigma(1)/sigma(2) selectivity (>600 fold). In vitro biotransformation of 7a with rat liver microsomes led to three main metabolites. N-Debenzylation was inhibited by introduction of an N-phenylethyl residue (7 g). The PET tracer [(18)F]7a was synthesized by nucleophilic substitution of the tosylate 13 with K[(18)F]F-K222-carbonate complex. The decay corrected radiochemical yield of [(18)F]7a was 35-48% with a radiochemical purity of >99.5% and a specific activity of 150-238 GBq/micromol. The radiotracer properties were evaluated in female CD-1 mice by organ distribution and ex vivo brain autoradiography. The radiotracer uptake in the brain was fast and sufficient, with values of approximately 4% injected dose per gram. Target specificity of [(18)F]7a was validated in blocking studies by preapplication of haloperidol, and significant reduction in the uptake of radioactivity was observed in the brain and peripheral organs expressing sigma(1) receptors.