Synthesis, Characterization, Computational and Biological Activity of Some Schiff Bases and Their Fe, Cu and Zn Complexes

: Four new symmetrical Schiff bases derived from 2,2 (cid:48) -diamino-6,6 (cid:48) -dibromo-4,4 (cid:48) -dimethyl- 1,1 (cid:48) -biphenyl or 2,2 (cid:48) -diamino-4,4 (cid:48) -dimethyl-1,1 (cid:48) -biphenyl, and 3,5-dichloro-or 5-nitro-salicylaldehyde, were synthesized and reacted with copper, iron-and zinc-acetate, producing the corresponding complexes. The Schiff bases and their metal complexes were characterized by 1 H, 13 C-NMR, IR and UV-Vis spectroscopy and elemental analysis. The structures of one Schiff base and the two zinc complexes were resolved by X-ray structure determination. Density functional theory (DFT) calculations at the B3LYP/6-31G(d) level of the latter compounds were carried out to optimize and examine their molecular geometries. The biomedical applications of the Schiff bases and their complexes were investigated as anticancer or antimicrobial agents.


Introduction
Schiff bases are a well-documented class of ligands that are capable of bonding to almost all metals of the periodic table [1].They are generally formed through a condensation reaction of carbonyl compounds with primary amines, in which mono-, di-or tri-functional amines lead to bi-, tri-, tetra-or poly-dentate Schiff bases.Symmetrical Schiff bases are formed when diamines are reacted with the same aldehydes or ketones in a 1:2 molar ratio.On the other hand, unsymmetrical Schiff bases may be obtained when two different aldehydes or ketones are reacted with diamines.
Due to the presence of N/O donor atoms in any Schiff base, they can coordinate to metals, leading to new metal complexes.Two main methods are used to synthesize Schiff base complexes.The first involves basic conditions, in which the Schiff base reacts with the metal ion in alcoholic or aqueous solutions [2].The second methodology depends on a template reaction of primary amines, aldehydes or ketones to react simultaneously with metal ions [3].This method is used to synthesize Schiff base complexes derived from macrocyclic and cyclic ligands.
Motivated by all of the aforementioned applications, we are interested in the synthesis of new Schiff base ligands and their metal complexes.The reaction of M(OAc) 2 (M = Cu(II), Co(II) or Ni(II) with 2,2 -bis(2-hydroxybenzylideneamino)-4,4 -dimethyl-1,1 -biphenyl allowed the synthesis of their complexes.The ligands are in tetradentate binding mode via ONNO motif of the two phenolic oxygen atoms and two azomethine nitrogen atoms [43].
In our previous studies, new Schiff base ligands derived from 2,2 -diamino-4,4dimethyl-1,1 -biphenyl-salicylaldehyde were prepared and characterized.These ligands were then reacted with Cu(II), Mn(II) or Zn(II) acetate, forming tetra-coordinate metal complexes.The anticancerous and antiproliferative activities of one representative ligand and its metal complexes were reported [22].Additionally, 4-thiazolidinone derivatives were prepared, and their biological activity, along with their metal complexes, was tested as a fungicide and found to have good activities against fungi [44].The preparation and characterization of substituted 2,2 -bis(2-oxidobenzylideneamino)-4,4 -dimethyl-1,1 -biphenyl complexes of zinc, potassium and titanium were studied [45][46][47].Recently, we undertook the evaluation and molecular modelling of bis-Schiff base derivatives as potential leads for the management of diabetes mellitus [48].
As an extension of our research in the area of Schiff base complexes, we report herein the synthesis of two new symmetrical Schiff bases by the condensation reactions of 2,2 -diamino-6,6 -dibromo-4,4 -dimethyl-1,1 -biphenyl and 2,2 -diamino-4,4 -dimethyl-1,1biphenyl with salicylaldehyde derivatives to produce the desired tetradentate Schiff base ligands.The copper, zinc and iron complexes of these new Schiff bases were obtained.To obtain a qualitative understanding of the structural characteristics and relative energies of the prepared Schiff bases and metal complexes, a density functional theory (DFT) computational analysis was carried out.Moreover, by certain biological testing, these Schiff bases and their complexes were demonstrated to be antimicrobial and anticancer agents.
