Simple SummaryPD-L1 plays a crucial role in the immune responses against cancer. Only around 30% of cancer patients respond to an anti-PD-L1 immune checkpoint inhibitor therapy. Noninvasive molecular imaging techniques such as positron emission tomography (PET) would allow identification of patients likely to respond. Here we report on the synthesis of nine PET radioligands targeting PD-L1, based on small-molecule inhibitors. We introduced a chelator for radiolabeling and water-soluble groups to aim for clearance through the kidneys. The compounds showed binding affinities toward PD-L1 in the lower nanomolar range and stability in vitro. Mouse experiments showed moderate accumulation in tumor tissue but mainly clearance through the liver. Additionally, the compounds showed unexpected long circulation times due to strong binding to albumin in blood. Nevertheless, our compounds are a starting point for further development of PD-L1 small molecule PET radiotracer to support therapy decisions.AbstractNoninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker–chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers.