Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging

Pretargeted imaging can be used to visualize and quantify slow-accumulating targeting vectors with short-lived radionuclides such as fluorine-18 – the most popular clinically applied Positron Emission Tomography (PET) radionuclide. Pretargeting results in higher target-to-background ratios compared to conventional imaging approaches using long-lived radionuclides. Currently, the tetrazine ligation is the most popular bioorthogonal reaction for pretargeted imaging, but a direct 18F-labeling strategy for highly reactive tetrazines, which would be highly beneficial if not essential for clinical translation, has thus far not been reported. In this work, a simple, scalable and reliable direct 18F-labeling procedure has been developed. We initially studied the applicability of different leaving groups and labeling methods to develop this procedure. The copper-mediated 18F-labeling exploiting stannane precursors showed the most promising results. This approach was then successfully applied to a set of tetrazines, including highly reactive H-tetrazines, suitable for pretargeted PET imaging. The labeling succeeded in radiochemical yields (RCYs) of up to approx. 25%. The new procedure was then applied to develop a pretargeting tetrazine-based imaging agent. The tracer was synthesized in a satisfactory RCY of ca. 10%, with a molar activity of 134 ± 22 GBq μmol−1 and a radiochemical purity of >99%. Further evaluation showed that the tracer displayed favorable characteristics (target-to-background ratios and clearance) that may qualify it for future clinical translation.