Synthesis and preliminary evaluation of N-(16-¹⁸F-fluorohexadecanoyl) ethanolamine (¹⁸F-FHEA) as a PET probe of N-acylethanolamine metabolism in mouse brain

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, Narachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA). Herein, we report the design and synthesis of N-(16-¹⁸F-fluorohexadecanoyl)- ethanolamine (¹⁸F-FHEA) as a positron emission tomography (PET) probe for imaging the activity of N-acylethanolamine hydrolyzing enzymes in the brain. Following intravenous administration of ¹⁸F-FHEA in Swiss Webster mice, ¹⁸F-FHEA was extracted from blood by the brain and underwent hydrolysis at the amide bond and incorporation of the resultant ¹⁸F-fluorofatty acid into complex lipid pools. Pretreatment of mice with the FAAH inhibitor URB-597 (1 mg/kg IP) resulted in significantly slower ¹⁸F-FHEA incorporation into lipid pools, but overall ¹⁸F concentrations in brain regions were not altered. Likewise, pretreatment with a NAAA inhibitor, (S)-N-(2-oxo-3-oxytanyl)biphenyl-4-carboxamide (30 mg/kg IV), did not significantly affect the uptake of ¹⁸F-FHEA in the brain. Although evidence was found that ¹⁸F-FHEA behaves as a substrate of FAAH in the brain, the lack of sensitivity of brain uptake kinetics to FAAH inhibition discourages its use as a metabolically trapped PET probe of N-acylethanolamine hydrolyzing enzyme activity. (Chemical Equation Presented).