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. 2025 Jun 20;8(7):1986–1995. doi: 10.1021/acsptsci.5c00032

Radiosynthesis and Evaluation of 11C‑Labeled Imidazolyl Pyrimidine Derivatives for Positron Emission Tomography Imaging of Glycogen Synthase Kinase‑3

Yinlong Li 1, Kenneth Dahl 2,3, Charles S Elmore 4, Johan Sandell 4, Akihiro Takano 3, Christer Halldin 3,7, Lars Farde 3, Charlotte Ahlgren 3, Alison Cochrane 4, Jian Rong 1, Jiahui Chen 1, Chunyu Zhao 1, Xin Zhou 1, Jimmy S Patel 1,5, Zhendong Song 1, Ahmad Chaudhary 1, Yabiao Gao 1, Zhenkun Sun 6, Zachary Zhang 1, Siyan Feng 1, Achi Haider 1, Steven H Liang 1,*, Magnus Schou 2,3,*
PMCID: PMC12260939  PMID: 40672682

Abstract

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that regulates various biological processes by phosphorylating protein substrates. Dysregulation of GSK-3 is linked to a variety of diseases, including malignancies, diabetes, and neurodegenerative disorders. Moreover, GSK-3 hyperactivity is a potential contributing factor in Alzheimer’s disease, suggesting that GSK-3 inhibition may offer therapeutic benefits. Herein, we report the synthesis and evaluation of five 11C-labeled imidazolyl pyrimidine analogues [11C]13a–e (codenamed AZ12646326, AZ12646603, AZ12656261, AZ12977360, and AZ12943203) as novel radioligands for positron emission tomography (PET) imaging of GSK-3. Pharmacological assays showed that compounds 13a–e exhibited high in vitro binding affinity to GSK-3β, with Ki values ranging from 2.49 to 4.95 nM. In vitro autoradiography confirmed high levels of specific binding in GSK-3-rich regions of the rodent brain, highlighting the promising imaging properties of these analogues. Radiosynthesis of [11C]13a–e was achieved via palladium-promoted carbonylation reactions with [11C]carbon monoxide, with excellent radiochemical purity (>99%). However, PET imaging studies in nonhuman primates in vivo showed low brain uptake of these radioligands, and [11C]13e was identified as a P-glycoprotein substrate. This study offers valuable insights for optimizing future GSK-3-targeted PET tracers based on the imidazolyl pyrimidine scaffold.

Keywords: glycogen synthase kinase-3, imidazolylpyrimidine derivatives, carbon-11, positron emission tomography, radioligand

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Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase that plays a crucial role in various cellular processes, such as proliferation, cell signaling, metabolism, and immune responses. − GSK-3 comprises two highly homologous isoforms (GSK-3α and GSK-3β) that are highly distributed across various tissues and especially widespread in the central nervous system (CNS). A growing body of evidence links dysregulation of GSK-3β activity with a variety of pathologies, − including Alzheimer’s disease (AD), − Parkinson’s disease (PD), , type-II diabetes − and different malignancies. − For example, enhanced activity of GSK-3β prompts hyper-phosphorylation of Tau protein leading to neuronal degeneration, thus contributing to AD pathology. − In clinical studies, aberrant GSK-3 activity has been reported in patients with AD, major depressive disorder, or schizophrenia. − As such, altered activity may occur without changes in receptor expression, which constitutes a significant challenge for the noninvasive assessment of GSK-3 function. Nonetheless, several studies have reported changes in GSK-3 expression in pathological conditions, such as Huntington’s disease (HD) and diabetes, prompting calls for the development of suitable GSK-3-targeted PET radioligands. Along this line, selective inhibition of GSK-3 holds the potential to restore homeostatic GSK-3 function, rendering GSK-3 a promising target for the potential treatment of these diseases. , Indeed, lithium is considered the first naturally occurring GSK-3 inhibitor, but its clinical use is hampered by low target selectivity, adverse side effects, and high toxicity. ,

