Achim Hiller

Radiosynthesis and first evaluation in mice of [18F]NS14490 for molecular imaging of α7 nicotinic acetylcholine receptors

[(18)F]NS14490, a new potential radiotracer for neuroimaging of α7 nicotinic acetylcholine receptors (α7 nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using [(3)H]methyllycaconitine and NS14490 as competitor showed a good target affinity (K(i,α7) = 2.5 nM) and a high selectivity towards other nAChRs. Radiosynthesis of [(18)F]NS14490 was performed by two different labelling procedures: a two-step synthesis using a prosthetic group, which led to 7% labelling yield, and the convenient direct nucleophilic substitution of the corresponding tosylate precursor, which resulted in 70% labelling yield. After optimisation of the isolation, purification and formulation process, biodistribution studies were performed in CD-1 mice. The brain uptake of [(18)F]NS14490 was comparably low (0.16% ID g(-1) wet weight at 5 min p.i.). The radiotracer showed a high metabolic stability in plasma and brain. Also, the target specificity was proven by pre-administration of a highly affine α7 ligand providing a rationale basis for further in vivo evaluation.

Neuroimaging of the vesicular acetylcholine transporter by a novel 4-[18F]fluoro-benzoyl derivative of 7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazines

Phenylpiperidinyl-octahydro-benzo[1,4]oxazines represent a new class of conformationally restrained vesamicol analogues. Derived from this morpholine-fused vesamicol structure, a new fluorine-18-labeled 4-fluorobenzoyl derivative ([(18)F]FBMV) was synthesized with an average specific activity of 75 GBq/micromol and a radiochemical purity of 99%. The radiolabeling method included an exchange reaction of a 4-nitro group of the precursor by fluorine-18, a reduction procedure to eliminate excess of the nitro compound, followed by a high-performance liquid chromatography purification. [(18)F]FBMV demonstrates (i) a moderate lipophilic character with a logD(pH7.0) 1.8+/-0.10; (ii) a considerable binding affinity to the vesicular acetylcholine transporter (VAChT) (K(i)=27.5 nM), as determined using PC12 cells transfected with a VAChT cDNA, and a low affinity to sigma(1,2) receptors (K(i) >3000 nM); (iii) a good uptake into the rat and pig brains; (iv) a typical accumulation in the VAChT-containing brain regions; and (v) an approximately 20% reduction in cortical tracer binding after a specific cholinergic lesion using 192IgG-saporin. [(18)F]FBMV exhibits another PET marker within the group of vesamicol derivatives that demonstrates potentials in imaging brain cholinergic deficits, while its usefulness in clinical practice must await further investigation.

A new 18F-labeled fluoroacetylmorpholino derivative of vesamicol for neuroimaging of the vesicular acetylcholine transporter

With the aim of producing selective radiotracers for in vivo imaging of the vesicular acetylcholine transporter (VAChT) using positron mission tomography (PET), here, we report synthesis and analysis of a new class of conformationally constrained vesamicol analogues with moderate lipophilicity. The sequential ring opening on trans-1,4-cyclohexadiene dioxide enabled an approach to synthesize 6-arylpiperidino-octahydrobenzo[1,4]oxazine-7-ols [morpholino vesamicols]. The radiosynthesis of the [18F]fluoroacetyl-substituted derivative ([18F]FAMV) was achieved starting from a corresponding bromo precursor [2-Bromo-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone] and using a modified commercial computer-controlled module system with a radiochemical yield of 27+/-4%, a high radiochemical purity (99%) and a specific activity of 35 GBq/micromol. In competitive binding assays using a PC12 cell line overexpressing VAChT and [3H]-(-) vesamicol, 2-fluoro-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone (FAMV) demonstrated a high selectivity for binding to VAChT (K(i): 39.9+/-5.9 nM) when compared to its binding to sigma 1/2 receptors (Ki>1500 nM). The compound showed a moderate lipophilicity (logD (pH 7)=1.9) and a plasma protein binding of 49%. The brain uptake of [18F]FAMV was about 0.1% injected dose per gram at 5 min after injection and decreased continuously with time. Notably, an increasing accumulation of radioactivity in the lateral brain ventricles was observed. After 1 h, the accumulation of [18F]FAMV, expressed as ratio to the cerebellum, was 4.5 for the striatum, 2.0 for the cortical and 1.5 for the hippocampal regions, measured on brain slices using ex vivo autoradiography. At the present time, 75% of [18F]FAMV in the plasma was shown to be metabolized to various hydrophilic compounds, as detected by high-performance liquid chromatography. The degradation of [18F]FAMV was also detected in brain extracts as early as 15 min post injection (p.i.) and increased to 50% at 1 h postinjection. In conclusion, although the chemical properties of [18F]FAMV and the selectivity of binding to VAChT appear to be promising indicators of a useful PET tracer for imaging VAChT, a low brain extraction, in combination with only moderate specific accumulation in cholinergic brain regions and an insufficient in vivo stability prevents the application of this compound for neuroimaging in humans.

