Reduced ¹⁸F‑Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on ¹⁸F-labeled 6<i>S</i>- and 6<i>R</i>-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four ¹⁸F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6<i>S</i>-α, 6<i>S</i>-γ, 6<i>R</i>-α, and 6<i>R</i>-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated ¹⁸F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1–7% d.c.) and high molar activities (139–245 GBq/μmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. <i>In vitro</i>, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC₅₀ = 17.7–24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high <i>in vivo</i> stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6<i>S</i>- and 6<i>R</i>-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8–11% IA/g). Based on these results, we conclude that ¹⁸F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues

Radiosynthesis and Preclinical Evaluation of 3′-Aza-2′‑[¹⁸F]fluorofolic Acid: A Novel PET Radiotracer for Folate Receptor Targeting

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [¹⁸F]­fluoride (integrated approach). 3′-Azafolic acid and its 2′-halogenated derivatives (2′-chloro and 2′-fluoro) were evaluated in vitro to determine their binding affinity. 3′-Aza-2′-[¹⁸F]­fluorofolic acid ([¹⁸F]<b>6</b>) was obtained, starting from <i>N</i>²-acetyl-3′-aza-2′-chlorofolic acid di-<i>tert</i>-butylester (<b>2</b>), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35–127 GBq/μmol. Binding affinity to the FR was high (IC₅₀ = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 °C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 °C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [¹⁸F]<b>6</b> in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new ¹⁸F-labeled 3′-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue

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 α-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 γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-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 α- and γ-conjugated ¹⁸F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/μmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (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 α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-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 ¹⁸F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel ¹⁸F-labeled folate-based radiotracers