Synthesis, Copper(II) Complexation, ⁶⁴Cu-Labeling and Bioconjugation of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane

A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid (6), has been synthesized and its complexation behavior towards Cu²⁺ ions investigated. The ligand 6 has been characterized by spectroscopic methods and a molecular structure of a corresponding Cu(II) complex has been elucidated by single crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), βAla-βAla-[Cha¹³, Nle¹⁴]BN(7-14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with ⁶⁴Cu²⁺ and the resulting complexes, ⁶⁴Cu ^. 6 and ⁶⁴Cu ^. 8, were found to be stable in the presence of a large excess of a competing ligand (cyclam) or the copper-seeking superoxide dismutase (SOD), as well as in rat plasma.
Biodistribution studies of ⁶⁴Cu ^. 8 in Wistar rats showed a high activity uptake into the pancreas (5.76 ± 0.25 SUV, 5 min p.i.; 3.93 ± 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is over-expressed in a large number of breast and prostate carcinomas. The novel ⁶⁴Cu ^. 6 complex had a dominating influence on the non-specific activity biodistribution of its BN conjugate, since the distribution data of ⁶⁴Cu ^. 6 are similar to ⁶⁴Cu ^. 8. The ⁶⁴Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of ⁶⁴CuCl₂, suggesting that ⁶⁴Cu ^. 6 does not undergo significant demetalation, but rather exhibits high in vivo stability.