3.1- Characterization of the Schiff base ligand.
The Schiff base ligand prepared by the reaction of anthranilic acid with dibenzoyl methane in a molar ratio 2:1. The ligand was yellowish stable powder at room temperature. It was soluble in common organic solvents.
The results of elemental analysis obtained were in good agreement with those calculated for the suggested formula indicating that the ligand had the molecular formula C29H22N2O4, table 1.
The IR spectrum of H2L ligand showed the presence of new strong and sharp vibration band at 1612 cm-1 referring to the azomethine group ν(C=N), corresponding to the formation of the Schiff base .
1H NMR spectrum was used to ensure ligand structure and purity in d6-dimethylsulfoxide (DMSO-d6) solution using Me4Si (TMS) as internal standard . The multiple in the region 7.34-8.18 ppm may be assigned to aromatic ring protons while the carboxylic acid group was given as sharp singlet at 12.98 ppm.
The mass spectrum of the H2L ligand was characterized by the molecular ion peak appeared at (m/z) 461.78 amu confirming the proposed formula in which the ligand moiety was C29H22N2O4 with atomic mass 462 amu.
The structure of the symmetric Schiff base ligand under study was shown in Figure 1.
3.2-Characterization of metal complexes
All the complexes were colored and stable to air and moisture. They were soluble in DMF. The complexes were characterized by different techniques such as elemental analyses, IR spectra, mass, 1H NMR and thermal analyses.
3.2.1-Elemental analyses of complexes
Metal complexes were synthesized by the reaction of the Schiff base dissolved in hot acetone with an ethanolic solution of the corresponding metal salt in a molar ratio 1:1. The experimental elemental analysis of complexes was in good compliance with the theoretical calculations. The elemental analyses of metal complexes (C, H, N and M) with molecular formula and the melting point were illustrated in Table 1.
3.2.2. Spectral studies
184.108.40.206. IR spectral studies
The IR spectra of the complexes were compared with those of the free ligand to determine the involvement of coordination sites in chelation, listed in Table 2. The IR spectra of the ligand H2L exhibited a band at 1612 cm–1 due to the azomethine ν(C=N) group [13,14], which was shifted to lower frequencies in the spectra of all the complexes (1548 – 1605) cm–1 . These bands were shifted to lower wave numbers indicating the involvement of azomethine nitrogen in coordination to the metal ions . A carbonyl ν(C=O) vibration band which appeared in the IR spectrum of the ligand H2L at 1671 cm–1 similarly underwent a shift into lower or higher frequency (1606 – 1697) cm-1 in the complexes . Strong bands corresponding to νasym(COO-) and νsym(COO-) vibrations of the free ligand was observed at 1558 cm–1 and 1419 cm–1, respectively . On complexion in this study, the asymmetric carboxylate stretching νasym(COO-) was shifted to the (1480 - 1555) cm–1 and the symmetric carboxylate stretching νsym(COO-) was shifted (1400 - 1488) cm–1 ; indicating the linkage between the metal ions and carboxylate oxygen atom . The spectra of the complexes showed broad bands in the 3382-3499 cm–1 range attributed to the stretching vibration of the ν (OH) of the carboxylic group in the spectra of the metal complexes while appeared at 3372 cm-1 in the spectrum of the Schiff base ligand . The presence of bands in all complexes in the region 416-494 cm-1 originated from ν(M-N) azomethine vibrational mode . The bands present in the region 556-583 cm-1 in all the complexes were assignable to ν(M-O) stretching vibration . In addition, two bands of coordinated water molecules ν(H2O) appeared in the IR spectra of metal complexes at 917-950 and 834-871 cm-1, indicating the binding of water molecules to the metal ions . The new weak intensity bands of (M-O) stretching vibrations occur at 516-545 cm-1. Accordingly, the ligand acted as tetradendate chelating agent, bonded to the metal ion via two carboxylate oxygen and two azomethine nitrogen atoms of the Schiff base. The formation of octahedral complexes was brought through coordination by two water molecules in all complexes.
