Synthesis, Characterization and Toxicity Evaluation of Cu(II), Mn(II), Co(II), Ni(II), Pd(II) Complexes with Ligand Derived from Hydrazinecarbothioamide

ELENA PAHONTU1, LAURA ILEANA SOCEA2*, STEFANIA FELICIA BARBUCEANU2, DIANA CAROLINA ILIES2, MIHAELA BADEA3, OCTAVIAN TUDOREL OLARU4, AURELIAN GULEA5, BOGDAN SOCEA6, OVIDIU BRATU7 1University of Medicine and Pharmacy Carol Davila, Faculty of Pharmacy, General and Inorganic Chemistry Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 2University of Medicine and Pharmacy Carol Davila, Faculty of Pharmacy, Organic Chemistry Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 3University of Bucharest, Faculty of Chemistry, Inorganic Chemistry Department, 23 Dumbrava Rosie Str., 020462, Bucharest, Romania 4University of Medicine and Pharmacy Carol Davila, Faculty of Pharmacy, Pharmaceutical Botany and Cell Biology DepartmentDepartment, 6 Traian Vuia Str., 020956, Bucharest, Romania 5Moldova State University, Coordination Chemistry Department, 60 Mateevici Str., 2009, Chisinau, Republic of Moldova 6University of Medicine and Pharmacy Carol Davila, Faculty of General Medicine, St. Pantelimon Emergency Hospital, 340-342 Pantelimon Road, 021623, Bucharest, Romania 7University of Medicine and Pharmacy Carol Davila, Clinical Departament 3, Faculty of General Medicine, Universitary Emergency Central Military Hospital, 6 Traian Vuia Str., 020956, Bucharest, Romania


Materials and methods
All reactants and solvents were obtained commercially with the highest purity and were used without further purification.
C, H and N analyses were performed with the Carlo-Erba LA-118 microdosimeter and the AAS-1N Carl-Zeiss-Jena spectrometer was used for the determination of Cu(II), Ni(II) and Co(II).
Infrared spectra (4000-400 cm -1 ) were recorded on a Bruker Vertex 70 spectrophotometer, using KBr pellets. Diffuse reflectance spectra were recorded on a Jasco V-670 spectrophotometer, using MgO dilution matrices. The molar conductance of the complexes in dimethylformamide solutions (10 -3 M), at room temperature, were measured using a Consort type C-533 conductivity instrument. Magnetic susceptibility measurements were performed at room temperature in the polycrystalline state on a Faraday magnetic balance (home-made). The complexes were studied by thermogravimetry (TG) in static air atmosphere, with a sample heating rate of 10 o C/ min using a STA 6000 Perkin Elmer.
D. magna bioassay was performed using the method described by Socea et al. with some modifications [9,16]. The determination was performed in 4 mL 12-tissue culture wells (Greiner Bio-One), using 10 daphnids/well and each sample was tested in duplicate. The assay was performed using concentrations ranging from 0.3 to 50.0 µM. The concentration range was different for each compound due to the low solubility in the 1% DMSO in artificial medium used as solvent. The daphnids were considered dead if they did not move their appendages for 30 s. The survivors were counted after 24 and 48 h of incubation at 25°C, 75% RH, in the absence of light, using a climatic room chamber (Sanyo MLR351H, Sanyo). The control samples used were the solvent and the ligand HL. The median lethal concentration (LC50) was determined by interpolation on lethality curve constructed using the least square method. The 95% confidence interval (CI95%) for LC50 and goodness of fit were also calculated using GraphPad Prism version 5.01 software (GraphPad Software, USA).

