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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.

Pridopidine is an investigational drug in late stage development for the treatment of Huntington disease and originally postulated to act as dopamine stabilizer by modulating dopamine-dependent motor behavior. However, preclinical studies show pridopidine has highest affinity to sigma-1 receptors. Importantly, mediated by sigma-1 receptors, pridopidine has neuroprotective properties and enhances neuronal plasticity. The aim of our study was to determine the in-vivo the target engagement (receptor occupancy) of pridopidine at clinically relevant doses in healthy volunteers and Huntington disease patients. We used sigma-1 receptor-specific (S)-(-)-[18F]Fluspidine and dopamine D2/D3 receptor-specific [18F]Fallypride PET imaging to quantify the sigma-1 and dopamine D2/D3 receptor occupancy of pridopidine. Eleven male healthy volunteers (pridopidine 0.5 to 90 mg in six dose groups) and three male Huntington disease patients (pridopidine 90 mg) were studied twice before and 2h following single oral doses of pridopidine using S-(-)-[18F]Fluspidine PET (300 MBq, 0-90min p.i.). Distribution volume VT was quantified using kinetic modeling (One-tissue compartment model; metabolite correction). Four male healthy volunteers were studied twice using [18F]Fallpride PET (200 MBq, 0-210min p.i.) before and 2h after a single oral dose of pridopidine (90 mg). Binding potential BPND was assessed by the simplified reference model. Volume-of-interest analyses were performed. For each subject/tracer, the receptor occupancy was calculated by the Lassen plot analysis. In healthy volunteers, there was high sigma-1 receptor occupancy (87 to 91%) across all brain regions at doses ranging from 22.5 to 90 mg. The sigma-1 receptor occupancy was 43% at 1 mg pridopidine. In Huntington disease patients, very similar to healthy volunteers, at 90 mg pridopidine, there was high sigma-1 receptor occupancy (87±7%, n.s.). In contrast, in healthy volunteers, there was only negligible dopamine D2/D3 receptor occupancy (3±2%) at 90 mg pridopidine. We established a sigmoid-shaped dose/sigma-1 receptor occupancy relation (Hill equation) with Hill coefficient larger than 1 in healthy volunteers, suggesting a positive cooperative binding nature of the sigma-1 receptor. Using PET, we report for the first time in the living human brain that after a single dose of 90 mg, pridopidine acts as a selective sigma-1 receptor ligand showing near to complete sigma-1 receptor occupancy (~90%) but only minimal (~3%) dopamine D2/D3 receptor occupancy. Our findings provide significant clarification about pridopidine’s mechanism of action and support further use of the 45 mg bidaily dose to achieve full and selective targeting of the sigma-1 receptor in future clinical trials in Huntington disease and amyotrophic lateral sclerosis.

We report the design, synthesis, and evaluation of a new series of benzylpiperazine derivatives as selective σ1 receptor ligands. All seven ligands possessed low nanomolar affinity for σ1 receptors (Ki (σ1 ) = 0.31-4.19 nM) and high subtype selectivity (Ki (σ2 )/Ki (σ1 ) = 50-2448). The fluoroethoxy analogues also exhibited high selectivity toward the vesicular acetylcholine transporter (Ki (VAChT)/Ki (σ1 ) = 99-18252). The corresponding radiotracers [18 F]13, [18 F]14, and [18 F]16 with high selectivity (Ki (σ2 )/Ki (σ1 ) > 100, Ki (VAChT)/Ki (σ1 ) > 1000) were prepared in 42% to 55% radiochemical yields (corrected for decay), greater than 99% radiochemical purity (RCP), and molar activity of about 120 GBq/μmol at the end of synthesis (EOS). All three radiotracers showed high initial brain uptake in mouse (8.37-11.48% ID/g at 2 min), which was not affected by pretreatment with cyclosporine A, suggesting that they are not substrates for permeability-glycoprotein (P-gp). Pretreatment with SA4503 or haloperidol resulted in significantly reduced brain uptake (35%-62% decrease at 30 min). In particular, [18 F]16 displayed high brain-to-blood ratios and high in vivo metabolic stability. Although it may not be an optimal neuroimaging agent because of its slow kinetics in the mouse brain, [18 F]16 can serve as a lead compound for further structural modifications to explore new potential radiotracers for σ1 receptors.

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%).

Purine nucleotides such as ATP and ADP are important extracellular signaling molecules in almost all tissues activating various subtypes of purinoreceptors. In the brain, the P2Y1 receptor (P2Y1R) subtype mediates trophic functions like differentiation and proliferation, and modulates fast synaptic transmission, both suggested to be affected in diseases of the central nervous system. Research on P2Y1R is limited because suitable brain-penetrating P2Y1R-selective tracers are not yet available. Here, we describe the first efforts to develop an 18F-labeled PET tracer based on the structure of the highly affine and selective, non-nucleotidic P2Y1R allosteric modulator 1-(2-[2-(tert-butyl)phenoxy]pyridin-3-yl)-3-[4-(trifluoromethoxy)phenyl]urea (7). A small series of fluorinated compounds was developed by systematic modification of the p-(trifluoromethoxy)phenyl, the urea and the 2-pyridyl subunits of the lead compound 7. Additionally, the p-(trifluoromethoxy)phenyl subunit was substituted by carborane, a boron-rich cluster with potential applicability in boron neutron capture therapy (BNCT). By functional assays, the new fluorinated derivative 1-{2-[2-(tert-butyl)phenoxy]pyridin-3-yl}-3-[4-(2-fluoroethyl)phenyl]urea (18) was identified with a high P2Y1R antagonistic potency (IC50 = 10 nM). Compound [18F]18 was radiosynthesized by using tetra-n-butyl ammonium [18F]fluoride with high radiochemical purity, radiochemical yield and molar activities. Investigation of brain homogenates using hydrophilic interaction chromatography (HILIC) revealed [18F]fluoride as major radiometabolite. Although [18F]18 showed fast in vivo metabolization, the high potency and unique allosteric binding mode makes this class of compounds interesting for further optimizations and investigation of the theranostic potential as PET tracer and BNCT agent.

