8-Bicycloalkyl-CPFPX derivatives as potent and selective tools for in vivo imaging of the A1 adenosine receptor.

Imaging of the A1 adenosine receptor (A1R) by positron emission tomography (PET) with 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propyl-xanthine ([18F]CPFPX) has been widely used in preclinical and clinical studies. However, this radioligand suffers from rapid peripheral metabolism and subsequent accumulation of radiometabolites in the vascular compartment. In the present work, we prepared four derivatives of CPFPX by replacement of the cyclopentyl group with norbornane moieties. These derivatives were evaluated by competition binding studies, microsomal stability assays and LC-MS analysis of microsomal metabolites. In addition, the 18F-labeled isotopologue of 8-(1-norbornyl)-3-(3-fluoropropyl)-1-propylxanthine (1-NBX) as the most promising candidate was prepared by radiofluorination of the corresponding tosylate precursor and the resulting radioligand ([18F]1-NBX) was evaluated by permeability assays with Caco-2 cells and in vitro autoradiography in rat brain slices. Our results demonstrate that 1-NBX exhibits significantly improved A1R affinity and selectivity when compared to CPFPX and that it does not give rise to lipophilic metabolites expected to cross the blood-brain-barrier in microsomal assays. Furthermore, [18F]1-NBX showed a high passive permeability (Pc = 6.9 ± 2.9 × 10-5 cm/s) and in vitro autoradiography with this radioligand resulted in a distribution pattern matching A1R expression in the brain. Moreover, a low degree of non-specific binding (5%) was observed. Taken together, these findings identify [18F]1-NBX as a promising candidate for further preclinical evaluation as potential PET tracer for A1R imaging.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.

7-[18F]Fluoro-8-azaisatoic Anhydrides: Versatile Prosthetic Groups for the Preparation of PET Tracers.

18F-Fluorination of sensitive molecules is often challenging, but can be accomplished under suitably mild conditions using radiofluorinated prosthetic groups (PGs). Herein, 1-alkylamino-7-[18F]fluoro-8-azaisatoic anhydrides ([18F]AFAs) are introduced as versatile 18F-labeled building blocks that can be used as amine-reactive or "click chemistry" PGs. [18F]AFAs were efficiently prepared within 15 min by "on cartridge" radiolabeling of readily accessible trimethylammonium precursors. Conjugation with a range of amines afforded the corresponding 2-alkylamino-6-[18F]fluoronicotinamides in radiochemical conversions (RCCs) of 15-98%. In addition, radiolabeling of alkyne- or azide-functionalized precursors with azidopropyl- or propargyl-substituted [18F]AFAs using Cu-catalyzed click cycloaddition afforded the corresponding conjugates in RCCs of 44-88%. The practical utility of the PGs was confirmed by the preparation of three 18F-labeled PSMA ligands in radiochemical yields of 28-42%. Biological evaluation in rats demonstrated excellent in vivo stability of all three conjugates. In addition, one conjugate ([18F]JK-PSMA-15) showed favorable imaging properties for high-contrast visualization of small PSMA-positive lesions.

Validation of analytical HPLC with post-column injection as a method for rapid and precise quantification of radiochemical yields.

Accurate assessment of isolated radiochemical yields (RCYs) is a prerequisite for efficient and reliable optimization of labeling reactions. In practice, radiochemical conversions (RCCs) determined by HPLC analysis of crude reaction mixtures are often used to estimate RCYs. However, incomplete recovery of radioactivity from the stationary phase can lead to significant inaccuracies if RCCs are calculated based on the activity eluted from the column (i.e. the summed integrals of all peaks). Here, we validate a simple and practical method that overcomes problems associated with retention of activity on the column by determination of the total activity in the sample using post-column injection. Post-column injections were carried out using an additional injection valve, which was placed between the outlet of the HPLC column and the inlet of the detectors. 2-[18F]Fluoropyridine ([18F]FPy) and 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) were prepared with radiochemical purities of > 99.8% and mixed with [18F]fluoride at a ratio of 1:1 to simulate reaction mixtures obtained by radiolabeling reactions with an RCC of 50%. The samples were analyzed on three different C18 HPLC columns using neutral and acidic mobile phases. RCCs determined using the summed area of all peaks in the chromatograms were compared with those determined using post-column injection. Additionally, RCCs determined by post-column injection were corrected for activity losses before, during and after radiosyntheses to afford analytical RCYs, which were compared with isolated RCYs. Determination of RCCs based on the summed area of all peaks gave correct results under certain chromatographic conditions, but led to overestimation of the actual RCCs by up to 50% in other cases. In contrast, determination of RCCs using post-column injection provided precise results in all cases, and often significantly reduced analysis time. Moreover, analytical RCYs calculated from RCCs determined by post-column injection showed excellent agreement with isolated RCYs (<3% deviation). In conclusion, HPLC analysis using post-column injection enables reliable determination of RCCs independent of the chromatographic conditions and, together with a simple activity balance, rapid and accurate prediction of isolated RCYs.