The metal complexes Z1M-Z4M were colored complexes and characterized by molar conductivity, UV-Vis, IR, 1 H-and 13 C-NMR spectra where applicable.The complexes had non-electrolytic behavior, evident by the very low (~0) molar conductivity, suggesting that the complexes were indeed neutral.The UV-Vis absorption spectra of the Schiff bases and their metal complexes were measured in DMSO solutions.The spectra of the Schiff bases displayed two bands (310-359 and 451-452 nm), which may be attributed to intraligand absorptions (π-π* and n-π) of the conjugated system and the azomethine group [49].However, the metal complexes showed only one band in the range of 361-412 nm, which may be attributed to a metal-to-ligand charge transfer band.The metal complexes Z1M-Z4M were colored complexes and characterized by molar conductivity, UV-Vis, IR, 1 H-and 13 C-NMR spectra where applicable.The complexes had non-electrolytic behavior, evident by the very low (~0) molar conductivity, suggesting that the complexes were indeed neutral.The UV-Vis absorption spectra of the Schiff bases and their metal complexes were measured in DMSO solutions.The spectra of the Schiff bases displayed two bands (310-359 and 451-452 nm), which may be attributed to intraligand absorptions (π-π* and n-π) of the conjugated system and the azomethine group [49].However, the metal complexes showed only one band in the range of 361-412 nm, which may be attributed to a metal-to-ligand charge transfer band.
The 1 H-NMR spectra of the free Schiff bases presented the phenolic signal (12.61-13.63ppm).This peak disappeared in the spectra of the Zn(II) complexes.This proves that the two hydroxyl groups in the free Schiff base ligands lost their protons, and new bonds between the metal and the two oxygen atoms were formed.Moreover, the signal in the range of 8.04-8.58ppm of the free Schiff bases that was attributed to the azomethine proton (CH=N) was shifted upfield in the zinc complexes' spectra, thereby supporting its bond formation with the Zn center.These 1 H-NMR values for both the free ligands and zinc complexes were consistent with those published for similar compounds [22,[45][46][47]50,51].The 13 C-NMR spectra of the free Schiff bases displayed peaks in the ranges of 21.23-21.39ppm and 161.04-166.32 ppm, corresponding to the methyl and azomethine carbons, respectively. Thse peaks were shifted to 20.99-21.03ppm and 168.72-175.62 ppm upon complexation to zinc.Although a small shift was observed for the methyl peak, a big shift was observed for the azomethine carbon, another indication of the N-bonding to zinc.
In the IR spectra of the Schiff bases, the characteristic bands in regions 3467-3450, 2900-3100, 1627-1613, 1476-1413 and 1356-1323 cm −1 may be attributed to the absorption of ν(O-H), ν(C-H), ν(C=N), ν(C=C) and ν(C-O), respectively.The ν(C=N) band was shifted to a lower wavenumber in the corresponding spectra of the complexes.This indicated that this group was coordinated to the metal center.In addition, the disappearance of the weak and broad band of the hydroxyl group gave important evidence of the complexation of the phenolic groups.Moreover, two new bands, in the ranges of 400-450 and 500-550 cm −1 due to M-N and M-O stretching, appeared in the metal complexes' The 1 H-NMR spectra of the free Schiff bases presented the phenolic signal (12.61-13.63ppm).This peak disappeared in the spectra of the Zn(II) complexes.This proves that the two hydroxyl groups in the free Schiff base ligands lost their protons, and new bonds between the metal and the two oxygen atoms were formed.Moreover, the signal in the range of 8.04-8.58ppm of the free Schiff bases that was attributed to the azomethine proton (CH=N) was shifted upfield in the zinc complexes' spectra, thereby supporting its bond formation with the Zn center.These 1 H-NMR values for both the free ligands and zinc complexes were consistent with those published for similar compounds [22,[45][46][47]50,51].The 13 C-NMR spectra of the free Schiff bases displayed peaks in the ranges of 21.23-21.39ppm and 161.04-166.32 ppm, corresponding to the methyl and azomethine carbons, respectively. Thse peaks were shifted to 20.99-21.03ppm and 168.72-175.62 ppm upon complexation to zinc.Although a small shift was observed for the methyl peak, a big shift was observed for the azomethine carbon, another indication of the N-bonding to zinc.
In the IR spectra of the Schiff bases, the characteristic bands in regions 3467-3450, 2900-3100, 1627-1613, 1476-1413 and 1356-1323 cm −1 may be attributed to the absorption of ν(O-H), ν(C-H), ν(C=N), ν(C=C) and ν(C-O), respectively.The ν(C=N) band was shifted to a lower wavenumber in the corresponding spectra of the complexes.This indicated that this group was coordinated to the metal center.In addition, the disappearance of the weak and broad band of the hydroxyl group gave important evidence of the complexation of the phenolic groups.Moreover, two new bands, in the ranges of 400-450 and 500-550 cm −1 due to M-N and M-O stretching, appeared in the metal complexes' spectra and represented another proof of O/N coordination to metals.These results are in good agreement with those previously published for similar complexes [22,[52][53][54][55][56].

