Fluorinated cyclobutyl group for increased metabolic stability using a tyrosine model system

Objectives: [¹⁸F]Fluoroalkyl chains are known to be potential sites for metabolic instability and can result in suboptimal PET image quality [1]. The aim of this work was to develop the [¹⁸F]fluorocyclobutyl substituent as a novel ¹⁸F-labeled prosthetic group that should have an increased metabolic stability whilst maintaining its efficacy. The well-characterized tyrosine LAT transporter system was used as a model test system.
Methods:
The precursors (cis-cyclobutane-1,3-diol ditosylate and tert-butyl N-(tert-butoxycarbonyl)-O-[trans-3-(tosyloxy)cyclobutyl]-L-tyrosinate) and the reference compound, 3-(cis-fluorocyclobutyl)-tyrosine, for the indirect and direct radiosyntheses of 3-(cis-[¹⁸F]fluorocyclobutyl)tyrosine (3-[¹⁸F]FCBT) were synthesized. The radiosynthesis of the 3-[¹⁸F]FCBT via indirect and direct methods were successfully established as outlined in Scheme 1. In vitro studies were performed in A549 cells after incubation with 3-[¹⁸F]FCBT at 37°C for up to 60 min with/without inhibitors FET and non-radioactive 3-FCBT. In vivo PET imaging studies were performed on mice bearing human lung cancer xenografts (A549 carcinoma).
Results:
3-[¹⁸F]FCBT was synthesized via the direct method from precursor 3 in good yield (27-37% d.c.) and excellent radiochemical purity (>99%) in 57-73 minutes. In comparison, the indirect method from the precursor 1 and tyrosine gave only moderate yields (6-12% d.c.) and required longer synthesis times (140-158 minutes). The cell uptake studies showed an increase of 3-[¹⁸F]FCBT over time reaching a plateau of 5.87% at 30 min . In animal PET imaging the A549 lung carcinoma xenografts in mice were clearly visualized by the injection of 6 MBq 3-[¹⁸F]FCBT. The compound showed fast clearance from the blood and renal as well as hepatobiliary excretion. Preliminary in vitro stability investigations using 3-FCBT indicate excellent stability.
Conclusions:
The radiosynthesis of 3-[¹⁸F]FCBT via indirect and direct methods were established The introduction of fluorinated cyclobutyl group into the LAT-targeting tyrosine was shown to be tolerated and actively transported in the in vitro and in vivo setting using A549 cells and their xenografts. The principle of this fluorinated cyclobutyl group as stabilizing moiety will be further investigated.
References:
[1] Treble, (1962) Biochemistry, 82, 129-134