Positron emission tomography (PET) is a molecular imaging technique that enables noninvasively visualizing and quantifying biochemical and physiological functions by tracking the real-time distribution of radioactive tracers. − PET imaging of GSK-3 would allow for assessment of its density and to monitor changes in GSK-3 levels within living subjects, thereby enhancing our understanding of GSK-3 related physiology and pathologies. , In recent years, several PET radioligands have been reported for PET imaging of GSK-3 (Figure ). One of the first reported GSK-3-targeted radioligands, [11C]1 (AR-A014418), was not successfully translated due to its low permeability across the blood-brain barrier (BBB). [11C]2 (PyrATP-1), which was developed from a literature compound (Ki = 4.9 nM), revealed poor brain uptake in rodents and nonhuman primates (NHPs) in PET imaging studies. Similarly, oxadiazole-based probes [11C]3–5 exhibited low brain penetration in mice, despite apparently suitable physicochemical properties. [11C]6 (SB-216763) was the first GSK-3 radioligand reported to penetrate the BBB in rodents and NHPs. However, [11C]6 displayed a homogeneous uptake across the brain and there was no support for specific binding. , The Maleimide-based GSK-3β tracer [18F]7 (IC50 = 1.7 nM) demonstrated moderate brain uptake, although no saturable binding was observed in rodents. [11C]8 (PF-04082367) demonstrated promising brain uptake and binding specificity in NHPs, but further kinetic modeling analysis is needed for high species translation. [11C]9 (A1070722) was identified as a selective GSK-3 ligand (Ki = 0.6 nM) in the NHP brain, but it lacked sufficient brain exposure. A series of isonicotinamide derivatives, including [11C]10 (CMP), [18F]11 and [18F]12 , labeled with either carbon-11 or fluorine-18 were recently reported, but they showed insignificant uptake in the rodent brain and/or low-to-moderate specific binding.

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Potential GSK-3 PET ligands.

Despite the above-mentioned attempts, a clinically validated GSK-3 radioligand is currently lacking. As part of our continuous efforts on GSK-3-targeted PET tracer development, , we focused on imidazolyl pyrimidine derivatives guided by established structure–activity relationship (SAR) data, which highlight their potency and selectivity as GSK-3 inhibitors. , The imidazole ring serves as a critical pharmacophore, facilitating essential hydrogen bonding and π-π stacking interactions within the ATP-binding site of GSK-3. Similarly, the pyrimidine scaffold is a privileged structure in kinase inhibitor design by enhancing binding affinity and selectivity. Herein, we describe the radiolabeling and preclinical evaluation of a series of imidazolyl pyrimidine derivatives, [11C]13a–e, as potential GSK-3 PET ligands (Figure ).

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Chemical structures of novel GSK-3 PET ligands.

Result and Discussion

The pharmacology and physicochemical properties of compounds 13a–13e are depicted in Table . To evaluate binding affinities, saturation binding assays using tritiated ligands [3H]13a, [3H]13c, [3H]13d, [3H]13e (Scheme S1) were performed using the hippocampal tissue of Sprague–Dawley (SD) rat brains. As depicted in Table A, total binding (TB), nonspecific binding (NSB), and specific binding (SB) of the respective tritiated ligands were measured with increasing concentrations of the radioligands. The dissociation constant (Kd) and capacity (B max) of each radioligand were calculated to confirm their binding affinity.

1. (A) Representative Saturation Binding Curves and Scatchard Plots for Tritiated Ligands [3H]13a, [3H]13c, [3H]13d, and [3H]13e in the Hippocampus of SD Rat Brains; In Silico (B) Physicochemical and (C) Pharmacology Properties of 13a–e .

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a

Values were calculated with ChemDraw 21.0 software.

b

Values were predicted with ACD/laboratories. [3H]13b was not subjected to a saturation binding experiment; therefore, Kd and B max values are not provided.

