key: cord-0974292-rk5jf9cx authors: Mudi, Prafullya Kumar; Mahato, Rajani Kanta; Verma, Himanshu; Panda, Subhra Jyoti; Purohit, Chandra Sekhar; Silakari, Om; Biswas, Bhaskar title: In Silico Anti-SARS-CoV-2 Activities of Five-membered Heterocycle-substituted Benzimidazoles date: 2022-03-21 journal: J Mol Struct DOI: 10.1016/j.molstruc.2022.132869 sha: a523ee484490060b5ab51bedbb4e1c3030b9c007 doc_id: 974292 cord_uid: rk5jf9cx The manuscript deals with cost-effective synthesis, structural characterization and in silico SARS-CoV-2 screening activity of 5-membered heterocycle-substituted benzimidazole derivatives, 1-((1H-pyrrol-2-yl)methyl)-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole (L1), 2-(furan-2-yl)-1-(furan-2-ylmethyl)-1H-benzo[d]imidazole (L2), 2-(thiophen-2-yl)-1-(thiophen-2-ylmethyl)-1H-benzo[d]imidazole (L3). The benzimidazole compounds were synthesized through a green-synthetic approach by coupling of 5-membered heterocyclic-carboxaldehyde and o-phenylenediamine in water under an aerobic condition. The compounds were characterized by various spectroscopic methods and X-ray structural analysis. The suitable single-crystals of the methyl derivative of L3 were grown as L3′ which crystallized in a monoclinic system and the thiophene groups co-existed in a nearly a perpendicular orientation. Further, in silico anti-SARS-CoV-2 proficiency of the synthetic derivatives is evaluated against main protease (M(pro)) and non-structural proteins (nsp2 and nsp7) of SARS-CoV-2. Molecular docking and molecular dynamics analysis of the ligands (L1-L3) against M(pro) and nsp2 and nsp7 for 50 ns reveal that L3 turns out to be the superlative antiviral candidate against M(pro), nsp2 and nsp7 of SARS-CoV-2 as evident from the binding score and stability of the ligand-docked complexes with considerable binding energy changes. The appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and their uncontrolled expansion explodes the social and economic situations of the modern civilization to a great extent [1] . The living and non-living world are also seriously affected by the corona virus infection in direct and indirect ways. Literature survey proposes that 2019-nCoV virus belongs to the family of Coronaviridae (Scheme 1) and the beta-coronaviruses are believed to be originated from bats [2] [3] [4] . This disease exhibits various symptoms, from mild flu to lethal conditions [5] [6] [7] [8] [9] . Thus, World Health Organization (WHO) avowed this COVID-19 a pandemic on 12 th March 2020 (WHO, 2020). Moreover, the corona virus infection spectacles more worsening starting from acute respiratory failure to sepsis and death [10] [11] [12] [13] . Nevertheless, critical issues like aggravated seditious reaction, tenacious viral load, organ-specific indices, and flawed antiviral resistance routes [14, 15] are accountable to monitor the COVID-19 disease. Therefore, investigations on the underlying cellular mechanisms at molecular grabs a great deal of interests to the scientists for the development of physiopathology and consequent progress of suitable therapeutics. During the last two years, various scientific groups made a significant effort to design and development of molecular therapeutics and vaccines including treatment procedures to prevent, control, and cure this disease is praiseworthy [14, 15] . In the true sense, this immense effort results in significant progress of medical science to combat human coronaviruses, although, the huge population, consciousness, and inadequate therapeutics enforce a restriction in recovering from this pandemic quickly. Typically, the rational design of cost-effective antibiotic compounds is crucial to the development of suitable therapeutics. Noteworthy, benzimidazole derivatives are a widely recommended functional pharmacophores used in a large number of drugs (Scheme 2) [16] [17] [18] [19] . Benzimidazole based drugs have also been used as potent inhibitors for reverse transcriptase of HIV-1 virus [20] , NSSB polymerase of Hepatitis C virus [21] , thrombin inhibitors [22] and as antibacterial agents [23] . Although mono-substituted benzimidazole derivatives are mostly used in drug design but the use of di-substituted benzimidazoles are limited. Only few structure of the di-substituted benzimidazole derivatives are also reported in the literature [24] [25] [26] . Nevertheless, 5-membered heterocycle substituted benzimidazoles have a great impact in therapeutic actions against bacterial, fungal and viral species [27] [28] [29] . Like 2-(2-Furyl) benzimidazole (FBD) showed an excellent antifungal activity [27] . The compound imidazole-6-yl)methanone compounds turn out to show noteworthy antitubercular activity against M. tuberculosis H37Rv strain [28, 29] . 