Synthesis and Characterization of Some New Cu(II), Ni(II) and Zn(II) Complexes with Salicylidene Thiosemicarbazones: Antibacterial, Antifungal and in Vitro Antileukemia Activity

Thirty two new Cu(II), Ni(II) and Zn(II) complexes (1–32) with salicylidene thiosemicarbazones (H2L1–H2L10) were synthesized. Salicylidene thiosemicarbazones, of general formula (X)N-NH-C(S)-NH(Y), were prepared through the condensation reaction of 2-hydroxybenzaldehyde and its derivatives (X) with thiosemicarbazide or 4-phenylthiosemicarbazide (Y = H, C6H5). The characterization of the new formed compounds was done by 1H-NMR, 13C-NMR, IR spectroscopy, elemental analysis, magnetochemical, thermoanalytical and molar conductance measurements. In addition, the structure of the complex 5 has been determined by X-ray diffraction method. All ligands and metal complexes were tested as inhibitors of human leukemia (HL-60) cells growth and antibacterial and antifungal activities.

In a recent study [20], we have concluded that the in vitro HL-60 leukemia cell growth inhibitory activity is influenced by the nature and geometric structure of copper complexes. Indeed, copper complexes containing tridentate ONS Schiff bases as well as salicyliden thiosemicarbazones have been found as effective inhibitors of cell proliferation. We have started a program directed toward the synthesis of different classes of anticancer, antibacterial and antifungal agents designed with complexes of a transition metal and an organic ligand [21][22][23][24].
In continuation of this approach, the present paper describes the synthesis, characterisation and in vitro evaluation of inhibitors of HL-60 cell proliferation, antibacterial and antifungal activity using thirty two novel Cu(II), Ni(II) and Zn(II) complexes with the salicylidene thiosemicarbazones (H 2 L 1 -H 2 L 10 ), obtained from the condensation reaction of thiosemicarbazide or 4-phenylthiosemicarbazide with 2-hydroxybenzaldehyde derivatives. All ligands and metal complexes were tested as inhibitors of human leukemia (HL-60) cell growth. The Cu(II) complexes 21-25, 30 have also been tested for their in vitro antibacterial activity against Staphylococcus aureus Smith,, Staphylococcus saprophyticus, Streptococcus (group A), Enterococcus faecalis (Gram-positive), Escherichia coli (O-111), Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniaie, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis (Gram-negative) and antifungal activity against Aspergillus niger, Aspergillus fumigatus, Candida albicans and Penicillium strains.
The complexes obtained are microcrystalline solids which are stable in air and decompose above 310 °C (Table 1). They are insoluble in organic solvents such as acetone and chloroform but soluble in DMF and DMSO.
The elemental analyses data of Schiff bases (reported in the Experimental section) and their complexes (Table 1) are in agreement with the proposed composition of the ligands as shown in Scheme 1 and with the formulas of the complexes as shown in Figure 1a                The structure of crystals, obtained from ethanolic solution after recrystallization of (5), has been determined by means of X-ray analysis and is similar to the structure described in [26].

IR Spectra and Coordination Mode
The tentative assignments of the significant IR spectral bands of H 2 L 1 -H 2 L 10 and their Cu(II), Ni(II) and Zn(II) complexes are presented in Table 1. It has been established that the substituted salicylaldehyde thiosemicarbazones of complexes 1-14 behave as monodeprotonated tridentate ligands and are coordinated to the central ions through deprotonated phenolic oxygen atom, azomethinic nitrogen atom and sulphur atom forming five-and six-membered metalocycles [9,20,21].
The IR spectra of the free ligands shows a broad band at ca. 3600 cm −1 attributed to phenolic group, δ(OH). This band disappeared from IR spectra of complexes 1-14 [22,23,27]. Moreover, this is confirmed by the shift of ν(C-O) stretching vibration bands observed in the range of 1250-1240 cm −1 in the spectra of the free ligands, to lower frequency at around 1225-1210 cm −1 in the spectra of the complexes. This is further confirmed by the presence of the band appearing in the region 500-470 cm −1 assigned to the ν(M-O) frequency [28].
Likewise, the IR spectra of the ligands exhibits a strong band in the range 1620-1610 cm −1 assignable to ν(C = N). In the spectra of the complexes 1-14 this band is shifted to lower frequencies by ca. 25-15 cm −1 suggesting the coordination of the azomethine nitrogen to the central metal atom. Also, this coordination is supported of ν(M-N) vibration around 515-540 cm −1 [29].
In the IR spectra of the H 2 L 1 -H 2 L 10 , the ν(S-H) band at 2570 cm −1 [30][31][32][33] was absent, but the ν(C = S) bands at about 1560 and 822 cm −1 were present. These bands were shifted to lower wavenumbers in complexes 1-14 and this shift can be assigned to the thiocarbonyl ν(C = S) stretching and bending modes of vibrations and to the coordination of sulfur atom to metal ion [34][35][36].
In complexes 15-32, thiosemicarbazones behave as double deprotonated tridentate ligands, coordinating to the central ion through phenolic oxygen atom, azomethinic nitrogen atom and sulphur atom forming two five-and six-membered heterocycles. As much, the absorption bands ν(C-OH), ν(N-NH) and ν(C=S), observed in the spectra of the free thiosemicarbazones, in the range 1245-1240, 1540-1535 and 1125-1120 cm −1 , respectively, were shifted to lower frequencies in the spectra complexes. In the spectra complexes the absorption band ν(C-S) is observed in the range 750-740 cm −1 and the band ν(C-N) is shifted to small frequencies with 35-30 cm −1 , being accompanied by the splitting into two components [27][28][29].
In the IR spectra of complexes 15-32, an absorption band is observed in the range 1520-1518 cm −1 , conditioned by valence oscillations >C = N-N = C<. This character of IR spectra demonstrates the thiosemicarbazone enolization in the process of synthesized complexes formation [30][31][32][33].