All the tested radioligands demonstrated high binding affinity to GSK-3, with Kd values ranging from 1.09 nM to 6.46 nM (Table C). The enzyme density, B max, quantified by each radioligand ranged from 64.5 to 83.4 fmol/mg. Notably, all B max/Kd ratios were higher than 10, which is considered favorable for in vivo PET neuroimaging. Similarly, in silico physicochemical properties of 13a–13e such as molecular weight (MW< 500 g/mol), lipophilicity (clogP: 1.64–3.07 and LogD: 1.20–3.20) and topological polar surface area (TPSA: 81.89–91.12) indicated favorable brain permeability (Table B).

Multiparameter optimization (MPO) and brain/plasma concentration at steady-state (logBB) are widely used to predict BBB permeability. A desirability score of ≥ 4.0 for MPO and ≥ – 1 for logBB are generally considered thresholds for potential BBB penetration. , As shown in Table B, compounds 13a–13e met these thresholds, suggesting their potential to cross the BBB. Additionally, compounds 13a–13e exhibited high inhibitory potency toward GSK-3β, with low IC50 (3.31–6.61 nM) and Ki (2.49–4.95 nM) values (Table C). A detailed ADME/PK (absorption, distribution, metabolism, excretion and pharmacokinetics) profiling was conducted, and the results are listed in Table . The apparent permeability coefficient (Papp A–B) of 13b–13e was measured as 12.7–22.7 × 10–6 cm/s, along with the efflux ratio (Papp B–A/Papp A–B) ranging from 0.7 to 2.1. These values meet the standards that BBB permeability is considered high if Papp A–B is ≥ 3.0 × 10–6 cm/s and the efflux ratio is <3.0. All brain-to-plasma unbound fraction ratios (f u brain) > 1, indicating that a greater proportion of the compound could remain unbound (free) in the brain and available for pharmacological effect. The solubility of 13a–13e was also measured to be greater than 150 μM in DMSO.

2. ADME/PK Analysis of Compounds 13a–13e .

Property 13a 13b 13c 13d 13e
P app A-B (× 10–6 cm/s) -- 22.7 16.2 21.9 12.7
efflux ratio -- 1.2 2.1 0.7 1.4
fu brain (%) 3.6 13 19 26 12
solubility (μM) 234 155 >400 -- 362
Property 13a 13b 13d 13e 13f
mean Prot binding (% free) -- 5.6 11 11.1 7.6
median CLint (μ/min/106 cells) 34.2 17.5 15.4 12 11
mean t 1/2 (h) -- 2.32 3.65 1.73 2.27
mean bioavailability (%) -- 39.8 32.9 10.1 30.9
CYP1A2 IC50 (μM) >2 >2 >2 -- >2
CYP2C9 IC50 (μM) >2 >2 >2 -- >2
CYP2D6 IC50 (μM) >2 >2 >2 -- >2
CYP3A4 IC50 (μM) >2 >2 >2 -- >2
hERG IC50 (μM) 4.52 18.8 >33 20.5 >33
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a

Solubility in dry DMSO at pH = 7.4.

The pharmacokinetics assays including mean protein binding, median intrinsic clearance (CLint), mean half-life (t1/2) and mean bioavailability revealed that these compounds exhibit reasonable clearance and good bioavailability. The inhibitory properties of 13a–13e on four cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) were greater than 2 μM. On the other hand, no hERG channel inhibition was observed (IC50 ≥ 4.52 μM), indicating no cardiac safety concerns. Therefore, all pharmacology and ADME assays suggested that compounds 13a–13e may be considered as potential radioligands with brain penetration and high affinity for GSK-3.

In the next step, we performed in vitro autoradiography studies on rat brain tissue sections to investigate the binding selectivity and distribution of compounds [3H]13a–13e (Figure ). The results demonstrated that [3H]13a–13e exhibited heterogeneous distributions. Among these, [3H]13d displayed the lowest level of radioactive accumulation, while [3H]13e exhibited the highest and most regionally distinct uptake. Notably, [3H]13e showed high binding in the cortex and hippocampus, moderate uptake in the striatum and cerebellum, and relatively low binding in the thalamus. This distribution pattern closely correlates with previously reported GSK-3 expression profiles from immunohistochemistry and in situ hybridization studies. , Co-incubation with cold reference and validated GSK-3β inhibitors (AR-A014418 and SB-216763) significantly reduced the radioactive signal in all the brain regions, confirming specific binding to GSK-3β. These findings support the potential of this compound series for further development as GSK-3β-targeted neuroimaging probes.

