Efficient preparation of PET tracers for visualization of age-related disorders using emerging methods of radiofluorination

In 2013 novel compounds targeting alpha-synuclein oligomer aggregates in Parkinson’s disease were reported. Especially 5-(3-bromophenyl)-3-(piperonyl)pyrazole (1a) showed dramatic therapeutic effects since it was able to eliminate pathological alpha-synuclein aggregates. Therefore, it was assumed that 18F-labeled analogs of 1a are potentially useful for the detection of alpha-synuclein oligomers. The first 18F-labeled compound 5-(3-bromphenyl)-3-(6-[18F]fluorpiperonyl)pyrazol ([18F]1b) was prepared by 1,3 dipolar cycloaddition between 3'-bromophenyl acetylene (2a) and [18F]fluorophenyldiazomethane generated in situ from tosylhydrazone ([18F]3b). In order to establish optimized reaction conditions, the 1,3-dipolar cycloaddition between 4-[18F]fluorophenyldiazomethane and 4'-fluorophenyl acetylene (2b) was studied as a model reaction. Optimized reaction conditions enabled to obtain 3,5-bis(4-fluorophenyl)-1H-pyrazole ([18F]1d) in radiochemical yield (RCY) of 67%. The same reaction conditions delivered [18F]1b in RCYs of 27–34%. The second approach applied in this work to obtain the alpha-synuclein targeting compound 5-(3-[18F]fluorphenyl)-3-(piperonyl)pyrazol ([18F]1c) was based on copper mediated 18F-fluorodestannylation. The latter method was recently reported by Scott et. al. This method highly benefits from the fact that trialkylaryl tin compounds are routinely used as precursors for electrophilic radiofluorinations. Therefore, a method using stannyl precursors and “nucleophilic [18F]Fluoride” should be of high interest for the production of radiopharmaceuticals. Initially, trimethyl(phenyl)tin was used as a model substrate to establish the 18F-fluorodestannylation approach. After reaction optimization, this approach was used to label several electron-neutral, -rich and -poor stannyl substrates in RCYs of 16-88%. Additionally, 18F-destannylation was used to produce [18F]1c in RCY of 62%. Furthermore, this method was applied in the synthesis of 18F-labeled amino acids starting from commercially available precursors. Finally, a scalable automated synthesis of 3-O-methyl-6-[18F]fluoro-L-DOPA (6-[18F]OMFD), 6-[18F]Fluoro-L-m-tyrosine (6-[18F]FMT), 2-[18F]fluoro-L-tyrosine (2-[18F]F-Tyr) und 6-[18F]fluoro-L-3,4-dihydroxyphenylalanin (6-[18F]FDOPA) was established affording these compounds in high isolated RCY of 32-57%. Remarkably, the automated radiosynthesis of 6-[18F]FDOPA via Cu-mediated radiofluorination of an appropriate stannane precursor afforded RCYs of 57%. Starting from 9.2 GBq [18F]fluoride 3.5 GBq of 6-[18F]FDOPA was obtained within 65 min suitable for clinical applications

Metal-free cross-coupling reactions of radiofluorinated tosylhydrazones with different nucleophiles

Objectives Numerous metal-free cross-coupling reactions of tosylhydrazones with different reaction partners have been recently reported. These building blocks are easily available from carbonyl compounds by reaction with tosylhydrazide. The aim of this work was the preparation of 18F-labeled tosylhydrazone and its utilization as an 18F-labeled building block for the preparation of radiofluorinated compounds via cross-coupling reactions with different model reaction partners like boronic acids, alkenes, thiols and alkynes.Methods 4-[18F]Fluorobenzylidene tosylhydrazone ([18F]1) was synthesized by the reaction of [18F]FBA with tosylhydrazide. [18F]1 was allowed to react with aromatic boronic acids with different reactivity; internal and terminal alkene; aromatic and aliphatic thiols; terminal alkyne; to afford the corresponding radiofluorinated diarylmethanes [18F]2a-c, cyclopropanes [18F]3a,b, thioethers [18F]4a,b and pyrazole [18F]5a respectively. The cross-coupling step for the preparation of [18F]2-5 was optimized with respect to base, solvent, reaction temperature and reaction time. Additionally, the feasibility of a one-pot preparation of [18F]1 and [18F]2a were studied.Results [18F]2 was obtained from [18F]FBA under optimized reaction conditions (MeOH, 70 °C, 10 min) in 91±2% RCY and in 97±1% RCP. Using acetyl chloride to neutralize excess of base after the [18F]FBA synthesis [18F]2 was prepared via a one-pot two-step reaction sequence in DMSO in 62±10% RCY. Highest RCCs of [18F]3a-c (28-75%) were achieved in 1,4-dioxane at 105°C for 10 min using K2CO3 as a base. Disubstituted cyclopropanes in ca. 50% RCC were obtained by the reaction between [18F]2 and alkenes in toluene at 140°C for 10 min in the presence of K2CO3 and TEACl. Thioethers [18F]5a,b were prepared in 67±2% and 20±2% RCC, respectively (K2CO3/TBAB, pyridine, 100 °C, 10 min). Finally, 3,5-disubstituted pyrazoles were prepared in MeCN at 95 °C for 25 min using LiOtBu as base to deliver [18F]5a,b in RCC of 62% and 34% respectively.Conclusions 18F-labeled tosylhydrazone is an easily accessible building block for metal-free cross-coupling reactions with different reaction partners to provide a broad spectrum of radiofluorinated compounds. The newly developed method could be used for the radiosynthesis of clinically applicable PET imaging agents

