Synthesis and Labeling of Potential PET Radioligands for Receptor Interacting Protein Kinase 1

Objectives: Receptor interacting protein kinase 1 (RIPK1) crucially regulates upstream necroptosis by signaling innate immune receptor [1]. RIPK1 is recognized as a key driver of inflammation and tissue damage involving death receptors [2]. Although significant effort has been made to regulate necrotic cell death by inhibiting RIPK1 [3], no RIPK1 PET radioligands have yet been developed for the potential study of inflammation. Here we report our progress in developing such PET radioligands.

Methods: Molecular docking was used to estimate the binding energy of compounds to RIPK1 as a guide to synthesis efforts. IC₅₀ values were determined with a kinase ADP-glow assay using recombinant human RIPK1. Cyclotron-produced [¹⁸F]fluoride ion was dried in the presence of kryptofix 2.2.2 (K 2.2.2) and base (K₂CO₃). Aryl(mesityl)iodonium salts were prepared from corresponding iodo-compounds in the GSK’963 series and treated with K.2.2.2-K⁺-¹⁸F^- complex in the presence of (MeCN)₄CuOTf in DMF for 20 min at 130 °C. Tri-n-butyltin labeling precursors were synthesized by treating iodo-precursors with hexabutylditin and Pd(PPh₃)₄, and then treated with copper(II)triflate, pyridine, and K.2.2.2-K⁺-¹⁸F^- in DMA at 140 °C for 20 min. Crude radioactive products were analyzed with reversed phase radio-HPLC. Radiochemical yields (RCYs) were calculated from analytical HPLC radio-chromatograms.

Results: Molecular docking provided binding energies for more than 15 new ligands to RIPK1 (Figure 1). Several compounds in the GSK’963, RIPA-9, and RIPA-56 series were synthesized (Scheme 1, typically in 3 to 5 steps). Assays showed that both the RIPA-9 and RIPA-56 analogs were significantly less potent (IC₅₀ > 50 nM) than racemic GSK’963 analogs (IC₅₀ values of < 30 nM). Single enantiomers of potent GSK’963 analogs, separated by chiral-HPLC, showed yet greater potency (IC₅₀ ~ 10 nM). p-¹⁸F-labeled compounds were obtained in fair RCYs (~ 45%) from aryl(mesityl) iodonium tosylates. m-¹⁸F-labeled compounds were obtained in much lower RCYs (~ 6%) from iodonium salts but RCY improved markedly when tri-n-butylstannyl precursor was used (e.g., 60% from 1; Scheme 2).

Conclusions: Several potent RIPK1 inhibitors of recombinant human RIPK1 were identified from molecular docking and a kinase ADP-glow assay after synthesis. Radiolabeling precursors were synthesized and several inhibitors successfully labeled with fluorine-18 in acceptable RCY. Evaluation of radioligand candidates with PET imaging in animal models can now be pursued. Acknowledgements: The NIH Intramural Research Program (NIMH). References: [1] Berger et al. Cell Death Discovery 2015, 1, 15009. [2] Ren et al. J. Med. Chem. 2017, 60, 972. [3] Yoshikawa et al. J. Med. Chem. 2018, 61, 2384.