A benzimidazole scaffold as a promising inhibitor against SARS-CoV-2

Abstract The manuscript reports the green-chemical synthesis of a new diindole-substituted benzimidazole compound, B1 through a straightforward route in coupling between indolyl-3-carboxaldehyde and o-phenylenediamine in water medium under the aerobic condition at 75 ºC. The single crystal X-ray structural analysis of B1 suggests that the disubstituted benzimidazole compound crystallizes in a monoclinic system and the indole groups exist in a perpendicular fashion with respect to benzimidazole moiety. The SARS-CoV-2 screening activity has been studied against 1 × 10e4 VeroE6 cells in a dose-dependent manner following Hoechst 33342 and nucleocapsid staining activity with respect to remdesivir. The compound exhibits 92.4% cell viability for 30 h and 35.1% inhibition against VeroE6 cells at non-cytotoxic concentration. Molecular docking studies predict high binding propensities of B1 with the main protease (Mpro) and non-structural (nsp2 and nsp7-nsp8) proteins of SARS-CoV-2 through a number of non-covalent interactions. Molecular dynamics (MD) simulation analysis for 100 ns confirms the formation of stable conformations of B1-docked proteins with significant changes of binding energy, attributing the potential inhibition properties of the synthetic benzimidazole scaffold against SARS-CoV-2. Communicated by Ramaswamy H. Sarma


Introduction
The relentless expansion of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shatters human life and health to an extreme level across the world (Behera et al., 2021;Galani et al., 2021). The infection caused by the novel coronaviruses is not only limited to humans, nevertheless mammals, and birds are also sufferer with this disease. Among the identified different viral species, six coronavirus species are commonly known namely SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 (Arabi et al., 2017;Skariyachan et al., 2019;Varnaiite et al., 2020). In late 2019, a viral respiratory illness was caused and rapidly spread by SARS-CoV-2 as pointed out by the International Committee of Taxonomy of Viruses (ICTV). The new virus, 2019-nCoV actually belongs to subgenus Sarbeco virus of the genus beta-coronavirus of the family Coronaviridae. The 2019-nCoV adopts a positive-sense single strand ribonucleic acid (RNA) genome ( Figure 1) and the beta-coronaviruses are believed to be originated from bats (Coelho et al., 2020;Kahn & McIntosh, 2005;Monto, 1974). The clinical manifestations of COVID-19 suggest that the infection primarily spreads through the close contact of the infected individuals. This disease exhibits various symptoms, from mild flu to life-threatening conditions, including invasive lesions in the lungs leading to a severe threat to human civilizations and the living world (Channappanavar & Perlman, 2017;Huang et al., 2020;Li et al., 2020;Pedersen & Ho, 2020;Peiris et al., 2003;Yuan et al., 2021). Thus, World Health Organization (WHO) declared this coronavirus disease (COVID-19) a pandemic on 12 March 2020 (WHO, 2020).
Surprisingly, the disease in the present pandemic displays more aggravation leading to acute respiratory failure, sepsis and death (Hadjadj et al., 2020;Olagnier et al., 2020;Pedersen & Ho, 2020;Wang et al., 2020;Yan et al., 2020). Multiple factors such as an exacerbated inflammatory reaction, systemic and organ-specific manifestations, persistent viral load, and defective antiviral defense pathways (Saichi et al., 2021;Yoshino et al., 2020) are responsible. Therefore, exploring the underlying cellular and molecular mechanisms is of paramount importance to shed off the COVID-19 physiopathology and develop appropriate therapies. The effort of considerable scientific research during the last two years to develop vaccines, therapeutics and treatment processes aiming to prevent, control and cure this disease is praiseworthy (Saichi et al., 2021;Yoshino et al., 2020). In the true sense, this immense effort results in significant progress of medical science to combat human coronaviruses. However, the huge population, awareness and limited medicinal resources (medicines, vaccines and medical staffs) impose a restriction in recovering from this pandemic quickly. Therefore, every valuable input from the scientific community may advance medical science largely.
In this context, we have made a considerable attempt to synthesize a new disubstituted indole-benzimidazole scaffold through a straightforward green-synthetic approach. The crystal structure analysis of the compound suggests that disubstituted indole-benzimidazole crystallizes in a monoclinic system with P2 1 /n space group. Furthermore, in vitro SARS-CoV-2 screening activities of the benzimidazole compound have been evaluated against 1 Â 10e4 VeroE6 cells. The molecular docking and molecular dynamics (MD) simulation studies of the synthetic compound have also performed to reveal the bio-potency of the compound.

