3.2.3. Synthesis of [Zn2(ClQ)3(HClQ)3]I<sup>3</sup> (**3**)

ZnI<sup>2</sup> (159 mg, 0.5 mmol) in ethanol (10 mL) was added drop by drop to the solution of HClQ (89 mg, 0.5 mmol) in ethanol (10 mL) under stirring. After one month at room temperature, dark brown crystals of **3** were formed, filtered off and dried in air.

[Zn2(ClQ)3(HClQ)3]I<sup>3</sup> (**3**): Anal. Calc. for C54H33Cl6I3N6O6Zn<sup>2</sup> (1586.09 g·mol−<sup>1</sup> ): C, 40.89; H, 2.10; N, 5.30%. Found: C, 40.72; H, 2.29; N, 4.98%. Yield: 87.23 mg (66%). <sup>1</sup>H NMR (DMSO-d6): *δ* = 10.19 (1H, br s, OHHClQ), 8.95 (1H, br s, H-2HClQ), 8.66 (1H, br s, H-2ClQ), 8.52 (2H, br s, H-4HClQ, H-4ClQ), 7.74 (2H, br s, H-3HClQ, H-3ClQ), 7.60 (1H, br s, H-6HClQ), 7.51 (1H, br s, H-6ClQ), 7.08 (1H, br s, H-7HClQ), 6.72 (1H, br s, H-7ClQ) ppm. IR (ATR, cm−<sup>1</sup> ): 3062(w), 1633(w), 1580(m), 1500(m), 1463(m), 1453(w), 1393(m), 1361(m), 1319(m), 1261(m), 1239(w), 1206(w), 1155(w), 1107(w), 1059(w), 955(w), 868(w), 820(m), 807(w), 777(m), 730(m), 660(m), 629(m), 575(w), 534(m), 510(w), 485(m), 458(w), 430(m).

### 3.2.4. Synthesis of [Zn2(dClQ)2(H2O)6(SO4)] (**4**)

ZnSO4·7H2O (144 mg, 0.5 mmol) in methanol (10 mL) was added drop by drop to the solution of HdClQ (107 mg, 0.5 mmol) in DMF (10 mL) under stirring. After one month at room temperature, yellow crystals of **4** were formed, filtered off and dried in air.

[Zn2(dClQ)2(H2O)6(SO4)] (**4**): Anal. Calc. for C18H20Cl4N2O12SZn<sup>2</sup> (761.01 g·mol−<sup>1</sup> ): C, 28.41; H, 2.65; N, 3.68; S, 4.21%. Found: C, 28.22; H, 2.48; N, 3.39; S, 3.90%. Yield: 62.78 mg (33%). <sup>1</sup>H NMR (DMSO-d6): *δ* = 8.79 (1H, br s, H-2 of minor form), 8.55 (1H, br s, H-2), 8.50 (d, *J* 8.4 Hz, H-4), 7.70 (2H, m, H-3, H-6). <sup>13</sup>C NMR (DMSO-d6): *δ* = 158.2 (C-8), 146.1 (C-2), 140.0 (C-8a), 135.1 (C-4), 129.5 (C-6), 125.7 (C-4a), 122.7 (C-3), 114.8 (C-7), 108.5 (C-5). IR (ATR, cm−<sup>1</sup> ): 3056(vs), 1624(m), 1566(m), 1491(m), 1455(m), 1395(m), 1377(w), 1364(m), 1290(w), 1249(w), 1231(w), 1110(w), 1052(m), 963(m), 885(w), 862(m), 803(m), 780(m), 740(m), 665(m), 593(w), 503(w), 432(w).

### 3.2.5. Synthesis of [Zn2(dBrQ)2(H2O)6(SO4)] (**5**)

ZnSO4·7H2O (144 mg, 0.5 mmol) in methanol (10 mL) was added drop by drop to the solution of HdBrQ (151 mg, 0.5 mmol) in DMF (10 mL) under stirring. After one month at room temperature, yellow crystals of **5** were formed, filtered off and dried in air.

