3.2.6. Synthesis of [Cu(dNQ)2(DMF)2] (**5**)

HdNQ (23.5 mg, 0.1 mmol) was dissolved in DMF (10 mL) and warmed to 60 ◦C. While continuously stirring, 10 mL of DMF solution of CuCl<sup>2</sup> (8.5 mg of CuCl2·2H2O, 0.05 mmol) (warmed to 60 ◦C) was added. After 30 min of stirring, the beaker was laid

down at room temperature. After two days, green prisms of **5** had formed, and were filtered off, and dried in the air.

[Cu(dNQ)2DMF2] (**5**)—Calc. for C18H8N6O10Cu (531.84 g·mol−<sup>1</sup> ): C, 40.65; H, 1.52; N, 15.80%. Found: not measured, IR (ATR, cm−<sup>1</sup> ): *ν*(C–H)ar 3058 (vw), *ν*(C–H)al 2968 (vw), 2928 (vw), 2860 (vw), *ν*(C=O) 1651 (s), *ν*(C=C)ar 1601 (m), 1591 (m), 1525 (m), 1497 (m), *ν*(N–O)as 1569 (m), *ν*(C=N) 1454 (m), *ν*(C–C)ar 1399 (m), 1374 (m), 1260 (m), *ν*(N–O)sym 1323 (m), *β*(C–H) 1182 (m), 1151 (m), *ν*(C–O) 1122 (m), *δ*(CH3) 1098 (m), *δ*(NO2) 922 (m), 705 (m), *γ*(C–H) 831 (m), 801 (m), Ring breathing 756 (m), 733 (s), *β*(CCC) 660 (m), *β*(CNC) 591 (w), *β*(C–O) 527 (m), *γ*(CCC) 470 (w), 450 (w).

### 3.2.7. Synthesis of [Cu(ClNQ)2] (**6**)

HClNQ (22.5 mg, 0.1 mmol) was dissolved in 10 mL of DMF and warmed to 60 ◦C. While continuously stirring, 10 mL of DMF solution of CuCl<sup>2</sup> (8.5 mg of CuCl2·2H2O, 0.05 mmol) (warmed to 60 ◦C), was added. After 30 min of stirring, the beaker was laid down and the precipitate of **6**, that formed during stirring, was filtered off. The mother liquor was laid to crystallise at room temperature. After five days, green needles of **6** had formed, and were filtered off, and dried in the air. The identity of the powder and crystals was verified by IR spectroscopy.

[Cu(ClNQ)2] (**6**)—Calc. for C18H8Cl2N4O6Cu (510.73 g·mol−<sup>1</sup> ): C, 42.50; H, 1.78; N, 10.97%. Found: C, 42.33; H, 1.58; N, 10.97%. IR (ATR, cm−<sup>1</sup> ): *ν*(C–H)ar 3078 (vw), *ν*(C=C)ar 1599 (m), 1482 (s) *ν*(N–O)as 1561 (m), *ν*(C=N) 1434 (s), *ν*(C–C) 1384 (m), 1374 (m), 1249 (m), 1220 (s), *ν*(N–O)sym 1323 (s), *ν*(C–O) 1123 (m), *β*(C–H) 1148 (m), 1052 (m), *ν*(C5–Cl) 984 (m), *δ*(NO2) 934 (m), 679 (m), *γ*(C–H) 831 (m), 818 (s), Ring breathing 754 (s), *β*(CCC) 726 (m), 679 (m), 655 (s), *β*(CNC) 613 (w), *β*(C–O) 535 (m), *β*(C5–Cl) 510 (w), *γ*(CCC) 476 (m).

### *3.3. Physical Measurements*

Infrared spectra of prepared complexes were recorded on a Nicolet 6700 FT-IR spectrometer from Thermo Scientific with a diamond crystal Smart OrbitTM, in the range 4000–400 cm−<sup>1</sup> . Elemental analyses of C, H, and N were with a CHNS Elemental Analyzer varioMICRO from Elementar Analysensysteme GmbH. Absorption spectra were measured with a SPECORD 250 spectrophotometer (Analytik Jena, Jena, Germany), from 300 to 600 nm, in Nujol, and DMSO and DMSO/water (1:1) solutions at 24, 48 and 72 h intervals. IR and UV-Vis spectra were described in Origin 2022b [56]. The morphological characteristics of the samples were studied using a JEOL JSM 6510 scanning electron microscope (W cathode, 20 nm resolution at 1 kV). Semi-quantitative chemical analysis of the samples was determined using the attached Oxford Instruments EDS analyser INCA X act. Measurements were performed on native samples without any conductive coating.