Chemicals, solvents and starting materials
Highly pure o-phenylenediamine (TCI, Japan) and indole-3-carboxaldehyde (SRL, India) and other reagents were purchased from respective suppliers. All the chemicals and solvents were of analytical grade.

Physical measurements
FTIR-8400S SHIMADZU spectrometer (Shimadzu, Kyoto, Japan) was employed to record IR spectra (KBr) of the benzimidazole product ranging 400-3600 cm À1 . 1 H and 13 C NMR spectra of the benzimidazole product were obtained on a Bruker Advance 400 MHz spectrometer (Bruker, Massachusetts, USA) in CDCl 3 at 25 C. Steady-state absorption and other spectral data were obtained on a JASCO V-730 UV-Vis spectrophotometer (Jasco, Tokyo, Japan). A Perkin Elmer 2400 CHN microanalyser (Perkin Elmer, Waltham, USA) was used to perform the elemental analysis. The electron spray ionization mass spectral (ESI-Ms) measurement was carried out on a Q-TOF-micro quadruple mass spectrometer.

Structural refinement and Hirshfeld surface analysis
X-ray diffraction data of the benzimidazole product were collected using a Rigaku XtaLAB Mini diffractometer equipped with Mercury375R (2 Â 2 bin mode) CCD detector. The data were collected with a graphite monochromated Mo-Ka radiation (k ¼ 0.71073 Å) at 150 K using x scans. The data were reduced using CrysAlisPro 1.171.39.7f, and the space group determination was done using Olex2 (Dolomanov et al., 2009). The structure was resolved by the dual space method using SHELXT-2015 and refined by full-matrix least-squares procedures using the SHELXL-2015 software package through the OLEX2 suite (Sheldrick, 2015a(Sheldrick, , 2015b.

SARS-COV-2 antiviral activity of the benzimidazole compound
2.4.1. Cytotoxicity assay against 1 Â 10e4 VeroE6 cells The cytotoxicity assay of the synthetic compound B1 was performed in a 96-well plate format in a dose-dependent manner. 1 Â 10e4 VeroE6 cells were plated per well and incubated at 37 C in a humidified 5% CO 2 for overnight to develop the monolayer formation. After 24 h, 10 mM remdesivir and three different concentrations of the B1 (9 mM, 0.9 mM, 0.45 mM) and DMSO were added, and the plates were incubated for 30 h at 37 C in a humidified 5% CO 2 (Vatansever et al., 2021;Zhou et al., 2021). After removal of the cell supernatant, treated cells were stained with Hoechst 33342 and Sytox orange dye. The images were taken at 10Â, 16 photos per well, which covered 90% of the well area using ImageXpress Microconfocal molecular devices. Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counter stain that emits blue fluorescence when bound to ds-DNA. It stains all the live and dead cells. Sytox orange dye stains nucleic acids in cells with compromised membranes. This stain is an indicator of cell death. First, the software counted the total number of cells in the Hoechst image. The Sytox image was counted among Hoechst positive cells to determine the number of positive cells for sytox.

