Comparison of in†Silico, Electrochemical, in†Vitro and in†Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography.

The imaging of σ1 receptors in the brain by fluorinated radiotracers will be used for the validation of σ1 receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1'-benzyl-3-(ω-fluoromethyl to ω-fluorobutyl)-3H-spiro[2]benzofuran-1,4'-piperidine] ([18 F]1-4) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [18 F]1-4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC-MS) analysis allowed structural identification of non-radioactive metabolites. Radio-HPLC and radio-TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [18 F]2-4, but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [18 F]4 reached the brain, rendering [18 F]4 less appropriate as a PET tracer than [18 F]2 and [18 F]3. Compounds [18 F]2 and [18 F]3 possess the most promising properties for imaging of σ1 receptors in the brain. High σ1 affinity (Ki =0.59 nm), low lipophilicity (logD7.4 =2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [18 F]2 slightly over the fluoropropyl derivative [18 F]3 for human use.

Synthesis, radiofluorination and pharmacological evaluation of a fluoromethyl spirocyclic PET tracer for central σ1 receptors and comparison with fluoroalkyl homologs.

The spirocyclic σ(1) receptor ligand 1 (1'-benzyl-3-(fluoromethyl)-3H-spiro[[2]benzofuran-1,4'-piperidine]) was prepared in four steps starting from methoxy derivative 5. Due to its high σ(1) affinity (K(i)=0.74nM) and selectivity against several other relevant targets, 1 was investigated as (18)F-labeled PET tracer and its biological properties were compared with those of homologous fluoroalkyl derivatives 2-4. The fluoromethyl derivative 1 was faster metabolized in vitro than homologs 2-4. In contrast to the radiosynthesis of [(18)F]2-4, the nucleophilic substitution of the tosylate 15 using the K[(18)F]F-K(222)-carbonate complex required heating to 150°C in DMSO to achieve high labeling efficiencies. Whereas radiometabolites of [(18)F]2-4 were not detected in vivo in the brain of mice, two radiometabolites of [(18)F]1 were found. Analysis of ex vivo autoradiography images provided rather low target-to-nontarget ratio for [(18)F]1 compared with [(18)F]2-4. [(18)F]1 showed a fast uptake in the brain, which decreased continuously over time. The brain-to-plasma ratio of the radiotracer [(18)F]1 was only exceeded by the fluoroethyl tracer [(18)F]2.

A ¹8F-labeled fluorobutyl-substituted spirocyclic piperidine derivative as a selective radioligand for PET Imaging of sigma1 receptors.

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.

Enantioselective σ1 receptor binding and biotransformation of the spirocyclic PET tracer 1'-benzyl-3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,4'-piperidine].

It was shown that racemic (±)-2 [1'-benzyl-3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,4'-piperidine], WMS-1813] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ(1) receptors. To study the pharmacological activity of the enantiomers of 2, a preparative HPLC separation of (R)-2 and (S)-2 was performed. The absolute configuration of the enantiomers was determined by CD-spectroscopy together with theoretical calculations of the CD-spectrum of a model compound. In receptor binding studies with the radioligand [(3)H]-(+)-pentazocine, (S)-2 was thrice more potent than its (R)-configured enantiomer (R)-2. The metabolic degradation of the more potent (S)-enantiomer was considerably slower than the metabolism of (R)-2. The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap-LC-MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2.

Pharmacological and metabolic characterisation of the potent sigma1 receptor ligand 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine].

OBJECTIVES: The pharmacology and metabolism of the potent sigma1 receptor ligand 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] were evaluated. METHODS: The compound was tested against a wide range of receptors, ion channels and neurotransmitter transporters in radioligand binding assays. Analgesic activity was evaluated using the capsaicin pain model. Metabolism by rat and human liver microsomes was investigated, and the metabolites were identified by a variety of analytical techniques. KEY FINDINGS: 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] (compound 1) is a potent sigma1 receptor ligand (Ki 1.14 nM) with extraordinarily high sigma1/sigma2 selectivity (>1100). It was selective for the sigma1 receptor over more than 60 other receptors, ion channels and neurotransmitter transporters, and did not interact with the human ether-a-go-go-related gene (hERG) cardiac potassium channel. Compound 1 displayed analgesic activity against neuropathic pain in the capsaicin pain model (53% analgesia at 16 mg/kg), indicating that it is a sigma1 receptor antagonist. It was rapidly metabolised by rat liver microsomes. Seven metabolites were unequivocally identified; an N-debenzylated metabolite and a hydroxylated metabolite were the major products. Pooled human liver microsomes formed the same metabolites. Studies with seven recombinant cytochrome P450 isoenzymes revealed that CYP3A4 produced all the metabolites identified. The isoenzyme CYP2D6 was inhibited by 1 (IC50 88 nM) but did not produce any metabolites. CONCLUSIONS: 1'-benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,4'-piperidine] is a potent and selective sigma1 receptor antagonist, which is rapidly metabolised. Metabolically more stable sigma1 ligands could be achieved by stabilising the N-benzyl substructure.

Synthesis of spirocyclic sigma1 receptor ligands as potential PET radiotracers, structure-affinity relationships and in vitro metabolic stability.

Several 3H-spiro[[2]benzofuran-1,4'-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF(3).OEt(2) catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure-affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar sigma(1) affinity (K(i)=0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the sigma(2) subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [(18)F]2a and [(18)F]2e two different radiosynthetic approaches were followed.