Structural Characterization, DFT Calculations, Metal Uptake, Fluorescence, Antimicrobial and Molecular Docking Studies of Novel Co(II) and Ni(II) Complexes with NNS Tridentate Schiff base Ligand

A new Schiff base ligand N 2 -((5-methylthiophen-2-yl) methylene) pyridine-2,6-diamine (L) was prepared by condensation of 5-Methyl-2-thiophenecarboxaldehyde and 2,6-diaminopyridine in ethanol in a molar ratio 1:1. The ligand and its complexes were characterized based on elemental analyses, molar conductance, magnetic moment, IR, MS, 1 H NMR, solid reectance, and thermal analysis (TG and DTG) techniques. The complexes were found to have the formulas [CoL(H 2 O) Cl 2 ].H 2 O and [NiL(H 2 O)Cl 2 ].2H 2 O. From FTIR spectral data, the coordination between the ligand L to the central metal ion was through its nitrogen of pyridyl and azomethine and sulfur of 2-thiophenecarboxaldehyde. The metal complexes were found to be nonelectrolyte. Octahedral geometries of the Co(II) and Ni(II) complexes were investigated from electronic and magnetic data. The kinetic analysis of the thermogravimetric data was performed by using the Coats-Redfern equation. The uorescence properties of the ligand and its complexes in DMF were studied. The values of optical band gap energy (Eg) of the synthesized complexes suggested that these compounds could be used as semiconductors. The adsorption of Co 2+ and Ni 2+ in aqueous solutions on ligand under various conditions was studied. The maximum adsorption percentage of Co(II) and Ni(II) were found to be 68% and 65%, respectively. The Molecular docking studies were executed to consider the nature of binding and binding anity of the synthesized compounds with the receptor of Bacillus subtilis (gram +ve bacteria) and Escherichia coli (gram –ve bacteria) (PDB ID: 1fj4). The ligand and its complexes were tested as antimicrobial agents.


Introduction
Transition metal complexes with nitrogen, sulphur, or oxygen as ligand atoms were becoming more important because these Schiff bases can bind with various metal centers involving various coordination sites, allowing for the successful synthesis of metallic complexes with interesting stereochemistry [1,2]. Moreover, Sulphur and nitrogen-containing ligands and their transition metal complexes were employed as corrosion inhibitors [3,4], extreme pressure lubricant additives [5] and showed broad biological activity [6-10] due to the different ways in which they were bonded to metal ions. Heterocyclic compounds such as pyridine, 2,6-diaminopyridine, and related molecules are good ligands because of the presence of at least one ring nitrogen atom with a localized pair of electrons [11,12]. Heterocyclic compounds were the most abundant in nature and were required for a wide range of applications in biology including antibacterial, antifungal, anticancer, antioxidant, anti-in ammatory activity, and also in analytical processes [13][14][15][16][17]. These heterocycle-containing compounds have important properties in the elds of material science and biological systems [18,19].
In the present study, we reported the synthesis and characterization of a novel Schiff base derived from the condensation of 5-Methyl-2-thiophenecarboxaldehyde and 2,6-diaminopyridine, and its complexes with Co(II) and Ni(II). There were many spectroscopic tools utilized to characterize the synthesis complexes elemental analysis, molar conductivity, 1 H NMR, IR and solid re ectance, magnetic moment, thermal analyses (TG/DTG), and uorescence spectra studies.

Materials and reagents
5-Methyl-2-thiophenecarboxaldehyde, 2,6-diaminopyridine and the metal salts; CoCl 2 .6H 2 O and NiCl 2 .6H 2 O were brought from sigma Aldrich company. All solvents were of analytical reagent grade (AR) and had the highest purity available. They were used without further puri cation.

Instrumentation
2.3 Synthesis of the Schiff base ligand N 2 -((5-methylthiophen-2-yl) methylene) pyridine-2,6-diamine (L) The Schiff base, L ligand was synthesized by adding the ethanolic solution of 5-Methyl-2-thiophenecarboxaldehyde (1.13 g, 9 mmol) with a solution of 2,6-diaminopyridine (0.98 g, 9 mmol) in hot ethanol and stirring for 8 hours. The color of the reaction mixture was changed from pale yellow to brown color with the formation of the precipitate. The isolated brown Schiff base was washed several times with diethyl ether and dried in a vacuum.

