Development and molecular modeling of Co(II), Ni(II) and Cu(II) complexes as high acting anti breast cancer agents

2.1 Molecular docking

To ascertain the mode of action of the synthesized metal complexes molecular docking calculations are carried out using a biopredicta module of the V life MDS 4.3. Virtual analysis was carried out using the crystal structure of the Human CDK 7 (PDB ID: IUA2) downloaded from Protein Data Bank (www.rcsb.org/pdb) at a resolution of 3.02 Å. Protein structure was refined via deletion of all the heteroatoms including water molecules and addition of the polar hydrogen atoms to get a native conformation. All other bonds were allowed to be rotatable. Structures of the synthesized metal complexes are drawn in the builder module of the V life MDS 4.3 engine. The 2D structures of the molecules were converted into the 3D structures are optimized via application of the MMFF force field. These optimized structures were further utilized for the docking analysis. All calculations were performed on an Intel i3, based machine running windows 7 as operating system. The docked protein ligand complex was further analyzed via docking score of the each of the complex, which is nothing but the binding energy of the complex, for every derivative best 100 binding conformation was analyzed to select the best conformation having the minimum energy of the binding.

3.1 Elemental and physical data

The Schiff base used to be soluble in ethanol, methanol, acetone, DMF and DMSO. The entire metal complexes are colored, non-hygroscopic solid which might be steady in air, insoluble in water and organic solvents but soluble in DMSO and decomposed at higher temperature. The purity of ligands and their metal complexes has been checked with the aid of TLC. Based on elemental and spectral studies the geometry of synthesized compounds has been elucidated. By the elemental analysis, the stoichiometry of ligand and their metal complexes is confirmed. The elemental analyses of ligand and metal complexes are found in agreement with the proposed structure of ligand and the metal complexes. Analytical and physical data of the Schiff base(HL) and Co(II), Ni(II) and Cu(II) complexes are listed in Table 1.

3.2 Electronic spectral analysis and magnetic studies

The electronic spectra and magnetic moments of the compounds are very useful in the evaluation of results acquired by using different ways of structural investigation. The information regarding the geometry of the complexes around the Co(II), Ni(II) and Cu(II) ion was obtained from electronic spectral studies and magnetic moments. The electronic spectra of ligand and their metal complexes were recorded at room temperature utilizing DMSO as a solvent.

The electronic spectra of ligand exhibit band at 29,585 cm⁻¹ which will also be assigned to n → π^∗ transition of azomethine group. Within the spectra of complexes, the bands of azomethine chromophore because of n → π^∗ transition are shifted to lower frequencies indicate that imine nitrogen is concerned in the coordination of metal ion.

Co(II) complex exhibits two absorption bands at 10,810 cm⁻¹ (γ₁) and 21,276 cm⁻¹ (γ₃) which might be assigned to ⁴T_1g(F) → ⁴T_2g (F) (γ₁); ⁴T_1g(F) → ⁴T_1g (P) (γ₃) transitions (Avji et al., 2008; Kulkarni et al., 2011). These are attribute bands of high spin octahedral Co(II) complexes; γ₂ is not observed, but it may be calculated by using relation γ₂ = γ₁ + 10Dq, which may be very nearly (γ₃) transition. These transitions propose a octahedral environment around Co(II) ion which was once extra supported by using its magnetic moment value of (μeff) 4.89BM.

Ni(II) complex generally shows three absorption bands in octahedral environment corresponding to ³A_2g (F) → ³T_2g (F) (γ₁), ³A_2g (F) → ³T_1g (F) (γ₂), and ³A_2g (F) → ³T_1g (P) (γ₃) transitions (Cotton et al., 2003). Ni (II) complex exhibits above three transitions at 9891 cm⁻¹ (γ₁), 16,129 cm⁻¹ (γ₂), and 24,992 cm⁻¹ (γ₃) suggesting octahedral geometry which is further proven through its magnetic moment value of (μ_eff) 3.45 BM.

The electronic spectra of Cu(II) complex show large band at 17,993 cm⁻¹ which could also be cheap be assigned to ²B_1g → ²A_1g (γ₁) transition, consistent with a square planar geometry around Cu(II) (Osman et al., 2004) which is further verified with the aid of its magnetic of moment value (μeff) 1.92 BM. The ligand field parameters like crystal field stabilizing energy (Dq), Racah parameter (B), Nephelauxetic ratio (β) and β% have been calculated for Co(II), Ni(II), Cu(II) complex and observed data are shown in Table 2.

