Synthesis, characterization, crystal structure, and antimicrobial studies of novel thiourea derivative ligands and their platinum complexes

Abstract N,N-Di-R-N′-(4-chlorobenzoyl)thiourea (Di-R: diethyl, di-n-propyl, di-n-butyl and diphenyl) ligands (HL1–4) and their Pt(II) complexes (cis-[Pt(L1–4-S,O)2]) have been synthesized and structurally characterized by elemental analyses, FT-IR and NMR spectroscopy. HL2 ligand and cis-[Pt(L4-S,O)2] metal complex have been also characterized by a single-crystal X-ray diffraction study. HL2, C14H19ClN2OS, crystallizes in the monoclinic space group P21/n (no. 14), with Z = 4, and unit cell parameters, a = 11.1405(16) Å, b = 9.7015(12) Å, c = 14.790(2) Å, β = 106.547(7)°. The cis-[Pt(L4-S,O)2], C40H28Cl2N4O2PtS2: triclinic, space group P-1 (no. 2), a = 8.9919(3) Å, b = 14.7159(6) Å, c = 15.7954(6) Å, α = 113.9317(18)°, β = 97.4490(18)°, and γ = 105.0492(16)°. Single crystal analysis of complex, cis-[Pt(L1–4-S,O)2], revealed that a square planar coordination geometry is formed around the platinum atom by two sulfur and two oxygen atoms of the related ligands, which are in a cis configuration. In addition, the thiourea derivative ligands and their complexes were evaluated for both their in-vitro antibacterial and antifungal activity. The results have been reported, explained, and compared with fluconazole and ampicillin, used as reference drugs.

Thiourea ligands, which are selective ligands for the platinum group metals, have a strong affinity toward the platinum(II) center, which is coming from the sulfur atom. Thiourea derivatives can be acceptable as convenient sulfur-containing nucleophiles in order to react relatively fast with platinum(II) species in experiments, such as glutathione, which is present in biological systems [18,19]. More recently, there have been efforts to design platinum-acylthiourea complexes in order to investigate their antifungal activity and inhibitory activities against viruses [20].
The prominence of such studies is the possibility that thiourea derivatives may be more effective as antimicrobial agents. However, there is a need for a comprehensive investigation relating to the structure and activity of thiourea derivatives as well as their stability under biological conditions. These detailed investigations could be helpful in the design of more potent antimicrobial agents. Based upon literature research, we could find no synthesis or characterization of the title compound type thiourea derivative metal complexes. In an effort to contribute to these studies, here we report the synthesis, characterization and antimicrobial properties of new N,N-di-R-N′-(4-chlorobenzoyl) thiourea ligands and their platinum metal complexes.

Instrumentation
Melting points were recorded on an Electrothermal model 9200 apparatus. Carbon, hydrogen, and nitrogen analyses were carried out on a Carlo Erba MOD 1106 elemental analyzer. Infrared measurements were recorded from 400 to 4000 cm −1 on a Perkin Elmer Spectrum 100 series FT-IR/FIR/NIR Spectrometer Frontier, ATR instrument. The NMR spectra were recorded in CDCl 3 solvent on a Bruker Avance III 400 MHz NaNoBay FT-NMR spectrophotometer using tetramethylsilane as an internal standard.
The X-ray diffraction data were recorded on a Bruker APEX-II CCD diffractometer. A suitable crystal was selected and coated with Paratone oil and mounted onto a Nylon loop on a Bruker APEX-II CCD diffractometer. The crystal was kept at T = 100 K during data collection. The data were collected with MoKα (λ = 0.71073 Å) radiation at a crystal-to-detector distance of 40 mm. Using Olex2 [21], the structure was solved with the Superflip [22][23][24] structure solution program using the Charge Flipping solution method and refined by full-matrix least-squares on F 2 using ShelXL [25] with refinement of F 2 against all reflections. Hydrogens were located by difference maps and were refined isotropically, and all non-hydrogen atoms were refined anisotropically. The molecular structure plots were prepared using PLATON [26]. Geometric special details: all e.s.d. 's (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d. 's are taken into account individually in the estimation of e.s.d. 's in distances, angles, and torsion angles; correlations between e.s.d. 's in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d. 's is used for estimating e.s.d. 's involving l.s. planes.

