H₂CHXhox: Rigid Cyclohexane-Reinforced Nonmacrocyclic Chelating Ligand for [^nat/67/68Ga]Ga³

A rigid chiral acyclic chelator H₂CHXhox was synthesized and evaluated for Ga³⁺-based radiopharmaceutical applications; it was compared to the previously reported hexadentate H₂hox to determine the effect of a backbone reinforced from adding a chiral 1S,2S-trans-cyclohexane on metal complex stability, kinetic inertness, and in vivo pharmacokinetics. NMR spectroscopy and theoretical calculation revealed that [Ga(CHXhox)]⁺ showed a very similar coordination geometry to that of [Ga(hox)]⁺, and only one isomer in solution was observed by NMR spectroscopy. Solution studies showed that the modification results in a significant improvement in the exceptionally high thermodynamic stability of [Ga(hox)]⁺ with a 1.56 log unit increase in stability constant (logK_ML = 35.91(1)). More importantly, H₂CHXhox showed very fast Ga³⁺ complexation at physiological pH 7.4, and acid-assisted Ga³⁺ complex dissociation kinetic studies (pH 1) in comparison with H₂hox revealed a 50-fold increase of the dissociation half-life time from 73 min to 58 h. Fluorescence microscopy imaging study confirmed its cellular uptake and accumulation in endoplasmic reticulum and mitochondria. MTT studies indicated a quite low cytotoxicity of [Ga(CHXhox)]⁺ over a large concentration range. Dynamic PET imaging studies showed no accumulation in muscle, lungs, bone, and brain, suggesting no release of free Ga³⁺ ions. [⁶⁸Ga][Ga(CHXhox)]⁺ is cleared from the mouse via hepatobiliary and renal pathways. Compared to [⁶⁸Ga][Ga(hox)]⁺, the increased lipophilicity of [⁶⁸Ga][Ga(CHXhox)]⁺ enhanced heart and liver uptake and decreased kidney clearance. [⁶⁷Ga][Ga(CHXhox)]⁺ SPECT/CT imaging and biodistribution study revealed good clearance from liver to gallbladder after 90 min and finally into feces after 5 h. No decomposition or transchelation was observed over the 5 h study. These results confirmed H₂CHXhox to be an obvious improvement over H₂hox and an excellent candidate in this new "ox" family for the development of radiopharmaceutical compounds.