In many aggressive cancers, hypoxia is a characteristic hallmark and drives malignant progression and therapy resistance, e.g. chemotherapy and radiotherapy, adversely impacting overall patient survival. With increasing availability of therapeutic ⁶⁷Cu, interest is rising to harness the ⁶⁴Cu/⁶⁷Cu matched pair for hypoxia-directed radiotheranostic concepts.(Boulefthour et al., Med. Sci. Monit. 2021) In recent years, [⁶⁴Cu]Cu-ATSM (Copper (II) diacetyl-bis(4-methyl-3-thiosemicarbazone); Cu-ATSM) has emerged as a non-invasive hypoxia-selective PET agent (Pérès et al., EJNMMI Res. 2019), whose selectivity can be enhanced through hybrid structures with other hypoxia-responsive moieties, e.g. 2-nitroimidazoles (2-NIM).(Bonnitcha et al., J. Inorg. Biochem. 2010) Regardless, suboptimal pharmacokinetics, metabolic instability, and poor aqueous solubility of ATSM derivatives persist and must be overcome to ensure radiotheranostic translation.(Martínez-Camarena et al., Dalton Trans. 2023) This work systematically evaluates solubility profiles, complexation behaviors, and radiolabeling potentials of novel synthesized ATSM-2-NIM hybrids compared to ATSM references.

ATSM references were synthesized as published. Novel ATSM-2-NIM hybrids were prepared via HBTU-mediated coupling of H₂ATSM/en with N-(carboxy)-functionalized 2-NIMs, bearing different linker moieties, using DIPEA in DMF. Solubility was assessed in 0–100% (v/v) DMSO/H₂O and DMSO/buffer systems (HEPES, Tris, PBS) compared to ATSM references. Cu(II) complexation and impact of linker heteroatoms on overall chelator performance were investigated via ¹H NMR, UV/Vis, and ESI-MS.

ATSM references and novel ATSM-2-NIM hybrids were prepared in good yields (48-95%) and high purity (>95%). Solubility assessments of ATSM standards reveal that high DMSO ratios (>50%) are required to achieve sufficient solubility for chemical evaluations and compatibility with radiolabeling buffers. This is in stark contrast to reported DMSO ratios of up to 30% for buffered systems, complicating subsequent radiolabeling procedures and biological evaluations. The aqueous compatibility can be improved through linker variations, e.g. coupling of H₂ATSM/en with N-PEG-carboxy-2-NIMs, currently under investigation. NMR, ESI, and UV/Vis studies in DMSO verify 1:1 Cu(II) complexation modes, alongside clear preference of Cu(II) over Zn(II). First radiolabeling protocols are being established and will be reported.

Overcoming solubility limitations, while preserving hypoxia-directed chelator performance, is crucial for suitable design of novel ATSM hybrids to be used in ⁶⁴Cu/⁶⁷Cu cancer radiotheranostics.

We sincerely thank Claudia and Hans-Peter Opitz for their generous financial support of our group. Their contribution plays a crucial role in enabling our research and fostering scientific progress.