Synthesis, Structural Characterization and Biological Activity Evaluation of Novel Cu(II) Complexes with 3-(trifluoromethyl)phenylthiourea Derivatives

Copper complexes with 1,3-disubstituted thiourea derivatives, all containing 3-(trifluoromethyl)phenyl tail and 1-alkyl/halogen-phenyl substituent, were synthesized. The experimental spectroscopic studies and theoretical calculation revealed that two ligands coordinate to Cu(II) in a bidentate fashion via thiocarbonyl S and deprotonated N atoms of thiourea moiety. Such monomers are characteristic of alkylphenylthiourea complexes, whereas the formation of a sandwich-type dimer is observed for halogeno derivatives. For the first time, the structural identifications of CuN2S2-based complexes using experimental and theoretical X-ray absorption near edge structure are demonstrated. The dimeric halogeno derivatives showed higher antimicrobial activity in comparison with alkylphenylthiourea complexes. The Cu(II) complex of 1-(4-chloro-3-nitrophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea was active against 19 strains of methicillin-resistant Staphylococci (MIC = 2 µg/mL). This derivative acted as a dual inhibitor of DNA gyrase and topoisomerase IV isolated from Staphylococcus aureus. Additionally, complexes of halogenphenylthiourea strongly inhibited the growth of mycobacteria isolated from tuberculosis patients, even fourfold stronger than the reference isoniazid. The complexes exerted weak to moderate antitumor activity (towards SW480, SW620, and PC3) being non-toxic towards normal HaCaT cells.


Introduction
Thiourea derivatives are widely studied and applied as crucial reagents in organic synthesis, and as bioactive agents and drugs in medicine [1,2]. There is also a growing interest in pharmaceutical applications of metal complexes with thiourea ligands, notably copper coordination compounds [3][4][5]. Regarding copper complexes with ligands containing thiourea moiety, the ones containing thiosemicarbazone (NH 2 = NH-C(=S)-NH 2 ) or thiosemicarbazide (NH 2 -NH-C(=S)-NH 2 ) derivatives have been the most widely studied for their use as anticancer chemotherapeutics [6,7], antimicrobial drugs [5,8], neuroprotective agent in Alzheimer's disease [9,10], and radiopharmaceuticals [11,12]. The broad spectrum of bioactivity of these compounds can be correlated with copper's ability to participate in redox reactions, and to coordinate ligands in 3D configuration, thus enabling interaction with particular molecular targets. The thiourea moiety can bind Cu cations in several ways, e.g., act as monodentate ligands via S atom in mononuclear

Spectroscopic Characterization
The complexation reactions were followed by the infrared and UV-Vis spectroscopies. Results of the analysis of IR and UV-Vis spectra of complexes revealed the oxidation state of copper, symmetry of the metal-ligand interactions, and indicated the atoms coordinating to the metallic center. The comparative XANES analysis confirmed the oxidation state of the metallic center, whereas EXAFS analysis gave us information about the radial distributions of atoms around Cu(II). These experimental studies completed with DFT and XANES calculations allowed us to build and optimize structural models of new coordination compounds. The described studies are presented below.

ATR-IR Studies
The attenuated total reflection Fourier transform-infrared spectra of five copper(II) complexes with 1,3-disubstitued thioureas (Cu1-Cu5, Figure 1) and their parent ligands (1-5, Figure 1) were recorded over the range 4000-400 cm −1 . The fingerprint regions (1000-400 cm −1 ) have been compared for each pair of the organic ligand-Cu(II) complex. The notable differences in this part of the spectra confirmed that new compounds (Cu1-Cu5) have been formed.
The region between 3400 and 2800 cm −1 in organic ligand spectra contains two broad features with several maxima corresponding to the N-H and C-H stretching vibrations ( Figure 2a). The high intensity of these bands is due to the presence of hydrogen bonding between C/N-H groups and electronegative atoms such as S, N, Cl, and Br. In the spectra of the copper(II) complexes (Figure 2b), these two broad features merge into one wide band, extending from 3500 to 2600 cm −1 . This widening indicates the presence of water molecules in Cu1-Cu5, which is consistent with the results of the elemental analysis. Moreover, a decrease in the intensity of the bands >3200 cm −1 and their extension are observed in the high-energy part of the spectra of complex Cu1-Cu5. This confirms the partial deprotonation of the N-H groups of the ligand molecules as a result of the complexation reaction.