Computational Study
The optimized geometry of ZH4 is depicted in Figure 4.The selected structural parameters for the optimized ligand, along with those determined experimentally, are listed in Table 1.
The calculated C1-C15 bond length (1.492 Å) between the aryl units of the biphenyl backbone, which was estimated to be 1.498(4) Å by the crystallographic study, featured a typical single-bond value, thus ruling out interactions between the π-systems of these moieties.
This value was close to the calculated average value of 1.494 Å, for a series of twelve comparable complexes bearing the biphenyl backbone around the coordination sphere [18].
The aryl moieties of the biphenyl backbone were twisted with angles between the planes by 87.31°(exp.83.89°).The large torsion angle was attributed to the steric requirements enforced by bromine substituents.This was consistent with the experimentally reported aryl-aryl dihedral angle of 80 • for a similar dibromo-substituted biphenyl ligand [59].Smaller torsion angles (between 21 • and 68 • ) for the halide-free analogues, where there was flexible twisting between the two biphenyl planes, were observed [46]

Computational Study
The optimized geometry of ZH4 is depicted in Figure 4.The selected structural parameters for the optimized ligand, along with those determined experimentally, are listed in Table 1.The calculated C1-C15 bond length (1.492 Å) between the aryl units of the biphenyl backbone, which was estimated to be 1.498(4) Å by the crystallographic study, featured a typical single-bond value, thus ruling out interactions between the π-systems of these moieties.This value was close to the calculated average value of 1.494 Å, for a series of twelve comparable complexes bearing the biphenyl backbone around the coordination sphere [18].
The aryl moieties of the biphenyl backbone were twisted with angles between the planes by 87.31ᵒ (exp.83.89ᵒ).The large torsion angle was attributed to the steric requirements enforced by bromine substituents.This was consistent with the experimentally reported aryl-aryl dihedral angle of 80° for a similar dibromo-substituted biphenyl ligand [59].Smaller torsion angles (between 21° and 68°) for the halide-free analogues, where there was flexible twisting between the two biphenyl planes, were observed [46].The optimized geometries of Z1Zn and Z2Zn are elucidated in Figure 5.The results indicated that the monoligated complexes exhibited a penta-coordinated zinc ion with distorted trigonal bipyramidal geometry.The computed H2O•••M bond was 2.247 Å (exp.Such slight structural variations between the experimental and calculated values were attributed to the experimental data being acquired for crystalline materials with lattice interactions, such as packing effects and intermolecular van der Waals forces, whereas the calculated values corresponded to an isolated molecule in the gas phase. The calculated bond length between the aryl units of the biphenyl backbone was 1.489 Å (exp.1.489 Å) and 1.488 Å (exp.1.491 Å) for the Z1Zn and Z2Zn complexes, respectively, alluding to a typical single bond and excluding π-interactions between the aryl units.The calculated twist angle between the planes of the aryl moieties of the biphenyl backbone was 59.39°(exp.60.61°) and 59.19°(exp.55.81°) for the Z1Zn and Z2Zn complexes, respectively.Such large torsion angles were attributed to the steric and electronic requirements enforced within the coordination sphere.Such slight structural variations between the experimental and calculated values were attributed to the experimental data being acquired for crystalline materials with lattice interactions, such as packing effects and intermolecular van der Waals forces, whereas the calculated values corresponded to an isolated molecule in the gas phase.
The calculated bond length between the aryl units of the biphenyl backbone was 1.489 Å (exp.1.489 Å) and 1.488 Å (exp.1.491 Å) for the Z1Zn and Z2Zn complexes, respectively, alluding to a typical single bond and excluding π-interactions between the aryl units.The calculated twist angle between the planes of the aryl moieties of the biphenyl backbone was 59.39ᵒ (exp.60.61ᵒ) and 59.19ᵒ (exp.55.81ᵒ) for the Z1Zn and Z2Zn complexes, respectively.Such large torsion angles were attributed to the steric and electronic requirements enforced within the coordination sphere.

