Novel Cr(III), Ni(II), and Zn(II) complexes of thiocarbamide derivative: Synthesis, investigation, theoretical, catalytic, potentiometric, molecular docking and biological studies

2.1 Materials

Solvents, metal(II) chlorides, benzoyl isothiocyanate and 1H-1,2,4-triazol-3-amine have been purchased from Sigma-Aldrich.

2.2 Instrumentation

PerkinElmer-2400 series-II analyzer have been used for partial elemental analysis. Ordinary methods (Jeffery et al., 1989) were being used to estimate the content of chloride and metal in investigated complexes. The Infra-red spectra were measured by KBr pellets via FT-IR spectrophotometer “Mattson 5000″. The electronic spectra are analyzed by via UV / Vis Spectrophotometer (Unicam). The NMR spectra of ¹H and ¹³C with Brucker 400 MHz were handled on ligand H₂L that is identified in the solvent (DMSO). The photoluminescence spectra of investigated compounds were made in DMSO solution on excitation by using a LS50B PerkinElmer Fluorimeter. Jenway 4010- conductivity meter was used to assess the molar conductivity of prepared DMSO solution of metal complexes (0.001 mol/dm⁻³). The spectrum of X-ray diffraction was described in detail utilizing Cu, Wavelength 1.5406 Å source on diffractometer ”the Bruker AXS Advance“. Mass spectra were obtained with ionization mode (EI) in the range of m/z = 40–1000 with Varian Mat 311. The Gouy procedure for scientific magnetic susceptibility to Sherwood has been used to determine the effective magnetic moment µ_eff, at room temperature per metal atom. The zeta potential measurements for the H₂L and its metal complexes in water were performed via Malvern Zeta-size Nano at 25 °C. The thermal analyzer TGA-50H was used for thermal evaluation (TGA / DTG) under measured conditions such as the temperature rises by rate equal to 10 °C/min.

2.3 Synthetic procedures of ligand, H₂L and its complexes

The H₂L ligand derived from thiocarbamide moiety and metal chelates have been synthesized in accordance with the procedures described in Scheme 1. The products obtained were crystallised a number of times with absolute EtOH then with Et₂O and then desiccated over anhydrous calcium chloride. TLC was performed to verify the purity of the H₂L ligand. The physical and analytical results are summarized in Table 1.

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Scheme 1

The outline synthesis of ligand (H₂L) and its metal complexes.

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2.4 Molecular modelling

Unfortunately, we could not get single crystals from the investigated compounds, thus structure optimization data have been measured with DMOL³ application in the Materials Studio software (Delley, 2002). Optimized complex frameworks were estimated using the DFT (Modeling and Simulation Solutions for Chemicals and Materials Research and Studio, 2011) process. Computations of DFT semi-core pseudopods (dspp) have been generated via double number basis sets and functional polarisation (DNP) (Warren, 1986) that was much additional successful than the duplicate gaussian basis groups (Kessi and Delley, 1998). Furthermore, The optimistic interchange-correlation feature was based on the functional (GGA) and (RPBE) (Hammer et al., 1999).

2.5 Catalytic oxidative degradation of EBT dye

The organic dye was oxidatively degraded at a pH 7.0 (buffered aqueous solution) in an air atmosphere in the presence of known doses of green oxidizing agent H₂O₂. 0.2 mg of the Zn²⁺ complex as a catalyst was added to 10 ml of the dye solution (30 mg/l), followed by an appropriate dose of H₂O₂ (30%) and stirring. At the end of each experiment, the flask contents were filtered, and the concentration of the dye in each filtrate was measured at λ_max of 530 nm (Hassani et al., 2015). At a constant dose of H₂O₂ (0.2 ml) and constant dye concentration (30 ppm), the effect of time was investigated by running the reaction for 5-, 10-, 15-, 20-, and 30-minute intervals. Initially, the effect of temperature was investigated by performing the reaction at 30, 45, and 60 °C. Finally, the effect of H₂O₂ dose was investigated using 0.2, 0.3, 0.4, 0.5, and 0.6 ml of H₂O₂ and constant dye concentration (30 ppm and 30 °C, respectively).

2.6 Potentiometric study

Hana Instrument 8519 digital pH meter was used to perform pH-metric measurements. Titrations were carried out at 298, 308, and 318 K. The Van Uitert and Hass relation (Uitert and Haas, 1953) is used to correct pH-meter readings in a 50 percent (v/v) dioxane-water mixture: $$-\log \left[H^{+}\right]=B+\log U_H^o+\log {\gamma }_{\pm }$$

Where $\log U_H^o$ and $\log {\gamma }_{\pm }$ are the correction factors for the solvent composition and ionic strength, respectively and B is the reading.

The titrations of solution mixtures towards standardized free carbonate NaOH solution (9.65x10^-3 M) in 50 percent (v/v) water - dioxane at constant ionic strength are used in the experiment (1 M KCl solution). Figure S1, Supplementary Materials, depicts this. The solution mixtures were made in the following manner:

1. 1.25 ml HCl (1.04 × 10⁻² M) + 1.25 ml KCl (1 M) + 10 ml bidistilled H₂O + 12.5 ml dioxane.