Parkinson’s disease (PD) is known to involve the peripheral nervous system (PNS) and the enteric nervous system (ENS). Functional changes in PNS and ENS appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients like constipation, that can precede the appearance of motor symptoms by years. We have shown that environmental toxins can trigger the disease by acting on the ENS and on the autonomic nervous system. Oral exposure to the pesticide rotenone, a mitochondrial Complex I inhibitor, leads to decreased stool depositions in mice. Here we analyzed the effect of rotenone on the function and structure of the ENS by measuring intestinal contractility in a tissue bath and by analyzing related protein expression. Our results show that rotenone changes the normal physiological response of the intestine to carbachol, dopamine and electric field stimulation. The magnitude and direction of these alterations varies between intestinal regions and exposure times and is associated with an early up-regulation of dopaminergic, cholinergic and adrenergic receptors and an irregular reduction in the amount of enteric neurons in rotenone-exposed mice. The early appearance of these alterations makes them ideal candidates to be used as biomarkers for the detection of Parkinson’s disease in its early stages.

Nowadays, it is general consensus that the cholinergic transmission system in the brain is heavily involved in the development, progress, and therapy of certain neurodegenerative diseases. In particular cholinergic presynaptic components such as the acetylcholinesterase (AChE) or the vesicular acetylcholine transporter (VAChT) are considered to be affected by early changes in neuropathological processes as observed, e.g., in Alzheimer’s disease (AD). The VAChT is a transmembrane protein located at synaptic vesicles and responsible for the transport and storage of the neurotransmitter acetylcholine (ACh) in the vesicles. Therefore, the VAChT is regarded as a potential target for neuroimaging of cholinergic alterations with positron emission tomography. To date, the development of PET radioligands for this transporter is based on a single known lead compound named vesamicol. A challenge was arising due to the finding that vesamicol also binds to the sigma receptors which are partly co-localized with the VAChT in several brain regions. In the last three decades, a multitude of structural diverse vesamicol analogs has been designed resulting in a considerable number of 11C- and 18F-labeled PET as well as a few 123/125I-labeled SPECT tracers which were mainly preclinically evaluated. However, only very few of them had the potential for translation to human studies. Therefore, a routinely used VAChT PET imaging method could not be established in the clinics so far. However, first results of recently published studies using the potent candidate [18F]FEOBV in patients with neuropathologies are very promising and possibly a breakthrough in this field.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes &alpha, 3&beta, 4 and &alpha, 7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary &beta, 4-subunit of the &alpha, 4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the &alpha, 4 selectivity of the quinuclidine-triazole chemotype, explaining the 47&ndash, 327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the &alpha, 7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

In order to detect novel σ receptor ligands, the rigit spiro [[2]benzopyran-1,1'-cyclohexan]-4'-one was connected with amino moieties derived from σ2 receptor preferring lead compounds resulting in mixtures of trans- and cis-configured amines 6, 18, and 27. In a four step synthesis the methyl acetals 6 were converted into fluoroethyl derivatives 13 and 30. The most promising σ2 receptor ligand is the methyl acetal 6a bearing a 2,4-dimethylbenzylamino moiety. The fluoroethyl derivatives 13c and 13d reveal high σ1 affinity but moderate selectivity over the σ2 subtype. In mice 13c and 13d showed antiallodynic activity that is stronger than that of the reference σ1 antagonist BD-1063 (34). Since the antiallodynic activity of 13c could only be partially reversed by the σ1 agonist PRE-084 (35), it is postulated that a second mechanism contributes to its overall antiallodynic effect. In contrast, the antiallodynic effect of its diastereomer 13d can be totally explained by a σ1 antagonism.

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.

We have designed and synthesized novel piperazine compounds with low lipophilicity as σ1 receptor ligands. 1-(4-Fluorobenzyl)-4-[(tetrahydrofuran-2-yl)methyl]piperazine (10) possessed a low nanomolar σ1 receptor affinity and a high selectivity toward the vesicular acetylcholine transporter (>2000-fold), σ2 receptors (52-fold), and adenosine A2A, adrenergic α2, cannabinoid CB1, dopamine D1, D2L, γ-aminobutyric acid A (GABAA), NMDA, melatonin MT1, MT2, and serotonin 5-HT1 receptors. The corresponding radiotracer [18F]10 demonstrated high brain uptake and extremely high brain-to-blood ratios in biodistribution studies in mice. Pretreatment with the selective σ1 receptor agonist SA4503 significantly reduced the level of accumulation of the radiotracer in the brain. No radiometabolite of [18F]10 was observed to enter the brain. Positron emission tomography and magnetic resonance imaging confirmed suitable kinetics and a high specific binding of [18F]10 to σ1 receptors in rat brain. Ex vivo autoradiography show...

Bariatric surgery remains the single most effective long-term treatment modality for morbid obesity, achieved mainly by lowering caloric intake through as yet ill-defined mechanisms. Here we show in rats that Roux-en-Y gastric bypass (RYGB)-like rerouting of ingested fat mobilizes lower small intestine production of the fat-satiety molecule oleoylethanolamide (OEA). This was associated with vagus nerve-driven increases in dorsal striatal dopamine release. We also demonstrate that RYGB upregulates striatal dopamine 1 receptor (D1R) expression specifically under high-fat diet feeding conditions. Mechanistically, interfering with local OEA, vagal, and dorsal striatal D1R signaling negated the beneficial effects of RYGB on fat intake and preferences. These findings delineate a molecular/systems pathway through which bariatric surgery improves feeding behavior and may aid in the development of novel weight loss strategies that similarly modify brain reward circuits compromised in obesity.