Antimicrobial and Anticancer Assays
The results showed significant antimicrobial activity for both the Schiff bases and their metal complexes against Gram-positive bacterial strains (Micrococcus luteus and Staphylococcus aureus), which were demonstrated as a zone of inhibition (Table 2, Figure 6).These compounds showed different activity towards the two bacteria types.For instance, ZH2, ZH3, ZH4 and their zinc complexes showed antimicrobial activity against Micrococcus luteus and Staphylococcus aureus, while Z2Fe, Z4Fe and Z4Cu showed antimicrobial activity against Staphylococcus aureus only.The selectivity of these compounds raises an interesting concern about the exact mechanism of these compounds as antimicrobial agents.The Schiff bases and certain complexes showed limited antimicrobial activity on Gram-negative bacteria (Escherichia coli).

Antimicrobial and Anticancer Assays
The results showed significant antimicrobial activity for both the Schiff bases and their metal complexes against Gram-positive bacterial strains (Micrococcus luteus and Staphylococcus aureus), which were demonstrated as a zone of inhibition (Table 2, Figure 6).These compounds showed different activity towards the two bacteria types.For instance, ZH2, ZH3, ZH4 and their zinc complexes showed antimicrobial activity against Micrococcus luteus and Staphylococcus aureus, while Z2Fe, Z4Fe and Z4Cu showed antimicrobial activity against Staphylococcus aureus only.The selectivity of these compounds raises an interesting concern about the exact mechanism of these compounds as antimicrobial agents.The Schiff bases and certain complexes showed limited antimicrobial activity on Gram-negative bacteria (Escherichia coli).
Table 2.The antimicrobial activity as shown by the inhibition area in the tested compounds.

Staphylococcus aureus (ATCC 29213)
Escherichia coli The IC50 of the Schiff bases and their metal complexes showed a variable reduction in viability of the tested cancer cell lines compared to the normal fibroblasts.For instance, the IC50 of ZH1 showed significant inhibition of cancer cell line growth compared to the HDF cell line.In addition, the IC50 of the tested compounds showed a selective anticancer effect against MCF7 compared to the A549 and HDF cell lines, as shown in Table 3. ZH1, its zinc complexes and Z2Fe showed selective anticancer effects against the MCF7 cell line.Interestingly, Z2Cu showed the lowest IC50 values against all cell lines.The IC 50 of the Schiff bases and their metal complexes showed a variable reduction in viability of the tested cancer cell lines compared to the normal fibroblasts.For instance, the IC 50 of ZH1 showed significant inhibition of cancer cell line growth compared to the HDF cell line.In addition, the IC 50 of the tested compounds showed a selective anticancer effect against MCF7 compared to the A549 and HDF cell lines, as shown in Table 3. ZH1, its zinc complexes and Z2Fe showed selective anticancer effects against the MCF7 cell line.Interestingly, Z2Cu showed the lowest IC 50 values against all cell lines.

Computational Study
The ground-state molecular geometries of ZH4, Z1Zn and Z2Zn were fully optimized in the gas phase, without any constraints at the B3LYP/6-31G(d) level of theory [60][61][62].
The initial geometries were extracted from the experimentally determined crystal structure.All electronic structure calculations were performed using the Spartan'18 package [63].

Antimicrobial Assay
For the evaluation of the antibacterial activity of all synthesized compounds, two Gram-positive bacteria and Gram-negative bacteria were used in the current study: Micrococcus luteus and Staphylococcus aureus, and Escherichia coli, respectively.The antimicrobial assay procedures were performed according to the recommendation of the National Committee for Clinical Laboratory Standards (NCCLS) [64][65][66].Briefly, the synthesized compounds were dissolved in DMSO at a concentration of 10 mg/mL.After the inoculation of the bacterial strains in the culture media, 50 µL sized wells were generated in the agar media, followed by the addition of 50 µL of the tested compounds.After 24-48 h of incubation, the diameter of the inhibition zone was measured.

Cell Viability Assay (MTT)
To determine the IC 50 of the ZH1-ZH4 and Z1Zn-Z4Cu compounds on the selected cell lines, an MTT assay was performed.Approximately (8 × 10 3 cells/well) of MDA-MB-231, MCF7 and HDF cell lines were seeded into a 96-well plate (Corning, Burlington, VT, USA).All cell lines were treated with different concentrations of the ZH1-ZH4 and Z1Zn-Z4Cu compounds, ranging from 0.5 to 500 µg/mL.Then, the cells were incubated at 37 • C in a 5% CO 2 incubator for 72 h, after which the old media was aspirated, and the MTT assay salt (Bioworld, Visalia, CA, USA) in 100 µL of fresh media was added to each well.Following that, the plates were incubated at 37 • C for 3 h, and then 50 µL of solubilization solution (DMSO) was added to each well to determine viability.The absorbance of the solution was measured at 560 nm using a Glomax plate reader (Promega, Madison, WI, USA).

