Exploration of inhibitory action of Azo imidazole derivatives against COVID-19 main protease (Mpro): A computational study.

Four novel ionic liquid tagged azo-azomethine derivatives (L1-L4) have been prepared by the condensation reaction of azo-coupled ortho-vaniline precursor with amino functionalised imidazole derivative and the synthesized derivatives (L1-L4) have been characterized by different analytical and spectroscopic techniques. Molecular docking studies were carried out to ascertain the inhibitory action of studied ligands (L1-L4) against the Main Protease (6LU7) of novel coronavisrus (COVID-19). The result of the docking of L1-L4 showed a significant inhibitory action against the Main protease (Mpro) of SARS-CoV-2 and the binding energy (ΔG) values of the ligands (L1-L4) against the protein 6LU7 have found to be -7.7 Kcal/mole (L1), -7.0 Kcal/mole (L2), -7.9 Kcal/mole (L3), and -7.9 Kcal/mole (L4).The efficiency of the ligands has been compared with the FDA approved and clinically trial drugs such as remdesivir, Chloroquin and Hydroxychloroquin and native ligand N3 of main protease 6LU7 to ascertain the inhibitory potential of the studied ligands (L1-L4) against the protein 6LU7. Pharmacokinetic properties (ADME) of the ligands (L1-L4) have also been studied.

Introduction

After the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 from the Wuhan city of China, the infectious disease was declared pandemic by World Health Organization (WHO) on 11the March 2020 and subsequently, the whole world witnessed a global health emergency [1]. As of June 11th 2020, the COVID-19 disease has spread to the whole world with over 7.4 million confirmed cases and over 0.4 million confirmed deaths (worldometer, June 11th 2020). After the declaration of novel coronavirus (COVID-19) infection as pandemic, a number of countries took a preventive measure to slow down the spread of the coronavirus (COVID-19) by implementing mandatory lockdown or national quarantine and as a result of mandatory home quarantine or isolations, more than a third of the planet's population faced some form of restriction (Business Insider, April 17th 2020). Early research on COVID-19 have shown that the cross species and human to human transmission of the virus is regulated by spike protein receptor binding domain and its host receptor (ACE2) as similar to SARS-CoV which was outbreak in 2002 [2]. Till date, no specific vaccine or therapeutic agents are available for the treatment of COVID-19 infection and several existing FDA approved antmaliarial and antiviral drugs have been formulated as a supportive care for the treatment of this infection [3]. Therefore, a rapid formulation of novel compounds as a potentential therapeutic agent for COVID-19 infection is an important mission [4]. Recent studies on SARS-CoV-2 have shown that the 3CL hydrolase (chymotrypsin-like protease, PDB ID: 6LU7), also known as the main protease (Mpro) of novel coronavirus (SARS-CoV-2) plays an important role in the life cycle of coronavirus and thus, its inhibition could provide a promising therapeutic principle for developing strategic and specific treatment against COVID-19 infection [5, 6]. Apart from the existing FDA approved antiviral and antimalarial drugs, Traditional Chinese Medicine has gained a lot of interest to treat coronavirus (COVID-19) infection in China [7], [8], [9], [10], [11], [12]. Identification of potential inhibitors for specific disease by traditional methods are time consuming and expensive. However, in-silico techniques such as molecular docking and molecular dynamic have gained lot of interest for the identification of potential inhibitors for specific disease in recent years [13, 14]. Since, the main protease (3CLpro) of coronaviruses is conserved and therefore many research groups are engaged in finding the effective inhibitors for COVID-19 Protease (Mpro) by virtual screening approach using the library of small molecules (antiviral, antimalarial, plant derived, fungal metabolites and synthesized molecules) [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. Recently, computer aided drug design namely molecular docking and molecular dynamic study have gained a lot of interest in the field of drug designing for the treatment of specific disease [32, 33]. On the other hand, azo or diazene compounds containing conjugated chromophoric azo (-N = N-) group are an important class of organic colorants which are mostly studied during recent years [34]. These colorant compounds are widely used as a class of dyes due to their versatile application in various fields such as the dyeing of textile fiber, coloring of different materials, plastics, cosmetics industries, biological-medical studies and advanced application in organic synthesis [35, 36]. Furthermore, the azo compounds were found to possess a variety of biological activities such as antibacterial, antifungal, antiviral and anti-inflammatory etc., [37], [38], [39], [40]. Interestingly, azo compounds are found to involve in many biological processes such as nitrogen fixation, inhibition of DNA, RNA and protein synthesis, and carcinogenesis [41]. Moreover, Microorganisms, fungi, fungal endophytes, lichenized, ascomycetes, plant parts (bark, berries, leaves, roots, and wood), and marine invertebrates are found to posses natural diazine or azo alkaloids. In recent years, several azo metabolites have been isolated from actenomycetes, fungal species, terrestrial and marine sources [42]. Valanimycin and its derivatives (isloalted from Streptomyces viridifaciens, MG456-hF10), maniwamycins A and B (isolated from Streptomyces prasinopilosus), azoxybacilin (isolated from Bacillus cereus, NR2991), Elaiomycin (isolated from Streptomyces hepaticus), Elaiomycins K, L and amide elaiomycin K (isolated from Streptomyces sp., Tu¨ 6399), jietacins A and B (isolated from Streptomyces sp.), lyophyllin (isolated from mushroom Lyophyllum shimeji), calvatic acid (isolated from fungi Calvatia craniformis) etc., are the actenomycetes and fungal metabolites which have naturally occurring azo groups and they are found to posses antibiotic activities [43], [44], [45], [46], [47], [48], [49], [50], [51], [52]. Thus, it was thought worthwhile to investigate the inhibitory potential of the synthesized azo derivatives against the main protease of COVID-19 and therefore, in this article, we report the computational study on inhibitory action of synthesized Azo imidazole derivatives (L1-L4) against COVID-19 main Protease (Mpro).

