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Purposes We present the first in-human brain PET imaging data of the new α4β2 nicotinic acetylcholine receptor (nAChR)–targeting radioligand (+)-[18F]Flubatine. Aims were to develop a kinetic modeling-based approach to quantify (+)-[18F]Flubatine and compare the data of healthy controls (HCs) and patients with Alzheimer’s disease (AD); to investigate the partial volume effect (PVE) on regional (+)-[18F]Flubatine binding; and whether (+)-[18F]Flubatine binding and cognitive test data respective β-amyloid radiotracer accumulation were correlated. Methods We examined 11 HCs and 9 mild AD patients. All subjects underwent neuropsychological testing and [11C]PiB PET/MRI examination. (+)-[18F]Flubatine PET data were evaluated using full kinetic modeling and regional as well as voxel-based analyses. Results With 270-min p.i., the unchanged parent compound amounted to 97 ± 2%. Adequate fits of the time-activity curves were obtained with the 1 tissue compartment model (1TCM). (+)-[18F]Flubatine distribution volume (binding) was significantly reduced in bilateral mesial temporal cortex in AD patients compared with HCs (right 10.6 ± 1.1 vs 11.6 ± 1.4, p = 0.049; left 11.0 ± 1.1 vs 12.2 ± 1.8, p = 0.046; one-sided t tests each). PVE correction increased not only (+)-[18F]Flubatine binding of approximately 15% but also standard deviation of 0.4–70%. Cognitive test data and (+)-[18F]Flubatine binding were significantly correlated in the left anterior cingulate, right posterior cingulate, and right parietal cortex (r > 0.5, p 18F]Flubatine binding and [11C]PiB standardized uptake value ratios were negatively correlated in several regions; whereas in HCs, a positive correlation between cortical (+)-[18F]Flubatine binding and [11C]PiB accumulation in the white matter was found. No adverse event related to (+)-[18F]Flubatine occurred. Conclusion (+)-[18F]Flubatine is a safe and stable PET ligand. Full kinetic modeling can be realized by 1TCM without metabolite correction. (+)-[18F]Flubatine binding affinity was high enough to detect group differences. Of interest, correlation between white matter β-amyloid PET uptake and (+)-[18F]Flubatine binding indicated an association between white matter integrity and availability of α4β2 nAChRs. Overall, (+)-[18F]Flubatine showed favorable characteristics and has therefore the potential to serve as α4β2 nAChR–targeting PET ligand in further clinical trials.

TRPC6, the sixth member of the family of canonical transient receptor potential (TRP) channels, contributes to a variety of physiological processes and human pathologies. This study extends the knowledge on the newly developed TRPC6 blocker SH045 with respect to its main target organs beyond the description of plasma kinetics. According to the concentration-time course in mice plasma, SH045 is available in pharmacological effective plasma concentrations up to 24 h after administration of 20 mg/kg BW (i.v.) and up to the 5-fold of its IC50 until 6 hours, orally. The short plasma half-life and rather low oral bioavailability are contrasted by its high potency. Dosage limits were not worked out, but absence of safety concerns for 20 mg/kg BW supports further dose exploration. The disposition of SH045 is described. In particular, a high extravascular distribution, most prominent in lung, and a considerable renal elimination of SH045 were observed. SH045 is substrate of CYP3A4 and CYP2A6. Hydroxylated and glucuronidated metabolites were identified under optimized LC-MS/MS conditions. The results guide a reasonable selection of dose and application route of SH045 for target-directed preclinical studies in vivo with one of the rare high potent and subtype-selective TRPC6 inhibitors available.

