NOVEL SERIES OF THIAZOLE-BASED COMPLXES; CHARACTERIZATION, STRUCTURAL OPTIMIZATION, IN-VITRO AND IN-SILICO ASSESSMENTS AGAINST COVID-19 VIRUS COMPARED TO PALXLOVID DRUG

. New thiazole-based complexes were prepared and suggested to be 1:1 metal to ligand ratio. This formula was built depending on different analyses. Various spectroscopic tools were used to confirm the mode of bonding for the ligand towards the metal ions. The ligand behaved as a neutral bidentate mode towards Cu(II), Co(II) and Ni(II) ions. The DFT method was applied to confirm the nucleophilicity of O(13) and N(2) atoms which qualified for excellent coordination. The new compounds were in-silico tested against 6lu7 and 6lzg as the main protease complex of COVID-19 and the receptor of coronavirus spike, respectively. This is according to the recent use of a thiazole-based compound (ritonavir) in treatment of COVID-19. This study was extended to handle the paxlovid antiviral (nirmatrelvir and ritonavir) in comparing to new compounds. The ritonavir drug as well as the Co(II) complex appeared as promising inhibitors. To deepen the study, we tested the cytotoxicity of the complexes and interestingly, the Co(II) complex showed a vital inhibition for liver and prostate cancers which exceeds 5-fluorouracil drug.


INTRODUCTION
Corona virus is receiving attention from the whole world, and many researchers are placing special importance in their research to help overcome the risk of infection. In recent years, promising medicines for the corona family have been discovered and are presently being examined for COVID-19, even though the studies have not been finished, and all drugs are already being examined [1]. Because of the exceptionally high ratio of viral enzyme mistakes that are accountable for their genetic reproduction, RNA viruses are exceptionally adaptable to changes [2]. While several elements, such as genetic differences in viruses and some external conditions, have a role in the development of diseases, these viruses are accountable for several new and reemerging diseases [3]. This virus was shown to be vulnerable to high temperatures (heat) and to be inhibited by a modest number of lipid solvents [4]. Umifenovir is a wider antiviral agent that has been approved as an anti-influenza medication (arbidol). The Academy of Chemical and Pharmaceutical Science in Russia has been developing an antiviral medication for the treating of influenza A and B for about 25 years. It has been registered since 2004 for use as a SARS virus treatment [5]. In 2014, Japan produced favipiravir (avigan), as a medication for novel or persistent influenza viruses. Purine nucleosides are substituted for ribosyl binding and intracellular phosphorylation to stimulate and assemble them into viral RNA. RNA-dependent RNA polymerase will then destroy viruses, preventing RNA strain growth and virus propagation [6]. Idoxuridine is a herpes antiviral medication. It's a nucleoside derivative with a changed deoxyuridine structure that's similar enough to be absorbed into viral DNA replication, but the The reaction mixture was stirred at 0-5 o C for 2 h. The product was filtered, dried well and recrystallized from ethyl alcohol. Reddish brown crystals, yield = 90%, m.p. 210-212 °C. IR (KBr): νmax/cm -1 : 3341, 3337 (NH2), 1693 (C=O), 1650 (C=N). 1

Synthesis of metal ion complexes
Equi-molar fraction (5mmol) from each of CoCl2.6H2O (1.189 g), NiCl2.6H2O (1.188 g) or CuCl2. 2H2O (0.852 g) salt was dissolved in EtOH solvent and then mixed with thiazole derivative (CET) (1.760 g) to generate the new complexes. The reaction mixtures were kept under reflux (at 75 o C) for a long time without remarkable changes. As known, the used salts have relatively acidic nature in reaction mixture due to presence of chloride anion, so the addition of 1g NaOAc (dissolved in few drops of dist. water) could elevate the reaction pH slightly (~5). The colors of reaction mixtures are changed suddenly, which considered the preliminary evidence for complexation. Then the reaction mixtures were maintained under reflux overnight for precipitation. The slight basicity of acetate anion helps the reaction to complete and complex precipitation. Filtered colored precipitates which were removed and then washed with EtOH. Under CaCl2, the specimens were put in closed desiccators for drying. Table 1 shows the results of some analyses, for studying.

