Maleimide−Thiol Conjugation Enables the Optimization of 68Ga-Labeled CAIX Targeting PET Probes

Carbonic anhydrase IX (CAIX) is a well-established biomarker of tumor hypoxia and an attractive target for molecular imaging. Despite the development of small-molecule CAIX-targeting positron emission tomography (PET) probes, most exhibit high normal-organ uptake and suboptimal tumor-to-background contrast. Moreover, their application in hypoxic tumors remains insufficiently explored. Herein, we reported four 68Ga-labeled CAIX-targeting probes based on the acetazolamide (AAZ) scaffold, employing distinct conjugation strategies, including amide coupling and maleimide−thiol ligation. All probes demonstrated detectable tumor uptake in vivo. Among them, [68Ga]Ga-SMIC-2607 displayed the highest tumor uptake (14.67 ± 3.06 %ID/g) and tumor-to-kidney ratio (1.69 ± 0.41) at 1 h in subcutaneous OS-RC-2-bearing mice, along with high tumor uptake in HT-29-bearing mice (2.98 ± 0.28 %ID/g at 1 h). Overall, [68Ga]Ga-SMIC-2607 demonstrates favorable imaging characteristics, with high tumor uptake and high tumor-to-normal organ ratios, representing a promising candidate for imaging CAIX-overexpressing tumors and supporting clinical translation.