Reagents
Potassium tetrachloroplatinate(II) was purchased from Sigma Aldrich. 4-Chlorobenzoyl chloride, potassium thiocyanate, diethylamine, di-n-propylamine, di-n-butylamine, and diphenylamine were purchased from Merck and used as received. All other chemicals and solvents were obtained from commercial suppliers and used without purification.

General procedure for the synthesis of ligands
HL 1-4 ligands were prepared according to previously published methods [27][28][29]. A solution of 4-chlorobenzoyl chloride (5 mmol, 0.860 g) in dry acetone (50 mL) was added dropwise to a suspension of potassium thiocyanate (5 mmol, 0.49 g) in acetone (30 mL). The reaction mixture was heated under reflux for 30 min and then cooled to room temperature. A solution of secondary amine (5 mmol, diethylamine: 0.370 g; di-n-propylamine: 0.510 g; di-n-butylamine: 0.650 g; diphenylamine: 0.850 g) in acetone (10 mL) was added and the resulting mixture was stirred for 2 h. Afterward, the reaction mixture was poured into hydrochloric acid (0.1 M, 300 mL) and the solution filtered. The solid product was washed with water and purified by recrystallization from an ethanol:dichloromethane mixture (1:1, v:v) (Scheme 1).

Cytotoxicity studies
To evaluate cytotoxicity, HEp-2 human cell line (ATCC CCL23) was selected. In preparation of the cell cultures, EMEM (Eagle's minimum essential medium) was used with 10% fetal bovine serum (Seromed) as growth medium. Incubation of the cells was performed in an atmosphere of 5% carbon dioxide at 37 °C. To determine the effects of the compounds on HEp-2 cells effects of added compound versus control cells with no added compound were observed. The nontoxic concentration was determined to be up to 1024 μg mL −1 . We used this limit in all experiments to test bacterial growth inhibition. In order to test the effects of the compounds on HEp-2 cells, 5 × 10 4 cells were seeded into each well of 12-well plates, cultured for 6 h at 28 °C, and allowed to grow for an additional 48 h in the presence of increasing amounts of compound (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 and 2048 μg mL −1 ). Cytotoxicity of extracts was determined by a conventional haemocytometer using the trypan blue exclusion method [32][33][34][35]. The highest non-cytocidal (on HEp-2 cells) concentration of the chemical compounds was determined to be 1024 μg mL −1 . This limit was used for the determination of antimicrobial activities.
The fungal and bacterial cell inoculums were prepared from the stock culture grown in Tryptic Soy Agar (TSA) at 28 °C for 24 h and Mueller-Hinton Agar (MHA) 37 °C for 24 h, respectively. The micro-organism suspension concentrations were adjusted according to McFarland 0.5 turbidity tubes using sterilized saline. A stock solution of the title compound was prepared in DMSO at 1000 μg mL −1 . A modified microdilution test was applied for antimicrobial activity and the experiments were run in duplicates independently.
For antifungal activity testing, 100 μL of Tryptic Soy Broth (TSB) was added to each of 11 wells. 100 μL of chemical derivative solution was added to the first well and twofold dilutions were prepared. Then, 5 μL of fungal suspension was added to each tube except the last one, which acted as a control well.
For antibacterial activity testing, 100 μL of Mueller-Hinton Broth (MHB) was added to each of 11 wells. 100 μL of chemical derivative solution was added to the first tube and twofold dilutions were prepared. Then 5 μL of the bacterial suspension was added to each tube except the last control well. Only 5 μL of fungal and bacterial suspension were added in another control tube without chemical and used as a control for growing. All plates were incubated at 28 °C (for fungi) and at 37 °C (for bacteria) for 24 h. After the incubation, the minimal inhibitory concentrations (MIC) were noted by controlling the growth inhibition for title compound (Table 1). Fluconazole and ampicillin were used as reference drugs. The results were read visually and by measuring optical density for 24 h.

