Development of Novel 11C-Labeled Selective Orexin-2 Receptor Radioligands for Positron Emission Tomography Imaging

Orexin 2 receptors (OX2R) represent a vital subtype of orexin receptors intricately involved in the regulation of wakefulness, arousal, and sleep–wake cycles. Despite their importance, there are currently no positron emission tomography (PET) tracers available for imaging the OX2R in vivo. Herein, we report [11C]1 ([11C]OX2-2201) and [11C]2 ([11C]OX2-2202) as novel PET ligands. Both compounds 1 (Ki = 3.6 nM) and 2 (Ki = 2.2 nM) have excellent binding affinity activities toward OX2R and target selectivity (OX2/OX1 > 600 folds). In vitro autoradiography in the rat brain suggested good to excellent in vitro binding specificity for [11C]1 and [11C]2. PET imaging in rat brains indicated that the low brain uptake of [11C]2 may be due to P-glycoprotein and/or breast cancer resistance protein efflux interaction and/or low passive permeability. Continuous effort in medicinal chemistry optimization is necessary to improve the brain permeability of this scaffold.

Numerous drug discovery efforts have been devoted to the development of orexin antagonists for insomnia therapy. 18,19ecently, a number of orexin receptor antagonists have progressed into clinical trials.For example, Merck's dual orexin receptor antagonists (DORAs) named suvorexant the first marketed orexin antagonist for primary insomnia treatment, which was approved by the U.S. Food and Drug Administration (FDA) in 2014 (see Supporting Information, Figure S1, a list of orexin 2 receptor antagonists). 20rthermore, the development of novel and selective orexin-2 antagonists is considered as a promising topic of research in sleep disorder therapy, since evidence continues to validate that the selective inhibition of OX 2 R alone is more adequate for sleep promotion.−23 However, only a few of these molecules have entered clinical trials, and there is no selective orexin-2 antagonist on  the market to date.−26 To investigate the distribution of the OX 2 R and the evaluation of the OX 2 R-targeted neurotherapeutics, there have been continuous efforts in the development of radiolabeled orexin-2 antagonists in the past several years.For instance, [ 11 C]EMPA, 27 [ 11 C]BBAC, 28 [ 11 C]CW4, 29 [ 11 C]FFMMCC, 30 [ 11 C]MK-1064, 31 and [ 18 F]DAN-1 32 have been developed as potential PET radioligands for imaging of OX 2 R (Figure 1).Unfortunately, most of these radiolabeled tracers display insufficient brain uptake or relatively low binding affinity for OX 2 R, which limits further translation in clinical research studies.Therefore, it represents an urgent and unmet need to develop novel potent and selective probes for OX 2 R imaging with high binding affinity, high selectivity, favorable brain uptake, and washout.With the goal of developing a potent and selective OX 2 R PET tracer, we started with the EMPA scaffold, which has already demonstrated excellent antagonist potency (IC 50 = 2 nM). 33The in silico permeability across the BBB (log BB = −1.05) of EMPA was predicted with low possibility by ACD/ Percepta (software used to predict the most common physicochemical, pharmacokinetic, and toxicology properties; ACD/Laboratories, Toronto, ON, Canada, https://www.acdlabs.com/products/percepta).We then modified the structure of EMPA and designed two new analogs 1 and 2 with higher log BB and log P values (log BB = −0.01 and −0.20, and log P = 3.06 and 2.29 for 1 and 2, respectively) predicted by ACD/Percepta, aiming for an improved BBB penetration.The topological polar surface areas (tPSA's) for EMPA, 1, and 2 were predicted by ChemDraw 22.2, indicating the appropriate physicochemical properties for them.Herein, we report the radiosynthesis and preliminary evaluation of ) as novel and potential PET ligands for imaging the orexin 2 receptor (Figure 1).
With compounds 1 and 2 as the molecules of interest, we performed an efficient multistep synthesis of standards and the corresponding phenolic precursors for 11 C-labeling.The synthesis of compound 1 and its phenolic precursor 7 was summarized in Figure 2. Sulfonamide 4 was initially attempted from cross-coupling reactions of 3-methylpyridine-2-sulfonamide and 1-iodo-2-methoxybenzene (path A), but it was unsuccessful.Then, we rerouted with path B, and sulfonamide 4 was obtained from the reaction of amine 3 with sulfonyl chloride in 21% yield.The following nucleophilic substitution was successfully realized with methyl 2-bromoacetate, leading to key intermediate 5 in 57% yield.Hydrolysis of compound 5 under basic conditions gave carboxylic acid 6, which was directly used in the condensation reactions with Nbenzylethanamine without further purification, to generate compound 1 with 29% yield.The phenolic precursor 7 was prepared from the demethylation of compound 1 in the presence of boron tribromide in 41% yield.