Metal-free cross-coupling reactions of radiofluorinated tosylhydrazones with different nucleophiles

Objectives Numerous metal-free cross-coupling reactions of tosylhydrazones with different reaction partners have been recently reported. These building blocks are easily available from carbonyl compounds by reaction with tosylhydrazide. The aim of this work was the preparation of 18F-labeled tosylhydrazone and its utilization as an 18F-labeled building block for the preparation of radiofluorinated compounds via cross-coupling reactions with different model reaction partners like boronic acids, alkenes, thiols and alkynes.Methods 4-[18F]Fluorobenzylidene tosylhydrazone ([18F]1) was synthesized by the reaction of [18F]FBA with tosylhydrazide. [18F]1 was allowed to react with aromatic boronic acids with different reactivity; internal and terminal alkene; aromatic and aliphatic thiols; terminal alkyne; to afford the corresponding radiofluorinated diarylmethanes [18F]2a-c, cyclopropanes [18F]3a,b, thioethers [18F]4a,b and pyrazole [18F]5a respectively. The cross-coupling step for the preparation of [18F]2-5 was optimized with respect to base, solvent, reaction temperature and reaction time. Additionally, the feasibility of a one-pot preparation of [18F]1 and [18F]2a were studied.Results [18F]2 was obtained from [18F]FBA under optimized reaction conditions (MeOH, 70 °C, 10 min) in 91±2% RCY and in 97±1% RCP. Using acetyl chloride to neutralize excess of base after the [18F]FBA synthesis [18F]2 was prepared via a one-pot two-step reaction sequence in DMSO in 62±10% RCY. Highest RCCs of [18F]3a-c (28-75%) were achieved in 1,4-dioxane at 105°C for 10 min using K2CO3 as a base. Disubstituted cyclopropanes in ca. 50% RCC were obtained by the reaction between [18F]2 and alkenes in toluene at 140°C for 10 min in the presence of K2CO3 and TEACl. Thioethers [18F]5a,b were prepared in 67±2% and 20±2% RCC, respectively (K2CO3/TBAB, pyridine, 100 °C, 10 min). Finally, 3,5-disubstituted pyrazoles were prepared in MeCN at 95 °C for 25 min using LiOtBu as base to deliver [18F]5a,b in RCC of 62% and 34% respectively.Conclusions 18F-labeled tosylhydrazone is an easily accessible building block for metal-free cross-coupling reactions with different reaction partners to provide a broad spectrum of radiofluorinated compounds. The newly developed method could be used for the radiosynthesis of clinically applicable PET imaging agents

A Practical Method for the Preparation of 18F-Labeled Aromatic Amino Acids from Nucleophilic [18F]Fluoride and Stannyl Precursors for Electrophilic Radiohalogenation

In a recent contribution of Scott et al., the substrate scope of Cu-mediated nucleophilic radiofluorination with [18F]KF for the preparation of 18F-labeled arenes was extended to aryl- and vinylstannanes. Based on these findings, the potential of this reaction for the production of clinically relevant positron emission tomography (PET) tracers was investigated. To this end, Cu-mediated radiofluorodestannylation using trimethyl(phenyl)tin as a model substrate was re-evaluated with respect to different reaction parameters. The resulting labeling protocol was applied for 18F-fluorination of different electron-rich, -neutral and -poor arylstannyl substrates in RCCs of 16-88%. Furthermore, this method was utilized for the synthesis of 18F-labeled aromatic amino acids from additionally N-Boc protected commercially available stannyl precursors routinely applied for electrophilic radiohalogenation. Finally, an automated synthesis of 6-[18F]fluoro-l-m-tyrosine (6-[18F]FMT), 2-[18F]fluoro-l-tyrosine (2-[18F]F-Tyr), 6-[18F]fluoro-l-3,4-dihydroxyphenylalanine (6-[18F]FDOPA) and 3-O-methyl-6-[18F]FDOPA ([18F]OMFD) was established furnishing these PET probes in isolated radiochemical yields (RCYs) of 32-54% on a preparative scale. Remarkably, the automated radiosynthesis of 6-[18F]FDOPA afforded an exceptionally high RCY of 54 +/- 5% (n = 5)