Immunofluorescence assay
The anti-SARS-CoV-2 assay was carried out in a 96-well plate format in which three wells were used for a sample of three different concentrations as previously described by Vernaite et al. 1 Â 10 4 VeroE6 cells were plated per well and incubated at 37 C in a humidified 5% CO 2 for 24 h to form a monolayer. In the next day, 10 mM remdesivir and three different concentrations of the B1 (9mM, 0.9 mM, 0.45 mM) were added to the cells and the plates were incubated for 30 h at 37 C in a humidified 5% CO 2 (Vatansever et al., 2021;Zhou et al., 2021). The normal VeroE6 cells without B1 were considered as a control, while the remdesivir was used as a standard drug used for SARS-CoV-2 treatment as well as to make a comparison of the efficacy of the synthesized benzimidazole scaffold with remdesivir. The cells were infected with SARS-CoV-2 at an MOI of 0.1 and incubated at 37 C in a humidified 5% CO 2 for 30 h. After 30 h, the cells were fixed in 4% paraformaldehyde. Afterwards, the cells were permeabilized with 0.3% tween-20 and stained with primary and secondary antibodies. The primary antibody-SARS-CoV2 nucleocapsid mouse monoclonal antibody (Catalog Number: 40143-MM05) and the secondary antibody, anti-mouse alexafluor 568. For staining the nucleus, Hoechst 33342 stain was used. Images were captured and analyzed using ImageXpress Microconfocal devices. The SARS-CoV2 nucleocapsid (Alexa flour-568) and Hoechst nuclei stain images were captured at 10Â, 16 images per well, covering 85% of the well area. The nucleocapsid positive cells and total nuclei were counted and compared with the control through MetaXpress software using a multi-wavelength cell scoring module.

Molecular docking studies, ADME and molecular property prediction
The rationale behind this study is to uncover the nature of binding of the benzimidazole with main protease (M pro ) and non-structural proteins (nsp2 and nsp7-nsp8) of SARS-CoV-2.
Prior to the docking study with benzimidazole derivative, the CIF files of the receptors were fetched from the protein data bank as M pro (PDB ID: 6LU7), nsp2 (PDB ID: 7MSX) and nsp7-nsp8 (PDB ID: 6YHU). Before performing molecular interaction studies, the SARS-CoV-2 main protease M pro , non-structural proteins nsp2 and nsp7-nsp8 receptors were further curated for missing side-chain residues using What If interface (https://swift. cmbi.umcn.nl/servers/html/index.html). The protonation states of the participated amino acids of the protein receptor molecule were checked for neutralization before interaction studies. Molecular docking studies were carried out using Autodock v 4.2.6. Both the receptor and B1 were prepared by adding polar hydrogen bonds followed by Kollman charge and Gastegier charges. Then finally, merging the non-polar hydrogens, both receptor and ligand molecules were saved in pdbqt format. A grid box was created with parameters X ¼ 68, Y ¼ 58, and Z ¼ 64 Å with 0.3 Å spacing. Following the Lamarckian Genetic Algorithm (LGA), docking studies of the protein-ligand complex were performed to achieve the lowest free energy of binding (DG). The Lipinski's 'Rule of five' was predicted by theoretical in silico ADME calculations (Lipinski et al., 2001). A web tool of Swiss ADME was used to predict Lipinski's parameters (Ertl et al., 2000;Swiss, 2020).
2.6. Molecular dynamics simulations, and molecular mechanics generalized born surface area (MM-GBSA) calculations The MD simulations and MM-GBSA calculations were carried out to view the insights on the interactions between benzimidazole scaffold and main protease M pro (PDB ID: 6LU7), non-structural proteins nsp2 (PDB ID: 7MSX), and nsp7-nsp8 (PDB ID: 6YHU). The calculations were performed for 100 ns using Desmond 2020.1from the Schr€ odinger, LLC. The OPLS-2005 force field (Bowers et al., 2006;Chow et al., 2008;Shivakumar et al., 2010) and explicit solvent model with the SPC water molecules were used in this system (Jorgensen et al., 1996). Na þ ions were added to neutralize the charge. 0.15 M, NaCl solutions added to the system to simulate the physiological environment. The NPT ensemble was set up using the Nose-Hoover chain coupling scheme (Martyna et al., 1992) with temperature 27 C, the relaxation time of 1.0 ps and pressure of 1 bar were maintained in all the simulations. A time step of 2 fs was used. The Martyna-Tuckerman-Klein chain coupling scheme (Martyna et al., 1994) barostat method was used for pressure control with a relaxation time of 2 ps. The particle mesh Ewald method (Morris et al., 1996) was used for calculating long-range electrostatic interactions, and the radius for the coulomb interactions were fixed at 9 Å. RESPA integrator was used to calculate the non-bonded forces. The root mean square deviation (RMSD) was employed to monitor the stability of the MD simulations. The binding free energy (DG bind ) of the docked complexes during MD simulations of the proteins with B1 was estimated using the prime molecular mechanics generalized born surface area (MM-GBSA) module at the (Schrodinger suite, LLC, New York, NY, 2017-4). The OPLS 2005 force field, VSGB solvent model and rotamer search algorithms were used to define the binding free energy during the calculation. The MD trajectories frames were selected at each 10 ns interval after MD run. The following formula was used to calculate the total free energy binding: where, DG bind ¼ binding free energy, G complex ¼ free energy of the complex, G protein ¼ free energy of the target protein, and G ligand ¼ free energy of the ligand. The MM-GBSA outcome trajectories were analyzed further for post dynamics structure modifications.

