Synthesis of piperazine-based fluorinated fibroblast activation protein inhibitors for the development of a novel 18F-labeled PET imaging probe

Radiolabeled probes addressing the fibroblast activation protein (FAP) expressed among Others by Cancer associated fibroblasts in the microenvironment of tumors emerged as promising drugs for diagnostic imaging and therapy of tumors. ⁶⁸Ga-chelator-based FAP inhibitors are clinically used for the diagnosis of various tumor types. To enhance the imaging quality and improve the applicability, we started to develop covalently ¹⁸F-labeled PET tracers for imaging of FAP in various diseases. For this purpose, four fluorinated quinolinecarboxamides 4a-d were synthesized and biologically evaluated. The seven-step synthesis of 4a comprised a Pd-catalyzed Buchwald-Hartwig reaction of bromoquinoline 6 and a Cu-catalyzed 1,3-dipolar cycloaddition of alkyne 12 with 1-azido-2-fluoroethane (Click reaction) as key steps. The fluorinated quinolinecarboxamides 4a-d showed low nanomolar inhibitory activity on FAP and high selectivity against related Enzymes. Due to its low lipophilicity (logD_7.4 = 0.08) and high metabolic stability (78 % intact after incubation with murine liver microsomes for 90 min), fluoroethyltriazole 4a (IC₅₀ = 1.7 nM) was selected for radiosynthesis. The two-step radiosynthesis with [¹⁸F]-1-azido-2-fluoroethane provided the PET tracer [¹⁸F]4a in acceptable radiochemical yields (10.8 %) and high radiochemical purities (>97.0 %) within a total synthesis time of 156 min. The molar activities were 0.1-5.8 GBq/μmol. In vitro [¹⁸F]4a was stable in human and mouse serum over 90 min. In the biodistribution studies in mice [¹⁸F]4a showed fast renal and hepatobiliary elimination. In a mouse xenograft model with a tumor expressing FAP only very low accumulation in the tumor tissue was observed. This unexpected result was confirmed by the relative low uptake of [¹⁸F]4a by FAP expressing HT1080 cells.

Cu-catalyzed azide-alkyne cycloaddition (click chemistry); Fibroblast activation protein (FAP); Fluorinated PET tracers; In vivo tumor imaging; Lipophilicity; Pharmacokinetics; Positron emission tomography (PET); Radiochemistry; Structure activity relationships.