Gas Phase Transformations in Carbon-11 Chemistry.

The short-lived positron-emitter carbon-11 (t1/2 = 20.4 min; ß+, 99.8%) is prominent for labeling tracers for use in biomedical research with positron emission tomography (PET). Carbon-11 is produced for this purpose with a cyclotron, nowadays almost exclusively by the 14N(p,α)11C nuclear reaction, either on nitrogen containing a low concentration of oxygen (0.1-0.5%) or hydrogen (~5%) to produce [11C]carbon dioxide or [11C]methane, respectively. These primary radioactive products can be produced in high yields and with high molar activities. However, only [11C]carbon dioxide has some utility for directly labeling PET tracers. Primary products are required to be converted rapidly and efficiently into secondary labeling synthons to provide versatile radiochemistry for labeling diverse tracer chemotypes at molecular positions of choice. This review surveys known gas phase transformations of carbon-11 and summarizes the important roles that many of these transformations now play for producing a broad range of labeling synthons in carbon-11 chemistry.

On the Risk of 18F-Regioisomer Formation in the Copper-Free Radiofluorination of Aryliodonium Precursors.

Aryliodonium precursors are widely applied for copper-free labeling of positron emission tomography (PET) tracers with fluorine-18. We assessed 18F-fluoroarene regioisomer formation in examples of these labeling methods. Aryliodonium ylides derived from Meldrum's acid bearing para electron-donating groups react with [18F]fluoride in acetonitrile to produce regioisomeric 18F-fluoroarenes via a competing aryne pathway. Regioisomer formation is decreased or absent in DMF. Analytically checking for the absence of the 18F-regioisomer from any particular PET tracer radiosynthesis using these or similar methods is recommended.

An Empirical Quantitative Structure-Activity Relationship Equation Assists the Discovery of High-Affinity Phosphodiesterase 4D Inhibitors as Leads to PET Radioligands.

A positron emission tomography (PET) radioligand for imaging phosphodiesterase 4D (PDE4D) would benefit drug discovery and the investigation of neuropsychiatric disorders. The most promising radioligand to date, namely, [11C]T1650, has shown unstable quantification in humans. Structural elaboration of [11C]T1650 was therefore deemed necessary. High target affinity in the low nM range is usually required for successful PET radioligands. In our PDE4D PET radioligand development, we formulated and optimized an empirical equation (log[IC50 (nM)] = P1 + P2 + P3 + P4) that well described the relationship between binding affinity and empirically derived values (P1-P4) for the individual fragments in four subregions commonly composing each inhibitor (R2 = 0.988, n = 62). This equation was used to predict compounds that would have high inhibitory potency. Fourteen new compounds were obtained with IC50 of 0.3-10 nM. Finally, eight compounds were judged to be worthy of future radiolabeling and evaluation as PDE4D PET radioligands.

Broad Scope and High-Yield Access to Unsymmetrical Acyclic [11 C]Ureas for Biomedical Imaging from [11 C]Carbonyl Difluoride.

Effective methods are needed for labelling acyclic ureas with carbon-11 (t1/2 =20.4 min) as potential radiotracers for biomedical imaging with positron emission tomography (PET). Herein, we describe the rapid and high-yield syntheses of unsymmetrical acyclic [11 C]ureas under mild conditions (room temperature and within 7 min) using no-carrier-added [11 C]carbonyl difluoride with aliphatic and aryl amines. This methodology is compatible with diverse functionality (e. g., hydroxy, carboxyl, amino, amido, or pyridyl) in the substrate amines. The labelling process proceeds through putative [11 C]carbamoyl fluorides and for primary amines through isolable [11 C]isocyanate intermediates. Unsymmetrical [11 C]ureas are produced with negligible amounts of unwanted symmetrical [11 C]urea byproducts. Moreover, the overall labelling method tolerates trace water and the generally moderate to excellent yields show good reproducibility. [11 C]Carbonyl difluoride shows exceptional promise for application to the synthesis of acyclic [11 C]ureas as new radiotracers for biomedical imaging with PET.

PET ligands [18F]LSN3316612 and [11C]LSN3316612 quantify O-linked-ß-N-acetyl-glucosamine hydrolase in the brain.

