Utilization and simulation of innovative new binuclear Co ( II ) , Ni ( II ) , Cu ( II ) , and Zn ( II ) diimine Schi ﬀ base complexes in sterilization and coronavirus resistance ( Covid - 19 )

: This article aimed at the synthesis and molecular docking assessment of new diimine Schi ﬀ base ligand, namely 2 -(( E )-( 2 -(( Z )- 2 -( 4 - chlorophenyl )- 2 - hydroxyvinyl ) hydrazono ) methyl )- 6 - methoxyphenol ( methoxy - diim ) , via the condensation of 1 -( 4 - chloro - phenyl )- 2 - hydrazino ethenol compound with 2 -(( E )-( 2 -(( Z )- 2 -( 4 - chlorophenyl )-2 - hydroxy vinyl ) hydrazono ) methyl )- 6 - methoxyphenol in acetic acid as well as the preparation of new binuclear complexes of Co ( II ) , Ni ( II ) , Cu ( II ) , and Zn ( II ) . The following synthesized complexes were prepared in a ratio of 2:1 ( metal/ligand ) . The 1 H - NMR, UV - Vis, and FTIR spectro scopic data; molar conductivity measurements; and micro analytical, XRD, TGA/DTG, and biological studies were carried out to determine the molecular structure of these complexes. According to the spectroscopic analysis, the two central metal ions were coordinated with the diamine ligand via the nitrogen of the hydrazine and oxygen of the hydroxyl groups for the ﬁ rst metal ions and via the nitrogen of the hydrazine and oxygen of the phenol group for the second metal ions. Molecular docking for the free ligand was carried out against the breast cancer 3hb5 - oxi doreductase and the 4o1v - protein binding kidney cancer and COVID - 19 protease, and good results were obtained.


Introduction
Our society is currently dealing with one of the most serious health issues, the Coronavirus. Isatin compounds were found to possess antiviral properties and were used in the treatment of HIV [1]. After virus infection, MT-4 cells infected with HIV-1 or infected human cells were incubated with various concentrations of Isatin derivatives, and the number of practical cells was determined using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique [1]. In addition, the Isatin Schiff base compounds have been studied widely and were found to exhibit significant activity towards Moloney leukemia virus [2], vaccinia virus [3], rhinovirus [4], and SARS virus [5]. The mechanism of inhibition of N-methylisatin-b-40,40-diethylthiosemicarbazone (MIBDET) has been described based on the growth of the molecular leukemia virus (MLV): the MLV inhibition by M-IBDET included blocking the viral RNA translation rather than RNA interference during the viral transcription process [2]. Isatin and Isatin derivatives have also been examined for their antiviral effectiveness against acute cardiac acute respiratory syndrome [5]. The Isatin derivatives showed prohibition activity against SERS-CoV3C-like protein (3CLpro) at concentrations ranging from 0.95 to 17.50 mM. The prohibited properties were tested using the fluorescence resonance energy transfer method and confirmed by HPLC analysis. Isatin-thiosemicarbazone as a preventive compound has a substantial bioactive role against a variety of viral infections [6]. Numerous compounds of 5-fluoroisatin and its derivatives were synthesized, and they displayed a significant inhibition behaviour towards different viruses and inhibited the replication process of the vesicular stomatitis virus in Vero cell lines [7].
The biological activities of Schiff bases of oximes such as hydrazone, semicarbazones, and thiosemicarbazones have been previously described [8]. Only a few variants of these chemicals showed considerable antiviral activity. For example, the 5-acyl-3-methylsulfamyl-1,2,4-triazine molecule and its derivatives were found to have antiviral activity against the coxsackie virus B4 in Vero and HeLa cell cultures, with EC50 values of 20 and 45 mg/mL, respectively [8]. The Schiff base silver(I) complexes with glycine salicylaldehyde showed good antiviral activities against the Cucumber mosaic virus [9][10][11][12]. Additionally, nickel(II), platinum(II), and palladium(II) derivatives, especially with semicarbazone and thiosemicarbazone of p-tolualdehyde, have been documented to show antifungal activities in vitro against several types of fungi such as Alternaria alternata, Aspergillus niger, and Fusarium udum [10][11][12][13]. In comparison to the parent ligands, the metal chelates show an excessive antifungal activity, as determined by examining these derivatives for preventing the growth of fungus. This result is attributed to the improved delocalization of the p-electrons caused by chelation, which increased the diffusion and the entry of the metal complexes to the lipid membrane, where the metal derivatives then blocked the enzymes in the microorganisms' membranes. Salicylaldehyde and 1-amino-3-hydroxyguanidine were used to determine the Schiff base. Wang et al. [14] used hydroxyaminoguanidines to modify a few basic structures of Schiff bases, resulting in the formation of unused substituted salicylaldehyde Schiff base compounds. For the first time, all of these compounds were tested against coronavirus infection and mouse hepatitis virus [14].
At this point, [1-[(30 allyl-20-hydroxy benzylidene) amino]-3-hydroxyguanidine] is the most active compound in the development of mouse hepatitis virus, roughly 376 times more active than hydroxylguanidine and around 564 times more active than hydroxyaminoguanidines. Schiff bases are versatile carbon-based molecules that are widely used and produced via the condensation reaction between various amines and aldehydes or ketones, which is known as imine. It has a wide range of applications, including pharmaceuticals, medicine, and natural activities, among others. It was also used as an oxygen sensor [15][16][17]. Recently, many research articles focused on the Schiff base of thiosemicarbazide and metal derivatives because of its numerous applications in the pharmacology field as antiviral, antiparasitic, antimicrobial, anticancer, and anti-HIV [15][16][17]. We have, therefore, undertaken the synthesis, characterization, structural, thermogravimetric, and antimicrobial studies of a few binuclear complexes formed from Co(II), Ni(II), Cu(II), and Zn(II) containing tetradentate 2-((E)-(2-((Z)-2-(4-chlorophenyl)-2-hydroxyvinyl)hydrazono) methyl)-6-methoxyphenol (methoxy-diim) Schiff base ligand.

