Comparative Studies of Three Pairs of α- and γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18.

The folate receptor (FR) is upregulated in various epithelial cancer types (FR alpha-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the gamma-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated alpha- and gamma-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the alpha- and gamma-conjugated F-18-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/mu mol. In vitro, all folate derivatives showed high binding affinity to the FR-alpha (IC50 = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the alpha-regioisomers compared to the corresponding gamma-regioisomers. In contrast, kidney uptake was up to 50% lower for the gamma-regioisomers than for the alpha-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of F-18-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel F-18-labeled folate-based radiotracers.