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Six indole-based derivatives with a methoxy group at the indole ring were synthesized and evaluated as σ2 receptor ligands with nanomolar affinity (Ki (σ2) = 4.40-9.46 nM) and moderate subtype selectivity (Ki(σ2)/Ki(σ1) = 7-102). Radioligands 1-(4-(5,6-dimethoxyisoindolin-2-yl)butyl)-4-(2-[18F]fluoroethoxy)-1H-indole ([18F]3) and 1-(4-(5,6-dimethoxyisoindolin-2-yl)butyl)-5-(2-[18F]fluoroethoxy)-1H-indole ([18F]4) with high σ2 receptor affinity and subtype selectivity were synthesized through a direct SN2 displacement reaction, with radiochemical yields of 36-50% and 20-29%, radiochemical purity of >99%, and molar activities of 29-151 GBq/μmol and 55-72 GBq/μmol, respectively. Radioligand [18F]3 displayed high brain uptake, high brain-to-blood ratio and slow washout from the brain in male ICR mice. Administration of compound CM398 5 min prior to the radiotracer injection led to a significantly dose-dependent reduction of the brain accumulation (29-54%) and the brain-to-blood ratio (60-88%) at 30 min, indicating high specific binding of [18F]3 to the σ2 receptors in the brain. Ex vivo autoradiography in male ICR mice showed widely and heterogeneous distribution of [18F]3 in the brain. Small animal positron emission tomography imaging in rats confirmed different distribution and high specific binding of [18F]3 to σ2 receptors in rat brain. These findings warrant [18F]3 as a potential probe used for neuroimaging of the σ2 receptors in the brain.

Sigma (σ) receptors represent attractive targets for the development of potential agents for the treatment of several disorders, including Alzheimer's disease and neuropathic pain. In the search for multitarget small molecules (MSMs) against such disorders, we have re-discovered chromenones as new affine σ1/σ2 ligands. 6-(4-(Piperidin-1-yl)butoxy)-4H-chromen-4-one (7), a previously identified MSM with potent dual-target activities against acetylcholinesterase and monoamine oxidase B, also exhibited σ1/σ2 affinity. 6-(3-(Azepan-1-yl)propoxy)-4H-chromen-4-one (20) showed a Ki value for σ1 of 27.2 nM (selectivity (σ1/σ2) = 28), combining the desired σ1 receptor affinity with a dual inhibitory capacity against both acetyl- and butyrylcholinesterase. 6-((5-Morpholinopentyl)oxy)-4H-chromen-4-one (12) was almost equipotent to S1RA, an established σ1 receptor antagonist.

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.

A2A adenosine receptors (A2A-AR) have a cardio-protective function upon ischemia and reperfusion, but on the other hand, their stimulation could lead to arrhythmias. Our aim was to investigate the potential use of the PET radiotracer [18F]FLUDA to non-invasively determine the A2A-AR availability for diagnosis of the A2AR status. Therefore, we compared mice with cardiomyocyte-specific overexpression of the human A2A-AR (A2A-AR TG) with the respective wild type (WT). We determined: (1) the functional impact of the selective A2AR ligand FLUDA on the contractile function of atrial mouse samples, (2) the binding parameters (Bmax and KD) of [18F]FLUDA on mouse and human atrial tissue samples by autoradiographic studies, and (3) investigated the in vivo uptake of the radiotracer by dynamic PET imaging in A2A-AR TG and WT. After A2A-AR stimulation by the A2A-AR agonist CGS 21680 in isolated atrial preparations, antagonistic effects of FLUDA were found in A2A-AR-TG animals but not in WT. Radiolabelled [18F]FLUDA exhibited a KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein in cardiac samples of A2A-AR TG, whereas in WT, as well as in human atrial preparations, only low specific binding was found. Dynamic PET studies revealed a significantly higher initial uptake of [18F]FLUDA into the myocardium of A2A-AR TG compared to WT. The hA2A-AR-specific binding of [18F]FLUDA in vivo was verified by pre-administration of the highly affine A2AAR-specific antagonist istradefylline. Conclusion: [18F]FLUDA is a promising PET probe for the non-invasive assessment of the A2A-AR as a marker for pathologies linked to an increased A2A-AR density in the heart, as shown in patients with heart failure.

