Cr(III) and Ni(II) Complexes of Isatin-hydrazone Ligand: Preparation, Characterization, Dft Studies, Biological Activity, and Ion-Flotation Separation of Ni(II)

In the current work, a ligand N '1 -((E)-2-hydroxy-3H-indol-3-ylidene)-N '3 -((E)-2-oxoindolin-3-ylidene)malonohydrazide (H 4 MDI) and its Cr(III) and Ni(II) complexes have been synthesized and characterized by various conventional methods. For evaluating the optimal ligand structure and its complexes, calculations of DFT were applied. Magnetic measurements inherent to their electronic spectra show that both Cr(III) and Ni(II) chelates have octahedron coordination frameworks. On the other hand, the IR spectral data revealed that the ligand behaves as a binegtive hexadentate in [Cr 2 (H 2 MDI)(H 2 O) 2 Cl 4 ] and as a tetranegative hexadentate in [Ni 2 (MDI)(H 2 O) 6 ].4H 2 O. In addition, the behavior of thermal decomposition for prepared complexes was discussed. Two comparable methods (Coats-Redfern and Horowitz-Metzger) were used to calculate the kinetic parameters of the resulted thermal decomposition stages. Furthermore, the ion-otation process was used for the separation of Ni(II) from aqueous media via the prepared ligand as a chelating agent and oleic acid as a surfactant. Moreover, the antimicrobial behavior of the synthesized moieties was investigated against various bacterial and fungal strains. H 4 MDI has the most activity with minimum inhibitory concentration (MIC) of 0.78 µg/mL for both E. coli, and C. Albicans, while Ni(II) complex shows the activity against S. aureus, E. coli, and C. Albicans with MIC of 2.34, 4.68, and 1.17 µg/mL, respectively. Finally, the in-vitro cytotoxic activity of the prepared compounds against hepatocellular carcinoma human tumor cells (HePG-2) has been examined, and revealed that H 4 MDI and its Ni(II) complex show very strong activity against HePG-2 with IC 50 of 9.7 and 7.7 µmol/L, respectively.


Introduction
Today, chemistry and biology are part of our daily world. These two sciences reside at the crossroads of many sectors and industries. Chemists have started to realize that a great number of biochemical molecules are compounds involving one or more metal ions coordinated to groups sometimes large and complex organic. The architectural beauty of these coordination complexes occurs owing to the interesting ligand structures that contain distinct donor locations in heterocyclic rings as well as aliphatic moieties. Schiff bases, also known as azomethines due to they have RC = N group, play important roles in biological systems. They result from condensation reactions between primary amines with carbonyl compounds (aldehydes or ketones) [1,2]. Schiff base ligands are capable of coordination and stabilization of multiple metal ions in multiple states of oxidation [1,3]. Because of N-N bond length shortness, the hydrazone ligands mostly act as bi-/tri-or tetradentate moieties although they have the potential to act as bridging tetradentate ligands. Hydrazone ligands also discover countless applications in analytical chemistry, such as transition metal binders. Studies have also shown that the azomethane N with a single pair of electrons in sp 2 hybridized orbital accounts for the biological activity of the hydrazones. The coordination with metal ions also enhances the applications of Schiff Base Ligands. Therefore, in multiple areas, such as luminescent [4][5][6][7], catalysis [8,9], sensors [10][11][12], and medications [13], this type of compound can be employed. Recently, ion-otation has received signi cant attention as a separation technique because of its quickness, easiness, good separation yields, and exibility of operating and equipment for recovery purposes [14,15]. In continuation to previous work [16][17][18][19][20] the present work aims to synthesize and characterize Cr(III) and Ni(II) complexes of N '1 -((E)-2-hydroxy-3H-indol-3-ylidene)-N '3 -((E)-2-oxoindolin-3-ylidene) malonohydrazide (H 4 MDI). The modes of chelation and the geometry for the separated complexes are discussed based on DFT calculations and different spectroscopic methods. Moreover, the kinetics and thermodynamic characteristics of the thermal decomposition steps have been studied employing Coats-Redfern and Horowitz-Metzger models. Also, the application of ion-otation technique to separate Ni(II) from aqueous solutions was carried out using H 4 MDI as chelating agent and oleic acid as a surfactant under the recommended conditions.

