Abstract
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Objectives: We recently published a novel method for Cu-mediated ortho-C(sp2)-H radiofluorination of aromatic carboxylic acids that are protected as 8-aminoquinoline benzamides with K18F.1 In this work, we have further optimized the method to develop a fully automated synthesis of [18F]AC261066 (a labeled Retinoic Acid Receptor β2 (RARβ2) agonist). The method provides doses that are suitable for use in pre-clinical PET imaging studies.
Methods: K18F was produced in a cyclotron and azeotropically dried using an automated radiochemistry synthesis module. Once the K18F was dry, it was opened to the atmosphere for 1 min. To the K18F under atmosphere, a solution of pre-AC261066 (20 µmol, 1 equiv), (MeCN)4CuOTf (10 µmol, 0.5 equiv), DBU (40 µmol, 2 equiv), and N-methylmorphorine (NMM, 0.18 mmol, 9 equiv) in DMF (1 mL) was added. The reaction vial was open to the air for 1 min and pre-stirred at r.t for 5 min before heating at 100°C for 30 min. The resulting mixture of [18F]inter-AC261066 (Scheme) was diluted with 25mM EDTA solution (60 mL) and loaded on a C18 plus Sep-Pak. [18F]Inter-AC261066 was then eluted from the Sep-Pak using EtOH (2 mL) and transferred to the second reactor. To the [18F]inter-AC261066 solution in EtOH, 4M NaOH aq. solution (1 mL) was added for hydrolysis at 120°C for 20 min. The reaction mixture was then acidified by 6N HCl (0.7 mL) and diluted with 3 mL of HPLC buffer (acetonitrile/water, v/v 0.1% TFA) = 55/45). The mixture was purified by semi-preparative HPLC and [18F]AC261066 was collected and reformulated (C18) to obtain the final dose in 5 mL volume (10% EtOH in saline). Activity yield (AY), radiochemical purity (RCP) and molar activity (Am)2 were determined by Capintec dose calibrator, radio-TLC and analytical HPLC. Results: According to our previous studies,1 oxygen is the key oxidant for oxidation of Cu (I) to Cu (II) in the C-H radiofluorination. RCP after Sep-Pak purification of [18F]inter-AC261066 from automated synthesis by supplying air in to the synthesis module was considerably higher (21 ± 2%, n=3) when compared to the RCP obtained from the automated synthesis under Ar as reported in our recent publication (12 ± 2%, n=3)1. Hydrolysis of the amide formed the desired product [18F]AC261066 in 99 ± 1% RCP from [18F]inter-AC261066 (n=3). Isolated and formulated decay-corrected AY of [18F]AC261066 was found to be 9 ± 1% based upon starting [18F]fluoride (n=3). The product was obtained in high Am (0.80 ± 0.25 Ci/µmol, n=3).
Conclusions: We successfully developed a method for the automated synthesis of high specific doses of RARβ2 agonist [18F]AC261066 in two steps. Pre-clinical studies with [18F]AC261066 are underway and, overall, this operationally simple process holds great potential for the late-stage radiofluorination of bioactive molecules. Acknowledgements: This work was supported by NIH (R01EB021155) and DOE (DE-SC0012484). References: [1] S.J. Lee et al. Angew. Chem. Int. Ed.10.1002/anie.201812701; [2] H.H. Coenen et al. Nucl. Med. Biol. 2017, 55, v-xi.