Results and discussion
3.1. Synthesis and spectroscopic characteristics of the benzimidazole scaffold (B1) The benzimidazole scaffold was synthesized through a greenchemical straightforward coupling between o-phenylenediamine and indole-3-carboxaldehyde in a 1:2 mole ratio in a water medium under refluxing conditions. This synthesis is a pure example of a green-chemical approach where the reaction is carried out in the water with air bubbles. The atom economy of the synthetic procedure is high and shown in Scheme 2. This structural characteristics and formulation of the benzimidazole compound was determined with FT-IR, UV-Vis and 1 H NMR spectral analysis. FT-IR spectrum of the benzimidazole scaffold (B1) exhibits important characteristics peaks at 3390, 3164 and 1635 cm À1 corresponding to the stretching frequencies of -N-H, -CH 2 and -CH ¼ N-groups respectively in B1 ( Figure S1). Other characteristic stretching frequencies in the range from 1120 to 1570cm À1 suggests the presence of stretching and bending vibrations assignable to -C ¼ C-and -C-Ngroups. These IR spectral data are in well concordance with the previously reported values (Mudi et al., 2020(Mudi et al., , 2021. The UV-Vis spectrum of B1 in ethanol medium exhibits a high intensity absorbance band at 210 nm and three closely spaced electronic transitions of moderate intensity at 241, 262 and 300 nm ( Figure S2). These moderate intensity electronic bands suggest the p!p or n!p electronic transitions of the azomethine groups in B1 ( Figure S3). The observation of the appearance of the electronic bands is in high agreement with the previously reported data Pal et al., 2020;Roy et al., 2021). The 1 H NMR spectrum of B1 distinctly defines the location of protons in B1. The singlet signals at 11.66 and 10.99 ppm represent the indole-NH protons in B1. The methylene protons of B1 were also detected and confirmed from the appearance of the signal at 5.82 ppm. The entire protons rise in the region from 8.33 to 6.81 ppm can be assignable to the presence of aromatic protons in B1 ( Figure S3). The 13 C NMR was also recorded to assign the characteristics of the C-atom in the structure ( Figure S4). The signals at 150.08 and 56.49 ppm attribute the presence of azomethine-C and methylene-C, respectively while the signals in the range 143.79 to 105.58 ppm correspond to the aromatic-C in the benzimidazole compound ( Figure S4). The reported values of the benzimidazole compound match very well to the reported literature values (Mudi et al., 2021). The ESI-Ms of B1 was also measured in methanol ( Figure S5). The molecular ion peak of B1 was observed at m/z 363.1510 attributing the molecular integrity and stability of B1 in solution phase.