2. 1.25 ml HCl (1.04 × 10⁻² M) + 1.25 ml KCl (1 M) + 2.5 ml (5 × 10⁻³ M) H₂L + 10 ml bidistilled H₂O + 10 ml dioxane.

3. 1.25 ml HCl (1.04 × 10⁻² M) + 1.25 ml KCl (1 M) + 2.5 ml (5 × 10⁻³ M) H₂L + 9.5 ml bidistilled H₂O + 10 ml dioxane + 0.5 ml metal ion (Ni²⁺) (5 × 10⁻³ M).

2.7 Biological activity

2.8 Molecular docking with EGFR protein

A large number of studies have shown that epidermal growth factor receptor (EGFR) is a potential therapeutic target for the treatment of various tumors (such as colorectal and breast tumors) (Avdović et al., 2020). The inactivation of this receptor can affect the spread of cell cancer and promote cancer cell apoptosis. thus, we analyzed the potential inhibitory effects of ligand, H₂L and its metal complexes.

Molecule Operating Environment (MOE) software is used to evaluate the binding affinity between EGFR protein and investigated molecules (Integrated Computer-Aided Molecular Design Platform, Molecular operating environment, Chemical Computing Group, 2019). Use the specified minimization algorithm to minimize the energy of all 3D molecule structures. Then the charge of the atom is processed and the minimized potential energy is corrected. The inspected target substances are saved as a new database in MDB format (Shah et al., 2020). The crystal structure of the EGFR receptor (PDB: 3W2S) is taken from the protein database (Sogabe et al., 2013). MOE has been used to adjust the structure of molecularly bound proteins by removing ligands, adding hydrogen, and minimizing the energy of 3W2S. The minimized energy structure is used more as a binding receptor. The largest active center of 3W2S (PHE 856, LYS 745, ASP 855, CYS 797, ARG 841, ASN 842, MET 793, LEU 718, GLY 719, SER 720, VAL 726, ASP 800, and PHE 997) is the MOE position obtained by the site finder algorithm. The docking uses various features (initial re-scoring methodology: London dG with poses 10, final re-scoring methodology: GBVI/WSA dG with poses 5, placement: triangle matcher, and refining: rigid receptor) to identify and evaluate the compounds' connections with 3W2S. The S value is a rating value that measures the affinity of a compound to the receptor and is calculated by the standard MOE rating function. The RMSD is also used to compare the binding conformation with the binding reference configuration.

3.1 ¹H and ¹³C NMR spectra of H₂L

The H₂L ¹H NMR spectrum (Figure S2, Supplementary Materials) in DMSO solution showed two signals related to SH and NH protons at 11.855 and 8.445 ppm, respectively. the presence of SH signal indicated that H₂L solution was in the form of thiol. All previous signals have vanished by addition D₂O (Figure S3, Supplementary Materials). The aromatic ring protons have been found between 7.334 and 7.880 ppm.

The ¹³C NMR spectrum (Figure S4, Supplementary Materials) revealed three signals related to the C-S and C = O carbons at 168.30 and 166.00 ppm, respectively (Hosny et al., 2021; Hosny et al., 2018). Signals occurring at 151.86, 153.62 and 158.78 ppm attributed to the C = N carbon atoms of the hetero ring and the open chain, separately.

3.2 ¹H NMR spectra of Zn(II)-complex

The ¹H NMR spectrum of Zn(II) complex (Figure S5, Supplementary Materials) in DMSO solution showed the signal related to NH proton at 8.449 ppm as well as the disappearance of signal attributed to SH proton which confirmed the proposed structure of Zn(II) complex.

3.3 IR spectra of H₂L and its complexes

A brief evaluation has been made between the Infra-Red spectral data of the H₂L ligand and its complexes in order to investigate the coordination action of H₂L towards the metal ions. The most significant IR bands of absorption were displayed in Table 2 and Figure S6, Supplementary Materials. The IR spectrum of H₂L shows distinct vibrations at 1612, 1638, 1697 and 2053 cm⁻¹, related to ν(C = N)_ring, ν(C = N)*, ν(C = O) and ν(SH), respectively (Rakha, 2000; Abdel-Monem et al., 2018; Abdel-Monem and Abouel-Enein, 2017; Hosny et al., 2018; Abdel-Rhman et al., 2019). The intense vibration at 2053 cm⁻¹, due to the v(SH) group, indicates that, H₂L is existing as thiol form in the solid phase. The vibrations referring to ν(NH) groups cannot be determined since a large destruction of 3160–3400 cm⁻¹ overshadows their asymmetrical and symmetrical vibrations. The bending system δ of the (C = N)_ring appeared at 623 cm⁻¹, which has changed to a larger wavenumber when it is participate in complexing.

