**Low-Dimensional Compounds Containing Bioactive Ligands. Part XX: Crystal Structures, Cytotoxic, Antimicrobial Activities and DNA/BSA Binding of Oligonuclear Zinc Complexes with Halogen Derivatives of 8-Hydroxyquinoline**

**Michaela Harmošová 1 , Martin Kello <sup>2</sup> , Michal Goga <sup>3</sup> , L'udmila Tkáˇciková 4 , Mária Vilková 1 , Danica Sabolová 1 , Simona Sovová 1 , Erika Samol'ová 5 , Miroslava Litecká 6 , Veronika Kuchárová 7 , Juraj Kuchár <sup>1</sup> and Ivan Potoˇc ˇnák 1,\***

	- CZ-25068 Rež, Czech Republic ˇ

7


**Abstract:** Two tetranuclear [Zn4Cl<sup>2</sup> (ClQ)<sup>6</sup> ]·2DMF (**1**) and [Zn4Cl<sup>2</sup> (ClQ)<sup>6</sup> (H2O)<sup>2</sup> ]·4DMF (**2**), as well as three dinuclear [Zn<sup>2</sup> (ClQ)<sup>3</sup> (HClQ)<sup>3</sup> ]I3 (**3**), [Zn<sup>2</sup> (dClQ)<sup>2</sup> (H2O)<sup>6</sup> (SO<sup>4</sup> )] (**4**) and [Zn<sup>2</sup> (dBrQ)<sup>2</sup> (H2O)<sup>6</sup> (SO<sup>4</sup> )] (**5**), complexes (HClQ = 5-chloro-8-hydroxyquinoline, HdClQ = 5,7-dichloro-8-hydroxyquinoline and HdBrQ = 5,7-dibromo-8-hydroxyquinoline) were prepared as possible anticancer or antimicrobial agents and characterized by IR spectroscopy, elemental analysis and single crystal X-ray structure analysis. The stability of the complexes in solution was verified by NMR spectroscopy. Antiproliferative activity and selectivity of the prepared complexes were studied using in vitro MTT assay against the HeLa, A549, MCF-7, MDA-MB-231, HCT116 and Caco-2 cancer cell lines and on the 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. Unfortunately, all complexes showed only poor selectivity to normal cells, except for complex **5**, which showed a certain level of selectivity. Antibacterial potential was observed for complex **5** only. Moreover, the DNA/BSA binding potential of complexes **1**–**3** was investigated by UV-vis and fluorescence spectroscopic methods.

**Keywords:** crystal structure; Zn complexes; 8-hydroxyquinoline; cytotoxicity; antimicrobial activity; DNA/BSA binding

## **1. Introduction**

Cancer diseases represent one of the greatest challenges for society. Cancer is responsible of around 10.0 million deaths worldwide and 19.3 million new cases each year [1]. There are seven platinum-containing drugs with a great effect in a cancer treatment. Three of them (cisplatin, carboplatin and oxaliplatin) are approved worldwide while other four (nedaplatin, lobaplatin, heptaplatin and miriplatin) are approved in specific Asian countries [2]. Despite their success in a cancer treatment, there are not only several side effects, such as allergic reactions, kidney problems, ototoxicity and decreased immunity associated with the use of these drugs, but also platinum drugs cell resistance can be developed.

**Citation:** Harmošová, M.; Kello, M.; Goga, M.; Tkáˇciková, L'.; Vilková, M.; Sabolová, D.; Sovová, S.; Samol'ová, E.; Litecká, M.; Kuchárová, V.; et al. Low-Dimensional Compounds Containing Bioactive Ligands. Part XX: Crystal Structures, Cytotoxic, Antimicrobial Activities and DNA/BSA Binding of Oligonuclear Zinc Complexes with Halogen Derivatives of 8-Hydroxyquinoline. *Inorganics* **2023**, *11*, 60. https:// doi.org/10.3390/inorganics11020060