Magnetochemistry
The room temperature magnetic moment of the solid copper (II) complexes 1-24 was found in the range 1.75-2.00 BM, indicative one unpaired electron per Cu(II) ion [39]. These experimental data allow us to suppose that in these compounds the spin-spin interaction lacks and probably the investigated complexes have monomer structure. Also, the magnetic moment values in the range 1.22-1.45 BM for the copper (II) complexes 25-30 are of indicative anti-ferromagnetic spin-spin interaction through molecular association [40]. Complex 31 is diamagnetic and the central Ni 2+ ion is in a square planar environment [40].

Thermal Decomposition
All complexes studied were investigated by thermogravimetry analysis. The TG thermograms of complexes 1-14 are characterized by three degradation steps (50-100, 130-170, 310-530 °C). The weight loss between 50 and 100 °C corresponds to the elimination of water molecules of dehydration and is an endothermic effect. The second step, also an endothermic effect, corresponds to the elimination of coordinated water molecules ( Table 1). The following effect on DTA curve is exothermic and corresponds to the complete decomposition (TG, TGD curves) of the organic part of the complexes.
The TG and TGD curves of the complexes 15-32 are characterized by two steps of weight loss united (350-480 °C, 480-620 °C) and corresponds to the complete decomposition of the ligands. In addition, the TG and TGD curves of the complexes 21, 22 and 31 are characterized by a weight loss in the renge 50-100 °C.
By replacing the sulphate ion from complexes with nitrate ion or by changing the thiosemicarbazide fragment with 4-phenylthiosemicarbazide fragment, TG and TGD curves show weight loss at lower temperatures. The final residues were identified by IR spectroscopy as CuO, which provides %Cu values in the initial samples, by quantitative analyses. They were in agreement wich the theoretical obtained %Cu values.

NMR Spectra
The NMR spectra of ligands H 2 L 1 -H 2 L 10 were recorded in DMSO-d 6 . The 1 H-NMR and 13 C-NMR spectral data are reported along with the possible assignments [41]. All the protons were found to be in the expected regions. It was observed that DMSO did not have any coordinating effect on the ligands or their metal complexes.
If copper is capsulated with amine, the antiproliferative activity change in dependence of substituents R 1 and R 2 in the same series Y = H or Y = -C 6 (18). If Y = H, R 1 = R 2 = Br and A -is variable, the moderate influence of amine nature can be observed depending on the ability of amine(N)-copper bond force: 25 > 28 > 26 = 27 = 29 > 23 = 24 > 21 > 22. If Y = H, R 1 = R 2 = H, A = ethazole and copper ion is replaced by nickel or zinc (31,32), the antiproliferative activity dramatically decreases. Table 3. Schiff bases H 2 L 1 -H 2 L 10 and their antiproliferative activity on human leukemia (HL-60) cells at three concentrations.

Antibacterial Activity
Experimental results obtained from the study of antimicrobial activity (Table 5) demonstrate that compounds 21-25 and 30 display bacteriostatic and bactericide activity in the concentration range 0.03-4000 µg/mL towards both Gram-positive as well as Gram-negative bacteria. In comparison, the antimicrobial data characteristic for furacillinum used in medical practice are given. The antimicrobial activity displayed by the above mentioned compounds is 32-260 times higher towards staphylococcus and streptococcus than furacillinum and exceeds by 2-260 times her bacteriostatic activity towards the majority of Gram-negative bacteria. The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) are influenced by the nature of thiosemicarbazone and amine of the inner sphere of the coordination compound.
The data concerning the study of antimycotic properties of compounds 22-24 show that they also display selective bacteriostatic and bactericide activity in the concentration range 9.3-600 μg/mL towards investigated fungi stems. In order to make a comparison, we also added data regarding the activity of nystatine, a compound used in medicine at mycoses treatment. The results show that the synthesized substances have antimycotic activity against most fungi, higher than nystatine activity. Aspergillus fumigatus is an exception, being less sensible towards mentioned substances. The toxicity (LD 50 ) of complexes 24 and 30 (some of the most active in this group of substances) is 1,420 mg/kg and 4,250 mg/kg so it is 8.6-25.5 times lower than that of furacillinum (LD 50 = 166.7 mg/kg).