### *3.4. X-ray Structure Analysis*

The crystal structures of **3** and **5** were determined using an Oxford Diffraction Xcalibur2 diffractometer equipped with a Sapphire2 CCD detector, while the structures of **1a**, **1b** and **6** were determined using a Rigaku XtaLAB Synergy S diffractometer with Hybrid Pixel Array detector (HyPix-6000HE). CrysAlis Pro software was used for data collection and cell refinement, data reduction and absorption correction [57]. Structures of prepared complexes were solved by SHELXT [58] and refined by SHELXL [59], implemented in the WinGX program [60]. For all non-H atoms, anisotropic displacement parameters were refined. Hydrogen atoms of XQ and DMF molecules were placed in calculated positions and refined riding on carbon atoms. Presence of hydrogen bonds was analysed by using SHELXL, while PLATON [61], running under WinGX, was used to analyse π–π interaction. Diamond was used for molecular graphics [62]. The summary of crystal data and structure refinements for **1a**, **1b**, **3**, **5** and **6** is presented in Table 7.

*Inorganics* **2022**, *10*, 223


**Table 7.** Crystal data and structural refinement of **1a**, **1b**, **3**, **5** and **6**.

### *3.5. Cell Cultures*

The human cancer cell lines were purchased from ATCC (American Type Culture Collection; Manassas, VA, USA). HCT116 (human colorectal carcinoma), HeLa (human cervical adenocarcinoma) and Jurkat (human leukemic T cell lymphoma) were cultured in RPMI 1640 medium (Biosera, Kansas City, MO, USA) while A549 (human alveolar adenocarcinoma), MCF-7 (human Caucasian breast adenocarcinoma), Caco-2 (human colorectal adenocarcinoma) and MDA-MB-231 (human breast cancer cell line) were maintained in a growth medium consisting of high glucose Dulbecco's Modified Eagle Medium (DMEM) + sodium pyruvate (Biosera). The human kidney fibroblasts (Cos-7) were cultured in DMEM medium (Biosera). All media were supplemented with a 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), Antibiotic/Antimycotic Solution (Sigma, St. Louis, MO, USA) and maintained in an atmosphere containing 5% CO<sup>2</sup> in humidified air at 37 ◦C. Prior to each experiment, cell viability was greater than 95%.

### Screening of Antiproliferative/Cytotoxic Activity

The antiproliferative/cytotoxic effect of copper complexes (concentrations of 10, 50 and 100 µM) was determined by resazurin assay in HCT116, Caco-2, A549, MCF-7, MDA-MB-231, HeLa, Jurkat and Cos-7 cells. Tested cells (1 <sup>×</sup> <sup>10</sup>4/well) were seeded in 96-well plates. After 24 h, final copper complex concentrations, prepared from DMSO stock solution, were added, and incubation proceeded for the next 72 h. Ten microliters of resazurin solution (Merck, Darmstadt, Germany), at a final concentration of 40 µM, was added to each well at the end-point (72 h). After a minimum of 1 h incubation, the fluorescence of the metabolic product resorufin was measured by the automated CytationTM 3 cell imaging multi-mode reader (Biotek, Winooski, VT, USA) at 560 nm excitation/590 nm emission filter. The results were expressed as a fold of the control, where control fluorescence was taken as 100%. All experiments were performed in triplicate. The IC<sup>50</sup> values were calculated from these data.

### *3.6. Antibacterial Activity*

### 3.6.1. Microorganisms Used

The tested bacteria (S. aureus CCM 4223 and E. coli CCM 3988) were obtained from the Czech collection of microorganisms (CCM, Brno, Czech Republic).