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Representative in vitro autoradiograph of [3H]13a, [3H]13b, [3H]13c, [3H]13d, and [3H]13e under baseline (A) and blocking (B) conditions (blocker = 3 or 5 μM). (C) Quantitative analysis of autoradiograms with [3H]13e.

To further evaluate the in vivo property of compounds 13a–13e, we designed precursors 14–16 for radiolabeling with PET radionuclide carbon-11 (half-life = 20.4 min). As shown in Scheme a-c, the radiosynthesis of [11C]13a–d was achieved through palladium-mediated carbonylation reactions with [11C]carbon monoxide ([11C]­CO) and aryl iodides 14 and 15. In contrast, [11C]13e was synthesized via the coupling of [11C]­CO with iodomethane (MeI) and the amine precursor 16 (Scheme d). The use of MeI represents a distinct reaction pathway compared to the aryl iodides used for [11C]13a–d. In particular, the radiolabeling approach was enabled by a key N-acylation step on the piperazine moiety, which provided a suitable precursor for efficient [11C]­methylation using MeI. The resulting radioligands were produced in 1.6%–13.8% radiochemical yields (RCY, decay-corrected) with excellent radiochemical purities (RCP > 99%) and molar activities (Am = 22–99 GBq/μmol) at the end of synthesis (EOS).

1. Radiosynthesis of (a) [ 11 C]­13a–b, (b) [ 11 C]­13c, (c) [ 11 C]­13d, and (d) [ 11 C]­13e .

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a (i) [11C]­CO, palladium­(π-cinnamyl) chloride dimer, xantphos or Pd­(PPh3)4, piperidine or diethylamine, THF, 100 °C, 5 min. (ii) [11C]­CO, palladium­(π-cinnamyl) chloride dimer, xantphos, diethylamine, or (2-methoxyl)­methylamine, THF, 100 °C, 5 min. (iii) [11C]­CO, Pd­(PPh3)4, MeI, THF, 100 °C, 5 min

Then, we sought to assess the in vivo PET imaging performance characteristics of [11C]13a–e in NHPs, and the results are presented in Figure . To our surprise, none of the radioligands exhibited significant brain uptake and radioactivity in the brain cleared rapidly at 1–2 min postinjection. Time–activity curves (TACs) expressed in standard uptake value (SUV) indicated that [11C]13e exhibited relatively higher signals in the brain, although potential spillover effects cannot be excluded in dynamic PET scans (0–120 min). In addition, all five radioligands displayed a heterogeneous distribution pattern across brain regions (Figure S2). Despite favorable in silico predictions for CNS penetration, the low brain uptake observed in NHPs suggests additional factors may be limiting BBB permeability. One potential contributor is metabolic instability of those tracers may reduce the amount of intact tracers to cross the BBB. Another important consideration is efflux by ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) that is highly expressed at the BBB and can actively pump substrates out of the CNS. To investigate whether 13a–e are P-gp efflux substrates, a P-gp inhibition study was conducted. As shown in Figures A and B, inhibiting P-gp with Tariquidar (6 mg/kg) significantly increased the brain uptake of [11C]13e. Brain regional distribution volume (VT) values for [11C]13e increased by 138%–313% compared to the respective control studies, indicating that these radioligands may be P-gp substrates. Additionally, although the imidazolyl-pyrimidine scaffold generally exhibits physicochemical properties compatible with CNS penetration, specific structural features such as the presence of hydrogen bond donors, high tPSA, or ionizable groups may contribute to poor passive diffusion across the BBB. To enhance brain uptake in future analogs, various medicinal chemistry strategies could be employed, such as hydrogen bond donor/acceptor optimization, fine-tuning lipophilicity, steric shielding of efflux motifs, and bioisosteric replacements. A potential limitation of this study is the lack of blood sampling during the PET scans, which precluded assessment of [11C]13e plasma concentrations following tariquidar pretreatment. While the tariquidar protocol is a widely used standard to probe P-gp–mediated efflux in PET tracer development, alternative approaches such as studies in P-gp knockout animals may offer additional insight. However, genetic deletion of P-gp has been associated with broader alterations in BBB integrity, which may also confound interpretation of tracer distribution.