Spectroscopic characterization of the ligand and platinum complexes
Ligands were synthesized in two steps. In the first step, 4-chlorobenzoyl isothiocyanate was synthesized by reaction of 4-chlorobenzoyl chloride with an equimolar amount of potassium thiocyanate in dry acetone. In the second step, ligands (HL 1-4 ) were obtained from reaction of 4-chlorobenzoyl isothiocyanate with a secondary amine (diethylamine, di-n-propylamine, di-n-butylamine, and diphenylamine) in dry acetone. Scheme 1 outlines the synthesis of the series of thiourea derivatives. The ligands were purified by recrystallization from an Table 1. miC values (μg ml −1 ) of the ligands and metal complexes tested against the Gram-positive, Gram-negative bacterial and fungal and cytotoxicity values (μg ml −1 ) against the hep-2 human cell line. * * "-": effective in all concentrations used.  Figures S1, S3, S7, S9, S11, S15, S17, S19, S23, Supporting Information). The reaction of the ligands with potassium tetrachloroplatinate(II) at room temperature with an ethanol:water mixture as solvent yielded the four new complexes cis-[Pt(L 1-4 -S,O) 2 ] (Scheme 2). All the new metal complexes were recrystallized from an ethanol:dichloromethane mixture and characterized by elemental analysis, 1 H NMR, 13 C NMR, cozy, HMQC, and FT-IR techniques ( Figures  S2, S4, S5, 6S, S8, S10, S12, S13, S14, S16, S18, S20, S21, S22, and S24, Supporting Information). The proposed structures given in Schemes 1 and 2 are consistent with the analytical and spectroscopic data. IR spectral analysis confirms the presence of characteristic groups in the prepared compounds. The main vibrational bands of the investigated compounds are given in the Experimental section. The IR spectra of all prepared ligands showed characteristic bands approximately at ~3200 cm −1 corresponding to a ν(NH) stretching vibration. This band disappears upon metal complex formation. The strong ν(C=O) stretching vibration bands for the free ligands were observed in the range of 1640-1681 cm −1 . In the obtained complexes, the ν(C=O) stretching vibration frequency decreases by ~155-195 cm −1 , in agreement with the literature [38]. A thiocarbonyl ν(C=S) vibration band appeared at 1212-1251 cm −1 for the free ligands. These bands shifted to lower values (~1085-1090 cm −1 ) for complex compounds as a result of the coordination via sulfur and oxygen atoms to platinum ions ( Figures  S7, S8, S15, S20, and S23, Supporting Information). These results agree with the data supplied in the literature [38,39].
The 1 H NMR spectra of the ligands were recorded in CDCl 3 . The 1 H NMR data of the obtained compounds are given in the Experimental section and are consistent with the structural results. The signals belonging to the NH groups of the ligands were observed the most downfield as a singlet in the range of δ 8.51-8.85 ppm in the NMR spectra (Figures S1, S9, and S17, Supporting Information). But these signals did not appear in the 1 H NMR spectrum of the Pt(II) complexes ( Figures S2, S10, and S16, Supporting Information). The signals for the aromatic protons in the free ligands were observed at δ ~7-8 ppm; slight variations were observed as a result of the difference in their environments. After complexation occurred, the aromatic proton resonance values for protons number 2,2′,4,4′ shifted downfield (Δδ ~0.35 ppm). Protons number 1,1′,3,3′ are nearly identical (a slight difference in the shift of signals amounting to Δδ ~0.05 ppm). In 1 H NMR spectra of the HL 1-3 ligands, different 1 H resonances are observed for each of the two ethylene groups of the (S)CN(CH 2 ) 2 -moiety at room temperature in CDCl 3 (δ 4.02 and 3.59 (for HL 1 ); δ 3.91 and 3.47 ppm (for HL 2 ); δ 3.96 and 3.51 ppm (for HL 1 ); δ 4.02 and 3.59 ppm (for HL 3 )). Since, the resonance in C(O)-NH-C(S)-N part gives the single bond a double bond character and slows the rotation of the C-N bond [40][41][42][43][44][45]. For HL 1-3 ligands, this restricted rotation results interestingly in the formation of E/Z configurational isomerism in solution [43][44][45]. The coordination of Pt 2+ with the ligands does not change the main profile of the ligands NMR signals of the chemical shifts of the two ethylene groups get closer, indicating that the anisotropy of the two ethyl groups is less  )). In other words, a significant consequence of complexation is an increase of the C-N rotation [43][44][45]. The most upfield protons in 1 H NMR spectra of all the ligands appear as one sharp triplet between δ 0.85-1.00 ppm which is generated by six protons assigned to -CH 3 groups of ligands. The other aliphatic proton signals (-CH 2 -CH 3 , -CH 2 -CH 2 and N-CH 2 ) of the free ligands are shown relatively in downfield. These resonance values for the complexes 1-3 are almost identical with a slight difference in the shift of signals ( Figures S1, S2, S9, S10, S17, and S18, Supporting Information).
In 13 C NMR, the characteristic thiocarbonyl (C=S) and carbonyl (C=O) carbons for all free ligands appeared at δ 179-162 ppm range. The C=S and C=O carbon resonance values of the complexes shifted upfield (Δδ ~12 ppm) and downfield (Δδ ~5 ppm), respectively, when compared with the free ligand's resonances. Consequently, the shift of signals in NMR spectra of complexes, compared with free ligand, is the result of the complexation process.
The molecular geometry of cis-[Pt(L 4 -S,O) 2 ] was affected by intra-and intermolecular hydrogen bond interactions. In addition, the crystal structure is further stabilized by C-H•••Cg and Cg⋯Cg (π⋯π) interactions (Tables 5 and 6 (Table 5), and slipped face-to-face of two phenyl rings with distances in the range of 4.097-5.868 Å ( Table 6). The C-H⋯π edge-to-face (C38-H38⋯Cg7 iii ) and π⋯π face-to-face interactions between the centers of gravity (Cg1⋯Cg3 i , Cg2⋯Cg3 iii , and Cg5⋯Cg7 vi ) of the phenyl rings in molecular packing of cis-[Pt(L 4 -S,O) 2 ] are shown in Figure 3. These stacking interactions direct the packing of the multilayer structure into a 3-D structure.
The dihedral angles between Pt1-S1-C8-N1-C1-O1 and Pt1-S2-C28-N3-C21-O2 planes, C15-C20 and C9-C14 planes are 4.34 and 81.13°, respectively (Figure 4). The unit cell and packing of the compound viewed approximately along the c axis is given in Figure 5. It can be said that there is a charge accumulation in the diagonals of the unit cell. There is intense electron localization available in the diagonal points. This is suitable for triclinic unit cell.