ATR-IR Studies
The attenuated total reflection Fourier transform-infrared spectra of five copper(II) complexes with 1,3-disubstitued thioureas (Cu1-Cu5, Figure 1) and their parent ligands (1-5, Figure 1) were recorded over the range 4000-400 cm −1 . The fingerprint regions (1000-400 cm −1 ) have been compared for each pair of the organic ligand-Cu(II) complex. The notable differences in this part of the spectra confirmed that new compounds (Cu1-Cu5) have been formed.
The region between 3400 and 2800 cm −1 in organic ligand spectra contains two broad features with several maxima corresponding to the N-H and C-H stretching vibrations (Figure 2a). The high intensity of these bands is due to the presence of hydrogen bonding between C/N-H groups and electronegative atoms such as S, N, Cl, and Br. In the spectra of the copper(II) complexes (Figure 2b), these two broad features merge into one wide band, extending from 3500 to 2600 cm −1 . This widening indicates the presence of water molecules in Cu1-Cu5, which is consistent with the results of the elemental analysis. Moreover, a decrease in the intensity of the bands >3200 cm −1 and their extension are observed in the high-energy part of the spectra of complex Cu1-Cu5. This confirms the partial deprotonation of the N-H groups of the ligand molecules as a result of the complexation reaction. The thiourea moiety in all compounds yields two absorption bands corresponding to the C = S stretching vibrations (Table 1). These bands are observed in the regions 1351-1342 cm −1 and 858-844 cm −1 in spectra of the organic ligands (1)(2)(3)(4)(5). After complexation, their position was shifted to the lower values, around 1300 cm −1 and 764-754 cm −1 (Table  1), respectively, proving the coordination of the sulfur atom of the ligands [23,24]. The thiourea moiety in all compounds yields two absorption bands corresponding to the C = S stretching vibrations (Table 1). These bands are observed in the regions 1351-1342 cm −1 and 858-844 cm −1 in spectra of the organic ligands (1)(2)(3)(4)(5). After complexation, their position was shifted to the lower values, around 1300 cm −1 and 764-754 cm −1 (Table 1), respectively, proving the coordination of the sulfur atom of the ligands [23,24]. Moreover, the several bands in the region of 1600-1400 cm −1 (Figure 3) in free ligands as well as their Cu(II) complexes indicate the presence of C-C and C-N bonds within their molecules. Interestingly, in the spectra of Cu1-Cu5, this range is widened to 1700 cm −1 . This bandwidth extension was observed for the previous series of analogous complexes [17]. The calculated harmonics using DFT revealed that this is a consequence of increased intensities of antisymmetric ring C-C stretching modes within the molecules of complexes.

UV-Vis Analysis
The electronic spectra of thiourea complexes (Cu1-Cu5) are presented in Figu Around 250 nm there is a strong band corresponding to the transition of the organic of molecules. A ligand-to-metal charge transfer (LMCT) band is found in the regi 400-420 nm. This band is tailing into 600 nm, which allows us to assign it to S→C transition within the complexes [18,23,24]. The broad feature between 700 and 140 corresponds to d-d transitions. Within the studied complexes, there are no differences in the intensities of d-d bands (Figure 4), which are correlated with differe in the geometry around the metal cation. The low intensity of the d-d band indicates copper cations are coordinated in a centrosymmetric fashion, whereas the higher inte suggests the noncentrosymmetric geometry [18].

UV-Vis Analysis
The electronic spectra of thiourea complexes (Cu1-Cu5) are presented in Figure 4. Around 250 nm there is a strong band corresponding to the transition of the organic part of molecules. A ligand-to-metal charge transfer (LMCT) band is found in the region of 400-420 nm. This band is tailing into 600 nm, which allows us to assign it to S→Cu(II) transition within the complexes [18,23,24]. The broad feature between 700 and 1400 nm corresponds to d-d transitions. Within the studied complexes, there are notable differences in the intensities of d-d bands (Figure 4), which are correlated with differences in the geometry around the metal cation. The low intensity of the d-d band indicates that copper cations are coordinated in a centrosymmetric fashion, whereas the higher intensity suggests the noncentrosymmetric geometry [18]. 400-420 nm. This band is tailing into 600 nm, which allows us to assign it to S→Cu(II) transition within the complexes [18,23,24]. The broad feature between 700 and 1400 nm corresponds to d-d transitions. Within the studied complexes, there are notable differences in the intensities of d-d bands (Figure 4), which are correlated with differences in the geometry around the metal cation. The low intensity of the d-d band indicates that copper cations are coordinated in a centrosymmetric fashion, whereas the higher intensity suggests the noncentrosymmetric geometry [18].

XANES Comparative Analysis
The copper oxidation state in the synthesized compounds was determined by XANES spectra comparative analysis. Therefore, we measured the Cu K-edge spectra of the complexes as well as two reference oxides: Cu2O and CuO. The copper K-edge corresponds to dipole-allowed 1s-4p transition and shifts to the higher energy values with increasing metal oxidation state. Figure 5 presents the comparison of Cu K-edge spectra of two complexes: Cu2 (representative of alkylphenyltiourea derivatives) and Cu4 (representative of halogenphenylthiourea complexes), and the reference oxides. The absorption edge for Cu2O is at 8979 eV and for CuO at 8982 eV. The energy positions and

XANES Comparative Analysis
The copper oxidation state in the synthesized compounds was determined by XANES spectra comparative analysis. Therefore, we measured the Cu K-edge spectra of the complexes as well as two reference oxides: Cu 2 O and CuO. The copper K-edge corresponds to dipole-allowed 1s-4p transition and shifts to the higher energy values with increasing metal oxidation state. Figure 5 presents the comparison of Cu K-edge spectra of two complexes: Cu2 (representative of alkylphenyltiourea derivatives) and Cu4 (representative of halogenphenylthiourea complexes), and the reference oxides. The absorption edge for Cu 2 O is at 8979 eV and for CuO at 8982 eV. The energy positions and the shape of spectra of thiourea complexes are closer to CuO ( Figure 5), which proves that analyzed complexes contain copper at a +2 oxidation state.

EXAFS Studies
Extended X-ray Absorption Fine Structure spectra were collected for all complexes: Cu1-Cu5. The comparison of Fourier-transformed experimental EXAFS oscillations ( Figure 6), especially the region between 2.5 and 3 Å, indicates structural differences within the studied compounds. Therefore, the complexes were divided into two groups: A (Cu1-Cu3) without any peak between 2.5 and 3 Å and group B (Cu4, Cu5) with a peak in this region.

EXAFS Studies
Extended X-ray Absorption Fine Structure spectra were collected for all complexes: Cu1-Cu5. The comparison of Fourier-transformed experimental EXAFS oscillations ( Figure 6), especially the region between 2.5 and 3 Å, indicates structural differences within the studied compounds. Therefore, the complexes were divided into two groups: A (Cu1-Cu3) without any peak between 2.5 and 3 Å and group B (Cu4, Cu5) with a peak in this region.
( Figure 6), especially the region between 2.5 and 3 Å, in within the studied compounds. Therefore, the complexes A (Cu1-Cu3) without any peak between 2.5 and 3 Å and g in this region. The EXAFS analysis was performed in two steps. In models were checked. In this case, only single scattering p Data obtained from the preliminary fitting (such as dista atoms) pointed out four similar models for group A and models were checked and refined by DFT and XANES c Finally, the EXAFS analysis of complexes was performed multiple scattering paths were also used in the final fits. EXAFS oscillations for Cu1-representative of group A, an B are presented in Figure 7 (fits for all complexes are pre mentary Materials). The EXAFS analysis was performed in two steps. In the first step, several different models were checked. In this case, only single scattering paths were taken into account. Data obtained from the preliminary fitting (such as distances, and type and number of atoms) pointed out four similar models for group A and two for group B. Then, these models were checked and refined by DFT and XANES calculations (see next section). Finally, the EXAFS analysis of complexes was performed on the refined models and the multiple scattering paths were also used in the final fits. The best fits together with FT EXAFS oscillations for Cu1-representative of group A, and for Cu5-belonging to group B are presented in Figure 7 (fits for all complexes are presented in Figure S1 in Supplementary Materials). The study of complexes within group A indicates four atoms within the first coordination sphere (Table 2), two N and two S atoms. The Cu-N/S bond length is in the range 1.93-1.95 Å for nitrogen and 2.23-2.25 Å for sulfur atoms. Moreover, at a distance of around 2.5 Å, two carbon atoms were identified. The study of complexes within group A indicates four atoms within the first coordination sphere (Table 2), two N and two S atoms. The Cu-N/S bond length is in the range 1.93-1.95 Å for nitrogen and 2.23-2.25 Å for sulfur atoms. Moreover, at a distance of around 2.5 Å, two carbon atoms were identified. Table 2. The distance from central cation to next atom R, number of atoms n, Debye-Waller factor σ 2 and R-factor of the fit evaluated during the final EXAFS fitting procedure. For clarity, only the main parameters for the closest neighbors are presented.

Complex
Path For two other complexes (Cu4, Cu5), belonging to group B, the first coordination sphere is formed by two N and two S atoms, being at similar distances from Cu(II) cation as in the case of the previous group of compounds. However, in the next coordination sphere, the second copper ion at a distance of around 2.8 Å, as well as one S atom above 2.6 Å, were identified (Table 2). These results suggest a more complex dimeric structure of compounds belonging to group B.