To identify a potent and selective σ2 receptor ligand appropriate for development as a positron emission tomography (PET) tracer, several fluorinated analogues of the spirocyclic lead compounds trans- and cis-6 (N-(2,4-dimethylbenzyl)-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine) were designed. In multistep syntheses, a fluorine atom was introduced directly or as a 2-fluoroethoxy moiety on the 2-benzopyran scaffold, on the dimethylbenzylamino moiety, or on the central amino moiety. The σ1 and σ2 receptor affinity was determined in receptor binding studies with radioligands. With respect to σ2 affinity and σ2 /σ1 selectivity, cis-N-(2,4-dimethylbenzyl)-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-15 c, Ki (σ2 )=51 nm) and cis-N-[4-(fluoromethyl)-2-methylbenzyl]-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-28 e, Ki (σ2 )=57 nm) are the most promising ligands. The combination of both structural elements in one molecule, cis-N-[4-(fluoromethyl)-2-methylbenzyl]-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-28 c: Ki (σ2 )=874 nm), resulted in decreased σ2 and σ1 affinity. Methylation of secondary amines led to three tertiary methylamines with moderate affinity for both σ receptor subtypes.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Transient receptor potential channel subfamily C, member 6 (TRPC6), a non-selective cation channel that controls influx of Ca2+ and other monovalent cations into cells, is widely expressed in the kidney. TRPC6 gene variations have been linked to chronic kidney disease but its role in acute kidney injury (AKI) is unknown. Here we aimed to investigate the putative role of TRPC6 channels in AKI. We used Trpc6−/− mice and pharmacological blockade (SH045 and BI-749327), to evaluate short-term AKI outcomes. Here, we demonstrate that neither Trpc6 deficiency nor pharmacological inhibition of TRPC6 influences the short-term outcomes of AKI. Serum markers, renal expression of epithelial damage markers, tubular injury, and renal inflammatory response assessed by the histological analysis were similar in wild-type mice compared to Trpc6−/− mice as well as in vehicle-treated versus SH045- or BI-749327-treated mice. In addition, we also found no effect of TRPC6 modulation on renal arterial myogenic tone by using blockers to perfuse isolated kidneys. Therefore, we conclude that TRPC6 does not play a role in the acute phase of AKI. Our results may have clinical implications for safety and health of humans with TRPC6 gene variations, with respect to mutated TRPC6 channels in the response of the kidney to acute ischemic stimuli.

The σ2 receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ2 receptors in cancer cells and tissue in combination with the antiproliferative potency of σ2 receptor ligands motivates the research in the field of σ2 receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with 18F and evaluation regarding detection of σ2 receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [18F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ2 receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ2 receptor ligand [18F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ2 receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

Monoamine oxidases (MAOs) play a key role in the metabolism of major monoamine neurotransmitters. In particular, the upregulation of MAO-B in Parkinson’s disease, Alzheimer’s disease and cancer augmented the development of selective MAO-B inhibitors for diagnostic and therapeutic purposes, such as the anti-parkinsonian MAO-B irreversible binder l-deprenyl (Selegiline®). Herein we report on the synthesis of novel fluorinated indanone derivatives for PET imaging of MAO-B in the brain. Out of our series, the derivatives 6, 8, 9 and 13 are amongst the most affine and selective ligands for MAO-B reported so far. For the derivative 6-((3-fluorobenzyl)oxy)-2,3-dihydro-1H-inden-1-one (6) exhibiting an outstanding affinity (Ki MAO-B = 6 nM), an automated copper-mediated radiofluorination starting from the pinacol boronic ester 17 is described. An in vitro screening in different species revealed a MAO-B region-specific accumulation of [18F]6 in rats and piglets in comparison to L-[3H]deprenyl. The pre-clinical in vivo assessment of [18F]6 in mice demonstrated the potential of indanones to readily cross the blood–brain barrier. Nonetheless, parallel in vivo metabolism studies indicated the presence of blood–brain barrier metabolites, thus arguing for further structural modifications. With the matching analytical profiles of the radiometabolite analysis from the in vitro liver microsome studies and the in vivo evaluation, the structure’s elucidation of the blood–brain barrier penetrant radiometabolites is possible and will serve as basis for the development of new indanone derivatives suitable for the PET imaging of MAO-B. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4592]

Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application. Supplementary Information The online version contains supplementary material available at 10.1007/s00259-020-05164-4.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of Ne-acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M–1 s–1, which resulted in comprehensive structure–activity relationships. Structure–activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the...

