Direct Aromatic F-18-labeling of Tetrazines:A Rapid and Convenient Entry to Tetrazines for Pretargeted PET Imaging [Meeting Abstract]

<b>134</b> <h3><b>Introduction:</b></h3> Recently, tetrazines have been successfully developed as pretargeted imaging agents. Particularly, they have been of interest when used with trans-cyclooctene (TCO) modified mAbs for pretargeted immunoimaging due to their fast reaction kinetics <i>in vivo</i> as well as their high specificity. (1) Pretargeting is based on a two-step process in which a TCO-tagged nanomedicine, for example an antibody, is administered and allowed to accumulate followed by a short-lived radiolabeled tetrazine. The latter reagent is able to form a stable covalent conjugate with the TCO through a bioorthogonal reaction. (2,3) This approach results in an enhanced safety profile as well as a higher image quality when compared to conventional radioimmunoimaging. (4) Many attempts to label highly reactive tetrazines with ¹⁸F, the most ideal nuclide for PET imaging, have been attempted. However, no successful ¹⁸F-direct labeling approach has been reported so far with enough radiochemical yields (RCYs). (5) In this work, we successfully developed a direct aromatic ¹⁸F-labeling approach of tetrazines. <h3><b>Methods:</b></h3> Initially, a low reactive methyl-tetrazine was selected as a model compound to evaluate the feasibility of the synthesis and radiolabelling of a set of precursors through direct aromatic fluorinations (<i>Figure 1</i>). Thereafter, the best labeling approach was optimized in respect to labeling conditions varying temperature, time and base amount. In a next step, we studied the substrate scope of the reaction on a set of tetrazines including methyl-, H-, phenyl- and pyridyl- tetrazines (<i>Figure 2</i>). The effect of electron withdrawing and electron donating groups on the aromatic ring was evaluated in terms of synthetic accessibility, radiochemical conversion (RCC) and RCY (<i>Figure 3</i>). <h3><b>Results:</b></h3> Out of all tested labeling reactions, radiolabeling only succeeded through Cu-mediated ¹⁸F-fluorination using stannate precursors (<i>Figure 1)</i>. Moderate to good RCYs (10-24%) were obtained for methyl-, phenyl- and H-tetrazines. 2-Pyridyl structures were prohibitive, most likely due to a chelation effect of copper with the respective pyridyl moieties of the tetrazines <i>(Figure 2</i>). Subsequently, we investigated which substitution pattern of the H-tetrazine yielded in highest RCYs. Best results were obtained with a 3,5-substitution pattern (<i>Figure 3</i>). <h3><b>Conclusions:</b></h3> This work showed the first ¹⁸F-direct labeling strategy of highly reactive tetrazines, starting from organotin precursors via a Cu-mediated approach. The developed procedure is simple, short, reproducible as well as scalable and as such, superior to previously used ¹⁸F-multistep labeling strategies with regard to clinical applications. Currently, we are working on the first directly ¹⁸F-radiolabeled tetrazine structures, which will be used for pretargeted brain and cancer imaging. <b>Acknowledgments:</b> This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 813528. This project has also received funding from the European Union9s Horizon 2020 research and innovation program under grant agreement No 668532: Click-it.<b><i>Figure 1</i></b><i>. Radiolabeling strategies using different methyl-tetrazine precursors to probe the fluorination.</i><b><i>Figure 2</i></b><i>. Radiolabeling with Cu-mediated fluorination reaction of higher reactive tetrazines. *The radiolabeled compound was never isolated. †Preliminary studies based on n=2.</i><b><i>Figure 3</i></b><i>. Study of the RCCs when including in the aromatic ring different linkers. *The stannate precursor was never isolated. **The radiolabeled compound was never isolated.</i><i>†Preliminary studies based on n=2.</i>

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