Description of crystal structure and non-covalent interactions
The crystal structural analysis reveals that the diindole-benzimidazole compound (B1) crystallizes in a monoclinic system with a P2 1 /n space group. The ORTEP diagram of the compound is displayed in Figure 2(a). The crystallographic refinement parameter is summarized in Table 1 and bond angles and bond distances are given in Table S1. In the structure of B1, one indole directly links with the benzimidazole group while the second imidazole attaches to the benzimidazole moiety through a methylene-C. Furthermore, two indole rings co-exist in a perpendicular fashion with respect to benzimidazole moiety.
To understand the nature of propensity for hydrophobic and hydrophilic interaction with surrounding molecule, the interaction map was carefully evaluated (Figure 2b). The generated interaction landscape of a molecule from its 3D coordinates exhibits the interaction preferences by highlighting regions around the molecule (maps) where chemical functional groups (probes) are likely to come into contact. The red and blue regions of the maps denote the areas in which there is a high probability of locating a hydrogen bond acceptor and H-bond donor respectively. The brown areas of the map indicate the hydrophobic preferences. Two multidiamonded dark red zones closest to NH protons of the indole rings in B1 suggest its interactive preferences with a strong H-bond acceptor (Figure 2b). The details of the interaction map are reported in the supporting information file. A self-assembled diagram of the compound in crystalline phase ( Figure 2c) suggests that strong to very strong C-H … p and N … H interactions are operative to lead a 3D architecture along b axis. In the formation of 3D supramolecular structure, it is revealed that two indole N-H and C-H protons act as donors while the aromatic centroids of the phenyl rings in the indole moiety behave as acceptor. Furthermore, the Natom of the benzimidazole moiety forms strong intermolecular H-bonding with the NH of indole rings. The details of the non-covalent interactions for B1 are summarized in Table S2. The molecular dimensionality for B1 was further calculated and shown in Figure 2d.

Cytotoxicity of the B1
Cell viability and cell toxicity assays are essential for assessing the cellular responses to a tested compound during its screening activity in a biological experiment. Typically, cell viability assay provides an important readout of healthy cells by measuring metabolic activity or cell proliferation (Folgueira et al., 2021). Cell viability, which measures the proportion of live and healthy cells within a total cell population, can also be estimated by cell toxicity assay by examining cell growth replication. The cytotoxicity of the B1 and remdesivir was evaluated independently against 1 Â 10e4 VeroE6 cells (n ¼ 3) in a dose-dependent manner. The non-cytotoxic concentration was also determined for B1 and remdesivir under a similar experimental condition. It is Scheme 2. Synthetic route for B1 observed that the compounds exhibit a non-cytotoxic concentration against 1 Â 10e4 VeroE6 cells up to a dose of 9 mM and 10 mM for B1 and remdesivir, respectively. Furthermore, the percentage cell viability of the compounds was also estimated for 1 Â 10e4 VeroE6 cells ( Figure S6). In comparison to the control, the percentage cell viability of the 1 Â 10e4 VeroE6 cells was determined as 50, 64.1, 92.4 at 0.45, 0.9, 9.0 mM concentrations of B1while 99.23% cell viability was displayed by remdesivir at 10 mM (Table S2). The cytotoxic effect of B1 and remdesivir on 1 Â 10e4 VeroE6 cells is shown in Figure 3(A-D). Therefore, the high percentage of cell viability at a non-cytotoxic concentration of B1 against 1 Â 10e4 VeroE6 cells makes a good promise to develop a potential therapeutic for SARS-CoV-2.