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.

Background The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to update the readership on trends in the field of radiopharmaceutical development. Results This commentary of highlights has resulted in 23 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals and also a contribution in relation to MRI-agents is included. Conclusion Trends in (radio)chemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.

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]

Innovations in radiochemistry and pharmacology are opening new vistas for studies of nicotinergic acetylcholine receptors (nAChRs) in human brain by positron emission tomography (PET) and by single-photon emission computed tomography (SPECT). In parallel, instrumentation optimized for molecular imaging in rodents facilitates preclinical studies in models of human diseases with perturbed nAChR signalling, notably Alzheimer’s disease and other neurodegenerative conditions, schizophrenia and other neuropsychiatric disorders, substance abuse, and traumatic brain injury. The nAChRs are ligand-gated ion channels composed of five subunits forming a central pore for cation flux. The most abundant nAChRs in the central nervous system are heteropentamers (designated α4β2), followed by the α7 homopentamer. We present a systematic review of published findings with the various nAChR ligands using imaging techniques in vivo, emphasizing preclinical models and human studies.. Molecular PET imaging of the α4β2 nAChR subtype with the antagonist 2-[18F]fluoro-A-85380 is hampered by the long acquisition times. Newer agents such as (-)-[18F]flubatine, [18F]XTRA or [18F]nifene permit quantitation of α4β2 receptors with PET recordings lasting 90 minutes or less, and without the toxicity risk of earlier epibatidine derivatives. The early PET studies of α7 nAChRs suffered from low pharmacological specificity, further hampered by low natural abundance of the receptor. However, several good α7 nAChR ligands such as [18F]ASEM and [18F]DBT10 have emerged in the past few years. There are still no ligands selective for α6-containing nAChRs, despite their importance for nicotine-induced dopamine release in striatum. Selective α3β4 nAChR radioligands are under development, but remain untested in clinical studies of depression and addiction. Several nAChR ligands find use for pharmacological occupancy studies, and competition from endogenous acetylcholine reduces α4β2 binding site availability, a property that enables monitoring by PET of acetylcholine release in living brain.

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.

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.

Objective: Recently, we developed [18F]FACH as the first radiolabeled inhibitor of MCTs for potential tumor imaging (1). Encouraged by the very promising results in mice, showing the specific binding/transport of [18F]FACH in kidneys combined with high in vivo stability, herein we report on the biological evaluation of this radiotracer in piglets. Methods: Biological evaluation was performed in 6 piglets (Landrace, 19.9±3.0 kg). The blocking experiments (n=3) were conducted using sodium α-cyano-4-hydroxycinnamate (α-CHC-Na, 25 mg/kg) administered i.v. 10 min prior to tracer application. The animals were anesthesized (ketamin/midazolam) and scanned by a SIEMENS Biograph mMR PET/MRI-system up to 60 min p.i. of 295±28 MBq [18]FACH via ear vein. The reconstructed list-mode PET-data were analyzed utilizing the PMOD Software. In vivo metabolite analysis was performed using plasma isolated from arterial blood samples (5, 15, 30, 45 and 60 min) and samples of homogenized kidney by semi-preparative radio-HPLC after deproteinization by ACN/H2O (9:1). Results: In contrast to mouse, a rather fast metabolism of the radiotracer was observed in piglet. Five and 30 minutes after injection, the intact tracer was found to represent 50±13% and 12±6% of total plasma activity, respectively. Metabolite analysis revealed 48% of intact tracer in kidney cortex at 60 min p.i. Despite the fast metabolism, the PET scan results showed comparable selective kidney uptake of [18F]FACH in piglets as in mice. The blocking experiments revealed a reduction of this uptake to about 72% after pre-injection of α-CHC-Na. Conclusion: The high kidney uptake of [18F]FACH obtained in both mice and piglets together with high inhibition by α-CHC-Na, specific inhibitor of MCT, provide evidence that the new MCT-targeting radiotracer could be proven in ongoing studies to be useful for imaging of MCTs expression with PET.

