Spirocyclic Reconfiguration of the Piperazine Linker Improves Quinoline-Based FAPI PET Tracer Performance

Abstract Improving the tumor uptake and retention without compromising background clearance remains challenging for fibroblast activation protein inhibitor (FAPI) radiotracers. Current approaches usually introduce pharmacokinetic modules or reshape molecular scaffolds, increasing structural complexity and synthetic burden. Spirocyclic rigidification offers a distinct strategy by combining conformational constraint and well-defined three-dimensional geometry, yet has not been leveraged for designing FAPI radiopharmaceuticals. Herein, we synthesized FAPI-04 analogues by replacing the piperazine linker with spirocyclic diamines. All analogues were successfully 68Ga-labeled and retained FAP-specific recognition, hydrophilicity and serum stability, but differed in pharmacokinetics and imaging profiles. [68Ga]Ga-FAPI-S1 exhibited higher tumor uptake, longer tumor residence, and improved tumor-to-background contrast than [68Ga]Ga-FAPI-04 in both HT-1080-FAP and U-87 MG models. Molecular dynamics simulations revealed enhanced local protein−ligand interactions without significant changes in overall complex stability. These findings support spirocyclic replacement of piperazine linker as a feasible strategy for local three-dimensional reconfiguration of quinoline-based FAPI PET tracers.