**[Cu**(**mef**)**2**(**dena**)**2**(**H2O**)**2]** (**5**)**.**

Complex **5** was prepared as described for **1**. Dark green crystals of **5** suitable for X-ray analysis were obtained after two weeks.

Yield: 0.58 g (62%). Anal. calc. for C50H60CuN6O<sup>8</sup> (*M*<sup>r</sup> = 936.615): C 65.89, H 6.17, N 9.16 %. Found: C 65.38, H 6.36, N 9.15 %. IR (ATR, cm–1): 3473 (w), 3217 (w, br), 2991 (w), 2934 (w), 1612 (s), 1575 (s), 1561 (s), 1496 (s), 1449 (s), 1376 (s), 1281 (s), 1214 (m), 1186 (m), 1105 (m), 947 (w), 920 (w), 878 (w), 831 (m), 783 (m), 760 (s),739 (m), 699 (m), 636 (m), 530 (m), 415 (m). UV-Vis: λ/nm (ε/M−<sup>1</sup> cm−<sup>1</sup> ) as nujol mulls (nm): 216 (sh), 268 (sh), 346, 407 (sh), 600 (br); in DMSO/H2O: 288 (22420), 330 (sh, 11400), 801 (56).

### *3.2. Physical Measurements*

Carbon, hydrogen and nitrogen analyses were carried out on a CHNSO FlashEATM 1112 Automatic Elemental Analyzer. The electronic spectra (190–1100 nm) of the complexes were measured in a Nujol suspension with a SPECORD 250 Plus (Carl Zeiss Jena) spectrophotometer at room temperature. The infrared spectra (ATR technique, 4000–400 cm–1) were recorded on a Nicolet 5700 FT-IR spectrophotometer at room temperature. Roomtemperature EPR spectra of the powdered samples were recorded with an EPR spectrometer EMX Plus series (Bruker, Germany) operating at X-band (≈9.4 GHz) and simulated using Spin.exe software developed by Dr. Ozarowski [52].

### *3.3. X-ray Crystallography*

The data collection and cell refinement of **1–5** were carried out using the four-circle diffractometer Stoe StadiVari using the Pilatus3R 300K HPD detector and the microfocused X-ray source Xenocs Genix3D Cu HF (Cu Kα radiation). The diffraction intensities were corrected for Lorentz and polarization factors. The absorption corrections were made with

the LANA program [53]. The structures were solved using the ShelXT [54], Superflip [55] or Sir14 [56] program and refined using the full-matrix least-squares procedure of the Independent Atom Model (IAM) with ShelXL (version 2018/3) [57]. Hirshfeld Atom Refinement (HAR) was carried out using the IAM model as a starting point. The wave function was calculated using ORCA 4.2.0 software [58] with the basis set jorge-TZP [59] and hybrid exchange–correlation functional PBE0 [60]. The least-squares refinements of the HAR model were then carried out with olex2.refine (version 1.5) [61], while keeping the same constraints and restraints as those used for the ShelXL refinement. The NoSpherA2 implementation [62] of HAR is used for tailor-made aspherical atomic factors calculated on-the-fly from a Hirshfeld-partitioned electron density. For the HAR approach, all H atoms were refined isotropically and independently. All calculations and structure drawings were performed in the OLEX2 package [63]. Ortep-like representations of the independent part of the crystal structures of **1–5** are shown in Supplementary Figures S3–S7. The crystal data and parameters of structure refinement are listed in Table 9.

**Table 9.** Crystallographic data for compounds **1–5**.


The trifluoromethyl group of the niflumate ligand in the crystal structure of **2** was disordered in three parts (Supplementary Figure S4), represented by atoms with occupation factors of 0.51(2) (green lines), 0.29(2) (orange lines) and 0.20(2) (violet lines). The occupation factors were specified using the SUMP instruction. HAR refinement was carried out using restraints C–F and F···F distances using SADI instructions. All fluorine atoms were refined anisotropically with RIGU instruction restraints. Isostructural complexes **3** and **5** contained similarly disordered tolfenamate (**3**) (Supplementary Figure S5) or mefenamate (**5**) (Supplementary Figure S7) ligands in two positions (green lines for main parts, and violet lines for minor parts) with a ratio of occupancy factors of 0.850(1)/0.150(1) for **3** and 0.710(1)/0.290(1) for **5**. The disordered parts of the tolfenamate or mefenamate ligands of both compounds were modeled and refined with constraints and restraints using the SAME instructions, supplemented with SADI/DFIX instructions for C–H distances and RIGU and EADP instructions for non-hydrogen atoms.