(S)-[18F]fluspidine ((S)-[18F]1) has recently been explored for positron emission tomography (PET) imaging of sigma-1 receptors in humans. In the current report, we have used plasma samples of healthy volunteers to investigate the radiometabolites of (S)-[18F]1 and elucidate their structures with LC-MS/MS. For the latter purpose additional in vitro studies were conducted by incubation of (S)-[18F]1 and (S)-1 with human liver microsomes (HLM). In vitro metabolites were characterized by interpretation of MS/MS fragmentation patterns from collision-induced dissociation or by use of reference compounds. Thereby, structures of corresponding radio-HPLC-detected radiometabolites, both in vitro and in vivo (human), could be identified. By incubation with HLM, mainly debenzylation and hydroxylation occurred, beside further mono- and di-oxygenations. The product hydroxylated at the fluoroethyl side chain was glucuronidated. Plasma samples (10, 20, 30 min p.i., n = 5-6), obtained from human subjects receiving 250–300 MBq (S)-[18F]1 showed 97.2, 95.4, and 91.0% of unchanged radioligand, respectively. In urine samples (90 min p.i.) the fraction of unchanged radioligand was only 2.6% and three major radiometabolites were detected. The one with the highest percentage, also found in plasma, matched the glucuronide formed in vitro. Only a small amount of debenzylated metabolite was detected. In conclusion, our metabolic study, in particular the high fractions of unchanged radioligand in plasma, confirms the suitability of (S)-[18F]1 as PET radioligand for sigma-1 receptor imaging.

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.

Cyclic nucleotide phosphodiesterase 10A (PDE10A) regulates the level of the second messengers cAMP and cGMP in particular in brain regions assumed to be associated with neurodegenerative and psychiatric diseases. A better understanding of the pathophysiological role of the expression of PDE10A could be obtained by quantitative imaging of the enzyme by positron emission tomography (PET). Thus, in this study we developed, radiolabeled, and evaluated a new PDE10A radioligand, 8-bromo-1-(6-[18 F]fluoropyridin-3-yl)-3,4-dimethylimidazo[1,5-a]quinoxaline ([18 F]AQ28A). [18 F]AQ28A was radiolabeled by both nucleophilic bromo-to-fluoro or nitro-to-fluoro exchange using K[18 F]F-K2.2.2 -carbonate complex with different yields. Using the superior nitro precursor, we developed an automated synthesis on a Tracerlab FX F-N module and obtained [18 F]AQ28A with high radiochemical yields (33 ± 6%) and specific activities (96-145 GBq·μmol-1 ) for further evaluation. Initially, we investigated the binding of [18 F]AQ28A to the brain of different species by autoradiography and observed the highest density of binding sites in striatum, the brain region with the highest PDE10A expression. Subsequent dynamic PET studies in mice revealed a region-specific accumulation of [18 F]AQ28A in this region, which could be blocked by preinjection of the selective PDE10A ligand MP-10. In conclusion, the data suggest [18 F]AQ28A is a suitable candidate for imaging of PDE10A in rodent brain by PET.

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.

We report the design and synthesis of several 4-phenylpiperidine-4-carbonitrile derivatives as σ1 receptor ligands. In vitro radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ1 receptors (Ki(σ1) = 1.22–2.14 nM) and extremely high subtype selectivity (Ki(σ2) = 830–1710 nM; Ki(σ2)/Ki(σ1) = 680–887). [18F]9 was prepared in 42–46% isolated radiochemical yield, with a radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic 18F- substitution of the corresponding tosylate precursor. Biodistribution studies in mice demonstrated high initial brain uptakes and high brain-to-blood ratios. Administration of SA4503 or haloperidol 5 min prior to injection of [18F]9 significantly reduced the accumulation of radiotracers in organs known to contain σ1 receptors. Two radioactive metabolites were observed in the brain at 30 min after radiotracer injection. [18F]9 may serve as a lead compound to develop suitable radiotracers for σ1 receptor imaging with positron emission tomography.

We report the design, synthesis, and evaluation of a series of 1-oxa-8-azaspiro[4.5]decane and 1,5-dioxa-9-azaspiro[5.5]undecane derivatives as selective σ1 receptor ligands. All seven ligands exhibited nanomolar affinity for σ1 receptors (Ki(σ1) = 0.47 – 12.1 nM) and moderate selectivity over σ2 receptors (Ki(σ2)/ Ki(σ1) = 2 – 44). Compound 8, with the best selectivity among these ligands, was selected for radiolabeling and further evaluation. Radioligand [18F]8 was prepared via nucleophilic 18F-substitution of the corresponding tosylate precursor, with an overall isolated radiochemical yield of 12–35%, a radiochemical purity of greater than 99%, and molar activity of 94 – 121 GBq/μmol. Biodistribution studies of [18F]8 in mice demonstrated high initial brain uptake at 2 min. Pretreatment with SA4503 resulted in significantly reduced brain-to-blood ratio (70% − 75% at 30 min). Ex vivo autoradiography in ICR mice demonstrated high accumulation of the radiotracer in σ1 receptor-rich brain areas. These findings suggest that [18F]8 could be a lead compound for further structural modifications to develop potential brain imaging agents for σ1 receptors.

The adenosine A2A receptor (A2AR) is regarded as a particularly appropriate target for non-dopaminergic treatment of Parkinson&rsquo, s disease (PD). An increased A2AR availability has been found in the human striatum at early stages of PD and in patients with PD and dyskinesias. The aim of this small animal positron emission tomography/magnetic resonance (PET/MR) imaging study was to investigate whether rotenone-treated mice reflect the aspect of striatal A2AR upregulation in PD. For that purpose, we selected the known A2AR-specific radiotracer [18F]FESCH and developed a simplified two-step one-pot radiosynthesis. PET images showed a high uptake of [18F]FESCH in the mouse striatum. Concomitantly, metabolism studies with [18F]FESCH revealed the presence of a brain-penetrant radiometabolite. In rotenone-treated mice, a slightly higher striatal A2AR binding of [18F]FESCH was found. Nonetheless, the correlation between the increased A2AR levels within the proposed PD animal model remains to be further investigated.

