pubs.acs.org/IC Article Copper(II) N,N,O‑Chelating Complexes as Potential Anticancer Agents Quim Peña, Giuseppe Sciortino, Jean-Didier Maréchal, Sylvain Bertaina, A. Jalila Simaan, Julia Lorenzo, Merce ̀ Capdevila, Pau Bayón, Olga Iranzo,* and Ò scar Palacios* Cite This: Inorg. Chem. 2021, 60, 2939−2952 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (−H, −Cl, and −Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 μM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds. ■ INTRODUCTION Metals and their inorganic complexes show an enormous Tumors contain a more reducing environment with respect to healthy tissues. This is based on what is known as “the versatility in front of strictly organic compounds for the Warburg effect” and consequence of the fact that cancer cells development of therapeutic agents. The possibility of having do primarily generate energy by an atypical aerobic glycolysis several oxidation states, different coordination numbers, and pathway.10,11 This abnormal metabolic process (instead of the diverse geometries gives rise to a broader spectrum of tuneable usual oxidative phosphorylation) induces an imbalanced redox properties.1 Among them, Cu complexes have become homeostasis inside cancer cells, leading to an enhanced promising alternatives for cancer treatment during the two intracellular ROS production.12 Consequently, the interference last decades.2−5 Copper is a physiological metal, being widely with cellular redox homeostasis arises as an attractive and present in many biomolecules and playing a remarkable role in promising target for chemotherapy. Cancer cells exhibit a diversity of biochemical processes because of its interesting abnormal levels of ROS, and they show higher vulnerability Cu(II)/Cu(I) redox pair. 6 In fact, one of the main potentialities of Cu as an antiproliferative agent lies in its capability to form reactive oxygen species (ROS) inside the Received: October 2, 2020 cells. The generation of these entities (H2O2, O2•−, HO•, etc.) Published: February 17, 2021 is not only reported to damage DNA but also to offer a putative discrimination between healthy and cancer cells.7,8 The lack of selectivity in cancer therapy has always been a downside in this field, giving rise to severe side effects.9 © 2021 American Chemical Society https://dx.doi.org/10.1021/acs.inorgchem.0c02932 2939 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Scheme 1. General Synthesis of the N,N,O-Chelating Salphen-like Ligands H2L1 (R = H), H2L2 (R = Cl), and H2L3 (R = Br) and of Their Corresponding Cu(II) Complexes C1−C3 Figure 1. SQUID data obtained for complex C3: (A) susceptibility measured at 1 T. Red curve is the best fit for a dimeric complex, (B) magnetization measured at 2 K showing a fitting with the theoretical model for 2 coupled spins, and (C) ACmagnetic susceptibility. (D) Comparison of the magnetic susceptibility of C1−C3 complexes. to ROS level changes than healthy cells do; therefore, the be able to generate high ROS levels in cells. This should lead alteration of those levels may be a unique opportunity to to enhanced toxicity toward cancer cells with respect to healthy selectively target cancer cells.7,8 There is, hence, high potential ones. The impact of halogenated substituents has also been for the development of bioreducible metal complexes. evaluated and is discussed here. Speciation and the putative Up to date, several Cu(II) complexes have been reported to active species in solution are discussed hereby from a be redox-active,5,13−15 and indeed, some structure−activity theoretical approach, and the mechanism of action is relationships have been reported between the redox behavior thoroughly evaluated and related to the redox behavior of of N-donor aromatic Cu(II) complexes and their ROS- the Cu(II) complexes. ■ mediated cytotoxicity.16−18 In particular, Schiff-based Cu(II) complexes have attracted attention from researchers in this RESULTS AND DISCUSSION field and have been reported to show interesting cytotoxicity toward cancer cells and DNA cleavage.15,19−21 Not many This work is based on the design of a basic scaffold ((E)-N-(2- Cu(II) complexes with N,N,O-chelating Schiff base ligands (2-hydroxybenzylideneamino)phenyl)acetamide, H 2 L1), have been evaluated in cancer cells,22 in contrast to Cu(II) which specifically intends to chelate Cu(II) in a tridentate N,N,S-chelated thiosemicarbazone and bis(thiosemicarbazone) fashion having a fourth labile in-plane coordination position, complexes.23−27 with the idea of biologically attaining a fast Cu(II)/Cu(I) Here, we describe the synthesis, characterization, and redox cycle. evaluation of the biological activity of three novel Cu(II) Synthesis and Characterization of the Ligands and complexes bearing N,N,O-chelating salphen-like ligands as Their Corresponding Copper(II) Complexes. The three potential antitumoral agents. The aim is to obtain biologically N,N,O-chelating salphen-like ligands (H2L1, H2L2, and H2L3, accessible Cu(II)/Cu(I) redox cycling systems, which would Scheme 1) were synthesized based on a condensation reaction 2940 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article between the mono-protected benzene-1,2-diamine precursor Table 1. Experimental JCu−Cu (cm−1) and g-Factors (1) and the corresponding salicylaldehyde precursor. Pure Obtained from SQUID Measurements in the Solid State for ligands were obtained by column chromatography purification. Complexes C1−C3 Characterization data are reported in the experimental section JCu−Cu (cm−1) g and Supporting Information (Figures S1−S3). Mono-protected diamine (1) was obtained following the C1 3.5 2.19 procedures reported in the literature.28 For H2L1, commer- C2 5.7 2.15 cially available 2-hydroxybenzaldehyde was used as the starting C3 8.7 2.19 material. To obtain H2L2 and H2L3, halogen derivatization was carried out on the para-hydroxy position of the starting obtained points to an analogous conformation for all three material 2-hydroxybenzaldehyde following the reported complexes. Isothermal magnetization (T = 2 K) confirms the procedures. Chlorination was carried out under mild presence of two coupled spins (Figures 1B and S5). Finally, conditions using N-chlorosuccinimide (NCS) and an acid AC-susceptibility measurements (Figures 1C and S5) indicate catalyst.29 This reaction provided lower yields than those the absence of long-range order that would be due to the found with common chlorinating agents (Cl2 or sulfuryl presence of polymers. chloride), but a cleaner reaction.30 The final 4-chloro-2- The integrity of the species was evaluated in the DMSO hydroxybenzaldehyde (2) precursor was purified through solution. The dimeric structure of C1−C3 has been confirmed column chromatography. Alternatively, bromination of the by the observation of the corresponding peaks in HR-ESI-MS starting material was carried out using standard procedures (m/z 631.0456, 700.9661, and 786.8678, respectively, Figure with Br2, to obtain compound 3. S4A). These data indicate that the nuclearity is at least partially Complexation of pure H2L1−H2L3 was carried out using maintained in solution. At the same time, peaks attributed to Cu(OAc)2 as the metal precursor salt (Scheme 1). The use of the corresponding mononuclear species were also found the Cu(II) acetate salt allowed deprotonating −OH and −NH (Figure S4C), suggesting a solvent-dependent process at once. Complexes (C1−C3, Scheme 1) were isolated as involving partial breakage of the dinuclear species and brownish powders by precipitation from the reaction media. In coordination of the DMSO solvent in the fourth binding site all the cases, the solubility of the complexes was very poor in of the metal coordination sphere. Electron paramagnetic the common organic solvents, especially for C2 and C3, even if resonance (EPR) shows the presence of a single EPR-active this can be improved by the use of coordinating solvents such Cu(II) species in solution for all the three complexes (Figure as dimethyl sulfoxide (DMSO) and dimethylformamide. S4B and Table S1). The observed EPR signals for C1−C3 in The data recorded for the complexes (Experimental Section, the DMSO solution are typical for Cu(II) monomeric species Figure S4 and Table S1) suggest a 1:1 ligand to metal in square−pyramidal-derived geometries with the single stoichiometry in the solid state (elemental analysis, see electron in dx2−y2 orbitals (g∥ > g⊥ > ge). From the analysis of Experimental Section), with both −OH and −NH groups the EPR parameters derived from simulations (A and g- deprotonated and in the absence of any additional ligand or tensors), it appears that the three complexes mainly exist in a counterion. The IR bands of H2L (Figures S1C−S3C) nonsignificantly distorted square-planar or square-pyramidal assigned to the stretching of both O−H (phenol) and N−H geometries with the N2O2 coordination in the equatorial plane, (amide) bonds at 3500−3300 cm−1 as well as those related to as expected based on the N,N,O-chelating ligands and on the bending mode of the N−H bond (about 1660 cm−1, scissor solvent coordination. In order to provide further insights into bending) and of the O−H (1500 cm−1) bond have disappeared the monomer/dimer coexistence of the complexes in DMSO, in the corresponding Cu(II) complexes (Figure S4A). This quantification of the Cu(II) mononuclear species through confirms the deprotonation of both the amide (−NH) and the double integration of the EPR spectra of C1, as the model phenol (−OH) groups upon metalation. No peaks for compound, was carried out at different time points (Table S2). additional counterions or ligands have been observed. The EPR spin quantification data demonstrate that the dissolution Cu(II) coordination sphere in the solid state is composed of of C1 in DMSO gives rise to 30% of the mononuclear Cu(II) the N,N,O-chelating salphen-like ligand and a fourth oxygen signal, thereby pointing to the presence of EPR-silent from the carbonyl of the amide group (CO) of the second magnetically coupled dinuclear species. The Cu(II) signal entity of the dimer. evolves over time reaching about 50% of the mononuclear The magnetic properties of the complexes C1−C3 were species after 24 h. This confirms the dimeric cleavage process studied using a conventional SQUID magnetometer. The in a solvent and time-dependent manner. In addition, results for the three complexes are very similar. The results for increasing the ionic strength by the addition of salts seems C3 are presented in Figure 1, while those for C1 and C2 are to slightly contribute to the cleavage of the dimeric form given in the Supporting Information (Figure S5). Figure 1A (Table S2) reaching up to 60% after several days. The overall shows the isofield (H = 1 T) χT as a function of temperature data are thus in concordance with those of ESI-MS analysis (T). The red curve is the best fit using the Bleaney−Bowers (Figure S4C), and suggest the coexistence of both dimer and equation of a coupled S = 1/2 dimer.31 The fit shows a monomer species in solution. ferromagnetic coupling for the three complexes, with small DFT Studies for the Evaluation of the Active Species exchange coupling constant (J) values ranging from 3.5 to 8.7 in Solution. Density functional theory (DFT) computational cm−1 and a g-factor of 2.15 to 2.2 coherent with the presence studies of the parent ligand H2L1 and its corresponding Cu(II) of Cu(II) (Table 1) and the absence of monomers. The complex C1 have been carried out to model and rationalize the increase in the J values (Table 1) with the increase of the size speciation in solution of C1−C3 complexes. Two solvents of the R substituent (Scheme 1) suggests that the were chosen to be computed: DMSO to compare with the functionalization influences the Cu(II)−Cu(II) distance in beforehand obtained experimental values and water because of the dinuclear structure. Additionally, the very similar g values its biological relevance. Dimeric and monomeric Cu(II) 2941 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Figure 2. (A−C) Optimized geometry of the three main conformations of the dimeric complex [CuII(L1)]2, and the monomeric species (D) [CuII(L1)(H2O)] and (E) [CuII(L1)(DMSO)]. Cu−Cu distances are reported in angstrom. The deviation from square-planar toward tetrahedral geometry is also reported in degrees as the dihedral angle (θ) between the fourth equatorial donor atom and the donors of the tridentate chelating L1 ligand (D−Ncis−O−Ntrans). complexes formed by the ligand H2L1 (Scheme 1) were Table 3. Simulated JCu−Cu (cm−1) and ΔG Values for the examined and their proposed structures were simulated: Different Conformations of the Dimeric [CuII(L1)]2 [CuII(L1)]2, [CuII(L1)(H2O)], and [CuII(L1)(DMSO)] Speciesa (Figure 2). Concerning the dimeric species, among the seven Cu−Cu different conformations considered with relative orientation of JCu−Cu distance ΔGDMSO/ΔGaq L1 ligands and the coordination position (axial or equatorial) [CuII(L1)]2 (cm−1) coupling (Å) (kcal·mol−1)b of their donors, only three have been characterized as minima conf. A 12.0 ferromagnetic 2.741 0.0/0.0 in the potential energy surface (Figure 2A−C). conf. B −43.2 antiferromagnetic 4.128 0.7/7.4 In all the cases, the Cu(II) centers present a square-planar conf. C 22.3 ferromagnetic 2.766 5.3/9.7 arrangement with different grades of distortion, ranging from a J has been determined with a reported method.33,34 bDFT level using almost pure square-planar geometries for [CuII(L1)(H2O)] B3LYP-D3 combined with the basis-set def2-TZVP for the main (170.0°, Figure 2D) to highly distorted for [CuII(L1)]2 group elements and the quadruple-ζ def2-QZVP basis set for Cu. (114.8° in conformation C, Figure 2C). The Gibbs energy calculations for the dissociation reactions of the dimeric forms to monomeric species (Table 2) suggest a experimentally (Table 1). Computed values show that only conformation B (Figure 2B) has an antiferromagnetic coupling Table 2. ΔG Values for the Dissociation of the Dimeric (J = −43.2 cm−1). In contrast, for conformations A and C Species [CuII(L1)]2 to the Monomeric Complex (Figure 2A,C), the predicted interaction is ferromagnetic (12.0 [CuII(L1)(solv)] in Different Solventsa and 22.3 cm−1, respectively). The reason behind the different magnetic behaviors can be found from the Cu−Cu distances [CuII(L1)]2 solv ΔEsolv ΔGsolv between the two radical spins, that is, ∼2.7 Å in conformations conformation A H2O −1.8 −1.8 A and C allowing a ferromagnetic coupling, versus 4.1 Å in DMSO 9.2 4.4 conformation B, in which the cores are well separated (Figure conformation B H2O −9.5 −9.2 2).32 According to the experimental ferromagnetic exchange DMSO 6.9 3.7 couplings for the three complexes (JCu−Cu, Table 1), the most conformation C H2O −12.6 −11.4 probable structure of C1−C3 would fit with conformation A, DMSO 2.8 −1.0 which shows the lowest Gibbs energy and JCu−Cu values (Table a Values reported in kcal mol−1. The corrections of RT ln V (1.89 3). kcal/mol) and RT ln([solv]/n) were applied. Values computed in the The EPR parameter simulations for the monomeric species SMD continuum model for H2O or DMSO. in DMSO, [CuII(L1)(DMSO)], are in the range of the experimental results (Table 4). The relative deviation of the different behavior depending on the solvent. In water, the calculated gz and Az values from the experimental ones is dissociation reaction appears to be favored with ΔGaq from −16.0% for Az and −1.9% for gz. The larger deviation of Az for −1.8 up to −12.6 kcal·mol−1, while in DMSO, data are [CuII(L1)(DMSO)] must be related to the significant consistent with the coexistence of the dimeric and monomeric distortion of the equatorial plane of the Cu(II) ion because forms with ΔGaq from 4.4 to −1.0 kcal·mol−1. of the coordination of DMSO (θ = 138.4°), and these To corroborate the structures obtained and to discriminate differences are common between the computed and exper- between the three dimeric forms, the J values were computed imental parameters, especially on the A tensor.35 in each case to determine the ferro or antiferromagnetic nature The UV−vis vertical excitation has also been computed for of the interaction between the two unpaired electrons on the both dimeric [CuII(L1)]2 (conformation A) and monomeric Cu(II) centers (Table 3), and compared with those obtained [CuII(L1)(H2O)] and [CuII(L1)(DMSO)] species and 2942 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Table 4. EPR Parameters Computed (Calc) for the Monomeric [CuII(L1)(DMSO)] Species in the DMSO Medium, and Comparison with the Experimental (Exp) Values [Relative Deviation (RD) Related to the Experimental Value], Extracted from Figure S4B and Table S1 a a species Acalc, z Aexp, z ARD z gcalc z gexp z gRD z II b b [Cu (L1)(DMSO)] 154.0 183.4 16.0% 2.201 2.244 1.9% a Values in 10−4 cm−1. bValues recorded in DMSO. Figure 3. (A) Cyclic voltammograms vs Fc+/Fc (Fc) of C1−C3 in DMSO with 0.1 M TBAP at a scan rate of 100 mV/s. (B) Representation of E1/2 values obtained for C1−C3 placed in the biological redox window at pH 7.36 compared with experimental values (Figures S6 and S7 and The determined Cu(II)/Cu(I) redox potentials (E1/2 = Table S3). Computed MLCT transition bands are in the range −1.07 V for C1 and −1.03 for C2−C3 vs Fc+/Fc, Table S4) of the experimental ones for the monomeric [CuII(L1)(solv)] are within the biological range of −1.1 to 0.2 V versus Fc+/Fc species (Figure S6 and Table S3), while computed Cu(II) d−d (Figure 3B). The presence of electrowithdrawing groups in C2 transitions could indeed fit with both forms (dimer and and C3 slightly favors the Cu(II) reduction to Cu(I) (Ered = monomer, Figure S7 and Table S3). The overall results are in −1.09 and −1.08 V, respectively) compared to C1 (Ered = concordance with the EPR values compared previously. Even if −1.15 V) (Table S4). Both the chloro- and bromo-derivatives the presence of the dimeric form has been widely have the reduction potential 60 and 70 mV higher than C1. demonstrated, all data point to a significant role of the Despite the fact that halogen groups make Cu(II) more prone monomeric Cu(II) form in the final activity of C1 in solution. to be reduced to Cu(I), the final E1/2 values for the three Evaluation of the Potentiality of the Complexes as complexes are similar (Figure 3B and Table S4). ROS Generators. The redox properties of the Cu(II) In order to characterize the center of the redox process, the complexes were evaluated by cyclic voltammetry (CV) Gibbs energy of the product of the monomeric C1 reduction experiments. CV was carried out with both the ligands process was calculated at the DFT theory level for two (H2L1−H2L3) and the complexes (C1−C3) in DMSO. different spin multiplicities: S = 1, corresponding to [CuI(L1)- Taking into account that the biological redox window (DMSO)]−, and S = 3, accounting for the L1 reduction approximately ranges from −1.1 to 0.2 V versus Fc+/Fc forming [CuII(L1•−)(DMSO)]− (Figure S9).18 The obtained (values arising from the oxidation and reduction of water at pH Gibbs free energy value of the reduction on the ligand is 32.7 7,36 respectively), we specifically analyzed in detail this region kcal·mol−1 higher than that on the metal center (Table S5). This difference highlights that the ligand participation in the (Figure 3). H2L1−H2L3 do not show any kind of redox redox process is negligible and the oxidation state of Cu in the activity in this specific range. On the contrary, all the Cu(II) two minima can be described as +II and +I. complexes are redox active and the signals observed on the The CV results suggest that the C1−C3 complexes can be cyclic voltammograms of C1−C3 have been ascribed to the thermodynamically reduced by biological redox buffers, and Cu(II) ⇄ Cu(I) redox process (Figure 3). perform a quasireversible redox process. Therefore, they seem The redox potentials (Table S4) were assigned to the redox to be capable of undergoing Cu(II)/Cu(I) redox cycling under couple Cu(II)/Cu(I) based on bulk electrolysis and EPR biological conditions. In order to confirm their capability to experiments (data not shown). The difference between biologically undergo Cu(II)/Cu(I) redox cycling, ascorbate cathodic and anodic peaks in C1−C3 cyclic voltammograms consumption at pH 7.2 was monitored by UV−vis (Figure 4). is higher than the theoretical 0.060 V for fully reversible redox Cu(II), in the presence of ascorbate and under aerobic processes (ΔEp ranging from 0.11 to 0.16 V), but in the range conditions, catalyzes the generation of ROS.38 Measuring the of the ΔEp (0.10−0.12 V) obtained for the ferrocene (Fc+/Fc) consumption of ascorbate at its maximum absorbance (265 reference compound under the same experimental conditions. nm) in the presence of the Cu(II) complexes provides an idea Successive scans were performed, and the lack of signal change of their capability to generate ROS inside cells. In the absence upon the successive collected scans indicates that no of any Cu catalyst (DMSO control), no decrease in the disproportion occurred after cycling between Cu(II) and absorbance at 265 nm can be observed (Figure 4), thus Cu(I) in none of the three Cu(II) complexes. The Ipa/Ipc ratio indicating that ascorbate (100 μM) is stable and the medium close to 1 and the calculated ΔEp values (Table S4) suggest a does not consume it. In contrast, the presence of a catalytic quasireversible one-electron process. The linear dependence of amount of free Cu(II) ions (2 μM of CuCl2 addition) clearly the peak currents Ipc and Ipa versus the square root of the scan shows a rapid decrease in the absorbance, and ascorbate has rate (ν1/2) is indicative of a diffusion-controlled process been almost totally consumed after just 20 min. Complex C1 (Figure S8).37 (2 μM concentration added) is able to consume it at similar 2943 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article show significant antiproliferative activity, yet bearing a nontoxic ligand (H2L1). The toxicity of the latter can then only be attributed to a conjoint contribution between the ligand H2L1 and the Cu(II) ion, that is, to the entire complex; and not solely to the simple addition of the Cu(II) ion plus the ligand toxicities. In the case of C1, this feature may imply an advantage in terms of drug metabolism because none of the frameworks that constitute the complex (H2L1 and Cu(II) ion) do separately exhibit cytotoxicity. Because of the low solubility in the biological culture medium exhibited by C2 and, at lesser extent, C3, and considering the similar IC50 values in both cancer cell lines with that of C1, the latter was chosen as the model scaffold to evaluate the cytotoxicity toward normal embryotic fibroblasts Figure 4. Consumption of ascorbate (100 μM) mediated by CuCl2 and complexes C1, C2, and C3 in NaCl and Tris−HCl buffer at pH (NIH 3T3), selected as nontumoral cell lines. As observed in 7.2 (5% DMSO). The four Cu(II) compounds were at a the dose−response cell viability diagram (Figure 5), complex concentration of 2 μM. rates than free copper(II) ions do, while C2 and C3 (at the same concentration as C1) exhibit a slower consumption rate than that of C1. One possible explanation for the different consumption rates could be related to solubility issues because C2 and C3 are less soluble in aqueous media than C1. Consequently, and based on the overall ascorbate consumption data, it is expected that C1−C3 can exert some kind of redox- mediated cytotoxicity through the generation of ROS inside cells. Cytotoxicity Assays in Cancer and Normal Cell Lines. The in vitro antiproliferative activity of the complexes C1−C3 Figure 5. Comparison of the dose−response cell viability diagrams of and their corresponding free ligands were determined on C1 in HeLa, MCF7, and NIH 3T3 (fibroblasts) cell lines (0−100 somatic HeLa and MCF7 cancer cell lines (Table 5 and Figure μM) at 72 h. The obtained values average at least three independent S10). experiments. Table 5. IC50 (μM) Values at 72 h of Complexes C1, C2, C3 and Their Corresponding Ligands in HeLa, MCF7, and NIH C1 exhibits lower toxicity toward normal fibroblasts with 3T3 Cultures, Using CuCl2·2H2O as the Reference respect to both HeLa and MCF7 cancer cells. This is Compounda interesting in terms of selective chemotherapy because it might provide less side effects. compound HeLa MCF7 NIH 3T3 Evaluation of the Interactions of the Complexes C1 26 ± 4 30 ± 6 ≥100 toward DNA. In order to evaluate the effect and interaction of C2 25 ± 2 b b the complexes C1−C3 with DNA, traditionally considered as C3 23 ± 10 29 ± 5 b one of the main targets of chemotherapy, several experiments H2L1 ≥200 ≥150 ≥200 have been carried out, namely gel electrophoresis, UV−vis, H2L2 ≥50 b b and/or circular dichroism (CD). H2L3 ≥50 ≥50 b First of all, the cleaving properties of complexes C1, C2, and CuCl2·2H2O18,39 ≥200 ≥200 ≥200 C3 were investigated by gel electrophoresis because many a The results shown are representative of at least three independent Cu(II) complexes have been reported to induce cell death experiments (N = 3). bExperiments were not carried out because of through DNA cleavage.5,19,43,44 The conversion of supercoiled poor solubility in the cell culture medium. In the case of the circular plasmid DNA to open DNA forms was followed nonassayed complexes, their corresponding ligands were not assayed (Figure 6) and the obtained results indicate that the three either. complexes are only able to partially cleave supercoiled plasmid DNA (ScdsDNA), leading into a minor band corresponding to The IC50 values obtained in HeLa cancer cells (Table 5 and its open circular form (ocDNA, form II). This confirms that Figure S10) for the ligands show that while ligand H2L1 they do not possess prominent cleaving capacity by themselves. presents poor or negligible toxicity, H2L2 and H2L3 display In contrast, the presence of a reductant species, such as significant cytotoxicity. This difference might be attributed to ascorbic acid (a biological reductant), enhances their cleaving the presence of the halogen substituents.40,41 Complexes C1, capacity (Figure 6, colored lines), and they are then able to C2, and C3 exhibit remarkable and dose-dependent cytotox- practically transform all the ScdsDNA into ocDNA and, to a icity in both HeLa and MCF7 cells (IC50 about 25 μM, Table lesser extent, into its linear form (form III). As already 5) when compared to CuCl2 and to the two commercially mentioned, the generation of Cu(I) stimulates the potential available Pt-drugs cisplatin (IC50,72h of 15 μM in HeLa39) and formation of ROS, which have DNA-cleaving abilities.3 In our carboplatin (IC50,72h of 39 μM in HeLa42). Both C2 and C3 particular case, the results clearly point to a redox-dependent are bearing toxic ligands (H2L2 and H2L3), whereas C1 does mechanism, triggered by the presence of ascorbic acid, which 2944 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Ao εG εG 1 = + · A − Ao εH−G − εG εH−G − εG Kb[DNA] (1) The plot of the relative variation of the absorbance (Ao/(A − Ao)) versus the inverse of the DNA concentration (1/ [DNA]) (Figure S11) allows the determination of Kb (Table 6). The Kb values obtained for complexes C1, C2, and C3 are in the order of 104 M−1, indicating a moderate interaction and lower than the values around 106 to 107 known for classical and strong mtallointercalators (DAPI, HOECHST, etc.).48,51,52 Figure 6. Agarose gel electrophoresis of a BlueScript Supercoiled In Vitro ROS Generation and Induction of Apoptosis. DNA (ScdsDNA) treated with complexes C1, C2, and C3. Incubation time of 24 h at 37 °C. Some samples were incubated The results obtained from the CV studies (Figure 3), ascorbate for an additional 1.5 h in the presence of ascorbic acid. consumption experiments (Figure 4), and DNA-cleaving activity (Figure 6) strongly indicate an oxidative dependent mechanism of action. In order to confirm the formation of promotes the Cu(I) generation, the potential formation of intracellular ROS in HeLa cancer cells, the 2′,7′-dichloro- ROS, and the concomitant DNA damage. fluorescin diacetate (DCFDA) assay was performed.14,53 Next, the binding of C1−C3 with calf thymus DNA (ct- DCFDA is a nonfluorescent and permeable dye that, after DNA) was studied. Covalent interactions with DNA are highly cleavage by intracellular esterases and subsequent oxidation by important in the case of cisplatin and Pt compounds,45,46 ROS, generates dichlorofluorescein (DCF), a fluorescent and whose