Letter pubs.acs.org/chemneuro
Fluorine-18-Labeled Antagonist for PET Imaging of Kappa Opioid Receptors Zhengxin Cai,*,† Songye Li,† Richard Pracitto,† Antonio Navarro,‡ Anupama Shirali,† Jim Ropchan,† and Yiyun Huang† †
PET Center, Department of Radiology and Biomedical Imaging, Yale University, New Haven, Connecticut 06520, United States Eli Lilly and Company, Indianapolis, Indiana 46285, United States
‡
S Supporting Information *
ABSTRACT: Kappa opioid receptor (KOR) antagonists are potential drug candidates for diseases such as treatment-refractory depression, anxiety, and addictive disorders. PET imaging radiotracers for KOR can be used in occupancy study to facilitate drug development, and to investigate the roles of KOR in health and diseases. We have previously developed two 11C-labeled antagonist radiotracers with high affinity and selectivity toward KOR. What is limiting their wide applications is the short half-life of 11C. Herein, we report the synthesis of a first 18F-labeled KOR antagonist radiotracer and the initial PET imaging study in a nonhuman primate. KEYWORDS: Kappa opioid receptor, PET, fluorination, nonhuman primate, iodonium ylide, microfluidic
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he kappa opioid receptor (KOR) is one subtype of the opioid receptors, and is expressed in the human brain, spinal cord, and immune cells.1 KOR plays important roles in depression,2 anxiety,3 drug abuse,4−7 Alzheimer’s disease,8 immunomodulation,9,10 tumor growth,11−13 and the sensation of pain.14,15 Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders worldwide, and is the leading cause of morbidity among other mental and behavioral disorders. Nearly half of MDD patients develop treatment-refractory depression (TRD), which represents a challenge in MDD management.16 Recently, the development of KOR antagonists as therapeutics for TRD has attracted great interest from both pharmaceutical industry and academia, leading to promising drug candidates in clinical trials such as JDTic (1) and CERC-501 (2, previously known as LY2456302, Figure 1).17−26 The availability of readily accessible KOR PET imaging probes will facilitate the development of KOR antagonist therapeutics, and provide diagnostic and prognostic tools in disease research.27 Over the years, we have developed generations of highly selective 11 C-labeled KOR ligands as PET imaging probes.28−32 For example, we reported the synthesis and evaluation of the first 11 C-labeled KOR antagonist 11C-LY2795050 (3, Figure 1) using 11 C-hydrogen cyanide radiochemistry in 2013.33,34 In 2014, we reported a second generation KOR antagonist as a PET imaging © XXXX American Chemical Society
Figure 1. Selected KOR antagonists as drug candidates and PET imaging probes.
Received: August 26, 2016 Accepted: October 14, 2016
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DOI: 10.1021/acschemneuro.6b00268 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience probe, 11C-LY2459989 (4, Figure 1).35 By replacing the chlorine with fluorine on the aromatic ring, the binding affinity toward KOR increased by 4-fold, without compromising the selectivity (Ki values are 0.18, 7.68, and 91.3 nM for κ, μ, and δ opioid receptors, respectively). Consistent with the in vitro data, dynamic PET imaging using 11C-LY2459989 in nonhuman primates showed higher specific binding signals than 11CLY2795050, with more than 2-fold increase in nondisplaceable binding potential (BPND). However, due to the short half-life of 11 C (20.38 min), the use of these radiotracers requires an on-site cyclotron, which limits their wide applications in clinical setting. Thus, an 18F-labeled PET tracer, which is accessible through distribution from a centralized production, is desirable. The presence of a fluorine on the aromatic ring of LY2459989 opened the possibility for 18F-labeling. Our initial strategy to synthesize the 18F-labeled LY2459989 was to apply the traditional nucleophilic aromatic substitution reaction using the iodo or nitro precursors. These radiolabeling precursors were readily synthesized from known compounds 4-hydroxybenzaldehyde (5) and 4-fluoro-3-iodobenzonitrile (6) or 4-fluoro-3nitrobenzonitrile (7), via a nucleophilic aromatic substitution reaction, followed by reductive amination of 4-(4-formylphenoxy)-3-iodobenzonitrile (8) or 4-(4-formylphenoxy)-3-nitrobenzonitrile (9) with (S)-2-(pyrrolidin-2-yl)pyridine (10) to form 11 or 12 (Scheme 1). Both traditional heating and
Scheme 2. Radiosynthesis of 18F-LY2459989 (14) via the Iodonium Ylide Precursor (16)
With the labeling precursor 16 in hand, a screening of radiolabeling conditions showed N,N-dimethylformamide (DMF) as the preferred reaction solvent. While radiolabeling using acetonitrile (MeCN) only produced minimal incorporation of 18F-fluoride (Table S1, entries 1−3); 18F-fluorination in DMF at 120 °C for 5 min yielded the cyanide intermediate 13 in 19% radiochemical yield (RCY), with 10% hydrolyzed to 14 (Table S1, entry 4). Prolonged heating did not increase the overall yields (Table S1, entries 5 and 6). The incorporation yields were not improved by using K222 as complexing agent and K2CO3 as the base (Table S1, entries 7 and 8). By reducing the amount of base from 10 mg to 2 mg, RCY was raised to 49%, when heating at 80 °C for 5 min (Table S1, entry 9). Neither longer reaction times nor elevated temperatures improved the overall yields (Table S1, entries 10−17). Radiofluorination conditions were further optimized using a commercially available continuous flow microfluidic reactor, NanoTek, by varying the flow rates of reagent solutions and temperatures. Optimization runs in the microfluidic reactor indicated that although low incorporation was obtained at 80 °C (5%) or 100 °C (21%), excellent incorporations were achieved at 120−160 °C, with 120 °C being optimal (76% incorporation). Higher temperatures led to decomposition of the precursor (Tables S2 and S3). The optimal flow rate was 20 μL/min for both the precursor and fluoride solutions. As we found that a significant amount of the cyanide intermediate 13 was hydrolyzed to a more polar product, presumably the corresponding carboxylic acid, with only 52% of 13 converted to 14 (Table S4, entry 1) when sodium hydroxide was used as the base for hydrolysis, we optimized this step by testing different bases and temperatures. Using DMSO as an additive, potassium carbonate as the base, and hydrogen peroxide as the oxidant, 13 was converted to 14 at ambient temperature in 10 min or at 80 °C in 5 min (Table S4, entries 2 and 3).27 Without DMSO, the hydrolysis was slower at ambient temperature (72% after 10 min), but quite efficient at 80 °C over 5 min (94%) without any observable side reactions (Table S4, entries 4 and 5). After extensive optimization, we settled on the final conditions for the preparation of 14 by heating a mixture of 18F-fluoride and the iodonium ylide precursor in DMF at 80 °C for 5 min, followed by hydrolysis of the intermediate 13 to the final product 14. More than 30-fold higher yields were obtained with the iodonium ylide precursor 16 (30−43% RCY) than with the iodo (11) or nitro (12) precursor (