Antiviral efficacy of B1and remdesivir following immunofluorescence assay
The in vitro antiviral activities of the synthetic B1 and remdesivir at non-cytotoxic concentration were further evaluated through immunofluorescence assay (IFA) against VeroE6 cells to understand the viral screening efficacy. It is well documented that remdesivir is a globally prescribed antiviral therapeutic agent used for SARS-CoV-2 inhibition. A comparison of the antiviral activity for B1 and remdesivir was also drawn to evaluate the potential candidature of B1 as a therapeutic. The anti-SARS-CoV-2 activity was further quantified using primary (mouse monoclonal antibody) and secondary antibodies (anti-mouse alexafluor 568) using IFA. It is observed that 10 mM of remdesivir can significantly inhibit 99.1% of the SARS-CoV-2 infection (Figure 3-1B) while DMSO (Dimethyl sulfoxide) as a control didn't exhibit any inhibition (Figure 3-1D). In contrast, B1 at 0.45 mM and 0.9 mM didn't display any inhibition property against the replication of SARS-CoV-2. However, B1 behaved an effective therapeutic property which showed 35.1% inhibition activity at 9 mM (Figure 3-1A). The uninfected VeroE6 cells are shown in Figure  3-1C. With an increase of the dose (concentration) of B1 results  a high degree of interaction propensity with main protease proteins. This leads to the ceasing of RNA genome replication and results in the prevention of viral attachment to the cells (Martyna et al., 1994). However, a detail experiment needs to be carried out to account the dose-dependent activity of the B1 with the %cell viability against 1 Â 10e4 VeroE6 cells.

Molecular docking studies
Molecular docking studies were performed to decipher the mechanistic aspects and the nature of interactions between the SARS-CoV-2 protein and B1. The B1 as a ligand interacts significantly with the binding pocket of main protease (M pro ). The images of docked complex, molecular surface, 3D and 2D interactive plots for B1 À with M pro are given in Figure 4. The B1 showed a significant number of non-covalent interactions with the amino acid residues His164, Arg188 and Cys145 of M pro . The effective change of free energy for binding, DG was estimated to be À9.36 kcal/mol and a high inhibition efficacy was evident from the inhibition constant, Ki ¼ 1.37 mM (Table S4). The binding interaction of B1with the binding pocket of nsp2 (Figure 4), also showed a significant binding effect with the change of free energy for binding (DG ¼ À7.11 kcal/mol) as well as high inhibition (Ki) 6.14 mM (Table S4). The principal amino acid, Leu60, Val66 and Gly113 at the binding pocket of nsp2 involved in ligand binding through the formation of hydrophobic interactions (Figure 4). The binding effect of B1 with nsp7-nps8 is also evaluated and displayed in Figure 4. The change of binding energy of B1 with nsp7-nsp8 was found to be À7.98 kcal/mol with a significant inhibitory constant (Ki) 1.92 mM. The B1 compound formed an important network with Leu60, Ala110 and Arg111 at the binding site of nsp-2 through p-sigma and p-alkyl type interactions (Figure 4). The details of the overall interaction between B1 and main protease, and non-structural proteins are summarized in Table S4. Interesting, the molecular docking studies of B1 with main protease and non-structural proteins of SARS-CoV-2 didn't exhibit any intermolecular H-bonding. The entire interactions between B1 with the proteins are of p … p, p … sigma and p … alkyl types that strongly recommend very ordered binding interactions (Table S4). Therefore, from molecular docking study, it might be predicted with a high promise that B1 shows a high binding propensity towards the binding sites of M pro , nsp2 and nsp7-nsp8 proteins of SARS-CoV-2 with a significant change of free energy. Keeping the overhead results of molecular docking in mind, the tested drug-like nature of B1 against SARS-CoV-2 was further estimated through the calculation of ADME values (Table S5). The cytotoxic effect of B1was well recognized as passing Lipinski's 'Rule of 5' with 0 violation which strongly recommends the acceptance of B1 as a potential therapeutic against SARS-CoV-2. Therefore, it might be recommended that B1 may turn out to be a good inhibitor against M pro , nsp2 and nsp7-nsp8 proteins of SARS-CoV-2.