An investigation on the selective binding of six quinuclidine-triazole enantiomeric pairs to nicotinic acetylcholine receptor (nAChR) subtypes, (S)-enantiomers for a3b4-nAChR and its (R)-counterpart for a7-nAChR, was performed in silico to provide the insight into the molecular basis for subtype discrimination of the quinuclidine-triazole enantiomers. The homology modeling and molecular docking analyses revealed that unique amino acid residues in the complementary subunit of nAChR subtypes are related to a high subtype selectivity profile. One non-conserved residue AspB173 in a complementary b4-subunit of the a3b4-nAChR binding pocket was found to be a primary determinant for the a3b4 selectivity of the quinuclidine-triazole chemotype as evidenced by the more pronounced enantioselectivity of (S)-enantiomers for the a3b4 nAChR, 47-326 times greater than its corresponding (R)-enantiomers. For (R)-enantiomers toward the a7 subtype, the interacting amino acid residues were the conserved TyrA93 and TrpA149 and TrpB55, leading to a lesser degree of stereoselectivity. The interaction with non-conserved amino acid residues in the complementary subunit of nAChR subtypes appeared to be the determinant for the nAChR subtype-selective binding, particularly at the heteropentameric subtype.

By structure–activity relationship studies on the tilorone scaffold, the ‘one armed’ substituted dibenzothiophenes and the fluoren-9-ones were identified as the most potential α7 nAChR ligands. While the suitability of dibenzothiophene derivatives as PET tracers is recognized, the potential of fluoren-9-ones is insufficiently investigated. We herein report on a series of fluoren-9-one based derivatives targeting α7 nAChR with compounds 8a and 8c possessing the highest affinity and selectivity. Accordingly, with [18F]8a and [18F]8c we designed and initially evaluated the first fluoren-9-one derived α7 nAChR selective PET ligands. A future application of these radioligands is facilitated by the herein presented successful implementation of fully automated radiosynthesis.

To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the brain by PET, we systematically modified this molecule and investigated five different groups of derivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of 67 different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as σ1 and σ2 receptors, we performed an extensive structure-affinity relationship (SAR) study regarding both affinity and selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to > 10 µM and selectivity factors from 0.1 to 73 regarding σ1 and σ2 receptors, respectively. We could confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in particular to the σ1 receptor. Finally, none of the various vesamicol analogs in all five groups showed an in vitro binding profile suitable for specific VAChT imaging in the brain.

We have designed and synthesized a series of cyclopentadienyl tricarbonyl 99mTc/Re complexes containing a spirocyclic piperidine moiety as sigma receptor ligands. Rhenium compound 3a (4-(3H-spiro(2-benzofuran-1,4’-piperidin)-1’-yl)butylcarbonylcyclopentadienyl tricarbonyl rhenium) showed high affinity for both sigma-1 (Ki = 13.8 ± 0.7 nM) and sigma-2 (Ki = 15.1 ± 3.5 nM) receptors. In the MTT assay, 3a displayed significant and comparable antiproliferative activity in DU145, MCF7 and MCF7/Adr tumor cells to siramesine, indicating 3a is an agonist of sigma-2 receptors and a potential antitumor agent. The corresponding radiolabeled compound [99mTc]3b was prepared via double-ligand-transfer reaction from the corresponding ferrocene precursor with a radiochemical yield of 48% and a radiochemical purity of more than 99%. Studies of the cellular accumulation of [99mTc]3b in C6 and DU145 tumor cells indicated that the total binding and the intracellular association is notably high. Incubation with haloperidol significantly reduced the radiotracer accumulation dose-dependently. The biodistribution of [99mTc]3b in nude mice bearing DU145 tumor xenografts showed high tumor uptake (4.27%ID/g) and high tumor-to-blood (12) and tumor-to-muscle (5) ratios at 2 h postinjection. Pretreatment with haloperidol resulted in a remarkable reduction of tumor accumulation, indicating the specific binding of [99mTc]3b to sigma receptors in the tumor. These findings highlight the further evaluation of cyclopentadienyl tricarbonyl 99mTc/Re complexes and prospectively the 188Re-labeled analogs containing a spirocyclic piperidine moiety as sigma ligands for tumor imaging and therapy.

The increasing incidence of Alzheimer's disease (AD) worldwide is a major public health problem. Current treatments provide only palliative solutions with significant side effects. Therefore, new efficient treatment options and novel early diagnosis tools are urgently needed. Recently, strong pre-clinical evidences suggested that phosphodiesterase 5 (PDE5) may be clinically relevant both as biomarker and drug-target in AD. In this study, we intended to develop a new radiofluorinated tracer for the visualisation of PDE5 in brain using PET imaging. Based on currently known PDE5 inhibitors, a series of novel fluorinated compounds bearing a quinoline core have been synthesised via multi-steps reaction pathways. Their affinity for PDE5 and selectivity over other PDE families have been investigated. According to the data collected from this in vitro screening, fluorinated derivatives 24a, b bearing a fluoroethoxy group at the C-3 position of the quinoline core appeared to be the most promising structures and will be further radiolabelled with fluorine-18 for in vitro and in vivo evaluations as PET radiotracer for neuroimaging of PDE5.

Cannabinoid type 2 receptors (CB2 receptors) are involved in various pathological processes, and the visualization of their in vivo availability with positron emission tomography (PET) is of high interest. The study focuses on the introduction of fluorine into the structure of the highly affine and selective CB2 receptor ligand N-(adamantan-1-yl)-5-ethyl-2-methyl-1-phenyl-1H-imidazole-4-carboxamide (5). A novel series of compounds was developed by modifying (i) the adamantane-3-position, (ii) the imidazole-N-phenyl ring, and (iii) the imidazole-2-position, and the impact on the CB2 binding affinity and selectivity toward cannabinoid type 1 receptors (CB1) was evaluated. This study identified compound 15 as one of the most potent (Ki(CB2) = 0.29 nM) and selective (CB1/CB2 > 10000) CB2 receptor ligands discovered so far, eligible for the development of an 18F-labeled PET radiotracer.

