Diastereomerically Pure 6R- and 6S-3'-Aza-2'-^sup 18^F-Fluoro-5-Methyltetrahydrofolates Show Unprecedentedly High Uptake in Folate Receptor–Positive KB Tumors

The aim of this study was to develop the radiosyntheses of diastereomerically pure 6R- and 6S-3'-aza-2'-18F-fluoro-5-methyltetrahydrofolate (MTHF) (6R-18F-1 and 6S-18F-1) using the integrated approach and to compare the in vitro and in vivo performance characteristics of both radioligands with the previously reported 3'-aza-2'-18F-fluorofolic acid tracer (18F-2), the oxidized form. Methods: 6R-18F-1, 6S-18F-1, and 18F-2 were radiolabeled with 18F using aromatic nucleophilic substitution reaction. In vitro cell uptake studies and binding affinity assays were performed using folate receptor (FR)-a–expressing KB cells. PET/CT imaging and biodistribution experiments were performed with KB tumor–bearing mice. Results: Reference compounds 6R-1 and 6S-1 were obtained after acidic hydrolysis of the corresponding protected intermediates 6R-3 and 6S-3 in high chemical yields (81%–87%) and chemical purities of more than 95%. 6R-18F-1, 6S-18F-1, and 18F-2 were obtained after a 2-step radiosynthetic procedure in a decay-corrected radiochemical yield of up to 5% and molar radioactivities ranging from 20 to 250 GBq/µmol. In vitro binding affinity studies using FR-a–positive KB cells gave half-maximal inhibitory concentrations of 27.1 ± 3.7 and 23.8 ± 4.0 nM for 6R-1 and 6S-1, respectively, which were higher than for the previously reported 3'-aza-2'-fluorofolic acid 2 (1.4 ± 0.5 nM). Comparably high cell uptake values in FR-a–expressing KB cells were found for all 3 radiofolates. In biodistribution studies, exceptionally high KB tumor uptake value of over 32% injected activity per gram of tissue for both 6R-18F-1 and 6S-18F-1 was observed at 180 min after injection, whereas for 18F-2 only 15% injected activity per gram was found in the KB tumors. Radioactivity uptake in the kidneys, liver, salivary glands, and spleen was substantially different for the 6R- and 6S-diastereoisomers and 18F-2. Excellent KB tumor visualization was found in PET/CT images with 6R-18F-1 and 6S-18F-1, both of which outperformed the corresponding oxidized 18F-2. Conclusion: We have successfully radiolabeled 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF with 18F using the integrated approach. Our results suggest that both 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF are promising reduced radiofolates for imaging FR-a–expressing cancers.