MD simulation and MMGBSA calculations
MD simulation of the benzimidazole scaffold bound main protease, M pro and non-structural proteins nsp2 and nsp7-nsp8 complexes of SARS CoV-2 were studied in details to realize the nature of possible binding motifs and their conformations. The snapshots of the interactions between B1 and M pro , nsp2 and nsp7-nsp8 for 20 ns interval are displayed in Figure S7. The RMSD of the 100 ns MD simulation trajectories displayed a stable conformation of B1 bound M pro ( Figure 5A, red) complex with 0.1 Å deviations. The nsp2 bound complex with B1 showed a 0.1 Å displacement of the RMSD ( Figure 5A, pink) while nsp7-nsp8 bound B1 displayed a 0.5 Å deviation ( Figure 5A, blue). The RMSD plots signify that the benzimidazole scaffold afforded more stable conformation of B1 bound complex with M pro protein compared to the non-structural proteins, nps2 and nsp7-nsp8. Root mean square fluctuation (RMSF) of the amino acid residue position of 100 ns simulation trajectories of B1 bound proteins displayed in Figure 5B. Least fluctuations of amino acid residues were observed in the main M pro and nsp2 proteins in the complexed forms ( Figure 5B, red and pink, respectively). However, the maximum fluctuations were observed for nsp7-8 proteins at the residue positions 50 and 100 ranging from 3 to 6 Å ( Figure 5B-blue). The radius of gyration (Rg) was also determined to verify the size and compactness of the proteins in the ligand-bound state. The Rg plots are displayed in Figure 5C. The Rg plots of Ca-backbone indicate that M pro proteins and nsp2 ( Figure 5C, red and pink) have the least fluctuations having significant compactness with an average of $21Å and $24 Å, respectively from the beginning to end of the 100 ns simulation. In contrast, a lower Rg score was observed in nsp7-nsp8 protein from $25 Å to $24 Å ( Figure 5C, blue) to achieve the final stability of the B1 bound complex. The average hydrogen bonding observed between B1and the respective proteins during the 100 ns simulation were also noted and recorded in Figure 5D. It is revealed from Fig. 5D that on an average, 1.5 numbers of Hbonds were formed between B1 with M pro (Figure 5D, red) and B1 and nsp7-nsp8 ( Figure 5D, blue), and while B1 showed highest interactive propensity with nsp2 ( Figure 5D, pink) adopting an average of 2.5 numbers of hydrogen bonding. A comparison of RMSD, RMSF and Rg trajectories of the free ligand and docked ligand-protein complexes is shown in Figure S8. Overall, this simulation results ensure well in favor of high binding propensities for B1 with the main protease and other structural proteins leading to stable structures.
The binding free energy and other contributing energies for B1 complexed with M pro , nsp2 and nsp7-nsp8 were determined based on MM-GBSA calculations employing MD simulation trajectory analysis. The results ( Figure 6) suggest that the maximum contribution to dG bind in the simulated complexes stability was due to dG bind Coulomb, dG bind vdW and dG bind Lipo. In contrast, dG bind Solvation energies contributed to the instability of the corresponding complexes. The ligand, B1 displayed good binding energy contributions with nsp-7-nsp8, nsp2 and M pro . The highest binding energies from every 10 ns of M pro trajectories displayed the high contribution of coulombic energy (blue) toward more negative free energy as well as vdW ( Figure  6G). While dGLipo contributed to stability, dG solvation energy contributed to more destabilization with positive free energies ( Figure 6H). Similar patterns were also observed in nsp2 ( Figure   6I and J) and nsp7-nsp8 ( Figure 6K and L). These non-covalent contributions of energies lead to high affinity for the ligand B1 toward the proteins with dG Bind (kcal/mol) À36.40 ± 9.20, À28.68 ± 7.36 and À32.02 ± 5.95 for M pro , nsp2 and nsp7-nsp8, respectively. Therefore, high negative free energies suggest higher binding of B1 with the structural and non-structural proteins leading to conformationally stable structures.
Nowadays, computer-aided drug design based on structural chromophores of different chemical compounds is growing as an emerging field of research. A large number of scientists are actively engrossed in searching for potential therapeutics against SARS-CoV-2 (Badavath et al., 2020;Choudhary & Silakari, 2020;Culletta et al., 2020;Juki c et al., 2020 and references their in). Tutone and group explored the inhibition properties of a large number of designed structure-based pharmacophores against the proteins encoded by SARS-CoV-2. They considered 26 are experimental drugs, 5 investigational drugs and 3 approved drugs to study and carried out molecular docking, and MM-GBSA calculations using MD simulations for 100 ns. The drug molecules showed a significant change of dG Bind energy ranging from À35 to À90 kcal/mol with the interaction of main protease and different non-structural proteins. Bren and co-workers performed a virtual screening study to develop potential inhibitors against SARS-CoV-2 and considered two small synthetic molecules namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1ethanone and  Among the studied molecules, A_BR4, A_BR9, A_BR18, A_BR22 were highly interactive with spike proteins and A_BR5, A_BR6, A_BR9 and A_BR18 were effective against main protease of the SARS-CoV-2. The group further reported the DG MM-GBSA energies (kcal/mol) for the main protease docked complexes ranging from À2 to À47 kcal/mol. Similarly, Badavath et al. forecasts a computer-aided drug design for the anti-SARS screening activity of 118 isatin derivatives comprising 16 distinct heterocyclic compounds, 5 natural products and 7 repurposed drugs. The binding propensities of the compounds towards the main protease of SARS-CoV-2 reveal their potential inhibition properties turn out to be excellent therapeutics against SARS-CoV-2. Nevertheless, on comparison of the binding propensities of Figure 6. The 3D contour plots of free energy contributions of non-bonded interactions during B1 bound state with M pro , (G) and (H); with nsp2, (I) and (J), and with nsp7-nsp8 (K) and (L). Color coding in the contour plots signify the of non-covalent interaction energies contribution to the total binding energy (dGb ind ) of protein and ligand complex. The color blue in the map signifies the highest positive contribution to high binding energy; In contrast, red color signifies the lowest contribution to binding energy. While gradient colors from green to orange signify high to low contributions to the dG Bind.
the molecules with main protease and non-structural proteins of SARS-CoV-2, it is evident that B1 shows a pronounced binding effect with main protease (À36.40 kcal/ mol), with nsp2 (À28.68 kcal/mol) and for nsp7-nsp8 (À32.02 kcal/mol) as revealed from dG Bind energies calculated through MM-GBSA. The significant changes of binding energies of B1 with M pro , nsp2 and nsp7-nsp8 proteins are well accepted and fall in the range of reported literature which further attributes the highly stable docked conformations of B1 and proteins of SARS-CoV-2.