Structural Models of Complexes-DFT and XANES Calculations
On the basis of the spectroscopic data presented above, four possible structural models for complexes within group A ( Figure 8) and two dinuclear models for complexes belonging to group B ( Figure 9) were constructed and optimized. All relevant DFT structures are included in Supplementary Materials.
The structural models of complex Cu1 (representative for group A) consist of two thiourea ligands coordinating via thiocarbonyl S and deprotonated N atoms to the Cu(II) cation, see Figure 8. These models differ in symmetry (C 2 or C i ) and the N atom binds to the metallic center. The coordination through the N(f) atom attached to the trifluoromethylphenyl substituent (Figure 8b,d) is more likely, as such structures are over 40 kJ/mol more stable (Table 3)  thiourea ligands coordinating via thiocarbonyl S and deprotonated N atoms to the Cu(II cation, see Figure 8. These models differ in symmetry (C2 or Ci) and the N atom binds t the metallic center. The coordination through the N(f) atom attached to th trifluoromethylphenyl substituent (Figure 8b,d) is more likely, as such structures are ove 40 kJ/mol more stable (Table 3)  All these models have Cu-N and Cu-S distances being about 2.0 and 2.3 Å respectively (Table 3), which are in very good agreement with EXAFS fitting for the firs coordination sphere (Table 2). Furthermore, the calculated Cu-C distances (around 2.6 Å coincide with the experiment ones (Table 2), which additionally confirms the validity o the assumed models.  Taking into account the EXAFS results for complexes belonging to group B, sandwich dimer was found to be the most plausible structure. Two structural models o Cu5 differing in symmetry of monomers (C2 or Ci) were constructed and optimized ( Figure 9). The calculation indicated that dimers composed of noncentrosymmetri monomers turned out to be more energetically stable (ΔE = 24 kJ/mol). The distanc between both mononuclear complexes in DFT-optimized dimeric models is much large than the values obtained from EXAFS data, i.e., equilibrium Cu-Cu distance amounts to 3.38 Å. However, if the dimer was optimized with a Cu-Cu distance fixed to the EXAF value (2.68 Å), the resulting structure was only 10 kJ/mol less stable than the fully optimized model. This small energy difference is of the order of intermolecular interaction energy in the condensed phase, which implies that in solutions or solids such mor compact dimers can be stabilized.
(a) (b) Figure 9. The structural dimeric models for representative Cu5 complex composed of (a noncentrosymmetric and (b) centrosymmetric monomers. The dimer as a whole belongs to the C group.
In contrast to EXAFS, the shape of a XANES spectrum strongly depends on the angle between the neighboring atoms. The XANES spectra were measured for all complexes Then, for two representative complexes from groups A and B (namely, for compound Cu1 and Cu5), the XANES spectra were calculated. The DFT-optimized structures wer Figure 9. The structural dimeric models for representative Cu5 complex composed of (a) noncentrosymmetric and (b) centrosymmetric monomers. The dimer as a whole belongs to the C i group. All these models have Cu-N and Cu-S distances being about 2.0 and 2.3 Å, respectively (Table 3), which are in very good agreement with EXAFS fitting for the first coordination sphere (Table 2). Furthermore, the calculated Cu-C distances (around 2.6 Å) coincide with the experiment ones (Table 2), which additionally confirms the validity of the assumed models.
Taking into account the EXAFS results for complexes belonging to group B, a sandwich dimer was found to be the most plausible structure. Two structural models of Cu5 differing in symmetry of monomers (C 2 or C i ) were constructed and optimized ( Figure 9). The calculation indicated that dimers composed of noncentrosymmetric monomers turned out to be more energetically stable (∆E = 24 kJ/mol). The distance between both mononuclear complexes in DFT-optimized dimeric models is much larger than the values obtained from EXAFS data, i.e., equilibrium Cu-Cu distance amounts to 3.38 Å. However, if the dimer was optimized with a Cu-Cu distance fixed to the EXAFS value (2.68 Å), the resulting structure was only 10 kJ/mol less stable than the fully optimized model. This small energy difference is of the order of intermolecular interaction energy in the condensed phase, which implies that in solutions or solids such more compact dimers can be stabilized.
In contrast to EXAFS, the shape of a XANES spectrum strongly depends on the angles between the neighboring atoms. The XANES spectra were measured for all complexes. Then, for two representative complexes from groups A and B (namely, for compounds Cu1 and Cu5), the XANES spectra were calculated. The DFT-optimized structures were used as inputs; however, the distances between the absorbing and neighboring atoms were adjusted to the experimental values obtained from the EXAFS analysis. Comparison with the experimental XANES spectra ( Figure 10) shows that the main features are reproduced confirming the validity of the proposed models.