Several spirocyclic piperidine derivatives were designed and synthesized as σ 1 receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ 1 receptors and subtype selectivity. Particularly for ligand 1′-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3 H -spiro[2-benzofuran-1,4′-piperidine] ( 2 ), high σ 1 receptor affinity ( K i = 2.30 nM) and high σ 1 /σ 2 subtype selectivity (142-fold) as well as high σ 1 /VAChT selectivity (234-fold) were observed. [ 18 F] 2 was synthesized using an efficient one-pot, two-step reaction method in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8–10%, a radiochemical purity of higher than 99%, and specific activity of 56–78 GBq/μmol. Biodistribution studies of [ 18 F] 2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 μmol/kg) 5 min prior to injection of [ 18 F] 2 significantly decreased the accumulation of radiotracer in organs known to contain σ 1 receptors. Ex vivo autoradiography in Sprague–Dawley rats demonstrated high accumulation of radiotracer in brain areas with high expression of σ 1 receptors. These encouraging results prove that [ 18 F] 2 is a suitable candidate for σ 1 receptor imaging with PET in humans.

We report the design, synthesis, and evaluation of a series of novel cyclopentadienyl tricarbonyl 99mTc complexes as potent σ1 receptor radioligands. Rhenium compounds 3-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-propylcarbonylcyclopentadienyl tricarbonyl rhenium (10a) and 4-(4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl)-butylcarbonylcyclopentadienyl tricarbonyl rhenium (10b) possessed high in vitro affinity for σ1 receptors and moderate to high selectivity for σ2 receptors and the vesicular acetylcholine transporter. Biodistribution studies in mice demonstrated high initial brain uptake for corresponding 99mTc derivatives [99mTc] 23 and [99mTc]24 of 2.94 and 2.13% injected dose (ID)/g, respectively, at 2 min postinjection. Pretreatment of haloperidol significantly reduced the radiotracer accumulation of [99mTc]23 or [99mTc]24 in the brain. Studies of the cellular uptake of [99mTc]23 in C6 and DU145 tumor cells demonstrated a reduction of accumulation by incubation with haloperidol, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine (SA4503), or 1,3-di-otolyl-guanidine (DTG). Furthermore, blocking studies in C6 glioma-bearing mice confirmed the specific binding of [99mTc]23 to σ1 receptors in the tumor.

Based on the potent phosphodiesterase 10 A (PDE10A) inhibitor PQ-10, we synthesized 32 derivatives to determine relationships between their molecular structure and binding properties. Their roles as potential positron emission tomography (PET) ligands were evaluated, as well as their inhibitory potency toward PDE10A and other PDEs, and their metabolic stability was determined in vitro. According to our findings, halo-alkyl substituents at position 2 of the quinazoline moiety and/or halo-alkyloxy substituents at positions 6 or 7 affect not only the compounds' affinity, but also their selectivity toward PDE10A. As a result of substituting the methoxy group for a monofluoroethoxy or difluoroethoxy group at position 6 of the quinazoline ring, the selectivity for PDE10A over PDE3A increased. The same result was obtained by 6,7-difluoride substitution on the quinoxaline moiety. Finally, fluorinated compounds (R)-7-(fluoromethoxy)-6-methoxy-4-(3-(quinoxaline-2-yloxy)pyrrolidine-1-yl)quinazoline (16 a), 19 a-d, (R)-tert-butyl-3-(6-fluoroquinoxalin-2-yloxy)pyrrolidine-1-carboxylate (29), and 35 (IC50 PDE10A 11-65 nM) showed the highest inhibitory potential. Further, fluoroethoxy substitution at position 7 of the quinazoline ring improved metabolic stability over that of the lead structure PQ-10.

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.

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.

In order to increase the polarity of the potent CB2 ligand 1a, the homologous hydroxyalkyl carbazoles 2a-c were prepared and pharmacologically evaluated. An important step in the synthesis is the hydroxyalkylation of carbazole with cyclic sulfates providing the 2-hydroxyethyl and 3-hydroxypropyl derivatives 5a and 5b in a one-step reaction. The final propionamides 2a-c were prepared using the recently reported coupling reagent COMU®. The X-ray crystal structure of 2c displays an almost coplanar arrangement of the 3-phenyl-1,2,4-oxadiazole biaryl system. The increased polarity of 2a is associated with an almost 100-fold reduced CB2 affinity. The 3-hydroxypropyl derivative 2b represents the best compromise between lipophilicity and CB2 affinity (Ki = 33 nM).

The enantiomers of the potent σ1 ligand fluspidine (1) were prepared by using chiral preparative HPLC. Synthesis of racemic tosylate 2 and subsequent separation of enantiomers yielded (R)-2 and (S)-2 in excellent enantiomeric purities. The fluspidine enantiomers (R)-1 and (S)-1 were synthesized from (R)-2 and (S)-2 by nucleophilic substitution with tetra-n-butyl-ammonium fluoride, affording (R)-1 with 99.6% ee and (S)-1 with 96.4% ee. Tosylates (R)-2 and (S)-2 can also serve as precursors for the radiosynthesis of enantiomerically pure radiotracers [18F](R)-1 and [18F](S)-1. The absolute configuration of the pure enantiomers was elucidated by comparison of their CD spectra with a calculated CD spectrum of a simplified model compound. In receptor binding studies, both enantiomers displayed very high σ1 receptor affiniy and selectivity against the σ2 receptor. (R)-Fluspidine ((R)-1) is the eutomer, with a K1 value of 0.57 nM and a eudysmic ratio of 4. Incubation of (R)-1 and (S)-1 with rat liver microsomes led to the identification of seven and eight metaboites, respectively. Although the S-configured enantiomer formed additional metabolite (S)-1-3, it ist metabolically more stable than (R)-1.

