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We present a detailed investigation of the coordination chemistry toward [natCu/64Cu]copper of a series of H2DEDPA derivatives (H2DEDPA = 6,6′-((ethane-1,2-diylbis(azanediyl))bis(methylene))dipicolinic acid) containing cyclohexyl (H2CHXDEDPA), cyclopentyl (H2CpDEDPA) or cyclobutyl (H2CBuDEDPA) spacers. Furthermore, we also developed a strategy that allowed the synthesis of a H2CBuDEDPA analogue containing an additional NHBoc group at the cyclobutyl ring, which can be used for conjugation to targeting units. The X-ray structures of the Cu(II) complexes evidence distorted octahedral coordination around the metal ion in all cases. Cyclic voltammetry experiments (0.15 M NaCl) evidence quasi-reversible reduction waves associated with the reduction of Cu(II) to Cu(I). The complexes show a high thermodynamic stability, with log KCuLvalues of 25.11(1), 22.18(1) and 20.19(1) for the complexes of CHXDEDPA2–, CpDEDPA2–and CBuDEDPA2–, respectively (25 °C, 1 M NaCl). Dissociation kinetics experiments reveal that both the spontaneous- and proton-assisted pathways operate at physiological pH. Quantitative labeling with 64CuCl2was observed at 0.1 nmol for CHXDEDPA2–and CpDEDPA2–, 0.025 nmol for CBuDEDPA2–and 1 nmol for CBuDEDPA-NHBoc2–, with no significant differences observed at 15, 30, and 60 min. The radio-complexes are stable in PBS over a period of 24 h.

We synthesized, thanks to the regiospecific N-functionalization using an orthoamide intermediate, two 1,4,7-triazacyclononane derivatives containing an acetate arm and either a methylpyridine or a picolinic acid group, respectively, Hnoapyand H2noapa, as new Ga3+chelators for potential use in nuclear medicine. The corresponding Ga3+complexes were synthesized and structurally characterized in solution by 1H and 13C NMR. The [Ga(noapy)]2+complex appears to exist in solution as two diasteroisomeric pairs of enantiomers, as confirmed by density functional theory (DFT) calculations, while for [Ga(noapa)]+, a single species is present in solution. Solid-state investigations were possible for the [Ga(noapa)]+complex, which crystallized from water as a pair of enantiomers. The average length of the N–Ga bonds of 2.090 Å is identical with that found for the [Ga(nota)] complex, showing that the presence of the picolinate arm does not hinder the coordination of the ligand to the metal ion. Protonation constants of noapy–and noapa2–were determined by potentiometric titrations, providing an overall basicity ∑log KiH(i= 1–4) that increases in the order noapy–< noapa2–< nota3–with increases in the negative charge of the ligand. Stability constants determined by pH-potentiometric titrations supplemented with 71Ga NMR data show that the stabilities of [Ga(noapy)]2+and [Ga(noapa)]+are lower compared to that of [Ga(nota)] but higher than those of other standards such as [Ga(aazta)]−. 67Ga radiolabeling studies were performed in order to demonstrate the potential of these chelators for 67/68Ga-based radiopharmaceuticals. The labelings of Hnoapyand H2noapawere nearly identical, outperforming H3nota. Stability studies were conducted in phosphate-buffered saline and in the presence of human serum transferrin, revealing no significant decomplexation of [67Ga][Ga(noapy)]2+and [67Ga][Ga(noapa)]+compared to [67Ga][Ga(nota)]. Finally, all complexes were found to be highly hydrophilic, with calculated log D7.4values of −3.42 ± 0.05, −3.34 ± 0.04, and −3.00 ± 0.23 for Hnoapy, H2noapa, and H3nota, respectively, correlating with the charge of each complex and the electrostatic potentials obtained with DFT.

Radioisotopes of Cu, such as 64Cu and 67Cu, are alluring targets for imaging (e.g., positron emission tomography, PET) and radiotherapeutic applications. Cyclen-based macrocyclic polyaminocarboxylates are one of the most frequently examined bifunctional chelators in vitro and in vivo, including the FDA-approved 64Cu radiopharmaceutical, Cu(DOTATATE) (Detectnet); however, connections between the structure of plausible reactive intermediates and their stability under physiologically relevant conditions remain to be established. In this study, we share the synthesis of a cyclen-based, N,N-alkylated spirocyclic chelate, H2DO3AC4H8, which serves as a model for N-protonation. Our combined experimental (in vitro and in vivo) and computational studies unravel complex pH-dependent speciation and enable side-by-side comparison of N- and O-protonated species of relevant 64Cu radiopharmaceuticals. Our studies suggest that N-protonated species are not inherently unstable species under physiological conditions and demonstrate the potential of N,N-alkylation as a tool for the rational design of future radiopharmaceuticals.