Techniques used
The majority of the instruments used for analysis are found in Mansoura and Cairo Universities. However the measuring conditions were mostly included in the discussion section. While, the 1 H NMR spectra of the organic compounds were obtained using a Bruker WP (300 MHz) in DMSO-d6 as the solvent and TMS as an internal reference in Mansoura University. In addition, each metal percentage was determined using a conventional volumetric titration method under the most appropriate conditions as described in the textbook [17]. Also, the qualitative test of chloride content was carried out by using AgNO3 [17] after complete digestion of the complexes using conc. HNO3. The negative result (no white precipitate) detects there is no presence of chloride with the three complexes completely. This is acceptable especially in presence of NaOAc in the reaction medium and the best ligational ability of acetate group which superior chloride ion.

Structural optimization
Gaussian 09 [18] is a well-known molecular dynamics simulation tool that has been approved for structural type validation and is comparable to the single crystal x-ray capability. Becke3-Lee-Yang-Parr (B3LYP) [18] and valence double zeta polarizing basis set (6-31G*) have indeed been included to the DFT process and adapted to the ligand and its complexes properly. This balanced basis set corresponds to the most well-known split-valence double-zeta plus polarization. This word denotes that the core-orbital is defined by a contract of six Gaussian orbitals, whilst the valence is defined by two. Polarization operations on non-hydrogen atoms are indicated by the star (*). The DFT method was performed at the B3LYP level using integral equation formalism variant (IEF-PCM) and polarizable continuous model to examine ground and excited state properties. The produced computing files (log and chk) were analyzed in Gauss-View and Gauss-Sum 2.2 [19] tools to obtain all quantum properties. The fchk file, on the other hand, was created from the chk file that modified in Gauss-prog panel.

In silico study A) Pharmacophore search
This simulation strategy aims to evaluate the behavior of new compounds towards COVID-19 depending on the therapeutic history of thiazole-based compounds, recently in COVID-19 treatments. Consequently, the ligand-based discovery model with grid -based model [20] was used to execute pharmacophore quiries (http://pharmit.csb.edu). This silico used to look at how thiazole derivative interacts with two COVID-19 proteins (6lu7 and 6lzg) that taken from Protein Database (PDB). Additionally, this study was elongated to handle Paxlovid drug which already now used for treating COVID-19. This drug contains two antivirals as nirmatrelvir and ritonavir that is from thiazole family, for comparison with our compounds. The two proteins chosen for this simulation were 6lu7 and 6lzg as the co-crystals of main protease complex of COVID-19 and the receptor of coronavirus spike, respectively according to PDB. Each PDB file was imported as a source of receptors and then the features were loaded by inserting hydrogen atoms over receptors. After that, each tested compound was loaded as a mol2 file into program screen to start the simulation and obtaining the targeted interaction patterns. Finally, run to search on analogues drugs within MolPort and Zn libraries that updated regularly and aggregating 112,939,594 and 123,399,574 compounds, respectively [21]. Then, we could estimate the number and the types of H-bonding inside the interaction patterns obtained.

B) Molecular operating environmental docking (MOE)
The MOE module (version 2018) was used to capture all interactions features of synthesizes or the approved antivirals (nirmatrelvir and ritonavir) with COVID-19 proteins (6lu7 and 6lzg). Regulation of these proteins, which have a multiplication function in COVID-19 cells [22], is a useful target. Furthermore, one of the two antivirals (ritonavir) is a thiazole derivative, and it has recently been employed in treatment of COVID-19. As a result, this in-silico study was designed to compare the antiviral efficiency of the novel thiazole-based compounds with the two drugs and rank their activity. To be appropriate, both of the tested compounds and the COVID-19 proteins should be orientated independently before beginning each docking operation.
The atomic charges and potential energy of each compound were measured after that they were exposed to energy minimization. The consecutive orientation processes were proceeded by creating new database to save the compound as MDB format [23]. Before adding H-atoms, the water must be removed from protein receptors and dummies which adapt to MMFF-force field. After that, attach receptor classes, regulate potential energy, and then run the site-finder to locate allosteric binding receptors or dummies. As a result, after selecting the protein helix, the docking operation on the saved MDB-file can begin. The approach progresses from 30 poses explored to the proper docking pose [24]. Some poses were rejected because of the unfavorable collisions between protein pockets and inhibitors. The qualified hit score and the H-bonding time, which must be ≤ 3.5 Å, were used to determine the interacting capability of all chemicals examined. The energy score values were calculated using known formula [25]. Additionally, the docking structures as well as the interaction parameters were assessed to identify the inhibitory activity against coronavirus.

