Synthesis and Evaluation of 68Ga-Labeled (2 S,4 S)-4-Fluoropyrrolidine-2-Carbonitrile and (4 R)-Thiazolidine-4-Carbonitrile Derivatives as Novel Fibroblast Activation Protein-Targeted PET Tracers for Cancer Imaging

Abstract

Fibroblast activation protein α (FAP-α) is a cell-surface protein overexpressed on cancer-associated fibroblasts that constitute a substantial component of tumor stroma and drive tumorigenesis. FAP is minimally expressed by most healthy tissues, including normal fibroblasts. This makes it a promising pan-cancer diagnostic and therapeutic target. In the present study, we synthesized two novel tracers, [⁶⁸Ga]Ga-SB03045 and [⁶⁸Ga]Ga-SB03058, bearing a (2S,4S)-4-fluoropyrrolidine-2-carbonitrile or a (4R)-thiazolidine-4-carbonitrile pharmacophore, respectively. [⁶⁸Ga]Ga-SB03045 and [⁶⁸Ga]Ga-SB03058 were evaluated for their FAP-targeting capabilities using substrate-based in vitro binding assays, and in PET/CT imaging and ex vivo biodistribution studies in an HEK293T:hFAP tumor xenograft mouse model. The IC₅₀ values of ^natGa-SB03045 (1.59 ± 0.45 nM) and ^natGa-SB03058 (0.68 ± 0.09 nM) were found to be lower than those of the clinically validated ^natGa-FAPI-04 (4.11 ± 1.42 nM). Contrary to the results obtained in the FAP-binding assay, [⁶⁸Ga]Ga-SB03058 demonstrated a ~1.5 fold lower tumor uptake than that of [⁶⁸Ga]Ga-FAPI-04 (7.93 ± 1.33 vs. 11.90 ± 2.17 %ID/g), whereas [⁶⁸Ga]Ga-SB03045 (11.8 ± 2.35 %ID/g) exhibited a tumor uptake comparable to that of [⁶⁸Ga]Ga-FAPI-04. Thus, our data suggest that the (2S,4S)-4-fluoropyrrolidine-2-carbonitrile scaffold holds potential as a promising pharmacophore for the design of FAP-targeted radioligands for cancer diagnosis and therapy.

Keywords: FAP inhibitors (FAPIs); Gallium-68; PET imaging; cancer-associated fibroblasts (CAFs); fibroblast activation protein α (FAP-α); targeted radiopharmaceuticals.