220.127.116.11. 1H NMR spectral studies of H2L and its complexes
The 1H NMR spectra of the Schiff base ligand and its Zn(II) and Cd(II) complexes were recorded in DMSO-d6 by using tetramethylsilane (TMS) as internal standard. The chemical shifts of the different types of protons in the 1H NMR spectra of the Schiff base and its complexes were listed in Table 3. The protons of an aromatic ring showed in the range of 7.34 -7.54 ppm , while appeared at 6.46 - 7.51 and 6.26 - 7.53 ppm in [Zn(L)(H2O)2] and [Cd(L)(H2O)2] complexes, respectively. The disappearance of the COOH signal in the spectra of the [Zn(L)(H2O)2] and [Cd(L)(H2O)2] complexes relative to free Schiff base at 12.98 ppm indicated that the ligand acted as di-negative ligand which undergoes deprotonation during complexation process .
The mass spectra of the Cu(II) and Fe(III) complexes revealed the molecular ion peaks at m/z 576.77 and 586.58 amu, respectively, which were consistent with the calculated weight 577.55 and 587.34 amu, respectively. These data confirmed the stoichiometry of these complexes as being of [ML] type.
18.104.22.168. Electronic spectral studies and magnetic susceptibility
Electronic spectra of H2L and its complexes were recorded at room temperature. The spectrum of H2L showed band at 263 nm, assigned to π → π* transitions of the aromatic rings. In addition, strong bands also appeared at 342 nm assignable to n → π* transition of the azomethine group. Due to the coordination of azomethine nitrogen to the metal centers showed bands which shifted to 253 - 346 nm for π → π* transition and 341 - 537 nm for n → π* transition .
The diffused reflectance spectrum of Cr(III) complex exhibited three bands at 28,530 25,252 and 19,157 cm-1 which may be assigned to the 4A2g(F)→4T2g(F), 4A2g(F)→4T1g(F) and 4A2g(F)→4T2g(P) spin allowed d-d transitions, respectively. The magnetic moment value was found to be 4.10 B.M. which indicated the presence of Cr(III) complex in octahedral geometry .
Manganese(II) complex exhibited four intensity absorption bands in the ranges 16,366; 22,371; 25,445; 37,174 cm-1, which may be assigned to the transitions: 6A1g→4T1g (4G), 4A1g(4G) (10B + 5C), 6A1g→4Eg, 6A1g→4Eg (4D) (17B + 5C), 6A1g→4T1g (4P) (7B + 7C) , respectively . The magnetic moment value was found to be 5.98 B.M; which indicated the presence of Mn(II) complex in octahedral structure .
From the diffuse reflectance spectrum, it was observed that the Fe(III) chelate exhibited a band at 37,453 cm-1, which may be assigned to the 6A1g→T2g(G) transition in the octahedral geometry of the complex . The 6A1g→5T1g transition appeared to be split into two bands at 26,666 cm-1 and 22,883 cm-1. The spectrum also showed a band at 47,846 cm-1, which may be attributed to ligand to metal charge transfer. The observed magnetic moment of Fe(III) complex was 5.08 B.M which confirmed the octahedral geometry.
The diffused reflectance spectrum of cobalt(II) complex showed three bands in the 16,366, 17,376, and 21,008 cm-1 regions assignable to the 4T1g)F(→4T2g)F(; 4T1g)F(→4A2g)F(; and 4T1g)F(→4T1g)P( transitions, respectively, corresponding to the octahedral geometry around the Co(II) ion . The observed magnetic moment value of 4.97 B.M. correspond to high spin state and further support the octahedral geometry around Co(II) ion.
The diffused reflectance spectral data of Ni(II) complex showed d-d bands in the region 16,694,18.115 and 25,740 cm-1, respectively , assigned to 3A2g(F)→3T2g(F), 3A2g(F)→3T1g(F) and 3A2g(F)→3T2g(F) transitions, which were characteristic of Ni(II) in octahedral geometry . The Ni(II) complex exhibited magnetic moment value of 2.44 BM attributed to two unpaired electrons per Ni(II) ion suggesting that this complex has an octahedral geometry .
Diffused reflectance spectrum of Cu(II) complex showed the d-d transition bands in the range of 16,406, 19,569 and 26,809 cm-1 . These bands correspond to 2B1g→2A1g (dx2-y2 → dz2), 2B1g→2B2g (dx2-y2 → dxy) and 2B1g→2Eg (dx2-y2 → dxz, dyz) transitions, respectively. On the basis of electronic transitions, a distorted octahedral geometry was suggested for Cu(II) complex . The obtained magnetic moment value of 2.68 BM for Cu(II) complex was indicative of one unpaired electron per Cu(II) ion for d9-system suggesting spin-free distorted octahedral geometry . Both of Zn(II) and Cd(II) complexes were diamagnetic, which according to the empirical formulae, an octahedral geometry was proposed for these chelates.