Cytotoxicity by MTT method
At the Research Centre, the toxicity of the complexes was tested against tumor cell lines, including liver carcinoma (HepG2), prostate cancer (PC3), and breast cancer (MCF-7) using 4,5-methyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromid MTT assay [26,27]. The toxicity of 5-fluorouracil (5-Fu), as a common anticancer medication, was measured in terms of 50% inhibition of cell viability (IC50 values). On the basis of the mentioned study [27], the synthesized materials were submitted to MTT viability assay. This practical method relies on mitochondrial reductase activity inside living cells to convert yellow 3-(4,5-methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide into purple formazan product. The cells were grown in RPMI 1640 medium that completed with 10% fetal bovine serum for assay. 96-well culture plates were loaded with cells immersed in media (2×10 4 cells/mL) and incubated for 12 hr at 37 °C in 5% CO2 incubator. The tested sample (2 μL) was mixed with 2×10 4 cells in 96-well plates and grown for 3 days at 37 °C. The cultivated cells were combined with 20 μL of MTT solution and placed at 37 °C for 4 h. After removing the supernatant from each well, the formazan crystals were decomposed after adding 100 μL of DMSO to each well to generate the cellular reduction of MTT. The absorbance of each well was evaluated using a microplate reader at λmax = 570 nm after mixing with a mechanical plate mixer. The data were recorded as IC50 values, which indicate that the treating cells' proliferation is inhibited by 50% when compared to control cells' development.

Analytical data
The molar ratio inside the complexes was 1:1 (M:L) depending on the analytical results ( Table  1). The complexes are stable during storing in desiccators and insoluble in common solvents while are completely soluble in DMSO. The conductivity of 1 mmol in DMSO was measured for the three complexes and the values point to their electrolytic nature ( Table 1). The measured values (Λm) were from 33.7 to 42.1  -1 cm 2 mol -1 range, detecting the mono-anionic property of the complexes [28]. As we told previously (synthesis section) the chloride did not found in the complexes completely. This indicates that the electrolytic nature may be due to acetate anion which found in the reaction medium.

Synthesis of thiazole-based ligand (CET)
The key of our research paper, 2-amino-5-(4-ethoxycarbonyl-phenylazo) thiazole (3) was prepared by the electrophilic diazo-coupling reaction of 2-aminothiazole (1) and the diazotized ethyl 4-aminobenzoate (Scheme 1). Diazotization of ethyl 4-aminobenzoate (2) has been achieved by treatment with sodium nitrite and conc. HCl at 0-5 °C. The obtained diazonium chloride was coupled with 2-aminothiazole in ethyl alcohol and sodium acetate at the most reactive site of thiazole, the fifth position (C-5) rather than the fourth one (C-4). The IR spectrum of compound 3 revealed absorption bands at 3341; 3337 and 1693 cm -1 corresponding to amino (NH2) and carbonyl (C=O) groups, respectively. Its 1 H NMR spectrum exhibited signals at 1.32 ppm (triplet) and 4.31 ppm (quartet) for the ethoxy group of ester (-OCH2CH3). The aromatic protons resonated as two doublet signals at 7.70 and 8.01 ppm while the singlet signal at 8.15 ppm indicated the proton of thiazole-C4. The singlet signal at 8.61 ppm (exchangeable after addition of D2O) indicated the protons of amino group (NH2).
Chloroacetylation of 2-aminothiazole derivative 3 was achieved by stirring with chloroacetyl chloride in dry dimethylformamide and anhydrous K2CO3 to afford 2-chloroacetamido-5-(ethoxycarbonyl-phenylazo) thiazole (4). The structure of compound 4 was confirmed by spectroscopic techniques including IR and 1 H NMR. Its IR spectrum exhibited absorption bands at 3160, 1723 and 1688 cm -1 corresponding to the NH, carbonyl-ester (COOEt) and carbonyl amide (NCOCH3) groups, respectively. Its 1 H NMR spectrum revealed triplet and quartet signals at 1.34 and 4.35 ppm for the ethoxy group (-OCH2CH3), singlet at 4.48 ppm for two protons (NCOCH2Cl). The aromatic protons resonated as two doublet signals at 7.84 and 8.08 ppm while the singlet signal at 8.53 ppm indicated the proton of thiazole-C4. The singlet signal at 12.99 ppm (exchangeable by addition of D2O) for one proton of chloroacetamide moiety (NHCOCH2Cl).

