Synthesis and Pharmacological Evaluation of Fluorinated Quinoxaline‐Based κ‐Opioid Receptor (KOR) Agonists Designed for PET Studies

Abstract κ‐Opioid receptors (KORs) play a predominant role in pain alleviation, itching skin diseases, depression and neurodegenerative disorders such as multiple sclerosis. Therefore, imaging of KOR by a fluorinated PET tracer was envisaged. Two strategies were followed to introduce a F atom into the very potent class of cis,trans‐configured perhydroquinoxalines. Whereas the synthesis of fluoroethyltriazole 2 has already been reported, fluoropyrrolidines 14 (1‐[2‐(3,4‐dichlorophenyl)acetyl]‐8‐[(R)‐3‐fluoropyrrolidin‐1‐yl]‐perhydroquinoxalines) were prepared by SN2 substitution of a cyclic sulfuric acid derivative with hydroxypyrrolidine and subsequent transformation of the OH moiety into a F substituent. Fluoropyrrolidines 14 showed similar low‐nanomolar KOR affinity and selectivity to the corresponding pyrrolidines, but the corresponding alcohols were slightly less active. In the cAMP and β‐arrestin assay, 14b (proton at the 4‐position) exhibited similar KOR agonistic activity as U‐50,488. The fluoro derivatives 14b and 14c (CO2CH3 at the 4‐position) revealed KOR‐mediated anti‐inflammatory activity as CD11c and the IFN‐γ production were reduced significantly in mouse and human dendritic cells. Compounds 14b and 14‐c also displayed anti‐inflammatory and immunomodulatory activity in mouse and human T cells. The PET tracer [18F]‐2 was prepared by 1,3‐dipolar cycloaddition. In vivo, [18F]‐2 did not label KOR due to very fast elimination kinetics. Nucleophilic substitution of a mesylate precursor provided [18F]‐14c. Unfortunately, defluorination of [18F]‐14c occurred in vivo, which was analyzed in detail by in vitro studies.


Materials
The guinea pig brains for KOR, MOR, and σ1receptor binding assays, rat brains for DOR binding assay and rat liver for σ2 binding assay were commercially available (Harlan- Chemicals and reagents were purchased from different commercial sources and of analytical grade.

Preparation of membrane homogenates from guinea pig brain 1-3
Five guinea pig brains were homogenized with the potter (500-800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 23500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23500 x g (20 min, 4 °C). This procedure was repeated twice.
The final pellet was resuspended in 5-6 volumes of buffer and frozen (−80 °C) in 1.5 mL portions containing about 1.5 mg protein/mL.

Preparation of membrane homogenates from rat brain 4-6
5 rat brains (Sprague Dawley rats) were homogenized with the potter (500-800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes S3 of buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500 x g (20 min, 4 °C). This procedure was repeated twice. The final pellet was resuspended in 5-6 volumes of buffer and stored at -80,°C in 1.5 mL portions containing about 1.5 mg protein/mL.

Preparation of membrane homogenates from rat liver 1-3
Two rat livers were cut into small pieces and homogenized with the potter (500-800 rpm,

Protein determination
The protein concentration was determined by the method of Bradford, 7 modified by Stoscheck. 8 The Bradford solution was prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in 2.5 mL of EtOH (95%, v/v). 10 mL deionized H2O and 5 mL phosphoric acid (85%, m/v) were added to this solution, the mixture was stirred and filled to a total volume of 50.0 mL with deionized H2O. The calibration was carried out using bovine serum albumin as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg/mL). In a 96-well standard multiplate, 10 µL of the calibration solution or 10 µL of the membrane receptor preparation were mixed with 190 µL of the Bradford solution, respectively. After 5 min, the UV absorption of the protein-dye complex at  = 595 nm was measured with a platereader (Tecan Genios, Tecan, Crailsheim, Germany).

General procedures for the binding assays
The test compound solutions were prepared by dissolving approximately 10 µmol (usually 2-4 mg) of test compound in DMSO so that a 10 mM stock solution was obtained. To obtain the required test solutions for the assay, the DMSO stock solution was diluted with the respective assay buffer. The filtermats were presoaked in 0.5 % aqueous

Determination of MOR affinity (guinea pig brain) 4-6
The assay was performed with the radioligand

Determination of DOR affinity (rat brain) 4-6
The assay was performed with the radioligand