PET Ligands for Imaging Mutant Huntingtin Aggregates: A Case Study in Non-For-Profit Scientific Management.

Positron emission tomography imaging of misfolded proteins with high-affinity and selective radioligands has played a vital role in expanding our knowledge of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The pathogenesis of Huntington's disease, a CAG trinucleotide repeat disorder, is similarly linked to the presence of protein fibrils formed from mutant huntingtin (mHTT) protein. Development of mHTT fibril-specific radioligands has been limited by the lack of structural knowledge around mHTT and a dearth of available hit compounds for medicinal chemistry refinement. Over the past decade, the CHDI Foundation, a non-for-profit scientific management organisation has orchestrated a large-scale screen of small molecules to identify high affinity ligands of mHTT, with lead compounds now reaching clinical maturity. Here we describe the mHTT radioligands developed to date and opportunities for further improvement of this radiotracer class.

Synaptic Loss in Frontotemporal Dementia Revealed by [11 C]UCB-J Positron Emission Tomography.

OBJECTIVE: Synaptic loss is an early feature of neurodegenerative disease models, and is severe in post mortem clinical studies, including frontotemporal dementia. Positron emission tomography (PET) with radiotracers that bind to synaptic vesicle glycoprotein 2A enables quantification of synaptic density in vivo. This study used [11 C]UCB-J PET in participants with behavioral variant frontotemporal dementia (bvFTD), testing the hypothesis that synaptic loss is severe and related to clinical severity. METHODS: Eleven participants with clinically probable bvFTD and 25 age- and sex-matched healthy controls were included. Participants underwent dynamic [11 C]UCB-J PET, structural magnetic resonance imaging, and a neuropsychological battery, including the revised Addenbrooke Cognitive Examination, and INECO frontal screening. General linear models compared [11 C]UCB-J binding potential maps and gray matter volume between groups, and assessed associations between synaptic density and clinical severity in patients. Analyses were also performed using partial volume corrected [11 C]UCB-J binding potential from regions of interest (ROIs). RESULTS: Patients with bvFTD showed severe synaptic loss compared to controls. [11 C]UCB-J binding was reduced bilaterally in medial and dorsolateral frontal regions, inferior frontal gyri, anterior and posterior cingulate gyrus, insular cortex, and medial temporal lobe. Synaptic loss in the frontal and cingulate regions correlated significantly with cognitive impairments. Synaptic loss was more severe than atrophy. Results from ROI-based analyses mirrored the voxelwise results. INTERPRETATION: In accordance with preclinical models, and human postmortem evidence, there is widespread frontotemporal loss of synapses in symptomatic bvFTD, in proportion to severity. [11 C]UCB-J PET could support translational studies and experimental medicine strategies for new disease-modifying treatments for neurodegeneration. ANN NEUROL 2023;93:142-154.

The inverse electron demand Diels-Alder cycloaddition with carbon-11 and fluorine-18: A gateway to pretargeted imaging across the blood-brain barrier.

There is increased focus on developing tools to image large biomolecules, such as antibodies, within the brain using positron emission tomography (PET). The inverse electron demand Diels-Alder cycloaddition (IEDDA) reaction has offered the greatest prospect of achieving such a feat and has gained much interest over the past decade. The fast reaction kinetics of the IEDDA reaction opens up the possibility of utilising a pretargeted approach, whereby the subject is pretreated with a biomolecule that has high specificity for its target. A radiolabelled second component is then administered to the subject, enabling the biomolecule to be visualised by PET. However, for this to become common practice, there is a need for the development of either radiolabelled trans-cyclooctenes (TCOs) or tetrazines that can cross the blood-brain barrier (BBB). This review highlights the advancements in the development of both radiolabelled TCOs and tetrazines, which have been radiolabelled with either carbon-11 or fluorine-18 and show promise or have been evaluated for use in pretargeted PET imaging across the BBB.

Validation of a combined image derived input function and venous sampling approach for the quantification of [18F]GE-179 PET binding in the brain.

