Structure–Property Investigation of New “KetoFAPI” Inhibitors of Fibroblast Activation Protein (FAP): Discovery of Highly Potent, Selective Compounds with Prolonged Residence Times and Promising Radiotheranostic Potential

Fibroblast activation protein α (FAP) is a serine protease that has emerged as an attractive, pan-cancer radiotheranostic target. The development of effective FAP-targeting radiotheranostics critically relies on the identification of FAP inhibitors that achieve prolonged tumor retention, thereby maximizing therapeutic efficacy and ensuring sustained tumor irradiation, regardless of radionuclide half-life considerations. Here, we present the design, synthesis, and biological evaluation of potent and selective FAP inhibitors bearing an electrophilic α-ketoamide warhead (ketoFAPIs). Through structure–activity relationship studies, we identified highly potent compounds with selectivity over prolyl oligopeptidase (PREP) by up to 3 orders of magnitude. Moreover, this manuscript shows for the first time that ketoFAPIs can exhibit significantly longer target residence times than first-generation FAPIs comprising an electrophilic carbonitrile warhead, which in turn translates into extended tumor retention in a mouse cancer model. This work lays the groundwork for the use of ketoFAPIs as a versatile platform for the development of FAP-targeted radiopharmaceuticals.