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

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

Synthesis, Radiolabeling, and Biological Evaluation of 5‑Hydroxy-2‑[¹⁸F]fluoroalkyl-tryptophan Analogues as Potential PET Radiotracers for Tumor Imaging

Aiming at developing mechanism-based amino acid ¹⁸F-PET tracers for tumor imaging, we synthesized two ¹⁸F-labeled analogues of 5-hydroxy-l-[β-¹¹C]­tryptophan ([¹¹C]­5HTP) whose excellent in vivo performance in neuroendocrine tumors is mainly attributed to its decarboxylation by aromatic amino acid decarboxylase (AADC), an enzyme overexpressed in these malignancies. Reference compounds and precursors were synthesized following multistep synthetic approaches. Radiosynthesis of tracers was accomplished in good radiochemical yields (15–39%), high specific activities (45–95 GBq/μmol), and excellent radiochemical purities. In vitro cell uptake was sodium-independent and was inhibited ≥95% by 2-amino-2-norbornanecarboxylic acid (BCH) and ∼30% by arginine. PET imaging in mice revealed distinctly high tumor/background ratios for both tracers, outperforming the well-established <i>O</i>-(2-[¹⁸F]­fluoroethyl)­tyrosine ([¹⁸F]­FET) tracer in a head-to-head comparison. Biological evaluation revealed that the in vivo performance is most probably independent of any interaction with AADC. Nevertheless, the excellent tumor visualization qualifies the new tracers as interesting probes for tumor imaging worthy for further investigation

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

Synthesis and Pharmacological Evaluation of [¹¹C]Granisetron and [¹⁸F]Fluoropalonosetron as PET Probes for 5‑HT₃ Receptor Imaging

Serotonin-gated ionotropic 5-HT₃ receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT₃ receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the “setrons”) that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (<b>15</b>) that has a binding affinity (<i>K</i>_i = 0.26 ± 0.05 nM) similar to the parent drug (<i>K</i>_i = 0.21 ± 0.03 nM). We radiolabeled <b>15</b> by nucleophilic ¹⁸F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-¹¹<i>C</i>)-<i>N</i>-granisetron ([¹¹C]<b>2</b>) through <i>N</i>-alkylation with [¹¹C]­CH₃I, respectively. Both compounds [¹⁸F]<b>15</b> (chemical and radiochemical purity >95%, specific activity 41 GBq/μmol) and [¹¹C]<b>2</b> (chemical and radiochemical purity ≥99%, specific activity 170 GBq/μmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [¹⁸F]<b>15</b> and [¹¹C]<b>2</b> revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT₃ receptors at significant levels. Subsequent PET experiments suggested that [¹⁸F]<b>15</b> and [¹¹C]<b>2</b> are of limited utility for the PET imaging of brain 5-HT₃ receptors in vivo

Studies toward the Development of New Silicon-Containing Building Blocks for the Direct ¹⁸F‑Labeling of Peptides

Silicon-containing prosthetic groups have been conjugated to peptides to allow for a single-step labeling with ¹⁸F radioisotope. The fairly lipophilic di-<i>tert</i>-butylphenylsilane building block contributes unfavorably to the pharmacokinetic profile of bombesin conjugates. In this article, theoretical and experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional theory calculations were used to predict the hydrolytic stability of di-<i>tert</i>-butylfluorosilanes <b>2</b>–<b>23</b> with the aim to improve the in vivo properties of ¹⁸F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile, hydrophilic linkers were introduced between the lipophilic di-<i>tert</i>-butylphenylsilane building block and the bombesin congeners. Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of ¹⁸F-labeled silicon–bombesin conjugates

Design, Synthesis, and Initial Evaluation of a High Affinity Positron Emission Tomography Probe for Imaging Matrix Metalloproteinases <b>2</b> and <b>9</b>

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of <i>N</i>-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (<i>R</i>)-2-(4-(4-Fluorobenzamido)­phenylsulfonamido)-3-(1<i>H</i>-indol-3-yl)­propanoic acid (<b>7</b>) exhibited the best in vitro binding properties: MMP2 IC₅₀ = 1.8 nM, MMP9 IC₅₀ = 7.2 nM. Radiolabeling of <b>7</b> with no carrier added ¹⁸F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO₄^– > Br^– > TFA^– > tosylate). ¹⁸F-<b>7</b> was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/μmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo