Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC₅₀ = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a¹⁸F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC₅₀ = 8.8 nM) and 3g (IC₅₀ = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [¹⁸F]3f and [¹⁸F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

胆固醇 24-羟化酶 (CH24H, CYP46A1) 是一种细胞色素 P450 家族酶，可维持脑胆固醇的稳态。Soticlestat 是一种有效的选择性 CH24H 抑制剂，正在开发中作为 Dravet 综合征和 Lennox-Gastaut 综合征的治疗剂。在这里，我们报告了芳基哌啶衍生物作为有效和选择性 CH24H 正电子发射断层扫描 (PET) 示踪剂的发现，可用于临床 CH24H 抑制剂的剂量指导和 CH24H 相关病理学的诊断工具。从化合物1 (IC ₅₀  = 16 nM, logD = 1.7) 开始，据报道它是一种亲脂性较低的 CH24H 抑制剂，¹⁸F-标记位点 (3-fluoroazetidine) 通过基于结构的药物设计 (SBDD) 利用化合物1类似物的共晶结构进行整合。随后优化调整 PET 示踪剂的关键参数，例如效力、亲脂性、脑渗透和未结合的血浆蛋白结合，使化合物3f (IC ₅₀  = 8.8 nM) 和3g (IC ₅₀  = 8.7 nM) 成为 PET 成像候选物。这些化合物对 CH24H 的选择性通过使用 CH24H-WT 和 KO 小鼠的脑分布研究得到验证。在非人类灵长类动物 PET 成像中，[ ¹⁸ F] 3f和 [ ¹⁸ F] 3g在大脑中显示出相似的区域摄取，表明这些示踪剂对 CH24H 表达区域具有特异性，并通过不同的示踪剂验证了 CH24H 在活体大脑中的表达。