The theranostic pair mercury-197m and mercury-197g (197m/gHg, t1/2 = 23.8 h/64.14 h), through their γ rays and Meitner-Auger electron emissions, have potential use as constituents in radiopharmaceuticals to treat small metastatic tumours. However, the use of this pair of nuclear isomers in radiopharmaceuticals requires the development of suitable [197m/gHg]Hg2+ chelators as currently there is a lack of established ligands for radiometals in the field. Herein, this work studies the natHg/197m/gHg coordination of three thiacrown 18-membered N2S4 macrocycles with pendant arms of varying chemical "softness". Following the synthesis and characterization of the N2S4 ligand series (6,6'-((1,4,10,13-tetrathia-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid (N2S4-Pa), 7,16-bis(pyridin-2-ylmethyl)-1,4,10,13-tetrathia-7,16-diazacyclooctadecane (N2S4-Py) and 7,16-bis(2-(methylthio)ethyl)-1,4,10,13-tetrathia-7,16-diazacyclooctadecane (N2S4-Thio)), Hg2+ complexes were studied through mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations, revealing successful complexation of all ligands with the Hg2+ ion. Radiolabeling studies demonstrated the effect of the pendant arm on [197m/gHg]Hg2+ coordination, as N2S4-Thio and N2S4-Py had the highest radiochemical yield, similar to that of previously reported N-benzyl-2-(1,4,7,10-tetrathia-13-azacyclopentadecan-13-yl)acetamide (NS4-BA), while N2S4-Pa had the lowest. The complex integrity of [197m/gHg][Hg(N2S4-Py)]2+ and [197m/gHg][Hg(N2S4-Thio)]2+ in both human serum and glutathione was notably lower compared to the [197m/gHg][Hg(NS4-BA)]2+ complex. However, the [197m/gHg][Hg(N2S4-Py)]2+ and [197m/gHg][Hg(N2S4-Thio)]2+ complexes remained above 70% intact over 82 h when competed against biologically relevant metals (ZnCl2, FeCl3, CuCl2, MgCl2 and CoCl2), suggesting the selectivity of the ligands for Hg2+. This study illustrates the importance of the macrocyclic backbone size and electron-donor groups of the donor pendant arms in the design of chelators for 197m/gHg-radiopharmaceuticals, as both affect the radiolabeling properties and complex inertness.
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