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Radiolabeling and evaluation of cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl) carbamate (PK68), a potent inhibitor for receptor interacting protein 1 kinase (RIP1)
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Objectives: The receptor interacting protein 1 kinase (RIP1) is well-known as a key enzyme to regulate neuronal cell death, indicating relationship with several central nervous system disorders. Recently, Hou J et al has identified potent inhibitors for RIP1 [1]. Among, cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl)carbamate (PK68) showed the highest inhibitory efficacy (mIC50 = 13 nM) for RIP1. The aim of this study is to radiolabel PK68 with 11C and to evaluate its distribution in healthy mice.
Methods: [11C]PK68 was synthesized by the reaction of an amine precursor with [11C]acetyl chloride ([11C]AcCl) that was prepared by the reaction of methyl magnesium bromide with [11C]CO2, followed by chlorination with oxallyl chloride and distillation (Scheme 1), using an automated synthesis system developed in house [2]．Dynamic PET scans were conducted for 60 min (1 min × 4 frames, 2 min × 8 frames, and 5 min × 8 frames) using healthy mice. For the blocking study, the mouse was intravenously administrated with unlabeled PK68 (1 mg/kg) before staring a PET scan. Metabolite analysis of [11C]PK68 was performed using the plasma and liver samples by radio-HPLC system. Biodistribution of [11C]PK68 in mice was evaluated by measuring radioactivity in tissues of mice sacrificed at several time points (1, 5, 15, 30, and 60 min) after the injection.
Results: At the end of synthesis, 670 ± 68 MBq (n = 6) of [11C]PK68 was obtained with >99% radiochemical purity and > 37 GBq/μmol of molar activity, starting from about 24 GBq of [11C]CO2. The fully-automated synthesis time was 30 min from the end of irradition. This radioactive product showed > 95% radiochemical purity within 60 min after the formulation. PET imaging with [11C]PK68 showed high uptakes in the liver and kidney of mouse in early time after the injection. Although the uptake of radioactivity in the liver was slightly decreased by treatment with unlabeled PK68, there was no significant difference between control and blocking subjects. Metabolite analysis exhibited relative high stablilty of [11C]PK68 in the plasma and liver in vivo. In ex vivo biodistribution study, it was suggested that [11C]PK68 was metabolised in the liver and removed from body via the hepatobiliary excretion and intestinal reuptake pathway.
Conclusions: In this study, we successfully radiosynthesized [11C]PK68 and evaluated its dynamics and stability in vivo.
Radiolabeling and evaluation of cyclohexyl (5-(2-acetamidobenzo[d]thiazol-6-yl)-2-methylpyridin-3-yl) carbamate (PK68), a potent inhibitor for receptor interacting protein 1 kinase (RIP1)