Experimental

Materials and methods

All the reagents used were of analytical grade and used without further purification. orthovaniline, Aniline, 2 chloroaninline, 4-chloroaniline, and 4-fluoro aniline were procured from Sd fine chemical company, India.1-methylimidazole, KPF6 and 2-bromoethylaminehydrobromide were procured from sigma Aldrich and used without further purification. All the solvents used were of spectroscopic grade.

Instrumentation

The IR spectra of the synthesized compounds were analyzed using Perkin-Elmer Spectrum FT-IR spectrometer (RX-1) operating in the region 4000 to 400 cm−1 in KBr. 1H NMR spectra were recorded at room temperature on a FT-NMR (Bruker Advance-II 400 MHz) spectrometer using DMSO‑d6 as solvents and chemical shifts are quoted in ppm downfield of internal standard tetramethylsilane (TMS). Elemental microanalyses (C, H and N) were conducted by using Perkin–Elmer (Model 240C) analyzer. The purity of the prepared compounds was confirmed by thin layer chromatography (TLC) on silica gel plates and the plates were visualized with UV-light and iodine as and when required.

Preparation of protein and ligand for docking study

The X-ray crystallographic structures of main protease (Mpor, PDB ID 6LU7) of SARS-CoV-2 has been retrieved from the Protein Data Bank (PDB) (http://www.pdb.org.) database. Graphical User Interface program “Auto Dock Tools (ADT) 1.5.6″ from Molecular Graphics Laboratory (MGL) developed by Scripps Research Institute has been employed for the preparation of protein for docking study [53]. Input file of receptor protein for the docking study is created by taking specific china (Chain A) of the protein (6LU7). In a typical receptor protein preparation, water molecules and hetero atoms along with the co-crystallised ligands in PDB crystal structures were removed and subsequently, the receptor .pdbqt file has been created by adding polar hydrogen atoms and Kollman united atom charges [54]. The three dimensional (3D) structures of ligands (L1-L4) were drawn using Chemsketch (ACD/Structure Elucidator, version 12.01, Advanced Chemistry Development, Inc., Toronto, Canada, 2014, http://www.acdlabs.com.) and geometry optimization of the ligands (L1-L4) were carried out using MM2 program incorporated in Chem. Draw Ultra 8.0 and further optimization of geometry of each molecule were carried out with the MOPAC 6 package using the semi-empirical AM1 Hamiltonian [55]. The input .pdbqt file of the ligands was generated using Auto Dock Tools (ADT). As the ligand molecules (L1-L4) are non peptides, therefore, Gasteiger charge was assigned and then non-polar hydrogen was merged. The structure of the ligands L1-L4 is given in Fig. 1 .

Fig. 1

Structure of Azo Imidazole ligands (L1-L4).

