Synthesis and physicochemical studies of a series of mixed-ligand transition metal complexes and their molecular docking investigations against Coronavirus main protease

Abstract A novel series of mixed-ligand complexes of the type, [M(L1)(L2)Cl]·2H2O [L1 = 2-(α-methyl salicylidene hydrazine) benzimidazole (primary ligand), L2 = 2,2′-bipyridine (bipy; secondary ligand), M = Co(ii), Ni(ii), Cu(ii) and Zn(ii)], were based on the physicoanalytical studies. The spectroscopic findings revealed tridentate nature of the Schiff base ligand (L1) and its coordination to the metal ions via azomethine nitrogen, ring nitrogen and the deprotonated phenolic oxygen atoms. Furthermore, the synthesized compounds were evaluated for antimicrobial activity against Bacillus subtilis, Escherichia coli and Salmonella typhi microorganisms. In addition, molecular docking studies were carried out against Middle East respiratory syndrome coronavirus (PDB ID: 4ZS6) and severe acute respiratory syndrome coronavirus 2 main protease (PDB ID: 6W63). Graphical abstract


Introduction
Benzimidazole, an important precursor of the heterocyclic compounds, exhibits a wide range of biological applications, i.e., antiviral, antifungal, antipyretic, antidepressant and inhibitory activities in various cancers [1][2][3][4][5]. The broad biological activity of these compounds is supposed to be due to the presence of sp 2 hybridized nitrogen donor atoms [6]. The ligational behavior of benzimidazole and its derivatives has been explored in coordination chemistry to form stable complexes through various modes of coordination [7]. The benzimadazolebased metal complexes exhibit broad spectrum of pharmaceutical activities, such as zinc complexes of benzimidazole find usage in anticancer activity, and reveal remarkable antimicrobial activity [8]. In addition, several benzimadazole complexes have been reported as anticancer agents [9,10]. It is also reported that the transition metal ions form stable complexes and have displayed several properties [11,12]. Over the years, bipy and its derivatives have received immense importance as binding blocks and ligands in the construction of various homo-and heteroleptic metal complexes with broad spectrum of applications in the area of both material and biological science [13][14][15][16][17].
Mixed-ligand complexes, which have two different ligands, find significant consideration in coordination chemistry because of their structural variation and diverse applications [18,19]. Besides, mixed-ligand complexes also exhibit various biological applications [20]. Recent outbreak due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has destroyed the economy and various socioeconomic sectors worldwide [21][22][23]. So far, not a single approved drug is available to treat the caused infection. Therefore, scientists are trying to find a drug to combat the COVID-19 pandemic. Several reports suggest that the progression of this pandemic can be controlled by targeting the main protease (M pro ) to develop the potential inhibitor. Molecular docking, a computational strategy to predict the binding site to assist drug repositioning for several diseases, plays a significant role in the pharmaceutical industry to bring new drugs to the market [24]. The HIV-1 protease inhibitors as the repurposed drugs for SARS-CoV-2 M pro are discussed in literature [24][25][26]. Therefore, considering the versatile nature of mixed-ligand complexes, we are reporting four new mixed-ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) ions with benzimidazole-based Schiff base ligand (L 1 ) as the primary ligand, and bipy as the co-ligand, (L 2 ) in 1:1:1 molar ratio, and investigated them by various physicochemical studies. The synthesized metal complexes exhibited moderate antibacterial activity when screened against Bacillus subtilis, Escherichia coli and Salmonella typhi. In addition, we analyzed the molecular docking of the complexes of protein obtained from Middle East respiratory syndrome coronavirus (MERS-CoV; PDB ID: 4ZS6) and SARS-CoV-2 M pro (PDB ID: 6W63). The precursor, 2-hydrazinobenzimidazole, was prepared following the previously reported protocol [27]. The primary ligand, L 1 , was prepared by the condensation reaction of 2-hydrazinobenzimidazole with o-hydroxyacetophenone in equimolar ratio in ethanol [27].

Preparation of mixed-ligand complexes
An ethanolic solution of the Schiff base ligand, L 1 (0.01 mol and 20 mL), metal(II) chloride (0.01 mol and 20 mL) and bipy, and L 2 (0.01 mol and 20 mL) were mixed in 1:1:1 molar ratio and refluxed for 2.5 h at pH = 7-8 by adding catalytic amount of solid NaOH. A colored precipitate was obtained, which was separated by filtration. The precipitate was washed with methanol and dried in vacuo. The schematic representation of the synthesis of mixed-ligand complexes is given in Scheme 1.

In vitro antibacterial activity
The in vitro antimicrobial activity of the studied mixedligand complexes was reported by Agar Well diffusion method [27] at 100 μg mL −1 concentrations against B. subtilis, E. coli and S. typhi with ciprofloxacin as the standard antibacterial drug.

