Revisiting dithiadiaza macrocyclic chelators for copper-64 PET imaging

Abstract

Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii) complexes were calculated using density functional theory (DFT) and validated by both NMR and EPR spectroscopy. DFT calculations revealed a switch in the orientation of tetragonal distortion upon protonation, which might be responsible for poor stability of the Cu(II)NSNS2A complex in aqueous media, whilst the same switch in tetragonal distortion was experimentally observed by changing the solvent. The chelator was radiolabeled with 64Cu and evaluated using PET/MRI in rats. Despite a favorable redox potential to stabilize the cuprous state in vivo, the 64Cu(II)NSNS2A complex showed suboptimal stability compared to its tetraazamacrocyclic analogue, 64Cu(TE2A), with a significant 64Cu uptake in the liver.

Figure 1.

Coordination environment of the Type 1 copper binding site in azurin.

Figure 2.

Structures of ligands discussed in the paper.

Figure 3.

The thermal ellipsoid diagram showing the structure of the cationic unit for [Cu(1)](ClO₄)₂. Hydrogen atoms and counteranions are omitted for clarity. Thermal ellipsoids are set at 35% probability.

Figure 4.

Spin density contour plots (contour value 0.003) of Cu^(II)(NSNS2A) on the left and [Cu^(II)(H₂NSNS2A)]²⁺ on the right based on optimised geometry from DFT calculations. Atoms are colour-coded as follows: Cu – orange, S – yellow, O – red, N – blue, C – dark grey, H – light grey, hydrogens attached to carbons are omitted for clarity.

Figure 5.

Experimentally observed UV-vis spectra of Cu^(II)(NSNS2A) in water and acetonitrile (top) and corresponding calculated spectra for Cu^(II)(NSNS2A) and [Cu^(II)(H₂NSNS2A)]²⁺ (bottom). The calculated spectra were shifted to match the corresponding experimental spectra.

Figure 6.

CW X-band EPR spectrum at 77K (solid line) with a fitted curve (dashed lines) for Cu^(II)(NSNS2A) (left). 45% glycerol/55% 0.1 HEPES solution (pH = 7.0). CW X-band EPR spectrum at 298K for Cu^(II)(NSNS2A) in water (right).

Figure 7.

Cyclic voltammograms of copper complexes Cu(NSNS2A) upon variation of scanning rate (A, 0.1M HEPES, 0.5M KNO₃, pH = 7.0) and pH (B, 0.1M HEPES, 0.5M KNO₃). The inset in Fig.7A shows a linear dependence between the anodic peak current and the square root of scan rates.

Figure 8.

A least-square fit of the binding curve for Cu^(II)(NSNS2A) (42 μM ClO₄⁻, 0.2 mM Cu²⁺, pH = 5.0, left) and dissociation curves for Cu^(II)(NSNS2A) in the presence of 1.0 and 12.5 equivalents of EDTA (0.1M MES, pH = 5.5, right).

Figure 9.

Optimised structures of Cu^(I/II)(NSNS2A)^-/0 and its protonated version [Cu^(I/II)(H₂NSNS2A)]^+/2+. Geometry was optimized using D3-B3LYP/def2-TZVP in a continuum model of water (SMD parametrization).

Figure 10.

Coronal PET/MR (A) and PET (B) images of a rat injected with ⁶⁴Cu^(II)(NSNS2A) 30 min post injection and a corresponding time-activity curve (E). Coronal PET/MR (C) and PET (D) images of a rat injected with ⁶⁴Cu(TE2A) 30 min post injection and a corresponding time-activity curves for blood, liver, and kidneys (F).

Figure 11.

Biodistribution data (%ID/g) for ⁶⁴Cu^(II)(NSNS2A) (N=3) and ⁶⁴Cu(TE2A) (N=3) at 90 min post injection.

Scheme 1.

Synthetic pathways for copper complexes discussed in the paper. Crystal structures of 1 and 2·2HCl can be found in ESI.