Preparation of the metal complexes
A general procedure was employed for the synthesis of the reported complexes by adding equimolar amounts of a methanolic solution of the selected metal salts to THF solution of Schiff base L ligand in the molar ratio 1:1. The reaction mixtures were heated to re ux for 6 hours. The resulting solution was concentrated by evaporation and the precipitate was then isolated, washed with hot petroleum ether several times and then the complex was left to dry in vacuo for several hours. Table 1 gave the reaction period, the color of complex and % yield.

Metal ion uptake study
The metal ion uptake study was done by batch technique. A batch experiment was carried out to investigate the adsorption of Co(II) and Ni(II) ions using ligand L. The adsorption tests were performed by introducing 5 mg ligand in a ask containing 10 mL of the prepared aqueous solution of each of CoCl 2 .6H 2 O and NiCl 2 .6H 2 O (0.01M, 0.001M and 0.0001M) in a ask at room temperature and ltered after different times 1, 2 and 5 hours. Then, the supernatant was ltered and the concentration of the residual metal was determined complexometrically by titration against a standard solution of EDTA-Na using the suitable indicator at a suitable pH value [20]. The removal percentage % of metal was calculated using the following equation: Where: C o is the initial concentration of metal ions (mg/L). C e is the concentration of metal ions at time t (mg/L).

DFT calculations
Density functional theory (DFT) for geometry optimization and frontier molecular orbitals were calculated for the ligand and its complexes. B3LYP functional was employed with a 6-311G++(d,p) basis set for C, H, N, O, S and Cl and LANL2DZ for cobalt and nickel in the gas phase [21]. All calculations were performed by using the Gaussian 09 program package [22].

Microbiological investigation
The synthesized metal complexes were screened for antibacterial activity against two local Gram-positive bacterial species (Streptococcus pneumonia and Bacillus subtilis) and two local Gram-negative bacterial (proteus vulgaris and Escherichia coli) on nutrient agar medium (NA). Also, the antifungal activities were tested against two local fungal species (Aspergillus fumigatus and Candida albicans) on Sabouraud Dextrose Agar (SDA) medium. Gentamycin and Ketoconazole were used as standard references for Gram-positive & Gram-negative bacteria and fungi, respectively served as positive controls. In the agar, well diffusion method, the wells (6 mm in diameter) were dug in the media with the help of a sterile borer with centers at least 24 mm apart [23]. The concentration of the test samples (1 mg/ml in DMF) was introduced in the respective wells. The plates were then incubated at 37 ºC and 25 ºC for bacteria and fungi, respectively.
Generally, the antimicrobial agent diffuses in the agar medium and inhibits the germination and growth of the test microorganism and then the diameters of inhibition growth zones were measured in millimeters, after 24 h for bacteria and 48 h for fungi as an indicator for antimicrobial activity and the obtained results are represented as no activity, low activity, intermediate activity and high activity. The assay was carried out in triplicate. The zone diameters values were averaged and the mean values were tabulated.

Molecular docking
Molecular docking studies were achieved with MOA2019 software [24], to nd out the possible binding modes of the ligand and the complexes with the receptor of gram -ve bacteria (Escherichia coli) (PDB ID: 1fj4) [25] and gram +ve bacteria (Bacillus subtilis) (PDB ID: 1QD9) [26]. The optimized structure of ligand and complexes from the output of Gaussian09 calculations were created in PDB le format. The crystal structures of the receptors were downloaded from the protein data bank (http://www.rcsb.org/pdb).

Results And Discussion
In this work, The Schiff base ligand N 2 -((5-methylthiophen-2-yl) methylene) pyridine-2,6-diamine and its Co(II) and Ni(II) complexes were synthesized and characterized by several analytical and spectroscopic techniques. Several attempts to create single crystals of complexes suitable for crystal analysis failed due to their poor solubility in common organic solvents. Elemental analyses and some physical properties of the ligand and its complexes were listed in Table 1.