22.214.171.124. Electron spin resonance spectrum of Cu(II) complex
The ESR spectra of metal complexes provided information about hyperfine and superhyperfine structures, which are of importance in studying the metal ion environment in the complex, i.e., the geometry, nature of ligating sites of Schiff base and metal, and the degree of covalency of metal–ligand bonds . The ESR spectrum for copper(II) complex at room temperature consisted of two signals, one with four hyperfine-structure lines at low magnetic field (the g║ signal) and the other at high field (g⊥ signal) . The trend g║ > g⊥ > free electron-spin (2.0023), indicated that the unpaired electron was localized in dx2-y2 orbital of the Cu(II) ion  and the spectral figure was characteristic for distorted octahedral sites (D4h) . The spectrum of [Cu(L)(H2O)2]H2O complex was given in Figure 2 which exhibited a broad g⊥ component, with splitting of g║ component, reflecting the coupling with the Cu(II) nucleus ( I= 3/2 ), the g║ value at 2.12 and g⊥ at 1.96 . It had reported that g║ was less than 2.3 for covalent character and greater than 2.3 for ionic character of the metal ligand bond in the complexes . According to Hathaway [37, 38], the parameter G, determined as G = (g║ −2) / (g⊥ −2), if G was greater than 4, the exchange interaction may be negligible; however, if G is less than 4, a considerable exchange interaction was indicated in the solid complex [39, 40]. The calculated G value for the Cu(II) complex was 3.2, indicative of considerable exchange interaction between the copper centers in the solid .
3.2.3. Molar conductance measurements
The measured molar conductance values of 10-3 M metal complexes in DMF at 25 oC were listed in Table 1. The Cr(III) and Fe(III) complexes had molar conductance values of 70 and 67 Ω-1 mol-1 cm2, respectively, indicating the ionic nature of these complexes and they were electrolytes. The rest of prepared complexes were non-electrolytes (Table 1).
3.2.4. Thermal analysis studies (TG and DTG)
The thermal studies of ligand and its metal complexes were carried out using the thermogravimetric technique (TG) and differential thermogravimetric (DTG) analyses. The thermal analysis gives useful data for the thermal stability of the metal complexes. The TG and DTG was recorded within the temperature range from 30 to 1000 °C (Table 4).
The Schiff base ligand (H2L) with the molecular formula (C29H22N2O4) was thermally decomposed in two successive decomposition steps. The first and second steps with estimated mass loss of 99.92% (calculated mass loss = 98.71%) within the temperature range 152 - 367 oC may be attributed to the loss of C29H22N2O4 molecule. The DTG curve gave two maximum peaks temperature at 182 - 352 oC. The overall weight loss amounts to 99.92% (calculated mass loss = 98.71%).
Thermogravimetric (TG) curve for [Cr(L)(H2O)2]Cl complex showed three weight loss events. The first and second steps of decomposition occurred within the range of 134 – 250 oC, with two maxima at 150 and 222 oC and corresponded to the loss of two molecules of coordinated water, HCl molecule, CO2 gas and C7H3N fragment with an estimated mass loss of 36.54% (calculated mass loss = 37.27%). The final step occurred within the range of 385 - 470 oC which corresponded to loss of C18H16NO0.5 fragment with estimated mass loss 43.38% (calculated mass loss = 43.53%) leaving behind ½Cr2O3 contaminated with carbons as the product of decomposition. The overall weight loss amounts to 79.93% (calculated mass loss = 80.80%).
The TG curve of [Mn(L)(H2O)2] complex was thermally decomposed in two successive decomposition steps. The first and second stages of decomposition occurred in the range of 152-499 °C with two maximum temperatures at 189 and 453 oC represented the loss of two H2O molecules, carbon dioxide gas and C25H20N2O fragment with an estimated weight loss of 80.19% (calculated mass loss = 80.58%). MnO contaminated with carbon was the residue of decomposition.