IR and 1 H NMR spectroscopy
The new thiazole-based ligand (CET) was coordinated with Co(II), Ni(II) and Cu(II) ions to yield three corresponding complexes. These complexes were basically scanned by IR-spectroscopy, to investigate the shifts recorded to the functional groups. This information could being obtained via comparing the spectrum of the ligand with its complexes, to estimate the affected functional groups. The ligand behaved as a neutral bidentate during the coordination with the metal ions used. This proposal was confirmed firstly by the significant shift of (C=O)amide and (C=N) groups from 1688 and 1600 cm -1 , respectively, in the three complexes. On the other side, the vibration of NH was shifted to higher wavenumber, referring to its ruling out from the coordination [29]. The crystal water molecules that suggested with [Ni(OAc)(CET)](OAc).2H2O and [Cu(OAc) (CET)(H2O)2](OAc).2H2O complexes, lead to broadness noticed around 3400 cm -1 . This suggestion will be confirmed later through thermogravimetric analysis (TGA).
Regarding the complexes spectra, new bands were observed at ≈ 1480 and 1440 cm -1 which attributed to as(OAc) and s(OAc) vibrations respectively [30]. The difference between the two bands (˂ 100 cm -1 ) signifies the bidentate binding of the group. While, the ionic acetate group was confirmed via the molar conductivity values that reported in the previous section. Further, the metal-ligand bonds were recorded at ≈ 600 and ≈ 520 cm

Electronic spectra and magnetism
To promote transition of electrons inside the chemical compounds, you must scanned the samples under UV-Vis radiation either in its dissolved form (in DMSO) or in its solid form (nujol mull).
Here, we scanned the dissolved samples of the complexes, after detecting the negligible role of the solvent on the complexes due to no noticeable change in their colors after dissolving. The scan was carried out over 200-1000 nm range, to facilitate all transitions belong intraligand, charge transfer (CT) and d-d transitions (ligand field) within the complexes ( Figure 1) [31].  [32]. While, the band appeared at 18,530 cm -1 assigned to LF transition of 1 A1g→ 1 B1g type inside the square-planer geometry centered by Ni(II) ion. The diamagnetic property appeared (μeff = 0) reflects the paring of electrons in d 8 system during formation of dsp 2 hybridization [33]. The [Cu(OAc)(CET)(H2O)2](OAc).2H2O complex showed a magnetic moment value (1.70 BM), which is comparable to that of d 9 -systems. In addition, its UV-Vis spectrum ( Figure 1) reveals a band at 26,525 cm -1 (377 nm) that is assigned to CT in a tetragonally distorted octahedral structure. The LF transitions, on the other hand, were seen at 20,964 cm -1 (477 nm) and 14824 cm -1 (broad band at 675 nm), which could being to 2 B1g→ 2 Eg and 2 Eg→ 2 A1g transitions, respectively [34,35]. Finally, Scheme 2 displayed the suggested structures of the three complexes.

TGA and kinetics
This analysis was performed for Ni(II) and Cu(II) complexes to assess presence of water molecules that suggested to be found with these complexes based on elemental and IR analyses. The degradation curves were performed at a fixed rate of heating (10 o C min -1 ) and over the range of 20-800 o C. Also, the plausible thermal degradation of the two complexes were tabulated. The curves of the two complexes exhibited four degradation steps starting from ≈ 31 o C, which detects the thermal instability of the two complexes. The first step showed the expel of the crystal water molecules in the two complexes in addition to the coordinating molecules in the Cu(II) complex. The successive three steps showed a complete distortion for the structural building of the complexes until ≈ 790 o C. At this point, we recorded the residual parts which agree with NiO and CuO+6C in the two complexes, respectively. The parameters of the kinetic or thermodynamic estimated from thermal decomposition of Ni(II) and Cu(II) complexes were calculated by utilizing known equations. The relations were drawn according to Coats-Redfern and Horowitz-Metzger equations. Then the values of parameters were calculated for the first three degradation steps in the two curves and tabulated. Consequently from the data displayed we could conclude the following information; 1-For all decomposition stages of complexes, first-order rate was the best fit in every case. 2-The activation enthalpy for the first degradation was determined to be lower because of ejection for water molecules that occurs comparatively fast. 3-When negative activation entropy (ΔS) is found, complexes are more stimulated than reactants [29]. 4-The positive value of ΔH denotes the endothermic character of decomposition processes. 5-The reduction in the decomposition rate from one stage to another was shown via increasing the ΔG value. 6-Furthermore, the positive sign of ΔG detects that the ultimate residual quantity was bigger than the starting stage, indicating a non-spontaneous decomposition process.