Compound (2)
An equimolar solution of p-chlorophenacyl bromide (0.01 M) and hydrazine hydrate (0.01 M) was prepared in 50 mL of ethanol; then the mixture was refluxed for 2 h with heating at 70°C. After cooling the mixture, dilute acetic acid was added to neutralize it. The resulting compound was filtered, washed with water, and then recrystallized in ethanol to obtain compound (2).

Compound (3)
A solution was prepared in 30 mL of acetic acid from the mixture of hydrazine derivative (2) (0.01 M) and 2-hydroxy-3-methoxybenzaldehyde (0.01 M). Then, the solution was heated under reflux for 2 h, cooled, and added into water. The resulting compound was separated out and recrystallized to give The suggested structural formula for the ligand (methoxy-diim) ( Figure 1)  Then, each metal solution was added gradually to the above solution of the methoxy-diim ligand. Ammonia solution was used to neutralize the formed mixtures (pH = 8-9), refluxed, stirred well for 2 h at 75°C, and evaporated to half its initial volume. The resulting complex was allowed to cool for a day, separated and washed with a mixture of methanol and diethyl ether. Then it was recrystallized in methanol and dried using anhydrous CaCl 2 . Thin layer chromatography was performed to confirm its purity.

Instruments
A Jenway 4010 conductivity meter was used to measure the conductance of complex solutions (10 −3 mol/cm 3 in DMF). The ratio of metal in the synthesized metal complexes was determined using the gravimetric method by oxidizing it to its most stable form as well as chloride ions. The results confirmed the proposed theoretical structure for the complexes. A magnetic balance (Sherwood Scientific Cambridge, England)was used for magnetic measurements. A Bruker FT-IR spectrophotometer was used to record the infrared spectra in the range of 4,000-400 cm −1 ; the electronic, 1 H-NMR (400 MHz), and thermal studies were carried out at the Cairo University. The elemental analyses (e.g. carbon, hydrogen and nitrogen) were carried out using an elemental analyser (Vario EL III Germany) at the Microanalytical Center, Cairo University. The X-ray diffraction patterns were obtained using a Bruker AXS configuration X-ray powder diffractometer.

Molecular docking
Autodock software has been utilized for the docking study.      Figure 2).