We aimed to develop effective radioligands for quantifying brain O-linked-ß-N-acetyl-glucosamine (O-GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O-GlcNAc through the enzyme pair OGA and O-GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O-GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC50) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [3H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [18F]LSN3316612 and [11C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [18F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (VT) values by 110 min of scanning. Overall, [18F]LSN3316612 is preferred over [11C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

[11 C]Carbonyl Difluoride-a New and Highly Efficient [11 C]Carbonyl Group Transfer Agent.

Herein, the synthesis and use of [11 C]carbonyl difluoride for labeling heterocycles with [11 C]carbonyl groups in high molar activity is described. A very mild single-pass gas-phase conversion of [11 C]carbon monoxide into [11 C]carbonyl difluoride over silver(II) fluoride provides easy access to this new synthon in robust quantitative yield for labeling a broad range of cyclic substrates, for example, imidazolidin-2-ones, thiazolidin-2-ones, and oxazolidin-2-ones. Labeling reactions may utilize close-to-stoichiometric precursor quantities and short reaction times at room temperature in a wide range of solvents while also showing high water tolerability. The overall radiosynthesis protocol is both simple and reproducible. The required apparatus can be constructed from widely available parts and is therefore well suited to be automated for PET radiotracer production. We foresee that this straightforward method will gain wide acceptance for PET radiotracer syntheses across the radiochemistry community.

Synthesis and evaluation of two new candidate high-affinity full agonist PET radioligands for imaging 5-HT1B receptors.

INTRODUCTION: The serotonin 1B receptor subtype is of interest in the pathophysiology and treatment of depression, anxiety, and migraine. Over recent years 5-HT1B receptor binding in human brain has been examined with PET using radioligands that are partial but not full agonists. To explore how the intrinsic activity of a PET radioligand may affect imaging performance, two high-affinity full 5-HT1B receptor agonists (AZ11136118, 4; and AZ11895987, 5) were selected from a large compound library and radiolabeled for PET examination in non-human primates. METHODS: [11C]4 was obtained through Pd(0)-mediated insertion of [11C]carbon monoxide between prepared iodoarene and homochiral amine precursors. [11C]5 was obtained through N-11C-methylation of N-desmethyl precursor 6 with [11C]methyl triflate. [11C]4 and [11C]5 were studied with PET in rhesus or cynomolgus monkey. [11C]4 was studied with PET in mice and rats to measure brain uptake and specific binding. Ex-vivo experiments in rats were performed to identify whether there were radiometabolites in brain. Physiochemical parameters for [11C]4 (pKa, logD and conformational energetics) were evaluated. RESULTS: Both [11C]4 and [11C]5 were successfully produced in high radiochemical purity and in adequate amounts for PET experiments. After intravenous injection of [11C]4, brain radioactivity peaked at a low level (0.2 SUV). Pretreatment with tariquidar, an inhibitor of the brain P-gp efflux transporter, increased brain exposure four-fold whereas pretreatment with a high pharmacological dose of the 5-HT1B antagonist, AR-A000002, had no effect on the binding. Ex-vivo experiments in rats showed no radiometabolites entering brain. [11C]5 also failed to enter monkey brain under baseline conditions. CONCLUSIONS: [11C]4 and [11C]5 show too low brain uptake and specific binding to be useful PET radioligands. Low brain uptake is partly ascribed to efflux transporter action as well as unfavorable conformations.

[O-methyl-11C]N-(4-(4-(3-Chloro-2-methoxyphenyl)-piperazin-1-yl)butyl)-1H-indole-2-carboxamide ([11C]BAK4-51) Is an Efflux Transporter Substrate and Ineffective for PET Imaging of Brain D3 Receptors in Rodents and Monkey.

Selective high-affinity antagonists for the dopamine D3 receptor (D3R) are sought for treating substance use disorders. Positron emission tomography (PET) with an effective D3R radioligand could be a useful tool for the development of such therapeutics by elucidating pharmacological specificity and target engagement in vivo. Currently, a D3R-selective radioligand does not exist. The D3R ligand, N-(4-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-2-carboxamide (BAK4-51, 1), has attractive properties for PET radioligand development, including full antagonist activity, very high D3R affinity, D3R selectivity, and moderate lipophilicity. We labeled 1 with the positron-emitter carbon-11 (t1/2 = 20.4 min) in the methoxy group for evaluation as a radioligand in animals with PET. However, [11C]1 was found to be an avid substrate for brain efflux transporters and lacked D3R-specific signal in rodent and monkey brain in vivo.

[Carboxyl-11 C]Labelling of Four High-Affinity cPLA2α Inhibitors and Their Evaluation as Radioligands in Mice by Positron Emission Tomography.

