Synthesis, Radiolabeling, and Evaluation of a Dimeric Fibroblast Activation Protein Inhibitor with a Triazine Linker

Dimeric fibroblast activation protein inhibitor (FAPI) radioligands are promising candidates for theranostic applications because of their enhanced tumor uptake and prolonged retention compared to monomeric radioligands. Several linker strategies have been investigated to connect two FAPI motifs and a radiometal chelator in a dimer design. In this study, we report the development of a novel dimeric FAPI radioligand, [⁶⁸Ga]Ga-1, which utilizes a triazine core as a trifunctional linker. The ligand [^natGa/⁶⁸Ga]Ga-1 was synthesized by chelating gallium or gallium-68 to 2-(DOTAGA-NH-ethyl-NH)-4,6-bis(Gly-FAPI)-1,3,5-triazine (1). [^natGa]Ga-1, a nonradioactive ligand, exhibited FAP-binding affinity comparable to that of FAPI-04 (IC₅₀ = 2.42 nM vs 1.57 nM). [⁶⁸Ga]Ga-1 was synthesized in high decay-corrected radiochemical yields (91.7-94.7%) with high molar activities (40.8-50.0 GBq/μmol) and showed good stability in phosphate-buffered saline and fetal bovine serum. In vitro studies confirmed FAP-specific cellular uptake of [⁶⁸Ga]Ga-1. Positron emission tomography (PET) imaging and ex vivo biodistribution of [⁶⁸Ga]Ga-1 in U87MG tumor-bearing mice demonstrated high tumor uptake and retention, which were significantly blocked by FAPI-04, confirming FAP specificity. Notably, [⁶⁸Ga]Ga-1 demonstrated superior and more sustained tumor uptake than monomeric [⁶⁸Ga]Ga-FAPI-04, indicating improved pharmacokinetics. Altogether, the triazine linker is well suited for constructing dimeric FAPI radioligands, and [⁶⁸Ga]Ga-1 holds potential as a FAP-targeted radioligand for theranostic applications.

DOTAGA; FAP inhibitor (FAPI); PET; dimer; fibroblast activation protein (FAP); gallium-68; triazine.