Materials
All the chemicals used in this study were purchased from Merck Chemical Company, Darmstadt, Germany, and used without any further puri cation.

Instrumentation
Elemental analysis (C, H, and N) for the H 4 MDI ligand and its complexes were proceeded using a Perkin-Elmer 2400 Series II analyzer, while the content of chloride and metal was done with the referenced procedures [21]. FTIR of H 4 MDI and related complexes were recognized on Mattson 5000 FTIR spectrophotometer (4000-400 cm -1 ). The electronic spectra of the prepared ligand and its complexes mull were done using UV 2 Unicam UV/Vis spectrophotometer using 1-cm stopper quartz cells. The magnetic moment was estimated thru a Sherwood magnetic susceptibility balance. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were done on a Shimadzu TGA-50H thermogravimetric analyzer at (20-800 o C) with a nitrogen flow rate of 15 mL/minute and heating rate of 10 o C/minute. The 1 H-NMR and 13 C-NMR of the prepared ligand were obtained on an Advance DRX 500 Bruker spectrometer (400 MHz) with a 5 mm probe head in d 6 -DMSO and chemical shifts are given in parts per million relatives to internal tetramethylsilane (TMS). In the ionotation procedure of Ni(II), the concentration of the metal ion was determined using Flame Atomic Absorption Spectrometry (GBC, Sensaa Series) with air-acetylene ame at a wavelength of 232 nm. The otation cell was a cylindrical tube of 15 mm internal diameter and 290 mm length [22]. The pH of the studied solutions was adjusted using Hanna 8519 digital pH meter.

Synthesis of ligand
The malonohydrazide was synthesized by the addition of diethyl malonate and hydrazine with a ratio of (1:2) in absolute ethanol placed in an ice bath for 30 min with non-stop stirring, then the resultant substance ltered and washed several times with absolute ethanol and dried in a vacuum desiccator. To prepare the H 4 MDI ligand, the prepared material mixed with isatin with a ratio of (1:2) in presence of glacial acetic acid, and the mixture was re uxed on a water bath for a time of (2-4 hours). The products obtained were crystallized several times from absolute ethanol and dried in a vacuum desiccator.

Synthesis of complexes
Cr(III) and Ni(II) complexes were synthesized via the re ux of H 4 MDI ligand with metal salt (CrCl 3 .6H 2 O or NiCl 2 .6H 2 O) with a ratio of (1:2) in a solution of absolute ethanol placed in a water bath for (2-3 hours). The resulted materials were ltered off, washed several times with ethanol followed by diethyl ether, and nally dried in a vacuum desiccator. H 4 MDI ligand and its metal complexes were prepared according to procedures explained in Scheme 1.
2.5 Molecular modeling DMOL 3 program was applied in the Materials Studio package to estimate the cluster calculations [23]. The optimized configurations of the complexes were predicted by applying the DFT method [24]. DFT calculations and semi-core pseudopods calculations (dspp) were made using the duplicate numerical basis sets plus functional polarization (DNP). The DNP basis was more exact than the Gaussian basis groups of the duplicate extent [25]. The exchange and correlation functional between electrons were described using the revised-Perdew-Burke-Ernzerhof (RPBE) [26] based on the generalized gradient approximation (GGA) functional [27].

Analytical application (Ion-otation of nickel)
Suitable aliquots containing a recognized quantity of Ni(II) and H 4 MDI ligand were mixed for each investigation.
The pH was adjusted to the required value with HNO 3 or NaOH. After that, the solution was moved to the cell of otation with a total volume of 10 mL adjusted with distilled H 2 O and shacked well for 2 minutes to con rm completion of complexation followed by adding 2 mL of surfactant with identi ed concentration. Then, the cell was shacked well with hand and left 5 minutes standing for con rmation of complete otation. Finally, the removal % (Re %) of Ni(II) was calculated as follows after its determination by FAAS in the mother liquor: where: M i and M f are referred to the initial and the nal concentration of Ni(II), respectively.