Mass spectroscopy
This analytical framework adds further supporting for the molecular formula proposed for two new complexes (Co(II) and Ni(II)), especially with the inability to separate or studying single crystals. This scanning was carried out at a heating rate of m/z = 50-1000 and a temperature of less than 40 °C/min at 70 eV. After evaporating the sample, a beam of electrons was used for collision-induced ionization, which leads to produced molecular ion that effectively fractionated into smaller masses. The mass/charge values of these particles were registered in the patterns exhibited, and they were sorted accordingly. The molecular ion peak of Co(II)-CET complex was appeared at m/z = 532.21 (calcd. 529.82; I = 10), which fits exactly to [M + +2] ion. This difference in mass value is owing to isotopes of chloride which may be found. When this ion is exposed to accelerated electrons, it produces a series of fragmentation peaks (Figure 2), including the base peak (m/z = 377.69; I = 100%), which corresponds to [C14H13N4O3S] + moiety. Furthermore, cobalt isotopes produce peaks below to m/z = 65.
In addition, the spectrum of Ni(II)-CET complex exhibited the molecular ion peak at m/z = 532.35 (I = 8, calcd. 565.61), which could be related to ([M + +2]-2H2O) ion. The observed difference between the found and calculated mass due to expel of crystal water molecules during the vaporization process prior the bombardment of electrons [36]. Lastly, it's worth noting that the base peak recorded at m/z = 56.98 corresponds to the metal isotope that was primarily visible during the complex's fragmentation.

EDX and XRD studies
Energy-dispersive X-ray microanalysis (EDX) has being used to confirm the production of selected complexes (i.e. Figures 3) as well as to assess the quantity of elements in the compound (wt %) [37]. The solid specimens (complexes) were placed to a beam of accelerated X-ray photons. The intense photons could enter the inner shells (K-or L-shell) and interact directly with core-electrons. These electrons were easily dragged from their ground orbitals and stimulated to higher levels, leaving positive holes that could be filled with electrons from upper shells such as the L-or M-shell. During relaxation the lost photons able to draw K-(Ka1, Ka2, or Kb) or Lseries. The L-series was crucial in this assessment since it allowed us to assess the percentage of each element. As we all know, each element has its own electronic configuration, which corresponds to the development of a singlet-set of peaks that is characteristic for such element. In addition, the heavier element (metal and Cl) was utilized as the basic marker for all percentages (wt%), while the lighter elements (C, H, and O) were omitted. The validation was carried out over a long period of time at several sites in order to obtain better signals. The computed elemental molar ratios (wt/At %) appeared to be close to those derived from elemental analysis.  The X-ray diffraction methodology (XRD) is useful for determining the degree of crystallinity in solid substances. The patterns were produced throughout the range of 10 o < 2θ < 80 o (Figures 4) [38]. The X-ray beam was generated from Cu/Kα source. Sharp peaks could be seen in the patterns, indicating that these compounds have well-defined crystalline particles. Therefore, the crystal parameters were determined using FWHM approach, including the crystallite size from the Deby-Scherrer equation [β = 0.94 λ/(S cos θ)]. Where B: is the line thickness at half maximum height, θ: is the intense peak diffraction angle, S: is the crystallite size, and Cu/Kα (λ) = 1.5406 Å [39]. The d-spacing between the interior layers, on the other hand, was calculated using Bragg equation

Structural optimization
The quantum calculations take importance in the last decades and caused revolutionary in the scientific researches based on high credibility of equations used. Gaussian 09 program achieved such importance particularly with unavailability of X-ray single crystallography in our labs. All structures were optimized under 6-31G * basis set and the exported files were visualized based on standard scheme of numbering to extract parameters (Tables 2) and maps (Figures 5) for investigation and analysis.

