Following the synthesis of the best virtual candidates, in vitro assays revealed that one member of this chemical series, compound 4, was more active than any of our previous inhibitors, exhibiting a Ki of 1.0 nM. Additional assays also showed that the scaffold of this potent inhibitor, in contrast to the series based on compound 2, is highly metabolically stable. However, upon in vitro testing of 3 and 4 against recombinant FAP, they were completely inactive. Analysis of docking poses revealed a lack of stabilizing interactions with the two glutamic acid residues in the active site of FAP (Glu203 and Glu204).