Nicotinic α4β2 receptor imaging agents. Part IV. Synthesis and biological evaluation of 3-(2-(S)-3,4-dehydropyrrolinyl methoxy)-5-(3'-¹⁸F-fluoropropyl)pyridine (¹⁸F-Nifrolene) using PET

Abstract

Imaging agents for nicotinic α4β2 receptors in the brain have been under way for studying various CNS disorders. Previous studies from our laboratories have reported the successful development of agonist, ¹⁸F-nifene. In attempts to develop potential antagonists, ¹⁸F-nifrolidine and ¹⁸F-nifzetidine were previously reported. Further optimization of these fluoropropyl derivatives has now been carried out resulting in 3-(2-(S)-3,4-dehydropyrrolinylmethoxy)-5-(3'-Fluoropropyl)pyridine (nifrolene) as a new high affinity agent for nicotinic α4β2 receptors. Nifrolene in rat brain homogenate assays--labeled with ³H-cytisine--exhibited a binding affinity of 0.36 nM. The fluorine-18 analog, ¹⁸F-nifrolene, was synthesized in approximately 10%-20% yield and specific activity was estimated to be >2000 Ci/mmol. Rat brain slices indicated selective binding to anterior thalamic nuclei, thalamus, subiculum, striata, cortex and other regions consistent with α4β2 receptor distribution. This selective binding was displaced >90% by 300 μM nicotine. Thalamus to cerebellum ratio (>10) was the highest for ¹⁸F-nifrolene with several other regions showing selective binding. In vivo rat PET studies exhibited rapid uptake of ¹⁸F-nifrolene in the brain with specific retention in the thalamus and other brain regions while clearing out from the cerebellum. Thalamus to cerebellum ratio value in the rat was >4. Administration of nicotine caused a rapid decline in the thalamic ¹⁸F-nifrolene suggesting reversible binding to nicotinic receptors. PET imaging studies of ¹⁸F-nifrolene in anesthetized rhesus monkey revealed highest binding in the thalamus followed by regions of the lateral cingulated and temporal cortex. Cerebellum showed the least binding. Thalamus to cerebellum ratio in the monkey brain was >3 at 120 min. These ratios of ¹⁸F-nifrolene are higher than measured for ¹⁸F-nifrolidine and ¹⁸F-nifzetidine. ¹⁸F-Nifrolene thus shows promise as a new PET imaging agent for α4β2 nAChR.

Figure 1

Chemical Structures of α4β2 radioligands: (a) Nicotine 1; (b) 2-F-A85380 2; (c) Nifene 3; (d) Nifrolidine 4; (e) Nifzetidine 5; (f) Nifrolene 6.

Figure 2

Synthesis scheme for nifrolene 6 synthesis.

Figure 3

(A) Reaction scheme showing the radiosynthesis of ¹⁸F-N-BOC-nifrolene ¹⁸F]13 and ¹⁸F-nifrolene ¹⁸F]6. (B) HPLC purification of ¹⁸F-nifrolene using C₁₈ reverse-phase semi-preparative column eluted with 60% acetonitrile-0.1M ammonium formate at a flow rate of 2.5 mL/min. Retention of ¹⁸F-nifrolene was found to be 15 min.

Figure 4

In vitro autoradiographic studies of ¹⁸F-nifrolene ¹⁸F]6 in rat brain slices. Binding of ¹⁸F-nifrolene in 20 µm horizontal slices (1.6 µCi/cc at 37°C. Horizontal brain slices of rat brain showing binding of ¹⁸F-nifrolene (red = highest binding and white = lowest binding). (A) Total binding (COR: frontal cortex; STR: striatum; TH: thalamus; SUB: subiculum; CB: cerebellum). (B) Binding in the presence of 1 µM nicotine showing partial displacement of ¹⁸F-nifrolene [¹⁸F]6. (C) Binding in the presence of 300 µM nicotine showing >80 displacement of ¹⁸F-nifrolene [¹⁸F]6. (D). Plot showing amount of ¹⁸F-nifrolene binding in brain regions in total, 10⁻⁷ and 10⁻⁶ nicotine.

Figure 5

In vivo MicroPET rat brain slices of ¹⁸F-nifrolene: (A) coronal, (B) sagittal. The thalamus (TH) shows the highest binding while the cerebellum (CB) exhibits little binding. (C) Time-activity curves of the thalamus and the cerebellum show initial rapid uptake in both regions, and greater retention is seen in the thalamus compared to the cerebellum.

Figure 6

In vivo MicroPET rat brain slices (coronal) of ¹⁸F-nifrolene before and after the administration of nicotine. (A) Time-activity curves for the thalamus and cerebellum show initial rapid uptake of ¹⁸F-nifrolene and a lack of retention in the thalamus post-injection of nicotine. (B) Pre-nicotine scans show uptake in the thalamus. (C) Post-nicotine scans exhibit displacement of selective binding of ¹⁸F-nifrolene in the thalamus.

Figure 7

MR-PET Coregistered Monkey Images of ¹⁸F-nifrolene. Distribution of ¹⁸F-nifrolene in the rhesus monkey brain: (A–C) MRI, (D–F) PET. Male rhesus monkey anesthetized with ketamine/isoflurane. Approximately 4 mCi of ¹⁸F-nifrolene injected iv. Summed PET images (100–200 mins) showing binding of ¹⁸F-nifrolene in select brain slices.

Figure 8

PET images of the distribution of ¹⁸F-nifrolene in the rhesus monkey brain (G-R). Brain regions include TH: thalamus; STR: striatum; LG: lateral geniculate; TC: temporal cortex; SB: subiculum; OC: occipital cortex; CB: cerebellum.

Figure 9

(A) Time-activity curves of the binding of ¹⁸F-nifrolene in select areas of the monkey brain corresponding to the regions identified in Figure-7, 8. (B) Ratio plot of brain regions to cerebellum.