New Mn(II), Ni(II), Cd(II), Pb(II) complexes with 2-methylbenzimidazole and other ligands. Synthesis, spectroscopic characterization, crystal structure, magnetic susceptibility and biological activity studies

2.1 Material and measurements

All chemicals used in this study were obtained from commercial sources and used without further purifications (MnCl₂ 4H₂O, NiCl₂ 6H₂O, CdCl₂ H₂O, Pb(NO₃)₂ obtained from BDH. (2-Methylbenzimidazole, benzotriazole, N-acetylglycine, ethanol, DMSO) obtained from Merck.

Measurement of UV–Vis spectra was carried out using a Shimadzu UV–Vis spectrophotometer UV-160 in 10⁻³ M DMSO solution. IR spectra (KBr disc) in the region of 4000–400 cm⁻¹ were recorded using a Shimadzu FTIR 8400 S Fourier transform infrared spectrophotometer. ¹H NMR, ¹³C NMR spectra were recorded using a Bruker DPx-400 spectrophotometer relative to the internal standard, tetramethylsilane (TMS). Philips pw 9526 digital conductivity meter was used to measure molar conductivity. The magnetic susceptibility was measured at 25 °C using a balance magnetic susceptibility apparatus MK1. Elemental analysis (C, H, N) was performed on a Perkin Elmer B-240 Elemental Analyzer. Determination of metals was carried out using an Atomatic absorption spectrometer A-Analysis 800 Perkin Elmer. Finally, the melting points were determined using a stuart-melting point apparatus. Crystal data collection: APEX2 (Bruker, 2006); Cell refinement: SAINT (Bruker, 2006); data reduction: SAINT; program used to solve structure: SHELXS 97 (Sheldrick, 2008); program used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: xp in SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97 and publCIF (Westrip, 2010).

2.2 Preparation of [Cd₂(B)₄(NCS)₄]

An ethanolic solution (15 ml) of 2-methylbenzimidazole (1.32 g, 10 mmol) was added to an aqueous solution (20 ml) of CdCl₂ H₂O (0.5 g, 2.5 m mol) followed by the addition of an aqueous solution (15 ml) of KSCN (0.49 g, 5 mmol). The mixture was heated in a water bath for 30 min with constant stirring. The resulting precipitate was filtered off and recrystallized from ethanol to give the white crystals of Cd(II) complex (Shayma et al., 2010b).

2.3 Preparation of [Mn(B)₂(Gly)]Cl and [Pb(B)₂(Gly)] NO₃

An ethanolic KOH solution (10 ml) of N-acetylglycine (Gly) (0.176–0.295 g) (1.5–2.51 mmol) and an ethanolic solution (12 ml) of 2-methylbenzimidazole (0.399–0.667 g) (3–5 mmol) were added respectively to an aqueous solution of metal salts, after constant stirring on water bath. The product precipitates immediately, filtered off, washed then recrystallized with 12 ml of ethanol and dried in an oven at 65 °C.

2.4 Preparation of [Ni(B)₂(BT)(Gly]Cl

An ethanolic solution (12 ml) of 2-methylbenzimidazole (0.556 g, 4 mmol) and an ethanolic solution (14 ml) of benzotriazole (0.25 g, 2 mmol) were added respectively to an aqueous solution of NiCl₂ 6H₂O. An ethanolic KOH solution (12 ml) (0.246 g, 2 mmol) was added to the mixture with constant stirring. The product was immediately precipitated which was then filtered off, washed and recrystallized with ethanol (12 ml) and dried in an oven at 65 °C.

2.5 Cell culture

All the cells used in this study were obtained from American Type Cell Collection (ATCC) and maintained in a 37 °C incubator with 5% CO₂ saturation. A375 human melanoma, MCF-7 human breast adenocarcinoma cells and WRL-68 normal hepatic cells were maintained in Dulbecco's modified Eagle's medium (DMEM). Whereas A549 non-small cell lung cancer cells were maintained in RPMI medium. Both medium were supplemented with 10% fetus calf serum (FCS), 100 units/mL penicillin, and 0.1 mg/ml streptomycin.

2.6 Cellular viability

Different cell types from above were used to determine the inhibitory effect of compounds CdB, MnB, NiBT and PbT on cell growth using the MTT assay. The MTT assay was modified as described by (Mosmann, 1983; Cheah et al., 2011). Briefly, cells were seeded at a density of 1 × 10⁵ cells/mL in a 96-well plate and incubated for 24 h at 37 °C, 5% CO₂. Next day, cells were treated with the compounds respectively and incubated for another 24 h. After 24 h, MTT solution at 2 mg/mL was added for 1 h. Absorbance at 570 nm was measured and recorded using Plate Chameleon V microplate reader (Hidex, Turku, Finland). Results were expressed as a percentage of control giving percentage cell viability after 24 h exposure to test agent. The potency of cell growth inhibition for each test agent was expressed as an EC₅₀ value, defined as the concentration that caused a 50% loss of cell growth. Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells.