Academic Editors: Peter Segl'a and Ján Pavlik

Received: 4 January 2023 Revised: 19 January 2023 Accepted: 24 January 2023 Published: 26 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Therefore, new drugs need to be synthesized to overcome the drug-resistance and unwanted side effects [3]. New transition metal complexes could be a suitable choice because of their bioactivity, biocompatibility, varying coordination numbers and geometries; they can also bind with DNA through covalent, noncovalent and electrostatic interactions, which can lead to cellular death [4]. There are many classes of organic compounds, including heterocyclic compounds, which have been synthesized as suitable bioactive ligands, and their complexes were tested as possible anticancer drugs. Growing interest is noted for 8-hydroxyquinoline (H8-HQ) and its derivatives (HXQ) [5,6]. H8-HQ, as well as HXQ, are known to exhibit anti-SARS-CoV-2 [7], antimicrobial [8,9], antifungal [10], anti-inflammatory [11], anticancer [12–14] and antioxidant activity [15,16]. They also have significant effects in the treatment of malaria, HIV and neurological diseases [6]. The lipophilic derivative of H8-HQ, clioquinol (HCQ = 5-chloro-7-iodo-8-hydroxyquinoline), has been studied as a treatment for Alzheimer's disease [17]. The halogenation of H8-HQ at various positions makes it more lipophilic, which leads to better absorption and better activity. In addition, the complexation of HXQ with metal ions shows better anticancer activity [6 and citations therein]. Recently, we published several papers describing the biological activities of both transition and non-transition metal complexes with HXQ. Some of them, such as K[PdCl2(dClQ)], Cs[PdCl2(dClQ)] (HdClQ = 5,7-dichloro-8-hydroxyquinoline) [18] and [Ga(ClQ)3] (HClQ = 5-chloro-8-hydroxyquinoline) [19], exhibited strong and selective cytotoxic effects against tested cancer cell lines. On the other hand, although mononuclear zinc complexes with HXQ, such as K[Zn(dClQ)3]·2DMF·H2O, (HdClQ)2[ZnCl4]·2H2O or [Zn(dBrQ)2(H2O)]2·DMF·H2O (HdBrQ = 5,7-dibromo-8-hydroxyquinoline), have shown significant cytotoxic activity against colon cancer HCT116 cell lines, they were not selective. However, we have observed strong antimicrobial and antifungal activity of these complexes [20]. These results motivated us to continue our work, and we have prepared a series of di- and tetranuclear zinc complexes with mono and dihalogen derivatives of H8-HQ with the aim of studying their cytotoxic and antimicrobial activity. In this work, we describe preparation of five new zinc(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**), which were characterized by IR and NMR spectroscopy, as well as elemental and single crystal X-ray structure analysis. Moreover, the results of cytotoxic and antimicrobial assays and their DNA/BSA binding properties are presented.

### **2. Results and Discussion**

### *2.1. Syntheses*

In this work, we have focused on the preparation of zinc complexes with halogen derivatives of H8-HQ. We have used several zinc salts, solvents, different synthetic strategies and different procedures of crystallization. Regarding solvents, DMF was used to dissolve respective HXQ ligands, while ethanol was used to dissolve zinc(II) chloride or iodide. Because of the better solubility of ZnSO4·7H2O in methanol, for the preparations of sulphato complexes **4** and **5**, methanol was used instead of ethanol. The preparation of **1**, **3**, **4** and **5** was performed at room temperature by a simple one-pot synthesis. After the mixing of reactants, complexes were isolated after several months of crystallization at room temperature. On the other hand, the preparation of **2** was performed at low temperatures; moreover, KOH was used to improve deprotonation of the HClQ hydroxyl group and crystallization was carried out at a low temperature in a fridge. It has to be noted that we attempted to prepare complexes with all three HXQ derivatives at room temperature and also under reflux either with or without added KOH; however, no other products were obtained. Figure 1 schematically describes the synthetic procedure used for the synthesis of **1**–**5**.

**Figure 1.** Synthesis and formulae of **1**–**5**. **1**: bis(µ<sup>3</sup> -5-chloro-8-hydroxyquinolinato)-tetrakis(µ<sup>2</sup> - 5-chloro-8-hydroxyquinolinato)-dichloride-tetra-zinc(II) bis-dimethylformamide solvate, [Zn4Cl<sup>2</sup> (ClQ)<sup>6</sup> ]· 2DMF; **2**: bis(µ<sup>3</sup> -5-chloro-8-hydroxyquinolinato)-tetrakis(µ<sup>2</sup> -5-chloro-8-hydroxyquinolinato)-diaquadichloride-tetra-zinc(II) tetra-dimethylformamide solvate, [Zn4Cl<sup>2</sup> (ClQ)<sup>6</sup> (H2O)<sup>2</sup> ]·4DMF; **3**: tris(5-chloro-8-hydroxyquinolinato)-tris(5-chloro-8-hydroxyquinoline)-di-zinc(II) triiodide, [Zn<sup>2</sup> (ClQ)<sup>3</sup> (HClQ)<sup>3</sup> ]I3 ; **4**: catena-(µ<sup>2</sup> -sulphato)-haxaaqua-bis(5,7-dichloro-8-hydroxyquinolinato)-di-zinc (II), [Zn<sup>2</sup> (dClQ)<sup>2</sup> (H2O)<sup>6</sup> (SO<sup>4</sup> )]; **5**: catena-(µ<sup>2</sup> -sulphato)-haxaaqua-bis(5,7-dibromo-8-hydroxyquinolinato) di-zinc(II), [Zn<sup>2</sup> (dBrQ)<sup>2</sup> (H2O)<sup>6</sup> (SO<sup>4</sup> )].