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(A) Summed (0 to 120 min) PET images and (B) TACs of [11C]13a, [11C]13b, [11C]13c, [11C]13d, and [11C]13e in NHP.

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PET imaging study of PgP inhibition effects (pretreatment with 6 mg/kg tariquidar) on (A) radioactivity levels of [11C]13e and (B) TACs and (C) VT values in NHP brains.

Conclusions

In summary, we identified a series of imidazolyl pyrimidine analogues [11C]­13a–e as potential radioligands for PET imaging of GSK-3. Pharmacological assays demonstrated that compounds 13a–e possess high in vitro specific binding affinity with nanomolar IC50 and K i values. Autoradiography studies using tritium-labeled radioligands on rodent brain tissue sections demonstrated high specific binding to brain regions known to highly express GSK-3. Co-incubation with cold-validated GSK-3β inhibitors (AR-A014418 and SB-216763) confirmed specific binding to GSK-3β. The radiosynthesis was accomplished through palladium-promoted carbonylation reactions with [11C]­CO, yielding [11C]13a–e in excellent radiochemical purity and high molar activity. Although in vivo PET imaging in the NHP brain did not exhibit sufficient brain uptake, insights from this study suggest the limitation of the current chemical scaffold and improved brain permeability is necessary for developing future GSK-3 targeted PET tracers.

Experimental Section

Materials and Methods

The imidazolyl pyrimidine-based analogues 13a–13e and corresponding precursors were synthesized following the procedure described in our patent. High-performance liquid chromatography (HPLC) solvents were sourced from Fisher (Sweden). Unless specified otherwise, all additional reagents and solvents were provided by Sigma-Aldrich (Sweden) and used as received. Analytical HPLC was conducted with a Hitachi L-6200 gradient pump (Tokyo, Japan) equipped with a variable wavelength UV detector (λ = 254 nm) and a Bioscan β+ flow detector. A reverse phase column (Zorbax eclipse, 5 μm, 3.9 × 150 mm) with an eluent of acetonitrile/0.1 M ammonium formate (gradient method, 10–90% CH3CN in 7 min) at a flow rate of 3 mL/min was used. All radioligands were confirmed by coinjection with the corresponding cold compounds.

Radiochemistry

No-carrier-added [11C]­CO2 (approximately 50 GBq) was generated through the 14N­(p,α)11C nuclear reaction, by irradiating a PETtrace 800 cyclotron (GE, Uppsala, Sweden) target with a mixture of nitrogen and 1% oxygen using a 16.4 MeV proton beam (35 μA, 30 min). The produced [11C]­CO2 was then converted to [11C]­CO using a preheated quartz glass column (6 × 4 × 180 mm; outer diameter × inner diameter × length) heated in a Carbolite oven 850 °C and packed with molybdenum powder (1.5 g, < 150 μm, 99.99% trace metals basis, Sigma-Aldrich, Sweden). [11C]­CO was subsequently treated with coupling reagents (halide precursor, Pd-source, supporting ligand, and amine precursor) in anhydrous THF using a high-pressure microautoclave system (GE, Uppsala, Sweden) following a previously established method. For specific reaction details, e.g., reagent amounts, Pd-catalyst, and supporting ligand, see the following sections below. Purification and formulation were carried out with a computer-controlled automated system (DM Automation, Sweden). Semipreparative HPLC was equipped with a reversed-phase C-18 column (μBondapak, 10 μm, 10 × 300 mm, Waters) with a UV detector wavelength of 254 nm in line with a radiation detector. The purified radioligands underwent further purification via solid phase extraction (SepPak, C18 plus short, Waters) and were then formulated in a solution of 10% ethanol in phosphate-buffered saline (PBS, pH 7.4).

