Synthesis and Biological Investigation of A Novel Fluorine-18 Labeled Benzoimidazotriazine: A Potential Radioligand for In Vivo Phosphodiesterase 2A (PDE2A) PET imaging

Objectives: Cyclic nucleotide phosphodiesterase 2A (PDE2A), an enzyme which hydrolyzes the second messengers cAMP and cGMP, is highly enriched in distinct areas of the brain. Accordingly, PDE2A is involved in important signaling pathways related to normal brain function but also to
neurodegeneration and neuro-oncology [1]. To enable the visualization of this protein in the brainwith PET, we developed a novel fluorine-18 radioligand for PDE2A.
Methods: Based on the benzoimidazotriazine (BIT) tricyclic scaffold, several fluorine-containing derivatives were synthesized via a multi-step synthesis route, and their inhibitory profiles were assessed by PDE isoenzyme-specific activity assays. The most potent and selective PDE2A ligand
BIT1 was radiolabeled via nucleophilic aromatic substitution of the corresponding 2-nitro pyridine precursor by [18F]fluoride in DMSO with thermal heating (Figure 1). [18F]BIT1 was isolated using semi-preparative HPLC (Reprosil-Pur C18-AQ column, 250 x 10 mm, 46% ACN/aqu. 20 mM
NH4OAc, flow 5.5 mL/min) followed by final purification with solid-phase extraction and formulation in isotonic saline containing 10% ethanol. Preliminary in vitro autoradiography and in vivo PET studies (60 min dynamic PET imaging, nanoScan® PET/MRI, MEDISO, Budapest, Hungary) of [18F]BIT1 were performed using pig brain slices and female CD-1 mice, respectively. The in vivo metabolism of [18F]BIT1 was investigated by radio-HPLC analysis of mouse plasma and brain samples at 30 min p.i.
Results: From the series of BIT derivatives, BIT1 was selected as candidate for PET imaging of PDE2A based on the most suitable inhibitory potential and profile (IC50 PDE2A3 = 3.3 nM;16-fold selectivity over PDE10A). [18F]BIT1 was successfully synthesized with a radiochemical yield of 54 ± 2% (n = 3, EOB), molar activities of 155 – 175 GBq/μmol (EOS) and radiochemical purities of ≥99%. [18F]BIT1 was stable in saline, pig plasma, and n-octanol up to 60 min at 37 °C. The distribution pattern of [ 18F]BIT1 in pig brain cryosections corresponds to the spatial distribution of PDE2A with accumulation in the striatal regions caudate nucleus and nucleus accumbens. Additionally, the displacement of [18F]BIT1 with BIT1 as well as TA1 (a potent PDE2A ligand) indicated saturability and selectivity of these binding sites. Uptake of [18F]BIT1 in the brain was shown by subsequent imaging studies in mice (SUVwhole brain = 0.7 at 5 min p.i.); however, more detailed analyses revealed nonspecific distribution of the tracer in the brain (78% parent compound at 30 min p.i.).
Conclusions: The potent and selective PDE2A inhibitor [18F]BIT1 binds in vitro in brain regions known to express PDE2A. Further structural modifications will be performed to develop radiotracers with improved brain uptake and target-selective accumulation in vivo.
Acknowledgement
1. Deutsche Forschungsgemeinschaft (German Research Foundation, SCHE 1825/3-1).
2. Scholarship Program for Research and Innovation in Science and Technology Project
(RISET-PRO)-Indonesia Ministry of Research, Technology and Higher Education (World
Bank Loan No: 8245-ID)

References
[1] S. Schröder, B. Wenzel, W. Deuther-Conrad, M. Scheunemann, P. Brust, Novel Radioligands
for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update
on Developments Since 2012, Molecules 21 (2016) 650–685.