Thermogravimetric (TG) curve for [Fe(L)(H2O)2]Cl chelate showed three weight loss events. The first step of decomposition occurred in the range of 121 - 232 oC with maximum temperature at 169 oC and corresponded to the loss of two molecules of coordinate water, carbon dioxide gas, HCl molecule and C6H3N fragment with estimated mass loss 35.25% (calculated mass loss = 34.98%). The second and third steps occurred within the range of 366 – 586 °C with two maxima temperatures at 401 and 557 oC which corresponded to loss of C19H16NO0.5 fragment with estimated mass loss 45.18% (calculated mass loss = 45.28%) leaving metal oxide ½Fe2O3 contaminated with carbons as residue. The overall weight loss amounts were 80.43% (calculated mass loss = 80.26%).
[Co(L)(H2O)2] complex gave decomposition pattern of two stages. The first stage within the temperature range of 144 – 208 °C with maximum temperature 173 °C was related to the evolution of two H2O molecules, CO2 gas and C13H9N fragment with an estimated mass loss of 46.86 % (calcd. = 46.67 %). The final decomposition stage occurred in the temperature range from 360 to 432 oC with one maximum at 392 oC. The estimated mass loss of 31.55% (calculated mass loss = 31.53%) was reasonably for complete decomposition of CO gas and a part of ligand C10H13N and leaving CoO contaminated with carbons as final product. The overall weight loss amounted to 78.41 % (calcd. 78.20 %).
The [Ni(L)(H2O)2] complex decomposed from temperature 144 to 425 oC with two steps as follows. The first step occurred within the temperature range of 144 - 265 oC with maximum temperature at 172 oC and correspond to elimination of two water molecules, carbon dioxide gas and a part of ligand molecule (C14H11N) with a found mass loss of 49.32 % (calcd. = 49.21 %). The second step represented the loss of CO gas and C10H9N molecule with a mass loss of 31.40% (calcd. = 30.82%) and the temperature range 366 - 425 oC with maxima at 400 oC. At the end of the thermogram, the metal oxide NiO contaminated with carbons was the residue with total estimated mass loss of 80.72% (calcd. = 80.03%).
[Cu(L)(H2O)2]H2O complex was thermally decomposed in three steps within the temperature range from 66 to 346 oC. The first decomposition step with an estimated mass loss of 3.22 % (calcd. = 3.11%) occurred within the temperature range from 66 to 115 oC with maximum temperature at 89 oC. The second and third decomposition steps occurred within the range of 135 – 346 °C with two maximum temperatures at 168 and 304 oC. These steps may be attributed to elimination of two molecules of coordination water and CO2 gas and C24H20N2O fragment leaving CuO contaminated with carbons as residues. The overall weight loss amounts to 77.83 % (calcd. 77.90 %).
The [Zn(L)(H2O)2] chelate gave decomposition pattern of three stages. The first stage occurred within the temperature range of 172-211°C with maximum temperature at 190 oC. This step related to the evolution of two molecules of coordinate water, CO2 gas and a part of ligand (C13H9N) molecule with a found mass loss of 45.71% (calcd. = 46.13%). The last two decomposition stages within the temperature range of 341-651 °C with two maxima temperature at 352 and 623 oC, in which the complex losses carbon monoxide gas and C10H11N fragment with estimated mass loss 30.38% (calcd. = 30.81%). At the end of the thermogram, the metal oxide ZnO and four carbons as final product with total estimated mass loss of 76.10% (calcd. = 76.94%).
[Cd(L)(H2O)2] complex was thermally decomposed in one step within the temperature range from 155 to 239 oC with maximum temperature at 195 oC. This step involved complete evaluation of two molecules of coordinate water, carbon dioxide gas and ligand C27H20N2O molecule with estimated mass loss of 76.52% (calcd. = 76.92%) and leaving CdO contaminated with carbon as residue.
The structures of the Schiff base ligand (H2L) with Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes were characterized by elemental analyses, molar conductance, magnetic, solid reflectance and thermal analysis data. From IR spectra, it could be concluded that H2L behaved as a di-negatively tetradentate ligand coordinated to the metal ions via two nitrogen atoms of azomethine group and two oxygen atoms of carboxylate group. From the molar conductance data, it was found that the complexes are non-electrolytes expect Cr(III) and Fe(III) complexes. The structure was given in Figure (3).
