Pycup--a bifunctional, cage-like ligand for (64)Cu radiolabeling

In developing targeted probes for positron emission tomography (PET) based on (64)Cu, stable complexation of the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward (64)Cu dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3-4 synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 °C (t1/2: 1.5 h for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)](2+) complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of (64)Cu labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed. Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with (64)Cu for successful PET imaging of arterial thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.