Development of the First Aliphatic ¹⁸F-Labeled Tetrazine Suitable for Pretargeted PET Imaging—Expanding the Bioorthogonal Tool Box

Abstract

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18 F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18 F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positronemission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [ 18 F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratiosas early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging

“…The tumor-to-liver (T/L) ratio was 0.3. Therefore, the uptake and target-to-background contrast found for [ 18 F]45 were at comparable levels to previous findings [17,32,34]. However, there is a general need to improve the target-to-background contrast before clinical translation.…”

“…Bispyridyl scaffolds could not be labeled with Cu-mediated approaches due to the chelating effect of the pyridyl nitrogen [32,35]. Aliphatic labeling was only applied on monosubstituted Tzs [34]. Bispyridyl Tzs are, however, very appealing subjects to be used for pretargeted experiments (Figure 2).…”

“…We have previously reported that fluoroethyl moieties can be used to introduce 18 Ffluoride into H-Tz employing non-nucleophilic bases [33,34]. We believed that these conditions could also be suitable to label bispyridyl-based Tzs.…”

“…We recently developed a blocking assay that allowed us to assess the in vivo ligation performance of unlabeled tetrazine derivatives, omitting the time-consuming development of radiolabeled tetrazines for each tested ligand. It was based on the ability of Tzs to block the binding of the literature-known pretargeted imaging agent [ 111 In]47 to the pretargeting vector CC49-TCO (administered 72 h prior) in tumor-bearing mice [17,32,34]. In this setup, 45 showed almost complete blocking.…”

“…In a similar way, we were able to develop the first direct aliphatic 18 F-radiolabeled Tz (2) (Figure 2). This was possible using ultralow basic conditions [33,34]. Both compounds (1 and 2) showed high imaging contrast during in vivo experiments, and are currently subjects in further studies to evaluate their potential to be translated into the clinic.…”