Infrared spectral studies
The infrared spectrum of the methoxy-diim free ligand  Figure 4 in Table 2. The mediumstrong peak (1,680 cm −1 ) of the carbonyl group found in the free methoxy-diim ligand disappeared after complexation. This result can be due to the tautomerism  change in the methoxy-diim ligand from the keto to the enol form in alkaline media through the chelation process ( Figure 5). In the case of the free diim ligand, the very strong peak at 1,622 cm −1 is due to the C]N stretching frequency of the azomethine group [19]. This stretching absorption band is withdrawn to a lower frequency range of 1,603-1,607 cm −1 in the prepared metal complexes, probably suggesting the coordination of the azomethine C]N group by the lone pair of electrons on its nitrogen towards the centre metal ions. The peak of the phenolic -OH group is slightly shifted to higher average frequencies than those in the spectrum of the diim ligand (3,448 cm −1 ) because of the hydrogen bond formed among the oxygen atoms of the phenolic and nitrogen of azomethine groups ( Figure 5). In the spectra of the synthesized complexes ( Figure 4 I-IV), the broad bands in the frequency range 3,422-3,450 cm −1 are assigned to the presence of coordinated and uncoordinated water molecules [19]. From the spectrum of the free ligand and its metal complexes, the wavenumber of ν(COCH 3 ) (2,842 cm −1 ) is slightly [19] changed, indicating that the COCH 3 group does not take part in the complexation with the metal ion. The average weak absorption peaks found in the range 855-735 cm −1 refer to bending vibrations δ(H 2 O), emphasizing the presence of coordinated water. From the spectra of the methoxy-diim ligand, the peaks found at 1,397 and 1,253 cm

Electronic and magnetic studies
In the wavelength range of 300-800 nm, the electronic absorption spectra of the methoxy-diim free ligand and its metal complexes were recorded in DMSO as a solvent. The electronic spectra of the free methoxy-diim ligand showed two peaks at 315 and 355 nm, referring to π-π* and n-π* electronic transitions [20][21][22]. The Co(II) complex showed three main bands at 300, 355, and 448 nm in its UV-Vis absorption spectrum: the first absorption peak was due to the intra-ligand transition of the organic moiety, and both the second and third electronic transitions were due to 4 T 1g → 4 T 1g (P) and 4 T 1g (F) → 4 T 2g (F), respectively [23,24]. The octahedral geometry of the Co(II) complex was confirmed from the UV-Vis spectrum and the recorded value of its magnetic moment (5.10 B.M.) [21]. The electronic spectrum of the Ni(II) complex was found to have four electronic transition peaks at 300, 320, 360 and 400 nm. The first and second peaks may be assigned to the ligand-to-metal charge-transfer transitions, while the next two transition bands were assigned to d-d transitions of 3 A 2g (F) → 3 T 1g (P) and 3 A 2g → 3 T 1g (F), respectively. The magnetic moment value of the Ni(II) complex was recorded at 2.74 B.M. From these results, the octahedral geometry was confirmed for this complex [21,22]. The electronic UV-spectrum of the copper(II) complex displayed four electronic peaks at 300, 350, 385, and 410 nm. The first two absorption peaks were assigned to  the ligand-to-metal charge-transfer transitions, while the second two absorption peaks, assigned to the 2 E g → 2 T 2g d-d transition, were due to a distorted octahedral geometrical structure of the Cu(II) complex [21,22]. The Cu(II) complex recorded a magnetic moment of 1.66 B.M., confirming the deformed octahedral geometry [13]. The electronic spectrum of the Zn(II) complex was found to have two electronic transition peaks at 300 and 356 nm. According to the conductivity, microanalytical studies and data from the spectrum of the zinc(II) complex, its diamagnetic behaviour and tetrahedral geometry were confirmed [21,22].   all of the peaks became weaker and shifted to slightly lower frequencies. The predicted structural formula of the zinc(II) complex is debated using the results from the analysis performed, confirming its tetrahedral geometry as shown in Figure 2.

Thermogravimetric studies
The TG and DrTGA thermal decomposition curves of the free methoxy-diim ligand and its metal complexes [Co(II), Ni(II), Cu(II) and Zn(II)] under a N 2 atmosphere at a heating rate of 10°C/min ( Figure 6) are assigned and summarized in Table 3.