Biological Evaluation
The newly synthesized complexes Cu1-Cu5 were evaluated against a panel of standard and clinical bacterial strains, as well as towards mycobacteria. To establish the mechanism of bactericidal action, one complex was examined in both the topoisomerase IV decatenation assay and the DNA gyrase supercoiling assay. In addition, the cytotoxic Figure 10. Calculated XANES spectra for the most energy-stable optimized models vs. experimental data for (a) Cu1-representative for monomeric alkylphenyl complexes (left) and for (b) Cu5-belonging to the dimeric complexes with halogenphenyl moiety.

Biological Evaluation
The newly synthesized complexes Cu1-Cu5 were evaluated against a panel of standard and clinical bacterial strains, as well as towards mycobacteria. To establish the mechanism of bactericidal action, one complex was examined in both the topoisomerase IV decatenation assay and the DNA gyrase supercoiling assay. In addition, the cytotoxic effect of thiourea derivatives on different types of cancer cells was determined.

In Vitro Antimicrobial Activity
The antimicrobial properties of title thiourea coordinates were examined against a broad series of microorganisms, using the method described before [16,17]. The investigation of standard bacterial strains revealed that the antibacterial potency varied from weak (compounds Cu1-Cu3) to considerable (for complex Cu4) and high (for Cu5) ( Table 4). Alkylphenylthiourea complexes Cu1-Cu3 inhibited the growth of staphylococcal isolates at the level of 16-64 µg/mL. However, derivatives with halogenphenyl moiety exerted markedly stronger antimicrobial activity, with MIC values from 8 µg/mL (complex Cu4) to 4 µg/mL (compound Cu5). The biological studies of (trifluoromethyl)phenylthiourea coordinates also included tests on therapeutically difficult clinical strains: methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), isolated from patients of clinical hospitals at the Medical University of Warsaw (Table 5).
Against these bacterial strains, the complex of 4-chloro-3-nitrophenylthiourea Cu5 was the most active. As the only one, it exerted very high potency for above half of tested Staphylococci (MIC = 2 µg/mL). A bit weaker, but still strong, it inhibited the growth of S. aureus 537, 585, and 586 strains, as well as half of the S. epidermidis isolates (MIC = 4 µg/mL). The compound Cu5 showed higher effectiveness (up to 128 times) toward half of the clinical pathogens than the control antibiotic. The activity of other thiourea complexes against hospital staphylococcal strains was weak (derivatives Cu1, Cu3) to moderate (for Cu2, Cu4), and remained at the level of 8-128 µg/mL. Among the tested series, no relevant potency against Gram-negative strains of E. coli (MIC ≥ 128 µg/mL) and fungal species was detected. Although studied complexes are weaker antistaphylococcal agents than their starting ligands [20][21][22], the coordinate Cu5 has retained the strong growth-inhibitory action against hospital isolates, as the corresponding unbound thiourea.

Antitubercular Activity
The structural element of currently used tuberculostatic agents, e.g., isoxyl and its analogs, is the thiourea system combined with the flexible alkylaryl substituent. In this spirit, the evaluation of the in vitro antituberculosis activity of the synthesized derivatives was planned. The experiment was performed on the standard M. tuberculosis strain H 37 Rv and two "wild" strains isolated from patients suffering from tuberculosis: Spec 210 resistant to p-aminosalicylic acid (PAS), isoniazid (INH), ethambutol (ETB), and rifampicin (RMP), as well as the Spec. 192 strain, fully sensitive to the applied tuberculostatics. New complexes of 3-(trifluoromethyl)phenylthiourea showed weak activity toward H 37 Rv and Spec.192 strains; however, three compounds (Cu2, Cu4, and Cu5) strongly inhibited the growth of the resistant Spec. 210 isolate ( Table 6). In relation to it, the inhibitory properties of the 3-bromophenyl-(Cu4) and 4-chloro-3-nitrophenylthiourea (Cu5) complexes were 4-8-fold stronger than the reference tuberculostatics. Additionally, the antitubercular potential of the molecule containing the 1-phenylethyl moiety (Cu2) was 2-4 times as strong as the control drugs. The other derivatives containing alkylphenyl moieties (Cu1, Cu3) reached the same or twice activity against Spec 210 as references. Due to the fact that the parental ligands 1-5 were not able to produce a noticeable growth inhibitory effect against the mentioned M. tuberculosis isolates [20,22], the complexation reaction improved their antimycobacterial profile. To eliminate potential carcinogens, complexes Cu1-Cu5 were tested for DNA-damaging potency by rec-assay. For this aim, two genetically modified Bacillus subtilis strains were used: M45 (rec − ; devoid of the recombinant-based DNA repair mechanism) and H17 (rec + ), which is more sensitive to mutagenic substances than the H17 isolate. As shown in Table S1 (Supplementary Materials), no significant differences were found between the diameters of the inhibition zones denoted for both strains. This led to the conclusion that the tested thiourea complexes are non-genotoxic-they are not a source of genetic mutations. What is more, their biological action is not connected with a genotoxic activity.