Blood-based kinetic analysis of PET data relies on an accurate estimate of the arterial plasma input function (PIF). An alternative to invasive measurements from arterial sampling is an image-derived input function (IDIF). However, an IDIF provides the whole blood radioactivity concentration, rather than the required free tracer radioactivity concentration in plasma. To estimate the tracer PIF, we corrected an IDIF from the carotid artery with estimates of plasma parent fraction (PF) and plasma-to-whole blood (PWB) ratio obtained from five venous samples. We compared the combined IDIF+venous approach to gold standard data from arterial sampling in 10 healthy volunteers undergoing [18F]GE-179 brain PET imaging of the NMDA receptor. Arterial and venous PF and PWB ratio estimates determined from 7 patients with traumatic brain injury (TBI) were also compared to assess the potential effect of medication. There was high agreement between areas under the curves of the estimates of PF (r = 0.99, p<0.001), PWB ratio (r = 0.93, p<0.001), and the PIF (r = 0.92, p<0.001) as well as total distribution volume (VT) in 11 regions across the brain (r = 0.95, p<0.001). IDIF+venous VT had a mean bias of -1.7% and a comparable regional coefficient of variation (arterial: 21.3 ± 2.5%, IDIF+venous: 21.5 ± 2.0%). Simplification of the IDIF+venous method to use only one venous sample provided less accurate VT estimates (mean bias 9.9%; r = 0.71, p<0.001). A version of the method that avoids the need for blood sampling by combining the IDIF with population-based PF and PWB ratio estimates systematically underestimated VT (mean bias -20.9%), and produced VT estimates with a poor correlation to those obtained using arterial data (r = 0.45, p<0.001). Arterial and venous blood data from 7 TBI patients showed high correlations for PF (r = 0.92, p = 0.003) and PWB ratio (r = 0.93, p = 0.003). In conclusion, the IDIF+venous method with five venous samples provides a viable alternative to arterial sampling for quantification of [18F]GE-179 VT.

Synaptic Loss in Primary Tauopathies Revealed by [11 C]UCB-J Positron Emission Tomography.

BACKGROUND: Synaptic loss is a prominent and early feature of many neurodegenerative diseases. OBJECTIVES: We tested the hypothesis that synaptic density is reduced in the primary tauopathies of progressive supranuclear palsy (PSP) (Richardson's syndrome) and amyloid-negative corticobasal syndrome (CBS). METHODS: Forty-four participants (15 CBS, 14 PSP, and 15 age-/sex-/education-matched controls) underwent PET with the radioligand [11 C]UCB-J, which binds to synaptic vesicle glycoprotein 2A, a marker of synaptic density; participants also had 3 Tesla MRI and clinical and neuropsychological assessment. RESULTS: Nine CBS patients had negative amyloid biomarkers determined by [11 C]PiB PET and hence were deemed likely to have corticobasal degeneration (CBD). Patients with PSP-Richardson's syndrome and amyloid-negative CBS were impaired in executive, memory, and visuospatial tasks. [11 C]UCB-J binding was reduced across frontal, temporal, parietal, and occipital lobes, cingulate, hippocampus, insula, amygdala, and subcortical structures in both PSP and CBD patients compared to controls (P < 0.01), with median reductions up to 50%, consistent with postmortem data. Reductions of 20% to 30% were widespread even in areas of the brain with minimal atrophy. There was a negative correlation between global [11 C]UCB-J binding and the PSP and CBD rating scales (R = -0.61, P < 0.002; R = -0.72, P < 0.001, respectively) and a positive correlation with the revised Addenbrooke's Cognitive Examination (R = 0.52; P = 0.01). CONCLUSIONS: We confirm severe synaptic loss in PSP and CBD in proportion to disease severity, providing critical insight into the pathophysiology of primary degenerative tauopathies. [11 C]UCB-J may facilitate treatment strategies for disease-modification, synaptic maintenance, or restoration. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Automated radiosynthesis of [11 C]UCB-J for imaging synaptic density by positron emission tomography.

An automated radiosynthesis of carbon-11 positron emission tomography radiotracer [11 C]UCB-J for imaging the synaptic density biomarker synaptic vesicle glycoprotein SV2A was established using Synthra RNPlus synthesizer. Commercially available trifluoroborate UCB-J analogue was used as a radiolabelling precursor, and the desired radiolabelled product was isolated in 11 ± 2% (n = 7) nondecay corrected radiochemical yield and formulated as a 10% EtOH solution in saline with molar activities of 20 to 100 GBq/µmol. The method was based upon the palladium(0)-mediated Suzuki cross-coupling reaction and [11 C]CH3 I as a radiolabelling synthon. The isolated product was cGMP compliant as demonstrated by the results of quality control analysis.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline.

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24's activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz' activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

Catalytic Nucleophilic Allylation Driven by the Water-Gas Shift Reaction.

