Multifunctional Desferrichrome Analogues as Versatile ⁸⁹Zr(IV) Chelators for ImmunoPET Probe Development

New bifunctional hexa- and octadentate analogues of the hydroxamate-containing siderophore desferrichrome (DFC) have been synthesized and evaluated as ⁸⁹Zr-chelating agents for immunoPET applications. The in vitro and in vivo inertness of these new ligands, Orn3-hx (hexadentate) and Orn-4hx derivatives (octadentate), was compared to the gold standard hexadentate, hydroxamate-containing chelator for ⁸⁹Zr desferrioxamine (DFO). Density functional theory was employed to model the geometries of the resulting Zr­(IV) complexes and to predict their relative stabilities as follows: Zr­(Orn4-hx) > Zr­(DFC) > Zr­(Orn3-hx). Transchelation challenge experiments of the corresponding radiochemical complexes with excess ethylenediaminetetraacetate (EDTA) indicated complex stability in accordance with DFT calculations. Radiolabeling of these ligands with ⁸⁹Zr was quantitative (0.25 μmol of ligand, pH 7.4, room temperature, 20 min). For antibody conjugation, the isothiocyanate (NCS) functional group was introduced to the N terminus of Orn3-hx and Orn-4hx. An additional trifunctional derivative that bears a silicon-rhodamine fluorophore on the C-terminus and NCS on the N terminus was also furnished. As proof of concept, all NCS derivatives were conjugated to the HER2-targeting antibody, trastuzumab. Radiolabeling of immunoconjugates with ⁸⁹Zr was accomplished with radiochemical yields of 16 ± 2% to 95 ± 2%. These constructs were administered to naive mice (male, C57BL/6J, n = 4) to assess in vivo inertness, which is inversely correlated with uptake of ⁸⁹Zr in bone, after 96 h circulation time. We found bone uptake to range from 7.0 ± 2.2 to 10.7 ± 1.3% ID/g, values that compare well to the corresponding DFO conjugate (7.1 ± 0.8% ID/g). In conclusion, we have rationally designed linear, bifunctional and trifunctional desferrichrome analogues suitable for the mild and inert radiolabeling of antibodies with the radionuclide ⁸⁹Zr.