The synthesis of compound 2 and its phenolic precursor 12 was summarized in Figure 3.The synthesis of sulfonamide 9 was initially designed from the reaction of 6-methoxypyridin-3amine and pyridine-2-sulfonyl chloride (path A), but it was not successful.Alternatively, sulfonamide 9 was then obtained from the cross-coupling reaction of 3-methylpyridine-2-sulfonamide 8 and 5-iodo-2-methoxypyridine in 60% yield (path B).The following nucleophilic substitution was successfully realized with methyl 2-bromoacetate, leading to key intermediate 10 in 99% yield.Hydrolysis of compound 10 under basic conditions gave carboxylic acid 11, which was directly used in the condensation reactions with N-benzylethanamine without further purification, to achieve compound 2 in 40% yield.The phenolic precursor 12 was prepared from the demethylation of compound 2 in the presence of hydrogen bromide in 45% yield.
To determine the binding affinities of compounds 1 and 2 toward human orexin 2 receptor, we performed the radioligand ([ 3 H]EMPA) competition assays.As shown in Figure 4, compound 1 had an IC 50 value of 7.1 nM and a K i value of 3.6 nM.Compound 2 had a better potency and binding affinity toward OX 2 (IC 50 = 4.4 nM and K i = 2.2 nM).In a selected list of CYP450-mediated drug metabolism panels, low inhibition (IC 50 > 5 μM) was observed for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP19A for both compounds 1 and 2 (Figure S2).Both compounds 1 and 2 displayed low hERG inhibition (IC 50 > 100 μM; Figure S2).Furthermore, we investigated the off-target binding of compounds 1 and 2 in vitro toward more than 50 major transporters, ion channels, and GPCR enzymes in the CNS (supported by the NIMH PDSP).As shown in Figures S3 and  S4, no significant off-target binding (all <50% inhibition) toward 54 CNS targets was observed for both compounds 1 and 2 at a concentration of 10 μM, except the peripheral benzodiazepine receptor for compound 1 (K i = 253 nM).
Based on the favorable in vitro pharmacological and ADME properties, compounds 1 and 2 were labeled with carbon-11 and evaluated as OX 2 PET radioligands.The radiosynthesis of    Sprague−Dawley (SD) rats for 60 min.As depicted in Figure 7, ligand [ 11 C]2 had poor brain uptake, and the highest radioactivity levels in the hippocampus and cerebellum were around 0.4 SUV (2 min postadministration of tracer), followed by washout to 0.2 SUV.Preadministration of OX 2 antagonist, EMPA (1 mg/kg, i.v.), prior to tracer injection, did not remarkably reduce the bound radioactivity levels in the hippocampus and cerebellum (Figure S5).To investigate the effect of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux on brain uptake, we subsequently performed PET imaging of [ 11 C]2 in SD rats with Elacridar (pretreatment), an inhibitor of Pgp and BCRP.Specifically, the SD rats were treated with Elacridar (3 mg/kg) 20 min before injection of the [ 11 C]2. 35The highest radioactivity levels in the hippocampus and cerebellum increased to 0.9 SUV, followed by a quick washout to 0.3 SUV.Although the ligand [ 11 C]2 may be the substrate of Pgp and/or BCRP, low passive permeability could be another confounding factor for the relatively low SUV in the brain.
In conclusion, we have developed two novel OX 2 R PET ligands, compounds 1 and 2. Pharmacological evaluation identified compounds 1 and 2 with high binding affinity (K i = 3.6 nM and 2.2 nM, respectively) and excellent target selectivity (OX 2 /OX 1 > 600 folds for both 1 and 2).The PET ligands [ 11
[ 11 C]1 and [ 11 C]2 was conducted by11 C-methylation34 of phenolic precursors with [ 11 C]CH 3 I, utilizing NaOH or Cs 2 CO 3 as the base in DMF at 80 °C for 5 min (Figure5).The reaction mixtures were then diluted with the HPLC buffer and purified by a semipreparative reverse HPLC to give [11 C]1 and [ 11 C]2 in 71% and 20% radiochemical yields (decaycorrected) with the molar activities of 210 GBq/μmol and 185 GBq/μmol, respectively.The purities of both tracers were greater than 99%, and both [ 11 C]1 and [ 11 C]2 were stable in saline within 90 min.With the ligands [ 11 C]1 and [ 11 C]2 in hand, we subsequently conducted in vitro autoradiography studies using rat brain sections to investigate the binding specificity of [ 11 C]1 and [ 11 C]2 toward orexin 2 receptors.As shown in Figure 6, in the baseline experiments with [ 11 C]1 and [ 11 C]2, relatively high radioactivity accumulation was observed in cortical layer 6 (L6), cornu ammonis 3 (CA3), the central bed nucleus (CM), and medial hypothalamus (MH).Blocking studies with compounds 1 (10 μM), 2 (10 μM), and EMPA (10 μM) remarkably diminished the radioactivity in the OX 2rich brain regions.These results suggested good to excellent in vitro binding specificity for both [ 11 C]1 and [ 11 C]2, in which ligand [ 11 C]2 had superior specific binding in the rat brain.Besides our blocking studies using EMPA analogs, it is