Malignant tumors have become a significant public health challenge threatening human health. Although significant progress has been made in early cancer screening technologies, immunotherapy and targeted therapy in recent years, the high recurrence rate, strong metastatic potential and treatment resistance of tumors remain the main reasons that limit the therapeutic effect and long-term survival rate. There is an urgent need to develop novel and effective treatment strategies to enhance the precision and efficacy of cancer therapies.

CD73 (Ecto-5′-Nucleotidase, NT5E), as a significant extracellular nucleotidase, plays a core role in regulating the immunosuppressive aspects of tumor microenvironment. CD73 mainly catalyzes the hydrolysis of extracellular adenosine monophosphate (AMP) to adenosine. Adenosine, as a potent immunosuppressive factor, inhibits the anti-tumor functions of effector T cells, natural killer cells and other immune cells by activating A2A and A2B adenosine receptors, thereby promoting the immune escape of tumor cells [1]. In addition to inhibiting tumor immunity by regulating the adenosine signaling pathway, CD73 itself can mediate cell adhesion, promote angiogenesis, and induce epithelial-mesenchymal transition (EMT). These processes contribute to tumor cell proliferation, invasion, and metastasis, further highlighting the multifaceted role of CD73 in cancer progression [2,3]. Studies have shown that CD73 is significantly overexpressed in various solid tumors such as non-small cell lung cancer [4], breast cancer [3,5,6], colorectal cancer [7,8], and pancreatic cancer [9,10]. The expression level of CD73 is correlated with the invasive and metastatic characteristics of tumors. Due to its crucial role in tumor immune regulation and microenvironment remodeling, CD73 is regarded to possess significant clinical value and has attracted much attention as a promising drug target in tumor immunotherapy in recent years.

At present, the detection of CD73 mainly relies on histopathological methods. However, due to tumor heterogeneity, the limitations of biopsy puncture and invasiveness, the effective monitoring of CD73 expression is restricted. Radio-labeled molecular probes may provide an alternative solution for the in vivo dynamic detection of CD73 expression based on SPECT or PET technologies. In 2022, Hitomi Sudo et al. first reported a111In-labeled CD73-specific monoclonal antibody (067-213), showing higher radiotracer uptake in MIAPaCa-2 tumors with high CD73 expression compared with low-CD73–expressing tumors and isotype controls in SPECT images [11]. Due to the relatively low resolution of SPECT imaging, in 2024, Kyung-Ho Jung et al. used PET/CT to image the 89Zr-labeled anti-CD73 antibody [12]. This study further confirmed that the 89Zr-CD73 probe could specifically bind to the CD73 and visualize the expression of CD73 in vivo.

Currently, only a limited number of radionuclide probes targeting CD73 have been reported, and most of them are antibody-based probes. Small molecules that hold more flexible pharmacokinetic properties have not been fully investigated and reported as CD73 radiotracers. Moreover, the exploration of radio-ligand therapies that target CD73 have not been reported yet.

To develop novel CD73-targeted PET tracers, a non-nucleotide small-molecule inhibitor of CD73 (compound 1, Fig. 1) with high affinity toward CD73 (reported IC50 = 12 nM) attracted our attention [13]. In this investigation, structural modifications and introduction of DOTA moiety were performed on the scaffold of compound 1, which led to a series of non-nucleotide small molecule inhibitors that hold potent affinity against CD73 and possess the potential to be developed as radio-metal labeled radiotracers. To the most potent compound in enzymatic assay, further radio-labeling and in-depth evaluations for anti-CD73 activities both in vitro and in vivo were performed and discussed.