Methoxy-diim free ligand
The melting point of the diim ligand was 100°C after it underwent concurrent degradation. It was found that the ligand decomposition takes place in two main degradation steps. The first degradation phase was in the temperature range 46-205°C with equal weight loss (obs. = 10%, calc. = 11%). The second degradation phase was in the temperature range 205-536°C with equal weight loss   (obs. = 66.30%, calc. = 65.40%). The remaining residue until 700°C was accompanied by weight loss (obs. = 23.7%, calc. = 23.6%). All of the thermal fragments in all stages were organic moieties that were converted to gaseous phases.

Cobalt(II) complex I
From the thermal degradation curve of the Co(II) complex, it was found that it decomposes as follows: the first decomposition phase is in the temperature range 35-217°C with weight loss (obs. = 10.6 %, calc. = 10.4%), attributed to the loss of coordinated 2H 2 O + CO. The second phase degradation takes place in the temperature range 217-544°C with a weight loss (obs. = 63.6%, calc. = 62.9%), attributed to the loss of chloride, ammonia, nitrogen, hydrogen, and acetylene gas molecules. The final residue was pure cobalt metal produced from the reduction of cobalt carbonate to cobalt(II) oxide and remained stable up to 700°C.

Nickel(II) complex II
The thermal degradation of the Ni(II) complex occurred in two main decomposition phases. The first step in the decomposition with weight loss (obs. = 8%, calc. = 8.2%) was due to the loss of three uncoordinated water molecules and two hydrogen molecules in the temperature range 40-196°C. The second degradation phase with weight loss (obs. = 69%, calc. = 69.1%) was due to the loss of chloride, ammonia, water, nitrogen gas, acetylene gas, and hydrogen gas molecules in the temperature range 196-562°C. The final residue obtained was 2NiO molecules until 700°C.

Copper(II) complex III
The thermal degradation phase of the Cu(II) complex undergoes three decomposition phases with temperature maximum values (DTG max ) of 60 (37-180°C), 255 (180-354°C), and 423 (354-530°C). The first phase with weight loss (obs. = 5.9%, calc. = 5.8%) was due to the loss of two water molecules with a DTG max = 60°C. The phases from second to the third degradation with weight loss (obs. = 58.8%, calc. = 58.7%) due to the loss of two water molecules, ammonia gas, chloride, nitrogen, hydrogen, carbon oxide, and acetylene gas molecules with DTG max values of 255 and 423°C. The final residue was CuO.

Zinc(II) complex IV
The thermal degradation of the Zn(II) complex was found to take place in two main steps with DTG max = 170 (40-250°C) and 399.6 (250-580)°C. The first degradation phase with weight loss (obs. = 8.7%, calc. = 8.1%) was   II  15  18  11  NA  NA  NA  III  8  13  13  NA  NA  NA  IV  19  20  15  NA  NA  NA  Gentamycin  26  24  30 25 16 20 due to the loss of water and carbon oxide with a DTG max = 170°C. The second thermal decomposition with weight loss (obs. = 56.5%, calc. = 57.1%) was due to the loss of chloride, ammonia, nitrogen, and acetylene in the temperature range 250-580°C. The residual product was 2ZnO until 700°C.

Kinetic thermodynamic parameters
From the Arrhenius plots between the rate of decomposition (ln K) and 1/T, the kinetic values such as activation energy (E*) were computed. The Coats-Redfern and Horowitz-Metzger equations were utilized to calculate other thermodynamic parameters such as free energy ΔG*, the enthalpy ΔH*, and the entropy ΔS* of the process [23,24]. Since ΔG*, ΔH*, and ΔS* are relevant to the highest ratios, they are computed by the peak temperature T s ( Table 4). The prepared activated metal complexes were found to be more orderly structured than the reactants and this was proved by the negative value of ΔS* [25] obtained.
3.12 Powder X-ray diffraction studies of the free ligand and its complexes with Co(II) and Ni(II) The powder XRD pattern of the free ligand (diim) and complexes prepared with different ions are shown in Figure 7.
We have recorded the pattern within the range of 2θ between 5°and 60°. In the case of methoxy-diim, nine peaks have been obtained at different diffraction angles, summarized in Table 5, and therefore diim is crystalline in nature [25,26]. On the other hand, the complex of diim with Co(II) does not show any sharp peak in the XRD pattern; instead, it has noise signals that confirm its amorphous behaviour. It was noted from the data that only two peaks at diffraction angles 2θ of 8.75°and 44.12°are observed in the Ni(II) complex as shown in Table 5.