In the Cr(III) complex, the H₂L coordinates the Cr(III) ion as neutral bidentate via the recently formed groups of (C = N)* and (C = N) _{hetero ring}. This type of complexation is indicated by the presence of ν(SH) and ν(C = O) at the higher and same wavenumbers, separately. This shows that these locations are not participate in coordination. Furthermore, the change of ν(C = N)* to the lower wavenumber and the change of ν/δ(C = N)_{triazole ring} to lower and higher wavenumbers (Rastogi and Sharma, 1974), respectively, indicated mutual coordination between these locations. In addition, in Zn(II) chloride complex, the H₂L works as mono-negative tridentate via deprotonated-SH, (C = O) and (C = N) ring groups. This statement was concluded by the absence of ν(SH) (Pandey et al., 1993), the change of ν(C = O) to less wavenumber (Abdel-Monem and Abouel-Enein, 2017) and the change of δ and ν of (C = N) ring to higher and lower wavenumbers, respectively (Abdel-Monem et al., 2018). Finally, the H₂L operates as a binegative tetradentate throughout the binuclear Ni(II) complex. This proposal is verified by the disappearance of both ν(C = O) and ν(SH) (El-Sawaf et al., 2020) with the parallel presence of ν(C = N)^** and ν(C-O) (Liu et al., 2012) as well as the changing of ν/δ(C = N) vibrations (Rastogi and Sharma, 1974; Pandey et al., 1993).

Recent vibrations in regions 410–470 and 511–585 cm⁻¹ were related to ν(M−N) (Ferraro and Walker, 1965) and ν(M−O), respectively. As well, the wide vibrations at ≈ 3432–3380 cm⁻¹ confirm the existence of H₂O in the complex (Chubar et al., 2003). The values of weight loss from the TGA data were used to distinguish between coordinated and crystallised H₂O.

3.4 Electronic spectra, optical band gap and magnetic properties

The electronic spectra of ligand (H₂L) as well as metal complexes were recorded in DMSO solution have been revealed in (Table S1, supplementary materials and Figure S7, supplementary materials). The ligand H₂L presented bands in the ranges 242 and 216 nm, that could have been allocated to the (π → π*)_Ar and (π → π*)_ring transitions, respectively (Tossidis et al., 1987). The band at a value of 300 nm is due to the carbonyl moiety n → π* transition (Tossidis et al., 1987). The electronic spectra of Cr(III) complex shows three absorption bands at 610, 450 and 336 nm attributable to ⁴A_2g(F)→⁴T_2g(F)(ν₁), ⁴A_2g(F)→⁴T_1g(F)(ν₂) and ⁴A_2g(F)→⁴T_1g(P)(ν₃) transitions, respectively characteristic for octahedral Cr(III) complexes (Parmar et al., 2010). Furthermore, the magnetic moment value, (µ_eff. = 3.36B.M.) can be taken as an extra indication for the octahedral geometry of Cr(III) complex. The complex, [Ni₂(HL₁)Cl₂(H₂O)₂].4H₂O complex have a magnetic moment values 2.63B.M., which is lesser than the measured value for a single nickel atom of d⁸-octaedral and/or tetrahedral complexes and larger than the diamagnetic square-planar complexes. This value may indicate the presence of Ni(II) complex in mixed stereochemistry (El-Asmy et al., 1990). This interpretation is also verified by the two bands at 346 and 484 nm assignable to ³T₁(F)→³T₁(P) and ³T₁(F)→³T₂(F) transition, respectively compatible with the tetrahedral configuration as well as one band at 396 nm is appearing because of forbidden d–d transition, reliable with the square planar geometry of Ni(II)-complex (Saha et al., 2016).

Tauc’s equation, αhυ = A(hυ - E_g)^r was used to calculate optical band gaps for Cr(III) and Ni(II) metal complexes, where (r = ½ or 2 for indirect, and direct transitions, respectively), (A): energy independent constant, and (E_g): optical band gap (Hosny et al., 2020). E_g Values are estimated from the plot of (αhυ) with (hυ) (Fig. 1). According to the curves, the direct band gaps for Cr³⁺, and Ni²⁺ complexes are 4.64, and 4.66 eV, respectively. while, the indirect band gaps are 4.22, and 4.26 eV, respectively This information reveals that these complexes are magnetic insulators with insulating properties, high-spin frameworks, and antiferromagnetic ordering on a regular basis, i.e., metal cations in close proximity have opposite spin. Furthermore, the electronic structure of these complexes is distinguished by a predominance of metal d-orbitals in both the valence and conduction bands. (Cipriano et al., 2020).

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Fig. 1

Optical band gaps (a: indirect, and b: direct band gap) for A: Cr³⁺, and B: Ni²⁺ complexes.

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3.5 Mass spectra

The mass spectrum of H₂L (Figure S8, Supplementary Materials) revealed that the molecular ion peak [M]⁺ appeared at the value of m / z equal to 247, that was identical to the M.wt. of ligand. The fragmentation path of the H₂L ligand was given in Scheme 2.

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Scheme 2

The main fragments in mass spectrum of H₂L.