Docking study using AutoDock Vina

All molecular docking simulations were carried out in the AutoDock Vina programe 1.1.2 developed by Scripps Research institute [56]. and the results of the docking study and the intermolecular interactions between receptors and the ligand molecules were analyzed using BIOVIA Discovery Studio 2020 (DS), version 20.1.0.0 (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016) and Edu pymol version 1.7.4.4 [57]. The three dimensional (3D) affinity (grid) maps and electrostatic a grid boxes of 50×50×50 Å grid points and grid center (X, Y, Z) of −26.283 12.599 58.966 with a spacing of 1.00 Å generated by AutoGrid auxiliary program for each of the receptor for blind docking were generated to cover the entire active site of the receptor protein in order to eliminate any biasness arising during the docking simulation [58]. Lamarckian genetic algorithm and a standard protocol with default setting of other run parameters were used for docking simulation. For each docking experiments, several runs were performed by the program with one predicted binding mode with each run. All the torsions were allowed to rotate. The predicted inhibitory constant (pK) has been calculated using the following standardized equations [59], [60], [61].

Synthesis

General procedure for the synthesis of azo imidazole derivatives, L1-L4

azo imidazole derivatives has been synthesized by following the literature procedure given elsewhere [37, [62], [63], [64], [65]. In a typical synthetic procedure 1-(2-Aminoethyl)−3-methylimidazolium hexafluoro-phosphate, ([2aemim] [PF6]) (5 mmol) in absolute ethanol was pour in a stirring solution of azo-coupled o-vaniline precursors (5 mmol) in 20 ml of absolute ethanol during a period of 10 min. The reaction mixture was then refluxed in an oil bath for 6 h at 90 °C with constant stirring with the help of magnetic stirrer and the progress of the reaction monitored by TLC (10% ethyl acetate in hexane as eluent). The solution was cooled to room temperature and the solid product thus obtained was filtered, washed with little ethanol and diethyl ether respectively and finally recrystallized from hot ethanol solution and dried over silica under vacuum to get the pure products (Scheme 1 ).

Scheme 1

Syntheses of Azo Imidazole derivatives L1-L4.

The analytical and spectroscopic data for each of the synthesized azo imidazole derivatives (L1-L4) are given in supplementary data file SD1.

Results and discussion

Azo imidazole derivatives has been successfully prepared by the condensation reaction of 1-(2-Aminoethyl)−3-methylimidazolium hexafluoro-phosphate, ([2aemim] [PF6]) with substituted azo-coupled o-vaniline precursors in ethanol. All the isolated compounds (L1-L4) were found to be air stable solid, soluble in common organic solvents like methanol, ethanol, acetonitrile, DMF, DMSO etc., and intensely colored compounds.

IR spectral studies

In order to ascertain the binding mode in the synthesized azo imidazole derivatives L1-L4, the IR spectra of the compounds were closely analyzed. In the solid state IR spectrum of the Azo imidazole derivatives (L1-L4), appearance of bands in the IR spectra of L1-L4 in the range 1620–1647 cm−1 can be assigned to the stretching vibration of azomethine linkage ν(-C = N) [64, 66]. Astrong band at 33,100–3436 cm−1 can be assigned to stretching frequency of OH group, ν(OH) group [67]. A band appearing in the range 1593–1616 cm-1 in the IR spectra of L1-L4 derivatives can be assigned to ν(C = C) stretching frequency. Also, the N = N stretching frequency of the synthesized compounds are found at 1542–1552 cm−1 and 1453–1500 cm−1 and the N = N bending vibrations are found in the range 1153–1176 cm−1 and 956–969 cm−1. IR spectra of the representative compound L1 is depicted in Fig. 2 . A band in the range 838–842 cm−1 in the IR spectrum of derivatives (L1-L4) can be assigned for stretching vibration of PF6 group [65]. Thus the IR spectral data therefore supports the formation of the compounds (L1-L4). The IR spectra of L2, L3 and L4 are depicted in supplementary figures (Fig. S1.1-Fig. S1.3).

Fig. 2

Infrared Spectra of Compound L1.

