Development of ¹²³I-labelled acrylamides as radiotracer candidates for transglutaminase 2

Objectives: The development of radiotracers for transglutaminase 2 (TGase 2) based on irreversible inhibitors appears highly attractive to further uncover the role of this enzyme for the emergence and progression of various tumour entities. However, the present imaging probes labeled with carbon-11 or fluorine-18 suffer from unfavourable pharmacokinetic properties such as fast blood clearance and metabolism. [1] Furthermore, incorporation of radionuclides with a longer half-life is desirable as the irreversible reaction between the molecular target and the probe might lead to a better signal-to-noise ratio at later time points. To tackle these two aspects, we developed the potent inhibitors 1 and 2 (Scheme) containing pyridine-3-yl residues with electron withdrawing groups and a iodinated phenylacetyl moiety. Herein, we present the radiosynthesis of [¹²³I]1 and [¹²³I]2 using their respective boronic acid pinacol esters as precursors.
Methods: Compounds 1 and 2 and the precursors 1-P and 2-P were synthesised starting from Nα-Boc-L-lysine in a sequence consisting of Nε-acrylation, PyBOP-mediated amide bond formation, Boc deprotection and HATU-mediated Nα-acylation. [2] For optimisation of radioiodination, reactions were performed in HPLC vials as reaction vessels applying 0.5-2 MBq [¹²³I]iodide in 50-100 µL of solvent. In addition to varying additive/precursor/water concentrations, the storage life and premixing time of reagent solutions were investigated. Radiolabelling at higher activity levels (up to 600 MBq) was conducted using 15-50 µL [¹²³I]iodide in total reaction volumes of 200-300 µL. Radiotracers were separated by semi-preparative HPLC. Solid-phase extraction was performed to provide [¹²³I]1 and [¹²³I]2 in suitable formulations for preclinical studies.
Results: 80-100 mg of each reference and precursor compound were obtained in sufficient purities (>95%). While chloramine T did not lead to the formation of [¹²³I]1 and [¹²³I]2, both compounds were accessible by the use of copper(II) trifluoroacetate and 1,10-phenanthroline [3] or tetrakis(pyridine)copper(II) triflate (Cu(OTf)₂(py)₄) [4]. In this context, Cu(OTf)₂(py)₄ in a 4-fold excess to precursor appeared to be most favourable leading to radiochemical conversions of >90% already after 10 min at room temperature (Scheme). These conditions allow for efficient radioiodinations even at 0.1 mM of precursor as well as with a water portion of up to 26%. Furthermore, the reagent solutions can be stored up to 3 month (at -20°C) without significant loss of radiochemical conversion. [¹²³I]1 and [¹²³I]2 were obtained as solutions in ethanol (2 MBq/µL) with (radio)chemical purities of ≥98% and radiochemical yields of >80%.
Conclusion: ¹²³I-labelled irreversible inhibitors of TGase 2 were prepared for the first time. The use of boronic acid pinacol ester as precursor and Cu(OTf)₂(py)₄ as catalyst facilitated the efficient radiolabelling with [¹²³I]iodide. This enables the radiopharmacological characterisation of these potential radiotracers towards their target TGase 2 in vitro and in vivo.
Acknowledgments: The authors thank ROTOP Radiopharmacy for continuously providing [¹²³I]iodide. Financial support by “Europäischer Fonds für regionale Entwicklung (EFRE)” (ML, HJP, and RW) is gratefully acknowledged.
References: [1] van der Wildt et al. Amino Acids 2017, 49, 585; [2] Wodtke et al. J. Med. Chem. 2018, 61, 4528; [3] Wilson et al. Chem. Comm. 2016, 52, 13277; [4] Reilly et al. Org. Lett. 2018, 20, 1752.