[Zn2(dBrQ)2(H2O)6(SO4)] (**5**): Anal. Calc. for C18H20Br4N2O12S1Zn<sup>2</sup> (938.82 g·mol−<sup>1</sup> ): C, 23.03; H, 2.15; N, 2.98; S, 3,42%. Found: C, 23.45; H, 2.13; N, 2,97; S, 3.31%. Yield: 110.31 mg (47%). <sup>1</sup>H NMR (DMSO-d6): minor form: *δ* = 8.76 (1H, br s, H-2), 8.44 (1H, br s, H-4), 7.92 (1H, s, H-6), 7.75 (1H, br s, H-3) ppm. <sup>1</sup>H NMR (DMSO-d6): major form: *δ* = 8.47 (1H, br s, H-2), 8.41 (1H, d, *J* 8.5 Hz, H-4), 7.92 (1H, s, H-6), 7.70 (1H, br s, H-3) ppm. <sup>13</sup>C NMR (DMSO-d6): minor form: *δ* = 159.8 (C-8), 145.9 (C-2), 140.0 (C-8a), 137.6 (C-4), 134.6 (C-6), 127.3 (C-4a), 123.2 (C-3), 105.3 (C-7), 97.7 (C-5) ppm. <sup>13</sup>C NMR (DMSO-d6): major form: *δ* = 159.8 (C-8), 145.9 (C-2), 140.0 (C-8a), 137.4 (C-4), 134.7 (C-6), 127.3 (C-4a), 123.2 (C-3), 105.3 (C-7), 97.7 (C-5) ppm. IR (ATR, cm−<sup>1</sup> ): 3186(m), 3072(vw), 1632(m), 1556(m), 1484(m), 1455(m), 1389(m), 1359(s), 1284(w), 1247(w), 1221(w), 1121(w), 1106(w), 1050(m), 985(w), 943(m), 863(m), 800(w), 777(m), 741(m), 684(m), 660(m), 577(w), 500(w), 428(w).

### *3.3. Physical Measurements*

The infrared spectra of the prepared complexes were recorded on a Nicolet 6700 FT-IR spectrophotometer from Thermo Scientific equipped with a diamond crystal Smart Orbit™ in the range 4000–400 cm−<sup>1</sup> . Elemental analyses of C, H, N and S were obtained on CHNOS Elemental Analyzer Vario MICRO from Elementar Analysensysteme GmbH. NMR spectra were recorded at room temperature on a Varian VNMRS spectrometer operating at 599.87 MHz for <sup>1</sup>H and 150.84 MHz for <sup>13</sup>C. Spectra were recorded in DMSO-d<sup>6</sup> and the chemical shifts were referenced to the residual solvent signal (1H NMR 2.50 ppm, <sup>13</sup>C NMR 39.5 ppm). The 2D gCOSY, gHSQC and gHMBC (optimized for a long-range coupling of 8 Hz) methods were employed.

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

The data collection for **1**, **2**, **3** and **4** was carried out on SuperNova diffractometer from Rigaku OD equipped with Atlas2 CCD detector; for **5** a Rigaku XtaLAB Synergy, Dualflex diffractometer equipped with Hybrid Pixel Array Detector (HyPix-6000HE) was used. CrysAlisPro software was used for data collection and cell refinement, data reduction and absorption correction [38]. Crystal structure of **3** was solved and refined by JANA2020 [39], other structures were solved and refined by SHELXT [40] and subsequent Fourier syntheses using SHELXL-2018 [41], respectively, implemented in WinGX program suit [42]. Anisotropic displacement parameters were refined for all non-H atoms, except for heavily disordered triiodide anion in the canals formed in the crystal structure of **3**. The maxima found in the difference Fourier map were assigned as iodine and their thermal movement is described by refinement of the 4th order anharmonic ADP for each atom using JANA2020 program. Hydrogen atoms of HXQ and DMF molecules were placed in calculated positions and refined riding on their parent C atoms. H atoms of hydroxyl groups in **3** and water molecules in **2** and **5** involved in hydrogen bonds were found in a Fourier difference map and refined as riding model. A geometric analysis was performed using SHELXL-2018, PLATON [43] was used to analyze π-π interaction, while DIAMOND [44] was used for molecular graphics. A summary of crystal data and structure refinement for all complexes is presented in Table 11.


**Table 11.** Crystal data and structural refinement for **1**–**5**.


### **Table 11.** *Cont*.

<sup>a</sup> Only isotropic model.

### *3.5. DNA/BSA Binding Studies*

### 3.5.1. UV-vis Absorption Study

UV-vis spectrophotometric measurements were carried out on a Varian Cary 100 UV-vis spectrophotometer at room temperature (24 ◦C) in 10 mM Tris buffer (pH 7.4) using quartz cuvettes with 10 mm light path in the range of 230-500 nm. The complexes were dissolved in DMSO, while the stock solution of ctDNA was prepared in Tris–EDTA buffer (pH 7.4). The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M−<sup>1</sup> cm−<sup>1</sup> ) at 260 nm. An equal volume of nucleic acid was added to both the sample and reference cuvettes to eliminate any interference due to the absorbance of DNA itself.

