Radiosynthesis and evaluation of an ¹⁸F-labeled radioligand for imaging metabotropic glutamate receptor 3 with positron emission tomography

Abstract

The metabotropic glutamate receptor 3 (mGluR3) is a G-protein-coupled receptor (GPCR) involved in modulating glutamatergic neurotransmission and maintaining neural homeostasis. By inhibiting adenylyl cyclase activity, mGluR3 negatively modulates the activity of adenylyl cyclase via Gi/o protein coupling, reducing cyclic AMP (cAMP) levels and modulating downstream signaling pathways. Dysfunction of mGluR3 is associated with a range of neurological and psychiatric disorders, including depression, autism, cognitive impairment, bipolar affective disorder, schizophrenia, and neurodegenerative diseases. Despite its therapeutic relevance, no selective mGluR3 positron emission tomography (PET) radioligand is currently available to image this target in vivo. In this study, we report the radiosynthesis and preclinical evaluation of [¹⁸F]VU6010572 - a novel PET tracer based on a therapeutical drug candidate. VU6010572 exhibits potent binding affinity (IC₅₀ = 39.9 nM) and exceptional selectivity (>100-fold over other mGluR subtypes). Radiolabeling with fluorine-18 yielded [¹⁸F]VU6010572 with high radiochemical yield (48%, decay-corrected) and molar activity (59 GBq/µmol). While in vitro autoradiography demonstrated heterogeneous brain distribution, dynamic PET imaging in rodents revealed reasonable brain uptake in vivo yet modest binding specificity and rapid brain washout. While these findings support the potential of [¹⁸F]VU6010572 as a lead structure, further medicinal chemistry optimization is warranted to enhance the metabolic and pharmacokinetic properties.

Introduction

Glutamate is one of the primary excitatory neurotransmitters in the central nervous system and plays a critical role in regulating synaptic plasticity, learning, memory, and overall neural development and communication under physiological conditions [1,2]. Glutamate exerts its effects through two major classes of receptors: ionotropic glutamate receptors (iGluRs), which are ligand-gated ion channels, and metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors (GPCRs). Based on sequence homology, pharmacological profiles, and signaling mechanisms, mGluRs are further subdivided into three groups (I-III) [3]. Among these, group II mGluRs - comprising mGluR2 and mGluR3 - are negatively coupled to adenylyl cyclase and function to suppress excitatory synaptic transmission through the inhibition of cyclic adenosine monophosphate (cAMP) production. In particular, mGluR3 is widely expressed in both neurons and glial cells, where it orchestrates complex intercellular signaling involved in neuroprotection, synaptic plasticity, and neuroinflammation. Its modulation of neuron-glia communication has been implicated in maintaining central nervous system homeostasis, particularly under conditions of stress or injury. mGluR3 dysfunction has been associated with several neuropsychiatric and neurodegenerative conditions, including schizophrenia, major depressive disorder, autism spectrum disorder, and Alzheimer’s disease [4,5]. These findings emphasize the relevance of mGluR3 as a therapeutic target.

Positron emission tomography (PET) is a powerful, noninvasive imaging modality that enables real-time monitoring of molecular and cellular processes in vivo with high sensitivity [6-8]. The development of PET radioligands targeting key neuroreceptors has reshaped our ability to assess disease pathophysiology, as well as pharmacodynamic responses, and guide the development of central nervous system therapeutics. Despite the biological relevance of mGluR3, efforts to develop selective PET tracers for this receptor subtype have yielded limited success. To date, only three mGluR3-targeted PET radioligands, [¹¹C]CMG, [¹¹C]CMGDE, and [¹¹C]mG3N001 have been reported, yet most of them exhibit dual affinity for mGluR2 and mGluR3, limiting their utility for subtype-specific imaging (Figure 1) [9,10]. To address this unmet need, we report on the radiolabeling and in vivo evaluation of [¹⁸F]VU6010572, a novel ¹⁸F-labeled PET radioligand with high affinity and high selectivity for mGluR3. Indeed, VU6010572 was identified through structure-activity relationship (SAR) optimization and demonstrated >100-fold selectivity over other mGluR subtypes. VU6010572 was successfully radiolabeled with fluorine-18 and subsequently evaluated in a series of preclinical studies, including in vitro autoradiography, dynamic PET imaging, whole-body biodistribution, and radiometabolite analysis in rodents. These efforts represent an important step toward enabling subtype-selective mGluR3-targeted imaging to support translational drug development.