Molecular docking study
In order to evaluate the biological activity, molecular docking was analyzed on CLC Drug Discovery Workbench Software to obtain accurate predictions about the structure and interactions with a protein/enzyme receptor [28]. Some protein/enzyme receptors were imported from protein data bank (http://www.rcsb.org/:PDB): MERS-CoV (PDB ID: 4ZS6 [29]) and SARS-CoV-2 M pro (PDB ID: 6W63 [30]).
In the docking simulation, the compounds ( Figure 1) were placed into a predictable binding site on the surface of a protein target. The CLC Drug Discovery Work bench utilized MMFF94 (Merck Molecular Force Field [MMFF]) force field to generate 3D structure on import. Rotation around bond generates several conformations. Thus, the ligand optimizer was realized by geometry minimization using MMFF94 force field [31], conforming the binding  pocket geometry. The protein-ligand interaction was scored, and the best score-binding mode was returned for individual ligand and collected with the score. The ligand-binding mode search is effectuated inside the binding site (green sphere with a radius large enough to comprise the ligands docked to the receptor protein). After the import of the protein receptor from the PDB bank, the next step involved the setting up of the binding site and the binding pockets; binding pockets are necessary to guide the docking simulation. After the setup of the binding site and the binding pocket, the co-crystallized-natural ligand was extracted and redocked in the active binding site of the protein receptor to validate the method and the docking parameters obtained from the molecular docking studies.
Ethics approval: The conducted research is not related to either human or animal use.

Results and discussion
All the synthesized mixed-ligand complexes are stable at room temperature, non-hygroscopic and soluble in dimethyl sulfoxide and N,N-dimethylformamide. The physicoanalytical data are shown in Table 1.

Fourier-transform-infrared (FT-IR) spectra
The FT-IR spectra revealed the coordination of the metal ion through the deprotonated phenolic oxygen atom by the disappearance of the band at ∼3,300 cm −1 due to the phenolic -OH stretching vibration [32,33]. This was further confirmed by the hypsochromic shift of phenolic v (C-O) band at ∼1,280 cm −1 in the free ligand and L 1 to ∼1,500 cm −1 in the spectra of the mixed-ligand complexes [33,34]. However, the position of the band due to v N-H (exocyclic) remains practically unaltered in the spectra, suggesting its noninvolvement in coordination [33] ( Figure S1). Furthermore, noncoordination of ring nitrogen atom v (-C═N) of benzimidazole moiety in the spectra of the complexes is also ascertained by finding no change in the positions of the characteristic IR bands at ∼1,540 and ∼1,320 cm −1 due to v (C═N) (cyclic) and v (C-N) (cyclic) modes of vibration, respectively [33]. On the other hand, the position due to benzimidazole v (N-H) at ∼3,150 cm −1 reduced to lower frequency of ca. 20-25 cm −1 , suggesting participation of -NH group of benzimidazole in coordination [33,35]. However, the bands due to v (C═N) and v (N-N) vibrations also show negative shift of ca. 10-20 cm −1 in the spectra of the complexes, suggesting their role in coordination [33,36]. Moreover, the vibration at ∼3,450 cm −1 is assigned to v (O-H) of the lattice water [33,34]. In addition, a sharp band at 655-680 cm −1 due to ν (C═N) of pyridine is observed in the spectra of the complexes [33,37].

Electronic spectra
The electronic spectral data and the magnetic moment values of the mixed-ligand complexes are listed in Table S1. The electronic spectrum of Co(II) complex showed two main bands at ∼10,745 cm −1 (broad) and ∼22,136 cm −1 (strong) due to 4 [38,39]. However, 4 T 1g (F) → 4 A 2g (F) (v 2 ) transition was not observed because of its association with a large amount of energy cause by two electron transitions (t 5 2g e 2 g → t 3 2g e 4 g ) [39,40]. Furthermore, the observed magnetic moment, µ eff (4.72 BM), supported an octahedral geometry around Co(II) ion [39,41]. The studied mixed-ligand complex of Ni(II) complex showed split bands at ∼8,850 and ∼ 10,420 cm −1 assigned to 3 B 1g → 3 E g and 3 B 1g → 3 B 2g transitions, respectively. In addition, two characteristic bands at ∼15,350 and ∼24,575 cm −1 were assigned to 3 transitions, respectively, and suggested an octahedral geometry around Ni(II) ion, which was confirmed by the observation of magnetic moment at 2.98 BM [39,42]. The mixed-ligand complex of Cu(II) displayed two bands at ∼14,320 and ∼16,775 cm −1 attributed to 2 B 1g → 2 B 2g (v 2 ) and 2 B 1g → 2 E g (v 3 ) transitions, respectively, thus suggesting a distorted octahedral geometry [39,43]. However, the band due to 2 B 1g → 2 A 1g was not observed in the studied copper complex [39,44]. The µ eff value for copper complex at 1.88 BM also suggested an octahedral geometry [39,45].

