Synthesis and characterization of the novel amino acid (S)-[F-18]FAHep for brain tumor imaging: effect of alpha-carbon substitution on brain availability

Objectives Radiolabeled amino acids such as [C-11]methionine, [F-18]FET, and [F-18]FDOPA are substrates for system L transport which is active at the blood-brain barrier (BBB) and is upregulated in many tumors including gliomas. The ability to cross the BBB via system L is critical for visualizing the entire tumor volume, and this property contributes to the efficacy of these tracers for brain tumor imaging. As part of an effort to develop optimized PET tracers for neuro-oncology and better define structure-activity relationships for system L substrates, we have synthesized and characterized two long chain fluoroalkyl amino acids, (S)-2-amino-7-[F-18]fluoro-2-methylheptanoic acid (FAMHep) and (S)-2-amino-7-[F-18]fluoroheptanoic acid (FAHep). These tracers differ only by their alpha-carbon substitution (methyl for FAMHep, hydrogen for FAHep) but were found to have dramatically different biological properties.

Methods Enantiomerically pure tosylate precursors for the radiosynthesis of [F-18]FAMHep and [F-18]FAHep as well as non-radioactive standards were prepared from commercially available starting materials. Incorporation of [F-18]fluoride was performed in 2-methylbutan-2-ol at 102-104°C for 10 min and provided the target compounds in a radiochemical purity greater than 97% and enantiomeric purity greater than 98% after high performance liquid chromatography (HPLC) purification followed by deprotection. Biological evaluation of [F-18]FAMHep and [F-18]FAHep was performed in the mouse DBT glioma model through in vitro competitive uptake inhibition assays as well as in vivo biodistribution and small animal PET studies in male Balb/c mice implanted with subcutaneous and intracranial DBT tumors, respectively. The biodistribution data were expressed as percent of injected dose per gram (%ID/g). The biological properties of these novel tracer were also compared to [F-18]FET, a well-established system L substrate.

Results Both [F-18]FAHep and [F-18]FAMHep were readily radiolabeled (47-83% decay corrected yields, 22-37% end of synthesis yields). In vitro uptake assays demonstrated that the alpha-hydrogen substituted compound [F-18]FAHep was a good and selective substrate for system L amino acid transport with an uptake profile very similar to [F-18]FET. In contrast, the alpha-methyl analogue [F-18]FAMHep was a poor system L substrate. Biodistribution studies demonstrated higher tumor uptake with [F-18]FAHep (9.1 %ID/g) than with [F-18]FAMHep (4.8 %ID/g) at 60 min after injection. The tumor uptake of [F-18]FET was 7.4 %ID/g at this time point. Normal brain uptake was much higher at all time points with [F-18]FAHep (3.6 to 4.2 %ID/g) and [F-18]FET (1.5 to 2.8 %ID/g) than with [F-18]FAMHep (0.4 to 0.5 %ID/g), consistent with the in vitro uptake assays demonstrating that [F-18]FAHep and [F-18]FET are effective system L substrates while [F-18]FAMHep is not. Small animal PET studies in mice with intracranial DBT tumors confirmed the biodistribution results and demonstrated that [F-18]FAHep had higher absolute uptake in DBT tumors and normal brain than both [F-18]FET and [F-18]FAMHep, although tumor to brain ratios for [F-18]FAHep and [F-18]FET were similar.

Conclusions Comparison of the long chain fluoroalkyl amino acids [F-18]FAHep and [F-18]FAMHep demonstrate that the alpha-methyl group plays a key role in the recognition of this class of amino acids by system L amino acid transporters. In vitro and in vivo studies demonstrated that [F-18]FAHep is a very good substrate for system L amino acid with similar properties to [F-18]FET while (S)-[F-18]FAMHep is not an effective system L substrate. This difference in transport is somewhat surprising given the reported brain availability of radiolabeled aromatic amino acids with α-methyl substitutions and is an important consideration for future amino acid tracer design.

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