Radiosynthesis of racemic and enantiomerically pure (-)-[18F]flubatine--a promising PET radiotracer for neuroimaging of α4β2 nicotinic acetylcholine receptors.

(-)-[(18)F]flubatine is a promising agent for visualization by PET of cerebral α4β2 nicotinic acetylcholine receptors (nAChRs), which are implicated in psychiatric and neurodegenerative disorders. Here, we describe a substantially improved two-step radiosynthesis strategy for (-)-[(18)F]flubatine, based on the nucleophilic radiofluorination of an enantiomerically pure precursor followed by deprotection of the intermediate. An extensive leaving group/protecting group library of precursors was tested. Application of a trimethylammonium-iodide precursor with a Boc-protecting group provided the best results: labeling efficiencies of 80-95%, RCY of 60±5%, radiochemical purity of >98%, and a specific activity of >350GBq/μmol. The radiosynthesis is easily transferable to an automated synthesis module.

Synthesis and biological evaluation of both enantiomers of [18F]flubatine, promising radiotracers with fast kinetics for the imaging of α4β2-nicotinic acetylcholine receptors

Both enantiomers of the epibatidine analogue flubatine display high affinity towards the α4β2 nicotinic acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level) of 6.2μg/kg for (-)-flubatine and 1.55μg/kg for (+)-flubatine. We developed syntheses for both flubatine enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylammonium precursors allowed for highly efficient (18)F-radiolabelling in radiochemical yields >60% and specific activities >750GBq/μmol, thus making the radioligands practical for clinical investigation.

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 (Ki=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.

Distinctive In Vivo Kinetics of the New σ1 Receptor Ligands (R)-(+)- and (S)-(–)-18F-Fluspidine in Porcine Brain

Because of their involvement in growth and survival signaling cascades, the σ1 receptors (σ1Rs) represent a novel target for the treatment of cancer and several brain diseases such as depression and neurodegeneration. From a series of σ1R-specific 18F-fluoroalkylated spirocyclic piperidines, we have chosen 18F-fluspidine for detailed investigation of the in vivo kinetics of the (R)-(+)- and (S)-(–)-enantiomers to identify their potential for imaging in humans. Methods: Enantiopure tosylate precursors for radiolabeling were obtained using chiral preparative high-performance liquid chromatography and used for radiosynthesis of both 18F-fluspidine enantiomers by nucleophilic substitution with K-18F-F-Kryptofix 222-carbonate complex in a synthesis module. Brain pharmacokinetics were investigated by dynamic PET studies in piglets under baseline and blocking conditions using the highly selective σ1R agonist SA4503. Standardized uptake values (SUVs) were calculated for 24 MR-defined brain regions. Total distribution volume (VT) and binding potentials (k3′/k4) of (S)-(–)- and (R)-(+)-18F-fluspidine were estimated. Furthermore, VT values were estimated by graphical analysis using Logan plots. Results: The (S)- and (R)-tosylates were obtained in excellent enantiomeric purities (>98% and >96% enantiomeric excess, respectively). (S)-(–)- and (R)-(+)-18F-fluspidine were synthesized within approximately 70 min (radiochemical yield, 35%–45%; specific activity, 650–870 GBq/μmol; radiochemical purity, >99%). Both radiotracers displayed different brain uptake kinetics. Although the initial brain uptake was similar, the SUV at the end of the study differed significantly (P < 0.05), with (R)-(+)-18F-fluspidine showing about 60%–150% higher values. Administration of SA4503 reduced SUV almost equally for both radiotracers by approximately 65%. Furthermore, k3′ was significantly decreased under blocking conditions in almost all regions ((S)-(–)-18F-fluspidine, −90%–95%; (R)-(+)-18F-fluspidine, −70%–90%) whereas effects on k4 differed according to the particular brain region. VT estimated by both graphical analysis using Logan plots and full nonlinear kinetic analysis revealed significant inhibition for both radiotracers under blocking conditions. Conclusion: Both (S)-(–)- and (R)-(+)-18F-fluspidine appear to be suitable for σ1R imaging in humans. The different pharmacokinetics of (S)-(–)-18F-fluspidine and (R)-(+)-18F-fluspidine may have the potential for application in the diagnostics of different pathologic conditions.