Antimicrobial activity
The antimicrobial behavior of the H 4 MDI ligand and its corresponding chelates were investigated versus grampositive bacteria: Staphylococcus Aureus (ATCC 12600) and gram-negative bacteria: Escherichia Coli (ATCC 11775) as well as Candida albicans fungus (ATCC 7102) by an adjusted disc diffusion technique [28]. The solution of each compound such as free ligand, metal complex, and standard drug (Fluconazole "antifungal agent" and Cipro oxacin "antibacterial agent") was dissolved in DMSO and prepared for testing versus spore germination.  [29,30] The cell line declared above were utilized to investigate the inhibition effects of the isolated ligand and its complexes on cell growth utilizing the MTT inspection [31]. The colorimetric test has been evaluated and recorded by the plate reader (EXL 800, USA) at 570 nm wavelength. The percentage of relative cell viability was calculated as follows:

Results And Discussion
Analytical and spectroscopic data for the prepared ligand and its complexes indicate a 1:2 (ligand-metal) stoichiometry for all the complexes. The physical and analytical data of H 4 MDI ligand and its metal complexes are given in Table 1. The molecular structures of the metal complexes were con rmed using the data of elemental analyses. All the isolated solid complexes are quite stable in air and insoluble in most of the organic solvents except in dimethylformamide (DMF) and dimethylsulphoxide (DMSO). The thermogravimetric studies of the complexes con rmed high stability which agreed with the high melting points.

IR spectra of H 4 MDI and its complexes
A comparison between the IR spectral data of the H 4 MDI ligand and its metal complexes was made to study the coordination behavior of H 4 MDI toward the studied metal ions. The most important assignments of FTIR absorption bands of H 4 MDI and its complexes are displayed in Table 2 and denoted graphically in Figure 1S. with the consecutive appearance of new bands due to (C-O) [16] and (C=N), and (ii) the shift of (C=N) azomethine to higher wavenumber.

Electronic spectra and magnetic properties
The most important assignments of the electronic spectral bands of H 4 MDI and its complexes and the values of magnetic moments of the prepared complexes are displayed in Table 3 and denoted graphically in Figure   2S. The spectrum of H 4 MDI exhibits an absorption band at 32258 cm -1 assigned to (π→π*) of phenyl rings overlapped with that of (C=N) azomethine group. In the region of 22935-27932 cm -1 , there is a second intense band that is assigned to (n→π*) of carbonyl groups that undergo a redshift that suggests the coordination of the oxygen atom of the carbonyl group and the central metal ion [33]. ion of the complex is also compatible with the suggested geometry.

Thermogravimetric Analysis
Estimation of coordinated or crystallized water molecules can be achieved using TG data [35,36] ( Figure 3S).
One can say that there is an agreement between the TG data and the suggested formula. Cr(III) complex has one degradation step in the range (220.  Table 4.

Kinetic parameters
Coats-Redfern [37] and Horowitz-Metzger [38] methods were used to estimate the thermodynamic and kinetic parameters for the prepared metal complexes as shown in Figures (3-6). Table 5 shows the different parameters (A, E a , ΔS*, ΔH*, and ΔG*) of the prepared complexes. From the results obtained and listed in Table 5, the following remarks can be pointed out: 1. There is a similarity between the data obtained by the two methods.
3. The high value of the activation energies revealing the high stability of the remaining part of the chelate.
1. The negative ΔS* values of some degradation stages show that the activated fragments have more ordered structure than the undecomposed one and the degradation reactions are slow [39], while the positive values may suggest that the disorder of the decomposed fragments increases much more rapidly than that of the undecomposed one [40].
2. According to the positive values of ΔH*, the decomposition stages are endothermic.
3. According to the positive values of ΔG*, all the decomposition steps are nonspontaneous.
4. Moreover, the values of ΔG* increase signi cantly for the subsequent decomposition stages as a result of the increasing of TΔS* which re ects that the rate of removal of the subsequent species is lower than that of the precedent one [41,42].
According to all previous results, the thermal stability of the metal complexes decreases in the following order: [ The optimized structures of the ligand and its metal complexes with atomic numbering are given in Figure 7 and recorded in Tables (1S-6S