### 3.6.2. Agar Well-Diffusion Method

The antibacterial properties of the four complexes, **1b**, **3**, **4** and **6**, and their ligands, HClBrQ, HBrQ, HdNQ and HClNQ, were evaluated by the agar well diffusion method using a slightly modified process compared to [63]. Firstly, each compound was dissolved in a small amount of 100% DMSO and then dissolved to 33.6 µM solution. As a positive control, gentamicin sulfate (Biosera, Nuaille, France), with concentration 50 µg/mL, was used.

Bacteria were cultured overnight, aerobically, at 37 ◦C in LB medium (Sigma-Aldrich, Saint-Louis, MO, USA), with agitation. The inoculum from these overnight cultures was prepared by adjusting the density of the culture to equal that of the 0.5 McFarland standard (1–2 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL), by adding a sterile saline solution. These bacterial suspensions were diluted 1:300 in liquid plate count agar (HIMEDIA, Mumbai, India) resulting in a final concentration of bacteria approximately 5 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL, and 20 mL of this inoculated agar was poured into a Petri dish (diameter 90 mm). Once the agar was solidified, five mm diameter wells were punched in the agar and filled with 50 µL of samples. Gentamicin sulfate, with a concentration of 50 µg/mL, was used as a positive control. The plates were incubated for 18–20 h at 37 ◦C. Afterwards, the plates were photographed, and the inhibition zones were measured by ImageJ 1.53e software (U. S. National Institutes of Health, Bethesda, MD, USA). The values used for the calculation were mean values calculated from 3 replicate tests.

The antibacterial activity was calculated by applying the formula reported in [63]:

%RIZD = [(IZD sample − IZD negative control)/IZD gentamicin] × 100

where RIZD is the relative inhibition zone diameter (%) and IZD is the inhibition zone diameter (mm).

As a negative control, the inhibition zones of 5% DMSO equal to 0 were taken. The inhibition zone diameter (IZD) was obtained by measuring the diameter of the transparent zone.

### 3.6.3. Determination of the Minimum Inhibitory Concentration (MIC) by the Microdilution Method

The minimum inhibitory concentration was determined by the microdilution method with a slight modification of the procedure described in [64].

Stock solutions of the test samples prepared in 5% DMSO (Sigma-Aldrich, USA) were two-fold diluted (1:1 to 1:8) in wells of a 96-well plate (Greiner Bio-One, Germany): the wells of the microtiter plate were filled with 50 µL of Mueller–Hinton Broth (MHB, HIMEDIA, Mumbai, India), and 50 µL of stock solution of the test substance was added to the first well (1:1 dilution). After mixing, 50 µL of this solution was transferred to the next well (1:2 dilution), etc. Each sample was tested in triplicate.

Bacteria were cultured overnight, aerobically, at 37 ◦C in MHB, with agitation. The inoculum from the overnight cultures was prepared by adjusting the density of bacterial suspension to 0.5 McFarland standard (1–2 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL) by adding sterile saline and then diluting 1:150 in MHB. Subsequently, 50 µL of this inoculum was added to each well with 50 µL of diluted test samples (final concentration of bacteria in the well ca. <sup>5</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL). Wells filled only with MHB medium and bacterial suspension were used as a positive control, and wells filled with sterile MHB alone were used as a negative control. The plates were covered with a lid and incubated for 18–20 h at 37 ◦C. The evaluation was made by measuring the absorbance at 600 nm on a microplate reader (Synergy HT, Biotek, Santa Clara, CA, USA).

### *3.7. Radical Scavenging Experiments*

Radical scavenging activity of complexes and free ligands was estimated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (diammonium 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) radicals, according to slightly modified methods described in literature [65,66]. Compounds were dissolved in DMSO and the resulting solutions were diluted with MeOH in 1:3 ratio. The prepared solutions were mixed with methanolic solutions of the respective radicals and incubated in the dark at room temperature (7 min ABTS and 30 min DPPH). The absorbance was recorded at 734 nm (ABTS) and 517 nm (DPPH), respectively. L-ascorbic acid was used as a standard, and experiments were performed in triplicate. The IC<sup>50</sup> parameters were calculated from a linear plot of the inhibition percentage against the concentration of compounds.