Objective: MCT1-4 are involved in several diseases, particularly in cancer. [18F]FACH has recently been developed as a novel MCT-targeting imaging agent (1), which could be used for monitoring MCT-based treatment approaches. With the aim to develop a similar potent radiotracer with higher brain permeability for future brain tumor studies, we designed structurally modified analogs of FACH possessing increased lipophilicity. Methods: Two analogs of FACH (I and II) were synthesized by introduction of a 2-fluoropyridinyl moiety via Buchwald-Hartwig cross coupling reaction. Inhibition of MCT1 was measured by [14C]lactate uptake assay using rat brain endothelial cells and analog I with higher inhibition was selected to synthesize corresponding precursor used for radiofluorination by aromatic nucleophilic substitution. LogD7.4 of [18F]I was experimentally determined in the n-octanol-PBS system. In vitro autoradiography and dynamic PET studies of [18F]I were performed in CD-1 mice. Results: The analogs I and II showed a moderate MCT1 inhibition with IC50 values of 118 and 274 nM, respectively. [18F]I was obtained with radiochemical yields of 73±12% (n=4, non-isolated) and a high radiochemical purity of > 98%. A logD7.4 value of 0.816 was achieved for [18F]I, which was 2-fold higher than that for [18F]FACH. By in vitro autoradiography in cryosections of the mouse kidney, nearly complete displacement of [18F]I by 10-5 M CHC-Na was observed. In vivo, similar to [18F]FACH, a low uptake of [18F]I in the brain without significant washout was found with an almost constant SUV of 0.15 between 15 and 60 min p.i. Conclusion: Despite a higher lipophilicity of [18F]I compared to [18F]FACH, the brain uptake of [18F]I was in a similar low range. However, the high and specific uptake of the new radiotracer in the kidneys suggests suitability of [18F]I for detecting MCTs’ expression in vivo. References: (1) Sadeghzadeh M, et al. J Label Compd Radiopharm.2019; 62: 411-424.

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.

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.

We have designed and synthesized a series of indole-based σ2 receptor ligands containing 5,6-dimethoxyisoindoline or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline as pharmacophore. In vitro competition binding assays showed that all ten ligands possessed low nanomolar affinity (Ki=1.79-5.23nM) for σ2 receptors and high subtype selectivity (Ki (σ2)/Ki (σ1)=56-708). Moreover, they showed high selectivity for σ2 receptor over the vesicular acetylcholine transporter (>1000-fold). The corresponding radiotracers [18F]16 and [18F]21 were prepared by an efficient one-pot, two-step reaction sequence with a home-made automated synthesis module, with 10-15% radiochemical yield and radiochemical purity of >99%. Both radiotracers showed high brain uptake and σ2 receptor binding specificity in mice.

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...

The σ1 receptors (S1Rs) are implicated in a variety of diseases including Alzheimer disease and cancer. Previous PET S1R radiotracers are characterized by slow kinetics or off-target binding that impedes their use in humans. Here, we report the first PET imaging evaluation in rhesus monkeys of 4 18F-labeled spirocyclic piperidine-based PET radiotracers (18F-1 to 18F-4). Methods: Baseline scans for the 4 radiotracers were obtained on an adult male rhesus monkey. Blocking scans were obtained with the S1R-selective agonist SA4503 to assess binding specificity of 18F-2 and 18F-4 Arterial input functions were measured, and binding parameters were determined with kinetic modeling analysis. Results: In the rhesus brain, all 4 radiotracers showed high and fast uptake. Tissue activity washout was rapid for 18F-2 and 18F-4, and much slower for 18F-1 and 18F-3, in line with their respective in vitro S1R-binding affinities. Both the 1-tissue-compartment and multilinear analysis-1 kinetic models provided good fits of time-activity curves and reliable estimates of distribution volume. Regional distribution volume values were highest in the cingulate cortex and lowest in the thalamus for all radiotracers. 18F-4 showed greater differential uptake across brain regions and 3-fold-higher binding potential than 18F-2 SA4503 at the dose of 0.5 mg/kg blocked approximately 85% (18F-2) and 95% (18F-4) of radiotracer binding. Conclusion: Tracers 18F-2 and 18F-4 displayed high brain uptake and fast tissue kinetics, with 18F-4 having higher specific binding signals than 18F-2 in the same monkey. Taken together, these data indicate that both 18F-2 and 18F-4 possess the requisite kinetic and imaging properties as viable PET tracers for imaging S1R in the human brain.

