Synthesis and Preliminary Studies of a Novel Negative Allosteric Modulator, 7-((2,5-Dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[11C]methoxyphenyl) quinoline-2-carboxamide, for Imaging of Metabotropic Glutamate Receptor 2

Abstract

Metabotropic glutamate 2 receptors (mGlu₂) are involved in the pathogenesis of several CNS disorders and neurodegenerative diseases. Pharmacological modulation of this target represents a potential disease-modifying approach for the treatment of substance abuse, depression, schizophrenia, and dementias. While quantification of mGlu₂ receptors in the living brain by positron emission tomography (PET) would help us better understand signaling pathways relevant to these conditions, few successful examples have been demonstrated to image mGlu₂ in vivo, and a suitable PET tracer is yet to be identified. Herein we report the design and synthesis of a radiolabeled negative allosteric modulator (NAM) for mGlu₂ PET tracer development based on a quinoline 2-carboxamide scaffold. The most promising candidate, 7-((2,5-dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[¹¹C]methoxyphenyl) quinoline-2-carboxamide ([¹¹C]QCA) was prepared in 13% radiochemical yield (non-decay-corrected at the end of synthesis) with >99% radiochemical purity and >74 GBq/μmol (2 Ci/μmol) specific activity. While the tracer showed limited brain uptake (0.3 SUV), probably attributable to effects on PgP/Bcrp efflux pump, in vitro autoradiography studies demonstrated heterogeneous brain distribution and specific binding. Thus, [¹¹C]QCA is a chemical probe that provides the basis for the development of a new generation mGlu₂ PET tracers.

Keywords: 11C; Positron emission tomography; mGlu2; metabotropic glutamate receptor 2; negative allosteric modulator.

Figure 1

PET tracers targeting mGlu₂. While the majority of compounds in this figure have no selectivity data available in the primary literature, compound 2 showed mGlu₂ selectivity greater than 350 fold over the other mGlu receptors.

Figure 2

In vitro evaluation of the potencies of QCA, FEQCA, FPQCA, and the control mGlu_2/3 NAM MNI-137 in mGlu₂ GIRK (A) or mGlu₃ GIRK (B) functional assays.

Figure 3

QCA noncompetitively right-shifts the glutamate concentration-response for mGlu₂ and decreases the maximal glutamate response (A) but has no effect on the glutamate concentration-response for mGlu₃ (B).

Figure 4

Ex vivo biodistribution in mice at five different time points (1, 5, 15, 30 and 60 min) post [¹¹C]QCA injection. Data are expressed as %ID/g.

Figure 5

In vitro autoradiography of [¹¹C]QCA binding in rat brain sections. (A) Brain sections were treated with [¹¹C]QCA in the absence (baseline) or presence of QCA, MNI-137, LY-487379 (1 µM each). Cer, cerebellum; Hip, hippocampus; Cx, cortex; Str, striatum. (B) The radioactivity distribution was quantified in regional rat brain. The data are expressed as radioactivity per mm² (n = 4). (C) Blocking studies. The data are normalized to % of radioactivity vs control (n = 4).

Figure 6

PET/MRI fused images of [¹¹C]QCA in rat brain: (A) baseline and (B) self-blocking with QCA (1 mg/kg). (C) Time-activity curves in whole brain under baseline and QCA self-blocking.

Figure 7

Percentages of unchanged [¹¹C]QCA in rat brain tissue and plasma (n = 3) at 5, 20 and 60 min post injection.

Figure 8

PET/MRI fused images in the whole brain of (A) wild-type and (B) Pgp/Bcrp knockout mouse. (C) Time-activity curves of whole brain in wildtype and Pgp/Bcrp knockout mouse after [¹¹C]QCA injection.

Scheme 1

Synthesis of quinoline 2-carboxamide analogs. (i) mCPBA, DCM, 1h; (ii) TMSCN, dimethylcarbamic chloride, DCM, 12 h, 74% for two steps; (iii) mCPBA, DCM, 40°C, 4 h; (iv) POCl₃, DMF, CHCl₃, 70°C, 6 h, 64% yield for two steps; (v) NBS, Benzoyl peroxide, CCl₄, 85°C, 4 h, 60% yield; (vi) succinimide, Cs₂CO₃, DMF, 30 min, 80% yield; (vii) arylboronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane, H₂O, 100°C; (viii) Cs₂CO₃, DMF, 12 h, IC₂H₄F for 14, 45% yield over 2 steps from 11; IC₃H₆F for 15, 53% yield over 2 steps from 11; (ix) sodium percarbonate, 55% yield for QCA (16) over 2 steps from 11, 51% yield for precursor 17 over 2 steps from 11, 60% yield for FEQCA (18), and 53% yield for FPQCA (19).

Scheme 2

Radiosynthesis of [¹¹C]QCA (A) potential labeling methods; (B) ¹¹CH₃I labeling method.