1H NMR spectral studies

The 1H NMR spectra of the synthesized azo imidazole derivatives L1-L4 recorded in DMSO‑d6 at ambient temperature, displays a group of signals corresponding to the hydrogen of each molecule. In the 1H NMR spectrum of L1-L4, the signal corresponds to OH proton exhibited a slightly broad singlet peak at δ 13.3–13.50 ppm and the signal for CH N proton of the compounds L1-L4 appeared as a singlet in the range δ 8.56–8.61 ppm. The appearance of the following peaks, triplet in the range δ 7.62–7.68 ppm for (NCH) proton, triplet in the range δ 7.73–7.76 for (NCH) proton and singlet in the range δ 9.11–9.17 ppm for N (H) CN proton respectively is in good agreement with the proposed structure for the synthesized compounds L1-L4. Signals for NCH3 and OCH3 protons appeared in the range δ 3.37–3.76 and δ 3.91–3.97 respectively. The aromatic protons of all the synthesized compounds (L1-L4) appeared as multiplate in the range δ 7.31–7.95 ppm. The NMR spectra of representative compound L1is depicted in Fig. 3 and the NMR spectra of other compounds (L3 and L4) are listed in supplementary figures (Fig. S1.4 & Fig. S1.5).

Fig. 3

1H NMR spectra of compound L1.

Pharmacokinetic properties of L1-L4

Pharmacokinetics provides information about drug concentrations in the different parts of the organism with respect to time. Drugs when administered into the body of an organism, it needs to cross different biological barriers. Thus, the main properties like absorption, distribution, metabolism and excretion (ADME) are very important parameters for any compounds to be considered as a drug and therefore prior to any clinical and animal studies it is relatively important to ascertain their pharmacokinetic properties of the molecules under consideration [68]. The properties such as lipophilicity (LogPo/W), gastrointestinal absorption (GI), water soluble capability (Log S), and CYP1A2 inhibitor, Blood - Brain Barrier (BBB) are very important pharmacokinetic parameters for any compounds [69]. Therefore, we also determine the pharmacokinetic properties such as lipophilicity (LogPo/W), gastrointestinal absorption (GI), water soluble capability (Log S), and CYP1A2 inhibitor, Blood - Brain Barrier (BBB) of the studied compounds (L1-L4) to ascertain their drug likeness character using SwissADME database (http://www.sib.swiss) and the Lipinski's properties and pharmacokinetic properties of compounds (L1-L4) and standard drug remdesivir are depicted in Table 1 [70].

Table 1

Lipinski's properties and pharmacokinetic properties (ADME) of the ligands L1-L4 and drug Remdesivir.

Properties	L1	L2	L3	L4	Remdesivir
Molecular weight (gm/mole)	364.42	398.87	398.87	382.41	602.58
Rotatable bonds	7	7	7	7	14
H-bond donor (5)	1	1	1	1	4
H-bond acceptor	5	5	5	6	12
Violations	0	0	0	0	2
Log Po/W	2.41	2.89	2.89	2.70	1.81
Log S	−3.83(MS)	−4.42(MS)	−4.42(MS)	−3.99(MS)	−4.12(MS)
GI	High	High	High	High	Low
BBB	No	No	No	No	No
CYP1A2	No	No	No	No	No
Bioavailability Score	0.55	0.55	0.55	0.55	0.17
Topological Surface Area (Å2)	76.40	76.40	76.40	76.40	213.36

*MS: Moderately Soluble, BBB: Blood-Brain Barrier, CYP: Cytochrome P450, GI: Gastrointestinal tract.

From Table 1, it is evident that the compounds (L1-L4) with bioavailability 55% have consensus lipophilicity (LogPo/W) value in the range 2.41–2.89. Thus the high and positive lipophilicity values (LogPo/W) which indicates that the compounds are more lipophilic. Lipophilicity is an important phartmacokinetic property of drug candidate in the development of its dosage form, because drug molecules must pass through the lipid bilayer of most cellular membranes. Therefore, generally it is believed that for a drug molecule to have good absorption, it must be lipophilic. According to Lipinski's Rule of Five, the partition coefficient should be positive, but less than 5 i.e., log10PC=5 [71]. Therefore, the studied compounds (L1-L4) with positive and less than 5 (LogPo/W) values can have good absorption in the body and the studied molecules fall on the category of good lipophilic compounds. However, the compounds (L1-L4) are moderately soluble in water as indicated by their solubility (LogS) values which ranges from −3.83 to −4.42. All the studied compounds have high gastrointestinal absorption. The compounds (L1-L4) have no Blood-Brain Barrier and CYP1A2 properties. Thus from the pharmacokinetic analysis, it can be seen that the compounds (L1-L4) qualifies the drug likeness criteria with no Lipinski rule violations and these compounds can serves as a potential drug candidate.