Thermal analysis
The thermogravimetric analysis (TGA) data for the studied mixed-ligand complexes displayed similar pattern of thermal decomposition. The lattice water degraded at temperature below 100°C in all the studied complexes. This is followed by the degradation of the anhydrous complexes in two distinct stages at temperatures 250-280°C and 360-390°C. However, the degradation of organic constituents continued until the formation of stable metal oxide as the end product. The temperature ranges of decomposition, peak temperature and the possible fragments removed are presented in (Table 2).

Proton nuclear magnetic resonance ( 1 H-NMR) spectra
The 1 H-NMR spectra of Schiff base ligand, L 1 , and its mixed-ligand complex of Zn(II) displayed a multiplet due to the aromatic protons at δ 7.3-8.1 ppm. The signals at δ 6.7 and δ 9.0 ppm were attributed to the ring NH and exocyclic NH protons, respectively ( Figure S2). However, the observed de-shielding in the ring -NH proton at δ 7.0 ppm indicated its participation in coordination. Furthermore, disappearance of phenolic -OH proton in the metal complexes confirmed its role in coordination. Moreover, -CH 3 signal was also observed at δ 2.4 ppm.

Antibacterial activity
The in vitro antibacterial activity was reported by Agar well diffusion method [27] against B. subtilis, E. coli and S. typhi at 100 μg mL −1 concentrations. The zone of inhibition data was studied using ciprofloxacin as the standard antibacterial drug as shown in Figure 2. The studied complexes exhibited better activity than the free ligand, which is likely due to the presence of -C═N linkage and its involvement in coordination. Furthermore, coordination through the metal ions reduces the electron density due to the partial sharing of its positive charge with the donor groups and possible π-electron delocalization [46]. In addition, solubility, dipole moment, the nature of the ligand and geometry are supposed to be the possible factor for the higher antibacterial activity displayed by the mixed-ligand complexes [47]. However, the mixedligand Zn(II) complex remained inactive against E. coli and S. typhi.

Docking evaluation against MERS-CoV
Docking studies were performed to obtain accurate predictions on the optimized conformations for both the ligands and protein target to form a stable complex. All      NAG was considered as the reference ligand to compare the docking results of the studied compounds. The docking studies revealed that the docking score of all the metal complexes are greater than the co-crystallized NAG (docking score: −21.63; root-mean-square deviation [RMSD]: 0.04 Å) but smaller than L 1 (docking score: −41.40; RMSD: 0.05 Å; Table 4). The L 1 showed the presence of six hydrogen bonds: three with GLU 382 . The docking pose of the compounds interacting with amino acid residues is presented in Figure 3. The amino acid residues that formed the interacting group of each compound are listed in Table 3. After analyzing the data, it was noticed that all the studied compounds were placed in the same binding site (represented by a green sphere) of 4ZS6 as the co-crystallized one and have the same orientation as the co-crystallized NAG (Figure 4).

Docking evaluation against SARS-CoV-2 M pro
All the compounds were docked on the crystal structure of SARS-CoV-2 M pro (PDB ID: 6W63). The docking pose of the co-crystallized X77 interacting with amino acid residues of the active site and the hydrogen bonds created with GLU 166 (2.721 Å) and GLY 143(3.202 Å) is shown in Figure 5. The co-crystallized X77 (N-(4-tert-butylphenyl)-N-[(1R)-2-(cyclohexylamino)-2-oxo-1-(pyridin-3-yl)ethyl]-1H-imidazole-4-carboxamide) was considered as a standard ligand to compare the docking results of the studied compounds. The docking studies revealed that the docking      Figure 5. The amino acid residues that formed the interacting group of each compound are listed in Table 4. After analyzing the data, it was noticed that the all studied compounds were placed in the same binding site (represented by a green sphere) of 6W63 as the cocrystallized one and have the same orientation as the cocrystallized X77 ( Figure 5). According to Lipinski's rule of five, the calculated parameters (Table S2) may predict the property of a molecule to turn into an active drug [48] on the basis of the number of violations made. It is observed that all metal complexes have two violations of Lipinski's rule of five (Lipinski violation is 2), namely, molecular weight >500 Da and octanol-water partition coefficient (log P) > 5.

Electrostatic potential (ESP) surface analysis
The ESP surface of Schiff base ligand, L 1 , was analyzed by using Argus Lab 4.0.1 software as shown in Figure 6. The surface contains a number of possible sites for electrophilic attack. The ESP surface displayed a specific data about the charge distribution. The negative regions were mainly over phenolic oxygen, azomethine nitrogen and benzimidazole ring nitrogen atoms as indicated in red and were involved in coordination. However, the other nitrogen atom was not involved in coordination likely due to the steric effect.  Research funding: The authors acknowledge the financial support through Researchers Supporting Project number (RSP-2020/147), King Saud University, Riyadh, Saudi Arabia.

Conclusion
Author contribution: Ranjan K. Mohapatra designed and supervised the study. V. P. Saikishore performed the study. Susanta K. Biswal supervised and was in charge of the initial draft. Mohammad Azam was involved in final editing and also revised the manuscript.

Conflict of interest:
There are no conflicts to declare.
Data availability statement: All data presented in the study are included in the article and supplementary material.