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Tetrazines (Tz) have been applied as bioorthogonal agents for various biomedical applications, including pretargeted imaging approaches. In radioimmunoimaging, pretargeting increases the target-to-background ratio while simultaneously reducing the radiation burden. We have recently reported a strategy to directly 18F-label highly reactive tetrazines based on a 3-(3-fluorophenyl)-Tz core structure. Herein, we report a kinetic study on this versatile scaffold. A library of 40 different tetrazines was prepared, fully characterized, and investigated with an emphasis on second-order rate constants for the reaction with trans-cyclooctene (TCO). Our results reveal the effects of various substitution patterns and moreover demonstrate the importance of measuring reactivities in the solvent of interest, as click rates in different solvents do not necessarily correlate well. In particular, we report that tetrazines modified in the 2-position of the phenyl substituent show high intrinsic reactivity toward TCO, which is diminished in aqueous systems by unfavorable solvent effects. The obtained results enable the prediction of the bioorthogonal reactivity and thereby facilitate the development of the next generation of substituted aryltetrazines for in vivoapplications.

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 vivochemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18F-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 positron-emission-tomography in vivoimaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [18F]15displayed 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.

Background: In our previous work, spiroxatrine was taken as reference compound to develop selective NOP ligands. Therefore, several triazaspirodecanone derivatives were synthesized. Here, we verify their selectivity towards other 5-HT1receptor subtypes and with respect to α2-AR (Adrenergic Receptors). Methods: Binding affinities were determined on cells expressing human cloned receptors for 5-HT1A/B/Dand α2A/B/Csubtypes. The Ki values were determined for those with at least 50% radioligand inhibition. Results: All our derivatives show a moderate affinity for α2subtypes, spanning from 5 to 7.5 pKivalues. Moreover, they show affinity values in a μM–nM range at the 5-HT1Areceptor, while they are practically inactive at 5-HT1Band 5-HT1Dsubtypes. Compound 11, the best of the series, has a 5-HT1ApKivalue of 8.43 similar to spiroxatrine but, notably, it has a 5-HT1Afavorable selectivity ratio of 52, 8 and 29, respectively over α2A, α2Band α2Cadrenoceptor subtypes. Conclusions: In this SAR study, a 5-HT1Aselective ligand has been identified in which a tetralone moiety replaced the 1,4-benzodioxane of spiroxatrine and the methylene linker to the triazaspirodecanone portion was maintained in position 2. Graphic abstract:

The choice of solvent is critical for a different regioselectivity in the reduction of 2,3‐dihydrobenzothiazines (I) with NaBH3CN.