Elemental analysis and physical properties
The analytical & physical data of the Schiff base ligand (L) and its metal(II) complexes were supported the proposed structure for all compounds as 1:1 metal to ligand stoichiometry ( Table 1). The presence of hydrated/ coordination water molecules was supported by TG/DTG analysis. Additionally, all the resulting complexes were colored, stable at room temperature, non-hygroscopic, and insoluble in the most common organic solvents.

Conductivity and magnetic measurements
The molar conductivities of Co(II) and Ni(II) complexes were measured in DMF (10 −3 M) as shown in Table 1. The observed values for Co(II) and Ni(II) complexes showed non-electrolyte natural. The observed magnetic moment values for Co(II) and Ni(II) complexes were found to be 4.6 and 3.85 B.M., respectively [27,28]. These values suggested a coordination number of six for the central metal ions and an octahedral geometry [27,29].

IR spectra studies
The IR spectra of the Co(II) and Ni(II) complexes were compared with those of the free ligand to determine the involvement of coordination sites in chelation ( Table 2). Table 2 Most important IR spectral bands of the ligand (L) and its metal complexes.
The IR spectrum of the Schiff base ligand ( Fig. 1) showed the appearance of two bands at 3464 and 3386 cm −1 which may be assigned to ν as (NH 2 ) and ν s (NH 2 ) [30]. The IR spectra for Co(II) and Ni(II) complexes exhibited broad band at 3379 and 3376 cm −1 , respectively assigned to ν(OH) of crystalline or coordinated water molecules associated with the complex. This band may be overlapped with the band corresponding to the stretching vibration of ν(NH 2 ) group [31]. The IR spectrum of the ligand exhibits a band at 1613 cm −1 due to ν(C=N) of the azomethine group [32]. After complexation, the peak assigned to ν(C=N) was moved to a higher region by about 17-31 cm −1 suggesting coordination through N atom of the azomethine group [33]. Also, the in-plane deformation band of pyridine was observed for Schiff base at 626 cm −1 and on complexation was shifted to 631 and 630 cm −1 for Co(II) and Ni(II) complexes which indicated the coordination via pyridyl nitrogen atom [34]. The existence of coordinated water was proved by the appearance of the non-ligand band in the 987 and 926 cm −1 regions for Co(II) and Ni(II) complexes [35]. Appearance new bands in the spectra of Co(II) and Ni(II) complexes at 468 and 457 cm −1 were due to the formation of ν(M-S) bonds [36]. The proof of coordination to the N atom was provided by the occurrence of the bands 530 and 532 cm −1 in the IR spectra of the Co(II) and Ni(II) complexes due to ν(M-N) [37].

1 H NMR study
1 H NMR spectrum of the synthesized ligand (L) was measured in CDCl 3 , with TMS as internal standard. The spectrum of the prepared Schiff base ligand L displayed singlet signal at 4.32 ppm which was attributed to methyl protons of ligand L. The signal at 8.12 ppm was assigned to the azomethine proton (-CH=N-) [38]. Also, the spectrum showed a multiplet in the range 6.23-8.72 ppm corresponding to the aromatic protons and thiophene protons [39], and the appearance of a singlet signal at 9.53 ppm due to the proton of the NH 2 group of the ligand [40].