2-(5'-Cyano-2,2'-bithiophen-5-yl)-1H-benzimidazole-5(6)-carbonitrile compound showed a remarkable antiparasitic activity against T. brucei rhodesiense [29] . Very recently, we have designed and synthesized one hydrated phenazine chloride explored their therapeutic characteristics with computational aided drug design and in vitro SARS-CoV-2 screening activities against Vero cell lines [30] . In this context, we have designed and developed three 5-membered heterocycle-substituted benzimidazoles and characterized them with a suite of spectroscopic methods and X-ray structural analysis. We also evaluated the effect of 5-member heterocycle-substituted benzimidazole derivatives against M pro , nsp2 and nsp7 through in silico SARS-CoV-2 screening to reveal the bio-potency of the compounds. synthesized following our established synthetic strategy with a little modification [31] . Aromatic amine, o-phenylene diamine (1 mmol, 0.108 g) was taken in 15 mL test tubes. Aromatic aldehydes, pyrrole-2-aldehyde (2 mmol, 0.190 g), furan-2-aldehyde (2 mmol, 0.192 g) and thiophene-2-aldehyde (2 mmol, 0.224 g) were added separately to the amine with the addition of 2 mL 10% ethanol-water mixture. The compound 6-methyl-2-(thiophen-2-yl)-1- carried out on a Q-tof-micro quadruple mass spectrometer. Suitable single-crystals of L3′ was selected for single crystal X-ray diffraction studies. The structural diffraction data of L3′ were collected on a Bruker-Kappa APEX II CCD diffractometer equipped with a 1 K charge-coupled device (CCD) area detector employing a graphite monochromated Mo-Ka radiation (k ¼ 0.71073 Å) at 100.0(2) K. The cell parameters and the reduction and correction of the collected data were determined by SMART SAINTPLus software, respectively [32] followed by SADABS absorption corrections [33] . Finally, the structure was solved by direct method with SHELXL-97 program package [34] . The refinement by full-matrix least-squares method was executed on all F2 data with SHELXL-97. For all non-hydrogen atoms, anisotropic refinement was performed. Subsequently the additional hydrogen atoms were positioned by riding model. The therapeutic potency of the synthetic benzimidazole derivatives was examined against main protease and non-structural proteins (Covid-19 main protease, NSP2 and NSP7) by molecular docking and dynamic simulations studies. Prior to carrying out the docking study, initially 3D structures of the target proteins of interest were retrieved from protein data bank (https://www.rcsb.org/)/. Later, the obtained structures corresponding to each of the protein were prepared and pre-processed to carry out further analysis. This step was performed using Flare protein preparation option available in the Flare version 4.0 of cresset software (https://www.cresset-group.com/software/flare/). The steps involved addition of missing hydrogen's, assigning optimal ionization states to residues, optimizing spatial positions of polar hydrogen's to improve hydrogen bonding, minimizing the steric strain and reconstructing unresolved side chains [35] . Followed by protein preparation, structures of each of the compounds to be analyzed for molecular docking analysis were prepared and minimized using 'Flare minimize selection calculation' option to remove clashes among atoms of the ligand and to develop a reasonable starting pose. Following protein and ligand preparation, each compound was docked with each of the prepared target protein mentioned above using Flare (Cresset) with default settings using the "Very accurate but slow" mode. The grid box was created around the selected key residues with the active binding site. Compounds were ordered according to the LF rank score, LF dG score and LF LE score of the best binding poses. As per the literature, amino acids Asp 5, Thr 9, Ala 65, Asp 67 and Glu 74 are crucial to facilitate the inhibition of NSP 7 by inhibitors. Therefore, considering these reports, grid was generated around these key residues [36] . Herein for carrying the docking analysis PDB ID: 7JLT was selected. In case of NSP2, PDB ID: 7EXM was selected because a x-ray crystallographer [37] suggested that Lys111, Lys112, Lys113 were key residues which are required for interacting with nucleic acid and further and regulating intracellular signaling pathways. Based on Ma and co-workers study, these three positively charged residues were considered for grid generation and molecular docking [37] . For the third target, PDB ID: 6LU7 was considered and docking was carried out