Evaluation of the 18F-labeled analog of the therapeutic all-D-enantiomeric peptide RD2 for amyloid ß imaging.

Positron emission tomography (PET) imaging with radiotracers that bind to fibrillary amyloid ß (Aß) deposits is an important tool for the diagnosis of Alzheimer's disease (AD) and for the recruitment of patients into clinical trials. However, it has been suggested that rather than the fibrillary Aß deposits, it is smaller, soluble Aß aggregates that exert a neurotoxic effect and trigger AD pathogenesis. The aim of the current study is to develop a PET probe that is capable of detecting small aggregates and soluble Aß oligomers for improved diagnosis and therapy monitoring. An 18F-labeled radioligand was prepared based on the Aß-binding d-enantiomeric peptide RD2, which is currently being evaluated in clinical trials as a therapeutic agent to dissolve Aß oligomers. 18F-labeling was carried out using palladium-catalyzed S-arylation of RD2 with 2-[18F]fluoro-5-iodopyridine ([18F]FIPy). Specific binding of [18F]RD2-cFPy to brain material from transgenic AD (APP/PS1) mice and AD patients was demonstrated with in vitro autoradiography. In vivo uptake and biodistribution of [18F]RD2-cFPy were evaluated using PET analyses in wild-type and transgenic APP/PS1 mice. Although brain penetration and brain wash-out kinetics of the radioligand were low, this study provides proof of principle for a PET probe based on a d-enantiomeric peptide binding to soluble Aß species.

[18F]ALX5406: A Brain-Penetrating Prodrug for GlyT1-Specific PET Imaging.

Selective inhibition of glycine transporter 1 (GlyT1) has emerged as a potential approach to alleviate N-methyl-d-aspartate receptor (NMDAR) hypofunction in patients with schizophrenia and cognitive decline. ALX5407 is a potent and selective inhibitor of GlyT1 derived from the metabolic intermediate sarcosine (N-methylglycine) that showed antipsychotic potential in a number of animal models. Whereas clinical application of ALX5407 is limited by adverse effects on motor performance and respiratory function, a suitably radiolabeled drug could represent a promising PET tracer for the visualization of GlyT1 in the brain. Herein, [18F]ALX5407 and the corresponding methyl ester, [18F]ALX5406, were prepared by alcohol-enhanced copper mediated radiofluorination and studied in vitro in rat brain slices and in vivo in normal rats. [18F]ALX5407 demonstrated accumulation consistent with the distribution of GlyT1 in in vitro autoradiographic studies but no brain uptake in µPET experiments in naïve rats. In contrast, the methyl ester [18F]ALX5406 rapidly entered the brain and was enzymatically transformed into [18F]ALX5407, resulting in a regional accumulation pattern consistent with GlyT1 specific binding. We conclude that [18F]ALX5406 is a promising and easily accessible PET probe for preclinical in vivo imaging of GlyT1 in the brain.

Rapid 18F-labeling via Pd-catalyzed S-arylation in aqueous medium.

We report radiolabeling of thiol-containing substrates via Pd-catalyzed S-arylation with 2-[18F]fluoro-5-iodopyridine, which is readily accessible using the "minimalist" radiofluorination method. The practicality of the procedure was confirmed by preparation of a novel PSMA-specific PET-tracer as well as labeling of glutathione, Aß oligomer-binding RD2 peptide, bovine serum albumin and PSMA I&S.

Baeyer-Villiger oxidation tuned to chemoselective conversion of non-activated [18 F]fluorobenzaldehydes to [18 F]fluorophenols.

A reaction pathway via oxidation of [18 F]fluorobenzaldehydes offers a very useful tool for the no-carrier-added radiosynthesis of [18 F]fluorophenols, a structural motive of several potential radiopharmaceuticals. A considerably improved chemoselectivity of the Baeyer-Villiger oxidation (BVO) towards phenols was achieved, employing 2,2,2-trifluoroethanol as reaction solvent in combination with Oxone or m-CPBA as oxidation agent. The studies showed the necessity of H2 SO4 addition, which appears to have a dual effect, acting as catalyst and desiccant. For example, 2-[18 F]fluorophenol was obtained with a RCY of 97% under optimised conditions of 80°C and 30-minute reaction time. The changed performance of the BVO, which is in agreement with known reaction mechanisms via Criegee intermediates, provided the best results with regard to radiochemical yield (RCY) and chemoselectivity, i.e. formation of [18 F]fluorophenols rather than [18 F]fluorobenzoic acids. Thus, after a long history of the BVO, the new modification now allows an almost specific formation of phenols, even from electron-deficient benzaldehydes. Further, the applicability of the tuned, chemoselective BVO to the n.c.a. level and to more complex compounds was demonstrated for the products n.c.a. 4-[18 F]fluorophenol (RCY 95%; relating to 4-[18 F]fluorobenzaldehyde) and 4-[18 F]fluoro-m-tyramine (RCY 32%; relating to [18 F]fluoride), respectively.