Mass spectra
The mass spectral analysis of the synthesized Schiff base ligand is essential as it is one of the methods to exactly con rm the proposed formula. The mass spectrum of the ligand, L (Fig. 2) showed an intense molecular ion peak at m/z 217.00 (35%) (calc. 217.28 amu) that indicates the formation of the desired compound with molecular formula C 11 H 11 N 3 S as suggested in Table 1. 3.6 Diffuse re ectance spectra The diffuse re ectance of the Schiff base ligand (L) exhibited band at 245 nm attributed to π-π* transitions in both aromatic benzene and thiophene rings and azomethine (-C=N) group [41]. Another band was displayed at 345 nm referred to n-π* transitions of the lone pair of the nitrogen in the azomethine group and sulfur in the thiophene ring [42].
In the complexes these bands were shifted to lower wavelengths as an outcome of coordination when binding with metal, thus con rming the formation of Schiff base metal complexes (Table 3) [43]. The diffuse re ectance spectrum of the Co(II) complex, exhibits two peaks at 614 and 698 nm assigned to the transition 4 T 1g (F)→ 4 T 2g (F) and 4 T 1g (F)→ 4 A 2g (F), respectively suggesting an octahedral geometry for this complex [44]. The diffuse re ectance spectrum of Ni(II) complex showed two peaks around 505 and 636 nm, attributed to 3 A 2g (F) → 3 T 2g (F) and 3 A 2g (F)→ 3 T 1g (P) transitions, respectively, indicating an octahedral geometry [45]. On the other hand, the diffuse re ectance spectra in both Co(II) and Ni(II) complexes showed two bands at 445 and 444 nm, respectively corresponding to charge transfer [46,47].

Fluorescence spectral studies
The uorescence properties of the ligand L and its metal complexes were investigated in DMF at room temperature (Table 4). Generally, Schiff base systems exhibited uorescence due to intra-ligand π-π* transitions, which showed an intense emission band at 347 nm upon photoexcitation at 309 nm. On the other hand, the excitation spectra of Co(II) and Ni(II) complexes exhibited uorescence emission bands at 370 and 455 nm when excited at 329 and 340 nm, respectively. It was found that the uorescence emission intensity of Schiff-base decreased on complex formation with metal ions as clear from Fig. 3 [48, 49]. In general, all the synthesized compounds can serve as potential photoactive materials, as indicated by their characteristic uorescence properties.

Thermal analyses
The thermal behaviors of the metal complexes were studied by using TG and DTG analyses, and the results were listed in Table 5 and represented in Fig. 4. The thermogram analysis (TGA/DTA) of the complexes is useful to prove the presence of water molecule inside or outside the coordination sphere and decomposition temperature of the compounds.

Kinetic data
The kinetic thermodynamic parameters of decomposition processes of complexes such as the energy of activation (E*), enthalpy (ΔH*), entropy (ΔS*) and free energy change (Gibbs free energy) of decomposition (ΔG*) were evaluated graphically by employing the Coats-Redfern [50,51] equation. The data were summarized in Table 6 of the supporting information.

Coats-Redfern equation
The Coats-Redfern equation, which is a typical integral method, can be represented as: For the convenience of integration, the lower limit T 1 is usually taken as zero. This equation on integration gives If a plot of the left-hand side against 1/T was drawn, E* (the energy of activation in kJ mol −1 ) was calculated from the slope and A in (s −1 ) from the intercept (Fig. 5). The entropy of activation ΔS* (in J K −1 mol −1 ) was calculated using the equation: where K B is the Boltzmann constant, h is the Planck constant and T s is the DTG peak temperature.
The entropy of activation was found to have negative values for Co(II) and Ni(II) complexes which indicated that decomposition reactions proceed with a lower rate than normal ones. Also, according to the kinetic data obtained, Co(II) and Ni(II) complexes have negative entropy, which indicated that activated complexes have more ordered systems than reactants.
The correlation coe cients of the Arrhenius plots of the thermal decomposition steps were found to range from 0.92-0.98, indicating good tness of the linear function. From all the previous data, the proposed structures of the ligand and its complexes were given in Fig. 6.

Optical properties
The optical band energy gap (E g ) for the Co(II) and Ni(II) complexes can be determined based on the dependence of absorption coef cient (α) on photon energy (hν). For transition, α is given by [52].
(αhν) 2/n = B (hν-E g ) Where α was the absorption coe cient and was calculated from the relation α = A/d (where A is the absorbance and d is the thickness of the cell), B is the optical constant, h is Planck's constant and was incident light frequency [53]. The optical absorption spectra of complexes were given in Fig. 7 and the values of bandgap energy for Co(II) and Ni(II) complexes were observed from the curves as 2.18 and 1.64, respectively. These results indicated that these complexes have highly e cient photovoltaic properties and could be used as semiconductors in solar cell programs [54,55].

