Clickable, Hydrophilic Ligand for <i>fac-</i>[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> (M = Re/<sup>99m</sup>Tc) Applied in an <i>S</i>‑Functionalized α‑MSH Peptide

The copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (<b>1</b> and <b>3</b>) for <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> (M = Re/<sup>99m</sup>Tc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (<b>3</b>) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of <i>fac</i>-[<sup>99m</sup>Tc<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The <i>fac</i>-[Re<sup>I</sup>(CO)<sub>3</sub>(<b>3</b>)] complex (<b>4</b>) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between <b>3</b> and <i>fac</i>-[<sup>99m</sup>Tc<sup>I</sup>(OH<sub>2</sub>)<sub>3</sub>(CO)<sub>3</sub>]<sup>+</sup>. The corresponding <sup>99m</sup>Tc complex (<b>4a</b>) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log <i>P</i> analysis compared to an analogous complex with nonfunctionalized pyridine rings (<b>2a</b>). An α-MSH peptide functionalized with an azide was labeled with <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> using both <i>click, then chelate</i> (CuAAC reaction with <b>1</b> or <b>3</b> followed by metal complexation) and <i>chelate, then click</i> (metal complexation of <b>1</b> and <b>3</b> followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexation within a peptide framework. The peptides from the <i>click, then chelate</i> strategy had different HPLC <i>t</i><sub>R</sub>’s and in vitro stabilities compared to those from the <i>chelate, then click</i> strategy, suggesting nonspecific coordination of <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> using this synthetic route. The <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup>-complexed peptides from the <i>chelate, then click</i> strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC<sub>50</sub> analyses, and led to moderately high uptake in B16F10 melanoma cells. Log <i>P</i> analysis of the <sup>99m</sup>Tc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (<b>10a′</b>) compared to the peptide with the unmodified DPA chelate (<b>9a′</b>). In vivo biodistribution analysis of <b>9a′</b> and <b>10a′</b> showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for <b>10a′</b> compared to predominantly hepatic accumulation for <b>9a′</b>. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexes in future studies.