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

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A rigid chiral acyclic chelator H 2 CHX hox was synthesized and evaluated for Ga 3+ -based radiopharmaceutical applications; it was compared to the previously reported hexadentate H 2 hox 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( CHX hox)] + 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 (log K ML = 35.91(1)). More importantly, H 2 CHX hox showed very fast Ga 3+ complexation at physiological pH 7.4, and acid-assisted Ga 3+ complex dissociation kinetic studies (pH 1) in comparison with H 2 hox 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( CHX hox)] + over a large concentration range. Dynamic PET imaging studies showed no accumulation in muscle, lungs, bone, and brain, suggesting no release of free Ga 3+ ions. [ 68 Ga][Ga( CHX hox)] + is cleared from the mouse via hepatobiliary and renal pathways. Compared to [ 68 Ga][Ga(hox)] +, the increased lipophilicity of [ 68 Ga][Ga( CHX hox)] + enhanced heart and liver uptake and decreased kidney clearance. [ 67 Ga][Ga( CHX hox)] + 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 H 2 CHX hox to be an obvious improvement over H 2 hox and an excellent candidate in this new “ox” family for the development of radiopharmaceutical compounds.