Metal uptake
The adsorption of Co(II) and Ni(II) ions with different concentrations (0.01M, 0.001M and 0.0001M) using ligand L was studied at different times 1, 2 and 5 hrs.
From the data, the results showed that the removal percentage of Co(II) and Ni(II) metal ions increased as time increased (Fig. 8). The highest removal percentage at 0.01 M of Co(II) and Ni(II) was found to be 45.6, 29.7% and at 0.001 M of the metal ion was 55.4, 53.2% and at 0.0001 M the removal was found 65.8, 68.6%, respectively. Also, the results appeared that the percentage of the removal of the metal ion increased as the concentration of metal ion decreased [56] (Fig. 9).    3.12.3 Molecular electrostatic potential and molecular orbital Figure 11 showed the MEP surface is to locate the positive (blue color) and negative (red color, it is bound loosely or excess electrons) charged electrostatic potential in the molecule. The computed total energy, the highest occupied molecular orbital (HOMO) energies, the lowest unoccupied molecular orbital (LUMO) energies and the dipole moment for the ligands and complexes were calculated, Table 9. The more negative values of the total energy of the complexes than that of the free ligand indicate that the complexes are more stable than the free ligands and the energy gaps (E g = E LUMO -E HOMO ) are smaller in case of complexes than that of ligand due to chelation of ligand to metal ions, Table 9. The lowering of E g in complexes compared to that of ligand explains the charge transfer interactions upon complex formation, Fig. 12.

Reactivity studies
Many reactivity descriptors such as ionization potential (I), electron a nity (A), Electronegativity (χ), chemical potential (µ), hardness (η), softness (S) and electrophilicity index (ω), all derived from the HOMO and LUMO energies, have been proposed for understanding various aspects of reactivity associated with chemical reactions, Table 9.

Antimicrobial activities
The antibacterial activity of the Schiff base ligand and its complexes were tested against two gram-positive bacteria (S. aureus and B. subtilis), two gram-negative bacteria (P. vulgaris and E. coli) and two fungi (A. avus and C. albicans) (Fig. 13). The inhibition zone diameter (mm) of all investigated compounds in addition to the calculated percent activity index data were listed in Table 10. The activity index was calculated by the following relation [57]: A=I/I *100, where A= Activity index, I= Inhibition zone of the compound (mm) and I = Inhibition zone of the standard drug (mm) (Fig. 14).
The test was done using the agar well diffusion method. *% Activity index are in parentheses.

Effect of bacteria
The results revealed that: The Schiff base ligand showed remarkable potential activity against all bacteria under study except E. coli. It was a moderate activity against B. subtilis which has an activity index of about (58%).
Co(II) and Ni(II) tested complexes, showed greater bacterial activities than the Schiff base ligand against all tested bacteria.
Co(II) complex has the highest activity against all the studied bacteria than Ni(II) complex.

Effect of fungi
The Schiff base ligand showed a zero activity index against the tested fungi.
The activity index of Co(II) and Ni(II) complexes against C. albicans are 60 and 65, respectively, which is moderate to the reference drug (Ketoconazole).
Ni(II) complex showed the highest activity against all tested fungi than Co(II) complex.

Molecular docking studies
Molecular docking studies were achieved with MOA2019 software [24]     Mass spectrum of the synthesized ligand (L).

Figure 3
Fluorescence spectra of the synthesized ligand (L) and its Co(II) and Ni(II) complexes.   The propose structure of the Schiff base ligand (L) and its Co(II) and Ni(II) complexes.  The e ciency of removal Co(II) and Ni(II) complexes at different times.

Figure 9
The e ciency of removal Co(II) and Ni(II) complexes at different concentrations of L.

Figure 10
Page 26/30 The optimized structure, the vector of the dipole moment, the natural charges on active centers and Molecular electrostatic potential (MEP) surface of ligand.   Effect of antimicrobial activity of the ligand and its metal complexes.
Page 28/30 Figure 14 Activity index of the Schiff base ligand and its complexes.