Radiosynthesis and Evaluation of ¹⁸ F-Labeled Deuterated Radioligand for Positron Emission Tomography Imaging of Cholesterol 24-Hydroxylase

Brain cholesterol homeostasis is critical for neuronal function and primarily regulated by cholesterol 24-hydroxylase (CYP46A1). Dysregulation of CYP46A1 has been implicated in Alzheimer's disease (AD) and Huntington's disease (HD). Building on the clinically validated positron emission tomography (PET) tracer [¹⁸F]-CHL-2205, we designed a deuterated isotopologue, CHL-2205<i>-d</i> ₃, targeting the amide <i>N</i>-methyl group to enhance stability and enable mechanistic studies. Compound <b>5</b> exhibited high CYP46A1 affinity (IC₅₀ = 0.38 nM; <i>K</i> _i = 0.22 nM). Radiosynthesis via copper-mediated [¹⁸F]-fluorination afforded [¹⁸F]<b>5</b> in 31.5 ± 1.5% non-decay-corrected radiochemical yield and high molar activity (>95 GBq/μmol). Autoradiography and PET imaging in mice demonstrated robust brain uptake, heterogeneous regional distribution, and specific target engagement. Radiometabolite analysis confirmed that brain radioactivity was mainly attributable to intact [¹⁸F]<b>5</b>, with a pharmacokinetics comparable to that of [¹⁸F]-CHL-2205. [¹⁸F]<b>5</b> preserves [¹⁸F]-CHL-2205 imaging performance and provides a deuterated PET tool for quantitative bioanalysis and integrated PET-deuterium metabolic imaging (DMI) studies of brain cholesterol metabolism.