The ruthenium-catalyzed allylation of aldehydes with allylic pro-nucleophiles has been demonstrated to be an efficient means to form carbon-carbon bonds under mild conditions. The evolution of this reaction from the initial serendipitous discovery to its general synthetic scope is detailed, highlighting the roles of water, CO, and amine in the generation of a more complete catalytic cycle. The use of unsymmetrical allylic pro-nucleophiles was shown to give preferential product formation through the modulation of reaction conditions. Both (E)-cinnamyl acetate and vinyl oxirane were efficiently used to form the anti-branched products (up to >20:1 anti/syn) and E-linear products (up to >20:1 E/Z) in high selectivity with aromatic, α,ß-unsaturated, and aliphatic aldehydes, respectively. Attempts to render the reaction enantioselective are highlighted and include enantioenrichment of up to 75:25 for benzaldehyde.

Quantitative positron emission tomography of mGluR5 in rat brain with [(18) F]PSS232 at minimal invasiveness and reduced model complexity.

Imaging the density of metabotropic glutamate receptor 5 (mGluR5) in brain by positron emission tomography (PET) is of interest in relation to several brain disorders. We have recently introduced [(18) F]PSS232, an F-18-labeled analog of the mGluR5-targeting [(11) C]ABP688. Quantitative PET requires kinetic modeling with an input function (IF) or an appropriate reference tissue model. We aimed at minimizing invasiveness of IF recording in rat and employing this protocol for mGluR5 quantitative PET with [(18) F]PSS232. We further aimed at defining models of low complexity for quantitative PET with [(18) F]PSS232. The IF was recorded in an arterio-venous shunt applied by minimally invasive cannulation. PET data were analyzed with a modified two-tissue compartment model including a single variable for radiometabolite correction in brain. We further evaluated a simple reference tissue model. Receptor-dependent accumulation was similar to [(11) C]ABP688 at lower unspecific accumulation of unchanged [(18) F]PSS232, in agreement with its higher plasma protein binding and lower lipophilicity. The minimally invasive protocol revealed similar results as the invasive shunt method and parameters calculated with the modified two-tissue compartment model were similar to those calculated with the standard model. The simple area under the curve ratios agreed with the Logan reference method. [(18) F]PSS232 is a promising radioligand for mGluR5 quantification. Methods were evaluated to quantify mGluR5 in rat brain by PET with [(18) F]PSS232. We present a minimally invasive protocol for input function recording. A two-tissue compartment model correcting for radiometabolites at reduced complexity is compared with the standard model. Finally, we demonstrate and explain why for [(18) F]PSS232 the area-under-the-curve ratio is a valid alternative to the Logan reference tissue analysis.

Preclinical evaluation and test-retest studies of [(18)F]PSS232, a novel radioligand for targeting metabotropic glutamate receptor 5 (mGlu5).

PURPOSE: A novel, (18)F-labelled metabotropic glutamate receptor subtype 5 (mGlu5) derivative of [(11)C]ABP688 ([(11)C]1), [(18)F]PSS232 ([(18)F] ]5), was evaluated in vitro and in vivo for its potential as a PET agent and was used in test-retest reliability studies METHODS: The radiosynthesis of [(18)F]5 was accomplished via a one-step reaction using a mesylate precursor. In vitro stability was determined in PBS and plasma, and with liver microsomal enzymes. Metabolite studies were performed using rat brain extracts, blood and urine. In vitro autoradiography was performed on horizontal slices of rat brain using 1 and 8, antagonists for mGlu5 and mGlu1, respectively. Small-animal PET, biodistribution, and test-retest studies were performed in Wistar rats. In vivo, dose-dependent displacement studies were performed using 6 and blocking studies with 7. RESULTS: [(18)F]5 was obtained in decay-corrected maximal radiochemical yield of 37 % with a specific activity of 80 - 400 GBq/µmol. Treatment with rat and human microsomal enzymes in vitro for 60 min resulted in 20 % and 4 % of hydrophilic radiometabolites, respectively. No hydrophilic decomposition products or radiometabolites were found in PBS or plasma. In vitro autoradiography on rat brain slices showed a heterogeneous distribution consistent with the known distribution of mGlu5 with high binding to hippocampal and cortical regions, and negligible radioactivity in the cerebellum. Similar distribution of radioactivity was found in PET images. Under displacement conditions with 6, reduced [(18)F]5 binding was found in all brain regions except the cerebellum. 7 reduced binding in the striatum by 84 % on average. Test-retest studies were reproducible with a variability ranging from 6.8 % to 8.2 %. An extended single-dose toxicity study in Wistar rats showed no compound-related adverse effects. CONCLUSION: The new mGlu5 radiotracer, [(18)F]5, showed specific and selective in vitro and in vivo properties and is a promising radioligand for PET imaging of mGlu5 in humans.

Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.

Preparation and structural analysis of (±)-threo-ritalinic acid.

Hydrolysis of the methyl ester (±)-threo-methyl phenidate afforded the free acid in 40% yield, viz. (±)-threo-ritalinic acid, C13H17NO2. Hydrolysis and subsequent crystallization were accomplished at pH values between 5 and 7 to yield colourless prisms which were analysed by X-ray crystallography. Crystals of (±)-threo-ritalinic acid belong to the P21/n space group and form intermolecular hydrogen bonds. An antiperiplanar disposition of the H atoms of the (HOOC-)CH-CHpy group (py is pyridine) was found in both the solid (diffraction analysis) and solution state (NMR analysis). It was also determined that (±)-threo-ritalinic acid conforms to the minimization of negative gauche(+)-gauche(-) interactions.

Development of [(18)F]-PSS223 as a PET tracer for imaging of metabotropic glutamate receptor subtype 5 (mGluR5).

Involvement of metabotropic glutamate receptor subtype 5 (mGluR5) in physiological and pathophysiological processes in the brain has been demonstrated, and hence mGluR5 has emerged as an important drug target. [(11)C]-ABP688 is clinically the most successful mGluR5 positron emission tomography (PET) tracer to date and it allows visualization and quantification of mGluR5. Due to the short half-life of carbon-11, clinical use of [(11)C]-ABP688 is limited to facilities with an on-site cyclotron and a fluorine-18 (half-life 110 min) analogue would be more practical. Based on the [(11)C]-ABP688 structural motif, a novel derivative [(18)F]-PSS223 was prepared and evaluated as a PET tracer for imaging of mGluR5 in vitro and in vivo. Our results show favourable in vitro binding properties; however rapid defluorination of [(18)F]-PSS223 does not allow visualization of mGluR5 in the rat brain.

Automated radiosynthesis and preclinical in vivo evaluation of [18F]Fluoroethylpuromycin as a potential radiotracer for imaging protein synthesis with PET.

PURPOSE: From a series of fluorinated analogues of puromycin, we recently identified [18F]fluoroethylpuromycin (FEPURO) as a potential candidate for imaging the rate of protein synthesis in vivo. Herein, we describe the automation of the radiosynthesis, and evaluation of [18F]FEPURO in vivo. PROCEDURES: [18F]FEPURO was radiosynthesised in an automated module. PET imaging was conducted in Wistar rats under control and blocking conditions using the protein synthesis inhibitor cycloheximide. Biodistribution and metabolite studies at 30, 60 and 120 min were conducted in healthy rats. RESULTS: Automation of the radiosynthesis resulted in reduction of the synthesis time by half from the manual method. A steady increase in the SUV was observed in the time-activity curves for the whole brain as expected for a protein synthesis marker. However, rapid in vivo metabolism of [18F]FEPURO within 15 min in plasma as well as the brain (4 % of parent 30 min p.i.) indicated formation of the [18F]FET radio-metabolite in >90 % thus suggesting that observed increase in the brain uptake was due to the radiometabolite. CONCLUSIONS: [18F]FEPURO is not a suitable PET radiotracer for imaging protein synthesis rates in brain in vivo due to its rapid metabolism. Further structural modifications to prevent in vivo metabolism are underway.

11C-UCB-J synaptic PET and multimodal imaging in dementia with Lewy bodies.

OBJECTIVE: Dementia with Lewy bodies (DLB) is a common cause of dementia, but atrophy is mild compared to Alzheimer's disease. We propose that DLB is associated instead with severe synaptic loss, and we test this hypothesis in vivo using positron emission tomography (PET) imaging of 11C-UCB-J, a ligand for presynaptic vesicle protein 2A (SV2A), a vesicle membrane protein ubiquitously expressed in synapses. METHODS: We performed 11C-UCB-J PET in two DLB patients (an amyloid-negative male and an amyloid-positive female in their 70s) and 10 similarly aged healthy controls. The DLB subjects also underwent PET imaging of amyloid (11C-PiB) and tau (18F-AV-1451). 11C-UCB-J binding was quantified using non-displaceable binding potential (BPND) determined from dynamic imaging. Changes in 11C-UCB-J binding were correlated with MRI regional brain volume, 11C-PiB uptake and 18F-AV-1451 binding. RESULTS: Compared to controls, both patients had decreased 11C-UCB-J binding, especially in parietal and occipital regions (FDR-corrected p < 0.05). There were no significant correlations across regions between 11C-UCB-J binding and grey matter, tau (18F-AV1451) or amyloid (11C-PiB) in either patient. CONCLUSIONS: Quantitative imaging of in vivo synaptic density in DLB is a promising approach to understanding the mechanisms of DLB, over and above changes in grey matter volume and concurrent amyloid/tau deposition. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41824-020-00093-9.

