Dimethyl sulfoxide (dmso)

TBAN and HEPES were purchased from Sigma-Aldrich
in puriss quality. HCl, KOH, KCl, DMSO, DMF, and KH-phthalate were
obtained from Molar Chemicals (Hungary) and used without further purification.
The stock solutions of metal ions were prepared by the dissolution
of CuCl₂, FeCl₃, and ZnCl₂ in water,
and the concentration was determined by complexometry using ethylenediaminetetraacetic
acid (EDTA). The Fe(II) stock solution was generated through the interaction
of iron powder and HCl in a purified, strictly oxygen-free argon atmosphere.
Subsequently, the solution was filtered and stored for use under anaerobic
conditions in a laboratory glovebox (GP(Campus), Jacomex), ensuring
that the O₂ level was kept below 1 ppm. The concentration
of the Fe(II) solution was determined through titrations with KMnO₄. All solutions were prepared using Milli-Q. If required,
the pH was adjusted to the desired level by adding of HCl or KOH.
All solvents used for the syntheses were dried and purified according
to standard procedures.

The effect of the complexes and of their ligands alone on the viability (on the metabolic activity) of human cancer cells was assessed by MTT assay. The tested compounds were dissolved in 90% (v/v) DMSO/H₂O at 10 mM concentration. Equimolar solutions of the ligand CuCl₂ salt were used for the cytotoxicity assays. The metal salts without ligands were also tested. For this, 1 × 10⁴ cells/well were seeded into 96-well plates and left to grow overnight. On the following day, cells were exposed to increasing concentrations of each compound (0; 2.5; 5; 10; 15; 20 µM). After a 24 h incubation, cells were washed with PBS buffer and then incubated with 0.5 mg/mL MTT (Sigma-Aldrich, St Louis, Missouri, MO, USA) for 1 h at 37 °C. The formazan crystals were solubilized in DMSO (Molar Chemicals, Halásztelek, Hungary), and the absorbance of samples was determined at 570 nm using a Synergy HTX plate reader (BioTek, Winooski, Vermont, VT, USA). Absorption corresponding to the untreated control samples was considered as 100%. The viability measurements were repeated three times using 3 independent biological replicates.

To quantify the violacein production of liquid E. coli cultures, we applied the modified protocol of Zhu et al. [47 (link)]. The investigated strain was grown in LB with or without selection for 24 h at 37 °C. One milliliter of the culture was spun at 13,000 rpm for 10 min. After discarding the culture supernatant, 1 mL of DMSO (Molar Chemicals Kft., Halásztelek, Hungary) was added to the pellet. The solution was vigorously vortexed for 30 s to completely solubilize violacein and was centrifuged again at 13,000 rpm for 10 min to remove the cell debris. Then, 200 μL aliquots of the violacein-containing supernatant were transferred to a 96-well flat-bottomed microplate (Greiner Bio-One International, Kremsmünster, Austria), making three technical replicates, and the absorbance was recorded at 585 nm using a Synergy2 microplate reader (BioTek, Winooski, VT, USA).