Figure 1.

Representative mGluR3 PET tracers. A. Previously reported mGluR3 PET tracers; B. [¹⁸F]VU6010572.

Material and methods

General information

Chemicals were used directly in synthesis as purchased. The NMR data was recorded on a 300 or 400 MHz NMR spectrometer (see NMR spectra in Supplementary Materials). High-resolution mass (HRMS) data and low-resolution mass (liquid chromatography-mass spectrometry, LC-MS) data were recorded in APCI mode and ESI mode, respectively. Rodents were fed in a light/dark (12 h/12 h) cycle. Animal studies were conducted according to the ethical guidelines of Emory University.

Chemistry

Synthesis of 4-(benzyloxy)-1-(4-fluorophenyl)pyridin-2(1H)-one (2)

To a solution of compound 1 (1.8 g, 9.0 mmol, 1.0 eq.) in CH₂Cl₂ (45 mL), (4-fluorophenyl)boronic acid (2.5 g, 18.0 mmol, 2.0 eq.), pyridine (1.6 ml, 19.9 mmol, 2.2 eq.), and copper (II) acetate (1.8 g, 9.9 mmol, 1.1 eq.) were added. The mixture was stirred at room temperature for 48 hours. Then the reaction mixture was washed with saturated aqueous sodium bicarbonate solution and brine, dried over Na₂SO₄, concentrated, and purified by flash chromatography to afford 2 (white solid, 2.0 g, 75% yield). ¹H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 7.6 Hz, 1H), 7.48-7.37 (m, 7H), 7.32 (t, J = 8.8 Hz, 2H), 6.11 (dd, J = 7.6, 2.8 Hz, 1H), 5.98 (d, J = 2.8 Hz, 1H), 5.14 (s, 2H). LCMS (ESI): calcd for C₁₈H₁₅FNO₂⁺ (M + H⁺): 296.1, found 296.1.

Synthesis of 4-(benzyloxy)-1-(4-bromophenyl)pyridin-2(1H)-one (3)

Compound 3 was prepared through the same procedure described for 2, except that (4-bromophenyl)boronic acid was used instead of (4-fluorophenyl)boronic acid. Compound 3 was obtained in 68% yield (white solid, 1.2 g). ¹H NMR (400 MHz, DMSO-d6) δ 7.68 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.47-7.33 (m, 7H), 6.12 (dd, J = 7.6, 2.8 Hz, 1H), 5.98 (d, J = 2.8 Hz, 1H), 5.14 (s, 2H). LCMS (ESI): calcd for C₁₈H₁₅BrNO₂⁺ (M + H⁺): 356.0, found 356.0.

Synthesis of 1-(4-fluorophenyl)-4-hydroxypyridin-2(1H)-one (4)

To a solution of 2 (1.1 g, 3.7 mmol, 1.0 eq.) in MeOH (25 mL), Pd/C (240.0 mg, 10%) was added. The mixture was stirred at room temperature for 18 hours under the H₂ atmosphere. The mixture was filtered and concentrated to afford 4 (yellow solid, 0.7 g, 92% yield), which was used directly in the next step.

Synthesis of 1-(4-bromophenyl)-4-hydroxypyridin-2(1H)-one (5)

To a solution of 3 (1.42 g, 4.0 mmol, 1.0 eq.) in CH₂Cl₂ (120 mL), BBr₃ (3.0 g, 12.0 mmol, 3.0 eq.) was added at -10°C. The mixture was stirred at -10°C for 15 mins. Then the reaction was quenched with ice water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over Na₂SO₄, concentrated, and purified by flash chromatography to afford 5 (white solid, 0.8 g, 75% yield). ¹H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 7.66 (dt, J = 9.6, 2.8 Hz, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.32 (dt, J = 8.8, 2.8 Hz, 2H), 5.97 (dd, J = 7.6, 2.4 Hz, 1H), 5.65 (d, J = 2.4 Hz, 1H). LCMS (ESI): calcd for C₁₁H₉BrNO₂⁺ (M + H⁺): 266.0, found 266.0.