3.3 IR and ¹H NMR spectroscopic studies

The prominent bands observed in IR spectra of Schiff base(HL) and its complexes are represented in Table 3. The IR spectrum of free ligands shows a strong band at 1590 cm⁻¹ assigned to ν(N⚌CH) indicating the formation desired shift base ligand. This used to be further proven by the presence of a new band observed in 483–515 cm⁻¹ region assigned to ν(M—N), which is coordination of metal to azomethine nitrogen. The Schiff base (HL) (Fig. 1) a band at 2760 cm⁻¹ is assigned to ν(SH) vibration (Singh et al., 2006a,b). This band disappeared in the spectra of the metal complexes indicating deprotonation and complexation via sulfur. In the spectra of Schiff base a band due to tautomeric form of C⚌S is appeared at 1114 cm⁻¹, in metal complexes this peak was disappeared.

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Figure 1

IR spectra of Schiff base (HL).

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Co(II), Ni(II) and Cu(II) complexes (Fig. 2a–c) show band at 350–379 cm⁻¹ have been assigned to ν(M—S) (Liver, 1968). Within the spectra of all complexes, bands appeared at 718–785 cm⁻¹ and 350–379 cm⁻¹ were assigned to ν(C—S) and ν(M—S), respectively (Osman et al., 2004; Singh et al., 2006a,b). In the spectra of Co(II) and Ni(II) complexes, a broad band in the region 3206–3395 cm⁻¹ indicated the presence of coordinated water molecules. The presence of water molecule was also confirmed by thermal analysis.

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Figure 2

(a–c): IR spectra of Co(II), Ni(II) and Cu(II) complexes.

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The ¹H NMR spectrum of Schiff base was recorded in DMSO, using Tetramethylsilane (TMS) as an internal standard. The ¹H NMR spectrum of ligand shows characteristic azomethine proton singlet at δ 11.07 ppm. The signal at δ 13.27 ppm is ascribed to SH proton. The aromatic proton of Schiff base appeared as multiplate at δ 7.27–7.99 ppm and singlet at δ 2.28 ppm is due to methyl group. Since Co(II), Ni(II) and Cu(II) complexes are paramagnetic, their ¹H spectra could not be obtained (Canpolat and Kaya, 2005). ¹H NMR spectrum of Schiff base(HL) is indicated in Fig. 3.

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Figure 3

¹H NMR spectra of Schiff base(HL).

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3.4 Thermal analysis

Thermal decomposition of Co(II), Ni(II) and Cu(II) complexes has been studied as a function of temperature by means of TGA-DTA method. The decomposition temperatures, paralyzed products, percentage mass losses of the complexes and ash percentage are listed in Table 4. The Co(II) and Ni(II) complexes exhibit three steps of decomposition. In first step, decomposition takes place from 50 to 180 °C due to total cleavage of the base metal complex along with hydroxide to oxide transformation followed by concomitant release of water molecules corresponding to loss of coordinated water molecules. The second decomposition takes place from 175 to 425 °C attributed to the release of organic moiety. Third decomposition around 400–550 °C can be attributed to the release of triazole moiety. The decomposition of both the complexes ended with oxide formation above 550 °C. For Cu(II) complex, first step of decomposition at 50–425 °C corresponds to loss of organic moiety while second step corresponds to removal of triazole molecules at 425–750 °C. After 750 °C metal oxide is formed as a residue. Thermal curve analysis of Co(II) complex is shown in Fig. 4.

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Figure 4

TGA-DTA curve of Co(II) complex.

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3.5 Mass spectroscopic studies

The mass spectrum of ligand reveals parent peak due to molecular ion (M⁺). The proposed molecular formulation of this compound used to be proven through comparing their molecular formula weight with m/z value. The molecular ion peak used to be acquired at m/z 264. This worth is in excellent agreement with the proposed molecular components of compound. A mass spectrum of Schiff base (HL) is exhibited in Fig. 5.

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Figure 5

Mass spectrum of Schiff base (HL).