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3.6 Photoluminescence spectra

The photoluminescence spectrum of H₂L show emission broad band at 349 nm. Moreover, its Cr(III), Ni(II) and Zn(II) complexes, show emission broad bands at 356, 349 and 359 nm, respectively, Fig. 2. These bands could be assigned to as L-M charge transfer (Singh et al., 1999; Etaiw et al., 2018). The emission bands of Cr(III) and Zn(II) complexes indicate that both are traditional blue complexes. Furthermore, the intensity of fluorescence in all complexes is substantially lower than that of free ligand. This could be because the transition metal (M)–fluorophore (F) interaction is too strong, resulting in fluorescence quenching (Zhao et al., 2010).

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Fig. 2

Emission spectra of H₂L and its complexes in DMSO solution.

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3.7 X ray powder diffraction of [Ni₂(HL)Cl₂(H2O)₂].4H₂O complex

The XRD patterns of separated [Ni₂(HL)Cl₂(H₂O)₂].4H₂O complex is depicted in Figure S9, Supplementary Materials and its (2θ)° value for peaks, the peak indexing, and inter-planar spacing (d-values) were showed in Table S2, supplementary materials. The lattice parameters of Ni(II) complex has been evaluated by using match software ( https://www.crystalimpact.com/match/). The [Ni₂(HL)Cl₂(H₂O)₂].4H₂O complex has a triclinic space group with P −1 and lattice parameters a = 12.34 Å, b = 12.50 Å, c = 24.45 Å, α = 100.09° β = 90.31°, γ = 95.43° whose unit cell volume is 3772.94 Å³. The lattice parameters of [Ni₂(HL)Cl₂(H₂O)₂].4H₂O complex exhibits a good harmony with the Crystallography Open Database (COD) No. 4,332,969 (Hernández-Molina et al., 2006).

Such lattice parameters were determined by using the next relationship: $$\mathit{triclinic}\frac{1}{d_{\mathit{hkl}}^2}=\left[\frac{h}{a}\left|\begin{array}{ccc}\frac{h}{a}& \cos \gamma & \cos \beta \\ \frac{k}{b}& 1& \cos \alpha \\ \frac{l}{c}& \cos \alpha & 1\end{array}\right|+\frac{k}{b}\left|\begin{array}{ccc}1& \frac{h}{a}& \cos \alpha \\ \cos \gamma & \frac{k}{b}& \cos \alpha \\ \cos \beta & \frac{l}{c}& 1\end{array}\right|+\frac{l}{c}\left|\begin{array}{ccc}1& \cos \gamma & \frac{h}{a}\\ \cos \gamma & 1& \frac{k}{b}\\ \cos \beta & \cos \alpha & \frac{l}{c}\end{array}\right|\right].{\left|\begin{array}{ccc}1& \cos \gamma & \cos \beta \\ \cos \gamma & 1& \cos \alpha \\ \cos \beta & \cos \alpha & 1\end{array}\right|}^{-1}$$

The crystalline-particle parameters were calculated using standard equations in the FWHM method (Velumani et al., 2003). the parameters were particulate sizes = 0.2897 Å lying in the nanometer scale, (2θ)°=23.99, d spacing = 3.7102 Å, FWHM = 5.1099, the crystal strain (ε) = 1.5346 and the dislocation density (δ) = 11.9127 Å⁻². The regular-crystal lattice of the Ni(II) complex could be calculated from the minimised quantities of dislocation‐density (δ) and the crystal strain (ε) (El-Metwaly et al., 2020).

3.8 Zeta potential measurements

The Zeta-potential measurements provide information regarding the stability of the colloidal suspension; the colloidal suspension is stable when the forces generating particle mutual repulsion which plays a prominent role. The higher absolute value of the zeta potential exhibits the greater the repulsion between the particles forming the suspension and thus the higher stability of suspension, whereas low values of ZP (±5 mV) indicate more flocculation between the particles and thus a higher tendency for instability (Bhagat et al., 2019). In this research, [Ni₂(HL)Cl₂(H₂O)₂].4H₂O complex had the largest positive-positive repulsion with a potential of 20.3 mV suggesting the stability of this colloidal suspension. But, the ligand (H₂L) and Cr(III) complex displayed potential of −9.34 and −6.91 mV, respectively which are in the negative range, this results shows the lower stability of the suspension of the ligand (H₂L) and its Cr(III) complex. Furthermore, Zn²⁺ complex demonstrated a potential of 2.69 mV, indicating the instability of Zn(II) complex colloidal solution as the particles of this suspensions prefer to flocculate.

3.9 Thermogravimetric studies

Prediction of coordinated or crystallised H₂O molecules may be performed utilizing TGA data (Rakha et al., 1989; Zaky et al., 2014) (Figure S10, Supplementary Materials). It can be assumed that there is an alignment between the TGA data and the proposed molecular formula. For instance, the Cr(III) complex has 5-degradation stages. The first one seems to have a weight loss of 8.05 percent between 35 and 124 °C, suggesting the elimination of two hydrated water molecules. The step two has a weight loss of 19.67 percent in the 124–352 °C temperature range, demonstrating the elimination of the H₂O co-ordinating molecule and two HCl molecules. The 3rd step (from 352 to 448 °C) has a weight loss of 41.36 percent, referring to the elimination of the molecule C₄H₃ClN₅S. The fourth stage in the 448–547 °C range has a weight loss of 6.27 percent, relating to the elimination of C₂H₄. Finally, unoxidized carbon and metal oxide existed as a residue. Table S3, Supplementary Materials, demonstrates the steps of decomposition of complexes.