Type II Topoisomerase Inhibitory Activity
The mechanism of the antibacterial action of the most active complex of 4-chloro-4 nitrophenylthiourea (Cu5) was determined on a panel of bacterial topoisomerases including topoIV and DNA gyrase isolated from a S. aureus strain ( Figures S2 and S3, Supplementary material). The obtained results exhibited that the antimicrobial activity of the investigated derivative is the result of a dual inhibitory ability against topoIV (IC 50 = 6.20 µg/mL) and gyrase (IC 50 = 16.80 µg/mL) ( Table 7). The mentioned copper coordinate suppressed the activity of bacterial topoisomerase, and its antimicrobial profile is similar to other arylthioureas [20,22] and their metal coordinates [17] published by our group recently.

Cytotoxicity
Since the metal-containing compounds possess promising anticancer properties [25][26][27][28], we established the cytotoxic effects of the Cu1-Cu5 thiourea complexes. To determine the potential cytotoxicity of new compounds, the MTT assay was used. The complexes were tested against human carcinoma cell lines, such as SW480 (primary colon cancer), SW620 (metastatic colon cancer), and PC3 (metastatic prostate cancer), as well as against the normal cell line HaCaT (immortalized human keratinocytes). Table S2 (Supplementary Materials) gives the derivative concentrations that produced 50% of growth inhibition (IC 50 , µM), in contrast with two commonly used chemotherapeutics, doxorubicin, and cisplatin.
The complexes exerted weak to moderate antitumor activity, simultaneously being non-toxic toward normal HaCaT cells (Figure 11). The most potent derivatives belonged to the group of phenylethyl (Cu1, Cu2) and 4-chloro-3-nitrothiourea (Cu5). Compounds Cu1, Cu2, Cu4, and Cu5 were found to be up to severalfold less cytotoxic to the HaCaT cell line than the reference cytostatics. The highest selectivity (SI from 2.4 to 11.5) was observed for isomeric phenylethylthiourea coordinates Cu1 and Cu2 and halogenothiourea Cu5. The human primary (SW480) and metastatic (SW620) colon cancer cells appeared to be the most susceptible to the presence of the investigated arylthiourea derivatives-both in the aspect of IC 50 (23.34 µM for the complex Cu2) and selectivity (SI = 6-16 observed for compounds Cu1-Cu2). The most promising halogen-containing compound Cu5 has the lowest IC 50 value towards all investigated cancer cells (11.7-19.5 µM), simultaneously with the highest selectivity versus HaCaT cells. That complex was also more effective against the PC3 cell line than cisplatin alone.

Materials and Methods
The chemicals were of analytical grade and were purchased from Sigma-Aldrich. The thiourea ligand was synthesized according to the procedure described elsewhere [20][21][22] Elemental analyses were carried out on Vario Micro Cube.

Synthesis
The 1 mmol of thiourea ligand 1-5 was stirred in dimethylformamide (2 mL) until its dissolution; next 1 mmol of anhydrous copper(II) chloride was added to the prepared solution. The mixtures were stirred for 6 h at room temperature (approx. 21 °C). After evaporation of the solvent, the powder products were purified by washing thoroughly with water and dried at room temperature to yield complexes Cu1-Cu5.

Materials and Methods
The chemicals were of analytical grade and were purchased from Sigma-Aldrich. The thiourea ligand was synthesized according to the procedure described elsewhere [20][21][22]. Elemental analyses were carried out on Vario Micro Cube.

Synthesis
The 1 mmol of thiourea ligand 1-5 was stirred in dimethylformamide (2 mL) until its dissolution; next 1 mmol of anhydrous copper(II) chloride was added to the prepared solution. The mixtures were stirred for 6 h at room temperature (approx. 21 • C). After evaporation of the solvent, the powder products were purified by washing thoroughly with water and dried at room temperature to yield complexes Cu1-Cu5.

ATR-IR Studies
Infrared spectra were performed on Nicolet iS5 (Thermo Scientific) FTIR Spectrometer with diamond ATR sample accessory. The thiourea complexes Cu1-Cu5 as well as initial ligands 1-5 were recorded in the range of 400-4000 cm −1 .

UV-Vis Studies
The electronic reflectance spectra of free ligands 1-5 and thiourea complexes Cu1-Cu5 were collected on SHIMADZU UV-VIS Spectrophotometer UV-2600Plus with Integrating Sphere in the range of 220-1400 nm.

