Diastereomerically Pure 6<i>R</i>- and 6<i>S</i>-3'-Aza-2'-¹⁸F-Fluoro-5-Methyltetrahydrofolates Show Unprecedentedly High Uptake in Folate Receptor-Positive KB Tumors.

Abstract

The aim of this study was to develop the radiosyntheses of diastereomerically pure 6<i>R</i>- and 6<i>S</i>-3'-aza-2'-¹⁸F-fluoro-5-methyltetrahydrofolate (MTHF) (6<i>R</i>-¹⁸F-<b>1</b> and 6<i>S</i>-¹⁸F-<b>1</b>) 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-<b>2</b>), the oxidized form. <b>Methods:</b> 6<i>R</i>-¹⁸F-<b>1,</b> 6<i>S</i>-¹⁸F-<b>1,</b> and ¹⁸F-<b>2</b> were radiolabeled with ¹⁸F using aromatic nucleophilic substitution reaction. In vitro cell uptake studies and binding affinity assays were performed using folate receptor (FR)-α-expressing KB cells. PET/CT imaging and biodistribution experiments were performed with KB tumor-bearing mice. <b>Results:</b> Reference compounds 6<i>R</i>-<b>1</b> and 6<i>S</i>-<b>1</b> were obtained after acidic hydrolysis of the corresponding protected intermediates 6<i>R</i>-<b>3</b> and 6<i>S</i>-<b>3</b> in high chemical yields (81%-87%) and chemical purities of more than 95%. 6<i>R</i>-¹⁸F-<b>1,</b> 6<i>S</i>-¹⁸F-<b>1,</b> and ¹⁸F-<b>2</b> 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-α-positive KB cells gave half-maximal inhibitory concentrations of 27.1 ± 3.7 and 23.8 ± 4.0 nM for 6<i>R</i>-<b>1</b> and 6<i>S</i>-<b>1,</b> respectively, which were higher than for the previously reported 3'-aza-2'-fluorofolic acid <b>2</b> (1.4 ± 0.5 nM). Comparably high cell uptake values in FR-α-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 6<i>R</i>-¹⁸F-<b>1</b> and 6<i>S</i>-¹⁸F-<b>1</b> was observed at 180 min after injection, whereas for ¹⁸F-<b>2</b> 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 6<i>R</i>- and 6<i>S</i>-diastereoisomers and ¹⁸F-<b>2</b> Excellent KB tumor visualization was found in PET/CT images with 6<i>R</i>-¹⁸F-<b>1</b> and 6<i>S</i>-¹⁸F-<b>1,</b> both of which outperformed the corresponding oxidized ¹⁸F-<b>2. Conclusion:</b> We have successfully radiolabeled 6<i>R</i>- and 6<i>S</i>-3'-aza-2'-¹⁸F-fluoro-5-MTHF with ¹⁸F using the integrated approach. Our results suggest that both 6<i>R</i>- and 6<i>S</i>-3'-aza-2'-¹⁸F-fluoro-5-MTHF are promising reduced radiofolates for imaging FR-α-expressing cancers.