‘In-loop’ [¹¹C]CO₂fixation: Application to the synthesis of a ¹¹C-labeled cholesterol 24-hydroxylase inhibitor

Objectives: Cholesterol 24-hydroxylase (CH24H) is a monooxygenase, which is encoded by CYP46A1 and specifically expressed in the brain [1]. CH24H controls cholesterol elimination by generating 24S-hydroxylcholesterol (24-HC) as the major metabolite. Selective blockade of CYP46A1 activity may suppress neuronal cell death, Aβ deposition and p-tau accumulation by decreasing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer’s disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 by positron emission tomography study.

Methods: The CYP46A1 inhibitor 1 and the corresponding precursor 2 were synthesized according to literature procedure [2]. Briefly, the commercially available tert-butyl 4-(4-phenylpyrimidin-5-yl)piperazine-1-carboxylate 3 was used as the starting material. Trifluoroacetic acid (TFA)-promoted removal of the tert-butyloxycarbonyl (Boc) group proceeded smoothly to give free amine 2 in 99% yield, which served as the labeling precursor for follow-up [¹¹C]CO₂ fixation. Subsequent incorporation of benzylaminocarbonyl group into 2 readily delivered the desired CYP46A1 inhibitor 1 with the combination of benzylamine, triphosgene and N,N-diisopropylethylamine (DIPEA) in THF. Compound 1 was radiolabeled via our previously developed ‘in-loop’ [¹¹C]CO₂ fixation strategy with benzylamine and BEMP as [¹¹C]CO₂ trapping reagents, andPOCl₃ as dehydrating reagent [3]. The lipophilicity of [¹¹C]1 was measured using liquid-liquid partition between n-octanol and PBS (‘shake flask method’). The binding specificity was confirmed via in vivo PET studies in CD-1 mice. The uptake, biodistribution and elimination of [¹¹C]1 was further probed by whole-body ex vivo biodistribution studies in mice at four time points (5, 15, 30 and 60 min) post intravenous (IV) injection of the radioligand.

Results: The CYP46A1 inhibitor 1 was assembled in 51% yield over 2 steps and the precursor 2 were obtained in 99% yield. [¹¹C]1 was isolated in an average 21% radiochemical yield (decay corrected from starting [¹¹C]CO₂ at end of synthesis) within 35 min with high radiochemical purity (>99%) and molar activity (>37 GBq/μmol). No sign of radiolysis was detected up to 90 min after formulation in saline containing 5% ethanol. The lipophilicity of [¹¹C]1 (logD) was measured as 3.05 ± 0.02, which indicated reasonable likelihood to penetrate the blood-brain-barrier. Further in vivo PET studies demonstrated limited brain uptake for [¹¹C]1 with a maximum standard uptake value (SUV) of 0.42 at 0.5 min, followed by a steady washout. Pretreatment of 2-phenylcyclopropan-1-amine (3 mg/kg), a known CYP46A1 inhibitor, reduced the uptake of [¹¹C]1 (~31% decrease of AUC, area under curve). These experiments indicated medium level of in vivo binding specificity for [¹¹C]1. High radioactivity accumulation at 5 min post tracer injection was observed in several organs including lungs, pancreas, small intestine, kidneys and liver (>10% ID/g, injected dose per gram of wet tissue), followed by fast washout. The radioactivity in small intestine reached a climax at 30 min followed by fast elimination, which together with high residue radioactivity in the small intestine and liver, suggested the urinary and hepatobiliary elimination pathway for [¹¹C]1.

Conclusions: We have successfully radiolabeled a CYP46A1 inhibitor 1 in high radiochemical yield and high molar activity. Reasonable lipophilicity and in vivo binding specificity were demonstrated, which may pave the way for future development and evaluation of novel CYP46A1 PET tracers. References: [1] Neuropharmacology 2017, 123,465-476; [2] US20130090341; [3] J. Labelled Comp. Radiopharm. 2018, 61, 252-262.