Synthesis and evaluation of nucleoside radiotracers for imaging proliferation

Introduction: Uncontrolled proliferation is a fundamental characteristic of cancer, and consequently, imaging of tumor proliferative status finds interest clinically both as a diagnostic tool and for evaluation of response to treatment. Positron emission tomography (PET) radiotracers based on a nucleoside core, such as 3'-[¹⁸F]fluoro-3'-deoxythymidine ([¹⁸F]FLT), have been extensively studied for this purpose. However, [¹⁸F]FLT suffers from poor DNA incorporation leading to occasional poor correlation of [¹⁸F]FLT tumor uptake with other proliferation indicators such as Ki-67 immunostaining. Methods: N³-((1-(2-[¹⁸F]fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)thymidine ([¹⁸F]2) and N³-((1-(2-[¹⁸F]fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-4'-thio-β-thymidine ([¹⁸F]3) were synthesized by click chemistry from [¹⁸F]fluoroethyl azide and by direct nucleophilic substitution of a tosylate precursor. Metabolic stability and phosphorylation potential of the radiotracers were evaluated in vitro and compared to [¹⁸F]FLT. Further, metabolic stability and biodistribution analysis of [¹⁸F]2 and [¹⁸F]3 were evaluated in vivo. Results: Stable isotope standards and radiochemistry precursors were synthesized by modification of existing literature procedures. [¹⁸F]2 and [¹⁸F]3 were synthesized in a radiochemical yield of 8%-12% (end of synthesis, non-decay corrected). Both nucleosides were stable to metabolic degradation by thymidine phosphorylase, and in vivo stability analysis showed only one metabolite for [¹⁸F]3. No phosphorylation of [¹⁸F]2 could be detected in HCT116 cell homogenates, and in the same assay, only minor (~8%) phosphorylation of [¹⁸F]3 was observed. Biodistribution in Balb/c mice indicated rapid clearance for [¹⁸F]2 and [¹⁸F]3 to a lesser extent. Conclusions: The favorable biodistribution and metabolic profile of [¹⁸F]3 warrant further investigation as a next-generation PET proliferation marker.