Molecular docking study

Recent study on structural analysis of Mpro of SARS-CoV-2 have shown that it also has same location of active site as Mpro of SARS virus and it is located between the domain I and domain II of protein 6LU7 (Fig. 4 ).

Fig. 4

Structure of Mpro of SARS-Cov-2 with domain I, II and III (yellow circle represents catalytically active site).

The catalytic dyad (Cys145-His41) is present on the active site of the protein and both the domain contributes one residue to the catalytic dyad [72, 73]. The Cys-His-catalytic dyad of Mpro shows protease activity and thus inhibition the catalytic dyad in Mpro could become an attractive target for designing anti-CoV drug [74]. As of now, no treapeutic agents or vaccine have been developed specifically to treat the infection caused by SARS-CoV-2 [73]. However, several FDA approved antiviral, anti HIV and anti Malarial drugs have been used as supportive measures to treat and prevent further spread of infection caused by SARS-CoV-2 infection [75], [76], [77]. On the other hand, azo containing molecules such as Valanimycin, maniwamycins A and B, Elaiomycin etc., are ubiquitous in nature and these azo compounds are highly bioactive and some of them have been used as antibiotics to cure specific disease [43], [44], [45]. Prontosil and Phenazopyridine are FDA approved antibiotic drugs containg Azo group.Prontosil is widely used antibacterial drug which contains azo linkage and sulfonamide group and it has also been used to cure puerperal fever [78, 79]. Phenazopyridine is used as local analgesic effects on the urinary tract infection and also sometimes used with antibiotics for immediate symptomatic relief [80]. Since, the traditional method of drug formulation is time consuming and therefore, computational method such as molecular docking and molecular dynamic simulation study may be an alternative way to find out the potential drug candidates for the specific disease at relatively less time [81]. Study of protein- ligand interactions with the help of computer aided drug design method such as molecular docking can provide a promising and potential therapeutic agent for the treatment of specific disease. Therefore, in this study we are utilizing the molecular docking method to study the efficiency of azo imidazole derivatives (L1-L4) as inhibitors of Mpro of SARS-CoV-2. The results obtained from these docking studies indicated strong interactions of ligands (L1-L4) with (Mpro, 6LU7) of SARS-CoV-2 near the domain pocket I. A summary of the results obtained from successful docking of studied ligands (L1-L4) with main protease (Mpro, 6LU7) are summarized in Table 2 .

Table 2

Summary of docking of ligand (L1-L4) against COVID-19 Main Protease (Mpro, 6LU7) with their binding energy (ΔG), predicted inhibitory constant (pKi), interacting amino acid residues and types of interactions.

Ligands	Binding Energy (ΔG) (Kcal/mole)	Predicted inhibitory constant (pKi) µM	Amino Acid residues	Types of interactions
L1	−7.7	1.72	Gly143 and Ser144	H-bond
			Met165	π-Sulpher
			His163 and His172	π-alkyl
			Thr24, Thr25, Phe140 and Cys145	π-donor H-bond
			Thr26, Leu27, His41, Thr45, Met49, Leu141, Asn142, Glu166 and Gln189	Van der walls
L2	−7.0	5.76	Leu141, Ser144 and Cys145	H-bond
			His163 and His172	π-alkyl
			Thr24, Thr26 and Phe140	π-donor H bond
			Thr25, Leu27, His41, Met49, Asn142, Gln143, Met165, Glu166 and Gln189	Van der walls
L3	−7.9	1.22	Gly143 and Ser144	H-bond
			Met165	π-sulpher
			His163 and His172	π-alkyl
			Thr24, Thr25, Thr26, Phe140 and Cys145	π-donor H bond
			Leu27, His41, Thr45, Met49, Asn142, Glu166, Gln189 and Thr190	Van der walls
			Leu141	Unfavorable Acceptor- acceptor
L4	−7.9	1.22	His41, Leu141 and Cys145	H-bond
			Gln189 and Thr190	Halogen (F) bond
			His163, Met165 and His172	π-alkyl
			Thr24, Thr26 and Phe140	π-donor H bond
			Thr25, Leu27, Thr45, Met49, Asn143, Gln143, Ser144 and Glu166	Van der walls

Visualization of docking results

It has been reported that the amino acid residues His41, Cyd145 and Glu166 are important residues in substrate binding site and involvement of these residues in the formation of hydrogen bond could be prominent for the inhibitory effect of main protease (Mpro) [82]. After successful docking of all the ligands (L1-L4) with Mpro of SARS-CoV-2, the results showed significant interactions and binding of ligands with the protein 6LU7. The binding energies (ΔG) and predicted inhibitory constant (pK) of the ligands (L1-L4) are −7.7 Kcal/mole, −7.0 Kcal/mole, −7.9 Kcal/mole, and −7.9 Kcal/mole respectively and 1.72 µM, 5.76 µM, 1.22 µM and 1.22 µM respectively.

