Diastereomerically Pure 6 R - and 6 S -3'-Aza-2'- 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'-F-18-fluoro-5-methyltetrahydrofolate (MTHF) (6R-F-18-1 and 6S-F-18-1) 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'-F-18-fluorofolic acid tracer (F-18-2), the oxidized form. Methods: 6R-F-18-1, 6S-F-18-1, and F-18-2 were radiolabeled with F-18 using aromatic nucleophilic substitution reaction. In vitro cell uptake studies and binding affinity assays were performed using folate receptor (FR)-alpha-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-F-18-1, 6S-F-18-1, and F-18-2 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/mu mol. In vitro binding affinity studies using FR-alpha-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-aexpressing 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-F-18-1 and 6S-F-18-1 was observed at 180 min after injection, whereas for F-18-2 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 F-18-2. Excellent KB tumor visualization was found in PET/CT images with 6R-F-18-1 and 6S-F-18-1, both of which outperformed the corresponding oxidized F-18-2. Conclusion: We have successfully radiolabeled 6R- and 6S-3'-aza-2'-F-18-fluoro-5-MTHF with F-18 using the integrated approach. Our results suggest that both 6R- and 6S-3'-aza-2'-F-18-fluoro-5-MTHF are promising reduced radiofolates for imaging FR-alpha-expressing cancers.