From Speciation to Thermodynamic Stability: A Rigid HBED Analogue Controls Isomerization in Gallium-68 Complexes.

The hexadentate acyclic ligand N,N′-di(2-hydroxybenzyl)-(1,2-cyclohexanediamine)-N,N′-diacetic acid (HBCD) was designed to improve the coordination chemistry of the positron-emitting radiometal ⁶⁸Ga by introducing rigidity into the ligand backbone. Specifically, the flexible ethylenediamine spacer of the parent HBED ligand was replaced with a cyclohexanediamine (DACH) scaffold to limit the formation of multiple coordination isomers, a well-known issue of Ga³⁺–HBED systems that can impact in vivo performances. Here, we report the synthesis of HBCD and a comprehensive investigation of its solution behavior, including protonation equilibria, Ga³⁺ complexation, radiolabeling with generator-produced [⁶⁸Ga]Ga3+, and stability under physiological conditions. Speciation studies, carried out by means of UV-Vis/NMR spectroscopy and MS spectrometry, reveal that the rigid DACH framework directs the formation of a single, well-defined hexacoordinated Ga³⁺ complex, in contrast to the isomeric mixtures observed for HBED. At physiological pH (7.4), the predominant species is [GaL]⁻, with thermodynamic stability comparable to that of Ga³⁺–HBED and significantly exceeding that of the clinically workhorse DOTA chelator. HBCD efficiently coordinates [⁶⁸Ga]Ga3+ under highly diluted radiochemical conditions (CL = 10⁻⁶ M, 90 °C, pH 4.5–7), and the resulting complex [68Ga][Ga(HBCD)]− exhibits remarkable stability in biological media. Overall, these results demonstrate that ligand rigidification is an effective strategy to control solution speciation and suppress isomerization equilibria in Ga³⁺ complexes. HBCD thus emerges as a promising platform for the development of next-generation ⁶⁸Ga-based radiopharmaceuticals.