### *2.2. Infrared Spectroscopy*

The presence of HXQ ligands in **1**–**5** was first proven by IR spectroscopy. The IR spectra of pure HdClQ, HdBrQ and HClQ ligands have already been described [21,22]. The characteristic vibrations of the free ligands include the *ν*(O–H) vibration, which manifests as a broad band starting at 3360 cm−<sup>1</sup> and ending with a vibration band of *ν*(C–H)ar at 3060 cm−<sup>1</sup> , and its absence in **1**–**5** suggests the coordination of the respective ligand to the zinc atom through the oxygen atom after the deprotonation of a hydroxyl group. The coordination of the ligands to the zinc atom through pyridine nitrogen atom is supported by the characteristic shift of *ν*(C=N) bands to lower frequencies in the spectra of **1**–**5** (1462–1454 cm−<sup>1</sup> ) compared to the free ligands (1470–1460 cm−<sup>1</sup> ). The remaining ligands bands are still present in the spectra of the complexes. However, they are often shifted compared to the IR spectra of free ligands.

Tetranuclear complexes **1** and **2** have a similar composition with two extra water molecules in **2** and thus their IR spectra will be discussed together. Despite the presence of water molecules, in the spectrum of **2** the valence vibration of the OH group is not clearly visible (Figure 2). We attribute it to a wide band starting at 3305 cm−<sup>1</sup> , which smoothly passes into the *ν*(C–H)ar vibration observed at 3076 cm−<sup>1</sup> . On the other hand, the planar deformation vibration of H2O is clearly visible (1650 cm−<sup>1</sup> ); however, the bands of the rocking, twisting and wagging vibrational modes of coordinated water molecules, which should appear in the spectrum in the ranges of 970–930 cm−<sup>1</sup> and 660–600 cm−<sup>1</sup> [23], are again missing. DMF molecules in both complexes are proven by the weak bands of *ν*(C–H)al vibrations between 2990 and 2850 cm−<sup>1</sup> and by strong absorption bands of *ν*(C=O) vibration at 1672 cm−<sup>1</sup> . The absorption bands of ClQ ligands are at the same wavenumbers in both spectra and are close to those of free ligands with the exception of the above-mentioned *ν*(O–H) and *ν*(C=N) bands.

**Figure 2.** FT–IR spectra of **1**–**5**.

IR spectrum of **3**, which contains the same ClQ ligand, is similar to those of **1** and **2**; of course, the absorption bands of water and DMF molecules are missing.

The composition of **4** and **5** is again very similar; both complexes differ only by HXQ ligands. The presence of water molecules in these complexes is proven by strong and wide bands of *ν*(O–H) vibration starting at 3630 cm−<sup>1</sup> and ending by *ν*(C–H)ar vibrations observed at 3072 cm−<sup>1</sup> , as well as by deformation vibrations around 1630 cm−<sup>1</sup> .

We observed huge bands around 1110 and 1050 cm−<sup>1</sup> in the spectra of **4** and **5,** which can be attributed to the *ν*as(S–O) and *ν*s(S–O) valence vibrations, respectively, originating from the sulphato ligand (ZnSO4·7H2O was used in the synthesis). The weak bands of deformation vibrations of the SO<sup>4</sup> group around 624 cm−<sup>1</sup> are also observed in the spectra [24]. The main difference in the bands of HdClQ and HdBrQ ligands present in **4** and **5**, respectively, are the bands of *ν*(C5–X) vibrations. In **4**, the *ν*(C5–Cl) vibration is observed at 964 cm−<sup>1</sup> (in the spectra of complexes **1**–**3** with HClQ ligand, corresponding bands are between 961 and 955 cm−<sup>1</sup> ) while *ν*(C5–Br) vibration is observed at 943 cm−<sup>1</sup> in the spectrum of **5**. On the other hand, *ν*(C7–X) vibrations that were absent in the spectra of **1**–**3** are in the spectra of **4** and **5** at the same wavenumber, 863 cm−<sup>1</sup> [21,25,26].