Synthesis of (S)-1-(4-fluorophenyl)-4-(2-phenoxypropoxy)pyridin-2(1H)-one (6 or VU6010572)

To a solution of 4 (205 mg, 1.0 mmol, 1.0 eq.) in THF (20 mL), (S)-2-phenoxypropan-1-ol (182 mg, 1.2 mmol, 1.2 eq.), PPh₃ (576 mg, 2.2 mmol, 2.2 eq.), DtBAD (di-tert-butyl azodicarboxylate, 368 mg, 1.6 mmol, 1.6 eq.) were added at 0°C. The reaction was stirred at room temperature for 12 h. Then the mixture was concentrated and purified by flash chromatography to afford 6 or VU6010572 (220 mg, 65% yield) as a white solid. ¹H NMR (400 MHz, Chloroform-d) δ 7.34-7.28 (m, 4H), 7.19-7.13 (m, 3H), 7.00-6.95 (m, 3H), 6.01 (dd, J = 7.6, 2.8 Hz, 1H), 5.97 (d, J = 2.8 Hz, 1H), 4.76 (m, 1H), 4.14 (dd, J = 10.0, 6.0 Hz, 1H), 4.04 (dd, J = 10.4, 4.4 Hz, 1H), 1.43 (d, J = 6.4 Hz, 3H). ¹³C NMR (100 MHz, Chloroform-d) δ 167.5, 163.8, 162.1 (d, J = 229.1), 157.5, 137.7, 136.5 (d, J = 3.3 Hz), 129.6, 128.5 (d, J = 8.7 Hz), 121.5, 116.3 (d, J = 22.8 Hz), 116.2, 101.7, 98.2, 71.8, 71.1, 16.8. LCMS (ESI): calcd for C₂₀H₁₉FNO₃⁺ (M + H⁺): 340.1, found 340.1.

Synthesis of (S)-1-(4-bromophenyl)-4-(2-phenoxypropoxy)pyridin-2(1H)-one (7)

Through the same procedure as 6, compound 7 was obtained (white solid, 0.8 g, 87% yield). ¹HNMR (400 MHz, DMSO-d6) δ 7.68 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.29 (dd, J = 8.8, 7.6 Hz, 2H), 6.99 (d, J = 7.6 Hz, 2H), 6.94 (t, J = 7.6 Hz, 1H), 6.06 (dd, J = 7.6, 2.4 Hz, 1H), 5.93 (d, J = 2.8 Hz, 1H), 4.83 (m, 1H), 4.17 (m, 2H), 1.32 (d, J = 6.4 Hz, 3H). LCMS (ESI): calcd for C₂₀H₁₉BrNO₃⁺ (M + H⁺): 400.1, found 400.1.

Synthesis of (S)-4-(2-phenoxypropoxy)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyridin-2(1H)-one (8)

To a solution of 7 (0.8 g, 2.0 mmol, 1.0 eq.) in 1,4-dioxane (110 mL), B₂Pin₂ (1.27 g, 5.0 mmol, 2.5 eq.), KOAc (588 mg, 6.0 mmol, 3.0 eq.), and Pd(dppf)Cl₂ (220 mg, 0.3 mmol, 0.15 eq.) were added. The mixture was stirred at 80°C for 12 hours under the N₂ atmosphere. The mixture was concentrated and purified by flash chromatography to afford 8 (200 mg, 22% yield) as a white solid. ¹HNMR (400 MHz, Chloroform-d) δ 7.91 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.30 (dd, J = 8.8, 7.6 Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 7.2, 1H), 6.96 (d, J = 7.6, 2H), 6.00 (dd, J = 7.6, 2.8 Hz, 1H), 5.96 (d, J = 2.4 Hz, 1H), 4.76 (m, 1H), 4.14 (dd, J = 10.0, 6.0 Hz, 1H), 4.04 (dd, J = 10.0, 4.0 Hz, 1H), 1.43 (d, J = 6.0 Hz, 3H), 1.35 (s, 12H). ¹³C NMR (75 MHz, Chloroform-d) δ 167.3, 163.6, 157.5, 143.0, 137.6, 135.8, 129.6, 125.8, 121.4, 116.2, 101.4, 98.2, 84, 71.8, 71.0, 24.8, 16.8 Hz. HRMS (APCI): for C₂₆H₃₁BNO₅ (M + H⁺): 448.2290, found 448.2294.

