An alkyne-conjugated quinoline for facile solid-phase construction of FAP-targeting ligands: synthesis, radiolabeling and in vivo evaluation

1049 Objectives: Fibroblast activation protein (FAP) expressed by tumor-associated fibroblasts in stroma has become a promising cancer imaging marker and therapeutic target. Developed by the Heidelberg group, Ga-68 labeled FAP-targeting FAPI-2 and FAPI-4 have been shown to generate excellent tumor-to-background contrast in various cancers. However; the synthetic schemes leading to FAPI-2, FAPI-4 and their derivatives are based entirely on multistep solution-phase synthesis which is time-consuming. In this study, we aimed to synthesize an alkyne-conjugated FAPI-2 pharmacophore which can be clicked on solid-phase for facile construction of DOTA-containing radiolabeling precursors and potentially facilitate the development and optimization of FAP-targeting tracers. Methods: The FAPI-2 pharmacophore, N-​[2-​[(2S)​-​2-​cyano-​1-​pyrrolidinyl]​-​2-​oxoethyl]​-​6-​hydroxy-4-​quinolinecarboxamide, was synthesis by multistep organic synthesis. The alkyne-conjugated derivative was obtained by Mitsunobu coupling of the FAPI-2 pharmacophore with 4-​pentyn-​1-​ol. For a proof-of-concept study, a DOTA-conjugated FAP-targeting ligand, Z06085, was constructed on solid-phase using Fmoc-Lys(ivDde)-Wang resin. Azidoacetic acid was first coupled to the α-amino group, following by the alkyne-conjugated FAPI-2 pharmacophore via the Cu(I) catalyzed click reaction. The metal chelator DOTA was coupled to the e-amino group. After cleavage with trifluoroacetic acid, Z06085 was purified by HPLC. Complexation of nonradioactive Ga and Ga-68 was conducted in acetate buffer (pH 4.5) and HEPES buffer (pH 5.0), respectively. PET imaging and biodistribution studies were carried out in NSG mice bearing FAP-expressing MIA PaCa-2 pancreatic ductal adenocarcinoma xenografts. Results: Z06085 and the nonradioactive standard Ga-Z06085 were obtained with 33% and 71% yields, respectively, and their identities were confirmed by MS analysis. Ga-68 labeled Z06085 was obtained with 63% average radiochemical yield (n= 2) after HPLC purification with > 99% radiochemical purity and 116 GBq/µmole average molar activity. Expression of FAP in MIA PaCa-2 cells was confirmed by Western blot. PET imaging study at 1-h post-injection (p.i.) showed that Ga-68 Z06085 was excreted mainly via the renal pathway (high bladder uptake). However, high radioactivity accumulation in the heart and high background radioactivity indicate that Ga-68 Z06085 was significantly retained in blood at 1-h p.i. Due to the high background radioactivity level the MIA PaCa-2 tumor xenograft could not be visualized in the PET images. The biodistribution data at 1-h p.i. were consistent with the observation from PET images with only moderate uptake in MIA PaCa-2 tumor xenograft (2.33 ± 0.72 %ID/g, n = 4) whereas the uptake in blood was high (6.58 ± 1.73 %ID/g). Conclusions: We successfully synthesized an alkyne-conjugated FAPI-2 pharmacophore, and coupled it to solid-phase via click chemistry for facile construction of an FAP-targeting ligand, Z06085. Such synthetic strategy can be used to facilitate the systematic refinement of FAP-targeting ligands with various pharmacophores and linkers to optimize binding affinity and selectivity. The cause of the unexpected high blood retention of Ga-68 Z06085 is currently being investigated, and this emphasizes that extra cautions are needed for the design of FAP-targeting tracers based on the reported FAPI-02 pharmacophore.