Synthesis of [¹⁸F]OGA1 and [¹¹C]OGA1 as novel O-linked-β-N-acetyl-glucosamine hydrolase PET radioligands

Objectives: Deregulation of O-GlcNAc modification of nucleocytoplasmic proteins has been linked to several human diseases, including neurodegenerative diseases. Literature evidence suggests that the increase of brain O-GlcNAcylation after treatment with O-linked-β-N-acetyl-glucosamine hydrolase (OGA) inhibitors reduces tau phosphorylation and aggregation in rodents and hinders tau-driven neurodegeneration in a mouse model of tauopathy. In this work, we found that the ligand OGA1 (LSN3316612) is a high-affinity inhibitor for OGA with other promising features for development as a PET radioligand. We synthesized [¹⁸F]OGA1 ([¹⁸F]LSN3316612) and [¹¹C]OGA1 ([¹¹C]LSN3316612) to aid PET imaging studies in animal models and human subjects on the role of OGA in Alzheimer’s disease.

Methods: Affinity of OGA1 for hOGA was determined by assay in vitro. Brain uptake of OGA1 and effect of preblock with thiamet-G were assessed with LC-MS-MS analysis ex vivo in male Sprague Dawley rats. [¹⁸F]OGA1 was synthesized from its nitro-precursor by aromatic nucleophilic substitution with cyclotron produced [¹⁸F]fluoride ion in DMSO on an automated TRACERlab FX_FN apparatus. [¹¹C]OGA1 was synthesized on a modified Synthia platform starting with Pd-mediated insertion of ¹¹CO (generated from cyclotron-produced ¹¹CO₂) into iodomethane in THF within an autoclave, followed by treatment in a V-vial with amine precursor in THF. Each radioligand was purified with HPLC and formulated for intravenous injection.

Results: hOGA IC₅₀ for OGA1 was 1.91 nM. OGA1 rapidly entered rat brain. In frontal cortex brain concentration reached 2.77 SUV at 40 min after administration of OGA1 (10 μg/kg, i.v.). Thiamet-G at >10 mg/kg greatly reduced uptake of OGA1 in brain (~ 60%). [¹⁸F]OGA1 was produced in 90 min from [¹⁸F]fluoride ion in 24 ± 5% (n = 4) yield, and 99.5 ± 0.1% (n = 4) radiochemical purity. Molar activity was 52 ± 17 GBq/μmol (n = 4) at the end of production. Radiochemically pure (> 98%) doses of [¹¹C]OGA1 were obtained in yields of 3.5 ± 1.3% (n = 4) from initial radioactivity and molar activities of 74 ± 39 GBq/μmol (n = 4) at 50 min after radionuclide production. Radioligand identities were confirmed by observation of co-mobility with OGA-1 on radio-HPLC and also by LC-MS of carrier ([M+H]⁺ (observed: m/z = 365.0; calculated: 365.1). HPLC analysis showed that formulated [¹⁸F]OGA1 and [¹¹C]OGA1 were > 97% unchanged after 2 h at room temperature.

Conclusion: [¹⁸F]OGA1 and [¹¹C]OGA1 are promising radioligands and now available in satisfactory radiochemical yields, molar activities, purities, and stabilities to initiate PET imaging studies of O-GlcNAc hydrolase. Research Support: This work was supported by the Intramural Research Program of the National Institutes of Health (NIMH) and by a Cooperative Research and Development Agreement with Eli Lilly & Co.