Development of highly affine 18F-labelled radiotracers for PET imaging of the adenosine A2A receptor

Objectives: The adenosine A2A receptor (A2AR) is a G-protein-coupled-receptor which is mainly expressed in the basal ganglia (including striatum) of the brain and in cells of the immune system. Radiotracers for A2AR imaging have emerged as promising candidates for the diagnosis of neurodegenerative and neurooncological diseases. Aiming at the development of such radiotracer with improved molecular imaging properties, a library of 21 fluorinated pyrazolo[2,3-d]pyrimidine derivatives was synthesized based on a recently published lead compound [1]. Among those, the highly affine 4 fluorobenzyl derivate 1 (Ki(hA2A) = 5.3 nM; Ki(hA1) = 220 nM) and the 2 fluorobenzyl derivate 2 (Ki(hA2A) = 2.1 nM; Ki(hA1) = 147 nM) were chosen for 18F isotopoic labelling although the introduction of 18F at non-activated aromatic positions is challenging. Herein, we report on the radiosyntheses of [18F]1 and [18F]2 via an alcohol-enhanced copper-mediated one-step radiofluorination and their first biological evaluation.

Methods: Three different labelling strategies for the synthesis of [18F]1 have been investigated (Fig. 1). The first two were using [18F]fluorobenzaldehyde ([18F]B) as intermediate, which was produced by nucleophilic radiofluorination of a trimethylammonium precursor of type A (step a). Compound [18F]B was used either in a reductive amination reaction (step b) or it was further reduced to the corresponding alcohol (step c) followed by an Appel bromination to get [18F]C (step d) which was finally used in a benzylation reaction (step e). The third strategy, a one-step approach, started from the boronic acid pinacol ester precursor of type D employing [18F]TBAF and Cu(OTf)2(py)4 in n-BuOH/DMA (step f). The specific binding of [18F]1 and [18F]2 was evaluated in vitro by autoradiography of mice brain slices using 1, 2 and ZM241385 as different blocking agents.

Results: The two- and four-step labelling strategies resulted in an overall radiochemical yield of only 1.4% and 10%, respectively for [18F]1 (non-isolated). Therefore, [18F]1 and [18F]2 were prepared by an alcohol-enhanced copper-mediated one-step radiolabelling approach starting from the corresponding boronic acid pinacol ester precursor D. Compound [18F]1 was obtained with a radiochemical yield of 52+7% (n = 5, EOB), a molar activity of 135+64 GBq/µmol (n = 4, EOS) and a radiochemical purity of >98%. Compound [18F]2 was synthesized with a radiochemical yield of 9+1% (n = 2, EOB), a molar activity of 132 GBq/µmol (n = 1, EOS) and a radiochemical purity of >98%. In vitro autoradiography performed with [18F]2 showed high binding in the striatum, which could be blocked by selective A2AR ligands thus proving the specificity of the new radiotracer (Fig. 1).

Conclusions: An efficient copper-mediated one-step radiolabelling procedure was established for two new highly affine A2AR radiotracers. In a first in vitro study on mice brain slices, [18F]2 demonstrated excellent imaging properties. Further biological in vitro and in vivo investigations are needed to completely evaluate the potential of both A2AR radiotracers.

Acknowledgments: This work has been supported by the the European Regional Development Fund and Sächsische Aufbaubank (project no. 100226753).

References: [1] Gillespie et al., Bioorg. Med. Chem. Lett. 2008, 18, 2924-2929.