### *2.3. NMR Characterization and Stability Studies*

The assignment of resonances for individual proton and carbon atoms within all complexes were made based on <sup>1</sup>H, <sup>13</sup>C and <sup>1</sup>H, <sup>13</sup>C-COSY, <sup>1</sup>H, <sup>13</sup>C-HSQC, <sup>1</sup>H and <sup>13</sup>C-HMBC spectra. These studies were performed at 298 K. The data obtained from these spectra are listed in Table 1 and Table S1. It should be noted that the same sets of proton and carbon signals were detected for each quinoline ligand of **1** and **2** because their composition is almost the same. They only differ by the presence of two water molecules in the coordination sphere of complex **2** whose signals are overlapped by the signals of water from DMSO-d6. On the other hand, the separate and broadened signals were detected for complexes **3**, **4** and **5**.

Based on the crystal structures, we assume that complexes **1** and **2** should have symmetric structures in solution and <sup>1</sup>H NMR spectra display only one set of chemical shifts (Table 1), corresponding to the appropriate units, and no fluxional phenomena were observed for these complexes.


**Table 1.** <sup>1</sup>H NMR (600 MHz, DMSO-d<sup>6</sup> ) chemical shifts *δ*<sup>H</sup> [ppm] for **1**–**5**.

\* 7.95 (s, HDMF), 2.89 (s, MeDMF) and 2.73 (s, MeDMF); \*\* chemical shifts for major form are in the first line and for the minor form in the second line.

Figure S1 displays the <sup>1</sup>H NMR spectrum for **3**. At 25 ◦C, the <sup>1</sup>H NMR spectrum consists of broadened signals attributable to the protons of ClQ and HClQ ligands. At first sight, one could think that this effect arises from a dynamic within a system. However, it is most likely that broadened signals of **3** arise from its low solubility and the presence of some particulate matter in the solution. For the same reason, it was not possible to measure <sup>13</sup>C and 2D NMR spectra and to assign carbon chemical shifts.

We assumed that in the solution, **4** and **5** would give well-resolved signals like the above-described complexes **1** and **2**. <sup>1</sup>H NMR experiments confirmed the opposite to be true. <sup>1</sup>H NMR spectra of complexes **4** and **5** contained broadened peaks consistent with the presence of a dynamic exchange process. As illustrated by Figure 3 and Figure S2, in the <sup>1</sup>H NMR spectrum of **4** only, signals belonging to proton H-2 are broadened, and in the <sup>1</sup>H NMR spectrum of **5,** almost all proton signals H-2, H-3 and H-4 are broadened. All broadened proton signals are shifted to the low field. This observation indicated that protons described as major must exchange positions with protons signed as minor. It is a consequence of the slow proton exchange of **4** and **5** within the NMR time scale. Similarly, <sup>13</sup>C signals of **4** and **5** (Figure 3 and Figure S2) exhibited very interesting changes. At ambient temperature, signals belonging to carbons C-8, C-2, C-8a, C-4a and C-5 of **4** are doubled. We expected a similar pattern for the complex **5**, but the very low solubility of the complex in DMSO-d<sup>6</sup> caused the minor form signals to not be well distinguished (Figure S2). Only the signals of carbons C-4, C-6 and C-4a are visibly doubled.

Because of the different structures of all studied complexes, complexes **4** and **5** are fluxional and are characterized by the proton position exchange without the dissociation of the complex. If the process occurred with dissociation, the process should be associated with the presence of free ligand peaks in NMR spectra, which is not the case.

The stability of the complexes **1**–**5** in DMSO-d<sup>6</sup> solution was tested using <sup>1</sup>H NMR time-dependent spectra to evaluate their suitability for biological testing.

As shown in Figure 4 and Figure S3, at ambient temperature, the time-dependent <sup>1</sup>H NMR spectra of complexes **1** and **2** show peaks corresponding to the one set of chemical shifts, an indication of the presence of complexes that are structurally the same for 72 h. However, as shown in Figure 4 and Figure S3, the <sup>1</sup>H NMR spectra after 48 h show new peaks at *δ*<sup>H</sup> 10.20 (s, OH), 9.03 (dd, J 4.7, 1.7, H-2), 8.31 (dd, J 7.9, 1.7, H-4) and 7.86 (dd, J 7.9, 4.7, H-3). Whereas the signals belonging to protons H-6 and H-7 are not detectable. By comparing chemical shifts with free ligand shifts (1H NMR (600 MHz, DMSO-d6): *δ*<sup>H</sup> 10.18 (s, OH), 8.94 (dd, J 4.1, 1.6, H-2), 8.48 (dd, J 8.6, 1.6, H-4), 7.71 (dd, J 8.6, 4.1, H-3), 7.60 (d, J 8.3, H-6) and 7.08 (d, J 8.3, H-7)), we cannot completely exclude complex decomposition. The ratio of the complex and newly formed structure is 90:1 after 48 h and 70:1 after 96 h for **1** and 56:1 after 48 h and 46:1 after 72 h for **2** (based on integral values

