A new series of FAP inhibitors for cancer imaging based on 8-substituted (4-quinolinoyl)-glycyl-2-cyanopyrrolidine.

Fibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts across most epithelial tumors but FAP is nearly absent in normal tissues, making it an attractive target for cancer imaging and therapy. Small-molecule FAP inhibitors have emerged as potent imaging tracers due to their rapid tumor uptake and high tumor-to-background ratios (TBRs), although their short tumor residence limits therapeutic applications. To improve pharmacokinetics and expand theranostic potential, we developed a novel library of FAP inhibitors (eFAPs) based on an 8-quinoline-cyanopyrrolidine scaffold. Structural modifications were introduced by varying linker length between the pharmacophore and chelator, altering global charge, and modulating hydrophilicity. All eFAPs were highly hydrophilic (LogD7.4: -3.2 to -3.7) and demonstrated strong inhibition of both human and murine FAP (IC50, human FAP < 3 nM and IC50, murine FAP < 4 nM, respectively), with high selectivity against prolyl oligopeptidase (PREP) and dipeptidyl peptidase-4 (DPP4). Radiolabeling with [111In]In achieved >96% radiochemical yield for the majority of the derivatives, with overall good stability in PBS and serums up to 24 h. In HT-1080.hFAP cells, eFAPs showed specific uptake (10.2 ± 1.9%-25.8 ± 4.5% AD), with eFAP-9 exhibiting the highest uptake, superior to the clinical reference FAPI-46 (17.5 ± 4.4% AD). In vivo SPECT/CT imaging revealed rapid blood clearance and tumor accumulation, resulting in clear tumor visualization sustained for up to 48 h for eFAP-12. Biodistribution confirmed strong tumor uptake of eFAP-9 (7.95 ± 3.68%ID/g), comparable to FAPI-46 (8.55 ± 1.28%ID/g) at 1 h p.i., and prolonged tumor half-life of eFAP-12 (6.01 h), which was 1.7-fold longer than FAPI-46 (2.84 h).