Synthesis and Biological Evaluation of a Novel 18F-Labeled Radiotracer for PET Imaging of the Adenosine A2A Receptor

Abstract

The adenosine A_2A receptor (A_2AR) has emerged as a potential non-dopaminergic target for the treatment of Parkinson's disease and, thus, the non-invasive imaging with positron emission tomography (PET) is of utmost importance to monitor the receptor expression and occupancy during an A_2AR-tailored therapy. Aiming at the development of a PET radiotracer, we herein report the design of a series of novel fluorinated analogs (TOZ1-TOZ7) based on the structure of the A_2AR antagonist tozadenant, and the preclinical evaluation of [¹⁸F]TOZ1. Autoradiography proved A_2AR-specific in vitro binding of [¹⁸F]TOZ1 to striatum of mouse and pig brain. Investigations of the metabolic stability in mice revealed parent fractions of more than 76% and 92% of total activity in plasma and brain samples, respectively. Dynamic PET/magnetic resonance imaging (MRI) studies in mice revealed a brain uptake but no A_2AR-specific in vivo binding.

Keywords: adenosine A2A receptor; fluorine-18; positron emission tomography; tozadenant.

Figure 1

Representative adenosine A_2A receptor (A_2AR) antagonists and the corresponding radiotracers in clinical trials.

Figure 2

Design of a novel ¹⁸F-labeled radiotracer based on the lead compound tozadenant and its fluorinated derivatives.

Scheme 1

Synthesis of novel fluorinated TOZ derivatives.

Figure 3

(A) Overlay of the docking poses of tozadenant (blue) and TOZ1 (green) in the binding site of crystal structure of A_2AR (Protein Data Bank (PDB) ID: 4EIY) and (B) 2D interaction diagram of TOZ1 with key interactions in green.

Figure 4

Investigated reaction conditions for the radiosynthesis of [¹⁸F]TOZ1: (A) solvent, heating mode (RCY = radiochemical yield, TH = thermal heating, MW = microwave heating), reaction time, complex and (B) precursor amount, solvent volume, reaction time.

Figure 5

(A) Radiosynthesis of [¹⁸F]TOZ1 by radiofluorination of the nitro precursor 9. (B) Representative radio- and UV-chromatograms obtained for the isolation of [¹⁸F]TOZ1 by semi-preparative reversed-phase (RP)-HPLC (ReproSil-Pur 120 C18-AQ (250 × 10 mm), 36% acetonitrile (MeCN)/H₂O/0.05% trifluoroacetic acid (TFA), flow rate: 4 mL/min); and (C) radio- and UV-chromatograms of formulated [¹⁸F]TOZ1 co-injected with the corresponding reference compound TOZ1 (ReproSil-Pur 120 C18-AQ column (250 × 4.6 mm), 10-90-10% MeCN/20 mM NH₄OAc_aq. flow rate: 1 mL/min).

Figure 6

Representative radio-chromatograms of the in vivo metabolism study of extracted mouse (A) brain and (B) plasma samples at 30 min post injection (p.i.) of [¹⁸F]TOZ1 (ReproSil-Pur 120 C18- AQ column (250 × 4.6 mm, 5 µm), 10-90-10% MeCN/20 mM NH₄OAc_aq., flow rate: 1 mL/min).

Figure 7

Representative autoradiographic images of (A) the transversal plane of mouse and (B) the sagittal plane of pig brain slices after incubation with 1.1 nM [¹⁸F]TOZ1: (i) Nissl staining; (ii) total binding; and (iii, iv) nonspecific binding in the presence of 1 µM ZM241385; St = striatum, Cb = cerebellum.

Figure 8

(A) Time–activity curves (TACs) of CD-1 mice in different brain regions after injection of [¹⁸F]TOZ1 (n = 7); (B) representative horizontal positron emission tomography (PET) images after pre-treatment with vehicle; (C) TACs of standardized uptake value ratio (SUVr) of striatum-to-cerebellum after pre-treatment with vehicle (n = 7) or tozadenant (2.5 mg/kg, n = 3); and (D) TACs after pre-treatment with vehicle (n = 7) or cyclosporine A (CsA, 50 mg/kg, n = 4). Mean SUV ± SEM.

Figure 9

Biodistribution of [¹⁸F]TOZ1 at different time points based on PET imaging (n = 7, mean SUV ± SEM).