The vesicular acetylcholine transporter (VAChT) is an important target for in vivo imaging of neurodegenerative processes using positron emission tomography (PET). So far the development of VAChT PET radioligands is based on the single known lead compound vesamicol. In this study we investigated a recently published spiroindoline based compound class (Sluder et al., 2012), which was suggested to have potential in the development of VAChT ligands. Therefore, we synthesized a small series of N,N-substituted spiro[indoline-3,4'-piperidine] derivatives and determined their in vitro binding affinities toward the VAChT. In order to investigate the selectivity, the off-target binding toward σ1 and σ2 receptors was determined. The compounds possessed VAChT affinities with Ki values in the range of 39-376nM. Binding affinities toward the σ1 and σ2 receptors are in a similar range indicating that the strong structural difference between the spiroindolines and vesamicol did not improve the selectivity. The observed potential to additionally bind to σ receptors let us assume that the herein investigated spiroindolines are not suitable to replace vesamicol as lead compound for the development of VAChT ligands.

Herein, new ligands for the vesicular acetylcholine transporter (VAChT), based on a benzovesamicol scaffold, are presented. VAChT is acknowledged as a marker for cholinergic neurons and a positron emission tomography tracer for VAChT could serve as a tool for quantitative analysis of cholinergic neuronal density. With an easily accessible triflate precursor, aminocarbonylations were utilized to evaluate the chemical space around the C5 position on the tetrahydronaphthol ring. Synthesized ligands were evaluated for their affinity and selectivity for VAChT. Small, preferably aromatic, N-substituents proved to be more potent than larger substituents. Of the fifteen compounds synthesized, benzyl derivatives (±)-7i and (±)-7l had the highest affinities for VAChT. Compound (±)-7i was chosen to investigate the importance of stereochemistry for binding to VAChT and selectivity toward the σ1 and σ2 receptors. Enantiomeric resolution gave (+)-7i and (-)-7i, and the eutomer showed seven times better affinity. Although racemate (±)-7i was initially promising, the affinity of (-)-7i for VAChT was not better than 56.7nM which precludes further preclinical evaluation. However, the nanomolar binding together with the ready synthesis of [11C]-(±)-7i shows that (-)-7i can serve as a scaffold for future optimizations to provide improved 11C-labelled VAChT PET tracers.

We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives as potential σ(1) receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ(1) receptors (K(i)=1.85 ± 1.59 nM) and high subtype selectivity (σ(2) receptor: K(i)=291 ± 111 nM; K(i)σ(2)/K(i)σ(1)=157). [(18)F]6 was prepared in 30-50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic (18)F(-) substitution of the corresponding tosylate precursor. The logD(pH 7.4) value of [(18)F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ(1) receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [(18)F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [(18)F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ(1) receptors. These findings suggest that [(18)F]6 is a suitable radiotracer for imaging σ(1) receptors with PET in vivo.

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.

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.

The function of gamma-aminobutyric acid type A receptors (GABA(A) receptors) is enhanced by various clinically important drugs including benzodiazepines that act on an allosteric site formed at the interface between the alpha and gamma subunits. In contrast to classical benzodiazepines, the novel pyrazolopyrimidine indiplon (N-methyl-N-{3-[7-(thiophene-2-carbonyl)-1,5,9-triazabicyclo[4.3.0]nona-2,4,6,8-tetraen-2-yl]phenyl}acetamide; N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide) demonstrates relative binding selectivity for the alpha1 subunit containing receptor subtypes, which are the most frequently expressed in the mammalian central nervous system. To investigate the pharmacological properties at GABA(A) receptors and to promote the development of alpha1 subunit selective radiotracers for positron emission tomography imaging, we have started with the evaluation of various fluorinated indiplon derivatives. Binding affinities were determined in homogenates from newborn and adult rats suggesting an alpha1 preference of the reference compounds indiplon, zaleplon as well as for all newly synthesized indiplon derivatives. In homogenated cerebellar tissue obtained from adult rat brain, known to primarily express alpha1 containing GABA(A) receptors, the high affinity of the basic indiplon structure was only slightly affected by an elongation of the alkyl substituent of the amide N from methyl (indiplon; K(i) 3.1 nM) via ethyl (2a, N-(2-fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 5.4 nM) to propyl (2b, N-(3-fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 2.4 nM). Whole cell patch-clamp recordings at neuronal and recombinant GABA(A) receptors indicated that the fluorinated derivatives 2a and 2b have a high potency at alpha1beta3gamma2L isoforms comparable to indiplon (EC(50): 105, 158, and 81 nM, respectively), with 2b displaying the most pronounced efficacy at alpha3beta3gamma2L subtypes. In conclusion, the affinity profiles and functional properties of the newly synthesised fluorinated indiplon derivatives make compounds 2a and 2b suitable for the development of [(18)F]-labelled ligands at GABA(A) receptors containing the alpha1 subunit.

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 [ 18 F]fluoroacetyl-substituted derivative ([ 18 F]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/μmol. In competitive binding assays using a PC12 cell line overexpressing VAChT and [ 3 H]-(−) 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 ( K i >1500 nM). The compound showed a moderate lipophilicity (logD (pH 7) =1.9) and a plasma protein binding of 49%. The brain uptake of [ 18 F]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 [ 18 F]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 [ 18 F]FAMV in the plasma was shown to be metabolized to various hydrophilic compounds, as detected by high-performance liquid chromatography. The degradation of [ 18 F]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 [ 18 F]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.