A) Common features
The structures of CET ligand and its complexes were cleared to recognize the orientation of the functional groups before and after the complexation. The appearance of C(7)=O(13) and C(1)=N(2) groups is favorable for their coordination with the metal ions. Although, the cisorientation of N(6) and S(5) atoms but they did not contribute in the coordination with the metal ions. This suggestion was supported by Mullikan's charges on the donor atoms. The high negativity of O13 and N2 atoms is helpful for their coordination perfectly. While, the positivity of N6 and S5 prevents their coordination due to high electrophilic property [40,41]. The charges of O13 and N2 atoms are significantly reduced in the Co(II)-CET and Ni(II)-CET complexes. Whereas, their charges were elevated in the Cu-CET complex due to metal-ligand CT which is known for the Cu(II) complexes through π-back donation [41]. Also, the charges of the metal ions were efficiently shifted from the starting charge (+2) which considered as a legal feature after coordination.
The bond lengths (Å) for some functional groups within the structures of the ligand and its corresponding complexes, were determined and tabulated ( Table 2). The lengths of C(7)=O (13)

B) Global reactivity
To predict the bioavailability of the studied substances, relevant indices were determined using conventional formulae [44] depending on ELUMO and EHOMO values. The indicators that calculated were absolute-softness (σ), global softness (S), global hardness (η), chemical potential (µ), electrophilicity (ω), and electronegativity (χ). We can draw the following conclusions based on the projected values: (i) if the complexes' softness property exceeds than that of their ligand, we can expect their superiority in biomedical field. (ii) The electrophilicity scores of the complexes are likewise higher than those of the ligand, indicating that these compounds have a great propensity to take electrons from their surroundings. (iii) Furthermore, the complexes' high energy gabs (∆E) represent their tolerance towards electronic transitions. Table 2 shows that the dipole moment (Debye) values are less; this could be attributed to decreased polarity over the molecules, which is preferable in biological efficiency. The formation energy values (a.u.) show that the produced complexes are quite stable [44].

C) 3-D maps
Over cubic surfaces of the compounds, the HOMO and LUMO levels were created ( Figure 5). The following were the characteristics of the two frontier orbitals; regarding the ligand, the functional groups which included the donor atoms, were well covered by the two frontier orbitals. This shows how adaptable their electronic-clouds during the coordination. The complexes imaging, on the other hand, showed the two levels focused on the core metal atoms and their environment [42].
The location of electronic cloud on the functional groups was also differentiated using electrostatic potential maps. By differentiating nucleophilic, electrophilic, and neutral zones in the ligand, this map can be utilized to investigate the nucleophilicity of groups towards the metal ions then analyzing the binding mechanism. The colors red, blue, and green were used to depict these three parts. The electrostatic potential of the free ligand showed brightly the nucleophilic and electrophilic zones. The nucleophilicity of O (13) and N(2) atoms was clearly visible, showing their capacity to coordinate with metal ions. While, the electrophilicity of N(6) and S(5) was clearly noticed, indicating their inability to coordinate with the metal ions. Regarding the maps of the three complexes, the nucleophilicity of O (13) and N(2) atoms was notably reduced. Whereas, the overall nucleophilicity in the complexes was improved due to the coordination of acetate or water molecules as a secondary ligands [43,44].

In-silico assessments versus COVID-19
This simulation aims to evaluate the behavior of CET ligand and its complexes versus COVID-19 depending on the therapeutic history of thiazole-based compounds, recently in COVID-19 treatments. The goal of this research is to look at how thiazole derivative interacts with two COVID-19 proteins (6lu7 and 6lzg). Also, we oriented our study for comparison with Paxlovid drug which contains two antivirals (nirmatrelvir and ritonavir) and recently used for treating COVID-19. The ritonavir drug is a thiazole-based compound as our ligand which may success also in coronavirus treatment or one of its complexes. The two proteins chosen for this simulation were 6lu7 and 6lzg as the co-crystal structures of main protease complex of COVID-19 and the receptor of coronavirus spike, respectively. This selection is based on their role in cell growth of virus and their inhibition is a perfect target.