of H-4 signals). Nevertheless, we believe that the low concentration of the newly formed structures will not significantly affect the results of biological activity tests. Contrary to our findings, Zhang et al. [27] prepared tetranuclear [Zn4(8-HQ)6Ac2] (Ac = acetate) and [Zn4(MeQ)6Ac2] (MeQ = 2-methyl-8-hydroxylquinoline) complexes, which are very similar to complexes **1** and **2** in our work, and tested them for their stability in the physiological conditions within 48 h. Based on the results of UV-Vis spectroscopy, they concluded that both complexes were stable.

**Figure 3.** <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 **4**.

**Figure 4.** Time-dependent <sup>1</sup>H NMR (600 MHz, DMSO-d6) spectra of complex **1**.

As shown in Figure S3, the <sup>1</sup>H NMR spectrum measured the after dissolving of the complex in DMSO-d<sup>6</sup> shows broad peaks corresponding to complex **3**. After 24 h, spectrum becomes somewhat better resolved but remains broad. It can be confirmed that all time-dependent spectra (24–72 h) are consistent with the structure of complex **3** observed immediately after dissolving. Likewise, complexes **4** (Figure S5) and **5** (Figure S6) are stable in the DMSO-d<sup>6</sup> solution for 72 h. Minor peaks at δ<sup>H</sup> 8.75 and 8.29 ppm appeared in the spectra (Figure S6) of the complex **5** and can be attributed to the dynamics rather than the decomposition of complex **5**.

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

The [Zn4Cl2(ClQ)6]·2DMF (**1**) and [Zn4Cl2(ClQ)6(H2O)2]·4DMF (**2**) complexes are similar tetranuclear molecular compounds with a complicated type of structure. They crystallize in the triclinic *P*1 space group. In both complexes, there are two pairs of crystallographically independent zinc atoms. Zn1 atoms are pentacoordinated in both structures by N2 and O2 atoms of one chelate ClQ ligand, with O2 atom being a bridge between Zn1 and Zn2 atoms. Other two coordination places are occupied by bridging O1 and O3 atoms of the other two ClQ chelate-bridging ligands, while the fifth place is occupied by the chloride ligand, Cl1. Zn2 atoms are hexacoordinated in both structures; however, coordination spheres around the Zn2 atoms in both structures are different. In both structures, two chelate-bridging ClQ ligands coordinate to the Zn2 atoms through N1 and N3, and O1 and O3 atoms, which are bridges to the Zn1 atoms. The last two coordination places in **1** are occupied by two bridging oxygen atoms of another two ClQ ligands (Figure 5), while only one place is occupied by the bridging oxygen atom of another ClQ ligand in **2** and the last coordination place is occupied by the O4 atom of a water molecule (Figure 5). In summary, there are two tridentate and four bidentate oxygen atoms of the six ClQ ligands in **1** and each Zn1-Zn2 pair is bridged by two oxygen atoms of two different ClQ ligands. Thus, there are four double-oxygen bridges between Zn1 and Zn2 atoms in **1** (Figure 5). On the other hand, all six ClQ oxygen atoms are bidentate in **2** and thus there are only two double-oxygen bridges between Zn1 and Zn2 atoms, while the remaining two bridges are single-oxygen bridges only.

Based on bond angles (Table 2), the shapes of coordination polyhedra around Zn2 atoms in **1** and **2** are distorted octahedrally. On the other hand, Zn1 atoms are coordinated in a trigonal bipyramidal or square pyramidal shape, respectively, as suggested by the τ parameter [28] and program SHAPE [29] (Table S2).

Bond distances around the zinc atoms in both structures are listed in Table 2. It can be seen that the Zn-O and Zn-N distances within the chelate rings are in the range of 2.0789(12)–2.1525(16) Å, while distances between Zn atoms and bridging oxygen atoms are shorter, 2.0134(12)–2.0463(13) Å, except for the Zn1<sup>i</sup> -O2 bond (2.2779(13) Å; i = −*x* + 1, −*y* + 1, −*z* + 1) in the structure of **1** which length is comparable to Zn1-Cl1 in **1** and **2** (2.2766(5) and 2.3149(5) Å, respectively). In addition, a water molecule in **2** is coordinated with a Zn2-O4 bond length being 2.2044(13) Å. All mentioned distances and angles are similar to those described in the literature for other tetranuclear zinc complexes [27,30].