The involvement of gamma amino butyric acid (GABA) receptors in a variety of neurological and psychiatric diseases has promoted the development and use of radiolabelled benzodiazepines (BZ) for brain imaging by PET. However, these radioligands are unable to distinguish between the various subtypes of GABAA receptors. Novel non-BZ such as the pyrazolo-pyrimidine indiplon proved to be selective for the α1-subunit of the GABAA receptor. Here, we describe the syntheses of four novel 18F-labelled indiplon derivatives. Radiosyntheses were performed via n.c.a. 18F-nucleophilic substitution starting from the tosyl, bromo, and 4-nitrobenzoyl precursors to obtain fluorine substituted N-alkylamide side chain derivatives of indiplon, followed by multistep purification using semi-preparative high-performance liquid chromatography and solid phase extraction. Tosyl and bromo precursors were converted into 18F-labelled indiplon derivatives with good and reproducible radiochemical yield (RCY) (35–70%, decay corrected), high radiochemical purity (≥98.5%), and high specific activity ( > 150 GBq/µmol). By contrast, a low RCY (5–10%) and specific activity (10–15 GBq/µmol) were achieved for the 4-nitrobenzoyl precursor. Copyright © 2008 John Wiley & Sons, Ltd.

A series of fluorinated diphenylchalcogen derivatives, possessing a sulfur or an oxygen bridge, has been prepared with the aim to get a suitable radiotracer to image the SERT in vivo using positron emission tomography (PET). The compounds were synthesized and assayed toward the serotonin (SERT), dopamine (DAT), and norepinephrine (NET) transporters. Among the developed series, five compounds display a high SERT affinity (Ki: 0.27–2.91 nM range) and can be labeled either with carbon-11 or fluorine-18.

The cholinergic system is highly vulnerable to traumatic brain injury (TBI). However, limited information is available to what extent the degrading enzyme acetylcholinesterase (AChE) is involved. The present study addresses this question.Thirty-six anaesthetized Sprague-Dawley rats were subjected to sham operation or to TBI using controlled cortical impact (CCI). The AChE activity was histochemically determined in frozen brain slices at 2, 24 and 72 hours after TBI.High enzyme activity was observed in regions rich in cholinergic innervation such as the olfactory tubercle, basal forebrain, putamen and superior colliculi. Low activity was found in the cortex, cerebellum and particularly in the white matter. A decrease of AchE activity (20-35%) was found in the hippocampus and hypothalamus already at 2 hours after TBI. An increase of approximately 30% was found in the basal forebrain at 2 and 24 hours. No changes occurred at 72 hours.The findings are consistent with impairment of the cholinergic neurotransmission after TBI and suggest the involvement of the AChE in short-term regulatory mechanisms.

Radiopharmacological investigations of [(18)F]NS14490 have proven that this radiotracer could be a potential PET radiotracer for imaging of alpha7 nicotinic acetylcholine receptor particularly with regard to vulnerable plaques of diseased vessels. For further optimisation of the previously automated one-pot radiosynthesis of [(18)F]NS14490 using a tosylate precursor, precursors with other leaving groups (nosylate and mosylate) were synthesized and compared with the tosylate with respect to their reactivities towards [(18)F]fluoride. The use of these different precursors resulted in comparable labelling yields of [(18)F]NS14490. A novel mosylate precursor was synthesized and evaluated, which has revealed a higher stability during a storage period of five months compared to the corresponding tosylate and nosylate.

A novel 99mTc labeled complex, [N-[2-((2-oxo-2-(4-(3-phenylpropyl)piperazin-1-yl)ethyl) (2-mercaptoethyl)amino)acetyl]-2-aminoethanethiolato]Technetium(V) oxide (PPPE-MAMA’-99mTcO) ([99mTc]-2) has been designed and prepared based on the integrated approach. The corresponding rhenium complex (PPPE-MAMA’-ReO)(Re-2) has been prepared and characterized. In vitro competition binding assays show moderate affinity of Re-2 towards σ1 and σ2 receptors with Ki values of 8.67 ± 0.07 and 5.71 ± 1.88 μmol, respectively. Planar images obtained at 0.5 h, 4 h, 20 h after i.v. injection indicate the accumulation of [99mTc]-2 in MCF-7 human breast tumor bearing mice at 20 h. Furthermore, the accumulation of [99mTc]-2 has been inhibited at 20 h after co-injection of [99mTc]-2 plus haloperidol (1 mg/kg). Biodistribution studies of [99mTc]-2 display an in vivo tumor uptake of 0.14% ± 0.01% ID/g at 24 h post i.v. injection with a tumor/muscle ratio of 6.02 ± 0.87. The above results suggest that [99mTc]-2, derived from a previously published lead compound, retains certain tumor uptake and affinity for σ receptors. [99mTc]-2 may be used as a basis for further structural modifications to develop tumor imaging agents with high affinity for σ receptors.

The radiolabeled serotonin transporter (SERT) ligand [(11)C](+)-McN5652 has recently been used in clinical positron emission tomography (PET) studies for SERT imaging. However, this radioligand offers disadvantages in routine clinical settings because of its short radioisotope half-life (eg PET facilities within hospitals without a cyclotron need to acquire such radioligands from distant cyclotron units for clinical use). S-([(18)F]fluoromethyl)-(+)-McN5652 ([(18)F](+)-FMe-McN5652) is an analogue which has been synthesized newly, and has a significantly longer radioisotope half-life. In the porcine brain, it demonstrates the same characteristic distribution pattern of serotonin-uptake sites like the (11)C-labeled congener with the highest binding in the midbrain and thalamus and the lowest in the cerebellum and occipital cortex. It shows a 30% higher blood-brain transfer and a slower peripheral metabolism than [(11)C](+)-McN5652. Rather uniform brain binding was observed after injection of the pharmacologically inactive radiolabeled enantiomer, or after pretreatment with the highly selective SERT inhibitor citalopram. The norepinephrine uptake inhibitor maprotiline did not show any inhibitory effect. Using a one-tissue compartment model (K(1), k"(2)) or a two-tissue compartment model (K(1) to k(4)) with or without constraints for calculation, the regional binding parameters of [(11)C](+)-McN5652 and [(18)F](+)-FMe-McN5652 are highly correlated among each other and with the SERT density, as determined by in vitro binding of [(3)H]citalopram. Using constraints to correct for the free fraction and nonspecific binding of the radiotracers, a considerable increase of the midbrain-occipital cortex ratios with higher values for [(18)F](+)-FMe-McN5652 compared to [(11)C](+)McN5652 was revealed. It is concluded that [(18)F](+)-FMe-McN5652 has better features than [(11)C](+)McN5652 for SERT imaging with PET.