Pharmacology

The mGlu3 human glutamate (metabotropic) GPCR cell-based cAMP leadhunter assay with compound VU6010572 was performed by Eurofins. VU6010572 was tested in negative allosteric modulator mode.

Radiochemistry

An aliquot of [¹⁸F]fluoride was added to a vial with tetraethylammonium bicarbonate (TEAB, 1 mg) and dried with nitrogen gas flow at 110°C. When the vial was dry, acetonitrile (1 mL) was added, and the drying process was repeated. Then precursor 8 (2 mg) and [Cu(Py)₄(OTf)₂] (7 mg) in DMAc/nBuOH (0.2/0.1 mL) were added and heated at 110°C under the air atmosphere for 20 min. After that, the reaction mixture was diluted with water and passed through Sep-Pak light C18, which was eluted with MeCN and diluted with water for HPLC purification (see HPLC radio-chromatograms in Supplementary Materials).

In vitro stability

According to a previous report [11], [¹⁸F]VU6010572 was incubated with saline, serums of mouse, rat, NHP, and human at 37°C. At 5, 30, 60, or 120 minutes after incubation with [¹⁸F]VU6010572, the samples were mixed with cold MeCN and centrifuged, and the supernatants were analyzed by radio-HPLC.

In vitro autoradiography

According to a previous report [12], the rat brain sections were incubated with Tris-HCl buffer (50 mM) and then [¹⁸F]VU6010572 at room temperature. For the blocking study, incubation was conducted with VU6010572 (10 μM) and [¹⁸F]VU6010572. After that, the brain sections were washed with cold Tris-HCl buffer (2 minutes) and distilled water (10 seconds). Then the dried brain sections were positioned on an imaging screen and scanned by the Typhoon biomolecular imager.

PET imaging

As previously reported [13,14], PET imaging of [¹⁸F]VU6010572 (0.04 mCi) in CD-1 mice was performed in a Genisys G8 PET scanner for 1 hour. Mice were pre-treated with VU6010572 (3 mg/kg, i.v.) 10 minutes before administration of [¹⁸F]VU6010572 in the blocking study.

Whole-body biodistribution

According to the previous reports [7,15], CD-1 mice were administered with [¹⁸F]VU6010572 (10 μCi) and then euthanized at 5, 15, 30, and 60 minutes, respectively. Major organs were weighed and measured by a gamma counter.

Radiometabolite analysis

Radiometabolite analysis of [¹⁸F]VU6010572 was conducted according to a previous report [16]. CD-1 mice were administered with [¹⁸F]VU6010572 and then euthanized 30 minutes after administration of [¹⁸F]VU6010572. The brain samples were homogenized and centrifuged with cold MeCN and PBS buffer. After that, the supernatants were analyzed by radio-HPLC. The collected [¹⁸F]VU6010572 and its ¹⁸F-metabolites were measured by a gamma counter. The plasma samples were analyzed through the same procedure described for the brain samples.

Results

Chemistry

The synthesis of compound VU6010572 commenced with a copper-mediated cross-coupling reaction of 4-(benzyloxy)pyridin-2(1H)-one 1 and (4-fluorophenyl)boronic acid, generating compound 2 in 60% yield (Figure 2). After the deprotection of the benzyl group in the presence of Pd/C and hydrogen gas, intermediate 3 was obtained in 95% yield. Finally, the condensation reaction of compound 3 and (S)-2-phenoxypropan-1-ol afforded VU6010572 in 65% yield. In short, VU6010572 was synthesized in 37% overall yield over three steps. Similar to the synthesis of VU6010572, its borate precursor was prepared from the cross-coupling reaction of compound 1 and (4-bromophenyl)boronic acid, deprotection of the benzyl group, condensation reaction with (S)-2-phenoxypropan-1-ol, and the following Pd-catalyzed boration reaction. Finally, the borate precursor 8 was obtained in 12% overall yield over four steps.

Figure 2.