Objectives: The monocarboxylate transporters 1 and 4 (MCT1/4) are integral plasma membrane proteins that bi-directionally transport lactate as well as small monocarboxylated molecules. They are highly expressed in several tumors. BAY-8002 belongs to a class of compounds that have been identified as novel and specific MCT1 inhibitors based on functional high-throughput screening assays using a panel of cell lines highly sensitive towards MCT1 inhibition [1]. IC50 values of 1 to 12 nM and ca. 500–fold selectivity towards MCT4 have already been reported for BAY-8002 [1]. Here we designed an 18F-labeled analog of BAY-8002 ([18F]F-BAY-8002) aiming to image mainly MCT1 upregulation considering the fact that the absence of MCT4 expression in many types of cancer may not be necessarily sufficient as a single marker to predict treatment response [2]. Methods: BAY-8002 and its novel fluorinated analog (F-BAY-8002) were synthesized based on reported procedures [1]. As BAY-8002 already contains a chloro substituent which could serve as leaving group, the compound was considered as precursor for radiofluorination via a halogen-fluorine exchange approach (Figure 1A). [18F]F-BAY-8002 was radiolabeled via a one-step aromatic nucleophilic substitution reaction (SNAr) using 2-5 mg of precursor in the presence of the [18F]KF/K222/K2CO3 complex in dimethyl sulfoxide (DMSO) at 150 °C within 5 min (Figure 1B). Figure 1. (A) Synthesis of BAY-8002 and its novel fluorinated analog; (B) Radiosynthesis of [18F]F-BAY-8002. Separation of [18F]F-BAY-8002 from the chlorinated precursor was performed by semi-preparative HPLC. The tracer was finally purified via solid-phase extraction (Sep-Pak® C18 light cartridge) and formulated in 10% EtOH/saline solution to be ready for biological evaluations. Results: Despite using identical conditions [1], the novel fluorinated analog F-BAY-8002 was obtained in only 4% overall yield due to formation of by-products which have not been observed during the synthesis of BAY-8002 (35% overall yield). Due to the lack of commercially available radioligands, the MCT1 affinity (Ki) of F-BAY-8002 could not yet be determined and we therefore intend to measure the KD value of our new radiotracer by in-house established methods in near future. The novel radiotracer [18F]F-BAY-8002 was synthesized in 30 ± 9% radiochemical yields (n = 4, non-isolated, estimated by radio-HPLC) within 5 min reaction time. After purification and formulation, the final product was obtained with a radiochemical purity of > 99% (n = 1). Further radiochemical characterization of the radiotracer and the transfer of the radiosynthesis to an automated module are in progress. Conclusions: A novel 18F-labeled radioligand for potential specific MCT1-targeted imaging was developed via a straightforward fast approach in good radiochemical yields and high radiochemical purity. Notably, the labeling was successful even without protection of the carboxylic acid group resulting in a beneficial one-step instead of a two-step radiosynthesis procedure. In vitro and in vivo biological evaluation of the newly synthesized MCT1 radioligand are currently ongoing. References: [1] Quanz, M. et al. Mol Cancer Ther. 2018 17:2285-2296; [2] Le Floch, R. et al. Proc Natl Acad Sci USA. 2011 108:16663-8.