A) Pharmit profile
An introductory view about the therapeutic activity versus targeted proteins could being built through this in-silico way [53,54]. Consequently, we aimed to investigate the new thiazole-based ligand (CET) and its complexes versus receptors of COVID-19 (6lu7 and 6lzg) compared to selected antivirals (nirmatrelvir and ritonavir). The interaction patterns for all processes were yielded (Figures 6), for investigation. The magnitude of interaction for all compounds as well as the two antivirus with 6lu7 protein is lesser than their interactions with 6lzg protein which is highly effective. The best interaction was observed from ritonavir drug and the Co(II)-CET complex with the two proteins especially with 6lzg one. The interaction of the free CET ligand in very poor compared to its complexes. The inhibition activity of Co(II)-CET complex exceeds than the nirmatrelvir drug. So, this complex may be used instead of it in Paxlovid drug to increase its efficiency towards COVID- 19 virus, which still irritating the world. This inhibition activity recorded from Co(II)-CET complex which exceeds than the free ligand, may refer to the cobalt ion inserted in the organic compound. As known, the cobalt ion could bind to the hydrophobic pockets inside the enzymes and its complexes played as antivirals perfectly [47]. The types of H-bonding formed inside the interaction patterns of Co(II)-CET, nirmatrelvir and ritonavir with 6lzg protein were H-acceptor (acc) and H-donor (don) ( Table 3). On the other hand, a pharmacophore query was run for each new compound inside MolPort and Zn libraries to find any analogues drugs, but we did not reach to anyone.

B) Molecular operating environmental (MOE) docking
In the previous section (A) we introduced in-silico assay of two recommended antivirals in treatment of COVID-19 compared to our new compounds via Pharmit link. The outcomes suggest the superiority of ritonavir drug and our new Co(II)-CET complex in the interaction with two COVID-19 receptors. This suggests the successful use of Co(II)-CET complex instead of nirmatrelvir in the Paxlovid drug to treat recent coronavirus. While, the whole interaction features cannot obtained from this silico way, while the MOE-docking offers a deep information about the interaction validity [48]. The two antivirals (nirmatrelvir and ritonavir), CET, Co(II)-CET and Ni(II)-CET were the potential inhibitors treated in this simulation against receptors of COVID-19 (6lu7 and 6lzg). The interaction patterns and the docking parameters (Table 3) were obtained and investigated to display the following remarks; (i) The docking score values lead to this inhibition order; ritonavir-6lzg (-9.1276) ˃ ritonavir-6lu7 (-8.3398) ˃ Co(II)-CET-6lzg (-7.7579) ˃ Co(II)-CET-6lu7 (-6.7341) ˃ nirmatrelvir-6lu7 (-7.0578). This detects the high inhibitory effect of ritonavir drug and the Co(II)-CET complex towards the two proteins [49]. All the docking poses are formed from true allosteric bending with protein pockets via normal H-bond lengths ≤ 3.5 Å.
The scoring values for CET docking are significant but all its interaction features are missing so its inhibition validity cannot verified. (ii) The H-donor type of binding is predominant in addition to the appearance of H-acceptor type in some cases. The acidic residues (blue circle) were the main residues recorded in the docking patterns and bonded via side-chain donor type. Whereas the polar residue (pink circle) was slightly appeared and bonded via side-chain acceptor type. (iii) The binding sites from new compounds were N2, O14, O22 and O42, the binding sites from ritonavir drug were N9, N40 and N37, while the binding sites from nirmatrelvir drug were O13, N4, O6 and N10. (iv) The residue of amino acids that interacted as a receptor from 6lu7 protein was GLU166 (A) with Co(II)-CET, Ni(II)-CET and nirmatrelvir drug. While, the CYS 145 (A) was also interacted with Co(II)-CET complex and the ritonavir drug interacted with ASN 142(A) and GLN 189 (A) residues. On the other side, the GLU 406 (A) residue was interacted from 6lzg protein with Co(II)-CET and Ni(II)-CET only. Whereas, the residue interacted with ritonavir drug was ASP 206 (A) and the residues interacted with nirmatrelvir drug were HOH 1034 (A), HOH 833 (A), HOH 940 (A) and HOH 1046 (A). The inhibition of Glutathione (GLU) is very essential due to its vital role in the living cells, so its controlling is a good goal. So, the Co(II)-CET complex exhibited interaction with this amino acid which is favored [49]. (v) The ligand exposure surface was effectively reduced in most complexes, which detects the saturation of binding for all compounds and there is no probability for extra-bonding can be formed. (vi) The formation energy values were notably lowered in Co(II)-CET docking complexes with both 6lu7 and 6lzg proteins. The values reach to -21.5 kcal/mol with 6lu7 and -18.2 kcal/mol with 6lzg. Also, Ni(II)-CET docking complexes were highly stable the formation energy values were -16.4 and -18.5 kcal/mol with 6lu7 and 6lzg proteins, respectively. While, the stability of nirmatrelvir or ritonavir drug with the two proteins are moderate [49]. Finally, we could suggest the Co(II)-CET complex as antiviral towards COVID-19 which close to the activity of ritonavir drug while exceeds the activity of nirmatrelvir drug. This study may be a starting point for specialist to concentrate their clinical investigations on our new complex, which could being promising.