mechanism of action is usually conceived through the nonpermeable compound. formation of Pt-DNA adducts. In the case of Cu(II) The experiment was performed with C1 as the main scaffold, complexes, covalent adducts with DNA are less common and and to serve as a proof-of-concept to understand the normally they do not show this kind of binding.18 CD and mechanism of action of the N,N,O-chelating metallic core. UV−vis spectroscopies have been used to enlighten the After 4 h treatment, strong DCF fluorescence, of up to 3-fold putative DNA-complex binding modes of C1, C2, and C3 respect to control cells, was observed for C1 (Figure 8), (Figure 7). highlighting the ROS production capabilities of this Cu(II) CD spectroscopy allows assessing the possible structural complex. The ROS levels of C1 are equivalent to those alterations of the characteristic bands of ct-DNA (a positive produced by the positive control H2O2. On the contrary, H2L1 band around 280 nm and a negative band around 245 nm)47 was not able to increase the ROS levels (Figure 8) with respect upon complex interaction. ct-DNA (50 μM) was incubated to the control group. This is in concordance with the Cu(II)/ with C1, C2, or C3 (from 0 to 2 equivalents) overnight and Cu(I) redox potential of C1 (Figure 3) and with the results analyzed by CD spectroscopy (Figure 7A−C). In all cases, only obtained for the toxicity of H2L1 and C1 (Table 5). minor modifications of the initial CD signals were observed, These results confirm the relationship inferred between the pointing to slight structural changes in the helicity of the ct- Cu(II)/Cu(I) redox potential of C1, its ROS production DNA. This suggests some kind of noncovalent interaction, but inside the cells, and the exerted biological activity. without significant structural DNA modifications. Furthermore, this ROS cell death pathway might explain the Three main classes of noncovalent binding have been different toxicity profiles observed for C1 in HeLa and MCF7 proposed for metal complexes: intercalation, groove binding, cancer cells with respect to normal cell lines (NIH 3T3) and electrostatic interactions with the negatively charged (Figure 5). Taking into account that cancer cells have higher phosphate backbone of DNA. In order to assess the nature of radical levels than healthy ones, the production of ROS might the complex−DNA interactions, UV−vis spectroscopy has appear as a differentiating feature. Accordingly, C1 displays a been used to monitor the changes in the absorbance of C1− lower toxicity profile in fibroblasts than in the two tested C3 complexes upon increasing additions of ct-DNA to a cancer cell lines (Figure 5). solution of the corresponding metal complexes. Absorption Finally, the evaluation of the mechanism of cell death in spectra in the range of 225−550 nm were recorded at a HeLa cancer cells by C1 was carried out by using the standard constant complex concentration (30 μM) with increasing propidium iodide (PI)/Annexin V-Alexa Fluor 488 assay amounts of DNA. The results for complexes C1−C3 (Figure (Table S6). The induction of ROS has been related to the 7D−F) clearly show a hypochromic effect upon ct-DNA mechanism of apoptosis,54 and many research efforts have addition, but no significant bathochromism is observed in any been devoted to the synthesis of potential anticancer agents spectra. This points to an interaction with DNA via groove that induce an apoptotic cell death pathway.55,56 The results binding or electrostatic interactions rather than via inter- indicate that C1 is able to partially trigger apoptosis in HeLa calation.48,49 Compounds displaying high DNA-intercalating cancer cells, where at least about 12% of cells are in the early capabilities usually induce a bathochromic shift because of apoptotic stage (Table S6). This value is in the range of their π−π interactions with the aromatic bases of DNA, a cisplatin, which is well known to induce an apoptotic phenomenon that has not been observed in this case. pathway.57,58 The rest of death cells (24%) have high Quantitative data, that is, the intrinsic binding constant Kb, fluorescence values of PI, indicating that the membrane is can be obtained from the recorded absorption spectra using not intact. This might point to a necrosis, with loss of the Benesi−Hildebrand equation (eq 1).50 A o is the membrane integrity, or to an apoptotic necrosis (late absorbance of the complex in the absence of DNA, A is the apoptosis). This last mechanism involves an early apoptosis, absorbance at any given DNA concentration, and εG and εH−G which ends up (with time and in the absence of phagocytosis) are the extinction coefficients of the complex and the in the membrane lysis of the already formed apoptotic bodies complex−DNA, respectively. and in the organelle breakdown. 2945 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Figure 7. DNA-binding studies. On the left, the results of CD studies for C1 (A), C2 (B), and C3 (C) at 50 μM of ct-DNA and at 1:1 and 1:2 (DNA/complex) ratios in NaCl/Tris−HCl at pH 7.2. Samples were previously incubated overnight at 37 °C. On the right, the results of UV−vis studies for complexes C1 (D), C2 (E), and C3 (F) at 30 μM of each complex upon ct-DNA titration from 0 to 60 μM in NaCl/Tris−HCl at pH 7.2. Each spectrum was recorded after 15 min of stabilization time. The arrows indicate change upon increasing concentrations of ct-DNA. ■ CONCLUSIONS In summary, three novel Cu(II) complexes have been dimeric form coexists with the monomeric one in DMSO and water solutions, as clearly observed in ESI-MS and EPR, and synthesized with different N,N,O-chelating salphen-like ligands supported by computational studies. The computational data bearing varying substituents (−H, −Cl, and −Br) on the (together with the SQUID results) also suggest that for the aldehyde aromatic scaffold. Synthesis and characterization have three complexes (C1, C2, and C3), the most probable been carried out, and a dimeric structure was found in the solid dinuclear structure adopts conformation A, with a Cu−Cu state. Magnetic measurements indicated that there is a distance of about ∼2.7 Å, and a dihedral angle in the metal ferromagnetic coupling between both Cu centers in the coordination plane of 154°. dimeric state and that similar conformations can be expected The three Cu(II) complexes have the Cu(II)/Cu(I) redox for the three complexes. For the model complex C1, the potential inside the biological redox window, and therefore 2946 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry ■ pubs.acs.org/IC Article Table 6. Intrinsic Binding Constants (Kb) and EXPERIMENTAL SECTION Hypochromism for the Interaction of ct-DNA with Chemicals. Reagents like copper(II) chloride, copper(II) acetate, Complexes C1, C2, and C3 DCFDA, calf thymus DNA (ct-DNA) sodium salt, benzene-1,2- Complex Kb (M−1)a log Kb % hypochromism (λ in nm) diamine, N-chlorosuccinimide (NCS), 2-hydroxybenzaldehyde, p- toluenesulfonic acid (p-TsOH), bromine, and 2-amino-2- C1 2.2 × 104 4.34 25 (397) (hydroxymethyl)propane-1,3-diol (TRIS) were obtained from C2 6.2 × 104 4.79 27 (424) Sigma-Aldrich and Thermo Fisher. Solvents such as acetonitrile C3 7.2 × 104 4.86 28 (438) (ACN), methanol (MeOH), ethanol (EtOH), ether, chloroform a (CHCl3), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), Kb is obtained from the ratio of the intercept to the slope, according to the Benesi−Hildebrand equation (eq 1),48 after the fitting of the dichloromethane (DCM), acetic anhydride, and hexane were used UV−vis data (Figure 7D−F). The calculated Kb values arise from a at synthesis grade purity and directly from commercial sources DNA−drug interactions according to the Benesi−Hildebrand model (Scharlab, Panreac and VWR). (which gives approximated Kb values), and hence they should be Synthesis of Ligand Precursors. N-(2-Aminophenyl)acetamide compared in orders of magnitude, rather than with the exact numbers. (1).59 Acetic anhydride (5.12 mL, 51.2 mmol) was added dropwise at 0 °C under a N2 atmosphere to a solution of benzene-1,2-diamine (5.54 g, 51.2 mmol) in anhydrous DCM (75 mL). The mixture was stirred for 2 h at 0 °C and then stored at −35 °C overnight. The precipitate was filtered off and washed with cold DCM (3 × 5 mL) and ether (3 × 5 mL) to yield 1.01 g of a white solid. The filtrate was further concentrated to the half of its volume and stored at −35 °C for 48 h more. The new precipitate was filtered off to render additional 1.56 g of the product. Yield: 38% (2.57 g). 1H NMR (360 MHz, DMSO-d6): δ 9.12 (s, 1H), 7.14 (d, J = 7.8 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.52 (t, J = 7.3 Hz, 1H), 4.85 (s, 2H), 2.03 (s, 3H). 5-Chloro-2-hydroxybenzaldehyde (2).60 Water (4 mL) was added to 2-hydroxybenzaldehyde (488 mg, 4.0 mmol). Under magnetic stirring, NCS (536 mg, 4.01 mmol, 1 equiv), p-TsOH (764 mg, 4.0 mmol), and NaCl (355 mg, 6.1 mmol, 1.5 equiv) were added at room temperature. The final solution was stirred at 40 °C for 1 h. Water (3 mL) was added and the formed precipitate was filtered off and washed Figure 8. In vitro ROS production measured with the DCFDA assay with water (2 × 2 mL). Then, the solid was extracted with DCM and in HeLa cancer cells for complex C1 (25 μM), H2L1 (50 μM), and dried with sodium sulphate to afford an off-white solid. Titled H2O2 (100 μM) as positive control after treatment for 4 h. compound was obtained after column chromatography (hexane/ EtOAc 6:1). Yield: 14% (85 mg). 