Design, Synthesis, and Evaluation of 99mTc-Labeled Proline Isonitrile Derivatives with Optimized Linker Lengths for Tumor SPECT Imaging

Amino acid metabolic imaging is among the important research directions in tumor molecular imaging. The highly active metabolism of proline in tumors, together with the abnormal expression of its key enzymes, makes it a promising target for tumor molecular imaging. To develop new proline transport-targeted tumor imaging agents, four l-proline isonitrile ligands (CN5Pro, CN6Pro, CN7Pro and CN8Pro) were synthesized and labeled with technetium-99m. All the complexes achieved >95% labeling yield with good stability. Biodistribution in S180 tumor-bearing mice revealed that [99mTc]Tc-CN7Pro exhibited the highest tumor uptake (1.05 ± 0.09% ID/g), a tumor/muscle ratio of 5.03, and predominant renal excretion. With respect to U87MG models, tumor uptake reached 2.47 ± 0.39% ID/g, confirming tumor imaging applicability. SPECT/CT imaging clearly revealed tumors, with uptake inhibited by excess l-proline. Cellular studies indicated uptake primarily via system A transporters, and the complex remained intact in vivo. Collectively, [99mTc]Tc-CN7Pro is convenient for labeling, achieves excellent targeting, and is very stable, making it a promising SPECT tumor imaging agent.