Global reactivity descriptors
The DFT strategy explains the chemical reactivity and site selectivity of the molecular frameworks. The main parameters in quantum chemical studies are the energies of both HOMO and LUMO. Where HOMO is the highest occupied molecular orbital that acting as an electron donor, and LUMO is the lowest unoccupied molecular orbital that acting as an electron acceptor of electrons. HOMO and LUMO molecular orbitals are recognized as molecular frontier orbitals (FMOs). The energy gap between FMOs describes the stability of the molecule, and this has an important role in calculating electron conductivity, which helps to explain the molecular electrical transport properties. The gaseous phase energies of frontier molecular orbitals (E HOMO , E LUMO ), energy bandgap (ΔE) that explains the possible charge transfer interaction within the molecule, electronegativity (χ), global hardness (η), chemical potential (µ), global electrophilicity index (ω), global softness (S) and softness ( ) are calculated [46,47] according to the following equations and listed in Table 6.
The outcomes results indicated that: 1. The energy of HOMO and LUMO levels are almost negative showing the stability of the isolated compounds.
2. In the case of ligand, we found that the energy gap is small viewing that charge transfers easily in it and this in uences the biological potency, which agrees with experimental data of antibacterial, and antifungal activities. Moreover, the low value of the energy gap is due to the groups that enter into conjugation [48].

The low value of energy gap (E HOMO -E LUMO ) could be expected to indicate that ligand has a high tendency to
coordinate the metal ions, where the molecules with a small gap are known as soft molecules, they are more polarized and reactive than hard molecules as they readily offer electrons to an acceptor.
4. Global hardness and softness are signi cant properties to measure molecular stability and reactivity. A hard molecule has a large energy gap while the soft one has a small value. During complex formation, the ligand acts as a Lewis base while the metal ion acts as a Lewis acid. Metal ions are soft acids and thus soft base ligands are most effective for complex formation. For that reason, it is concluded that ligand with a proper value of softness has a good tendency to chelate metal ions effectively [49]. This is also con rmed by the calculated chemical potential. The 3D plots for HOMO and LUMO orbital via DFT technique for H 4 MDI are illustrated in Figure 8.

Ion-otation of Ni(II)
3.6.1 In uence of pH Several trials were done to test the in uence of pH on the removal % of 4×10 -4 mol/L of Ni(II) using 4×10 -4 mol/L of H 4 MDI and 1×10 -3 mol/L of HOL. The results were offered in Figure 9. It is clear that maximum removal of Ni(II) (~100%) was attained at the pH (6-9), which facilitates the application of the H 4 MDI ligand for the removal of Ni(II) from aqueous media. At pH values higher than 9, the separation e ciency decreased due to the formation of oleates. So, pH 7 was xed for all subsequent studies.

In uence of nickel concentration
Attempts to remove dissimilar concentrations of Ni(II) with 4×10 -4 mol/L of H 4 MDI and 1×10 -3 mol/L HOL at pH 7 were done. The results obtained in Figure 10 showed that the optimum removal (~100%) of Ni(II) was attained at the range of (2.5×10 -4 -4×10 -4 mol/L) and then decreased sharply and this behavior can be explained as follows: the prepared ligand gave complete removal of Ni(II) (~100%) which may be due to the presence of enough quantities of ligand to bind all Ni(II) and after 4×10 -4 mol/L of Ni(II) there is an insu cient ligand. Consequently, 4×10 -4 mol/L of Ni(II) was used for all subsequent studies.

In uence of H 4 MDI concentration
The effect of dissimilar concentrations of prepared ligand on the removal % of 4×10 -4 mol/L Ni(II) with 1×10 -3 mol/L HOL at pH 7 was tested. The results were displayed in Figure 11 showed that the removal of Ni(II) increased reaching its optimal removal (~100%) at 4×10 -4 mol/L of ligand. After that, any excess ligand does not affect the separation process. Consequently, 4×10 -4 mol/L of H 4 MDI was used for all subsequent studies.

