*3.1. Chelators for Copper*

The coordination chemistry of the transition metal copper in an aqueous solution is limited to three accessible oxidation states (I–III). The lowest oxidation state, Cu(I) allows formation of complexes with various ligands, which are however labile and lack sufficient stability for radiopharmaceutical applications. Cu(II) is less labile toward ligand exchange and is optimal for incorporation into radiopharmaceuticals. The Cu(III) oxidation state is relatively rare and difficult to attain and thus not of importance for radiopharmaceuticals [71,72]. To evaluate <sup>67</sup>Cu for radioimmunotherapy (RIT), the macrocyclic chelating agent 1,4,7,11-tetraazacyclotetradecane-*N*,*N*0 ,*N*00 ,*N*000-tetraacetic acid (TETA) was designed specifically to bind copper rapidly and selectively for conjugation to MoAbs. The synthesis of this "bifunctional" metal chelator, 6(*p*-bromoacetamidobenzyl)-1,4,8,11 tetraazacyclotetradecane-*N*,*N*0 ,*N*00 ,*N*000-tetraacetic acid, which can be covalently attached to proteins and which binds copper stably in human serum under physiological conditions, was reported by Moi et al., in 1985 [73]. In contrast, other chelators based on ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid rapidly lose copper to serum albumin under the same conditions.

Despite their use in several pre-clinical studies, the stability of Cu(II) metal complexes with common macrocyclic ligands, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid) and TETA was evaluated and considered not satisfactory for biomedical applications, demonstrating that nearly 70% of the <sup>64</sup>Cu trans-chelated in the liver 20 hours post injection [74–76].

The addition of a bond (or Cross-Bridge, CB) between two opposite nitrogen atoms in the tetraazamacrocyclic skeleton of the macrocycle gives greater stability. The Cu(II) complex with the ligand CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo [6.6.2] hexadecane) has been demonstrated to be particularly stable in vivo and to have exceptional inertness kinetics in aqueous solution thanks to the stabilizing effects given by the CBmacrocycle and by the two hanging carboxymethyl arms [77–80]. Unfortunately, the extreme labeling conditions at 95 ◦C do not make it a good candidate for the development of radiopharmaceuticals labile at high temperatures, e.g., antibodies [81]. The replacement

of one or both of the hanging carboxymethyl arms with groups derived from phosphonic acid (–CH2-PO3H2), CB-TE1A1P (4,8,11-tetraazacyclotetradecane-1-(methanophosphonic acid)-8-(methanocarboxylic acid)), and CB-TE2P (1,4,8,11-tetraazacyclotetradecano-1,8 bis(methanephosphonic acid)), has been shown to confer greater thermodynamic and kinetic stability, as well as greater selectivity and faster complexation kinetics [82].

Another class of potential sarcophagus-like cage structure chelators for copper are hexaazamacrobicyclic ligands [83,84]. Labeling conditions of copper complexes with sarcophagine-like ligands are particularly favorable in a wide pH range (4–9) with yields of 100% in a few minutes at room temperature [75]. The complexes obtained possess excellent in vitro and in vivo stability [85–87].

Some N2S2-type acyclic chelators have also been studied, such as PTSM (pyruvaldehydebis(N4-methylthiosemicarbazone)) and ATSM (diacetyl-bis(N4-methylthiosemicarbazone)). This class of chelators, bis (thiosemicarbazones), act as tetradentate ligands and coordinate Cu2+ forming stable and neutral complexes, with a square planar geometry, which show a high permeability of the cell membrane. The preparation of these complexes with radioactive copper isotopes can be carried out under mild conditions in high yield [74,87–89].
