Synthesis and Preclinical Evaluation of Novel ^99mTc-Labeled Glucose Derivatives Containing Different Proline Analogs as Linker Moieties for Tumor Imaging

2-[¹⁸F]Fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) is the most extensively utilized tumor imaging agent in clinical positron emission tomography (PET) applications. Nevertheless, no radiolabeled glucose derivatives for single-photon emission computed tomography (SPECT) imaging have achieved clinical usage comparable to that of [¹⁸F]FDG. Technetium-99m is a common single-photon-emitting radionuclide used in clinical practice and has potential for the development of novel radiopharmaceuticals. Proline is an advantageous linker because of its unique physicochemical profile. This study aimed to develop novel ^99mTc-labeled glucose derivatives containing different proline analogs as linker moieties for tumor imaging. Six ^99mTc-labeled glucose derivatives ([^99mTc]Tc-GL1-[^99mTc]Tc-GL6) incorporating different proline analog linkers were successfully prepared. These derivatives exhibited high hydrophilicity and good <i>in vitro</i> stability. Among them, [^99mTc]Tc-GL1 showed the highest tumor uptake (5.66 ± 0.46% ID/g) and tumor-to-nontarget ratios and was thus selected as a promising candidate radiotracer. Further mechanistic studies revealed that [^99mTc]Tc-GL1 was transported into cancer cells via glucose transporters (GLUTs). Additionally, a kit formulation for [^99mTc]Tc-GL1 was successfully developed and warrants further clinical exploration.