by choosing some of the important amino acids i.e. Asn 142, Gly 143, Ser 144 and Cys 145 [38] . For docking analysis, Lead Finder™ which is a dedicated algorithm and scoring function for virtual screening was employed. The docked complexes of each target protein with the designed compounds harboring best docking scores were further subjected to molecular dynamic simulations for a period of 50 ns using dynamic option available with Flare module of cresset software. To carry out the dynamic simulations, AMBER force-field and version GAFF2 was employed. AM1-BCC charge method was set up while system was build using explicit solvent with 10Å10Å10Å dimensions. The buid system was initially equilibrated for 200ps and 2fs was kept as time step [39] . The whole calculations were run on GPU Nvidia Tesla V100, 16 GB. Generally, small molecules interacts with their respective receptors via non-covalent interactions including hydrogen bonding, ionic, cation-, lone-pair sigma hole (halogen bonding), -, and orthogonal multipolar interactions (fluorine bonding). These non-covalent interactions are important contributors to the binding free energy ΔG (enthalpic term) of each protein-ligand complexes. We performed electrostatic complementarity analysis using Flare module of Cresset software to assess the match between each of the designed molecules and binding pockets in terms of electrostatics. This type of analysis provided deep insight into how these ligands bind within each of these three targets associated with SARs-CoV2. The five-membered heterocycle-substituted benzimidazole compounds were synthesized following a straightforward synthetic approach by coupling of aromatic amines and aldehydes. We have established this synthetic approach to a large number of benzimidazole derivatives in aqueous medium and aerobic condition. Here, we modified this synthetic method and used The signals for the aromatic protons of the L1-L3 and L3ʹ appeared in the same region ranging from δ7.8 to 6.2 ppm, respectively. The chemical shift values at δ 11.83-11.80 ppm and 10.95 ppm represent the -NH protons of the pyrrole heterocycle in L1 and the peak at δ 2.48 ppm represents the methyl protons of L3ʹ (Fig. S3, S5, S7, S9) . The 13 C NMR spectra were also recorded to confirm the structural composition of the C-atoms in L1-L3 and L3ʹ. The crystal structural analysis reveals that the methyl derivative of thiophene substitutedbenzimidazole (L3′) crystallized in a monoclinic system with a P21/c space group. The ORTEP diagram of the compound is displayed in Fig. 1(a) . The crystallographic refinement parameter is summarized in Table 1 and bond angles and bond distances are noted in Table S1 . In the structure of L3′, one thiophene directly links with the benzimidazole group while the second thiophene attaches to the benzimidazole moiety through a methylene-C. Furthermore, two thiophene rings co-exist in a perpendicular orientation. Self-assembly analysis of L3′ reveals that the thiophene-substituted benzimidazole grows through moderate distant C-H … π, S … H and N … H interactions in crystalline phase (Fig. 1b) leading to a 1D supramolecular frameworks along b axis. The supramolecular 1D chain interacts with another supramolecular 1D framework to develop a 3D supramolecular architecture (Fig. 1c) . The interconnecting 1D frameworks are further stabilized by C-H … π interactions. The details of the non-covalent interactions for 3 are summarized in Table S2 . To predict the best matching mode of the designed ligand to each of the selected target, molecular docking analysis was carried out. This approach assisted in analyzing all possible (Table S4) . Overall, it was observed that L3 manifested highest docking scores with Covid-19 main-protease than other two targets including nsp2 and nsp7. This observation directly indicates main-protease as the main target of these benzimidazole based molecules. Similarly rests of the two compounds were also observed to be promising compounds for main-protease. The 3D docked poses for L3 in each of the target including main protease, nsp2 and nsp7 have been displayed in Fig. 2 . The estimated molecular docking results are also compared with previously reported work [42, 43] . Hosseini et. al. reported the binding energy for Ramelteon, Levomefolic acid, Ketoprofen etc. with main protease of SARS-CoV-2, revealing -6.0 to -6.66 kcal/mol energy score [43] . We found the binding score for the synthetic benzimidazoles against the main protease and non-structural proteins in between -5.0 to -11.0 kcal/mol, suggesting the promising inhibition actions against SARS-CoV-2. It is noticed