Development and preparation of novel carbon-11/fluorine-18 containing radiotracers for non-invasive PET-imaging of EphB4

Objectives: Members of the Eph receptor tyrosine kinase family play an essential role in the pathogenesis of cancer and, therefore, they are promising candidates for noninvasive molecular imaging, e.g. by PET. Several potent Eph inhibitors are reported either based on high molecular weight compounds like peptides which block the extracellular domain or on small organic molecules which bind to the intramolecular ATP binding pocket of the appropriate receptor. To date, only peptide-based radiotracers containing Cu-64 or In-111 are known which are specific for Eph receptors [1]. Due to favourable chemical and biological properties, small organic inhibitors based on the benzodioxolylpyrimidine structural motif were developed in the past [2]. Unfortunately, these tracers showed only a moderate uptake in tumor cells (A375) and no uptake in vivo in the respective tumor (A375) bearing mice. To overcome these problems, a novel lead structure 1 (IC₅₀ = 1.3 nM) was chosen based on a indazolylpyrazole core [3]. In silico studies were done to determine the best position for the introduction of fluorine-18. Further, the development and preparation of novel ¹¹C/¹⁸F-radiotracers is described. First biological studies were accomplished regarding the stability of the new tracers to figure out the ex vivo and in vivo behavior.
Methods: The first tracer prepared by isotopically labeling with [¹¹C]methyl iodide. For this purpose and for the ¹⁸F-labeling, a protecting group strategy was developed for the regioselective labeling due to three secondary amino groups of this compound. In silico docking studies were performed using the X-ray structure of the EphB4 receptor including a similar inhibitor. Based on these results, two novel references and precursors were evaluated and
prepared for the labeling with fluorine-18.
Results: For both labeling purposes, a protecting group strategy based on the ethoxyethyl (EOE) group was applied. The EOE group was introduced to block the amino function of the indazole core and to avoid radiolabeling on this position.
In the first labeling case, the ¹¹C-labeling was accomplished in a remotely controlled synthesis module in three steps. The first step involved the preparation of [¹¹C]methyl idodide from [¹¹C]methane, the second step comprehended the radiolabeling of the EOE-protected precursor and the final step included the cleavage of the protecting group under acidic conditions leading to [¹¹C]1.
Attempts were made using docking experiments to evaluate the optimal position for the introduction of fluorine-18/-19. In the first case, the methyl group of the molecule was changed against the 3-fluoropropyl residue leading to 2. Secondly, one of the morpholine rings was replaced with the (3-fluoropropyl)piperazinyl moiety yielding 3. Based on these findings, a preparation route to precursors and references was successfully established.
Conclusions: A novel ¹¹C-radiotracer was prepared for the imaging of Eph receptors in cancer using a three step labeling procedure. For radiofluorination purposes, references (2 and 3) as well as precursors were prepared
successfully. Radiolabeling and first promising biological studies are ongoing.
Acknowledgements: Support by the Fonds der chemischen Industrie (FCI-Germany).
References: [1] Xiong C, et al. (2011) J Nucl Med, 52, 241-8.
[2] Mamat C, et al. (2012) ChemMedChem, 7, 1991-2003. Bardelle C, et al. (2010) Biorg Med Chem Lett, 20, 6242-5.