A simple solid-phase approach for the synthesis of DOTA-conjugated α-ketoamide-based fibroblast activation...

Several studies have shown that fibroblast activation protein (FAP)-targeted radioligands with an α-ketoamide as the warhead can lead to high affinity for FAP and prolonged tumor retention. However, the preparation of reported FAP-targeted ligands requires multiple solution-phase synthesis and purification steps. Herein, we report a simple solid-phase approach for synthesizing DOTA-conjugated α-ketoamide-based FAP-targeted ligands. The new ligand, FAPI-CRC, was constructed on Rink Amide MBHA resin by sequentially coupling Fmoc-protected amino acids and DOTA-tris(tert-butyl ester), oxidation of α-hydroxyamide to α-ketoamide by 2-iodoxybenzoic acid in dimethyl sulfoxide, and final cleavage/deprotection with trifluoroacetic acid, followed by HPLC purification. FAPI-CRC was labeled with ^natGa/⁶⁸Ga to investigate its ability to target FAP in vitro and in vivo, respectively. ^natGa-FAPI-CRC was obtained in 50% yield and [⁶⁸Ga]Ga-FAPI-CRC was obtained in 27% decay-corrected radiochemical yield with >99% radiochemical purity and a molar activity of 30.8 GBq/μmol. FAPI-CRC and ^natGa-FAPI-CRC exhibited high affinity for FAP with IC₅₀ values of 0.33 ± 0.09 and 0.42 ± 0.27 nM, respectively. Positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice showed that [⁶⁸Ga]Ga-FAPI-CRC was specifically taken up by HEK293T:hFAP tumor xenografts with good tumor-to-background contrast at 1 h post-injection. Therefore, our simple solid-phase approach is promising for the synthesis of potent α-ketoamide-based FAP-targeted ligands and can be used to greatly facilitate the development of radiopharmaceuticals for the management of FAP-expressing cancer.