3.10 Kinetic parameters

Coats–Redfern (Coats and Redfern, 1964) and Horowitz–Metzger (Horowitz and Metzger, 1963) techniques were accustomed to predict the kinetic and thermodynamic variables of the designed complexes (Figures S11-S16, supplementary materials). Tables 3 and 4 demonstrate the various kinetic parameters (A, E_a, ΔS*, ΔH* and ΔG*) of the separated complexes. we may notice that:

1. A resemblance was found between the data collected from both approaches.

2. The good stability of the complex was proved by the high value of the activation energy.

3. The positive value of ΔG* shows that the degradation stage is non-spontaneous process, also, the + ve value of ΔH* suggested endothermic operations (Abu-Dief et al., 2019; Abu-Dief et al., 2020).

4. The negative ΔS* of certain degradation steps indicate that the activated fragments have a more orderly composition than the un-decomposed fragment and the degradation reactions become slow (Moore and Pearson, 1961). Although + ve values can indicate that the disorder of the decomposed fragments rises much faster than the un-decomposed fragment (Kenawy et al., 2001).

3.11 Molecular modeling

The optimized ligand, H₂L and its metal complexes structures that labeled with the atom symbol and its number were shown in figure S17, supplementary materials and are listed in tables S4-S11, supplementary materials. Coordination induces a minor difference in bond angles and lengths existing in the thiocarbamide structure of H₂L; the major initiatives in the angles of H₂L were N(14)-C(13)-N(11), N(15)-N(14)-C(13), S(12)-C(10)-N(8), O(9)-C(7)-N(8), O(9)-C(7)-C(7)(2), N(11)-C(10)-N(8), N(17)-C(13)-N(11), S(12)-C(10)-N(11), C(13)-N(11)-C(10), N(8)-C(7)-C(7)-C(2), and N(17)-C(13)-N(14). Analyzing the result of H₂L and the separated complexes, the following observations can be stated:

1. As predicted, the Cr(III) structure has angles close with those predicted for octahedral complexes with sp³d² hybridization (El-Gammal, 2010; El-Morshedy et al., 2019). In addition, the optimized structure of the Zn(II) complex tends to be tetrahedral (Moore and Pearson, 1961). moreover, the Ni(II) complex produced mixed geometry (tetrahedral and square planar) including sp³ and dsp² hybridization.

2. (C = O), (C-O), (C-S), (C = N)_azomethine, and (C = N)_ring moities have larger bond lengths than those found in the ligand (H₂L) attributed to the formation of metal–oxygen and metal-nitrogen bonds (Moore and Pearson, 1961).

3. The bond angles of coordination atoms of ligand moiety will be changed in all complexes due to the formation of chelate rings (Fukui et al., 1954).

4. In the Zn(II) complex, metal ion is tri-coordinated to the H₂L ligand in a tetrahedral geometry with bond angles; Cl¹⁹-Zn¹⁸-S¹² = 132.55°, Cl¹⁹-Zn¹⁸-O⁹ = 117.666°,

5. N(14)-Zn¹⁸-O⁹ = 106.487°, S¹²-Zn¹⁸-O⁹ = 91.074°, N¹⁴-Zn¹⁸-S¹² = 92.107° and Cl¹⁹-Zn¹⁸-N¹⁴ = 112.157° which give a small deviation from tetrahedral geometry.

3.11.1

3.11.1 Vibrational calculation and geometry optimization

Fig. 3 displays the computed IR spectrum of the ligand, H₂L in the vacuum and its observed spectrum. A slight variation between the observed and the computed can be found since the observed spectrum was evaluated for the solid material. Figure S18, Supplementary Materials is the relationship chart between the computed and the observed wavenumbers demonstrate the linear relationship according to the given equation ν_cal = 0.878 ν_Exp + 119.321 whereas R² = 0.9866.

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Fig. 3

Comparison of experimental and theoretical IR spectra of Ligand (H₂L).

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By the aid of Density functional theory (DFT), we have been able to determine different quantum variables as E_LUMO, E_HOMO, dipole moment, binding energy, and compounds' total energy (Table 5) (Liu et al., 2012; Yousef et al., 2012; Govindarajan et al., 2012; Abu El-Reash et al., 2013; Pearson, 1989; Padmanabhan et al., 2007; Gaber et al., 2018). Figure S19, supplementary materials includes the energy of frontier molecular orbitals (FMOs, that includes both orbitals of HOMO and LUMO). The data designated that:

1. the stability of studied metal complexes was demonstrated by the high E_a value and it was verified by the negative E_HOMO and E_LUMO value (Gaber et al., 2018; Abu El-Reash et al., 2013).