Synthesis of VU6010572 (6) and its labeling precursor 8. Reaction conditions: (i) (4-fluorophenyl)boronic acid (for 2) or (4-bromophenyl)boronic acid (for 3), pyridine, Cu(OAc)₂, CH₂Cl₂, N₂, RT, 48 h, 60% yield for 2 and 65% yield for 3; (ii) Pd/C, H₂, MeOH, r.t., 18 h, 95% yield; (iii) BBr₃, -10°C, 15 min, 76% yield; (iv) (S)-2-phenoxypropan-1-ol, PPh₃, DtBAD, THF, RT, N₂, 12 h, 65% yield for 6 and 80% yield for 7; (v) B₂Pin₂, Pd(dppf)Cl₂, KOAc N₂, 80°C, dioxane, 12 h, 30% yield. DtBAD = di-tert-butyl azodicarboxylate, THF = tetrahydrofuran.

Pharmacology

The mGlu3 human glutamate (metabotropic) GPCR cell-based cAMP leadhunter assay was performed with compound VU6010572 in negative allosteric modulator mode and VU6010572 showed an IC₅₀ value of 39.9 nM (Figure 3). VU6010572 had more than 100-fold mGluR3 selectivity over all other mGluRs [17]. Its topological polar surface area (tPSA = 38.77) was predicted by ChemDraw and the logD (3.14) was measured by the shake flask method [18]. The blood-brain barrier (BBB) permeability for VU6010572 (logBB = -0.19) was predicted by ACD/Percepta. Furthermore, an off-target binding screen was conducted for VU6010572 (Figure S1), and no significant off-target binding (>50% inhibition) was observed against 61 major central nervous system (CNS) targets.

Figure 3.

Pharmacological profile of VU6010572. A. Representative pharmacological and physicochemical properties of VU6010572; B. Concentration-response curve of VU6010572 for inhibiting mGluR3 activity.

Radiochemistry and in vitro stability

As shown in Figure 4A, VU6010572 was labeled with fluorine-18 through Cu-mediated ¹⁸F-fluorination of precursor 8 using [¹⁸F]Et₄NF in DMAc/tBuOH at 110°C for 20 minutes [19]. [¹⁸F]VU6010572 was obtained in 48% decay-corrected radiochemical yield with molar activity of 59 GBq/μmol (1.6 Ci/μmol). The in vitro stability of [¹⁸F]VU6010572 was tested in saline and serum across different species. No decomposition was detected in saline for up to 120 minutes (Figure 4B). Furthermore, the stability of [¹⁸F]VU6010572 in the serum of rodents, NHP, and human revealed minimal radiometabolite presence for up to 60 minutes with incubation at 37°C (Figure 4C-F).

Figure 4.

Radiosynthesis of [¹⁸F]VU6010572 and in vitro stability tests. A. Radiosynthesis of [¹⁸F]VU6010572, conditions (i) [¹⁸F]Et₄NF, [Cu(Py)₄(OTf)₂], DMAc/nBuOH, 110°C, 20 min, 48% RCY (decay-corrected); B. Stability of [¹⁸F]VU6010572 in saline; C. Stability of [¹⁸F]VU6010572 in mouse serum; D. Stability of [¹⁸F]VU6010572 in rat serum; E. Stability of [¹⁸F]VU6010572 in NHP serum; F. Stability of [¹⁸F]VU6010572 in human serum.

In vitro autoradiography

To assess the regional binding distribution and specificity of [¹⁸F]VU6010572, in vitro autoradiography was performed using rat brain sections. As illustrated in Figure 5, [¹⁸F]VU6010572 exhibited heterogeneous binding across brain regions, with prominent accumulation observed in the cortex, thalamus, and striatum, regions known to express high levels of mGluR3, and lower levels of radiotracer uptake detected in the cerebellum and pons, consistent with the relatively sparse expression of mGluR3 in these areas [20]. To confirm target-specific binding, competition studies were performed by co-incubating brain slices with an excess of non-radioactive VU6010572 (10 µM). This resulted in a notable reduction in tracer binding within mGluR3-enriched regions, with signal intensity decreased by approximately 31-36% in the cortex, thalamus, and striatum. These findings support the conclusion that [¹⁸F]VU6010572 binds selectively to mGluR3 in vitro, establishing a foundation for its further evaluation as a PET radioligand in vivo.