XANES Comparative Analysis
The K-edges of Cu were measured at the XAFS beamline in Elettra, Trieste, Italy. The spectra were recorded in the transmission mode. XANES spectra were collected for complexes Cu2 and Cu4, and two reference oxides (Cu 2 O and CuO).

EXAFS Studies
The EXAFS spectra were collected for all complexes at the XAFS beamline in Elettra, Trieste, Italy. The spectra were recorded in the transmission mode. For the XAS analysis, the Athena and Artemis programs included in the Demeter package [29,30] were used. The quantitative EXAFS analysis of complexes was performed as follows: k 2 weighted χ(k) data were Fourier transformed in the k range 2.7 to 12 Å −1 . The fitting was performed in the R space in the range from 1 to 4 Å.
Theoretical ab initio XANES calculations were carried out using FEFF 9.6 code. The XANES, Self-Consistent Field, and Full Multiple Scattering cards were used. The Hedin-Lundqvist potential was chosen. Minimal Inhibitory Concentration (MIC) was tested by the twofold serial microdilution method (in 96-well microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson) for bacteria or RPMI-1640 medium for Candida species according to CLSI guidelines [35,36]. The stock solution of tested agent was prepared in DMSO and diluted in sterile water. Concentrations of tested agents ranged from 0.125 to 512 µg/mL. The final inoculum of all studied microorganisms was 10 5 CFU/ mL −1 (colony-forming units per mL). Minimal inhibitory concentrations (the lowest concentration of a tested agent that prevents visible growth of a microorganism) were read after 18 h (bacteria) or 24 h (yeasts) of incubation at 35 • C.

Antitubercular Activity
The synthesized compounds were examined in vitro for their tuberculostatic activity using MABA method (Microplate Alamar Blue Assay method) [37,38]. Investigations were performed by the twofold serial microdilution method (in 96-well microliter plates) using Middlebrook 7H9 Broth medium (Beckton Dickinson) containing 10% of OADC (Beckton Dickinson). The inoculum was prepared from fresh LJ culture in Middlebrook 7H9 Broth medium with OADC, adjusted to a no. 1 McFarland tube, and diluted 1:20. The stock solution of tested agent was prepared in DMSO. Each test compound stock solution was diluted in Middlebrook 7H9 Broth medium with OADC by fourfold, the final highest concentration to be tested. Compounds were diluted serially in sterile 96-well microtiter plates using 100 µL Middlebrook 7H9 Broth medium with OADC. Concentrations of tested agents ranged from 0.125 to 512 µg/mL. A growth control containing no antibiotic and a sterile control without inoculation were also prepared on each plate. The plates were incubated at 37 • C for a week. After the incubation period, 30 µL of Alamar blue solution was added to each well, and the plate was re-incubated for 24 h. Growth is indicated by a color change from blue to pink and the lowest concentration of compound that prevented the color change was noted as its MIC. Isoniazid (INH), Rifampicin (RMP), Streptomycin (SM), and Ethambutol (EMB) as reference drugs were used for comparison.

Genotoxicity Studies
DNA-damaging activity of compounds was tested by rec-assay using two genetically modified Bacillus subtilis strains: M45 (rec − ) and H17 (rec + ) [39,40]. Tested compounds were dissolved in DMSO, and 10 µL of each solution was dripped onto sterile cotton discs (Rotilabo) to load 256 µg of a given compound per 9 mm disc. Discs were placed on the surface Nutrient agar plates (Difco) inoculated with 100 µL of bacterial overnight culture and incubated for 24 h at 35 • C. After incubation, the growth inhibition zones were measured. 4-Nitroquinoline N-oxide (NOQ) was used as reference genotoxin (concentration 2 µg per disc). Results of the genotoxicity test were estimated after 18 h of incubation at 35 • C by comparing the diameter of the inhibition zone on the B. subtilis M45 (rec − ) strain with that on the B. subtilis H17 (rec + ) strain.
3.3.3. Type II Topoisomerase Inhibitory Activity S. aureus Topoisomerase IV Decatenation Assay The assay was performed using a S. aureus topoisomerase IV decantation kit (Inspiralis, Norwich, UK). Kinetoplast DNA (kDNA) was the substrate for topoisomerase IV. Firstly, 1 U of topoisomerase IV decatenated 200 ng of kDNA, in the dedicated decantation assay buffer supplied by the manufacturer. Enzyme activity was detected by incubation for 30 min at 37 • C in a total reaction volume of 30 µL and in the presence of different concentrations of the tested compound. The reactions were terminated by adding an equal volume of STEB buffer (40% sucrose, 100 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mg/mL bromophenol blue), followed by extraction with 1 volume of chloroform/isoamyl alcohol (24:1). Then, 20 µL of the aqueous phase of each sample was loaded onto a 1% agarose gel. Electrophoresis was conducted in Tris-acetate-EDTA buffer for 1.5 h at 80 V. Gels were stained with ethidium bromide and visualized under UV light in a transilluminator (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).