The upregulation of the CB2 receptors in neuroinflammation and cancer and their potential visualization with PET (positron emission tomography) could provide a valuable diagnostic and therapy-monitoring tool in such disorders. However, the availability of reliable CB2-selective imaging probes is still lacking in clinical practice. We have recently identified a benzothiazole-2-ylidine amide hit (6a) as a highly potent CB2 ligand. With the aim of enhancing its CB2 over CB1 selectivity and introducing structural sites suitable for radiolabeling, we herein describe the development of fluorinated and methoxylated benzothiazole derivatives endowed with extremely high CB2 binding affinity and an exclusive selectivity to the CB2 receptor. Compounds 14, 15, 18, 19, 21, 24 and 25 displayed subnanomolar CB2 Ki values (ranging from 0.16 nM to 0.68 nM) and interestingly, all of the synthesized compounds completely lacked affinity at the CB1 receptor (Ki > 10,000 nM for all compounds), indicating their remarkably high CB2 over CB1 selectivity factors. The fluorinated analogs, 15 and 21, were evaluated for their in vitro metabolic stability in mouse and human liver microsomes (MLM and HLM). Both 15 and 21 displayed an exceptionally high stability (98% and 91% intact compounds, respectively) after 60 min incubation with MLM. Contrastingly, a 5- and 2.8-fold lower stability was demonstrated for compounds 15 and 21, respectively, upon incubation with HLM for 60 min. Taken together, our data present extremely potent and selective CB2 ligands as credible leads that can be further exploited for 18F- or 11C-radiolabeling and utilization as PET tracers.

Recent pharmacologic data revealed the implication of α3β4 nicotinic acetylcholine receptors (nAChRs) in nicotine and drug addiction. To image α3β4 nAChRs in vivo, we aimed to establish the synthesis of a [ 18 F]-labelled analog of the highly affine and selective α3β4 ligand ( S )-3-(4-(4-fluorophenyl)-1 H -1,2,3-triazol-1-yl)quinuclidine (( S )- T1 ). ( S )-[ 18 F] T1 was synthesized from ethynyl-4-[ 18 F]fluorobenzene ([ 18 F] 5 ) and ( S )-azidoquinuclidine by click reaction. After a synthesis time of 130 min ( S )-[ 18 F] T1 was obtained with a radiochemical yield (non-decay corrected) of 4.3±1.3%, a radiochemical purity of >99% and a molar activity of >158 GBq/μmol. The brain uptake and the brain-to-blood ratio of ( S )-[ 18 F] T1 in mice at 30 min post injection were 2.02 (SUV) and 6.1, respectively. According to an ex-vivo analysis, the tracer remained intact (>99%) in brain. Only one major radiometabolite was detected in plasma and urine samples. In-vitro autoradiography on pig brain slices revealed binding of ( S )-[ 18 F] T1 to brain regions associated with the expression of α3β4 nAChRs, which could be reduced by the α3β4 nAChR selective drug AT-1001 . These findings make ( S )-[ 18 F] T1 a potential tool for the non-invasive imaging of α3β4 nAChRs in the brain by PET.

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1&ndash, 9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required.

International audience; With the aim to develop a specific radioligand for imaging the cyclic nucleotide phosphodiesterase 5 (PDE5) in brain by positron emission tomography (PET), seven new fluorinated inhibitors (3-9) were synthesized on the basis of a quinoline core. The inhibitory activity for PDE5 together with a panel of other PDEs was determined in vitro and two derivatives were selected for IC50 value determination. The most promising compound 7 (IC50 = 5.92 nM for PDE5A), containing a 3-fluoroazetidine moiety, was further radiolabeled by aliphatic nucleophilic substitution of two different leaving groups (nosylate and tosylate) using [18F]fluoride. The use of the nosylate precursor and tetra-n-butyl ammonium [18F]fluoride ([18F]TBAF) in 3-methyl-3-pentanol combined with the addition of a small amount of water proved to be the best radiolabeling conditions achieving a RCY of 4.9 ± 1.5% in an automated procedure. Preliminary biological investigations in vitro and in vivo were performed to characterize this new PDE5 radioligand. Metabolism studies of [18F]7 in mice revealed a fast metabolic degradation with the formation of radiometabolites which have been detected in the brain.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [

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] ([18F]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 [18F]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 [18F]2–4, but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [18F]4 reached the brain, rendering [18F]4 less appropriate as a PET tracer than [18F]2 and [18F]3. Compounds [18F]2 and [18F]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 [18F]2 slightly over the fluoropropyl derivative [18F]3 for human use.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N