Antimicrobial activity studies
Safety tests, including cytotoxicity assays, are required for all products to be used in contact with humans. Cytotoxicity tests using culture cells have been accepted as a first step in identifying active compounds and for biosafety testing. Samples were placed in contact with a monolayer of HEp-2 cells and incubated. The cells were then scored for cytopathic effects [34,35]. In the cytotoxicity assay, concentrations up to 1024 μg mL −1 of the compounds were not toxic for the replication HEp-2 cells. Thus, lower concentrations of all compounds (1000 μg mL −1 ) were used for the experiments.  Table 1.
All free ligands and their Pt(II) complexes inhibited the growth of bacteria strains with MIC values ranging between 3.90 and 62.50 μg mL −1 (Table 1). When the synthesized Pt(II) metal complexes compared with ampicillin, which is used as a reference drug, the synthesized complexes demonstrated lower activity against S. aureus, S. pneumonia (as Grampositive bacteria) and A. baumannii (as Gram-negative bacteria). In contrast, the synthesized complexes showed mostly better activity than ampicillin against E. coli and P. aeruginosa (as Gram-negative bacteria). This may be explained by the differences between structures of bacterial cells [12,55]. Table 6. Geometrical parameters of the π-stacking moieties involved in the π⋯π interaction for the cis-   (7)    According to data generated from this study, ligands and their Pt(II) complexes are observed to have different antimicrobial activity despite having similar structures. This is due to the fact that the zone of inhibition depends on both the diffusion of a compound into the agar medium and the solubility of compound. When the solubility is low, the diffusion is limited, resulting in the small zone, even for highly active derivatives presenting low MICs. On the other hand, high inhibition zone correlated with relatively high MIC could be explained by precipitation of a compound in the liquid medium and the changing of its real concentration [56]. Ligand HL 2 showed excellent inhibition against some bacteria species compared to other ligands. The structures of HL 1 through HL 3 vary gradually by increasing one carbon atom each. However, the antimicrobial results show significant different values for HL 2 . In comparison to HL 1 and HL 2 , HL 3 , which has the longest carbon chain, could have imitated the molecule in the lipid bilayer of organism and afforded less disruption in the bacteria membrane. Also, it is known that binding affinities (binding free energy) of compounds to micro-organisms through intermolecular interactions are effective on antimicrobial activity [57]. The increase of carbon chain in ligands can give rise to lesser binding affinity. Looking at the results obtained, we think that the binding affinity of HL 3 ligand to micro-organisms may be lower than that of the other two ligands [58,59].
Moreover, antibacterial efficiency is higher than antifungal activity. Antimicrobial activity against bacteria may be due to the difference between cell structures of bacteria and fungi. While the cell walls of fungi contain chitin, the cell walls of bacteria contain murein [55]. The cell walls of fungi contain chitin, which enhance the rigidity and structural support. In addition, fungi contain ergosterol in cell membrane instead of cholesterol in the cell membrane of animals [9,54,60].