Fully Automated, High-Dose Radiosynthesis of [18F]PARPi

[18F]PARPi is currently undergoing clinical trials as a PET tracer for many applications. However, only manual radiosynthesis was reported; this has several drawbacks, including an increased risk of contamination from the operator, and the need to limit the starting activity. The automation of the previously reported protocol for [18F]PARPi synthesis is challenging, as it requires transferring microvolumes of reagents, which many platforms cannot accommodate. We report a revised, high yield, and automated protocol for the radiosynthesis of [18F]PARPi, with final doses of over 20 GBq

Abnormal Spermatid Formation in the Presence of the Parasitic B24 Chromosome in the Grasshopper Eyprepocnemis plorans

6 páginas, 3 figuras, 2 tablas.Morphology and size of spermatids were analysed in the grasshopper Eyprepocnemis plorans by means of light and electron microscopy. At light microscopy, normal and abnormal (macro- and micro-) spermatids differed in size and number of centriolar adjuncts (CAs): 1 CA in normal spermatids and 2 or more CAs, depending on ploidy level, in macrospermatids. Males carrying the additional B24 chromosome showed significantly more macro- and microspermatids than 0B males. The frequency of macro- and microspermatids showed an odd-even pattern in respect to the number of B chromosomes, with a higher frequency of abnormal spermatids associated with odd B numbers. Transmission electron microscopy showed that macrospermatids carried more than one axoneme, depending on ploidy level: 2 for diploid, 3 for triploid, and 4 for tetraploid spermatids. In 0B males, the most frequent abnormal spermatids were diploid, whereas in 1B males they were the tetraploid spermatids and, to a lesser extent, triploid ones. This suggests that most macrospermatids derived from cytokinesis failure and nucleus restitution. The implications of aberrant spermatids on B chromosome transmission and male fertility are discussed.Peer reviewe

Fully Automated, High-Dose Radiosynthesis of [18F]PARPi

peer reviewed[18F]PARPi is currently undergoing clinical trials as a PET tracer for many applications. However, only manual radiosynthesis was reported; this has several drawbacks, including an increased risk of contamination from the operator, and the need to limit the starting activity. The automation of the previously reported protocol for [18F]PARPi synthesis is challenging, as it requires transferring microvolumes of reagents, which many platforms cannot accommodate. We report a revised, high yield, and automated protocol for the radiosynthesis of [18F]PARPi, with final doses of over 20 GBq

Fully Automated, High-Dose Radiosynthesis of [¹⁸F]PARPi

[18F]PARPi is currently undergoing clinical trials as a PET tracer for many applications. However, only manual radiosynthesis was reported; this has several drawbacks, including an increased risk of contamination from the operator, and the need to limit the starting activity. The automation of the previously reported protocol for [18F]PARPi synthesis is challenging, as it requires transferring microvolumes of reagents, which many platforms cannot accommodate. We report a revised, high yield, and automated protocol for the radiosynthesis of [18F]PARPi, with final doses of over 20 GBq

Demonstration of the Early Cardiac Bioavailability of a Non-Specific Cell-Targeted Peptide Using Radionuclide-Based Imaging In Vivo

The cardiac bioavailability of peptide drugs that inhibit harmful intracellular protein–protein interactions in cardiovascular diseases remains a challenging task in drug development. This study investigates whether a non-specific cell-targeted peptide drug is available in a timely manner at its intended biological destination, the heart, using a combined stepwise nuclear molecular imaging approach. An octapeptide (heart8P) was covalently coupled with the trans-activator of transcription (TAT) protein transduction domain residues 48–59 of human immunodeficiency virus-1 (TAT-heart8P) for efficient internalization into mammalian cells. The pharmacokinetics of TAT-heart8P were evaluated in dogs and rats. The cellular internalization of TAT-heart8P-Cy(5.5) was examined on cardiomyocytes. The real-time cardiac delivery of 68Ga-NODAGA-TAT-heart8P was tested in mice under physiological and pathological conditions. Pharmacokinetic studies of TAT-heart8P in dogs and rats revealed a fast blood clearance, high tissue distribution, and high extraction by the liver. TAT-heart-8P-Cy(5.5) was rapidly internalized in mouse and human cardiomyocytes. Correspondingly, organ uptake of hydrophilic 68Ga-NODAGA-TAT-heart8P occurred rapidly after injection with an initial cardiac bioavailability already 10 min post-injection. The saturable cardiac uptake was revailed by the pre-injection of the unlabeled compound. The cardiac uptake of 68Ga-NODAGA-TAT-heart8P did not change in a model of cell membrane toxicity. This study provides a sequential stepwise workflow to evaluate the cardiac delivery of a hydrophilic, non-specific cell-targeting peptide. 68Ga-NODAGA-TAT-heart8P showed rapid accumulation in the target tissue early after injection. The implementation of PET/CT radionuclide-based imaging methodology as a means to assess effective and temporal cardiac uptake represents a useful and critical application in drug development and pharmacological research and can be extended to the evaluation of comparable drug candidates