Outside the tetranuclear complex species there are two or four solvated molecules of DMF in **1** and **2**, respectively, which are joined with the complexes by hydrogen bonds stabilizing both structures. These and other hydrogen bonds present in the structure of both complexes are gathered in Table 3 and are shown in Figures 6 and 7. Due to these hydrogen bonds, a layered structure parallel with the (011) plane in **1** and a chain-like structure along the *c* axis in **2** is formed.

**Figure 5.** Molecular structure of **1** (up) and **2** (down). Solvated DMF molecules are omitted, symmetry code: i = −*x*, −*y* + 1, −*z* + 1. Displacement ellipsoids are drawn at 50% probability.

The further stabilization of both structures arises from π-π interactions between aromatic rings. Data characterizing π-π interactions are given in Table S3.

Complex **3** crystallizes in the monoclinic space group *I*2/*c*. In its structure, there is a binuclear [Zn2(ClQ)3(HClQ)3] + cation (Figure 8, Table 4) in which both Zn atoms are hexacoordinated by three N,O-chelate bonded ligands. Three of them (ClQ) are anionic with deprotonated hydroxyl groups, while remaining three ligands (HClQ) contain protonated hydroxyl groups and are neutral. The hydrogen atoms of these hydroxyl groups are involved in strong hydrogen bonds, which link the two mononuclear Zn complex units into the binuclear cation. One of the hydrogen atoms lies on a two-fold rotation axis and is thus shared equally by O1 and O1<sup>i</sup> oxygen atoms (i = <sup>−</sup>*<sup>x</sup>* + 1, *<sup>y</sup>*, <sup>−</sup>*<sup>z</sup>* + 1.5). The remaining two hydrogen atoms involved in hydrogen bonds are shared unequally, and corresponding covalent bonds are elongated and are thus only slightly shorter than hydrogen bonds (Table 5). The positive charge of the binuclear cation is balanced by a triiodide anion. Selected bond lengths and angles are gathered in Table 4 and are similar to those in **1**, **2** and **5** (below), as well as in other similar zinc complexes [31,32].


**Table 2.** Selected bond distances and angles [Å, ◦ ] for **1** and **2**.

Symmetry code: <sup>i</sup> <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, <sup>−</sup>*<sup>y</sup>* + 1, <sup>−</sup>*<sup>z</sup>* + 1. <sup>a</sup> Atom O4<sup>i</sup> from **2**.



Symmetry codes: <sup>i</sup> = *x* + 1, *y*, *z*; ii <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, <sup>−</sup>*<sup>y</sup>* + 1, <sup>−</sup>*<sup>z</sup>* (**1**); <sup>i</sup> <sup>=</sup> *<sup>x</sup>* + 1, *<sup>y</sup>*, *<sup>z</sup>*; iii <sup>=</sup> *<sup>x</sup>*−1, *<sup>y</sup>*, *<sup>z</sup>*; iv <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, <sup>−</sup>*<sup>y</sup>* + 1, <sup>−</sup>*<sup>z</sup>* + 1 (**2**).

**Figure 6.** Part of the two-dimensional structure of **1** viewed along the *a* axis with hydrogen bonds (red dashed lines). Only hydrogen atoms involved in the hydrogen bonds are shown for clarity.

**Figure 7.** Part of the one-dimensional structure of **2** viewed along the *c* axis with hydrogen bonds (red dashed lines). Only hydrogen atoms involved in the hydrogen bonds are shown for clarity.

**Figure 8.** Molecular structure of **3**, symmetry code: i = −*x* + 1, *y*, −*z* + 1.5. Displacement ellipsoids are drawn at 50% probability.


**Table 4.** Selected bond distances and angles [Å, ◦ ] for **3**.

**Table 5.** Hydrogen bond interactions [Å, ◦ ] for **3**.


Symmetry code: <sup>i</sup> <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, *<sup>y</sup>*, <sup>−</sup>*<sup>z</sup>* + 1.5.

The further stabilization of the structure arises from π-π interactions between parallel aromatic rings of neighboring complexes. Due to these interactions a *zig-zag* chain parallel with the *a* axis is formed (Figure S7). Data characterizing π-π interactions are given in Table S4.

The complexes [Zn2(dClQ)2(H2O)6(SO4)] (**4**) and [Zn2(dBrQ)2(H2O)6(SO4)] (**5**) crystallize in the triclinic *P*1 space group and their structures are very similar. They are formed by binuclear complexes in which two Zn-species are bridged by a sulphate group (Figure 9). Both Zn atoms in the binuclear complexes are hexacoordinated by a corresponding N,Obidentate chelate XQ ligand, three oxygen atoms of water molecules and one oxygen atom of the sulphate group. Interestingly, in **4** there are four crystallographically independent binuclear complexes. However, due to the extremely small crystals of **4**, their diffraction power was low, and thus the quality of the obtained data enabled us to present only an isotropic model of its structure without hydrogen atoms. Therefore, we discuss neither bond distances and angles nor nonbonding interactions in this complex.