Conclusions
In conclusion, we present the synthesis of an indole-substituted benzimidazole derivative through a cost-effective green approach and characterize the compound with a suite of spectroscopic methods and single crystal X-ray diffraction studies. We study in vitro SARS-CoV-2 screening activity, which exhibits a high percentage of cell viability, and moderate inhibition activity against 1 Â 10e4 VeroE6 cells at the non-cytotoxic concentration with respect to remdesivir at 10 mM. The molecular docking studies of B1 with main protease (M pro ) and non-structural proteins (nsp2 and nsp7-nsp8) suggest the high binding propensities of the compound with promising pharmacokinetic properties. The changes of binding energies for B1-M pro , B1-nsp2 and B1-nsp7-nsp8 complexes are determined as À9.36, À7.11 and À7.98 kcal/mol, attributing the highly stable docked conformations. The MD simulation studies for 100 ns strongly recommend good binding propensity of B1withthe proteins M pro , nsp2 and nsp7-nsp8 and leads to the conformationally stable structure. The binding energies of the molecular docked complexes derived in MD simulations are in well agreement with molecular docking results. A large number of molecular docking and MD simulation studies are reported in scientific database; however, this cost-effective synthesis and significant pharmaocokinetic properties of the benzimidazole compound (B1) may turn out to be one of the important antiviral therapeutic.