Cytosolic phospholipase A2α (cPLA2α) may play a critical role in neuropsychiatric and neurodegenerative disorders associated with oxidative stress and neuroinflammation. An effective PET radioligand for imaging cPLA2α in living brain might prove useful for biomedical research, especially on neuroinflammation. We selected four high-affinity (IC50 2.1-12 nm) indole-5-carboxylic acid-based inhibitors of cPLA2α, namely 3-isobutyryl-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (1); 3-acetyl-1-(2-oxo-3-(4-(4-(trifluoromethyl)phenoxy)phenoxy)propyl)-1H-indole-5-carboxylic acid (2); 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (3); and 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(3-(4-octylphenoxy)-2-oxopropyl)-1H-indole-5-carboxylic acid (4), for labelling in carboxyl position with carbon-11 (t1/2 =20.4 min) to provide candidate PET radioligands for imaging brain cPLA2α. Compounds [11 C]1-4 were obtained for intravenous injection in adequate overall yields (1.1-5.5 %) from cyclotron-produced [11 C]carbon dioxide and with moderate molar activities (70-141 GBq µmol-1 ) through the use of Pd0 -mediated [11 C]carbon monoxide insertion on iodo precursors. Measured logD7.4 values were within a narrow moderate range (1.9-2.4). After intravenous injection of [11 C]1-4 in mice, radioactivity uptakes in brain peaked at low values (≤0.8 SUV) and decreased by about 90 % over 15 min. Pretreatments of the mice with high doses of the corresponding non-radioactive ligands did not alter brain time-activity curves. Brain uptakes of radioactivity after administration of [11 C]1 to wild-type and P-gp/BCRP dual knock-out mice were similar (peak 0.4 vs. 0.5 SUV), indicating that [11 C]1 and others in this structural class, are not substrates for efflux transporters.

Pd(0)-Mediated 11C-Carbonylation of Aryl(mesityl)iodonium Salts as a Route to [11C]Arylcarboxylic Acids and Derivatives.

Pd(0)-mediated 11C-carbonylation of aryl(mesityl)iodonium salts followed by suitable quench provides a rapid room-temperature two-pot procedure for labeling arylcarboxylic acids and amide derivatives with the short-lived positron emitter carbon-11 (t1/2 = 20.4 min) in generally good to high yields (up to 71%). High product ring selectivity (≥13) was achieved when using mesityl as a spectator group in the diaryliodonium salt precursors. This process has potential for preparing new radiotracers for molecular imaging with positron emission tomography.

11C-Labeling of Aryl Ketones as Candidate Histamine Subtype-3 Receptor PET Radioligands through Pd(0)-Mediated 11C-Carbonylative Coupling.

Pd(0)-mediated coupling between iodoarenes, [11C]carbon monoxide and aryltributylstannanes has been used to prepare simple model [11C]aryl ketones. Here, we aimed to label four 2-aminoethylbenzofuran chemotype based molecules ([11C]1-4) in the carbonyl position, as prospective positron emission tomography (PET) radioligands for the histamine subtype 3 receptor (H3R) by adapting this methodology with use of aryltrimethylstannanes. Radiosynthesis was successfully performed on a platform equipped with a mini-autoclave and a liquid handling robotic arm, within a lead-shielded hot-cell. Candidate radioligands were readily formulated in saline containing ethanol (10%, v/v) and ascorbic acid (0.5 mg/10 mL). Yields for preclinical use were in the range of 5-9%, decay-corrected from cyclotron-produced [11C]CO2 and molar activities were >115 GBq/µmol at end of synthesis. Radiochemical purities exceeded >97%.

Crown Ether Nucleophilic Catalysts (CENCs): Agents for Enhanced Silicon Radiofluorination.

New bifunctional phase transfer agents were synthesized and investigated for their abilities to promote rapid fluorination at silicon. These agents, dubbed crown ether nucleophilic catalysts (CENCs), are 18-crown-6 derivatives containing a side-arm and a potentially nucleophilic hydroxyl group. These CENCs proved efficacious in the fluorination of hindered silicon substrates, with fluorination yields dependent on the length of linker connecting the metal chelating unit to the hydroxyl group. The efficacy of these CENCs was also demonstrated for rapid radiofluorination under mild conditions for eventual application in molecular imaging with positron emission tomography (PET). The hydrolysis-resistant aryl silicon fragment is promising as a convenient synthon for labeling potential PET radiotracers.