2. Generally, the HOMO orbital was dispersed on O(9), S(12), N(8), N(11), N(15), N(14), and N(17) atoms, which are the expected position for nucleophilic attacks in the metal ion.

3. The stability of metal complexes than the ligand, H₂L has been explained from the total energy measurements (Aljahdali and El-Sherif, 2013).

Table 5 Calculated gas phase energy, dipole moment, E_HOMO, E_LUMO, energy band gap (E_H – E_L), chemical potential (μ), electronegativity (χ), global hardness (η), global softness (S) and global electrophilicity index (ω) for ligand and its complexes.

Compound	EH (eV)	EL (eV)	(EH -EL) (eV)	χ (eV)	µ (eV)	η (eV)	S (eV−1)	(ω) (eV)	Ϭ (eV)	dipole moment (debye)	Total Energy kcal/mol
H2L	−5.271	−2.377	−2.894	3.824	−3.824	1.447	0.724	5.053	0.691	3.451	−7.12 × 1005
Cr(III)-Complex	−4.435	−2.857	−1.578	3.646	−3.646	0.789	0.395	8.424	1.267	9.818	−1.69 × 1006
Ni(II)-Complex	−4.294	−4.083	−0.211	4.189	−4.189	0.106	0.053	83.145	9.479	8.447	−1.63 × 1006
Zn(II)-Complex	−5.374	−3.498	−1.876	4.436	−4.436	0.938	0.469	10.489	1.066	2.767	−1.16 × 1006

(E_H, E_L): Frontier molecular orbitals energies of HOMO and LUMO, (E_H -E_L): energy band gap, (χ): electronegativity, (S): global softness, (µ): chemical potential, (η): global hardness, (ω) global electrophilicity index and (ϭ): softness.

3.12 Catalytic degradation of azo dye

The catalytic activity of [Zn(HL)Cl]0.0.5H₂O complex was investigated in the oxidative degradation of an organic dye, such as EBT dye, using H₂O₂ as the oxidizing agent “due to its green character”. The oxidation did not occur in the absence of the H₂O₂ or Zn(II) catalyst, but when both oxidant and catalyst were used, the dye degraded. As a result, the impact of H₂O₂ dose was studied in combination with the impact of time and temperature to determine the ideal conditions for the reaction. The effect of time was investigated at a constant dose of H₂O₂ (0.2 ml) and constant dye concentration (30 ppm) by running the reaction for 5-, 10-, 15-, 20-, and 30-minute intervals. Early, the effect of temperature was investigated by performing the reaction at room temperature (30 ˚C), 45, and 60˚C. Finally, the effect of H₂O₂ dose was investigated by using 0.2, 0.3, 0.4, 0.5, and 0.6 ml of H₂O₂ and constant dye concentration and temperature (30 ppm, and 30 ˚C, respectively). The results of the experiment are shown in (Fig. 4), which shows that the degradation of the dye increases with the time of the reaction, with about 45 percent of the dye removed after 30 min. By increasing the H₂O₂ dose, the degradation first improved and then began to be constant or decrease, as shown by self-quenching of OH radicals according to the succeeding OH equation: H₂O₂ + OH ̇→ H₂O + HO₂ HO₂ ̇ + OH ̇ → H₂O₂ + O₂

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Fig. 4

(a) Effect of time on catalytic degradation, and (b) Dye removal against temperature and H₂O₂ dose.

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After 10 min, at a constant dose of H₂O₂ (0.2 ml) and constant dye concentration, the effect of temperature on the reaction was investigated (30 ppm). The study indicate that the capacity of dye removal increases with temperature, with only 57 percent of the dye remaining after 10 min at 60 °C compared to 85 percent at 30 °C.

3.13 Potentiometric studies

3.13.1

3.13.1 Protonation constants of ligand (L)

Table 6 shows the stoichiometric protonation constants of the investigated Ligand (L). The ligand compound investigated here has two protonation constants, corresponding to the protonated N-triazol ring and C-SH groups. As shown in Scheme 3, the N-triazol ring has the highest pKa value (pK_a1 = 8.60, at 25 °C) and the Sulphur group has the lowest (pK_a2 = 4.74, at 25 °C). Fig. 5 depicts the species distribution of the ligand (L). The ligand (L²⁺) from Sulphur group to form HL⁺ tends to lose it protons after the pH is raised to the pH range of 4.71–4.74. As the conditions become more alkaline, the second proton aims to be deprotonated to a free ligand (L).

Table 6 Stability constants of Ni(II)- L in 50% (V/V) Dioxane-Water at different temperatures and I = 0.05 M KCl.

System	T(oC)	log10 β
L	25	8.60
		13.34
	35	8.54
		13.27
	45	8.52
		13.23
Ni- L	25	8.60
		13.40
	35	8.56
		13.35
	45	8.25
		13.02

L: (Z)-N-benzoyl-N'-(1H-1,2,4-triazol-3-yl)carbamimidothioic acid.

