Is Smaller Better? Cu 2+ /Cu + Coordination Chemistry and Copper-64 Radiochemical Investigation of a 1,4,7-Triazacyclononane-Based Sulfur-Rich Chelator.

The biologically triggered reduction of Cu ²⁺ to Cu ⁺ has been postulated as a possible in vivo decomplexation pathway in ^64/67 Cu-based radiopharmaceuticals. In an attempt to hinder this phenomenon, we have previously developed a family of S-containing polyazamacrocycles based on 12-, 13-, or 14-membered tetraaza rings able to stabilize both oxidation states. However, despite the high thermodynamic stability of the resulting Cu ^2+/+ complexes, a marked [ ⁶⁴ Cu]Cu ²⁺ release was detected in human serum, likely as a result of the partially saturated coordination sphere around the copper center. In the present work, a new hexadentate macrocyclic ligand, 1,4,7-tris[2-(methylsulfanyl)ethyl)]-1,4,7-triazacyclononane (NO3S), was synthesized by hypothesizing that a smaller macrocyclic backbone could thwart the observed demetalation by fully encapsulating the copper ion. To unveil the role of the S donors in the metal binding, the corresponding alkyl analogue 1,4,7-tris- n -butyl-1,4,7-triazacyclononane (TACN- n -Bu) was considered as comparison. The acid-base properties of the free ligands and the kinetic, thermodynamic, and structural properties of their Cu ²⁺ and Cu ⁺ complexes were investigated in solution and solid (crystal) states through a combination of spectroscopic and electrochemical techniques. The formation of two stable mononuclear species was detected in aqueous solution for both ligands. The pCu ²⁺ value for NO3S at physiological pH was 6 orders of magnitude higher than that computed for TACN- n -Bu, pointing out the significant stabilizing contribution arising from the Cu ²⁺ -S interactions. In both the solid state and solution, Cu ²⁺ was fully embedded in the ligand cleft in a hexacoordinated N ₃ S ₃ environment. Furthermore, NO3S exhibited a remarkable ability to form a stable complex with Cu ⁺ through the involvement of all of the donors in the coordination sphere. Radiolabeling studies evidenced an excellent affinity of NO3S toward [ ⁶⁴ Cu]Cu ²⁺ , as quantitative incorporation was achieved at high apparent molar activity (∼10 MBq/nmol) and under mild conditions (ambient temperature, neutral pH, 10 min reaction time). Human serum stability assays revealed an increased stability of [ ⁶⁴ Cu][Cu(NO3S)] ²⁺ when compared to the corresponding complexes formed by 12-, 13-, or 14-membered tetraaza rings.