### 3.5.2. Ethidium Bromide Displacement Assay

The fluorescence emission spectra were measured using a Varian Cary Eclipse spectrofluorometer at room temperature (24 ◦C) in 10 mM Tris buffer (pH 7.4). The fluorescence spectra were measured at an excitation wavelength of 510 nm and slit width 10 nm for the excitation and emission beams. The emission spectra were recorded in the range 565–700 nm and analyzed according to the classical Stern–Volmer equation:

$$\frac{F\_0}{F} = \mathbf{1} + \mathbf{K}\_{SV} \mathbf{[Q]}$$

where *F*<sup>0</sup> and *F* represent the fluorescence intensities in the absence and presence of the quencher, *KSV* is the Stern–Volmer quenching constant and [*Q*] is the concentration of the quencher [33].

### 3.5.3. BSA Binding Experiments

The fluorescence spectra were recorded using a Varian Cary Eclipse spectrofluorometer at a temperature of 25 ◦C in 0.01 M phosphate-buffered saline (pH 7.4). The excitation wavelength (*λ*ex) for BSA was 280 nm and slit width 10 nm for the excitation and emission beams. The spectroflorometric titrations with increasing concentrations of the zinc complexes were recorded in the range 300–450 nm.

### *3.6. In Vitro Antitumor Activity*

### 3.6.1. Cell Lines and Cell Culture

The human cancer cell lines purchased from ATCC (American Type Culture Collection; Manassas, VA, USA): HCT116 (human colorectal carcinoma) and HeLa (human

cervical adenocarcinoma) were cultured in RPMI 1640 medium (Biosera, Kansas City, MO, United States), and A549 (human alveoral adenocarcinoma), MCF-7 (human Caucasian breast adenocarcinoma), Caco-2 (human colorectal adenocarcinoma) and MDA-MB-231 (human breast adenocarcinoma) were maintained in 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), 1X HyClone™ antibiotic/antimycotic solution (GE Healthcare, Little Chalfont, UK) 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%.

### 3.6.2. Assessment of Cytotoxicity

The antiproliferative effect of zinc complexes (concentrations of 10, 50, 100 µM) was determined by resazurin assay in HCT116, Caco-2, A549, MCF-7, MDA-MB-231, HeLa and Cos-7 cells. Tested cells (1 <sup>×</sup> <sup>10</sup>4/well) were seeded in 96-well plates. After 24 h, final zinc 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.

### *3.7. Antimicrobial Activity*

### 3.7.1. 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).

### 3.7.2. Agar Well-Diffusion Method

The antibacterial properties of the complexes **1**–**5** and their ligands HClQ, HdClQ and HdBrQ were evaluated by the agar well diffusion method by slightly modified process compared to [45]. 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 sulphate (Biosera, Nuaille, France) with a concentration of 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 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 sulphate with a concentration of 50 µg/mL was used as a positive control. The plates were incubated for 18–20 h at 37 ◦C. Afterward, the plates were photographed, and the inhibition zones were measured by the ImageJ 1.53e software (U. S. National Institutes of Health, Bethesda, MD, USA). The values used for the calculation are mean values calculated from 3 replicate tests.

The antibacterial activity was calculated by applying the formula reported in [45]:

%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.