The docking result of L1 with the Protease (6LU7) showed that the ligand (L1) fits inside the core pocket region of the protease at the interface between domain I and domain II (Fig. 5 ).

Fig. 5

Visualization of docking of Ligand L1 docked in Mpro (6LU7) (A) Best binding mode of protein (Ligand L1 as green and red stick), (B) Amino acid residues involved in hydrogen bonding interaction (green dash line represents H-bonding) and (C) Binding interaction (2D) of ligand L1 with amino acid residues of protein 6LU7 (green dash line represents H-bonding and yellow dashed line represents π-sulpher interaction).

A closed analysis of the binding of L1 with the protein 6LU7 revealed that the L1 binds to the protein with binding energy (ΔG) −7.7 Kcal/mole. Ligand L1 forms two hydrogen bonds with protein 6LU7. The first hydrogen bond is formed by NH2 group of the amino acid residue Gly143 with O atom of OH group of ligand L1 at a distance 2.58 Å and the second hydrogen bond is formed between OH group of residue Ser144 and proton (H) of OH group of ligand L1 at a distance 2.50 Å. Apart from hydrogen bond, S atom of residue Met165 form π-Sulpher bond with π electrons of azobenzene moiety. π-alkyl interactions also exist between the π-electro of residues His163 and His172 and OCH3 group of L1. The amino acid residues Thr25, Thr25 Cys145 and Phe140 form π-donor hydrogen bonds with L1. The residues ThrR26, Thr45, Leu27, His41, Met49, leu141, Asn142 Glu160 and Gln189 are observed to interact with the ligand L1 through Van der walls interactions.

The visualization of docking result of L2 with main protease (6LU7) revealed that the L2 binds with the Protein at the interface between domain I and domain II with binding energy −7.0 Kcal/mole. It shows significant binding with three hydrogen bonding interactions between, C = O group of amino acid residue Leu141, OH group of residue Ser144 and proton (H) of OH group of L2 at a distance 1.74 Å and 2.88 Å respectively and NH(amide) group of residue Cys145 with O atom of OH group of L2 at a distance 2.59 Å (Fig. S1.6). The other types of interactions are π-alkyl interactions between the π-electron of residues His163 and His172 and OCH3 group of L2.The residues Thr24, Thr26 and Phe140 are found to interact with the ligand L2 through π-donor hydrogen bond. Apart from these, amino acid residues Thr25, Leu27, His41, Met49, Asn142, Gly143, Met165, Glu166 and Gln189 found to interacts with the ligand L2 through Van der walls interactions.

The docking of ligand L3 with the protein 6LU7 revealed that the ligand binds tightly with the protein residues with binding energy −7.9 Kcal/mole and the significant interactions are characterized by two hydrogen bonds. The first hydrogen bond is formed between the NH2 group of residue Gly143 and O atom of OH group of L3 at a distance 2.21 Å and second hydrogen bond exist between the OH group of amino acid residue Ser144 and proton (H) of OH group of L3 at a distance 2.81 Å. The residue Met165 form π-Sulpher interactions between the π-electron of ligand L3 and S atom of amino acid residue (Fig. S1.7). The other types of interactions are π-alkyl (π electron of His163 and His172 with OCH3 group) and π-donor hydrogen bonds (Thr24, Thr25, Thr26, Phe140 and Cys145). Apart from these interactions, the residues Leu27, His41, Thr45, Met49, Asn142, Glu166, Gln189 and Thr190 are found to interact with L3 through Van der walls interactions. An unfavorable acceptor-acceptor interaction between the ligand and residue Leu141 has also been observed.

