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.