Results and discussions
All complexes were synthesized by direct reaction of the inorganic salt solutions with the ligand solution. The obtained complexes are microcrystalline solids which are stable in air and decompose above 250 o C. The synthesis of the complexes is reproducible and the hydrazine carbothioamide coordinates as a mononegative bidentate ligand.
The molar conductance values of the soluble complexes in DMF (6-37 Ω -1 cm 2 mol -1 ) showed that all complexes are non-electrolytes [17]. The elemental analyses data of the metal complexes are in agreement with the proposed structures of the complexes (scheme 3).
The structures of the compounds synthesized were elucidated by IR, UV-Vis spectrometry and thermal analyses.
The IR spectra of the complexes are compared with that of the free ligand to determine the changes that might occur during complex formation.
The ligand HL having three vibrational frequency from 3321 to 3147 cm -1 which are assigned to the stretching vibration of the NH groups, a strong band at 1682 cm -1 corresponding to ν(C=O) stretching vibration [18,19]. In all IR spectra of complexes the ν(C=O) mode of ligand is not observed, but the presence of a new band at 1620-1640 cm -1 , due to the ν(N 1 =C) of N 1 =C-Ogroup, indicates the enolisation of C=O group followed by deprotonation and complexation with metal ions. This is further supported by the presence of band at 1277 cm -1 corresponding to ν(C=S) stretching vibrations in free ligand which have not presented considerable change in the complexes and ruling out the possibility of bonding through the C=S group [20].
So, IR spectral data suggests that the ligand HL act as mononegative bidentate ligand and coordinate through deprotonated enolic carbonyl oxygen (=C-O -) and hydrazinic N 2 H nitrogen atoms and form a five-membered chelate ring.
The perchlorate complex 4 shows a band at 1121 cm -1 assignable to ν 3 (ClO 4 -) and a strong band at 1107 cm -1 assignable to ν 4 (ClO 4 -). The splitting of this band in two components indicates the presence of a monodentate perchlorate group [23]. All the complexes (except 7) exhibit ν(OH) and γ(H 2 O) bands in the 3435-3468 and 1158-1161 cm -1 regions, which are indicative of coordinated water [24].
The geometry of the metal complexes has been deduced from the electronic spectra of the complexes. In the electronic spectra of the complexes the bands of ligand are shifted to lower energies ( fig. 1). Except for complex 2, all the copper(II) complexes show absorption band in the area 16120-18340 cm -1 which was attributed to the d-d transitions: 2 B 2 → 2 B 1 and 2 B 2 → 2 A 1 , suggesting a pseudo-tetrahedral geometry for the metal center. For complex 2, the electronic spectrum show an absorption bands at 17850 and 13510 cm -1 that were assigned to the d-d transitions: 2 B 1g → 2 E g and 2 B 1g → 2 B 2g , respectively. These transitions indicate an axial distorted octahedral geometry of the copper(II) ion [25,26]. The magnetic moment values (1.64-1.98 BM) for the copper(II) complexes corresponds to one unpaired electrons per metal ion.
The observed value of 5.92 BM for the Mn(II) complex is consistent with the high spin octahedrally coordinated Mn 2+ ion ( 6 A 1g ground state) with five unpaired electrons. The electronic spectrum of this complex exhibits peaks at 19.100 and 27.027, assigned to the 6 A 1g → 4 T 1g (G) and 4 T 2g (G) transitions, respectively [27].
The electronic spectrum of the cobalt(II) complex 6 show an intense band attributable to 4 A 2 (F)→ 4 T 1 (P) transition at 16.670 cm -1 indicating a tetrahedral geometry for the complex. The magnetic susceptibility value and the deep green color for complex 6 is more consistent with tetrahedral stereochemistry [28].
For complex 7, the electronic spectrum showed a two band association for square-planar geometry. The magnetic moment value (3.54 B.M) corresponding to two unpaired electrons per nickel (II) centres for square-planar configuration [29].
The electronic spectrum of complex 8 showed a square-planar geometry for the palladium ion. The very intense band at about 22.200 cm -1 are assignable to a combination of metal-ligand charge transfer and d-d bands [30].
The thermogravimetric analysis tool is the most essential for supporting the presence of solvent molecules in attachment with the complex molecule.
Thermal analyses data reveal that compounds 1-6 and 8 are hydrated, having a weight loss between 100 and 211 o C which corresponds to one molecule (1, 3, 4, 6, 8) or two molecules (2, 5) of water respectively directly bonded to the metal center. The second weight loss steps correspond to the release of small coordinated anions: OAc -, Cl -, ClO 4 -. The other weight loss refers to the decomposition of the ligand (fig. 2).
In the first 24 h, no significant lethality was observed for any compound. The ligand HL toxicity was predicted using Lazar software (lazy structure-activity relationships), a modular framework for predictive toxicology used widely in the risk assessment of new synthetized compounds [31]. The predicted LC50 for Daphnia magna was 123 µM with a CI95% of 1.68 -8990 µM. Experimental data at 48h, showed that HL induced at highest concentrations a lethality between 20 and 40% and an estimated value of LC50 higher than 3800 µM. Given the low solubility of the compound, no concentration above 50 µM could be tested. Along with the experimental results, the predicted values indicate a low toxicity of the compound. Even if the predicted values are different from the obtained data, the predicted CI95% includes our results. After 48 h, only complexes 3 -5, induced a lethality equal or higher than 50%. For these compounds LC50 values were calculated and presented in table 1. All compounds induced on Daphnia magna lethalities between 0 and 30%. These results show a higher toxicity of two Cu 2+ and one Mn 2+ complexes compared to the effect of ligand HL. In our previous work [29], we tested a series of complexes along with their precursor salts on Daphnia manga and demonstrated that the ligand lowered their toxicity. The Cu 2+ salts had high toxicity with a LC50 ND-Not determined due to the results obtained. ranging from 0.3 to 5 µM. In the present paper, complexes 1, 2, 5 and 7, induced no toxicity above 30% at 48 h, showing the capability of the ligand to significantly lower the toxicity of the metal ions.
The physico-chemical analyses (molar conductivity, magnetic susceptibility measurements, IR, UV-Vis., elemental and thermal analysis) confirmed the composition and the structures of the newly obtained complexes. In all the complexes, the ligand HL acted as ON bidentate donor forming mononuclear complexes. A pseudo-tetrahedral and octahedral geometry for Cu 2+ , octahedral for Mn 2+ , tetrahedral geometry for Co 2+ and a square-planar arrangement for Ni 2+ and Pd 2+ complexes were proposed, respectively.
The newly synthesized complexes are far less toxic for Daphnia magna than the metal ions and the ligand showed a good ability to lower the toxicity of Cu(II), Mn(II), Co(II), Ni(II), Pd(II) ions. These metal complexes with lower toxicity represents promising candidates for future investigation regarding their biological effects.