A Comparative Study on the Complexation of the Anticancer Iron Chelator VLX600 with Essential Metal Ions

As cancer cells exhibit an increased uptake of iron, targeting the interaction with iron has become a straightforward strategy in the fight against cancer. This work comprehensively characterizes the chemical properties of 6-methyl-3-{(2E)-2-[1-(2-pyridinyl)ethylidene]hydrazino}-5H-[1,2,4]triazino[5,6-b]indole (VLX600), a clinically investigated iron chelator, in solution. Its protonation processes, lipophilicity, and membrane permeability as well as its complexation with essential metal ions were investigated using UV–visible, electron paramagnetic resonance, and NMR spectroscopic and computational methods. Formation constants revealed the following order of metal binding affinity at pH 7.4: Cu(II) > Fe(II) > Zn(II). The structures of VLX600 (denoted as HL) and the coordination modes in its metal complexes [Cu(II)(LH)Cl2], [Cu(II)(L)(CH3OH)Cl], [Zn(II)(LH)Cl2], and [Fe(II)(LH)2](NO3)2 were elucidated by single-crystal X-ray diffraction. Redox properties of the iron complexes characterized by cyclic voltammetry showed strong preference of VLX600 toward Fe(II) over Fe(III). In vitro cytotoxicity of VLX600 was determined in six different human cancer cell lines, with IC50 values ranging from 0.039 to 0.51 μM. Premixing VLX600 with Fe(III), Zn(II), and Cu(II) salts in stoichiometric ratios had a rather little effect overall, thus neither potentiating nor abolishing cytotoxicity. Together, although clinically investigated as an iron chelator, this is the first comprehensive solution study of VLX600 and its interaction with physiologically essential metal ions.

Table S1.Equilibrium processes associated with the overall protonation () and proton dissociation constants (K a ) of VLX600 and the overall stability (formation) constants () of its complexes.HL denotes the neutral form of VLX600.(The coordinated solvent molecules are not labelled for simplicity.)equilibrium process SI-5

DFT calculations for the [Fe(II)(LH) 2 ] 2+ complexes
For the bis-chelated iron(II) complexes, the formation of four coordination isomers is plausible.Iron(II) coordinates via the pyridine, imine N and the N 2 of the ligand or the pyridine, imine N and the N 4 donor set.However, the donor atoms of pyridine and N 2 or N 4 of the second ligand can alter the axial positions of the octahedral iron(II) leading to the formation of further coordination isomers.These binding modes together with the optimized structures are shown in Figure S3 and the ∆G values for the isomerisation reactions are reported in Table S3.Cartesian coordinates and energy values are summarized in Table S4-S7.

SI-8
The relative energies clearly indicate that iron(II) prefers the pyridine, imine N and the N 2 coordination mode, the energy gap for the formation of N 4 coordinated species was calculated to be 37.5 kJ/mol.The results of the DFT calculations of Isomer 1 are in good agreement with the experimental data obtained by X-ray crystallography.The two structures overlap confirming that the geometry optimization was reasonably accurate (Figure S4).
Table S3 Selected bond lengths, angles and the relative energy values obtained from DFT calculations.Selected parameters from X-ray crystallographic studies are also shown (taken from Figure 6).S21).{c VLX600 = 0.5 mM; c Cu(II) = 0.34 mM; I = 0.10 M KCl; T = 77 K}

SI-39
The ability of VLX600 to induce ROS production (DCFH/DA assay) To monitor the cellular levels of ROS, SW480 and CH1/PA-1 cells were seeded into 96-well plates (2.5 x 10 4 cells/well) in volumes of 100 µL per well.After a 24 h pre-incubation, the cells were washed with 200 μl Hanks' balanced salt solution (HBSS, Sigma-Aldrich, containing 1% FBS) and incubated with 100 μL of 25 μM DCFH-DA (2′,7′-dichlorofluorescein diacetate, Sigma-Aldrich) in HBSS (containing 1% FBS) for 45 min at 37 °C.After the incubation period, DCFH-DA solution was removed, and cells were washed with 200 μL HBSS.Cells were exposed to VLX600 alone and in the presence of 1 equiv.CuCl 2 , 0.5 equiv.ZnCl 2 and 0.5 equiv.FeCl 3 at four different concentrations between the range of 0.2 and 25 μM diluted in 200 μL phenol-red-free Opti-MEM (Gibco; with 1% FBS) and applied as triplicates.As blank, phenol-red-free medium with 1% FBS was used; the negative controls were the non-drug-treated cells in phenol-red-free medium with 1% FBS and the positive controls were cells treated with 200 μM and 400 μM tert-butyl hydroperoxide (Sigma-Aldrich).Fluorescence emission was measured at 10 min intervals with the BioTek Synergy HT reader (excitation: 485/20 nm, emission: 516/20 nm) over 2 h.

Figure S8
Figure S8 Hydrogen bonds of the [Cu(LH)Cl 2 ] crystal, highlighted in red.Intramolecular bond from Cl1 to N5 and intermolecular bond between Cl2 and N7.

Figure
Figure S16 13 C NMR spectra of VLX600 in DMSO-d 6 at 151 MHz.

Table S2
Experimental, sample and crystal data, data collection and structure refinement parameters

Isomer 1 Isomer 2 Isomer 3 Isomer 4 X-ray
Figure S6Comparison of the structures obtained from DFT calculation (yellow) and X-ray crystallography (blue).

Table S8
Selected bond lengths, angles and the relative energy values obtained from DFT calculations.Selected parameters from X-ray studies are also shown.Table S9Cartesian coordinates and energy values of [Cu(LH)Cl 2 ] (N 2 ).

complexes of VLX600: EPR spectroscopy in DMF/H 2 O solvent mixture Figure S10 Frozen
solution EPR spectra recorded for Cu(II) -VLX600 system in 30% DMF/H 2 O solution (lower spectra) in addition to the calculated component EPR spectra (upper spectra) obtained by the simulation of the measured spectra (see EPR parameters in Table