Quinuclidine and DABCO Enhance the Radiofluorinations of 5-Substituted 2-Halopyridines.

Positron emission tomography (PET) is an important molecular imaging technique for medical diagnosis, biomedical research and drug development. PET tracers for molecular imaging contain ß+-emitting radionuclides, such as carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min). The [18F]2-fluoro-pyridyl moiety features in a few prominent PET radiotracers, not least because this moiety is usually resistant to unwanted radiodefluorination in vivo. Various methods have been developed for labeling these radiotracers from cyclotron-produced no-carrier-added [18F]fluoride ion, mainly based on substitution of a leaving group, such as halide (Cl or Br), or preferably a better leaving group, such as nitro or trimethylammonium. However, precursors with a good leaving group are sometimes more challenging or lengthy to prepare. Methods for enhancing the reactivity of more readily accessible 2-halopyridyl precursors are therefore desirable, especially for early radiotracer screening programs that may require the quick labeling of several homologous radiotracer candidates. In this work, we explored a wide range of additives for beneficial effect on nucleophilic substitution by [18F]fluoride ion in 5-subsituted 2-halopyridines (halo = Cl or Br). The nucleophilic cyclic tertiary amines, quinuclidine and DABCO, proved effective for increasing yields to practically useful levels (> 15%). Quinuclidine and DABCO likely promote radiofluorination through reversible formation of quaternary ammonium intermediates.

An Investigation of (Diacetoxyiodo)arenes as Precursors for Preparing No-Carrier-Added [(18)F]Fluoroarenes from Cyclotron-Produced [(18)F]Fluoride Ion.

Treatment of (diacetoxyiodo)arenes (1a-1u) with cyclotron-produced [(18)F]fluoride ion rapidly affords no-carrier-added [(18)F]fluoroarenes (2a-2u) in useful yields and constitutes a new method for converting substituted iodoarenes into substituted [(18)F]fluoroarenes in just two steps.

Exploration of the labeling of [11C]tubastatin A at the hydroxamic acid site with [11C]carbon monoxide.

We aimed to label tubastatin A (1) with carbon-11 (t1/2 = 20.4 min) in the hydroxamic acid site to provide a potential radiotracer for imaging histone deacetylase 6 in vivo with positron emission tomography. Initial attempts at a one-pot Pd-mediated insertion of [(11)C]carbon monoxide between the aryl iodide (2) and hydroxylamine gave low radiochemical yields (<5%) of [(11) C]1. Labeling was achieved in useful radiochemical yields (16.1 ± 5.6%, n = 4) through a two-step process based on Pd-mediated insertion of [(11)C]carbon monoxide between the aryl iodide (2) and p-nitrophenol to give the [(11)C]p-nitrophenyl ester ([(11)C]5), followed by ultrasound-assisted hydroxyaminolysis of the activated ester with excess hydroxylamine in a DMSO/THF mixture in the presence of a strong phosphazene base P1-t-Bu. However, success in labeling the hydroxamic acid group of [(11)C]tubastatin A was not transferable to the labeling of three other model hydroxamic acids.

[carbonyl-11C]4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([11C]FIMX) is an effective radioligand for PET imaging of metabotropic glutamate receptor 1 (mGluR1) in monkey brain.

INTRODUCTION: Metabotropic glutamate subtype receptor 1 (mGluR1) is implicated in several neuropsychiatric disorders and is a target for drug development. [(18)F]FIMX ([(18)F]4-fluoro--N-methyl-N--(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide) is an effective radioligand for imaging brain mGluR1 with PET. A similarly effective radioligand with a shorter half-life would usefully allow PET studies of mGluR1 at baseline and after pharmacological or other challenge on the same day. Here we describe the preparation of [(11)C]FIMX for evaluation in monkey with PET. METHODS: [(11)C]FIMX was prepared via Pd-promoted carbonylation of 1-fluoro-4-iodobenzene with [(11)C]carbon monoxide, aminolysis of the [(11)C]acyl-palladium complex with the requisite Boc-protected amine, and deprotection with HCl in THF. PET scans of [(11)C]FIMX injected into a monkey were performed at baseline and after preblock of mGluR1 with measurement of the arterial input function. RESULTS: The radiosynthesis required 42 min and gave [(11)C]FIMX in about 5% overall decay-corrected radiochemical yield and with a specific activity of about 100 GBq/µmol. PET in rhesus monkey at baseline showed that radioactivity peaked high in receptor-rich cerebellum and much lower in receptor-poor occipital cortex. Radioactivity in cerebellum declined to 32% of peak at 85 min. VT at baseline appeared stable in all brain regions after 60 min. Under mGluR1 pre-blocked condition, radioactivity uptake in all regions declined more rapidly to a low level. Receptor pre-block reduced VT from 13.0 to 1.5 in cerebellum and from 2.9 to 1.4 in occipital cortex. CONCLUSION: [(11)C]FIMX is an effective radioligand for imaging mGluR1 in monkey with PET.