**Figure 9.** Molecular structure of **5**. Displacement ellipsoids are drawn at 50% probability.

Bond distances around both Zn atoms in **5** are normal (Table 6) and are close to the bond lengths observed in the above-described complexes and also in other Zn–HXQ complexes described in the literature [20,31,32]. Coordination polyhedra around Zn1 and Zn2 atoms are distorted octahedra, as represented by N-Zn-O and O-Zn-O *trans*-angles spanning from 165.6(3) to 173.8(3)◦ , while *cis*-angles are in a range 79.1(3) to 97.8(3)◦ . The shape of the coordination polyhedra is also confirmed by octahedral distortion parameter Σ and the results of the program SHAPE (Table S2).


**Table 6.** Selected bond distances and angles [Å, ◦ ] for **5**.

Water molecules are involved in hydrogen bonds (Table 7), forming a hydrophilic layer parallel with the (001) plane (Figure 10). On the borders of this layer there are hydrophobic dBrQ ligands, and between neighboring ligands π-π interactions stabilize the two-dimensional structure of **5** (Figure 10, Table S5).

**Table 7.** Hydrogen bond interactions [Å, ◦ ] in **5**.


Symmetry codes: <sup>i</sup> <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 2, <sup>−</sup>*<sup>y</sup>* + 1, <sup>−</sup>*<sup>z</sup>* + 1; ii <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, <sup>−</sup>*<sup>y</sup>* + 1, <sup>−</sup>*<sup>z</sup>* + 1; iii <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 2, <sup>−</sup>*<sup>y</sup>* + 2, <sup>−</sup>*<sup>z</sup>* + 1; iv <sup>=</sup> <sup>−</sup>*<sup>x</sup>* + 1, −*y* + 2, −*z* + 1.

**Figure 10.** Part of the two-dimensional structure of **5** viewed along the *b* axis with hydrogen bonds (red dashed lines) and π-π interactions (black dashed lines).

### *2.5. The DNA Binding Potency*

The UV-vis spectrometry is one of the basic techniques for analyzing small molecule– DNA interactions. The UV-vis titrations were realized at a fixed concentration of investigated zinc complexes with HClQ ligand (**1**–**3**) and varying concentrations of calf thymus DNA (ctDNA). The absorbance maxima of complexes **1**–**3** show reduced absorbance intensity (hypochromism) upon titration with ctDNA (Figures S8 and S9 and Figure 11). Hypochromic effect is noticed when molecules bind into DNA-supporting helix stabilization by the insertion of the flat aromatic part between base pairs [2]. This determined hypochromicity is associated with the intercalative binding mode of studied complexes [33]. The most obvious hypochromicity was recorded for complex **3** (about 46%) (Table 8). This means that the dinuclear complex **3,** which shows a promising cytotoxic effect (see Section 2.7), binds better into DNA compared to the tetranuclear complexes **1** and **2**.

**Figure 11.** UV-vis spectrum of complex **<sup>3</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 **3**.


**Table 8.** Spectral binding parameters of complexes **1**–**3**.

We tried to examine the actual binding mode of the Zn(II) complexes with DNA by competitive fluorometric dye displacement assay with Ethidium bromide (EB) also. EB is often used as a DNA intercalating agent, which displays maximum emission at about 602 nm upon binding to double helical DNA [34]. The fluorescence emission spectra of the DNA-EB system was measured from 565 nm to 700 nm with incremental amounts of complexes **1**–**3** (Figures S10 and S11 and Figure 12).

**Figure 12.** Fluorescence spectrum of DNA–EB complex in the absence (black line) and presence of complex **3**. Inset: the corresponding Stern–Volmer plot for quenching process of EB by **3**.

Table 8 shows Stern–Volmer quenching constants (*Ksv),* which are estimated by the linear regression of the *Fo/F* against quencher concentration. Obtained *Ksv* are in the range from 3.4 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>M</sup>−<sup>1</sup> to 1.06 <sup>×</sup> <sup>10</sup><sup>4</sup> <sup>M</sup>−<sup>1</sup> . Similar *Ksv* constants were found for complexes [Zn(bpy)(Gly)]NO<sup>3</sup> and [Zn(phen)(Gly)]NO<sup>3</sup> [35]. Since the calculated *Ksv* values are quite low, we assume that the complexes are only weak intercalators.