Both (+)-[18F]flubatine and its enantiomer (−)-[18F]flubatine are radioligands for the neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). In a clinical study in patients with early Alzheimer’s disease, (+)-[18F]flubatine ((+)-[18F]1) was examined regarding its metabolic fate, in particular by identification of degradation products detected in plasma and urine. The investigations included an in vivo study of (+)-flubatine ((+)-1) in pigs and structural elucidation of formed metabolites by LC-MS/MS. Incubations of (+)-1 and (+)-[18F]1 with human liver microsomes were performed to generate in vitro metabolites, as well as radiometabolites, which enabled an assignment of their structures by comparison of LC-MS/MS and radio-HPLC data. Plasma and urine samples taken after administration of (+)-[18F]1 in humans were examined by radio-HPLC and, on the basis of results obtained in vitro and in vivo, formed radiometabolites were identified. In pigs, (+)-1 was monohydroxylated at different sites of the azabicyclic ring system of the molecule. Additionally, one intermediate metabolite underwent glucuronidation, as also demonstrated in vitro. In humans, a fraction of 95.9 ± 1.9% (n = 10) of unchanged tracer remained in plasma, 30 min after injection. However, despite the low metabolic degradation, both radiometabolites formed in humans could be characterized as (i) a product of C-hydroxylation at the azabicyclic ring system, and (ii) a glucuronide conjugate of the precedingly-formed N8-hydroxylated (+)-[18F]1.

Recent pharmacologic data revealed the implication of α3β4 nicotinic acetylcholine receptors (nAChRs) in nicotine and drug addiction. To image α3β4 nAChRs in vivo, we aimed to establish the synthesis of a [

Sharpless asymmetric dihydroxylation of styrene derivative 6 afforded chiral triols (R)-7 and (S)-7, which were cyclized with tosyl chloride in the presence of Bu2SnO to provide 2-benzopyrans (R)-4 and (S)-4 with high regioselectivity. The additional hydroxy moiety in the 4-position was exploited for the introduction of various substituents. Williamson ether synthesis and replacement of the Boc protective group with a benzyl moiety led to potent σ1 ligands with high σ1/σ2-selectivity. With exception of the ethoxy derivative 16, the (R)-configured enantiomers represent eutomers with eudismic ratios of up to 29 for the ester (R)-18. The methyl ether (R)-15 represents the most potent σ1 ligand of this series of compounds, with a Ki value of 1.2 nM and an eudismic ratio of 7. Tosylate (R)-21 was used as precursor for the radiosynthesis of [18F]-(R)-20, which was available by nucleophilic substitution with K[18F]F K222 carbonate complex. The radiochemical yield of [18F]-(R)-20 was 18%–20%, the radiochemical purity greater than 97% and the specific radioactivity 175–300 GBq/µmol. Although radiometabolites were detected in plasma, urine and liver samples, radiometabolites were not found in brain samples. After 30 min, the uptake of the radiotracer in the brain was 3.4% of injected dose per gram of tissue and could be reduced by coadministration of the σ1 antagonist haloperidol. [18F]-(R)-20 was able to label those regions of the brain, which were reported to have high density of σ1 receptors.

A specific radioligand for the imaging of cyclic nucleotide phosphodiesterase 2A (PDE2A) via positron emission tomography (PET) would be helpful for research on the physiology and disease-related changes in the expression of this enzyme in the brain. In this report, the radiosynthesis of a novel PDE2A radioligand and the subsequent biological evaluation were described. Our prospective compound 1-(2-chloro-5-methoxy phenyl)-8-(2-fluoropyridin-4-yl)-3- methylbenzo[e]imidazo[5,1-c][1,2,4]triazine, benzoimidazotriazine (BIT1) (IC50 PDE2A = 3.33 nM, 16-fold selectivity over PDE10A) was fluorine-18 labeled via aromatic nucleophilic substitution of the corresponding nitro precursor using the K[18F]F‐K2.2.2‐carbonate complex system. The new radioligand [18F]BIT1 was obtained with a high radiochemical yield (54 &plusmn, 2%, n = 3), a high radiochemical purity (&ge, 99%), and high molar activities (155&ndash, 175 GBq/&mu, mol, n = 3). In vitro autoradiography on pig brain cryosections exhibited a heterogeneous spatial distribution of [18F]BIT1 corresponding to the known pattern of expression of PDE2A. The investigation of in vivo metabolism of [18F]BIT1 in a mouse revealed sufficient metabolic stability. PET studies in mouse exhibited a moderate brain uptake of [18F]BIT1 with a maximum standardized uptake value of ~0.7 at 5 minutes p.i. However, in vivo blocking studies revealed a non-target specific binding of [18F]BIT1. Therefore, further structural modifications are needed to improve target selectivity.

