A New Highly Reactive and Low Lipophilicity Fluorine-18 Labeled Tetrazine Derivative for Pretargeted PET Imaging

Abstract

A new (18)F-labeled tetrazine derivative was developed aiming at optimal radiochemistry, fast reaction kinetics in inverse electron-demand Diels-Alder cycloaddition (IEDDA), and favorable pharmacokinetics for in vivo bioorthogonal chemistry. The radiolabeling of the tetrazine was achieved in high yield, purity, and specific activity under mild reaction conditions via conjugation with 5-[(18)F]fluoro-5-deoxyribose, providing a glycosylated tetrazine derivative with low lipophilicity. The (18)F-tetrazine showed fast reaction kinetics toward the most commonly used dienophiles in IEDDA reactions. It exhibited excellent chemical and enzymatic stability in mouse plasma and in phosphate-buffered saline (pH 7.41). Biodistribution in mice revealed favorable pharmacokinetics with major elimination via urinary excretion. The results indicate that the glycosylated (18)F-labeled tetrazine is an excellent candidate for in vivo bioorthogonal chemistry applications in pretargeted PET imaging approaches.

Keywords: Bioorthogonal chemistry; PET imaging; click chemistry; kinetics; tetrazine.

Scheme 1. Synthesis of [¹⁸F]1 and 1

Reagents and conditions: (a) HATU, DIEA, DMF, RT, 4 h, dark, under argon, yield 69%; (b) 1 M HCl in Et₂O/methanol, RT, 20 h, dark, yield 97%; (c) 0.3 M anilinium acetate buffer pH 4.6, RT, 10 min, yield 96%, ratio of E to Z was 3:1.

Figure 1

Isolated over 95% pure E (black) and Z (gray) isomers interconvert back into the original isomeric mixture (75% E) over time.

Figure 2

In vitro stability of [¹⁸F]1 in PBS pH 7.41 (n = 4) and 50% mouse plasma in PBS pH 7.41 (n = 4 until 4 h, after that n = 2) over a period of 6 h. No degradation products were observed in PBS. Values represent mean ± s.d.

Figure 3

Biodistribution of [¹⁸F]1 in BALB/c male mice at four time points after i.v. administration of 3.0 ± 1.2 MBq (specific activity 12–16 GBq/μmol). Values represent mean ± s.d. (n = 3).

Figure 4

Representative summed (40–60 min) PET-CT image of the biodistribution of radioactivity.