that in silico screening studies against nsp-2 are limited; therefore, the study against nsp2 inhibition is a novel addition. The best scored L3 was analyzed for electrostatic complementarity (EC scores) analysis. Among three of the selected targets, this compound could show better EC scores for target Covid-19 main protease (PDB ID: 6LU7) in comparison to other two targets nsp2 and nsp7. The obtained EC scores can be classified into EC, EC r and EC rho, where EC indicates first computed score, a normalized surface integral of the complementarity score that effectively display the average value of the score over the ligand's surface. Meanwhile, EC r and EC rho indicate pearson's correlation coefficient and the spearman rank correlation coefficient, respectively. These scores generally compute the ligand and protein electrostatic potential sampled on the surface vertices. These measures of these scores generally range from 1 (perfect complementarity) to -1 (perfect clash) but have different characteristics. Herein, the L3 with best docking scores was found to manifest better EC scores while this compound couldn't display favorable EC scores with nsp2 and nsp7, as observed in Table 3 . The negative values for EC r and EC rho indicate some steric clashes in case of docked complexes of L3 each with nsp2 and nsp7. Thus L3 is a more promising inhibitor of main-protease, while partial inhibition could be expected in case of nsp2 and nsp7. As observed in previous analysis that L3 exhibited not only good docking scores but also good complementarity especially with target main-protease. Furthermore, to analyse the stability of compound-3 within the active site of each target protein including main protease, nsp2 and nsp7, molecular dynamic analysis was carried out. In each of the case, only slight RMSD fluctuations were observed with RMSD within the limit of 1Å. These RMSD fluctuations can be observed in Fig. 4 . The docking interactions retained after MD simulations were further analyzed and noted some key observations. It can be observed in Table 4 that compound-3 manifested most prominent interactions in terms of sulfur-lone pair and aromatic-aromatic interactions with His 41 by 21.4% and 20.1, respectively. Though this compound mange to retain some interactions with target nsp2 and nsp7, however the percentage contact with crucial amino acids were lower than the 15%, as observed in Table S5 and Table S6 . From all the considered in-silico analysis, it would be concluded that L3 is most promising anti-SARs-CoV2 and has a potential to inhibit main-protease effectively than nsp2 and nsp7. The 3D interactions after MD for a period of 50ns considering each of the complex for compound-3 with main-protease, nsp2 and nsp7 are displayed in Fig. 5 . Currently, structural modification based on chromophore-based drug design gains a paramount attention among the scientific community [44] [45] [46] . Culletta and researchers [47] made an in silico inhibition activities of the designed pharmacophores against the different proteins of SARS-CoV-2. A significant change of dG Bind energy was observed (-35 to -90 kcal/mol) for those compounds against M pro and nsp proteins. Badavath and co-workers [48] performed the computer-aided anti-SARS-CoV-2 screening activity for 118 isatin derivatives and evaluated their binding propensities against main. Furthermore, Purwati and collaborators [49] In conclusion, we present the synthesis and spectroscopic characterization of a series of 5membered heterocycle substituted benzimidazoles employing a straightforward synthetic approach. We also prepared the single-crystals of methyl derivative of L3 and analyzed the structural and long-range supramolecular frameworks of L3′ through X-ray crystallography. The therapeutic properties of the designed compounds were evaluated through in silico screening activities with molecular docking, electrostatic complementarity analysis and molecular dynamics studies against main protease (M pro ) and non-structural proteins (nsp2 and nsp7 NMR, and ADME properties etc are given here. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 1-Hydrogen bonding with Gly 143 ***LF-Rank score indicates correct energy-ranking of docked ligand poses * LF-dG score symbolizes accurate binding energy predictions ** LF-VScore indicates correct rank-ordering of active and inactive compounds in virtual screening experiments. ****LF-LE score signifies estimated ligand efficiency. 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Biswas gratefully acknowledge the financial support received from the University of North Bengal, Darjeeling 734013.