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 have designed and synthesized a series of cyclopentadienyl tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ2 receptor ligands. Rhenium compound 20a possessed low nanomolar σ2 receptor affinity (K(i) = 2.97 nM) and moderate subtype selectivity (10-fold). Moreover, it showed high selectivity toward vesicular acetylcholine transporter (2374-fold), dopamine D2L receptor, NMDA receptor, opiate receptor, dopamine transporter, norepinephrine transporter, and serotonin transporter. Its corresponding radiotracer [(99m)Tc]20b showed high uptake in a time- and dose-dependent manner in DU145 prostate cells and C6 glioma cells. In addition, this tracer exhibited high tumor uptake (5.92% ID/g at 240 min) and high tumor/blood and tumor/muscle ratios (21 and 16 at 240 min, respectively) as well as specific binding to σ receptors in nude mice bearing C6 glioma xenografts. Small animal SPECT/CT imaging of [(99m)Tc]20b in the C6 glioma xenograft model demonstrated a clear visualization of the tumor at 180 min after injection.

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.

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.

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.

Contilisant, a permeable, antioxidant, and neuroprotectant agent, showing high nM affinity at H3R and excellent inhibition of the monoamine oxidases and cholinesterases, is an affine and selective S1R agonist in the nanomolar range, based on the binding affinity and functional experiment, a result confirmed by molecular modeling. In addition, contilisant significantly restores the cognitive deficit induced by Aβ in the radial maze assay in an in vivo Alzheimer's disease test, comparing very favorably with donepezil., J.M.-C. thanks MINECO (Grant SAF2015-65586-R), UCJC (Grants 2015-12, 2014-35, and 2015-21), and EU (COST Action CA15135) for support. J.M.-C. thanks Prof. Stark and Prof. Ramsay, as well as Prof. Jia (Beijing Normal University), Dr. Chioua (CSIC), and Dr. Romero (UCM) for their help and support.

Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ1 receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K(i) = 5.4 ± 0.4 nM) for σ1 receptors and selectivity for σ2 receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [(18)F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/μmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [(18)F]5a to σ1 receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ1 receptors could be used for tumor imaging.

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 report the synthesis and biological evaluation of a series of indole-based σ2 receptor ligands derived from siramesine. In vitro competition binding assays showed that these analogues possessed high to moderate affinity and selectivity for σ2 receptors. Structure–affinity relationship analyses of these indole-based σ2 receptor ligands were performed. In the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 1a and 1b displayed significant and comparable antiproliferative activity in DU145, MCF7 and C6 cells to siramesine. In cell cycle analyses, compounds 1a, 1b and siramesine were found to induce a G1 phase cell cycle arrest in DU145 cells using flow cytometry. The combination of 5,6-dimethoxyisoindoline scaffold and N-(4-fluorophenyl)indole moiety was identified as a new σ2 receptor ligand deserving further investigation as an antitumor agent.

[18F]NS10743, a promising and highly competitive α7 nAChR radioligand has been synthesized so far by microwave irradiation using a manual single-mode device followed by a palladium-catalyzed reduction of remaining nitro-precursor for HPLC separation purposes. For further preclinical and clinical use, regulated production of [18F]NS10743 by fully automated radiosynthesis is a crucial requirement. Therefore, we chose a commercial synthesis module and developed the automated radiosynthesis of [18F]NS10743. Besides evaluation of several radiosynthesis procedures, we performed an extensive HPLC study for quantitative separation of [18F]NS10743 from the corresponding nitro precursor. After implementation of the optimized procedure on a TRACERlabTM FX F-N synthesis module, [18F]NS10743 was obtained in high radiochemical purity (≥99%) with an overall radiochemical yield of 32.2±7% (n=3). The specific activities at the end of the synthesis were 571±17 GBq/µmol (n=3).

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.

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.

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.

Introduction (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Methods Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound+metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro , tracer distribution between these blood components was assessed for up to 90min. Results A fraction of 15%±2% of (−)-[ 18 F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[ 18 F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82±0.03 at 3min p.i. to 0.87±0.03 at 270min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[ 18 F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. Discussion (−)-[ 18 F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.

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

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.

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).