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer’s disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (−)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[18F]flubatine (specific activity > 350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.

The imaging of σ

Phosphodiesterases (PDEs) are enzymes that play a major role in cell signalling by hydrolysing the secondary messengers cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) throughout the body and brain. Altered cyclic nucleotide-mediated signalling has been associated with a wide array of disorders, including neurodegenerative disorders. Recently, PDE5 has been shown to be involved in neurodegenerative disorders such as Alzheimer’s disease, but its precise role has not been elucidated yet. To visualize and quantify the expression of this enzyme in brain, we developed a radiotracer for specific PET imaging of PDE5. A quinoline-based lead compound has been structurally modified resulting in the fluoroethoxymethyl derivative ICF24027 with high inhibitory activity towards PDE5 (IC50 = 1.86 nM). Radiolabelling with fluorine-18 was performed by a one-step nucleophilic substitution reaction using a tosylate precursor (RCY(EOB) = 12.9% ± 1.8%; RCP > 99%; SA(EOS) = 70–126 GBq/μmol). In vitro autoradiographic studies of [18F]ICF24027 on different mouse tissue as well as on porcine brain slices demonstrated a moderate specific binding to PDE5. In vivo studies in mice revealed that [18F]ICF24027 was metabolized under formation of brain penetrable radiometabolites making the radiotracer unsuitable for PET imaging of PDE5 in brain.

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

Background The level of expression of cannabinoid receptor type 2 (CB2R) in healthy and diseased brain has not been fully elucidated. Therefore, there is a growing interest to assess the regional expression of CB2R in the brain. Positron emission tomography (PET) is an imaging technique, which allows quantitative monitoring of very low amounts of radiolabelled compounds in living organisms at high temporal and spatial resolution and, thus, has been widely used as a diagnostic tool in nuclear medicine. Here, we report on the radiofluorination of N-aryl-oxadiazolyl-propionamides at two different positions in the lead structure and on the biological evaluation of the potential of the two tracers [18F]1 and [18F]2 as CB2 receptor PET imaging agents. Results High binding affinity and specificity towards CB2 receptors of the lead structure remained unaffected by the structural changes such as the insertion of the aliphatic and aromatic fluorine in the selected labelling sites of 1 and 2. Aliphatic and aromatic radiofluorinations were optimized, and [18F]1 and [18F]2 were achieved in radiochemical yields of ≥30% with radiochemical purities of ≥98% and specific activities of 250 to 450 GBq/μmol. Organ distribution studies in female CD1 mice revealed that both radiotracers cross the blood–brain barrier (BBB) but undergo strong peripheral metabolism. At 30 min after injection, unmetabolized [18F]1 and [18F]2 accounted for 60% and 2% as well as 68% and 88% of the total activity in the plasma and brain, respectively. The main radiometabolite of [18F]2 could be identified as the free acid [18F]10, which has no affinity towards the CB1 and CB2 receptors but can cross the BBB. Conclusions N-aryl-oxadiazolyl-propionamides can successfully be radiolabelled with 18F at different positions. Fluorine substitution at these positions did not affect affinity and specificity towards CB2R. Despite a promising in vitro behavior, a rather rapid peripheral metabolism of [18F]1 and [18F]2 in mice and the generation of brain permeable radiometabolites hamper the application of these radiotracers in vivo. However, it is expected that future synthetic modification aiming at a replacement of metabolically susceptible structural elements of [18F]1 and [18F]2 will help to elucidate the potential of this class of compounds for CB2R PET studies.