Biological assessments
The free diim ligand and its metal complexes were dissolved in DMSO to obtain a final solution with concentrations of  10 and 50 mg/mL, which was used to measure the inhibition zone to determine the antibacterial activity of these compounds. As DMSO was used as a solvent it was loaded for monitoring. This activity was determined by the agar well diffusion method three times to ensure consistency of the results. Bacillus subtilis and Staphylococcus aureus were the Gram-positive bacteria used, while Escherichia coli and Proteus vulgaris were Gramnegative bacteria. From the summarized data in Table 6, we can conclude that the diim metal complexes show low average activity against B. subtilis, S. aureus, and E. coli. There was no activity against P. vulgaris for both the free diim ligand and its metal complexes. Also, they recorded no activity against Aspergillus flavus and Candida albicans species during determining their antifungal activity.

Molecular docking
The docking calculations were performed using the methoxydiim protein model using docking server after merging the non-polar hydrogen atoms, defining rotatable bonds, adding Gasteiger charges, and other analytical parameters with the assistance of AutoDock tools [27,28].
The simulation for the docking was accomplished by Solis & Wets local search method [29,30] and Lamarckian genetic algorithm (LGA). The docking survey output results carried out for the 3hb5-oxidoreductase (breast cancer) protein, 4o1v-protein (kidney cancer), and COVID-19 protease binding vs the methoxy-diim free ligand are summarized in Table 7.
The methoxy-diim free ligand displayed good binding interactions with the amino acids of the protein molecules showing good stability with binding reaction energy values of −6.64, −3.54, and −4.58 kcal mol −1 for 3hb5-oxidoreductase protein, 4o1v-protein, and COVID-19 protease, respectively (Figure 8a-c).
It can be concluded that the free diim ligand can bind to the active positions in the 3hb5-oxidoreductase protein, 4o1v-protein, and COVID-19 protease binding protein, which means that it has a great binding affinity towards them.
The active binding site of COVID-19 was studied with the metal free ligand (diim) and all of the other complexes formed with different ions (Co, Ni, Cu, Zn). It was found from the docking study that the Ni(II) complex displays the best free energy of binding of −7.12 kcal/mol with the protease. The complex interactions of ligand and COVID-19 are shown in Figure 9.
The free energy of bindings (FEBs) with Co(II), Ni(II), Cu(II), and Zn(II) were found to be −6.19, −7.12, −6.89, and −6.79 kcal/mol, respectively. Further, the smaller binding energy shows better efficiency of binding, which is observed in the nickel complex [31], and the obtained interactions with amino acids are Glu-166 and Asn-142. So, it represents the best binding of COVID-19 protease. However, 3D interactions of Co(II), Ni(II), Cu(II), and Zn(II) complexes are shown in Figure 10 and the other docked information is summarized in Table 8.

Conclusion
The idea of the present research was based on preparing some of the new Schiff base compounds from the condensation reaction of the salicylaldehyde nucleus with  some aromatic amines, as these types of compounds possess biological properties that are anti-bacteria, antifungal, and anti-viral. The work of this research project also extends to a theoretical and interactive simulation of the prepared compounds and their effect on the Corona virus. We were interested in preparing and characterizing four different metal Cu(II), Ni(II), Zn(II), and Co(II) complexes of the diim ligand in a ratio of 2:1 (metal/ligand). The free ligand and its metal complexes were characterized by elemental analysis, electronic analysis, FTIR, 1 H-NMR, XRD, and mass spectroscopy. From the FTIR spectra, it was found that the coordination process took place with the nitrogen atom of the azomethine group and the oxygen atom of the hydroxyl group for the first metal ion, while the second one formed a coordination bond with the oxygen atom of the phenol group and the other nitrogen atom of the azomethine group forming (2:1) complexes. All complexes had octahedral geometry, except for Zn(II) which had tetrahedral configuration. The thermal decomposition of both free ligand and its metal complexes occurs through two main degradation steps, with metal oxides (CoO, NiO, CuO, and ZnO) as a final residual product. The conductivity values confirmed the non-electrolytic behaviour of all of the complexes. Finally, biological evaluations were run as antibacterial, antifungal, and molecular docking. The molecular docking study reveals that the Ni(II) complex shows the best binding site with COVID-19 protease (6LU7), with a free energy of binding of −7.12 kcal/mol.