### **4. Conclusions**

In this work, we present six new copper(II) complexes: [Cu(ClBrQ)2] (**1a**, **1b**), [Cu(ClBrQ)2]·1/2 diox (**2**) (diox = 1,4-dioxane), [Cu(BrQ)2] (**3**), [Cu(dNQ)2] (**4**), [Cu(dNQ)2(DMF)2] (**5**) and [Cu(ClNQ)2] (**6**), where HClBrQ is 5-chloro-7-bromo-8-hydroxyquinoline, HBrQ is 7-bromo-8-hydroxyquinoline, HClNQ is 5-chloro-7-nitro-8-hydroxyquinoline and HdNQ is 5,7-dinitro-8-hydroxyquinoline. Prepared complexes were characterised by IR spectroscopy (**1**–**6**), and elemental (**1b**–**4** and **6**) and X-ray structural (**1a**, **1b**, **3**, **5** and **6**) analysis. The sability of **1b**–**4** and **6** in DMSO and DMSO/water solutions was verified by UV-Vis spectroscopy.

Complexes **1a**, **1b**, **3**, **5** and **6** were molecular compounds. Crystal structures of **1a**, **1b**, **3** and **6** formed by square planar [Cu(XQ)2] complexes, in which copper atoms were coordinated by two pairs of oxygen and nitrogen atoms from two deprotonated XQ ligands. On the other hand, a tetragonal bipyramidal coordination of the copper atom in **5** was observed. The equatorial plane was occupied by the same atoms as in the mentioned [Cu(XQ)2] complexes, and oxygen atoms from DMF molecules occupied axial positions. Interestingly, despite similar structures, intramolecular interactions were observed only in

**5** and **6**, where hydrogen bonds existed. They created layers parallel with (01-1) (**5**) and (100) (**6**) plane.

After cytotoxic evaluation, only complex **3** showed promising potential as an anticancer agent, but, due to the low selectivity towards non-cancerous cells, modification of the complex was required.

Among the prepared compounds only **3** was active against both tested radicals (ABTS and DPPH). Antioxidant activity could be involved in the cytotoxic effects of **3** against the tested cell line; however, the free ligand showed even better antiradical properties. Apparently, the coordination of HBrQ ligand to Cu(II) decreased its antioxidant activity.

Only the HClNQ ligand showed antibacterial potential with concentration 33.6 µM.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/inorganics10120223/s1, Figure S1: SEM micrograph (a) of **1a** along with EDS elemental analysis (b); Figure S2: Molecular structure of **1a**; Table S1: Hydrogen bonds [Å and ◦ ] for **5** and **6**.

**Author Contributions:** Conceptualization, I.P. and M.K. (Martina Kepeˇnová); investigation, M.K. (Martina Kepeˇnová), M.K. (Martin Kello), R.S., M.G., R.F., L'.T., M.L., J.Š. and I.P.; resources, M.K. (Martin Kello), M.G., M.L. and I.P.; writing—original draft preparation, M.K. (Martina Kepeˇnová), M.K. (Martin Kello), R.S., M.G. and I.P.; Writing—Review & Editing, M.K. (Martina Kepeˇnová), R.S. and I.P.; visualization, M.K. (Martina Kepe ˇnová) and J.Š.; supervision, M.K. (Martin Kello), M.G. and I.P.; project administration, M.K. (Martin Kello), M.G. and I.P.; funding acquisition, M.K. (Martin Kello), M.L. and I.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by VEGA 1/0148/19, VEGA 1/0653/19, APVV-18-0016 and VVGS-PF-2022-2134. Moreover, this publication is the result of the project implementation, "Open scientific community for modern interdisciplinary research in medicine (OPENMED)", ITMS2014+: 313011V455, supported by the Operational Programme Integrated Infrastructure, funded by the ERDF. We also thank the Research Infrastructure NanoEnviCz project, supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project No. LM2018124 for instrumentation.

**Data Availability Statement:** CCDC 2216977-2216981 contain the supplementary crystallographic data for **1a**, **1b**, **3**, **5**, and **6**. These data can be obtained free of charge via http://www.ccdc.cam. ac.uk/conts/retrieving.html, (accessed on 5 November 2022) or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

**Acknowledgments:** Special thanks go to Petra Ecorchard. (Centre of Instrumental Techniques, Institute of Inorganic Chemistry of the CAS) for the preparing of SEM samples.

**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.

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