Synthesis and Preclinical Evaluation of Novel 68 Ga-Labeled ( R )-Pyrrolidin-2-yl-boronic Acid-Based PET Tracers for Fibroblast Activation Protein-Targeted Cancer Imaging.

Fibroblast activation protein (FAP) is a membrane-tethered serine protease overexpressed in the reactive stromal fibroblasts of >90% human carcinomas, which makes it a promising target for developing radiopharmaceuticals for the imaging and therapy of carcinomas. Here, we synthesized two novel ( R )-pyrrolidin-2-yl-boronic acid-based FAP-targeted ligands: SB02055 (DOTA-conjugated ( R )-(1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)glycyl)pyrrolidin-2-yl)boronic acid) and SB04028 (DOTA-conjugated (( R )-1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid). nat Ga- and 68 Ga-complexes of both ligands were evaluated in preclinical studies and compared to previously reported nat Ga/ 68 Ga-complexed PNT6555. Enzymatic assays showed that FAP binding affinities (IC 50 ) of nat Ga-SB02055, nat Ga-SB04028 and nat Ga-PNT6555 were 0.41 ± 0.06, 13.9 ± 1.29 and 78.1 ± 4.59 nM, respectively. PET imaging and biodistribution studies in HEK293T:hFAP tumor-bearing mice showed that while [ 68 Ga]Ga-SB02055 presented with a nominal tumor uptake (1.08 ± 0.37 %ID/g), [ 68 Ga]Ga-SB04028 demonstrated clear tumor visualization with ~1.5-fold higher tumor uptake (10.1 ± 0.42 %ID/g) compared to [ 68 Ga]Ga-PNT6555 (6.38 ± 0.45 %ID/g). High accumulation in the bladder indicated renal excretion of all three tracers. [ 68 Ga]Ga-SB04028 displayed a low background level uptake in most normal organs, and comparable to [ 68 Ga]Ga-PNT6555. However, since its tumor uptake was considerably higher than [ 68 Ga]Ga-PNT6555, the corresponding tumor-to-organ uptake ratios for [ 68 Ga]Ga-SB04028 were also significantly greater than [ 68 Ga]Ga-PNT6555. Our data demonstrate that ( R )-(((quinoline-4-carbonyl)-d-alanyl)pyrrolidin-2-yl)boronic acid is a promising pharmacophore for the design of FAP-targeted radiopharmaceuticals for cancer imaging and radioligand therapy.