1H NMR (360 MHz, CDCl3): δ 10.94 (s, 1H), 9.87 (s, 1H), 7.56 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H). they are thermodynamically able to biologically undergo a 5-Bromo-2-hydroxybenzaldehyde (3).61 To a solution of 2- Cu(II)/Cu(I) redox cycling. Their similar ascorbate con- hydroxybenzaldehyde (0.5 g, 4.0 mmol) in chloroform (10 mL), sumption rates compared to free Cu(II) confirms the bromine (0.65 g, 4.0 mmol) in chloroform (5 mL) was added dropwise over a period of 15 min at 0 °C. The resulting mixture was potentiality of the complexes as ROS generators. The presence stirred overnight at 50 °C. Then, the reaction was diluted in water (20 of the electrowithdrawing substituents on the aromatic ring (Cl mL) and extracted with chloroform (3 × 8 mL). The organic phases or Br) shifts the Cu(II)/Cu(I) redox potential, slightly were combined, extracted with water (8 mL) and brine (8 mL), dried favoring the reduction from Cu(II) to Cu(I). The three over Na2SO4, and the solvent removed under reduced pressure. The complexes exhibited significant cytotoxicity in HeLa and crude solid was powdered and washed with hexane (2 × 2 mL) and MCF7 cancer cells, in the range of cisplatin and improved ether (2 × 3 mL) and the solvents were decanted. 3 was obtained values with respect to carboplatin. The most interesting feature without further purification. Yield: 55% (430 mg). 1H NMR (250 MHz, DMSO-d6): δ 10.95 (s, 1H), 10.22 (s, 1H), 7.72 (d, J = 2.6 Hz, relies on the higher toxicity displayed by C1 in cancer cells 1H), 7.65 (dd, J = 8.8, 2.6 Hz, 1H), and 6.99 (d, J = 8.8 Hz, 1H). with respect to normal cells, most likely owing to its Synthesis of Ligands H2L1−H2L3. (E)-N-(2-(2- demonstrated high in vitro ROS production capabilities. In Hydroxybenzylideneamino)phenyl)acetamide (H2L1). 2-Hydroxy- terms of the biological target, the studies with DNA suggest benzaldehyde (43.5 mg, 0.36 mmol) in absolute EtOH (6 mL) was that complexes C1−C3 show a moderate noncovalent binding added dropwise to a solution of 1 (58.4 mg, 0.39 mmol, 1.1 equiv) in to the double-strand DNA, but an interesting redox-dependent absolute EtOH (22 mL) at 0 °C and under strong agitation. The final cleaving capacity. mixture was stirred for 15 min at 0 °C and then overnight (12 h) at room temperature. The solution was filtered and the solvent of the The results altogether place C1 as a promising cytotoxic filtrate removed under vacuum to afford a yellowish crude. Pure H2L1 agent to be further explored, whose ROS-mediated mechanism was obtained by silica gel column chromatography using a gradient of action might produce some inherent selectivity toward elution (from DCM/hexane 1:1 to EtOAc/hexane 1:1). Yield: 39% cancer cells against healthy cells, giving rise to less undesired (36 mg). Rf (EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for effects. Unfortunately, the nature of the halogen substituent [H2L1+H]+, 255.1104 (theoretical, 255.1128). 1H NMR (360 MHz, shows no influence on the in vitro cytotoxicity of the DMSO-d6): δ 12.76 (s, 1H), 9.54 (s, 1H), 8.87 (s, 1H), 7.77−7.57 complexes, and it also results in some solubility issues. (m, 2H), 7.49−7.33 (m, 2H), 7.27 (s, 2H), 6.97 (d, J = 7.7 Hz, 2H), 2.05 (s, 3H). 13C NMR (400 MHz, DMSO-d6): δ 168.8, 163.7, 160.7, Nonetheless, the promising in vitro outcome observed for 142.3, 133.8, 132.9, 132.6, 127.3, 126.2, 125.4, 120.2, 119.6, 119.4, C1 encourage us to keep working on improving the properties 117.1, 23.8. FTIR−ATR (wavenumber, cm−1): 3294.31, 3055.63, of this metallic core to position it as a promising anticancer 1662.29, 1613.70, 1589.52, 1573.64, 1515.92, 1443.20, 1365.24, candidate. 1304.59, 1278.20, 1225.08, 1180.92, 1150.51, 1108.70, 1033.13, 2947 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article 1005.30, 965.08, 939.91, 909.25, 854.86, 829.27, 779.77, 752.58, mg, 0.12 mmol, 1 equiv) in ACN/DCM (1:1, 12 mL) at room 723.25, 674.22, 642.06. temperature. The same procedure as for C1 was followed to obtain (E)-N-(2-(5-Chloro-2-hydroxybenzylideneamino)phenyl)- pure C3. Yield: 74% (35 mg). HR-MS (ESI+, DMSO−MeOH): for acetamide (H2L2). 2 (20 mg, 0.13 mmol) in absolute EtOH (4 mL) [C3 + H]+, 786.8678 (theoretical, 786.8673). Elemental Analysis was added dropwise to a solution of 1 (20 mg, 0.13 mmol) in absolute Calcd for C3 (C30H22Br2Cu2N4O4): C, 45.64; H, 2.81; N, 7.10. EtOH (10 mL) at 0 °C and under strong agitation. The final mixture Found: C, 45.41; H, 2.81; N, 6.82. FTIR−ATR (wavenumber, cm−1): was stirred for 15 min at 0 °C and at room temperature overnight. 1613.76, 1492.89, 1475.85, 1454.37, 1436.29, 1408.34, 1374.91, The solution was filtered, and the solvent of the filtrate was removed 1317.00, 1281.65, 1241.56, 1203.98, 1159.97, 1133.05, 1070.82, to afford a yellowish crude. Pure H2L2 was obtained by silica gel 1028.23, 988.61, 961.31. column chromatography using a gradient elution (from DCM/hexane Physical Measurements. Instruments and Experimental 3:4 to hexane/DCM:EtOAc, 1:1:0.5). Yield: 43% (16 mg). Rf Procedures. SQUID Data. Magnetic characterization has been (EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for [H2L2 + performed using a conventional SQUID magnetometer MPMS-XL H]+, 289.0708 (theoretical, 289.0738). 1H NMR (360 MHz, DMSO- from Quantum Design working at a magnetic field up to 5 T and d6): δ 12.66 (br s, 1H), 9.55 (s, 1H), 8.86 (s, 1H), 7.83 (s, 1H), 7.68 temperature down to 2 K. The samples (powder) are filled in (d, J = 7.4 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), polypropylene sleeves then sealed in order to remove the maximum of 7.28 (m, 2H), 7.01 (d, J = 8.8 Hz, 1H), 2.05 (s, 3H). 13C NMR (400 dioxygen, which give the signal around 50 K (antiferromagnetic MHz, DMSO-d6): δ 168.8, 161.9, 159.3, 142.2, 133.2, 132.7, 131.3, transition). However, despite such care, the oxygen signal is visible in 127.7, 126.1, 125.2, 123.0, 121.8, 119.2, 119.1, 23.9. FTIR−ATR the C1 sample, but C2 and C3 are rather clean. Diamagnetic (wavenumber, cm−1): 3274.78, 2361.39, 1662.26, 1515.30, 1593.63, contribution of the sample holder was removed. The susceptibility 1529.51, 1479.28, 1452.05, 1358.29, 1303.45, 1280.16, 1220.51, was fitted using the Bleaney−Bowers formula of two coupled S = 1/ 1176.62, 1109.38, 1090.29, 1048.24, 1011.05, 959.80, 922.81, 870.39, 2.31 819.90, 760.15, 739.44, 697.78, 654.92, 641.38. The isothermal (T = 2 K) magnetization was fitted using the (E)-N-(2-(5-Bromo-2-hydroxybenzylideneamino)phenyl)- Brillouin function with one S = 1 (equivalent to two coupled S = 1/2 acetamide (H2L3). Compound 3 (150 mg, 0.75 mmol, 1 equiv) in at T < 2J) and two uncoupled S = 1/2 absolute EtOH (5 mL) was added dropwise to a solution of 1 (123 NMR Spectrometry. NMR experiments were recorded on mg, 0.76 mmol, 1 equiv) in absolute EtOH (10 mL) at 0 °C and BRUKER DPX-250, 360, and 400 MHz instruments at the Servei under stirring. The final mixture was kept under the same conditions de Ressonància Magnètica Nuclear (UAB). Deuterated solvents were for 15 min at 0 °C and at room temperature for additional 24 h. The directly purchased from commercial suppliers. All spectra have been solution was filtered, the precipitate was washed with DCM (2 × 3 recorded at 298 K. The abbreviations used to describe signal mL), and the solvent of the filtrate was removed to afford the crude multiplicities are: s (singlet), bs (broad singlet), d (doublet), dd H2L3. Titled compound was obtained after purification by flash silica (double doublet), and m (multiplet). All 13C NMR acquired spectra gel column chromatography (DCM/EtOAc, 1:1). Yield: 52% (125 are proton decoupled. mg). Rf (EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for ESI-MS Measurements. HR ESI-MS measurements were recorded [H2L3 + H]+, 333.0193 (theoretical, 333.0233). 1H NMR (250 MHz, after diluting the corresponding solid complexes using a MicroTOF-Q DMSO-d6): δ 12.67 (s, 1H), 9.53 (s, 1H), 8.85 (s, 1H), 7.95 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 6.9 Hz, 1H), 7.56 (dd, J = 8.8, 2.5 Hz, 1H), (Brucker Daltonics GmbH, Bremen, Germany) instrument equipped 7.40−7.22 (m, 3H), 6.96 (d, J = 8.8 Hz, 1H), 2.07 (s). 13C NMR with an electrospray ionization source (ESI) in positive mode at the ́ Servei d’Anàlisi Quimica (UAB). The nebulizer pressure was 1.5 bar, (400 MHz, DMSO-d6): δ 171.0, 164.1, 161.9, 144.2, 138.2, 136.5, 134.9, 129.9, 128.4, 127.4, 124.5, 121.7, 121.4, 112.6, 26.1. FTIR− the desolvation temperature was 180 °C, flow rate of dry gas was 6 L ATR (wavenumber, cm−1): 3295.33, 1661.38, 1614.48, 1587.78, min−1, the capillary counter electrode voltage was 5 kV, and the 1566.65, 1528.15, 1472.56, 1451.73, 1368.30, 1355.88, 1307.60, quadrupole ion energy was 5.0 eV. 1277.36, 1219.00, 1175.25, 1130.46, 1111.42, 1076.79, 1038.90, EPR Experiments. EPR measurements were carried out on a 1016.55, 960.34, 937.67, 914.61. BRUKER ELEXSYS 500 X-band CW-ESR spectrometer, with an Synthesis of Cu(II) Complexes. Complex C1 ([Cu(L1)]2). ELEXSYS Bruker instrument equipped with a BVT 3000 digital Cu(OAc)2·2H2O (15.7 mg, 0.08 mmol, 1 equiv) in ACN (3 mL) temperature controller. The spectra were recorded at 120 K in frozen was slowly added to a solution of H2L1 (20 mg, 0.08 mmol, 1 equiv) DMSO solutions otherwise noticed. Typical parameters were: a in ACN (8 mL) at room temperature. The final mixture was stirred microwave power of 10−20 mW, a modulation frequency of 100 kHz, for 2 h and the formed precipitate was filtered off and washed with and a modulation gain of 3 G. EPR spectra were simulated using the ACN (2 × 3 mL) and with Et2O (2 × 3 mL). The solid so obtained EasySpin toolbox developed for Matlab.62 Copper spin quantification was identified as C1. Yield: 68% (17 mg). HR-MS (ESI+, DMSO− has been carried out for C1 in frozen DMSO solutions (0.5 mM, e.g., MeOH): for [C1 + H]+, 631.0456 (theoretical, 631.0462); for [C1 + 1 mM copper concentration, with or without 0.1 M [NBu4][PF6] Na]+, 653.0194 (theoretical, 653.0282). Elemental Analysis Calcd for (TBAP) electrolyte) through double integration of the EPR derivative C1 (C30H24Cu2N4O4): C, 57.05; H, 3.83; N, 8.87. Found: C, 56.61; signal, using standardized Cu(NO3)2 solutions as an external H, 3.81; N, 8.56. FTIR−ATR (wavenumber, cm−1): 2363.09, calibrator. 