The visualization of docking result of ligand L4 with the protein 6LU7 revealed that the ligand binds with the protein residues near the interface of domain I and domain II with binding energy (ΔG) −7.9 Kcal/mole. Three hydrogen bonding interactions exist between the protein residues and ligand L4. The two hydrogen bonds are found to exist at the catalytic dyad (Cys145-His41) between the NH (imidazole ring) of residue His41 and N atom of azomethine (C = N) group of ligand at a distance 2.91 Å and NH2 group of residue Cys145 and O atom of OH group of L4 at a distance 2.65 Å. The other hydrogen bond is formed by the C = O group of residue Leu141 with proton (H) of OH group of L4 at a distance 2.19 Å (Fig. S1.8). The C = O (amide) group of residue Gln189 and C = O group of residue Thr190 have been found to form halogen bond with F atom of L4 at a distance 3.60 Å and 3.56 Å respectively. Other interactions include π-alkyl (π electron of residues His163, Met165 and His172 and OCH3 group of L4) and π-donor hydrogen bond (Thr24, Thr26 and Phe140). The amino acid residues Thr25, Leu27, Thr45, Met49, Asn142, Glu143, Ser144 and Glu166 found to interact with the ligand through Van der walls interactions.

The binding energy (ΔG) value of ligands L1-L4 has been compared with the previously reported binding energy (ΔG) values of FDA approved and clinically trial antiviral and antimalarial drugs such as Remdesivir, choloroquin and hydroxychloroquine and native ligand N3 of protein 6LU7 in order to ascertain the inhibitory potential of the studied ligands (L1-L4) against the main protease (6LU7) of SARS-CoV-2 and it is represented in Table 3 .

Table 3

Comparison of binding energy (ΔG) of ligand (L1 –L4) with previously reported docking result of some FDA approved and clinically trial antiviral and anti malarial drugs and ligand N3 against main protease of Covid-19 (6LU7).

Compounds (drugs)	Binding energy, ΔG (Kcal/mol)
Remdesvir	−5.8 [83], −7.215 [84], −2.47 [30]−6.5 [85] −9.70 [87]
Chloroquine	−7.5 [83], −3.62 [30] −5.1 [85] −5.75 [86]
Hydroxychloroquine	−6.7 [85] −5.21 [86]
N3	−7.7716 [87], −8.396 [88]
L1	−7.7 [this work]
L2	−7.0 [this work]
L3	−7.9 [this work]
L4	−7.9[this work]

Therefore, it is evident from Table 3 that the clinically trial antiviral drug Remdesivir has binding energy (ΔG) in the range −2.47—9.70, antimalarial drugs, Chloroquin and Hydroxychloroquin has binding energy (ΔG) in the range −3.63—7.5 Kcal/mole respectively and the ligand N3 has binding energy (ΔG) value in the range −7.77—8.36 Kcal/mole. In our work the binding energy (ΔG) value of the ligands (L1-L4) have been found to be −7.7 Kcal/mole (L1), −7.0 Kcal/mole (L2), −7.9 Kcal/mole (L3) and 7.9 Kcal/mole (L4) respectively. Thus from this comparison, it may be conclude that the ligands (L1-L4) could act as a potential inhibitor molecule against SARC—CoV-2.

Conclusion

In this article, we have reported the molecular docking study of four ligands (L1-L4) against the main protease (6LU7) of SARS-CoV-2 to ascertain the inhibitory potential of these ligands. The docking results attributed various types of protein- ligand interactions and it is also seen that the lignads shows significant interactions at the interface between domain I and domain II. Furthermore, most of the ligands show interaction at the Cys-His-catalytic dyad. The pharmacokinetic study has revealed that the ligands (L1-L4) could act as a potential drug candidate. Thus, it is evident that various kind of favorable interactions exist between the ligands (L1-L4) and the main protease 6LU7. Therefore, on the basis of computational study, it may concluded that the ligands (L1-L4) could act as potential inhibitor against the main protease (6LU7) of SARS-CoV-2. Furthermore, in vivo and in vitro study is required to ascertain the proper binding mechanism and understanding the drug behavior of the studied ligands (L1-L4).

CRediT authorship contribution statement

Abhijit Chhetri: Conceptualization, Formal analysis, Funding acquisition, Software, Validation, Writing - original draft, Writing - review & editing. Sailesh Chettri: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Writing - original draft. Pranesh Rai: Investigation, Methodology, Project administration, Validation. Biswajit Sinha: Conceptualization, Methodology, Project administration, Supervision, Visualization, Writing - review & editing. Dhiraj Brahman: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.

Declaration of Competing Interest

The authors do not have any conflict of interest.