2.7 Statistical analyses

Each experiment was performed at least two times. Results are expressed as mean value ± standard deviation (SD). Log EC₅₀ calculations were performed using the built-in algorithms for dose–response curves with variable slope using GraphPad Prism software (version 4.0; GraphPad Software Inc., San Diego, CA). A fixed maximum value of the dose–response curve was set to the maximum obtained value for each drug.

3.1 Electronic spectral studies and magnetic measurements

The electronic spectra of 2-methylbenzimidazole (B) in DMSO show bands at 274, 280 nm and 350 nm which can be attributed to π → π^∗ and n → π^∗ respectively (Shayma, 2010a; Shayma et al., 2011). The benzotriazole (BT) spectrum shows two strong bands assigned to π → π^∗ and n → π^∗ at 273 nm and 312 nm respectively. The electronic spectra of N-acetylglycine (Gly) show band at 284 nm which is due to π → π^∗ and another band at 340 nm attributed to n → π^∗ (Shayma, 2010c).

The electronic spectra of Mn(II) complex displayed two bands in the visible region at 425 and 598 nm which attributed to the electronic transition ⁶A₁ → ⁴T_2(G) and ⁶A₁ → ⁴T_1(G) respectively. Thus the magnetic moment value is 4.652 B. M, demonstrates that the Mn(II) complex is paramagnetic and has high spin tetrahedral geometry (Lever, 1968).

The gray octahedral complex of Ni(II) shows three bands in the visible region at 450, 765 and 920 nm which are assigned due to ³A₂g_(F) → ³T₂g_(F) (ν1), ³A₂g_(F) → ³T₁g_(F) (ν2) and ³A₂g_(F) → ³T₁g_(P) (ν3) respectively. The ligand field parameter 10Dq is 10869 cm⁻¹ for (ν1). Moreover, the interelectronic repulsion parameter B was calculated and found to be 179 cm⁻¹, this value is less than the free Ni²⁺ ion value of 1040 cm⁻¹ which was due to overlapping and delocalization of electrons over the molecular orbital that encompasses both the metal and ligands, the nephelauxetic ratio β = B/B^ο = 0.172 indicates appreciable covalent character in this ligand (Vidyavati et al., 2008; Antonio et al., 2008). The μ_eff value of Ni(II) d⁸ was 2.451 B M, this value was in agreement with the expected value (Nicholls, 1984).

Finally, Cd(II) and Pb(II) complexes did not show any d–d electronic transition in the visible region but shows absorption bands at 279, 280 and 320, 345 and 350 nm which are attributed to the charge transfer MLCT. All the absorption bands were fully assigned in Table 2.

3.2 Infrared spectra studies

The important infrared frequencies exhibited by the ligands and their complexes are listed in Table 3. A comparison of IR spectra of the ligands with their complexes is based on earlier studies of similar ligand (Vidyavati et al., 2008). The spectrum of the free ligand B shows a broad strong band at 3160 cm⁻¹ which is due to ν(NH) (Overberger et al., 1965; Rabiger and Joullie, 1964). Other bands were observed at 3040 cm⁻¹ and 2830 cm⁻¹ which can be attributed due to the ν(C–H) aromatic and aliphatic vibrations respectively (Cross and Alan, 1969; Dyer, 1965; Williams and Fleming, 1973). Moreover, the ν(C⚌N) band of the ligand B was observed at 1660 cm⁻¹ and this band was shifted to the lower frequencies by 20–5 cm⁻¹ in the spectra of the complexes. This indicates that the ligand B was coordinated with the metal ions through the N atom. However, the spectrum of BT exhibited a weak band at 3247 cm⁻¹ which is due to ν(NH). Thus, the band appeared at 1500 cm⁻¹ belongs to ν(N⚌N) in BT and was shifted to lower frequencies by 55 cm⁻¹ in the spectra of Ni(II) complex (Shayma, 2010a; Cross and Alan, 1969; Dyer, 1965; Williams and Fleming, 1973). The infrared spectrum of (Gly) shows an absorption band at 3380 cm⁻¹ which is due to ν(NH). On Mn(II), Ni(II) and Pb(II) complexes formation, this band was shifted to the lower frequencies by 60–50 cm⁻¹. So, it is noted both of the ν(COO) asymmetric and symmetric bands of the ligand Gly exhibited at 1740 and 1370 cm⁻¹. On Mn(II), Ni(II) and Pb(II) complexes these two bands were shifted to the lower frequencies by 10–5 cm⁻¹. This indicates that the ligand Gly was a bidentate ligand which coordinated with the metal ions through the N atom and a terminal carboxyl oxygen atom (Shayma, 2010c).

The potassium thiocyanate spectrum showed a very strong band at 2048 cm⁻¹ which is a result of ν(C≡N). This band was shifted to 2060 and 2130 cm⁻¹ in Cd(II) complex which attributed to the bridge group ν (Cd–NCS–Cd) and terminal group ν (Cd–NCS). Because the NCS group forms a bridge between two metal atoms, and the stretching frequency of a bridging group is generally higher than of a terminal group (Nakamoto, 1995; Socrates, 1980; Shayma and Yang, 2009). Moreover, all the complexes appeared as weak bands in the region 450–490 cm⁻¹ which are due to ν (M–O) indicated that the ligand Gly was coordinated with the Mn(II), Ni(II) and Pb(II) complexes through the two O atoms. While, the bands between 500 and 550 cm⁻¹ which are attributed to ν (M–N) indicate that the free ligands B, BT and isothiocyanate were coordinated with the metal ions through the N atom.