In uence of HOL concentration
Experiments were done to remove Ni(II) with HOL only, but the removal does not exceed 36.8 %. So, another run of trials was achieved to remove 4×10 -4 mol/L Ni(II) in the presence of 4×10 -4 mol/L of H 4 MDI and dissimilar concentrations of HOL (1×10 -3 -5×10 -2 mol/L) at pH 7. The results attained and were displayed in Figure 12 showed that in the concentration range of (1×10 -3 -9×10 -3 ) mol/L of HOL, high removal % of Ni(II) was attained. At a higher concentration of surfactant, the incomplete removal of Ni(II) may be because the high concentration of HOL can change the state of Ni-ligand precipitates which formed through coagulation otation to redispersion form [50]. Furthermore, the decrease in otation at a higher concentration of HOL is produced by the formation of a stable, hydrated envelope of surfactant on the air bubble surface [51,52]. Hence, 1×10 -3 mol/L of HOL was used for all subsequent studies.

In uence of temperature
Trails were performed to remove Ni(II) in a temperature range of (5-80 o C) under the optimum parameters. To do this, a solution of Ni(II) and H 4 MDI and another one having surfactant were heated or cooled to the same temperature in a water bath. Then, the surfactant was transferred to Ni(II) solution. Then, the mixture was transferred to the otation cell followed by a removal procedure. The attained results were displayed in Figure 13 showed that removal % (>98%) of Ni(II) in the range 25-65˚C. Thus, 25˚C was used for all subsequent studies.

In uence of interfering ions
Under the ideal parameters estimated for this study, the removal of Ni(II) from aqueous solutions was tested in the presence of large concentrations of several positive and negative ions. The data were presented in Table 7 and indicated that all the studied interfering ions with high concentrations compared to that of Ni(II) do not affect the removal of Ni(II). Consequently, the proposed method can nd its applications on water samples with a different matrix.

Application to real water samples
For testing the application of the proposed method, trials were conducted to remove Ni(II) ions added to some real water samples. The removal tests were done using 50 mL clear and ltered solutions at pH 7. The results displayed in Table 8 showed that the removal was acceptable and quanti able under the optimum parameters of the proposed removal method.

Antimicrobial evaluation
Ligand and related complexes were tested for their antibacterial activity against S. aureus as gram-positive bacteria and E. coli as gram-negative bacteria and their antifungal activity against C. Albicans. Table 9 provides antibacterial and antifungal MIC (μg/mL) activities. As references for comparing the potency of the tested compounds under the same circumstances, Fluconazole fungicide and Cipro oxacin bactericide were used.
H4MDI has the most potent activity with MIC of 0.78 µg/mL for both E. coli and C. Albicans. While Ni(II) complex shows the potent activity against S. aureus with MIC of 2.34 µg/mL. Ni(II) complex also shows good MIC activity against E. coli with 4.68 µg/mL and C. Albicans with 1.17 µg/mL, which have the same reference compounds activities. While Cr(III) complex has the lowest activity with MIC of 37.5 µg/mL against E. coli, 12.5 µg/mL against S. aureus, and 4.68 µg/mL against C. Albicans.

Cytotoxicity assay
For such experiments, the values of IC50 were determined in a micromolar unit. The cytotoxicity of 5-Fluorouracil (5-FU) was compared to the cytotoxicity of the free ligand and its complexes. It was noticed that the cytotoxicity was not affected by that chelation with metal. It is important to emphasize that ligand showed almost high to that of Fluorouracil activity (15.10 µmol/L) for HePG-2. These agreeable results encourage us to conduct further experiments in vitro. Moreover, after extra analysis steps, the ligand also exhibited signi cant activity against the hepatocellular carcinoma human tumor cells screened (HePG-2) cells as a cell growth inhibitor. Therefore, it is essential to further in vitro biological assessment and to study the mechanism of action [53, 54]. From Table 10, (which gives a chance for application to hot wastewater) making the process economic. Moreover, the highest antimicrobial activity was detected in Ni(II) complex. Additionally, H 4 MDI and its Ni(II) complex show a very strong cytotoxic activity against HePG-2 (liver carcinoma) with IC 50 of 9.7 and 7.7 µmol/L, respectively.

Declarations
Funding Not applicable.
Con ict of interest The authors declare that they have no competing interests and non-nancial competing interests.
Availability of data and material The data that support the ndings of this study are available from the authors upon reasonable request.      Figure 1 1H-NMR spectra of H4MDI.