Identification and Development of a New Positron Emission Tomography Ligand 4-(2-Fluoro-4-[11C]methoxyphenyl)-5-((1-methyl-1 H-pyrazol-3-yl)methoxy)picolinamide for Imaging Metabotropic Glutamate Receptor Subtype 2 (mGlu2)

Abstract

Metabotropic glutamate receptor 2 (mGlu₂) is a known target for treating several central nervous system (CNS) disorders. To develop a viable positron emission tomography (PET) ligand for mGlu₂, we identified new candidates 5a-i that are potent negative allosteric modulators (NAMs) of mGlu₂. Among these candidates, 4-(2-fluoro-4-methoxyphenyl)-5-((1-methyl-1H-pyrazol-3-yl)methoxy)picolinamide (5i, also named as [¹¹C]MG2-1812) exhibited high potency, high subtype selectivity, and favorable lipophilicity. Compound 5i was labeled with positron-emitting carbon-11 (¹¹C) to obtain [¹¹C]5i in high radiochemical yield and high molar activity by O-[¹¹C]methylation of the phenol precursor 12 with [¹¹C]CH₃I. In vitro autoradiography with [¹¹C]5i showed heterogeneous radioactive accumulation in the brain tissue sections, ranked in the order: cortex > striatum > hippocampus > cerebellum ≫ thalamus > pons. PET study of [¹¹C]5i indicated in vivo specific binding of mGlu₂ in the rat brain. Based on the [¹¹C]5i scaffold, further optimization for new candidates is underway to identify a more suitable ligand for imaging mGlu₂.

Figure 1.

Chemical structures of some modulators of mGlu₂.

Figure 2.

Chemical structures of the targeted compounds 5a–5i and the radioligands [¹¹C]5i and [¹¹C]4 for mGlu₂ in this study.

Figure 3.

Concentration-inhibition relationships of compounds 5a–5i to glutamate responses reflecting mGlu₂ (A) and mGlu₃ (B) functions, compared to the lead compounds 3 and 4.

Figure 4.

Assays to determine the antagonistic activity of 5i. The glutamate-induced increases in thallium flux were evaluated for both mGlu₂ (A) and mGlu₃ (B) in the absence or in the presence (30, 10, 3.3, and 1.1 μM, and 370, 123, and 41 nM) of 5i.

Figure 5.

In vitro autoradiography with [¹¹C]5i and [¹¹C]4 using rat brain sections. Representative autoradiographs were obtained by incubating sagittal brain sections with [¹¹C]5i (A) and [¹¹C]4 (B) solutions including either DMSO (vehicle), unlabeled 5i or 4 (10 μM, self-blocking), or 20 (10 μM). Ratios of total binding against nonspecific binding on autoradiographs with [¹¹C]5i (C) and [¹¹C]4 (D) were estimated in the cortex, striatum, hippocampus, thalamus, cerebellum, and pons. ***p < 0.001, **p < 0.01, *p < 0.05 (vs. control).

Figure 6.

Summed (0–60 min) PET/MRI-fused images and time-activity curves of [¹¹C]5i (A and B) and [¹¹C]4 (C and D) in the whole brains of wild-type and Pgp/BCRP-knockout mice.

Figure 7.

Averaged parametric PET/MRI-fused images, time-activity curves (n = 4, mean ± SD) of [¹¹C]5i in control (A and D), self-blocking (B and E), and 4-blocked (C and F) rat brains, and quantitative data for distribution volume ratio (DVR) in brain regions (G). The DVR was estimated using the TAC from the reference region (pons). ***p < 0.001, *p < 0.05 (vs Control)

Figure 8.

Percentages (n = 3, mean ± SD) of the unchanged form of [¹¹C]5i in the plasma and brain homogenate at 5 min and at 20 min after the injection.

Scheme 1.

Synthesis of mGlu₂-targeted compounds 5a–5i. a) NaH, THF, 0°C to room temperature, 10–12 h; b) DMSO, NaOH, room temperature, 20 min.

Scheme 2.

Synthesis of desmethyl precursor 12 for radiolabeling: a) NaH, THF, 0°C to room temperature, 12 h; b). DMSO, NaOH, 20 min, room temperature; c) MeOH, 40°C, 10 h.

Scheme 3.

Synthesis of the desmethyl precursor 17 for radiolabeling: a) NaH, THF, 0°C to room temperature, 12 h; b). NaOH, DMSO, room temperature, 20 min; c) MeOH, 40°C, 10 h; d) NaH, THF, room temperature, 4 h; e) THF, room temperature, 3 h.

Scheme 4.

Radiosynthesis of [¹¹C]5i and [¹¹C]4. a) NaOH, DMF, 80 °C, 5 min; b) Cs₂CO₃, DMSO, 90 °C, 5 min.