Conclusion
N,N-Di-R-N′-(4-chlorobenzoyl)thiourea type ligands and their Pt(II) complexes have been synthesized and characterized by elemental analysis, FTIR, 1 H, 13 C, cozy, and HMQC NMR studies. HL 2 ligand and cis-[Pt(L 4 -S,O) 2 ] metal complex have been also characterized by a single-crystal X-ray diffraction study. The crystal data reveal that HL 2 crystallizes in space group P2 1 /n of the monoclinic crystal system while the cis-[Pt(L 4 -S,O) 2 ] complex crystallizes in space group P-1 of the triclinic crystal system. The central Pt(II) atom is coordinated in cis form by two S and O donor atoms. The Pt(II) ion in complex has a slightly distorted square planar geometry. The analysis of crystal structures shows that intermolecular interactions have effective role in stabilization of crystal structure of both compounds. Synthesized compounds were tested in vitro against Gram-positive and Gram-negative bacteria and fungi. The evaluation of in vitro antimicrobial activities of compounds against various bacteria and fungi revealed that the HL 2 ligand and its platinum metal complex showed excellent inhibition against some bacteria species compared to other ligands and complexes. HL 2 and its platinum metal complex show best antimicrobial activity against S. pneumoniae as Grampositive bacteria, P. aeruginosa and A. baumannii as Gram-negative bacteria. The activity of HL 2 became more pronounced when coordinated with the platinum ion. Hence, from all these observations, it was concluded that the cis-[Pt(L 2 -S,O) 2 ] complex could be exploited for the design of novel antimicrobial drugs.

Supplementary material
Crystallographic data for the structures reported in this paper have been deposited at the Cambridge Crystallographic Data Center (CCDC) with quotation number CCDC-1532704 for HL 2