Chemistry
All commercially available reagents and chemicals were of analytical-or reagent-grade purity and used as received. 1 H-NMR and 13 C-NMR spectra were recorded at room temperature on a Bruker DRX 400 spectrometer in DMSO-d 6 , using TMS as the internal standard. IR spectra were recorded on a Specord-M80 spectrophotometer in the 4000-400 cm −1 region using KBr pellets. The chemical elemental analysis for the determination of C, H, N and Br was done the Carlo-Erba LA-118 microdosimeter. Metal ions were determined following the method described by G. Schwarenbach and H. Flaschka [45]. The complexes were studied by thermogravimetry (TG), in a current of air, with a sample heating rate of 1 °C/min, using a SETARAM 92-1600 thermo-balance. Magnetic measurements were carried out on solid complexes using the Gouy's method [39].
X-ray diffraction analysis of compound 5 was carried out on a Nonius KappaCCD diffractometer (MoK α radiation, λ = 0.71069 Å) at room temperature. The structures of complex 5 was solved by the direct method using SHELXS-86 [46] and SIR-97 [47] software and refined by least squares in the anisotropic approximation for nonhydrogen atoms (CRYSTALS) [48]. The hydrogen atoms were refined isotropically. In complex 5, all hydrogen atoms were included in the refinement in geometrically calculated positions (except for water molecules in which hydrogen atoms were not located). The C-H and N-H bond lengths varied in the 0.93-0.98 and 0.86-0.89 Å ranges, respectively. The thermal factors U H were taken to be 1.2-1.5 times as high as the U eq values of the carbon and nitrogen atoms.   Synthesis of Compound 15. To CuCl 2 ·2H 2 O (10 mmol) dissolved in 20 mL ethanol was added salicyliden thiosemicarbazone (10 mmol) dissolved in 15 mL hot ethanol.The mixture was stirred continuously (1 h) and then pyridine alcoholic solution is added till pH=7.5-8. The dark green microcrystals was filtered, washed with ethanol, diethyl ether and dried in air.

Preparation of Test Solutions
Stock solutions of the investigated compounds (H 2 L 1 -H 2 L 10 ) and copper complexes 1-30 were prepared in dimethylsulfoxide (DMSO) at a concentration of 10 mM and diluted with nutrient medium to various working concentrations. DMSO was used instead of ethanol due to solubility problems.

Cell Proliferation Assay
The cell proliferation assay was performed using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Cell Titer 96 Aqueous, Promega, Madison, Wi, USA), which allowed us to measure the number of viable cells. In brief, triplicate cultures of 1 x 10 4 cells in a total of 100 µL medium in 96-well microtiter plates (Becton Dickinson and Company, Lincoln Park, NJ, USA) were incubated at 37 °C, 5% CO 2 . Compounds were dissolved in DMSO to prepare the stock solution of 1 × 10 −2 M. These compounds were diluted at the appropriate concentration (1 or 10 µM) with culture media, added to each well and incubated for 3 days. Following each treatment, 20 µL MTS was added to each well and incubated for 4 h. MTS is converted to water-soluble coloured formazan by dehydrogenase enzymes present in metabolically active cells. Subsequently, the plates were read at 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The results were reported as the percentage of cell proliferation inhibition compared to the control (basal cell proliferation=100%).

Antifungal Bioassay
Antimycotic properties of the complexes were investigated in vitro on laboratory stems: Aspergillus niger, Aspergillus fumigatusi, Candida albicans and Penicillium. The activity has been determined in liquid Sabouroud nutritive environment (pH 6.8). The inoculates were prepared from fungi stems which were harvested during 3-7 days. Their concentration in suspension is (2-4) × 10 6 colonies form unities in milliliter. Sowings for levures and micelles were incubated at 37 °C during 7 and 14 days, respectively.

Conclusions
Ten new salicyliden thiosemicarbazones ligands and their corresponding Cu(II), Ni(II) and Zn(II) complexes have been synthesized and characterized. The molecular structure of the complex 5 has been determined by single crystal X-ray diffraction method. The IR, 1 H-NMR and 13 C-NMR data were successfully used to elucidate the formation of the salicyliden thiosemicarbazones ligands. All ligands and their metal complexes were tested as inhibitors of HL-60 cells proliferation. The ligands have unsignificant inhibitor activity at 0.1 and 1.0 μmol/L, but at 10 μmol/L H 2 L 8 , H 2 L 9 and H 2 L 10 inhibit the cell proliferation. The copper complexes, including inner sphere water and tridentate ONS ligands, are more active than those containing inner sphere amine, which blocked the metal active centre. The most indicative criteria for future synthesis of biological active coordination compounds from the viewpoint of the inhibition of HL-60 cell proliferation, antibacterial and antifungal activity: use of copper (II) complexes and presence of sulphur atom in the tridentate organic ligand.