Binding Azaphilic Copper Radioisotopes with All-Nitrogen Macrocycles for Cancer Theranostics

Abstract

Copper radioisotopes constitute a true theranostic family, enabling cancer imaging and therapy with chemically identical metal-based radiopharmaceuticals. Developing chelators that provide copper complexes combining high thermodynamic stability, kinetic inertness, and redox robustness remains a key challenge. Herein, we investigated a cyclen-based chelator with aminoethyl side chains (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(2-ethylamine), DO4N) and its TACN analogue (1,4,7-triazacyclononane-1,4,7-tris(2-ethylamine), NO3N). Both chelators rapidly form Cu2+ complexes with high thermodynamic stability comparable to or exceeding that of their carboxylate counterparts (DOTA and NOTA), with DO4N displaying superior stability. Cu2+ complexes adopt an elongated octahedral (DO4N) or distorted square pyramidal (NO3N) geometry in solution. All macrocyclic amines coordinate the metal, while only one or two side chains participate, leaving additional -NH2 groups available for conjugation to biological vectors. Both ligands are also able to stabilize Cu⁺ upon reduction. Radiolabeling with [64Cu]Cu2+ demonstrated superior incorporation by both DO4N and NO3N compared to NODAGA under mild conditions, with DO4N achieving the highest labeling efficiency. Both [64Cu]Cu2+ complexes remained fully intact in human serum over 24 h. In vivo PET imaging with [64Cu]Cu-DO4N showed sufficient stability for imaging, with renal clearance dominating early biodistribution. The results indicate that these all-nitrogen macrocycles are highly promising scaffolds for next-generation copper-based theranostic radiopharmaceuticals.