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Scheme 3

Protonation process of Ligand.

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Fig. 5

Species distribution of the ligand (L).

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3.13.2

3.13.2 Stability constants of the Ni-L complexes

The stability constants of binary chelated ligand (L) with Ni(II) metal ion as an example of divalent transition metal ions. The comparison of the titration curves of free ligand with the complexed ligand shows that adding Ni(II) ion to the free ligand solution lowers the pH. As a result, the curves associated with complexes are found at lower pHs than that of the free ligand because they require more alkali to raise the pH to the level of the free ligand. The release of protons from the coordinated ligand is an implication of complex formation. The stoichiometric stability constants associated with the inspected ligand's Ni(II) complex were estimated in 50 % (V/V) Dioxane-Water at various temperatures and are shown in Table 6.

Table 6 shows the logarithms of the stability constants for all complex systems evaluated by potentiometric equilibrium titration process (1) and (2) (simplicity charges are omitted):

(1)

$$M+L\rightleftharpoons ML\beta =\frac{\left[ML\right]}{\left[M\right]\left[L\right]}$$

(2)

$$M+2L\rightleftharpoons M{L}_2\beta =\frac{\left[M{L}_2\right]}{\left[M\right]{\left[L\right]}^2}$$

The Ni-L process distribution diagram (Fig. 6) is investigated with the goal of investigating the changes in concentration of the Ni(II) complex with pH. The Ni-L complex is generated at pH 5.5 with a maximum of 95 %, while at pH 8, the complex Ni(L)₂ is formed.

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Fig. 6

Species distribution of the Ni(II)-L complex.

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3.13.3

3.13.3 Effect of temperature and thermodynamics

The protonation of the ligand and its Ni²⁺ complex is attributed to the data of thermodynamic parameters that are relative to the temperature data shown in Tables 7 and 8. ΔS and ΔH values were determined by establishing a correlation between equilibrium constant values (ln K) and temperature reciprocal values (1/T) (ln K = - ΔH/RT + ΔS/R) resulting in an intercept ΔS/R, and a slope –ΔH/R (Figs. 7 and 8). There are several conclusions, which are summarized below:

Table 7 Protonation constants of L in 50% (V/V) Dioxane-Water at different temperatures and I = 0.05 M KCl.

T(oC)		pK	Thermodynamic parameters
			ΔHo kJ mol−1	ΔSo JK−1 mol−1	ΔGo kJ mol−1
25	L + H+ HL+	8.60	−7.2949	0.14008	−50.4395
35		8.54
45		8.52
25	HL++H+ L+2	4.74	−2.7111	0.08169	−27.8744
35		4.73
45		4.71

Table 8 Stepwise stability constants of Ni- L in 50% (V/V) Dioxane-Water at different temperatures and I = 0.05 M KCl.

T(oC)		pK	Thermodynamic parameters
			ΔHo kJ mol−1	ΔSo JK−1 mol−1	ΔGo kJ mol−1
25	Ni + L NiL	8.60	−31.4787	0.05991	−49.9307
35		8.56
45		8.25
25	NiL + L NiL2	4.8	−2.6856	0.08285	−28.2027
35		4.79
45		4.77

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Fig. 7

Effect of temperature on the dissociation constant of ligand (L).

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Fig. 8

Effect of temperature on the formation constants of Ni(II)– L complex.

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a. The reaction involving ligand protonation is exothermic and has a net negative ΔG (Table 7).

b. The data in Table 8 show that the values of log₁₀ K₁ - log₁₀ K₂ for binary complexes are positive, implying that the first ligand molecule coordinates to the metal ion is preferable to its bonding to the second (El-Sherif et al., 2012). This may demonstrate the significance of the steric effects caused by the addition of the second molecule of the ligand i.e., the NiL₂ (1:2) species cannot be formed until the NiL(1:1) species is formed. This could be due to: (i) an improvement in free metal ion Lewis acidity (M⁺ⁿ) when compared to a 1:1 chelated ion (ML^{+ n–1}) and (ii) steric weakness resulting from the addition of a 2nd bulky ligand to the chelated ion ML^{+ n–1}.

c. All of the negative values correlated with the complex formation of the Gibb free energy reflect the random existence of the Ni-L complex formation reactions.

d. The negative heat content (ΔH) values indicate that the complex formation operation is exothermic, implying that the chelation operation works better at low temperatures.

e. The ligand complex (ΔS) values are positive, suggesting that the metal complex formation is entropically favorable (El-Sherif and Eldebss, 2011), and the complexation mechanism is related to the production of hydrogen ion (H⁺) and H₂O molecules (Jeragh et al., 2007). During the production of metal chelates, the ligand displaces water molecules from the metal ion's main hydration sphere. The number of particles in the process thus increases, implying that the system's randomness increases with the next equation. $${\left[M{\left(H_{2}O\right)}_n\right]}_{\left(aq\right)}^{2+}+L_{\left(aq\right)}^{-}\rightleftharpoons M{L}_{\left(aq\right)}^{+}+n{H}_{2}O$$

f. indicate that the complexation mechanism is spontaneous and exothermic, implying that the process of complex formation is entropically favorable.

