The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. The chelators most commonly utilized for labeling copper radionuclides to biomolecules are analogues of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); however, recent reports have communicated the instability of the radio-Cu(II)-TETA complexes in vivo. A class of bicyclic tetraazamacrocycles, the ethylene "cross-bridged" cyclam (CB-cyclam) derivatives, form highly kinetically stable complexes with Cu(II) and therefore may be less susceptible to transchelation than their nonbridged analogues in vivo. Herein we report results on the relative biological stabilities and identification of the resulting radiolabeled metabolites of a series of (64)Cu-labeled macrocyclic complexes. Metabolism studies in normal rat liver have revealed that the (64)Cu complex of 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane ((64)Cu-CB-TE2A) resulted in significantly lower values of protein-associated (64)Cu than (64)Cu-TETA [13 +/- 6% vs 75 +/- 9% at 4 h]. A similar trend was observed for the corresponding cyclen derivatives, with the (64)Cu complex of 4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane ((64)Cu-CB-DO2A) undergoing less transchelation than the (64)Cu complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ((64)Cu-DOTA) [61 +/- 14% vs 90.3 +/- 0.5% protein associated (64)Cu at 4 h]. These data indicate that the structurally reinforcing cross-bridge enhances in vivo stability by reducing metal loss to protein in both the cyclam and cyclen cross-bridged (64)Cu complexes and that (64)Cu-CB-TE2A is superior to (64)Cu-CB-DO2A in that regard. These findings further suggest that a bifunctional chelator derivative of CB-TE2A is a highly desirable alternative for labeling copper radionuclides to biological molecules for diagnostic imaging and targeted radiotherapy.

The kinetic inertness of copper(II) complexes of several carboxymethyl-armed cyclams and cyclens in 5 M HCl have been determined confirming that the complex derived from crossbridged cyclam (Cu-CB-TE2A) is by far the most resistant to acid decomplexation. FT-IR studies in D 2 O solution revealed its unique resistance to full carboxylate protonation and its retention of coordination by both pendant arms even in 1 M DCl. The X-ray structure of its monoprotonated form, + , also established full coordination by both