Review articleNicotinic α4β2 receptor imaging agents: Part II. Synthesis and biological evaluation of 2-[18F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) in rodents and imaging by PET in nonhuman primate
Introduction
The α4β2 nicotine acetylcholine receptor (nAChR) is a heteromeric neuronal-subtype receptor that has been implicated in Alzheimer's disease (AD), substance abuse, Parkinson's disease (PD), schizophrenia and other disorders [1], [2], [3], [4], [5]. Due to this clinical relevance of nicotinic α4β2 receptors, efforts on imaging these receptors using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are currently underway in several laboratories [6], [7]. In Part I of our work, we have reported the development of nifrolidine, a putative antagonist PET imaging agent for this receptor subtype [8].
A number of agonist-based potential imaging agents have been studied previously. Toxicity issues as well as slow binding kinetics of these agents hampered the process of selecting optimal agents that would be amenable for eventual human use [6]. After much effort, 18F-2-FA85380 (2-FA; for PET [9], [10]) and 123I-5-A85380 (5-IA; for SPECT [11]) were chosen for studies in humans. Several human studies using 18F-2-FA85380 (2-FA) and 123I-5-IA85380 (5-IA) have been underway. Both of these radiotracers require prolonged imaging times in order to acquire reasonable data for purposes of quantitation [12]. Our objective in this work was to develop an agonist PET imaging agent for α4β2 receptors with relatively rapid kinetics compared to 2-FA and 5-IA.
The four-membered azetidine ring (found in 2-FA and 5-IA) has been known to exhibit higher binding affinity than the five-membered pyrrolidine ring in this series of pyridylether compounds [13]. The relatively slow binding kinetics may be related to this high affinity of 2-FA and 5-IA for α4β2 receptors. Similar, slow binding kinetics were observed with the azetidine ring analog of nifrolidine [14]. Thus, in order to enhance the kinetics of binding in vivo, a moderate affinity agent may be preferable. In order to obtain a moderate affinity agonist agent, we chose to incorporate a five-membered pyrrolidine ring as reported previously in the case of nifrolidine [8]. Additionally, the five-membered ring was unsaturated by inclusion of a double bond in the ring. It was anticipated that inclusion of the double bond (a) may reduce the basicity of the secondary pyrrolidine nitrogen and therefore assist in vivo binding kinetics; (b) may alter the planarity of the five-membered ring to assist in the binding; and (c) that lowered basicity may also allow for a higher lipophilicity, since these compounds tend to have low log P values [15]. Therefore, in an effort to study the effect on binding properties as well as the effect on PET imaging scan times, we evaluated incorporation of a 3,4-dehydropyrrolidine ring instead of the azetidine ring found in 2-FA. Unsaturated amine-bearing ring structures in this class of compounds have hitherto not been reported.
Thus, we have now developed 2-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (abbreviated name: nifene; Fig. 1) as a potential nAChR α4β2 receptor imaging agent. Due to the lack of any substitution in the pyridine ring, this compound would be expected to maintain agonist properties, similar to previous reports on related compounds [16]. We report here the synthesis of nifene, in vitro binding affinity at the α4β2 receptor, radiolabeling with fluorine-18 by nucleophilic aromatic nitro to 18F-fluorine substitution to provide 2-18F-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene; Fig. 1), in vitro autoradiographic studies in rat brain slices with 18F-nifene and PET imaging study in rhesus monkey.
Section snippets
General
All chemicals and solvents were of analytical or HPLC grade from Aldrich Chemical and Fisher Scientific. N-Boc-l-3,4-dehydroproline was purchased from CNH Technologies (Woburn, MA, USA) and 2-fluoro-3-hydroxypyridine was purchased from Asymchem (Durham, NC, USA). 3H-Cytisine (0.99 GBq/μmol specific activity) was purchased from Perkin Elmer Life and Analytical Sciences (Boston, MA, USA). All water was deionized to specific resistance of <18 mΩ cm using a Millipore Milli-Q water purification
Chemistry
The synthesis of the labeling precursor 2-nitro-3-[2-(S)-N-tert-butoxycarbonyl-2-pyrrolinemethoxy]pyridine and reference standard compound 2-fluoro-3-[2-(S)-3-pyrroline) methoxy]pyridine is shown in Fig. 2. Esterification of the commercially available N-Boc-l-3,4-dehydroproline 1 using methanol in THF/triethylamine containing “BOP” reagent gave methyl ester 2 in high yields. The methyl ester group of pyrroline derivative was reduced by LAH in THF at 0°C to ambient temperature to give the (S)-N-
Discussion
We have identified the 3,4-dehydroproline ring as a unique ring system for binding to the α4β2 nAChRs. This ring system was incorporated into the pyridylether skeleton to provide 2-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (nifene). The synthesis of the compound involved a Mitsunobu reaction of the (S)-N-tert-butoxycarbonyl-2-hydroxymethyl-3-pyrroline (7) with 2-fluoro-3-hydroxypyridine. The reaction proceeded with modest yields due to the low-yielding nature of Mitsunobu reactions
Conclusions
A new imaging agent, 18F-nifene, has been prepared for α4β2 receptors. Binding in thalamic and extrathalamic brain regions was observed in both rodent and monkey brain. A unique feature of 18F-nifene compared to previously reported agents is its faster binding kinetics. It therefore has potential for use in animal and human PET studies to study α4β2 receptors.
Acknowledgments
This research was supported by Biological and Environmental Research Program (BER), US. Department of Energy, Grant No. DE-FG02-03ER63598. We would like to thank Dr. Frances Leslie and David Keator for helpful discussions.
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