Radiosynthesis of (R,S)-[18 F]GE387: A Potential PET Radiotracer for Imaging Translocator Protein 18†kDa (TSPO) with Low Binding Sensitivity to the Human Gene Polymorphism rs6971.

Translocator protein (TSPO) is a biomarker of neuroinflammation, which is a hallmark of many neurodegenerative diseases and has been exploited as a positron emission tomography (PET) target. Carbon-11-labelled PK11195 remains the most applied agent for imaging TSPO, despite its short-lived isotope and low brain permeability. Second-generation radiotracers show variance in affinity amongst subjects (low-, mixed-, and high-affinity binders) caused by the genetic polymorphism (rs6971) of the TSPO gene. To overcome these limitations, a new structural scaffold was explored based on the TSPO pharmacophore, and the analogue with a low-affinity binder/high-affinity binder (LAB/HAB) ratio similar (1.2 vs. 1.3) to that of (R)-[11 C]PK11195 was investigated. The synthesis of the reference compound was accomplished in six steps and 9 % overall yield, and the precursor was prepared in eight steps and 8 % overall yield. The chiral separation of the reference and precursor compounds was performed using supercritical fluid chromatography with >95 % ee. The absolute configuration was determined by circular dichroism. Optimisation of reaction conditions for manual radiolabelling revealed acetonitrile as a preferred solvent at 100 °C. Automation of this radiolabelling method provided R and S enantiomers in respective 21.3±16.7 and 25.6±7.1 % decay-corrected yields and molar activities of 55.8±35.6 and 63.5±39.5 GBq µmol-1 (n=3). Injection of the racemic analogue into a healthy rat confirmed passage through the blood-brain barrier.

Improved Syntheses of the Mglu5 Antagonists MMPEP and MTEP Using Sonogashira Cross-Coupling.

The Sonogashira cross-coupling, a key step in the syntheses of the mGlu5 antagonists MMPEP and MTEP, provided an improved three-step method for the preparation of MMPEP in 62% overall yield. Using Spartan molecular modeling kit an explanation for the failure to employ analogues method in the synthesis of MTEP was sought. The DFT calculations indicated that meaningful isolated yields were obtained when the HOMO energy of the aryl halide was lower than the HOMO energy of the respective alkyne.

Radiosynthesis of Carbon-11 Labeled Puromycin as a Potential PET Candidate for Imaging Protein Synthesis in Vivo.

In order to address the limitations associated with the present range of PET radiotracers used for imaging protein synthesis in vivo we have synthesized a candidate PET radiotracer based on Puromycin (3, PURO), a protein synthesis inhibitor. The desmethylPURO 9 precursor for radiolabeling with carbon-11 radioisotope was synthesized in two steps employing EDC/HOBt amide coupling in overall 76% yield. Optimal conditions for radiolabeling were then established via methylation/deprotection sequence. Under these conditions as determined by NMR analysis 9 showed partial stability (ca. 80%) under acidic conditions. Limited evidence of stereochemical stability of 3 was also found. The radiolabeling of intermediate [(11)C]12 was accomplished with up to 57% conversion from [(11)C]iodomethane. An automated method was then developed for high radioactivity radiosynthesis to produce [(11)C]3 ([(11)C]PURO) in 16 ± 6% (n = 3) decay corrected radiochemical yields.

Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging.

Serotonin-gated ionotropic 5-HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the "setrons") that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-11C)-N-granisetron ([11C]2) through N-alkylation with [11C]CH3I, respectively. Both compounds [18F]15 (chemical and radiochemical purity >95%, specific activity 41 GBq/µmol) and [11C]2 (chemical and radiochemical purity ≥99%, specific activity 170 GBq/µmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5-HT3 receptors in vivo.