1610.64, 1477.82, 1458.40, 1429.20, 1401.49, 1376.22, 1353.19, Cyclic Voltammetry. Cyclic voltammograms were taken on a 1326.39, 1281.82, 1244.19, 1217.10, 1173.53, 1145.83, 1126.54, BioLogic SP-150 potentiostat and using EC-Lab 5,40 software. 1026.08, 961.53, 922.74, 849.42, 793.31, 747.55, 679.15, 649.79, DMSO was used as a solvent with 0.1 M of [NBu4][PF6] (TBAP) as a 620.37. supporting electrolyte. Measurements were carried out with a three- Complex C2 ([Cu(L2)]2). Cu(OAc)2·2H2O (6.0 mg, 0.03 mmol, 1 electrode configuration cell: glassy carbon electrode as the working equiv) in ACN (2 mL) was slowly added to a solution of H2L2 (9 mg, electrode, Ag wire in a 0.1 M TBAP solution in DMSO 0.03 mmol, 1 equiv) in ACN (5 mL) at room temperature. The same (semielectrode) as the reference electrode, and Pt as the counter procedure as for C1 was followed to obtain pure C2. Yield: 73% (8 electrode. The ferrocene (Fc+/Fc) system was used as the internal mg). HR-MS (ESI+, DMSO−MeOH): for [C2 + H]+, 700.9661 standard. The scan rate (ν) varied between 300 and 25 mV·s−1. All (theoretical, 700.9839). Elemental Analysis Calcd for C2 the experiments were recorded under an argon atmosphere. (C30H22Cl2Cu2N4O4): C, 51.44; H, 3.17; N, 8.00. Found: C, 51.47; Elemental Analysis. C, H, and O analyses were performed at the H, 3.18; N, 7.66. FTIR−ATR (wavenumber, cm−1): 1614.62, ́ Servei d’Anàlisi Quimica (UAB) on a Flash EA 2000 CHNS Thermo 1492.27, 1475.85, 1406.65, 1375.34, 1318.32, 1281.53, 1240.53, Fisher Scientific equipment, with a TCD and a MAS 200 R 1201.84, 1159.66, 1129.77, 1027.64, 988.20, 960.72, 932.29. autosampler for solid samples. Complex C3 ([Cu(L3)]2). Cu(OAc)2·2H2O (24.0 mg, 0.12 mmol, 1 IR Spectroscopy. Attenuated total reflectance (ATR)−FTIR equiv) in ACN (3 mL) was slowly added to a solution of H2L3 (40 spectra were recorded on a PerkinElmer spectrometer, equipped 2948 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article with a universal ATR accessory, with a diamond window in the range (FITC) by using an Annexin V−FITC apoptosis detection kit 4000−650 cm−1. (Roche). Exponentially growing HeLa cells in 6-well plates (3 × 105 UV−Vis Characterization. All the spectra were recorded at room cells/well) were exposed to concentrations equal to the IC50 for 24 h temperature either on an Agilent HP 8453, Varian Cary 50 Bio, a (70 μM), determined prior to the experiment. After the cells had been Varian Cary 60 Bio, or a PerkinElmer Lambda 650 spectropho- stained with the Annexin V−FITC and propidium iodide, the tometer, using 1 cm quart-cuvettes. Noncovalent DNA−complex percentage of apoptotic cells was analyzed by flow cytometry (FACS interactions were studied by UV−vis measurements. Solutions of Calibur). complexes C1−C3 were prepared in 50 mM NaCl/5 mM Tris−HCl Computational Details. The geometry of the monomeric buffer (pH 7.2), containing a maximum of 5% DMSO to solubilize [CuII(L1)(DMSO)] and dimeric [CuII(L1)]2 complexes was them. ct-DNA stock solutions were prepared from their correspond- optimized with Gaussian 0963 at the DFT theory level using the ing sodium salt and the concentration was determined from its hybrid B3LYP functional combined with Grimme’s D3 correction64 absorbance at 260 nm (ε = 6600 cm−1). Blank and dilution effects for dispersion and the split-valence plus polarization function 6- were corrected. Ascorbate consumption experiments were monitored 31g(d,p) basis-set for the main group elements, SDD plus f- by UV−vis at the maximum absorption band of the ascorbic acid (100 functions65 and pseudopotential were applied for copper. The effect μM) at 265 nm for about 45 min. CuCl2 and the assayed complexes of solvation was taken into account using the SMD continuum model C1−C3 were added at a final concentration of 2 μM in 50 mM NaCl/ of Marenich et al.66 For all the structures, minima were verified 5 mM Tris−HCl buffer (pH 7.2), with a maximum of 5% of DMSO. through frequency calculations. Circular Dichroism. CD experiments were acquired on a JASCO The thermodynamic stability in solution was estimated computing 715 spectropolarimeter. Measurements were carried out at a constant the Gibbs free energy change using the implicit solvent continuum temperature of 20 °C. CD spectra were measured in 50 mM NaCl/5 model.67 Concerning the 1e− reduction products, the previously mM Tris−HCl buffer (pH 7.2). The ct-DNA concentration was 50 optimized geometry of the Cu(II) complexes [CuII(L1)(DMSO)] μM. Different samples with increasing amounts of the complexes to was reoptimized at the same level of theory imposing the multiplicity study (0, 50, 100 μM) were incubated at 37 °C for 24 h, containing a relative to the [CuI(L1)(DMSO)]− and [CuII(L1•−)(DMSO)]− maximum of 5% DMSO to solubilize them. Ct-DNA stock solutions forms. The Gibbs free energy values were obtained by the addition were prepared from their corresponding sodium salt (Sigma-Aldrich) of the thermal and entropic corrections (Gtherm), obtained in the and the concentration was determined from their absorbance at 260 optimization stage, to the potential energy value of single point nm (ε = 6600 cm−1). calculations with the extended basis-set def2-TZVP for the main DNA-Cleaving Experiments. Gel electrophoresis experiments were group elements67 and the quadruple-ζ def2-QZVP basis set for performed on agarose gel (1% in Tris−acetate EDTA (TAE) buffer), Cu.68,69 using a BIORAD horizontal tank connected to a variable potential The g and A tensors of the 63Cu center for each complex were power supply. Samples were stained with EB and revealed with a obtained using the method implemented into the Orca package.70,71 Super GelDoc PlusImager. Complexes C1−C3 were incubated with The A tensor is obtained as a sum of the three contributions: the the plasmid DNA (200 ng of BlueScript plasmid per well) in 20 mM isotropic Fermi contact (AFC), the anisotropic dipolar (ADx,y,z), and the NaCl/40 mM Tris−HCl buffer (pH 7.20) medium for 24 h at 37 °C spin−orbit coupling term (ASO x,y,z). A tensors were computed using the (<10% DMSO in the final mixture to solubilize the complexes). functional B3LYP, while for g PBE0 was used, both coupled with a Samples containing the reducing agent ascorbic acid were incubated triple-ζ basis set 6-311g(d,p).35 for 1.5 extra hours in the presence of ascorbic acid (100 μM). The exchange coupling constants J for the dinuclear C1 complex Cell Viability Assays. The IC50 values were evaluated using the were calculated with the functional B3LYP and the 6-311g basis set PrestoBlue Cell Reagent (Life Technologies) assay. Working with the software ORCA,70 according to the method reported in the concentrations of complexes C1−C3 (final amount <0.1% DMSO literature.72 Using S1 = S1 = 1/2 in the Heisenberg Hamiltonian Ĥ = in biological experiments) were prepared in the corresponding MEM Ŝ 1·Ŝ 2, the value of J can be expressed as: J = EBS − EHS, where EBS and (modified Eagle’s medium, Invitrogen) for each cell. Human cancer EHS are the energies of the broken-symmetry solution and the triplet cells (HeLa and MCF7) and nontumoral NIH 3T3 cells were state. obtained from American Type Culture Collection (ATCC, Manassas, UV−vis vertical excitations were simulated on the time-dependent VA, USA). HeLa cells were routinely cultured with MEM; MCF7, DFT framework using the solvent continuum model.73 The with DMEN-F12 (Dulbecco’s MEM/Nutrient Mixture F-12 Ham); simulations were carried out on the previously optimized geometries and NIH 3T3, with DMEM (Dulbecco’s MEM), all containing 10% in solvent using BH and HLYP functionals and the triple-ζ type def2- heat-inactivated fetal bovine serum at 37 °C in a humidified CO2 TZVP basis set, according to the method established previously.74 atmosphere. Cells were plated at a density of 3 × 103 cells/well in 100 The predicted electronic spectrum of C1 was generated using Gabedit μL of culture medium and allowed to grow overnight. After the software.75 required incubation time with different concentrations (0, 1, 5, 10, 25, 50, 100, or 200 μM) of each complex, 10 μL of PrestoBlue were added following the standard protocol. The fluorescence of each well was measured at 572 nm with a Microplate Reader Victor3 ■ * ASSOCIATED CONTENT sı Supporting Information (PerkinElmer). The relative cell viability (%) for each sample related The Supporting Information is available free of charge at to the control well was calculated. Each complex was tested per https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c02932. triplicate and averaged from three independent sets of experiments. Blank and complex controls were also considered. NMR, ESI-MS, FT-IR, EPR, UV−vis absorbance, and Intracellular ROS Production Assays. HeLa cells were plated and SQUID data of ligands and complexes; cell viability allowed to adhere overnight in a 96-well plate (2 × 104 cells/well). results; EPR parameters obtained; and Gibbs energy The DCFDA reagent (25 μM in DMSO) was then added and the values computationally calculated and flow cytometry of cells incubated at 37 °C in the dark for 30 min. The DCFDA solution HeLa cells (PDF) was removed and cells were treated with the compounds at the corresponding IC50 values (at 72 h) and incubated for 4 h. The experiments were run in triplicate. H2O2 was used as a positive control at 100 μM. The fluorescence of each well was measured at 535 nm with a Microplate Reader Victor3 (PerkinElmer) after ■ AUTHOR INFORMATION Corresponding Authors excitation at 485 nm. Olga Iranzo − Aix Marseille Univ., CNRS, Centrale Marseille, In Vitro Apoptosis Assays. Induction of apoptosis was determined iSm2, 13397 Marseille, France; orcid.org/0000-0001- by a flow cytometric assay with Annexin V−fluorescein isothiocyanate 8542-2429; Email: olga.iranzo@univ-amu.fr 2949 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article Ò scar Palacios − Departament de Química, Facultat de (II) Complexes of Phenanthroline Bases Showing DNA Photo- Ciències, Universitat Autònoma de Barcelona, 08193 cleavage Activity and Cytotoxicity. Inorg. Chem. 2011, 50, 8452− Cerdanyola del Vallès, Barcelona, Spain; orcid.org/0000- 8464. 