3.3 ¹H NMR spectral studies

The ¹H NMR spectra of the free ligand B show a signal proton at 12.3 ppm which is due to NH. This signal showed at 12 and 11.8 ppm in the Cd(II) and Pb(II) complexes respectively. Actually, the appearance of NH group in both the complexes confirmed that the coordination is impossible through N1. Thus the spectrum of the ligand B shows signal protons at 7.53, 7.17, 7.30 and 7.50 ppm which are attributed to H4, H5, H6 and H7 respectively. In the Cd(II) and Pb(II) complexes, the signals of H5 and H6 were shifted downward to 7.18–7.20 ppm and 7.32–7.39 ppm respectively. While the signals of H4 and H7 were shifted to higher values between 7.45–7.50 and 7.40–7.49 ppm respectively (Haggag, 2007; Belanger et al., 1997).

The N-acetylglycine shows single signal at 2.00 ppm which is due to the CH₃ group. Thus, the spectrum of the ligand shows two singlet signals at 6.34 and 4.01 ppm which could be due to NH and CH₂ groups respectively. In the Pb(II) complex the signal due to NH group was shifted downfield at 6.50 ppm. While, the signal of CH₂ group was shifted upfield at 3.35 ppm (Irene et al., 2006).

3.4 ¹³C NMR spectral studies

The ¹³C NMR spectra of the ligand B had shown signals at 130.1, 120.74, 113.62 and 140.9 ppm due to (C8, 9), (C5, 6), (C4, 7) and C2 respectively. On the Cd(II) and Pb(II) complexes, only the signal due to C2 was deshielded to the downfield by 11.60–11.53 ppm.

The ¹³C NMR spectra of the ligand Gly exhibited signals at 179.3, 175.98, 45.20 and 25.55 pm due to C1, C3, C2 and C4 respectively. The signals of C1 and C2 were shifted downfield at 180.05 and 46.3 ppm respectively on the Pb(II) complex. These signals indicated that the N-acetylglycine was coordinated with the Pb(II) ion through the N and O atoms (Ramadan et al., 1997; Noriko et al., 2006).

3.5 Crystal structure of [Cd₂(B)₄(NCS)₄]

Structure of the complexes is presented in Fig. 1 and the crystal data and structure refinement are listed in Table 4. Hydrogen bonding interactions in the crystal structure are given in Table 5.

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Figure 1

The molecular structure of [Cd₂(B)₄(NCS)₄] showing displacement ellipsoids at the 50% probability level.

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3.6 Cytotoxic activity

To evaluate the cytotoxic activity, different single compounds namely CdB, MnB, NiBT and PbT were tested with a series of different doses on A375, A549, MCF-7 and WRL-68, respectively Fig. 2. After 24 h, cell viability was determined by the MTT assay. Test agents induced cell cytotoxicity in a concentration dependent manner. These dose titration curves allowed determining EC₅₀ for the test agents toward different cell lines Table 6.

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Figure 2

Dose–response curves (using GraphPad Prism) tested with Mn(II), Ni(II), Cd(II) and Pb(II) complexes in the MTT assays toward A) A375, B) A549, C) MCF-7, D) WRL-68 where MnB = [Mn(B)₂(Gly)]Cl, NiBT = [Ni(B)₂(BT)(Gly]Cl, CdB = [Cd₂(B)₄(NCS)₄] and PbB = [Pb(B)₂(Gly)] NO₃.

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From Fig. 2 CdB showed cytotoxic effect on several of the cancer cell lines with different EC₅₀ values. Cd(II) complex induced cell cytotoxicity in a concentration dependent manner. These dose titration curves allowed determining EC₅₀ for the various compounds toward different cell lines. Cd(II) complex demonstrated dose-depended cytotoxic effects with EC₅₀ values of 8.404 ± 1.4, 45.36 ± 3.7, 5.7 ± 0.9, and 6.7 ± 0.7 μg/mL; in A375, A549, MCF-7 and WRL-68, respectively.

However, Mn(II), Ni(II) and Pb(II) complexes showed no cytotoxic effect on different cell lines. These results indicate that cell lines differ in their sensitivity to the same test agent, which may be determined by multiple cell type-specific signaling cascades and transcription factor activities.

Cytotoxic screening models provide important preliminary data to select plant extracts or natural compounds with potential anticancer properties. In this study, the cytotoxic effect of Cd(II) complex was investigated by the addition of the MTT tetrazolium salt to various cancer cell lines previously treated with Cd(II) complex. Taken together, the cytotoxic effects exerted by Kunstlerone as a promising compound suggest its potential as anti-proliferation agent. To our knowledge, the cytotoxic potentials of Cd(II) complex have not been examined and the underlying molecular mechanisms remain to be discovered (Mosmann, 1983; Cheah et al., 2011).