### *3.4. Hirshfeld Surface Analysis*

Crystal Explorer [64] was used to calculate the Hirshfeld surfaces [65] and associated fingerprint plots [39,66]. The Hirshfeld surface for complex **2** was calculated including all three orientations of the disordered –CF<sup>3</sup> group with their partial occupancies. The Hirshfeld surfaces of strongly disordered complexes **3** and **5** were calculated separately for the main and minor disordered components.

### *3.5. Electrochemical Study*

The redox behavior of the studied complexes (10−<sup>4</sup> M for all substances) was determined via a cyclic voltammetry study using an argentochloride reference electrode, platinum counter electrode and boron-doped diamond (BDD) working electrode (diameter of 3 mm, boron doping level of 1000 ppm, Windsor Scientific Ltd., UK). All voltammetric curves were registered at the potential range from –1.0 to +1.5 V using a scan rate of 100 mV/s.

### *3.6. Interactions with ct-DNA*

### 3.6.1. Absorption Titrations

Interactions of the prepared complexes **1–4** with DNA were studied with UV-Vis monitored absorption titrations. In this experiment, a DNA stock solution was prepared by dissolving 6 mg of DNA in 5 mL of citrate buffer (containing 15 mM of sodium citrate and 150 mM of NaCl at pH = 7.0). The concentration of DNA was then determined with UV-Vis spectroscopy, where 0.150 mL of stock solution of DNA was added to 2.85 mL of citrate buffer, using a molar absorption coefficient of DNA at 260 nm (6 600 M−<sup>1</sup> cm−<sup>1</sup> ) [67]. The ratio of absorbance at 260 nm and at 280 nm indicated that the DNA was sufficiently pure from proteins [68]. Increasing concentrations of DNA were then added to a buffer/DMSO solution (<1% DMSO) of the corresponding complex. The magnitudes of the binding strength of the complex–DNA interactions expressed as the intrinsic binding constant *K*<sup>b</sup> were calculated with the ratio of slope to the intercept in the plots [DNA]/(εA-ε<sup>f</sup> ) versus [DNA], according to the Wolfe–Shimmer equation (Equation (1)):

$$\frac{[DNA]}{\left(\varepsilon\_a - \varepsilon\_f\right)} = \frac{[DNA]}{\left(\varepsilon\_b - \varepsilon\_f\right)} + \frac{1}{\mathcal{K}\_b\left(\varepsilon\_b - \varepsilon\_f\right)}\tag{1}$$

where the meaning of all symbols can be found elsewhere [48]. Control measurements with DMSO were performed, and no changes in the spectra of DNA were observed.

### 3.6.2. Fluorescence Quenching of EB-DNA Adduct

The ability of the prepared complexes to displace the standard intercalator ethidium bromide (EB) from the EB-DNA adduct was investigated with fluorescence spectroscopy. The EB-DNA adduct was prepared by adding 20 µM EB and 54 µM DNA in a buffer (15 mM of sodium citrate and 150 mM of NaCl at pH = 7.0). The possible intercalating effect of the compounds was studied by adding increasing concentrations of a corresponding complex into a solution of the DNA-EB complex. The fluorescence emission spectra were recorded in the range of 550–800 nm with an excitation wavelength of 515 nm. A decrease in the intensity of the EB-DNA emission band at 615 nm was monitored for complexes **1–4** and were correlated with the same measurement with DMSO. The quenching of the EB-DNA emission band by compounds **1–4** and DMSO was calculated via the Stern–Volmer equation (Equation (2))

$$\mathbf{I}\_0/I = \mathbf{1} + k\_q \tau\_0 \begin{bmatrix} Q \end{bmatrix} = \mathbf{1} + K\_{SV} \begin{bmatrix} Q \end{bmatrix} \tag{2}$$

where *k*<sup>q</sup> (M−<sup>1</sup> s −1 ) is the quenching constant of complexes **1–4**, *K*SV (M−<sup>1</sup> ) is the value of the dynamic quenching constant, *τ<sup>0</sup>* is the average lifetime of the EB-DNA adduct in the absence of the quencher (23 <sup>×</sup> <sup>10</sup>−<sup>9</sup> s), and [*Q*] is the concentration of the quencher. *I*<sup>0</sup> is the initial fluorescence intensity of the EB-DNA adduct, and I is the fluorescence intensity of the EB-DNA adduct after the addition of the complexes. *K*SV can be obtained from the slope of the *I*0*/I* versus [*Q*] plot [21].