S. aureus DNA Gyrase Supercoiling Assay
The assay was performed using a S. aureus DNA Gyrase Supercoiling kit (Inspiralis, Norwich, UK). The activity of DNA topoisomerase II was determined by measuring the conversion of relaxed pBR322 DNA to its supercoiled form. The reaction mixture contained 500 ng of mMpBR322 plasmid DNA and 1 U of enzyme and different concentrations of the compound in a total reaction volume of 30 µL. The mixture was incubated for 30 min at 37 • C. The reactions were terminated by adding an equal volume of STEB buffer (40% sucrose, 100 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mg/mL bromophenol blue), followed by extraction with 1 volume of chloroform/isoamyl alcohol (24:1). Then, 20 µL of the aqueous phase of each sample was loaded onto a 1% agarose gel. Electrophoresis was conducted in Tris-acetate-EDTA buffer for 3 h at 50 V. Gels were stained with ethidium bromide and visualized under UV light in a transilluminator (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).
To determine IC 50 of the thiourea complexes, cells were seeded in 96-well plates (1 × 104 cells per well) and treated for 72 h with different concentrations of compounds. Cells without studied compounds in the medium were used as a control.
The cell viability was assessed by determination of MTT salt (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) conversion by mitochondrial dehydrogenase. MTT assay was performed as previously described [41]. Experiments were repeated three times. Cell viability was presented as a percentage of MTT reduction in the treated cells versus the control cells. The number of viable cells cultured without studied compounds was assumed to be 100%. Decreased relative MTT level means decreased cell viability. Thiourea complexes with the highest cytotoxic potential assessed by MTT determination (with the lowest IC 50 ) were chosen for subsequent assessments of cytotoxicity mechanisms.

Conclusions
The Cu(II) complexes with five 1,3-disubstituted thiourea derivatives were synthesized. All ligands contain 3-(trifluoromethyl)phenyl tail and differ in the second substituent. Three complexes contain an alkylphenyl group, the other two have a halogenphenyl part. The coordination compounds were structurally characterized by ATR-IR, UV-Vis, and XAFS spectroscopies. These studies indicated that copper is in the +2 oxidation state and revealed that the initial thioureas act as N,S-donor ligands (L). Upon the experimental studies, the structural models of complexes were proposed and optimized using molecular modeling. In these models, two thiourea ligands coordinate to the Cu(II) cation in bidentate fashion through thiocarbonyl S and deprotonated N atoms, thus forming a four-membered chelate ring with CuN 2 S 2 moiety. All studied complexes are hydrates with the formula CuL 2 *xH 2 O. Such monomers are representative of alkylphenylthiourea complexes, whereas halogenphenyl derivatives formed sandwich-type dimers based on CuN 2 S 2 units. Due to the continuous development of new theoretical and computational approaches to XANES, we decided to check its utility for the investigation of studied coordination complexes. Thus, the DFT structural models were used for XANES calculations. The theoretical XANES spectra agree with experimental ones, confirming the validity of the structures we have proposed.
Parallel to the structural studies, the biological activity evaluation of new compounds was performed. The research carried out showed that alkylphenylthiourea complexes inhibited the growth of staphylococcal isolates at the level of 16-64 µg/mL, whereas derivatives with halogenphenyl moiety exerted markedly stronger antimicrobial activity, with MIC values from 4 to 8 µg/mL. Moreover, Cu(II) complex with a chloronitrophenyl part exerted very high potency for 19 of tested methicillin-resistant Staphylococci (MIC = 2 µg/mL). A similar relationship between biological activity and the structural characteristic was observed in the antitubercular profile of the studied complexes. The alkylphenylthiourea complexes showed weak activity against Mycobacterium tuberculosis strains with MIC values of 8-16 µg/mL, as opposed to the much better activity of halogenphenyl derivatives (MIC = 4 µg/mL). Simultaneously, all complexes showed no genotoxic effects. Moreover, the thiourea complex Cu5 was found to be an effective inhibitor of both DNA gyrase and topoisomerase IV isolated from Staphylococcus aureus.
The complexes exerted weak to fair antitumor activity (towards SW480, SW620, and PC3), simultaneously being non-toxic towards normal HaCaT cells. In addition to its antimicrobial profile, the complex Cu5 was more potent and selective towards prostate cancer cells than the reference cisplatin. Data Availability Statement: All data generated or analyzed during this study are included in this published article; further inquiries can be directed to the corresponding author.