It can be concluded that log K₁ > log K₂ indicates that Ni(II) ion empty locations are more readily available for binding the 1st ligand than the 2nd one. The chelation mechanism-induced reaction is exothermic, spontaneous, and entropically favorable.

3.14 Biological studies

3.14.3

3.14.3 The cytotoxicity of H₂L and its metal complexes

2-various cell lines, HePG2 (liver carcinoma), and MCF-7 (breast carcinoma), have been used to determine the cytotoxicity of the prepared compounds under investigation (in vitro). The represented Fig. 9 demonstrates the relationship between concentration and cell viability. By such plots, IC₅₀ values (IC₅₀ is the concentration that inhibits 50 percent) can be determined as shown in Table 11. The results may be summarized in the following points:

1. The Ni(II)-complex demonstrates effective strong activity towards MCF-7 and HePG2 cells with IC₅₀ values of 10.96 ± 1.0 and 8.31 ± 0.9 µM, respectively, identifying that Ni(II) complex acts as chemo-therapeutically substantial (Yousef et al., 2014).

2. Cr(III)-complex exhibited moderate activity toward MCF-7 and HePG2 cell lines with IC₅₀ values 41.03 ± 2.9 and 27.71 ± 2.1 µM, respectively.

3. The weak action of Zn(II) complex towards both cancer cells with IC₅₀ = 54.30 ± 3.5 and 61.97 ± 3.7 µM.

4. Finally, H₂L exhibited weak action towards HePG2 cancer cells with IC₅₀ values equal to 54.14 ± 3.3 µM and also moderate potency towards MCF-7 cancer cells with IC₅₀ values 33.82 ± 2.5 µM.

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Fig. 9

In vitro antitumor activity of Doxorubicin, H2L and isolated compounds against (a) breast cancer (MCF7) (b) liver carcinoma (HePG2).

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Table 11 Cytotoxic activity of ligand (H₂L) and its metal complexes against human tumor cells.

Compounds	In vitro Cytotoxicity IC50 (µM)
	HePG2	MCF-7
DOX	4.50 ± 0.3	4.17 ± 0.2
H2L	54.14 ± 3.3	33.82 ± 2.5
[Cr(H2L)Cl3(H2O)].2H2O	27.71 ± 2.1	41.03 ± 2.9
[Ni2(HL)Cl2(H2O)2].4H2O	8.31 ± 0.9	10.96 ± 1.0
[Zn(HL)Cl]0.0.5H2O	61.97 ± 3.7	54.30 ± 3.5

DOX: Doxorubicin.

IC₅₀ (µM): 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate). 51 – 100 (weak) and above 100 (non-cytotoxic) (El-Sayed et al., 2018).

3.15 Molecular docking with EGFR protein

It is believed that molecular binding is very important in drug discovery. The investigated compounds related to the most suitable active site of 3W2S of EGFR (PHE 856, LYS 745, ASP 855, CYS 797, ARG 841, ASN 842, MET 793, LEU 718, GLY 719, SER 720, VAL 726, ASP 800, and PHE 997) that predicted by the site-finder algorithm in MOE (Fig. 10 and Figures S22-S24). The largest binding pocket was assigned and all hits were docked against the most active site using the MOE docking software, Table 12. As a glance in this table, the S values of investigated compounds are close to each other. Thus, the inhibitory activity may be compared according to the type and number of interaction bonds of the tested compounds with EGFR protein. According to the interaction with EGFR, the inhibitory activity order is [Ni₂(HL)Cl₂(H₂O)₂].4H₂O > H₂L > [Cr(H₂L)Cl₃(H₂O)].2H₂O > [Zn(HL)Cl]0.0.5H₂O. This order is harmonious with experimental data. It has been observed that the contact between H-donor and H-acceptor is the most common type of interaction with EGFR receptor while Zn(II)-complex doesn’t show any interactions with EGFR receptor. Based on the results tabulated, it can be deduced that the Ni(II)-complex (Figure S23, Supplementary Materials) has the highest inhibitory activity of the EGFR protein which is similar to experimental data. In this complex, two nitrogen atoms of ligand build two H-acceptor interactions with LYS 745 and LEU 858 of EGFR (with distances 2.70 and 3.49 Å) also oxygen atoms of water molecules build two H-donor interactions with ASP 837 and ASN 842 of EGFR (with distances 2.52 and 2.98 Å). While in EGFR- H₂L interaction (Fig. 10), there is one H-donor and one H- acceptor interactions with nitrogen and oxygen atoms with PHE 856, and LYS 745, respectively (with distances 3.21 and 2.96 Å). Finally, Cr(III)- complex shows only π-cation interaction of five-membered ring of ligand with LYS 745 of EGFR (distance = 4.01 Å).

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Fig. 10

3D and 2D molecular interaction of ligand, H₂L to inhibitory activity to the EGFR protein.

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