0002-2987-7303; Email: oscar.palacios@uab.cat (3) Leite, S. M. G.; Lima, L. M. P.; Gama, S.; Mendes, F.; Orio, M.; Bento, I.; Paulo, A.; Delgado, R.; Iranzo, O. Copper(II) Complexes of Authors Phenanthroline and Histidine Containing Ligands: Synthesis, Quim Peña − Departament de Química, Facultat de Ciències, Characterization and Evaluation of Their DNA Cleavage and Universitat Autònoma de Barcelona, 08193 Cerdanyola del Cytotoxic Activity. Inorg. Chem. 2016, 55, 11801−11814. Vallès, Barcelona, Spain; Aix Marseille Univ., CNRS, (4) Santini, C.; Pellei, M.; Gandin, V.; Porchia, M.; Tisato, F.; Marzano, C. Advances in Copper Complexes as Anticancer Agents. Centrale Marseille, iSm2, 13397 Marseille, France; Chem. Rev. 2014, 114, 815−862. orcid.org/0000-0001-6477-8127 (5) Maheswari, P. U.; Roy, S.; den Dulk, H.; Barends, S.; van Wezel, Giuseppe Sciortino − Departament de Química, Facultat de G.; Kozlevčar, B.; Gamez, P.; Reedijk, J. The Square-Planar Cytotoxic Ciències, Universitat Autònoma de Barcelona, 08193 [Cu(II)(pyrimol)Cl] Complex Acts as an Efficient DNA Cleaver Cerdanyola del Vallès, Barcelona, Spain; Institute of without Reductant. J. Am. Chem. Soc. 2006, 128, 710−711. Chemical Research of Catalonia (ICIQ), 43007 Tarragona, (6) Kraatz, H.-B.; Metzler-Nolte, N. Concepts and Models in Spain; orcid.org/0000-0001-9657-1788 Bioinorganic Chemistry; Wiley-VCH, 2006. Jean-Didier Maréchal − Departament de Química, Facultat (7) Pelicano, H.; Carney, D.; Huang, P. ROS Stress in Cancer Cells de Ciències, Universitat Autònoma de Barcelona, 08193 and Therapeutic Implications. Drug Resist. Updat. 2004, 7, 97−110. Cerdanyola del Vallès, Barcelona, Spain; orcid.org/0000- (8) Schumacker, P. T. Reactive Oxygen Species in Cancer Cells: 0002-8344-9043 Live by the Sword, Die by the Sword. Cancer Cell 2006, 10, 175−176. Sylvain Bertaina − Aix Marseille Univ., CNRS, IM2NP, (9) Oun, R.; Moussa, Y. E.; Wheate, N. J. The Side Effects of 13397 Marseille, France; orcid.org/0000-0002-6466- Platinum-Based Chemotherapy Drugs: A Review for Chemists. Dalton Trans. 2018, 47, 6645−6653. 8830 (10) Vander Heiden, M. G.; Cantley, L. C.; Thompson, C. B. A. Jalila Simaan − Aix Marseille Univ., CNRS, Centrale Understanding the Warburg Effect: The Metabolic Requirements of Marseille, iSm2, 13397 Marseille, France; orcid.org/ Cell Proliferation. Science 2009, 324, 1029−1033. 0000-0003-2537-0422 (11) Liberti, M. V.; Locasale, J. W. The Warburg Effect: How Does Julia Lorenzo − Institut de Biotecnologia i Biomedicina, It Benefit Cancer Cells? Trends Biochem. Sci. 2016, 41, 211−218. Departamento de Bioquímica i Biologia Molecular, (12) Jungwirth, U.; Kowol, C. R.; Keppler, B. K.; Hartinger, C. G.; Universitat Autònoma de Barcelona, 08193 Cerdanyola del Berger, W.; Heffeter, P. Anticancer Activity of Metal Complexes: Vallès, Barcelona, Spain; orcid.org/0000-0001-5659- Involvement of Redox Processes. Antioxid. Redox Signaling 2011, 15, 6008 1085−1127. Mercè Capdevila − Departament de Química, Facultat de (13) Trachootham, D.; Alexandre, J.; Huang, P. Targeting Cancer Ciències, Universitat Autònoma de Barcelona, 08193 Cells by ROS-Mediated Mechanisms: A Radical Therapeutic Cerdanyola del Vallès, Barcelona, Spain Approach? Nature Reviews Drug Discovery 2009, 8, 579−591. Pau Bayón − Departament de Química, Facultat de Ciències, (14) Ng, C. H.; Kong, S. M.; Tiong, Y. L.; Maah, M. J.; Sukram, N.; Ahmad, M.; Khoo, A. S. B. Selective Anticancer Copper(II)-Mixed Universitat Autònoma de Barcelona, 08193 Cerdanyola del Ligand Complexes: Targeting of ROS and Proteasomes. Metallomics Vallès, Barcelona, Spain 2014, 6, 892−906. Complete contact information is available at: (15) da Silveira, V. C.; Luz, J. S.; Oliveira, C. C.; Graziani, I.; Ciriolo, https://pubs.acs.org/10.1021/acs.inorgchem.0c02932 M. R.; Ferreira, A. M. d. C. Double-Strand DNA Cleavage Induced by Oxindole-Schiff Base Copper(II) Complexes with Potential Anti- Notes tumor Activity. J. Inorg. Biochem. 2008, 102, 1090−1103. The authors declare no competing financial interest. (16) Verduzco-Ramírez, A.; Graciela Manzanilla-Dávila, S.; Eugenia ■ Morales-Guillén, M.; Carlos García-Ramos, J.; Toledano-Magaña, Y.; ACKNOWLEDGMENTS Marin-Becerra, A.; Flores-Á lamo, M.; Antonio Ortiz-Frade, L.; Fernando Olguín-Contreras, L.; Ruiz-Azuara, L. Essential Metal- Experimental support of Sergi Montané and Sergi Rodriguez- Based Drugs: Correlation between Redox Potential and Biological Calado (Institut de Biotecnologia i Biomedicina, Dept. Activity of M2+ with a N2O2 Ligand. J. Mex. Chem. Soc. 2017, 2017, Bioquímica i Biologia Molecular, Universitat Autònoma de 109−119. Barcelona) is kindly acknowledged. This work was supported (17) Bravo-Gómez, M. E.; García-Ramos, J. C.; Gracia-Mora, I.; by the Spanish Ministerio de Ciencia e Innovación and FEDER Ruiz-Azuara, L. Antiproliferative Activity and QSAR Study of to the projects BIO2015-67358-C2-2-P, CTQ2015-70371- copper(II) Mixed Chelate [Cu(N-N)(acetylacetonato)]NO3 and [Cu(N-N)(glycinato)]NO3 Complexes, (Casiopeinas). ́ J. Inorg. REDT, and RTI2018-098027-B-C22. Q.P. acknowledges the Ministerio de Educación, Cultura y Deporte the financial Biochem. 2009, 103, 299−309. support of a FPU grant. The authors from the UAB are (18) Peña, Q.; Lorenzo, J.; Sciortino, G.; Rodríguez-Calado, S.; Maréchal, J.-D.; Bayón, P.; Simaan, A. J.; Iranzo, O.; Capdevila, M.; members of the “Grup de Recerca de la Generalitat de Palacios, Ò . Studying the Reactivity of “old” Cu(II) Complexes for Catalunya” ref. 2017SGR-864. The Servei d’Anàlisi Química “novel” Anticancer Purposes. J. Inorg. Biochem. 2019, 195, 51−60. (UAB) and Spectropole facilities (AMU) are also kindly (19) Chakravarty, A. R.; Anreddy, P. A. N.; Santra, B. K.; Thomas, A. acknowledged for allocating instrument time. ■ M. Copper Complexes as Chemical Nucleases. Proc.-Indian Acad. Sci., Chem. Sci. 2002, 114, 391−401. REFERENCES (20) Brissos, R. F.; Torrents, E.; Mariana dos Santos Mello, F.; (1) Mjos, K. D.; Orvig, C. Metallodrugs in Medicinal Inorganic Carvalho Pires, W.; de Paula Silveira-Lacerda, E.; Caballero, A. B.; Chemistry. Chem. Rev. 2014, 114, 4540−4563. Caubet, A.; Massera, C.; Roubeau, O.; Teat, S. J.; Gamez, P. Highly (2) Goswami, T. K.; Chakravarthi, B. V. S. K.; Roy, M.; Karande, A. Cytotoxic DNA-Interacting copper(II) Coordination Compounds. A.; Chakravarty, A. R. Ferrocene-Conjugated L -Tryptophan Copper- Metallomics 2014, 6, 1853−1868. 2950 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article (21) Zhao, F.; Wang, W.; Lu, W.; Xu, L.; Yang, S.; Cai, X.-M.; Zhou, (40) Vickers, A. E.; Sloop, T. C.; Lucier, G. W. Mechanism of Action M.; Lei, M.; Ma, M.; Xu, H.-J.; Cao, F. High Anticancer Potency on of Toxic Halogenated Aromatics. Environ. Health Perspect. 1985, 59, Tumor Cells of Dehydroabietylamine Schiff-Base Derivatives and a 121−128. copper(II) Complex. Eur. J. Med. Chem. 2018, 146, 451−459. (41) Greenlee, W. F.; Osborne, R.; Dold, K. M.; Hudson, L. G.; (22) Dankhoff, K.; Gold, M.; Kober, L.; Schmitt, F.; Pfeifer, L.; Toscano, W. A. Toxicity of Chlorinated Aromatic Compounds in Dü rrmann, A.; Kostrhunova, H.; Rothemund, M.; Brabec, V.; Animals and Humans: In Vitro Approaches to Toxic Mechanisms and Schobert, R.; Weber, B. Copper(II) Complexes with Tridentate Risk Assessment. Environ. Health Perspect. 1985, 60, 69−76. Schiff Base-like Ligands: Solid State and Solution Structures and (42) Al-Allaf, T. A. K.; Rashan, L. J.; Steinborn, D.; Merzweiler, K.; Anticancer Activity. Dalton Trans. 2019, 48, 15220−15230. Wagner, C. Platinum(II) and Palladium(II) Complexes Analogous to (23) García-Tojal, J.; Gil-García, R.; Fouz, V. I.; Madariaga, G.; Oxaliplatin with Different Cyclohexyldicarboxylate Isomeric Anions Lezama, L.; Galletero, M. S.; Borrás, J.; Nollmann, F. I.; García-Girón, and Their in Vitro Antitumour Activity. Structural Elucidation of C.; Alcaraz, R.; Cavia-Saiz, M.; Muñiz, P.; Palacios, Ò .; Samper, K. G.; [Pt(C2O4)(cis-Dach)]. Transit. Met. Chem. 2003, 28, 717−721. Rojo, T. Revisiting the Thiosemicarbazone Copper(II) Reaction with (43) Suntharalingam, K.; Hunt, D. J.; Duarte, A. A.; White, A. J. P.; Glutathione. Activity against Colorectal Carcinoma Cell Lines. J. Mann, D. J.; Vilar, R. A Tri-copper(II) Complex Displaying DNA- Inorg. Biochem. 