[18F]ALX5406: a brain-penetrating prodrug for GlyT1-specific PET imaging

Objectives ALX5407 (1) is a potent and selective inhibitor of glycine transporter type 1 (GlyT1) originallydeveloped for the treatment of certain neurologic disorders like cognitive decline or schizophrenia. While it didnot reach clinical trials, ALX5407 could provide a starting point for development of GlyT1-selective PET tracersand was previously radiolabeled with carbon-11, but no preclinical studies have been published so far. The aim ofthe present work was to prepare the 18F-labeled counterpart [18F]ALX5407 ([18F]1) as well as its methyl ester[18F]ALX5406 ([18F]2), and to subject both candidate tracers to a preclinical evaluation.Methods The radiolabeling precursor was prepared by asymmetric reduction of 4'-bromo-3-chloropropiophenoneinto the respective (R)-alcohol (97% ee), followed by etherification via Mitsunobu reaction with 4-phenylphenol(>95% ee), amination with sarcosine methyl ester and finally Miyaura borylation. The radiosynthesis wasperformed using the protocol for alcohol-enhanced Cu-mediated radiofluorination. To this end, a solution ofEt4NHCO3 in nBuOH (400 μL) was used to elute 18F– from a QMA anion exchange cartridge into a solution of theradiolabeling precursor and Cu(py)4(OTf)2 (30 μmol of each) in DMA (800 μL) and the reaction mixture washeated at 110 °C for 10 min under air to afford [18F]2. The latter was hydrolyzed with 6 M NaOH to give [18F]1.Both tracers were evaluated by in vitro autoradiography in rat brain slices, in vivo μPET imaging in healthy rats,and ex vivo radiometabolite analysis in rat brain tissue and blood.Results The precursor was obtained in 15% yield over four steps. [18F]1 and [18F]2 were prepared in a ready-touseform in radiochemical yields of 55±7% (n=8) and 62±5% (n=4) within 90120 min, respectively, with molaractivities of 14137 GBq/μmol. In vitro evaluations showed accumulation of [18F]1 in brain regions consistentwith the distribution pattern of GlyT1, but in vivo brain uptake of the probe was very low. In contrast, [18F]2showed no specific binding in brain slices, but rapidly crossed the blood brain barrier (BBB) and showed an invivo brain distribution pattern consistent with GlyT1 specific binding. Metabolite studies demonstrated rapidhydrolysis of [18F]2 to [18F]1 in rat brain tissue and blood (t1/2=12 min), confirming that it acts as a BBB-penetratingprodrug.Conclusion [18F]2 is a promising and readily available prodrug for preclinical PET imaging of GlyT1 in the brain.Figur