Figure 5.

Autoradiography of [¹⁸F]VU6010572 in rat brains. A. Representative images for baseline and blocking autoradiography studies with [¹⁸F]VU6010572; B. Quantification of autoradiography studies with [¹⁸F]VU6010572. Cx, cortex; Hp, hippocampus; Th, thalamus; St, striatum; Cb, cerebellum; Po, pons. All data are mean ± SD, n = 5.

PET imaging

To evaluate the in vivo pharmacokinetic profile and brain penetration of [¹⁸F]VU6010572, dynamic microPET imaging was performed in CD-1 mice over a 60-minute scan duration. As shown in Figure 6, [¹⁸F]VU6010572 successfully crossed the blood-brain barrier, achieving an initial brain uptake of 2.5 SUV at 1-minute post-injection, followed by a fast brain clearance.

Figure 6.

PET imaging of [¹⁸F]VU6010572 in mouse brains. A. Representative summed PET images (0-60 minutes) of [¹⁸F]VU6010572 in mouse brains; B. Time-activity curve of [¹⁸F]VU6010572 in the whole brain. All data are mean ± SD, n = 4.

To investigate binding specificity in vivo, blocking studies were conducted using an intravenous pretreatment with non-radioactive VU6010572 (3 mg/kg). No substantial reduction in overall brain uptake was observed (Figure S2), suggesting a high proportion of nonspecific signal or rapidly reversible binding. However, when blood radioactivity was accounted for, a modest reduction in brain-associated signal was detected - specifically, a 13% decrease in PET signal at 45 minutes post-injection - supporting partial target engagement by the blocker (Figure S3). These results demonstrate that [¹⁸F]VU6010572 is brain-penetrant and serves as an initial scaffold for further optimization as an mGluR3-selective PET radioligand, albeit additional refinements may be required to improve in vivo binding specificity [21].

Whole-body biodistribution

To characterize the peripheral distribution and clearance profile of [¹⁸F]VU6010572, whole-body biodistribution studies were performed in CD-1 mice at multiple time points following intravenous injection. As shown in Figure 7 and Table S1, the tracer exhibited initially high uptake (>5 %ID/g) in several organs, including the heart, lungs, pancreas, liver, small intestine, and kidneys - reflecting perfusion-related delivery and tissue-specific retention, respectively. Over the course of 60 minutes, a general decline in radioactivity levels was observed across most tissues. Notably, the small intestine and liver exhibited sustained uptake, with levels remaining elevated at 14.8 %ID/g and 5.6 %ID/g, respectively, at 60-minute post-injection. These findings suggest a hepatobiliary route of excretion, likely contributing to the prolonged retention in gastrointestinal tissues. Importantly, no appreciable accumulation of radioactivity was observed in the bone throughout the study duration, indicating limited in vivo defluorination.

Figure 7.

Whole-body biodistribution study of [¹⁸F]VU6010572 in CD-1 mice. All data are mean ± SD, n = 3.

Radiometabolite analysis

The metabolic stability of [¹⁸F]VU6010572 was assessed in CD-1 mice via ex vivo radiometabolite analysis of plasma and brain samples collected 30 minutes post-injection (Figure 8). In the brain, only 63% of the radioactivity corresponded to the intact parent compound, indicating a substantial fraction of radiolabeled metabolites within the CNS. In plasma, 38% of the radioactivity is attributable to [¹⁸F]VU6010572 at 30 min post-injection. For brain PET tracers, it is generally envisioned that the vast majority of the brain signal arises from the unchanged parent compound, as the presence of brain-penetrant radiometabolites can confound quantitative analysis and complicate interpretation of regional binding. The detection of significant metabolite-derived signal in the brain suggests that [¹⁸F]VU6010572 undergoes metabolic degradation with generation of radiometabolites capable of crossing the blood-brain barrier. In general, the presence of radiometabolites in peripheral compartments such as plasma can be addressed by correcting it using the arterial input function. Overall, the radiometabolite data raises important limitations regarding the suitability of [¹⁸F]VU6010572 for quantitative brain imaging, which emphasizes the need for further structural optimization to enhance metabolic stability and reduce the formation of brain-penetrant radiometabolites.

Figure 8.