### *2.6. BSA Interaction Study*

The bovine serum albumin (BSA) works as a model protein to explore drug–protein interactions due to its structural similarities with human serum albumin (HSA). In vivo, the BSA serves as a transporter for biologically efficient drugs and also for endogenous molecules. The binding of biologically effective drugs to serum albumins is an important criterion for pharmacokinetics [36].

The changes noticed in the fluorescence emission spectra of BSA with various concentrations of Zn(II) complexes are presented in Figures S12 and S13 and Figure 13.

**Figure 13.** Fluorescence quenching spectra of BSA in presence of complex **3**. Inset: the corresponding Stern–Volmer plot for **3** at 25 ◦C.

Upon the addition of **1**–**3,** we have observed a substantial quenching of the fluorescence intensity of BSA at 348 nm. The decrease in fluorescence intensity indicates the formation of certain complexes between the Zn(II) complexes and BSA. The fluorescence quenching is also described by the Stern–Volmer relationship. The values of the parameters *Ksv* for BSA binding are listed in Table 8. The highest *<sup>K</sup>sv* value (5.44 <sup>×</sup> <sup>10</sup><sup>6</sup> <sup>M</sup>−<sup>1</sup> ) was found for the complex **3**, which shows the highest cytotoxicity against all cell lines. All estimated *Ksv* values were about 100 times higher than that found by Butkus et al. for small zinc complexes [37].

### *2.7. Antiproliferative Activity*

The robust screening test was used to analyze antiproliferative activity of the zinc complexes in various cancer cell lines (Table 9). As the data clearly showed, based on IC<sup>50</sup> values, the most cytotoxic complexes were **3** and **4** but unfortunately with poor selectivity towards normal kidney fibroblasts. Good cytotoxic potential and higher selectivity was shown by complex **5** on all cancer cell lines except HeLa. Complexes **1** and **2** showed the lowest cytotoxic IC<sup>50</sup> values on MCF-7 and HCT116 cells and average IC<sup>50</sup> concentration on other cell lines. As can be seen from the IC<sup>50</sup> values, **3** and **4** exhibited higher cytotoxic effect than cisplatin against all cancer cell lines except HCT116. In general, HeLa cells were most resistant to the tested complexes. The ligands used in synthesis showed cell-dependent cytotoxicity in the range of 5 to 103 µM concentration. In general, binuclear complexes **3**–**5** showed higher cytotoxic activity compared to their free ligands against all cancer cell lines. Tetranuclear complexes **1** and **2** showed the lowest efficiency from the complexes and ligands, probably due to their bigger size preventing their entrance to the cells. As the results showed, changing the structure of the complexes did not lead to an improvement in their cytotoxicity or selectivity in comparison with our mononuclear zinc complexes [20].

**Table 9.** IC<sup>50</sup> values [µM] for complexes **1**–**5**, corresponding ligands and cisplatin in the various cell lines.


### *2.8. Antimicrobial Activity*

The antibacterial activity of the prepared complexes was tested against Gram-positive (*S. aureus*) and Gram-negative (*E. coli*) bacteria. The RIZD (percentage of relative inhibition zone diameter) was performed. Only **5** showed in RIZD test inhibition against Grampositive bacteria *S. aureus*. The inhibition was 66.97% (Table 10), whereas the inhibition of gentamicin sulphate as positive control was 100%. Oher complexes are not suitable for antibacterial activity in concentrations of 33.6 µM. Concentration was the same as gentamicin sulphate. In the case of **2** and **3**, we observed problems with dissolution and after a few minutes it started flocculation.

**Table 10.** Antibacterial activity of tested complexes. RIZD (%) means percentage of relative inhibition zone diameter. All complexes were tested against *E. coli* and *S. aureus* in three replicates (n = 3, ±SD). NA means no activity.


### **3. Materials and Methods**

### *3.1. Materials and Chemicals*

Reagents were obtained from the following commercial sources: HClQ—95% from Sigma-Aldrich (Darmstadt, Germany); HdClQ—99%, HdBrQ—98%, and N,N-dimethylform amide—99% from Alfa Aesar (Kandel, Germany); ethanol—96% from BGV (Hniezdne, Slovakia), zinc(II) iodide—98% from Fisher Chemical (Loughborough, UK); zinc sulphate heptahydrate— p.a. from Lachema (Neratovice, Czech Republic); KOH—p.a. from ITES (Vranov, Slovakia); methanol—p.a.; and zinc(II) chloride—98% from Centralchem (Bratislava, Slovakia). All mentioned chemicals were used as received.

### *3.2. Syntheses*
