¹⁸F-Labeled analogs of nisoxetine as PET imaging agents for norepinephrine transporters

Abstract

1822

Objectives: The norepinephrine transporter (NET) found presynaptically to the noradrenergic neurons is associated with several psychiatric and neurological disorders such as depression and Alzheimer’s disease. We have synthesized two ¹⁸F-labeled nisoxetine analogs for evaluation as NET agents, (R)-N-methyl-3-(¹⁸F-3’-fluoropropyl)phenoxy)-3-phenylpropanamine 1 and a less lipophilic analog, (R)-N-methyl-3-(¹⁸F-3’-fluoropropoxy)-3-phenylpropanamine 2.

Methods: The N-BOC protected tosylate precursors, (R)-N-methyl-3-(3’-tosyloxypropyl)phenoxy)-3-phenylpropanamine and (R)-N-methyl-3-(3’-tosyloxypropoxy)-3-phenylpropanamine were synthesized. Radiolabeling of the precursors was carried out using ¹⁸F-fluoride, Kryptofix/K₂CO₃ in acetonitrile at 95°C for 30 mins. Product mixture was purified on RP-HPLC (CH₃CN:H₂O 0.25%Et₃N, 68%:32%; flow rate 2.5 ml/min for 1 and 1.5 ml/min for 2). Brain slices (coronal 10 μm thick) from male Sprague-Dawley rats were used for in vitro binding studies. Slides were preincubated at room temperature for 10 minutes in Tris-HCl buffer (50 mM pH 7.4). Slices were then incubated with 1-2 μCi/cc of ¹⁸F-1 or ¹⁸F-2 at 37°C for 1 hr. Nonspecific binding was measured using 10μM nisoxetine. Slides were rinsed twice for 2 minutes in ice-cold buffer, dipped briefly in ice-cold water, blown dry and laid out for autoradiography. Slides with tissue samples were exposed to a multipurpose storage phosphor screen for 24 hours, developed, and quantified. Specific binding to thalamus (Th), striatum (St), cortex (Ctx) and cerebellum (Cer) were measured.

Results: Radiosynthesis of both ¹⁸F-1 and ¹⁸F-2 after deprotection were obtained in 10-20% yields in 2hrs; RP-HPLC (retention time of ¹⁸F-1 was 21 mins and ¹⁸F-2 was 35 mins) provided specific activites >1000 Ci/mmol. In vitro binding studies in brain slices showed little binding of ¹⁸F-2 to NET-rich regions suggesting the importance of the phenyl ring to NET binding. On the other hand, extensive binding was observed with ¹⁸F-1 in thalamus and cortex indicating the lipophilic nature of ¹⁸F-1. Thalamus, a region known to contain NET, showed higher binding compared to cortex (thalamus/cortex = 2.03) and this binding was marginally reduced in the presence of increasing concentrations of nisoxetine. Nonspecific binding observed with ¹⁸F-1 appears to be a significant factor in degrading analysis of specific NET binding.

Conclusions: Our results suggest that ¹⁸F-1 appears to show some selectivity to NET binding regions in brain slices. In vivo studies need to be performed and evaluated to examine if the high nonspecific binding observed in vitro will be a detriment for development of this agent as a suitable NET PET radiotracer.