Radiometabolite analysis of [¹⁸F]VU6010572 in CD-1 mice. All data are mean ± SD, n = 3.

Discussion

To date, only three ¹¹C-labeled PET radioligand have been reported for imaging mGluR3, yet most of them exhibit similar binding affinities toward mGluR2 and mGluR3, precluding selective imaging of the mGluR3 subtype. In an effort to address this gap, we explored [¹⁸F]VU6010572, a novel ligand with excellent in vitro selectivity for mGluR3 (>100-fold over all other mGluRs) [17]. In the human mGluR3 cAMP-based assay, VU6010572 demonstrated favorable potency and in silico prediction was projected the compound to be brain-penetrant. The compound also exhibited no off-target activity across a panel of 61 CNS targets, confirming its high selectivity in the brain. VU6010572 was efficiently labeled with fluorine-18 using a copper-mediated approach, yielding [¹⁸F]VU6010572 with high radiochemical yield (48%, decay-corrected) and molar activity (59 GBq/μmol). The radiotracer displayed excellent in vitro stability in saline and serum from mouse, rat, non-human primate, and human sources, indicating its suitability for preclinical imaging studies. In vitro autoradiography using rat brain sections confirmed regionally specific accumulation of [¹⁸F]VU6010572 in mGluR3-enriched regions, including the cortex, thalamus, and striatum, with lower uptake in the hippocampus, cerebellum, and pons [20]. Importantly, pretreatment with non-radioactive VU6010572 substantially reduced signal intensity in these high-expression areas, indicating high binding specificity under in vitro conditions.

In vivo PET imaging in CD-1 mice demonstrated that [¹⁸F]VU6010572 crossed the blood-brain barrier and exhibited reasonable brain uptake (SUV of ca. 2.5 at 1 min), followed by rapid washout. Pretreatment with the unlabeled ligand did not lead to a marked reduction in total brain uptake, suggesting limited target-specific binding in vivo. Notably, when blood radioactivity was accounted for, a modest 13% reduction in brain signal at 45 minutes post-injection was observed [21], which may be attributed to the blocking of mGluR3, leading to an increased concentration of free [¹⁸F]VU6010572 in circulation. The whole-body biodistribution profile revealed high early uptake in organs such as the small intestine, liver, and kidneys, consistent with a combination of hepatobiliary and renal elimination. The absence of bone uptake throughout the imaging period suggests metabolic stability of the radiolabel, with negligible in vivo defluorination.

Despite promising in vitro results, radiometabolite analysis revealed that only 63% of radioactivity in the brain corresponded to intact [¹⁸F]VU6010572 at 30 minutes post-injection. Indeed, the presence of a substantial fraction of brain-associated radiometabolites in this study indicates that metabolic degradation of [¹⁸F]VU6010572 generates radiolabeled species capable of crossing the blood-brain barrier - thereby limiting its suitability for quantitative PET imaging. Taken together, [¹⁸F]VU6010572 represents a starting point for the development of selective mGluR3 PET tracers, with favorable in vitro binding characteristics, blood-brain barrier permeability, and metabolic stability against defluorination. However, the partial metabolic instability in vivo, particularly with radiometabolites in the brain, highlights the need for further medicinal chemistry efforts to enhance metabolic resistance and eliminate confounding radiometabolite signals. Such refinements will be critical to realizing the full translational potential of mGluR3-targeted PET imaging.

Conclusion

VU6010572 was identified as a potent and selective negative allosteric modulator of mGluR3 and was successfully radiolabeled with fluorine-18, yielding a PET tracer with high radiochemical efficiency and molar activity. While in vitro autoradiography displayed regionally heterogeneous brain uptake consistent with mGluR3 expression, and exhibited specific binding in mGluR3-enriched regions, in vivo PET imaging confirmed that [¹⁸F]VU6010572 crossed the blood-brain barrier with limited binding specificity. The presence of brain-penetrant radiometabolites and low specific signal present limitations for its use in quantitative in vivo imaging. Continued medicinal chemistry optimization will be required to explore structure-activity relationships (SAR) and modify the phenoxy group, aiming to design novel candidates with enhanced metabolic stability and improved pharmacokinetic properties for the development of a suitable mGluR3 PET radioligand.

Disclosure of conflict of interest

None.