3.3.1. Biological activity
The higher activity of metal complex was probably due to greater lipophilic nature of the complex. It can be explained on the basis of Overtone’s concept and Tweedy’s chelation theory . According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favors the passage of only lipid soluble materials due to which liposolubility was considered to be an important factor that controls the antimicrobial activity. On chelation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of metal ion with donor groups. Further, it increased the delocalization of the π electrons over the whole chelate ring and enhanced the lipophilicity of the complex. This increased lipophilicity enhanced the penetration of the complexes into lipid membrane and thus blocked the metal binding sites on enzymes of microorganisms. These metal complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restrict further growth of the organism. There were other factors which also increased the activity as: solubility, conductivity and bond length between the metal and ligand [42 - 44].
Antifungal and antibacterial activity in vitro using four fungi species (Aspergillus fumigatus; Syncephalastrum racemosum; Geotricum candidum and Candida albicans) and two bacteria – Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive bacteria (Streptococcus pneumoniae and Bacillus Subtilis) were assessed for the Schiff base as well as its metal complexes .
Measurement of zone of inhibition against the growth of bacteria and fungi for the ligand (H2L) and its metal complexes was shown in Figures (4,5) and Table (5). DMSO was used as a negative control and amikacin and ketoconozole drugs were used as positive standards for antibacterial and antifungal studies .
The antibacterial studies showed that, using Streptococcus pneumoniae as Gram-positive bacteria, the biological activity of the Ni(II), Cu(II), Cd(II), Fe(III) and Cr(III) complexes were higher than that of the free H2L ligand. While the Mn(II) complex had the same biological activity of free H2L ligand. The biological activity of the Co(II) and Zn(II) complexes were lower than that of the free H2L ligand. Using Bacillus Subtilis as Gram-positive bacteria, the biological activity of Cu(II), Ni(II), Fe(III), Cr(III), Cd(II) and Co(II) complexes were higher than that of the free H2L ligand. While the biological activity of the Mn(II) and Zn(II) complexes were lower than free H2L ligand. The study on Pseudomonas aeruginosa as Gram-negative bacteria, revealed that the biological activity of Ni(II), Fe(III), Cu(II), Co(II), Cr(III) were higher than that of the free H2L ligand. While the Cd(II), Zn(II), Mn(II) had the same biological activity as that of free H2L ligand. Using Escherichia coli as Gram-negative bacteria, the biological activity of Fe(III), Ni(II), Cu(II), Cd(II), Co(II) and Cr(III) were higher than that of the free H2L ligand. For Zn(II) complex, its biological activity was the same like the free H2L ligand. While the Mn(II) complex had lower biological activity than that the free H2L ligand.
The antifungal studies showed, by using Candida albicans fungus, that the biological activity of the Cr(III), Ni(II), Cu(II), Mn(II), Zn(II) and Fe(III) complexes were higher than that the free H2L ligand. While the biological activity of Cd(II) and Co(II) complexes were lower than that of the free H2L ligand. Using Geotricum candidum fungus, the biological activity of all metal complexes was higher than that free H2L ligand. While the highest biological activity was found for Cu(II) complex and the lowest activity was Co(II) and Fe(III) complexes. Using Syncephalastrum racemosumfungus, there isn’t biological activity of the free H2L ligand but the Zn(II) complex had the highest activity and the Ni(II) complex had the lowest activity. Using Aspergillus fumigatus fungus, the highest biological activity was found for the Zn(II) complex and the lowest activity was the Cu(II) complex. However, no significant activity has been observed for the free H2L ligand.
The activities of the prepared Schiff base ligand and its metal complexes were confirmed by calculating the activity index according to the following relation [46, 47]:
Activity index (A) = Inhibition Zone of compound (mm) ×100
Inhibition Zone of standard drug (mm)
From the data, it was concluded that Cu(II) complex had the highest activity index, while Fe(III) complex had the lowest activity index as shown in Figure (6).
3.3.2. Anticancer activity evaluation
The ligand and its complexes were investigated for their anticancer activity against human breast cancer cell line MCF 7. The range of inhibition of cell growth of ligand and its complexes between 33-56%. The results of inhibition of cell growth lower than 70% so we didn’t make further testing with different concentration.