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3.6 X-ray diffraction analysis

The X-ray diffraction measurements were obtained using a X-ray diffractometer (Rigaku Ultima IV, Japan made). The Cu Kα radiation tube with line focus was operated at 40 kV and 40 mA. The X-ray powder diffraction patterns were obtained in the range of 20–80° in steps of 0.0990. X-ray powder diffraction analysis of the ligand and its metal complexes was carried out to determine the type of crystal system, lattice parameters and the cell volume. As shown in Fig. 6(a) and (b) the XRD patterns indicate a crystalline nature for the ligand and Cu(II) complex, while Ni(II) and Co(II) complexes have amorphous nature. Indexing of the diffraction patterns was performed using High Score Plus software. For the ligand and Cu(II) complex d values, FWHM and relative intensities are given in Tables 5 and 6. From the indexed data the unit cell parameters, volume, space group and average crystallite size were also calculated and are listed in Table 7. The powder XRD patterns of the Cu(II) complex are completely different from those of ligand. XRD studies reveal that ligand has an orthorhombic structure, while Cu(II) complex has tetragonal structure. The full width at the half-maximum (FWHM) of the diffraction peaks obtained from the refinement was used to calculate the particle size. The X-ray diffraction data were determined by using Cu Kα radiation (λ = 1.54060 Å). The average size of the samples was calculated with the help of the Debye-Scherrer equation, $$D=(0.90\lambda )/(\beta \text{Cos}\theta )$$ where λ is the wavelength (Cu Kα), β is the full width at the half maximum (FWHM) and θ is the diffraction angle. The diffraction peaks indicate that the synthesized materials are in the nanometer range.

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Figure 6

XRD pattern of (a) ligand and (b) Cu(II) complexes.

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3.7 Biological activity studies

All the newly synthesized compounds were screened for their anticancer activity using SRB assay on the MCF 7 breast cancer cell line. In the current protocol each cell line is inoculated on a pre-incubated microtiter plate. The test agents are added at a single concentration and the culture is incubated for 48 h. End point determinations are made with Sulforhodamine B, a protein binding dye. The results for each test agents are reported as the percentage growth of the tested cells. The compounds that reduce the growth of any one of the cell lines to 32% or less (negative numbers indicates cell kill) are passed on for evolution over a 5-log dose range. In the present screening program all the compounds possessed growth to less than 32% are regarded as active compounds (Holla et al., 2003, 2006). Adriamycin was served as positive control compound in the cytotoxic assay. The observed results are listed in Table 8.

3.8 Structure activity relationship (SAR studies)

In Fig. 7 of Co(II), Ni(II) and Cu(II) complexes are found to be active against MCF 7 cell line up to molar dose of 10⁻⁴ M which indicates their anticancer potential. SAR analysis of the synthesized complexes indicated following points,

• 1,2,4-Triazole shows moderate activity against MCF −7 cell line. HL shows GI₅₀ value, 64.1 which indicates the substitution of the good electron withdrawing group —NO₂ sufficiently affecting the polarity of the molecules.

• Cobalt metal complex showing profound activity than the corresponding nickel and copper complexes. Chemical properties of copper make it to take part in number of biological process such as electron transfer and catalysis. Cobalt complex has ability to kill cancer cells are correlated with the induction of the oxidative stress. Cobalt is an important trace elements for humans in the form of vitamin B12 (Cobalamin), and this metal plays a vital role in Protein synthesis.

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Figure 7

Growth curve: human breast cancer cell line MCF-7.

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3.9 Molecular docking studies

In order to understand the possible mode of action of the synthesized metal complex the molecular docking analysis was carried out using crystal structure of the Human CDK 7. Optimized structure of the metal complexes was utilized for the grip based docking simulation was protein structure is kept rigid and ligand structure is kept flexible. All the molecules were docked into the similar binding site of CDK 7 with binding energy ranging from −33.57 kcal/mol to 96.36 kcal/mol. Nickel complex is most active molecule having binding score of −84.54 kcal/mol having critical interaction with amino acids such as ASN142 (Hydrogen Bond interaction), GLU99 and ASP97 (Charge Interaction) and hydrophobic interaction with GLY21 and GLU99. Hydrogen Bond Interactions and Charge Interactions of Schiff base (HL) and Co(II) Complex are shown in Fig. 8(a) and (b) respectively. Binding energy and critical interaction of all the compounds are given in Table 9. Correlation of binding energy and GI₅₀ of derivatives of Schiff base and metal complexes are shown in Fig. 9.

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Figure 8

(a): Hydrogen bond interactions and charge interactions of HL; (b): hydrogen bond interactions and charge interactions of HL-Co.

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Figure 9

Correlation of binding energy and GI₅₀ of HL and its complexes.

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