Radioligand Saturation Binding Assay

For membrane preparation, hippocampal tissue from adult rats (Sprague–Dawley) was dissected out, weighed, and frozen in 0.32 M sucrose at −70 °C. On the experiment day, the tissue was thawed, homogenized in 50 mM Tris, 5 mM EDTA (pH 7.4) and then centrifuged at 48 000 xg for 15 min at +4 °C. The pellet was diluted in binding buffer (50 mM Tris, 4 mM MgCl2, 1 mM EDTA pH 7.4) to a final concentration of 2.5 mg w.w./mL. Saturation binding experiments were performed in duplicate, with each tube containing 1 mg w.w. in 0.5 mL binding buffer and 0.1 – 17 nM (11–12 concentrations) of radioligand. Nonspecific binding was measured by adding 10 μM AR-A014418. The assays were incubated for 2–3 h at room temperature and terminated by quick filtration using Whatman GF/B filters pretreated with 0.3% polyethylenimine on a Brandel cell harvester. Radioactivity was measured with a Packard Tri-Carb 2900TR liquid scintillation counter, and data were processed by nonlinear regression analyses using PRISM 4.03.

In Vitro Binding Assays

The inhibition of GSK3β activity is assessed using a scintillation proximity assay (SPA) in a 96-well microtiter plate format. The assay utilizes purified human recombinant GSK3β enzyme (∼1 nM active), [γ-33P]­ATP (0.04 μCi), and a biotinylated peptide substrate (Biotin-AAEELDSRAGS­(PO3H2)­PQL) at a final concentration of 2 μM. Reactions are carried out in an assay buffer containing MOPS, EDTA, β-mercaptoethanol, Brij 35, glycerol, and BSA, and preincubated for 10–15 min at room temperature. Inhibitor testing is conducted in duplicate using 10 different concentrations. The kinase reaction is initiated by adding ATP (1 μM final) in the presence of 50 mM Mg­(Ac)2. After a 15 min incubation at room temperature, the reaction is terminated by adding a solution (25 μL) containing 5 mM EDTA, 50 μM ATP, 0.1% Triton X-100, and 10 mg/mL streptavidin-coated SPA beads, followed by the addition of distilled water (50 μL). After a 6-h incubation, bound radioactivity is measured using a MicroBeta Trilux liquid scintillation counter (1450 MicroBeta Trilux, Wallac). Data are analyzed using nonlinear regression with variable slope (GraphPad Prism or Excel Fit), and results are reported as Ki values calculated via the Cheng-Prusoff equation.

In Vitro Autoradiography

In vitro autoradiography was conducted on 10 μm thick cry-cut tissue sections of rat tissue that had been thaw-mounted onto microscope slides. The brain sections were incubated in 50 mM Tris-HCl buffer at room temperature and then incubated with [3H]­ligand for 30 min. The sections were rinsed three times for 10 min each in Tris buffer at 1 °C, followed by a quick rinse in deionized water at 1 °C, and then air-dried at room temperature with a fan. The radiolabeled sections and Amersham plastic tritium standards were then exposed to phosphoimager plates (Fuji BAS-TR2040) overnight. Blocking studies were conducted using a 1000-fold excess of the cold reference compounds, and validated GSK-3β inhibitors AR-A014418 and SB-216763. These inhibitors were selected based on their well-established selectivity and potency for GSK-3β, as reported in prior pharmacological and imaging studies. , AR-A014418 and SB-216763 were applied at 3 μM, significantly exceeding the IC50 values (4.44 nM) of 13e, to ensure complete displacement of specific radioligand binding. This micromolar concentration is consistent with established methods for GSK-3 radioligand autoradiography blocking experiments.