### 3.6.3. Viscosimetric Studies

Changes in the viscosity of the DNA solution (0.1 mM) were measured in the presence of increasing concentrations of the compounds in a buffer solution (15 mM of sodium citrate and 150 mM of NaCl at pH = 7.0) at a constant temperature 25 ◦C. The measurements were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL ELVAS spindle, and the measurements were performed at 60 rpm. The relation between the relative solution viscosity (η/η0) and DNA length (*I*/*I*0) is given by Equation (3), where η and η<sup>0</sup> are the viscosities of DNA in the presence and absence of the studied complex.

$$\left(\frac{\eta}{\eta\_0}\right)^{1/3} = \frac{I}{I\_0} \tag{3}$$

### *3.7. SOD Mimetic Activity*

The ability of the copper complexes to scavenge superoxide radical anions was determined using the NBT (Nitro-Blue Tetrazolium) indirect colorimetric test, in which the xanthine/xanthine oxidase (X/XO) system was used as a superoxide-generating system [27]. The extent of NBT reduction was monitored spectrophotometrically by measuring the absorbance at 560 nm for 5 min. The reaction mixture contained 0.2 mM of xanthine and 0.6 mM of NBT in 0.1 mM of a sodium phosphate buffer at a pH of 7.8 and at 25 ◦C with a volume of 3 mL. The tested compounds were dissolved in DMSO. The concentration of xanthine-oxidase (XO) was experimentally designed to give an absorbance change (∆A/min) between 0.035 and 0.045. Inhibitory concentrations were calculated from the slopes of individual curves with Equation (4):

$$IC = \frac{b\_0 - b}{b\_0} \tag{4}$$

where *b<sup>0</sup>* is the slope of the non-inhibited system, and *b* is the slope of the inhibited system with a corresponding complex concentration. *IC*<sup>50</sup> values were obtained from the graphical dependence of the inhibitory concentration and the concentration of the complex [42]. An investigation of the formation of Cu(I) ions after reduction with the superoxide radical anion was conducted via UV-Vis spectroscopy using a specific Cu(I) chelating agent, neocuproine (2,9-dimethyl-1,10-phenanthroline). Potassium superoxide (KO2) was used as a source of the superoxide anion together with 18-crown-6-ether, which acted as a stabilizing agent. The absorption spectra were measured in the range of 400–800 nm after the addition of 500 µL of 1 mM of neocuproine and 500 µL of 1 mM of potassium superoxide DMSO solution to 500 µL of 1 mM of complex **1–4** DMSO solution using the Specord 250 plus UV/Vis spectrometer [27].

### *3.8. Bovine Serum Albumin (BSA) Binding Studies*

The albumin binding studies for complexes **1–4** were performed with tryptophan fluorescence emission quenching experiments using BSA (30 µM) in a buffer solution (containing 15 mM of trisodium citrate and 150 mM of NaCl at a pH of 7.0). The quenching of the emission intensity of the tryptophan residues of BSA at 336 nm was monitored using increasing concentrations of complexes **1–4** as quenchers [21]. The fluorescence emission spectra were recorded in the range of 300–420 nm with an excitation wavelength of 280 nm. The values of the Stern–Volmer constant *K*SV (in M−<sup>1</sup> ), the BSA quenching constant *k*<sup>q</sup> (in M−<sup>1</sup> s −1 ) and the BSA binding constant *KBSA* (in M−<sup>1</sup> ) for the interaction of the compounds with BSA were derived with the Stern–Volmer (Equation (2)) and Scatchard equations (Equation (5)).

$$\frac{\Delta I/I\_0}{[Q]} = nK - K\frac{\Delta I}{I\_0} \tag{5}$$

where *K* (in M−<sup>1</sup> ) is the value of the bovine serum albumin constant *K*BSA, *n* is the number of binding sites per albumin, and [*Q*] is the concentration of the quencher. *I<sup>0</sup>* is the initial fluorescence intensity of the tryptophan residues of albumin, and *I* is the fluorescence intensity of albumin after the addition of the complexes. *KBSA* can be obtained from the slope of the ∆*I/I*0/[*Q*] versus ∆*I/I*0, and *n* can be calculated from the intercept [29].