2018, 180, 69−79. Cleaving Properties and Antiproliferative Activity against Cancer (24) Ohui, K.; Afanasenko, E.; Bacher, F.; Ting, R. L. X.; Zafar, A.; Cells. Chemistry 2012, 18, 15133−15141. Blanco-Cabra, N.; Torrents, E.; Dömötör, O.; May, N. V.; Darvasiova, (44) Maheswari, P. U.; van der Ster, M.; Smulders, S.; Barends, S.; D.; Enyedy, É . A.; Popović-Bijelić, A.; Reynisson, J.; Rapta, P.; Babak, van Wezel, G. P.; Massera, C.; Roy, S.; den Dulk, H.; Gamez, P.; M. V.; Pastorin, G.; Arion, V. B. New Water-Soluble Copper(II) Reedijk, J. Structure, Cytotoxicity, and DNA-Cleavage Properties of Complexes with Morpholine−Thiosemicarbazone Hybrids: Insights the Complex [Cu(II)(pbt)Br2]. Inorg. Chem. 2008, 47, 3719−3727. into the Anticancer and Antibacterial Mode of Action. J. Med. Chem. (45) Samper, K. G.; Vicente, C.; Rodríguez, V.; Atrian, S.; Cutillas, 2019, 62, 512−530. N.; Capdevila, M.; Ruiz, J.; Palacios, Ò . Studying the Interactions of a (25) Easmon, J.; Pürstinger, G.; Heinisch, G.; Roth, T.; Fiebig, H. platinum(II) 9-Aminoacridine Complex with Proteins and Oligonu- H.; Holzer, W.; Jäger, W.; Jenny, M.; Hofmann, J. Synthesis, cleotides by ESI-TOF MS. Dalton Trans. 2012, 41, 300−306. Cytotoxicity, and Antitumor Activity of copper(II) and iron(II) (46) Guo, Z.; Sadler, P. J. Metals in Medicine. Angew. Chem., Int. Ed. Complexes of 4N-azabicyclo[3.2.2]nonane Thiosemicarbazones 1999, 38, 1512−1531. Derived from Acyl Diazines. J. Med. Chem. 2001, 44, 2164−2171. (47) Vorlíčková, M.; Kejnovská, I.; Bednárǒ vá, K.; Renčiuk, D.; (26) Palanimuthu, D.; Shinde, S. V.; Somasundaram, K.; Samuelson, Kypr, J. Circular Dichroism Spectroscopy of DNA: From Duplexes to A. G. Vitro and in Vivo Anticancer Activity of Copper Bis- Quadruplexes. Chirality 2012, 24, 691−698. (thiosemicarbazone) Complexes. J. Med. Chem. 2013, 56, 722−734. (48) Sirajuddin, M.; Ali, S.; Badshah, A. Drug-DNA Interactions and (27) Paterson, B. M.; Donnelly, P. S. Copper Complexes of Their Study by UV-Visible, Fluorescence Spectroscopies and Cyclic Bis(thiosemicarbazones): From Chemotherapeutics to Diagnostic and Voltametry. J. Photochem. Photobiol. B. 2013, 124, 1−19. Therapeutic Radiopharmaceuticals. Chem. Soc. Rev. 2011, 40, 3005− (49) Rajendiran, V.; Karthik, R.; Palaniandavar, M.; Stoeckli-Evans, 3018. H.; Periasamy, V. S.; Akbarsha, M. A.; Srinag, B. S.; Krishnamurthy, (28) Shirin, Z.; Thompson, J.; Liable-Sands, L.; Yap, G. P. A.; H. Mixed-Ligand copper(II)-Phenolate Complexes: Effect of Rheingold, A. L.; Borovik, A. S. C2-Symmetric Ligands Containing Coligand on Enhanced DNA and Protein Binding, DNA Cleavage, Hydrogen Bond Donors: Synthesis and Properties of Cu(II) and Anticancer Activity. Inorg. Chem. 2007, 46, 8208−8221. Complexes of 2,6-bis[N,N′-(2-Carboxamidophenyl)carbamoyl]- (50) Benesi, H. A.; Hildebrand, J. H. A Spectrophotometric pyridine. J. Chem. Soc., Dalton Trans. 2002, 8, 1714−1720. Investigation of Interaction of Iodine with Aromatic Hydrocarbons. (29) Gołebiewski, W.; Gucma, M. Applications of N -Chlorosucci- nimide in Organic Synthesis. Synthesis 2007, 23, 3599−3619. J. Am. Chem. Soc. 1949, 71, 2703−2707. (30) Larock, R. C. Comprehensive Organic Transformations: A Guide (51) Williams, A. K.; Dasilva, S. C.; Bhatta, A.; Rawal, B.; Liu, M.; to Functional Group Preparations, 3rd ed.; Wiley-VCH, 2018. Korobkova, E. A. Determination of the Drug-DNA Binding Modes (31) Bleaney, B.; Bowers, K. D. Anomalous Paramagnetism of Using Fluorescence-Based Assays. Anal. Biochem. 2012, 422, 66−73. Copper Acetate. Proc. R. Soc. London, Ser. A 1952, 214, 451−465. (52) Bazhulina, N. P.; Nikitin, A. M.; Rodin, S. A.; Surovaya, A. N.; (32) Kumar, A.; Sciortino, G.; Maréchal, J.-D.; Garribba, E.; Sanfui, Kravatsky, Y. V.; Pismensky, V. F.; Archipova, V. S.; Martin, R.; S. Stepwise Oxidations in a Cofacial Copper(II) Porphyrin Dimer: Gursky, G. V. Binding of Hoechst 33258 and Its Derivatives to DNA. Through Space Spin Coupling and Interplay between Metal and J. Biomol. Struct. Dyn. 2009, 26, 701−718. Radical Spins. Chem.Eur. J. 2020, 26, 7869−7880. (53) Bhattacharyya, A.; Dixit, A.; Banerjee, S.; Roy, B.; Kumar, A.; (33) Ginsberg, A. P. Magnetic Exchange in Transition Metal Karande, A. A.; Chakravarty, A. R. BODIPY Appended Copper(II) Complexes. 12.1 Calculation of Cluster Exchange Coupling Constants Complexes for Cellular Imaging and Singlet Oxygen Mediated with the Xα-Scattered Wave Method. J. Am. Chem. Soc. 1980, 102, Anticancer Activity in Visible Light. RSC Adv. 2016, 6, 104474− 111−117. 104482. (34) Noodleman, L. Valence Bond Description of Antiferromagnetic (54) Simon, H.-U.; Haj-Yehia, A.; Levi-Schaffer, F. Role of Reactive Coupling in Transition Metal Dimers. J. Chem. Phys. 1981, 74, 5737− Oxygen Species (ROS) in Apoptosis Induction. Apoptosis 2000, 5, 5743. 415−418. (35) Sciortino, G.; Lubinu, G.; Maréchal, J.-D.; Garribba, E. DFT (55) Lowe, S. W.; Lin, A. W. Apoptosis in Cancer. Carcinogenesis Protocol for EPR Prediction of Paramagnetic Cu(II) Complexes and 2000, 21, 485−495. Application to Protein Binding Sites. Magnetochemistry 2018, 4, 55. (56) Kerr, J. F. R.; Winterford, C. M.; Harmon, B. V. Apoptosis. Its (36) Hagen, W. R. Biomolecular EPR Spectroscopy; CRC Press, 2009. Significance in Cancer and Cancer Therapy. Cancer 1994, 73, 2013− (37) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: 2026. Fundamentals and Applications; Wiley-VHC, 2001. (57) Ormerod, M. G.; O’Neill, C. F.; Robertson, D.; Harrap, K. R. (38) Halliwell, B.; Gutteridge, J. M. C. Role of Free Radicals and Cisplatin Induces Apoptosis in a Human Ovarian Carcinoma Cell Catalytic Metal Ions in Human Disease: An Overview. Methods Line without Concomitant Internucleosomal Degradation of DNA. Enzymol. 1990, 186, 1−85. Exp. Cell Res. 1994, 211, 231−237. (39) Grau, J.; Renau, C.; Caballero, A. B.; Caubet, A.; Pockaj, M.; (58) Liu, Y.; Xing, H.; Han, X.; Shi, X.; Liang, F.; Cheng, G.; Lu, Y.; Lorenzo, J.; Gamez, P. Evaluation of the Metal-Dependent Cytotoxic Ma, D. Apoptosis of HeLa Cells Induced by Cisplatin and Its Behaviour of Coordination Compounds. Dalton Trans. 2018, 47, Mechanism. J. Huazhong Univ. Sci. Technol., Med. Sci. 2008, 28, 197− 4902−4908. 199. 2951 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952 Inorganic Chemistry pubs.acs.org/IC Article (59) Shirin, Z.; Thompson, J.; Liable-sands, L.; Yap, G. P. A.; (74) Sciortino, G.; Maréchal, J.-D.; Fábián, I.; Lihi, N.; Garribba, E. Rheingold, L.; Borovik, A. S. C2-Symmetric Ligands Containing Quantitative Prediction of Electronic Absorption Spectra of copper- Hydrogen Bond Donors: Synthesis and Properties of Cu(II). J. Chem. (II)−bioligand Systems: Validation and Applications. J. Inorg. Soc., Dalton Trans. 2002, 1714−1720. Biochem. 2020, 204, 110953. (60) Mahajan, T.; Kumar, L.; Dwivedi, K.; Agarwal, D. D. Efficient (75) Allouche, A.-R. Gabedita - A Graphical User Interface for and Facile Chlorination of Industrially-Important Aromatic Com- Computational Chemistry Softwares. J. Comput. Chem. 2011, 32, pounds Using NaCl/p-TsOH/NCS in Aqueous Media. Ind. Eng. 174−182. Chem. Res. 2012, 51, 3881−3886. (61) Sadanandam, P.; Sathaiah, N.; Jyothi, V.; A Chari, M.; Shobha, D.; Das, P.; Mukkanti, K. Synthesis and Characterisation of 5-(6- Substituted Phenyl-2H-Chromen-3-Yl) Oxazole Derivatives. Lett. Org. Chem. 2012, 9, 683−690. (62) Stoll, S.; Schweiger, A. EasySpin, a Comprehensive Software Package for Spectral Simulation and Analysis in EPR. J. Magn. Reson. 2006, 178, 42−55. (63) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision c.01; Gaussian, Inc.: Wallingford, CT, 2010. (64) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 2010, 132, 154104. (65) Ehlers, A. W.; Böhme, M.; Dapprich, S.; Gobbi, A.; Höllwarth, A.; Jonas, V.; Köhler, K. F.; Stegmann, R.; Veldkamp, A.; Frenking, G. A Set of F-Polarization Functions for Pseudo-Potential Basis Sets of the Transition Metals Sc-Cu, Y-Ag and La-Au. Chem. Phys. Lett. 1993, 208, 111−114. (66) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 2009, 113, 6378−6396. (67) Jaque, P.; Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Computational Electrochemistry: The Aqueous Ru3+|Ru2+ Reduc- tion Potential. J. Phys. Chem. C 2007, 111, 5783−5799. (68) Weigend, F.; Furche, F.; Ahlrichs, R. Gaussian Basis Sets of Quadruple Zeta Valence Quality for Atoms H−Kr. J. Chem. Phys. 2003, 119, 12753−12762. (69) Sanna, D.; Ugone, V.; Sciortino, G.; Parker, B. F.; Zhang, Z.; Leggett, C. J.; Arnold, J.; Rao, L.; Garribba, E. V IV O and V IV Species Formed in Aqueous Solution by the Tridentate Glutaroimide- Dioxime Ligand - An Instrumental and Computational Character- ization. Eur. J. Inorg. Chem. 2018, 1805−1816. (70) Neese, F. ORCAAn Ab Initio, DFT and Semiempirical Program Package; Max-Planck-Institute for Chemical Energy Con- version: Mülheim a.d. Ruhr, 2017. (71) Neese, F. The ORCA Program System. Wiley Interdiscip. Rev.: Comput. Mol. Sci. 2012, 2, 73−78. (72) Ruiz, E.; Cano, J.; Alvarez, S.; Alemany, P. Broken Symmetry Approach to Calculation of Exchange Coupling Constants for Homobinuclear and Heterobinuclear Transition Metal Complexes. J. Comput. Chem. 1999, 20, 1391−1400. (73) Casida, M. E. Time-Dependent Density-Functional Theory for Molecules and Molecular Solids. J. Mol. Struct.: THEOCHEM 2009, 914, 3−18. 2952 https://dx.doi.org/10.1021/acs.inorgchem.0c02932 Inorg. Chem. 2021, 60, 2939−2952