Radiosynthesis of [11C]13a–e

Palladium­(π-cinnamyl)­chloride dimer (1.4 mg), xantphos (3.4 mg), and halide precursor (2 mg) were added to a 4 mL vial. After flushing the vial with nitrogen, piperidine (5 μL) and 600 μL anhydrous THF was added. The reaction mixture was then loaded into the reagent loop of the module, from which it was directed into the microautoclave precharged [11C]­CO and subsequently reacted at 100 °C for 5 min. [11C]13a was purified at a flow rate of 6 mL/min with acetonitrile/0.1M ammonium formate (v/v= 40/60) as the eluting solvent (tR = 10 min). [11C]13a was reformulated with SPE (SepPak, C18 plus short, Waters) in a mixture of phosphate-buffered saline (10 mL, pH 7.4) and 1 mL EtOH. The resulting solution was sterilized by sterile filtration. Pure [11C]13a was synthesized in 4% RCY (decay-corrected) at EOS (RCP > 99%, Am = 62 GBq/μmol). Following a similar procedure, [11C]13b–d were synthesized in 1.6%–13.8% RCY (decay-corrected) with excellent radiochemical purities (RCP > 99%) and molar activities (Am = 22–99 GBq/μmol) at EOS.

[11C]13e Was Synthesized According to the Following Procedure

Tetrakis­(triphenylphosphine)­palladium­(0) (3.0 mg) and methyl iodide (20 μL) were placed in an oven-dried 4 mL vial. The vial was flushed with nitrogen gas before amine precursor 16 (3 mg) and 600 μL anhydrous THF was added. The resulting mixture was loaded into the reagent loop of the module from where it was transferred into the microautoclave precharged with [11C]­CO. The mixture was heated at 100 °C for 5 min. [11C]13e was purified with 30:70 acetonitrile/0.1% TEA as mobile phase at a flow rate of 6 mL/min (tR = 16 min). The purified products were reformulated using SPE (SepPak, C18 plus short, Waters) in a mixture of phosphate-buffered saline (10 mL, pH 7.4) and 1 mL ethanol. The resulting solution was sterilized by sterile filtration. Pure [11C]13e was synthesized in 12.9% RCY (decay-corrected) at EOS (RCP > 99%, Am = 24 GBq/μmol).

NHP PET Imaging

PET experiments were conducted on anesthetized healthy cynomolgus monkeys, following approval from the Stockholm Animal Research Ethical Committee (Dnr. 4820/06–600). Anesthesia was induced with an intramuscular injection of ketamine hydrochloride (∼10 mg/kg, Ketalar, Pfizer) and maintained via a mixture of sevoflurane (2–8%, Abbott Scandinavia AB), oxygen (∼40%), and medical air after endotracheal intubation. The monkeys’ heads were stabilized with a fixation device, and body temperature was regulated with a Bair Hugger model 505 (Arizant Healthcare, MN, USA) and continuously monitored using an esophageal thermometer. Each radioligand was administered ([11C]13a = 149 MBq; [11C]13b = 155 MBq; [11C]13c = 164 MBq; [11C]13d = 144 MBq; [11C]13e = 156 MBq) as an intravenous bolus injection and dynamic PET images were acquired for 125 min using the high-resolution research tomograph (HRRT; Siemens Molecular Imaging, Knoxville, TN, USA).

Supplementary Material

pt5c00032_si_001.pdf (461KB, pdf)

Acknowledgments

The authors thank Roger Simonsson and Ulrika Yngve for the preparation of the iodides leading to compound [ 3 H]13c and [ 3 H]13e, respectively, and Galina Bessidskaia for analytical support. We are grateful to all members of the PET group at the Karolinska Institutet.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00032.

  • Syntheses of [3H]13a–e (Scheme S1) and TACs of [11C]13a, [11C]13b, [11C]13c, [11C]13d, and [11C]13e in various NHP brain regions (Figure S1) (PDF)

The authors declare no competing financial interest.

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