### *3.9. Anticancer Studies*

### 3.9.1. Cell Culture

Human lung cancer cells (A549), human breast cancer cells (MCF-7) and human glioblastoma cells (U-118MG) were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD, USA) containing 10% fetal bovine serum, 100 µg/mL of streptomycin and 100 U/mL of penicillin G at 37 ◦C in a humidified atmosphere of 5% CO2/95% air. Human lung fibroblasts (MRC-5) (ECACC, Salisbury, UK) were cultured in MEM containing 10% fetal bovine serum, 1% non-essential amino acids and 1% Lglutamine and penicillin–streptomycin mixture at 37 ◦C in a humidified atmosphere of 5% CO2/95% air. For our experiments, cells were seeded on culture dishes or plates in the amounts described below. Cells at passage numbers 10–13 were used.

### 3.9.2. Cytotoxic Analysis

We determined the cytotoxic effects of four copper complexes **1**–**4** on carcinoma cells and healthy cells by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric technique [69]. Cells were seeded (8 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/200 µL well) in individual wells of 96-multiwell plates. We added different concentrations of copper complexes (20–1000 µmol/L) to the cells and incubated them for 24, 48 and 72 h at 37 ◦C (humidified atmosphere of 5% CO2/95% air). After 72 h, the cells were treated with the MTT solution (5 mg/mL) in PBS (phosphate-buffered saline) (20 µL) for 4 h. The dark crystals of formazan, formed in intact cells, were dissolved in DMSO (dimethyl sulfoxide) (200 µL). The plates were shaken for 15 min, and the optical density was determined at 490 nm using a MicroPlate Reader (Biotek, Winooski, VT, USA). All dye exclusion tests were performed three times.

### 3.9.3. Genotoxic Analysis

We determined DNA strand breaks in MCF-7 cells after 72 h of incubation with complex **4** at an *IC*<sup>50</sup> value. DNA strand breaks were measured using the alkaline comet assay [70]. Cells were resuspended in 400 µL of 0.8% low-melting-point agarose in PBS at 37 ◦C and pipetted onto a frosted microscope slide precoated with 100 µL of 1% normalmelting-point agarose. Slides with layers of cells in agarose were incubated in a refrigerator for 10 min (4 ◦C) and then immersed in a lysis solution (2.5 mol/L NaCl, 100 mmol/L, Na2EDTA, 10 mmol/l Tris, 1% Triton, pH of 10) for 1 h to remove cell membranes. After lysis, slides were placed in a horizontal electrophoresis tank containing an electrophoresis solution (1 mmol/L Na2EDTA, 300 mmol/L NaOH, pH of 13) at 4 ◦C for 40 min (DNA uncoiling). Electrophoresis measurements were performed in the same solution at 25 V, 300 mA and 4 ◦C for 30 min. The slides were washed three times for 5 min at 4 ◦C with a neutralizing buffer (0.4 mmol/L Tris, pH of 7.5) before staining with 20 µL of 40 ,6-diamidine-2-phenylindole dihydrochloride (DAPI, 1 µg/mL solution in distilled water). Comets were viewed with fluorescence microscopy after staining with DAPI.

### 3.9.4. Statistical Analysis

The results obtained from the comet assay are shown as the arithmetic means ± the standard deviation (SD). The significance of differences between values acquired with the comet assay was evaluated with Student's t-test to determine if the values were statistically different from those of the control: \* *p* < 0.05.

### **4. Conclusions**

In this report, we discuss the synthesis, structural and spectroscopic characterization and biological activity of five copper (II) complexes with *N, N*-dietlylnicotinamide and fenamate ligands. The following complexes were prepared: [Cu(fluf)2(dena)2(H2O)2] (**1**), [Cu(nifl)2(dena)2] (**2**), [Cu(tolf)2(dena)2(H2O)2] (**3**), [Cu(clon)2(dena)2] (**4**) and [Cu(mef)<sup>2</sup> (dena)2(H2O)2] (**5**). The complexes were characterized in terms of their elemental composition, structure, physico-chemical and biological properties. The crystal structures of the studied compounds were refined using a more accurate aspherical HAR method using data measured with a high redundancy at 100 K. The crystal structures revealed the different influences of benzene versus the pyridine ring on the possibility of the coplanarity of fenamate anions and thus also the possibility of forming hydrogen bonds and/or π–π stacking interactions. The studied complexes are monomeric, forming a distorted tetragonal bipyramidal stereochemistry around a central copper ion. Complex **1** and the isostructural complexes **3** and **5** crystallize in a monoclinic system with a *P*21/c (**1**) or *P*21/n (**3**,**5**) space group, while the nearly isostructural complexes **2** and **4** crystallize in a triclinic system with a *P*-1 space group. The fenamate ligands are coordinated to a copper atom either monodentately (**1**, **3**, **5**) or asymmetrical chelating bidentately (**2**, **4**). The complex molecules of **1–5** are connected in *1D* supramolecular chains by means of intermolecular hydrogen bonds and π–π stacking interactions. In addition, Hirshfeld surface analysis was used to quantitatively identify the intermolecular interactions in the crystal structures of all five compounds.