Cytotoxicity
Depending on the in-silico results which exhibited the desirable inhibition activity of Co(II)-CET complex towards the recent coronavirus, we intended to investigate the cytotoxicity of these new compounds towards tumors that are not less dangerous than the COVID-19. MTT method [26,27] was applied to estimate the cell viability of liver carcinoma (HepG2), prostate cancer (PC3), and breast cancer (MCF-7) cells after treatment with the new complexes compared to 5fluorouracil (5-Fu). The IC50 (µg/mL) values were calculated (Table 4) and the Co(II)-CET complex showed the excellent cytotoxicity for liver carcinoma (HepG2) and prostate cancer (PC3) cell lines. The value towards HepG2 cells appeared close to reference drug (5-Fu), while exceeds the reference in PC3 cells. Consequently, the promising biological activity of Co(II)-CET complex was confirmed via silico towards COVID-19 and via in-vitro towards cancer cell lines, particularly the liver and prostate cancers. It was known that the biological activity of the chemicals is strongly influenced by the structure of such compound. Consequently here, the existence of thiazole rings, N=N, C=O, and NH functional groups, improves the biological performance of the compounds. They have the potential to increase the penetration of substances across cell membranes, allowing for actively engaging with biological systems while also inhibiting cell development. According to Tweedy's chelation theory, the metal ion has a substantial effect on the degree of inhibition towards microorganisms. According to this idea, the metal charge is minimized due to coordination until it reaches a limit that allows the complex to be lipophilic which is could miscible with cell lipid and then interface with cell systems. On the other side, the inclusion of the complex molecule on the cell wall could impair the cell's capacity to produce protein, which is necessary for respiration, and so the cell's development was slowed [50]. Also, the quantum data that reported in the previous sections supported the activity of such structures based on the estimated global reactivity indices. Table 4. Cytotoxic activity against three human cancer cell lines. a IC50 (µg/mL): 1-10 (very strong), 11-20 (strong), 21-50 (moderate), 51-100 (weak), and above 100 (noncytotoxic). b 5-FU = 5-fluorouracil is the reference drug for anticancer tests.

CONCLUSION
New Co(II), Ni(II) and Cu(II) complexes were prepared from thiazole-based ligand. The ligand coordinated through neutral bidentate towards the metal ions within different geometries. All available analytical and spectral techniques were used to elucidate the formulae of the ligand and its complexes. The DFT/B3LYP method was applied to configure the structures of all compounds and then confirming the coordination features. The global reactivity indices were calculated and indicated the best physical properties of the complexes. In-silico assays were performed against COVID-19 receptors. This is based on the recent use of a thiazole-based compound (ritonavir) in coronavirus treatment. This antiviral was mixed with nirmatrelvir drug in COVID-19 protocol. Consequently, this study was conducted to the two antivirals for comparison with the new compounds. The Co(II)-CET complex as well as the ritonavir drug are appeared with excellent inhibition for COVID-19 receptors. Additionally, we performed in-vitro test for cytotoxicity against three carcinoma cell lines, liver carcinoma (HepG2), prostate cancer (PC3), and breast cancer