The [18F]fluoromethyl analog of (+)-McN5652 ([18F]FMe-McN) for imaging serotonin transporter (SERT) with positron emission tomography (PET) has recently been synthesized. We describe here the biological evaluation of [18F]FMe-McN in rats. Biodistribution studies of [18F]FMe-McN in rat brain ex vivo after an intravenous injection showed a high accumulation of radioactivity in the regions rich in SERT, such as raphe nuclei, hypothalamus, thalamus, substantia nigra, locus coeruleus, and amygdala. Region-to-cerebellum ratios reached a maximum value of 9 in raphe nuclei within 3.5 h after administration. The specificity and selectivity of [18F]FMe-McN binding to SERT was studied by preinjecting blocking doses of serotonin, norepinephrine, and dopamine transporter inhibitors. Fluoxetine, a specific inhibitor for SERT, decreased the specific binding of [18F]FMe-McN in raphe nuclei by 91 ± 4%; in other regions rich in SERT, similar results were obtained. GBR12909 and nisoxetine, selective inhibitors for dopamine transporter (DAT) and norepinephrine transporter (NET), respectively, showed no significant effects on the uptake of [18F]FMe-McN. Our studies show that [18F]FMe-McN has a clear potential as a tracer for studies with PET of SERT function in humans. Synapse 47:45–53, 2003. © 2002 Wiley-Liss, Inc.

There is evidence that the dopaminergic system is sensitive to altered p(O(2)) in the immature brain. However, the respective enzyme activities have not been measured in the living neonatal brain together with brain oxidative metabolism. Therefore 18F-labelled 6-fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) together with positron emission tomography (PET) was used to estimate the activity of the aromatic amino acid decarboxylase (AADC) in the brain of fifteen newborn piglets (2-5 days old). Two PET scans were performed in each piglet. Eleven animals underwent a period of normoxia and moderate hypoxia/hypercapnia (H/H). The remaining four animals were used as an untreated control group. Simultaneously, the brain tissue p(O(2)) was recorded, the regional cerebral blood flow (CBF) was measured with colored microspheres and the cerebral metabolic rate of oxygen (CMRO(2)) was determined. In addition, in four untreated and six H/H treated piglets the relative amounts of fluorodopamine and the respective metabolites were determined in brain tissue samples using HPLC analysis. H/H conditions were induced by lowering the inspired fraction of oxygen from 0.35 to 0.10 and adding CO(2) to the inspired gas resulting in an arterial p(CO(2)) between 74 and 79 mmHg. H/H elicited a more than 3-fold increase of the CBF (P

An improved synthesis of the anantiomerically pure thioester precursors of [11C](+)-McN-5652 ([11C](+)−1]), and [11C](−)-McN5652 ([11C](−)−1]) starting from racemic McN-5652 ((±)−1) is described. The synthetic method includes the resolution of (±)−1 by repeated crystallization of the (+)- and (−)-di-p-toluoyltartrates yielding (+)-McN-5652 ((+)−1) and (−)-McN-5652 ((−)-1), each with >98% enantiomeric purity. S-Demethylation of (±)−1, (+)−1, and (−)-1, respectively was achieved by treatment with sodium amide at low temperatures (−78°C) followed by conversion of the intermediate thiols 2 with acetyl chloride to give the corresponding thioester precursors (±)−3, (+)−3 or (−)−3. This demethylation method almost completely suppressed the isomerization of the pharmacologically active trans diastereomer into the inactive cis form. Chiral HPLC analyses confirmed that the S-demethylation proceeded without any racemization. 11C-Labelling of (+)−3 or (−)−3, yields enantiomerically pure [11C](+)-McN-5652 or [11C](−)-McN5652, each in 22% radiochemical yield (decay-corrected, related to [11C]CO2) and a specific radioactivity of 74 GBq/μmol (2 Ci/μmol) at the end of synthesis (EOS) Copyright © 1999 John Wiley & Sons, Ltd.

The present study investigated the effect of cytotoxic hypoxia on cerebral and non-cerebral endothelial cells. Hypoxia was induced by inhibiting the cellular respiratory chain with 1 mM sodium cyanide. Cerebral endothelial cells were damaged after 2 h of hypoxia as assessed by a decrease in cell viability by 25% and by a 2.7-fold higher lactate dehydrogenase release compared to controls. Additional glucose deprivation did not significantly exacerbate hypoxic injury. In addition, we found after 2 h of hypoxia an increase in the release of lactate of 1.02 and 0.42 mg/mg protein compared to 0.27 and 0.07 mg/mg protein in controls in the presence and absence of glucose, respectively. While the activity of ALP of cerebral endothelial cells was maintained at the control level, we found a significant decrease in the gamma-GT activity from 3.8 +/- 1.3 to 1.09 +/- 0.3 U/mg protein after 3 h of hypoxia in the presence as well as in the absence of glucose. The paracellular permeability of the cell monolayer decreased after 1 h and returned to control level after 3 h of hypoxia in the presence of glucose. Non-cerebral endothelial cells remained 98% viable with no change in the release of lactate dehydrogenase and lactate after 2 h of hypoxia in the presence and absence of glucose. The activities of ALP and gamma-GT in non-cerebral endothelial cells were 10 and 3 times lower and remained unchanged during hypoxia. We conclude from our experiments that sodium cyanide is useful to study hypoxic injury and that cerebral endothelial cells are more sensitive than non-cerebral endothelial cells to cytotoxic hypoxia.