### **4. Conclusions**

In this work, we have prepared five new Zn(II) complexes, [Zn4Cl2(ClQ)6]·2DMF (**1**), [Zn4Cl2(ClQ)6(H2O)2]·4DMF (**2**), [Zn2(ClQ)3(HClQ)3]I<sup>3</sup> (**3**), [Zn2(dClQ)2(H2O)6(SO4)] (**4**) and [Zn2(dBrQ)2(H2O)6(SO4)] (**5**) (HClQ = 5-chloro-8-hydroxyquinoline, HdClQ = 5,7 dichloro-8-hydroxyquinoline and HdBrQ = 5,7-dibromo-8-hydroxyquinoline), which were characterized by IR spectroscopy, elemental analysis and single crystal X-ray structure analysis. Complexes **1** and **2** are tetranuclear molecular complexes with a complicated type of structure. Dinuclear complex **3** is an ionic compound and dinuclear complexes **4** and **5** have a similar molecular type of structure. The complexes **1** and **2** have symmetric structures in solution and <sup>1</sup>H NMR spectra display only one set of signals, corresponding to the appropriate units, and no fluxional phenomena were observed for these complexes. The broadened <sup>1</sup>H NMR signals of **3** arise from its low solubility and the presence of some particulate matter in the solution, but, on the other hand, the broadened <sup>1</sup>H NMR signals of **4** and **5** are due to the presence of a dynamic exchange process in solution. The time-dependent <sup>1</sup>H NMR spectra confirmed the stability of the studied complexes within 72 h. This study also provides information about the DNA binding mode and BSA binding potency of zinc complexes. According to the fairly low *Ksv* constant for EB displacement, we conclude that these complexes are only weak intercalators. We have also found that complex **3**, which shows considerable cytotoxic capacity, has the highest affinity to BSA of all the measured complexes. This outcome can be important for further potential pharmacokinetic measurements. The antiproliferative activity of the prepared complexes was studied using in vitro MTT assay against the HeLa, A549, MCF-7, MDA-MB-231, HCT116 and Caco-2 cancer cell lines and on Cos-7 non-cancerous cell line. The most sensitive to the tested complexes was Caco-2 cell line. Among the tested complexes, complex **3** showed the highest cytotoxicity against all cell lines. Complexes **3** and **4** showed better activity than cisplatin in almost all cases. Unfortunately, all complexes showed only poor selectivity to normal cells, except for complex **5**, which showed a certain level of selectivity. Only complex **5** showed antibacterial potential with a concentration of 33.6 µM.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/inorganics11020060/s1, Figure S1. <sup>1</sup>H NMR (600 MHz, DMSOd6 ) spectrum of complex **3**; Figure S2. <sup>1</sup>H (600 MHz, DMSO-d<sup>6</sup> ) and <sup>13</sup>C (150 MHz, DMSO-d<sup>6</sup> ) NMR spectrum of complex **5**; Figure S3. Time-dependent <sup>1</sup>H NMR (600 MHz, DMSO-d<sup>6</sup> ) spectra of complex **2**; Figure S4. Time-dependent <sup>1</sup>H NMR (600 MHz, DMSO-d<sup>6</sup> ) spectra of complex **3**; Figure S5. Time-dependent <sup>1</sup>H NMR (600 MHz, DMSO-d<sup>6</sup> ) spectra of complex **4**; Figure S6. Time-dependent <sup>1</sup>H NMR (600 MHz, DMSO-d<sup>6</sup> ) spectra of complex **5**; Figure S7. Part of the one-dimensional structure of **3** viewed along the *c* axis with π-π interactions (black dashed lines); Figure S8. UV-vis spectrum of complex **<sup>1</sup>** (6.14 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M) with ctDNA. The arrow indicates changes in absorbance upon increasing DNA concentration. Inset: UV-vis absorption spectrum of **1**; Figure S9. UV-vis spectrum of complex **<sup>2</sup>** (6.14 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M) with ctDNA. The arrow indicates changes in absorbance upon increasing DNA concentration. Inset: UV-vis absorption spectrum of **2**; Figure S10. Fluorescence spectrum of DNA-EB complex in the absence (black line) and presence of complex **1.** Inset: The corresponding Stern-Volmer plot for quenching process of EB by **1**; Figure S11. Fluorescence spectrum of DNA-EB complex in the absence (black line) and presence of complex **2**. Inset: The corresponding Stern-Volmer plot for quenching process of EB by **2**; Figure S12. Fluorescence quenching spectra of BSA in presence of complex **1**. Inset: The corresponding Stern-Volmer plot for **1** at 25 ◦C; Figure S13. Fluorescence quenching spectra of BSA in presence of complex **2**. Inset: The corresponding Stern-Volmer plot for **2** at 25 ◦C; Table S1. <sup>13</sup>C NMR (150 MHz, DMSO-d<sup>6</sup> ) chemical shifts δC [ppm] for complexes **1**–**5**. Table S2. Data describing different polyhedral distortions for **1**–**5**; Table S3. Cg···Cg distances and angles (Å, ◦ ) characterizing π-π interactions in **1** and **2**; Table S4. Cg···Cg distances and angles (Å, ◦ ) characterizing π-π interactions in **3**; Table S5. Cg···Cg distances and angles (Å, ◦ ) characterizing π-π interactions in **5**.

**Author Contributions:** Conceptualization, I.P. and M.H.; investigation, M.H., M.K., M.G., L'.T., M.V., D.S., S.S., E.S., M.L., V.K., J.K. and I.P.; resources, M.K., M.G., E.S., M.L. and I.P.; writing—original draft preparation, M.H., M.K., M.G., M.V., D.S., E.S. and I.P.; Writing—Review and Editing, M.H. and I.P.; visualization, M.H., M.V. and D.S.; supervision, I.P.; project administration, I.P.; funding acquisition, M.K., E.S., M.L. and I.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The financial support of Slovak grant agencies, VEGA 1/0126/23, 1/0037/22, 1/0653/19 and APVV-18-0016, as well as P.J. Šafárik University in Košice (VVGS-PF-2020-1425, VVGS-2021-1772 and VVGS-PF-2022-2134), are gratefully acknowledged. 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. CzechNanoLab project LM2018110 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at LNSM Research Infrastructure. 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 2219114–2219118 contain the supplementary crystallographic data for **1**–**5**. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/ retrieving.html, 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.

**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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