The EPR spectra of solid-state and frozen DMSO solutions of **1–5** were monomeric with axial symmetry and with a relative ordering of axial *<sup>g</sup>* factors of g<sup>k</sup> > g<sup>⊥</sup> ~ ge, showing either resolved or unresolved copper parallel hyperfine interactions. The similarity between the solid and solution EPR data suggests a resemblance in the geometries of the studied complexes in accordance with the observed crystal structures.

The SOD mimetic activity of the complexes was studied indirectly using an NBT assay, and the complexes were characterized by means of *IC*<sup>50</sup> (1.41–3.46 µM). The obtained inhibition concentrations showed that the complexes are good SOD mimetics, with the best results obtained for **2** and **4**. The cyclic voltammetry results confirmed the quasireversible nature of the redox processes on the studied complexes, with values of *E*1/2 that are in agreement with the SOD mimetic activity of the complexes. The interactions of complexes **1–4** with neocuproine and KO<sup>2</sup> were, again, in agreement with the SOD data and support the hypothesis of the redox cycling mechanism between the studied copper complexes and superoxide.

The potential of complexes **1–4** to interact with DNA was also investigated. Absorption titration studies pointed to the intercalative binding of our complexes to DNA with a relatively strong binding constant of *K*<sup>b</sup> (10<sup>5</sup> ), especially in cases of **4** and **1**. In the viscosity measurements, we observed a continual increase in the relative DNA viscosity for all four complexes, indicating a possible intercalation mechanism of interaction between the complexes and DNA. The intercalating ability of the complexes toward DNA was also studied with an ethidium-bromide-displacement-fluorescence-based method. The results revealed moderate intercalative ability toward DNA. In addition, the affinity of the complexes to interact with bovine serum albumin was studied, showing tight and reversible mutual interactions, as revealed by relatively high binding constants *K*BSA (of order 10<sup>5</sup> ) and quenching constants *k*<sup>q</sup> (of order 1013) for all four complexes. In the case of comparing the structures and biological activity of structurally similar complexes **2**, **4** vs. **1**, **3**, no distinct trend was observed.

The cytotoxic activity of the studied complexes revealed that only complex **4** exhibited cytotoxic activity on the MCF-7 tumor cell line after 72 h of exposure with an *IC*<sup>50</sup> value of 0.57 <sup>×</sup> <sup>10</sup>−<sup>4</sup> M.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/inorganics11030108/s1: Figures S1–S20: IR spectra; UV-Vis spectra; crystal structures, 3D Hirshfeld surfaces, 2D fingerprints plots; Scatchard plots; Table S1: Hydrogen bond parameters. checkCIF and crystallographic data (excluding structure factors) for the structures reported in this paper were deposited into the Cambridge Crystallographic Data Centre as supplementary publications, nos. CCDC-2202673–2202677. Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: (internat.) +44 1223/336033; e-mail: deposit@ccdc.cam.ac.uk].

**Author Contributions:** Conceptualization, J.Š. and M.P.; methodology, M.P., J.V. and J.Š.; investigation, M.P., M.S., K.K., L'.Š., J.M., M.V. and J.Š.; writing—original draft preparation, J.Š.; writing review and editing, J.V., J.M. and M.V.; visualization, J.Š. and M.P.; supervision, J.Š. All authors have read and agreed to the published version of the manuscript.

**Funding:** Slovak grant agencies (VEGA 1/0482/20, VEGA 1/0159/20, VEGA 1/0686/23, APVV-19- 0087, APVV-18-0016 and VEGA 1/0145/20) are acknowledged for their financial support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**


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