The Inhibitor Ko143 Is Not Specific for ABCG2.

Imaging ATP-binding cassette (ABC) transporter activity in vivo with positron emission tomography requires both a substrate and a transporter inhibitor. However, for ABCG2, there is no inhibitor proven to be specific to that transporter alone at the blood-brain barrier. Ko143 [[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4- b]indole-3-propanoic acid 1,1-dimethylethyl ester], a nontoxic analog of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although its specificity in mouse and human systems is unclear. This study examined the selectivity of Ko143 using human embryonic kidney cell lines transfected with ABCG2, ABCB1, or ABCC1 in several in vitro assays. The stability of Ko143 in rat plasma was measured using high performance liquid chromatography. Our results show that, in addition to being a potent inhibitor of ABCG2, at higher concentrations (≥1 µM) Ko143 also has an effect on the transport activity of both ABCB1 and ABCC1. Furthermore, Ko143 was found to be unstable in rat plasma. These findings indicate that Ko143 lacks specificity for ABCG2 and this should be taken into consideration when using Ko143 for both in vitro and in vivo experiments.

Enhanced Nucleophilic Fluorination and Radiofluorination of Organosilanes Appended with Potassium-Chelating Leaving Groups.

Here we aimed to explore the feasibility of enhancing the fluorination of organosilanes by appending potassium-chelating groups to the substrates. For this purpose, eight organosilanes were prepared in which a linear or cyclic leaving group, with putative potassium-chelating ability, was attached covalently to a congested silicon atom via an ether linkage to serve as a potential nucleophilic assisting leaving group (NALG). Organosilicon-NALGs with expected strong potassium-chelating capability enhanced reactions with potassium fluoride in acetonitrile to produce organofluorosilanes without any need to separately add phase transfer reagent. Similar rate enhancements were also observed with cyclotron-produced [18F]fluoride ion (t1/2 = 109.7 min, ß+ = 97%) in the presence of potassium carbonate in MeCN-0.5% H2O. This study found that metal-chelating NALG units can accelerate fluorination and radiofluorination reactions at sterically crowded silicon atoms.

(11)C-CUMI-101, a PET radioligand, behaves as a serotonin 1A receptor antagonist and also binds to α(1) adrenoceptors in brain.

UNLABELLED: The PET radioligand (11)C-CUMI-101 was previously suggested as a putative agonist radioligand for the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor in recombinant cells expressing human 5-HT1A receptor. However, a recent study showed that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. The current study examined the functional properties and selectivity of CUMI-101, both in vitro and in vivo. METHODS: The functional assay was performed using (35)S-GTPγS (GTP is guanosine triphosphate) in primate brains. The cross-reactivity of CUMI-101 with α1 adrenoceptors was performed using in vitro radioligand binding studies in rat, monkey, and human brains as well as in vivo PET imaging in mouse, rat, and monkey brains. RESULTS: CUMI-101 did not stimulate (35)S-GTPγS binding in primate brain, in contrast to 8-OH-DPAT, a potent 5-HT1A receptor agonist. Instead, CUMI-101 behaved as a potent 5-HT1A receptor antagonist by dose-dependently inhibiting 8-OH-DPAT-stimulated (35)S-GTPγS binding. Both in vitro and in vivo studies showed that CUMI-101 had significant α1 adrenoceptor cross-reactivity. On average, across all 3 species examined, cross-reactivity was highest in the thalamus (>45%) and lowest in the neocortex and cerebellum (<10%). PET imaging further confirmed that only preblocking with WAY-100635 plus prazosin decreased (11)C-CUMI-101 brain uptake to that of self-block. CONCLUSION: CUMI-101 behaves as a 5-HT1A receptor antagonist in primate brain, with significant, regional-dependent α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.