Abstract View

Introduction

Inverse electron demand Diels-Alder (IEDDA) click reaction has become a prominent tool for pretargeted PET imaging and for radiolabelling in the production of PET radiopharmaceuticals.¹ In the latter case, a significant drawback of IEDDA is caused by the formation of reduced metastable dihydropyridazine (DHP) intermediate products, which present a challenge in quality control.¹ As the DHPs pose a risk in terms of chemical identity, reproducibility, and purity, oxidation is required to convert these DHPs to a stable and easily identifiable pyridazine product (PP).

The potential of ultraviolet (UV) light and increased temperatures in the promotion of oxidation of DHPs has been under scrutiny, however, current literature lacks research on the rapid conversion of DHPs to PP using radioactivity.^1,2,3

Objectives

The main objective was to explore and verify radioactive decay induced oxidation of DHPs in the synthesis process of radiopharmaceuticals for PET imaging. Apart from radioactivity induced oxidation, it was desired to study the oxidation of DHPs using elevated temperatures and UV light.

Methods

IEDDA click reaction was achieved by conjugating a tetrazine [^18/19F]Tz-AmBF₃ 1/[¹⁸F]1 with TCO-PSMA 2 in a mixture of water, ethanol, and pyridazine buffer (pH 2.0) for 10 minutes at 40°C. Subsequently, the mixture containing DHPs 3/[¹⁸F]3 was subjected to three different conditions: temperature (r.t., 40°C, 80°C), UV light (365 nm, 5 W), and radioactivity (1, 3, 6, and 40 GBq using fluorine-18). The reaction mixture was analysed using HPLC after specific oxidation time intervals.

Results

A direct correlation was found between the oxidation rate and amount of radioactivity. The DHP conversion with 1 GBq at the start of synthesis was 82% after 260 minutes, while 3, 6, and 40 GBq reached full conversion within 244, 124, and 60 minutes, respectively (Fig. 1). When the radioactive source was removed from the reaction mixture and placed next to it, gamma radiation had no significant oxidation effect, indicating that positrons are responsible for the oxidation. UV oxidation had superior oxidation efficiency, resulting in a full conversion within 10 minutes of irradiation. Room temperature and 40°C resulted in 56% and 33% yield after 240 minutes, respectively, while 80°C oxidation resulted in sample degradation.

Figure 1. IEDDA click reaction of Tz-AmBF₃ 1/[¹⁸F]1 and TCO-PSMA 2 and the yield (%) of the oxidised PP 4/[¹⁸F]4 over time after the reaction mixture was subjected to radioactivity, increased temperatures, and UV light. The results are averages of triplicate measurements.

Conclusion

Under our conditions, UV light and radioactivity induce rapid conversion of DHPs into PP while increased temperatures do not. Increasing the amount of radioactivity increases the rate of oxidation and positron particles are the main cause of oxidation. This novel and catalyst-free oxidation approach could facilitate more efficient and rapid PET tracer production, significantly advancing PET therapies.

References

[1] Otaru, S.; Martinmäki, T.; Kuurne, I.; Paulus, A.; Helariutta, K.; Sarparanta, M.; Airaksinen, A. J., RSC Adv, 2023, 13 (32), 22606-22615

[2] Karaki, F.; Kiguchi, T.; Itoh, K.; Sato, N.; Konishi, K.; Fujii, H., Tetrahedron, 2021, 97, 132411

[3] Herth, M. M.; Battisti, U. M.; Poulie, C. B. M.; Shalgunov, V. Method for Rapid Oxidation of Dihydropyridazines to Pyridazines; Patent WO2025051721A1, March 13, 2025.