Fully automated radiosynthesis of both enantiomers of [18F]Flubatine under GMP conditions for human application

A fully automatized radiosynthesis of (+)- and (-)-[(18)F]Flubatine ((+)- and (-)NCFHEB) by means of a commercially available synthesis module (TRACERlab FX FN) under GMP conditions is reported. Radiochemical yields of 30% within an overall synthesis time of 40 min were achieved in more than 70 individual syntheses. Specific activities were approximately 3000 GBq/μmol and radiochemical purity was determined to be at least 97%.

Use of 3-(18F)fluoropropanesulfonyl chloride as a prosthetic agent for the radiolabelling of amines: Investigation of precursor molecules, labelling conditions and enzymatic stability of the corresponding sulfonamides

Summary 3-[18F]Fluoropropanesulfonyl chloride, a recently proposed prosthetic agent for fluorine-18 labelling, was prepared in a two-step radiosynthesis via 3-[18F]fluoropropyl thiocyanate as an intermediate. Two benzenesulfonate-based radiolabelling precursors were prepared by various routes. Comparing the reactivities of 3-thiocyanatopropyl nosylate and the corresponding tosylate towards [18F]fluoride the former proved to be superior accounting for labelling yields of up to 85%. Conditions for a reliable transformation of 3-[18F]fluoropropyl thiocyanate to the corresponding sulfonyl chloride with the potential for automation have been identified. The reaction of 3-[18F]fluoropropanesulfonyl chloride with eight different aliphatic and aromatic amines was investigated and the identity of the resulting 18F-labelled sulfonamides was confirmed chromatographically by comparison with their nonradioactive counterparts. Even for weakly nucleophilic amines such as 4-nitroaniline the desired radiolabelled sulfonamides were accessible in satisfactory yields owing to systematic variation of the reaction conditions. With respect to the application of the 18F-fluoropropansulfonyl group to the labelling of compounds relevant as imaging agents for positron emission tomography (PET), the stability of N-(4-fluorophenyl)-3-fluoropropanesulfonamide against degradation catalysed by carboxylesterase was investigated and compared to that of the analogous fluoroacetamide.

3-(4-(6-Fluoroalkoxy-3,4-dihydroisoquinoline-2(1H)-yl)cyclohexyl)-1H-indole- 5-carbonitriles for SERT imaging: Chemical synthesis, evaluation in vitro and radiofluorination

Aminocyclohexyl indoles bind with high affinity and specificity toward the serotonin transporter (SERT). Based on this structural lead, we designed fluoroalkoxydihydroisoquinoline-cyclohexyl indole carbonitriles for future application as (18)F-labeled tracers for SERT imaging by PET. Six compounds, three pairs of cis- and trans-isomer derivatives, respectively, were synthesized and evaluated in vitro. The chemistry of the new compounds, their affinity and specificity data, the general route to the phenolic precursor for labeling, and the successful (18)F-fluoroalkylation of one pair of compounds are described herein.