X-ray Structure Determination of ZH4, Z1Zn and Z2Zn
The intensity data of the X-ray diffraction peaks were collected on a Rigaku XtaLAB (Japan) P200 diffractometer, using Mo Kα radiation (λ = 0.71073 Å) fitted with SHELXTL for structure determination [67].A direct method using SHELXS-2014 was employed to solve the structure, and Fourier transformation was carried out using SHELXL-2014, employing full-matrix least-square refinement calculations [67].

Conclusions
New Schiff bases and their Fe, Cu and Zn complexes were synthesized and characterized by various spectroscopic methods.The structure of the ZH4, Z1Zn and Z2Zn were determined by DFT calculations and X-ray diffraction measurements.The crystallographically determined geometry of the ligands, as well as the measured vibrational spectra of ZH4, Z1Zn and Z2Zn, are in good agreement with the theoretical results.The antimicrobial and anticancer activities of the Schiff bases and their metal complexes were evaluated.The compounds ZH2-ZH4 and their zinc complexes showed antimicrobial activity against Micrococcus luteus, while ZH2, ZH3, Z2Zn, Z3Zn and Z2Fe showed antimicrobial activity against Staphylococcus aureus.An anticancer assay showed that most of our tested compounds had variable reduction in viability of the tested cancer cell lines compared to the normal fibroblasts.Moreover, ZH1, Z2Fe and all zinc complexes showed selective anticancer effects against the MCF7 cell line, and Z2Cu showed the lowest IC 50 values against all cell lines.

Figure 1 .
Figure 1.Molecular structure of ZH4 determined by X-ray diffraction at 100 K. White: H, gray: C, blue: N, red: O, and orange: Br.

Figure 1 .
Figure 1.Molecular structure of ZH4 determined by X-ray diffraction at 100 K. White: H, gray: C, blue: N, red: O, and orange: Br.

Figure 2 .
Figure 2. Molecular structure of Z1Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, light green: Cl, and blue-gray: Zn.

Figure 3 .
Figure 3. Molecular structure of Z2Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, and blue-gray: Zn.

Figure 2 .
Figure 2. Molecular structure of Z1Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, light green: Cl, and blue-gray: Zn.

Figure 2 .
Figure 2. Molecular structure of Z1Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, light green: Cl, and blue-gray: Zn.

Figure 3 .
Figure 3. Molecular structure of Z2Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, and blue-gray: Zn.

Figure 3 .
Figure 3. Molecular structure of Z2Zn determined by X-ray diffraction at 100 K.The ellipsoids are drawn with a 50% probability.White: H, gray: C, blue: N, red: O, and blue-gray: Zn.

Figure 4 .
Figure 4. Different views of the optimized ground-state geometry for the ZH4 ligand at the B3LYP/6-31G(d) level of theory.(O: red, N: blue, C: gray, and Br: brick).Dotted line for O-H•••N hydrogen bonding.

Figure 4 .
Figure 4. Different views of the optimized ground-state geometry for the ZH4 ligand at the B3LYP/6-31G(d) level of theory.(O: red, N: blue, C: gray, and Br: brick).Dotted line for O-H•••N hydrogen bonding.

Figure 6 .
Figure 6.A representative bacterial culture showing the inhibition zone in Gram-positive (Staphylococcus aureus and Micrococcus luteus) bacteria by compounds ZH1, ZH2, ZH3 and ZH4 ((left) and (middle)) compared to lack of antimicrobial activity against Gram-negative bacteria (Escherichia coli) (right).

Figure 6 .
Figure 6.A representative bacterial culture showing the inhibition zone in Gram-positive (Staphylococcus aureus and Micrococcus luteus) bacteria by compounds ZH1, ZH2, ZH3 and ZH4 ((left) and (middle)) compared to lack of antimicrobial activity against Gram-negative bacteria (Escherichia coli) (right).

Table 3 .
IC 50 values (µg/mL) of the tested compounds after the cell viability assay (MTT), showing variable responses of the treated cell lines with different specificity.