Synthesis and evaluation of bifunctional tetrahydroxamate chelators for labeling antibodies with Zr for imaging with positron emission tomography.

Two novel bifunctional tetrahydroxamate chelators 3 and 4 were synthesized and evaluated for labeling antibodies with Zr for positron emission tomography imaging. Compared to previously reported tetrahydroxamate chelators 1 and 2 with an iminodiacetamide backbone, 3 and 4 were based on an extended iminodipropionamide and dipropylenetriamine backbone, respectively. Trastuzumab conjugates of 3 and 4 were efficiently labeled with Zr (>95% radiochemical yield). The in vitro plasma stability of Zr-4-Trastuzumab and especially Zr-3-Trastuzumab was greatly improved over previously reported Zr-1-Trastuzumab and Zr-2-Trastuzumab, but their demetalation remained higher and faster than Zr-deferoxamine (DFO)-Trastuzumab. These observations were confirmed by PET imaging and biodistribution in mice, with significant higher bone uptake for Zr-4-Trastuzumab, followed by Zr-3-Trastuzumab, and to a lesser extent for Zr-DFO-Trastuzumab. Molecular modeling showed that 3 and 4 with an extended backbone could form eight-coordinate Zr-complexes as compared to only seven-coordinate Zr-complexes of 1 and 2. Our data suggest further elongation of linker length between hydroxamate motifs of this class of chelators is needed to reach a better Zr-coordination configuration and improve in vivo stability.