key: cord-0821949-uudm8zw6
authors: Singh, Gurjaspreet; Pawan; Mohit; Diksha; Suman; Priyanka; Sushma; Saini, Anamika; Kaur, Amarjit
title: Design of New Bis-triazolyl Structure for Identification of Inhibitory Activity on COVID-19 Main Protease by Molecular Docking Approach
date: 2021-11-03
journal: J Mol Struct
DOI: 10.1016/j.molstruc.2021.131858
sha: cc9e765fb1f5ccffadd86fc07875eef346bc2673
doc_id: 821949
cord_uid: uudm8zw6

In the rapidly growing COVID-19 pandemic, designing of new drugs and evaluating their inhibitory action against main targets of corona virus could be an effective strategy to accelerate the drug discovery process and their efficacy towards corona virus disease. Herein, we design new bis-triazolyl probe for an investigation of inhibitory activity towards COVID-19 main protease by Molecular docking approach. The formulated compound has been thoroughly characterized by elemental analysis, NMR ((1)H and (13)C) and complete structure elucidation was achieved via X-ray crystallography. Docking study reveals that newly synthesized compound confers good inhibitory response to COVID-19 main protease as supported by calculated docking score and binding energy. Strong hydrogen bonding and hydrophobic interactions of the newly synthesized compound with several important amino acids of the main protease also helps to explain the potency of the compound to inhibit the main protease. We hope that the present study would help the researcher in the field of Medicinal chemistry and to develop potential drug against the novel corona virus.

The ongoing outbreak of novel corona virus pandemic has claimed more than 2 million human lives worldwide and has posed notable threat to international health and economy [1] [2] [3] , but an effective therapeutic intervention has yet to be developed. COVID-19 is caused by new corona virus called severe acute respiratory syndrome corona virus 2 (SARS- and was first reported in December 2019 in Wuhan City, China. The crisis has motivated the researchers from different fields of science towards vaccine finding against this novel disease.

Drug designing in the pharmaceutical industry is largely based on the use of different organic moieties as a constructing unit because it can provide novel libraries of biologically active molecules with improved pharmacokinetic properties 4 . There are many heterocyclic ring structures, which have been designed in such a way that their binding efficiency with the receptors increases after structural modifications 5-6 . One of the most requisite moieties is triazoles, which have been explored and still its scope is inevitable. These are five-membered heterocyclic frameworks which can be readily obtained in good to excellent yield on the multi-gram scale by click chemistry via reaction of aryl/alkyl halides, alkynes and sodium azide (NaN 3 ) under ambient conditions 7 . These motifs are effective amide surrogates in bioactive molecules because of their strong dipole moments. Triazoles can also be used as a linker and shows bioisosteric effects on peptide linkage, aromatic ring, double bonds and an imidazole ring. Some unique features like hydrogen bond formation, dipole-dipole and pistacking interactions of triazole compounds have increased their importance in the field of medicinal chemistry as they bind with the biological target with high affinity due to their improved solubility. It has been an emerging area of interest for many researchers throughout the globe owing to its immense pharmacological scope as it exhibits anti-HIV, anti-malarial, anticancer, antibacterial, antifungal, and anti-TB properties. [8] [9] [10] [11] [12] [13] [14] . Some drugs currently in use are based on triazoles especially 1,2,3-triazole moiety such as anti-HIV agent TSAO, Cefatrizine (antibiotic), antibacterial agent Tazobactum, anti-TB agent I-A09 and anti cancer agent CIA 15 . Furthermore, Pantane-3-yl group containing substances play a pivotal role in drug chemistry and also have been reported to display potent binding inhibition activity against a corticotrophin-releasing factor 1 (CRF1) receptor and in vitro antagonistic activity, strong anti-proliferative activity against several tumor cell lines, including multi-drug resistant phenotypes. 16 For many viruses, the protease enzyme plays a critical role in viral protein maturation by cleaning pro-proteins after their translation into the host cell cytosol. As a result, viral proteases are often potential drug targets 17 . The inhibition of viral protease can reduce the assembly of mature viral particles. Molecular docking is one of the best and fast techniques in the scientific community for rational design of drugs. Docking addresses the binding between drugs and proteins via active sites determination. Keeping the above perspectives in mind, herein, we report the synthesis, spectral characterization and crystal structure of new bistriazolyl probe for an investigation of inhibitory activity towards COVID-19 main protease (M pro ) by Molecular docking approach. The formulated compound has been thoroughly characterized by elemental analysis NMR ( 1 H and 13 C) and complete structure elucidation was achieved via X-ray crystallography.

The development of a candidate drug into an approved drug is a long and costly process. Hence, designing a drug molecule on the basis of selected important functional moieties and blind identification of their inhibitory activity against main viral protease with the help of computational methodologies such as molecular docking, virtual screening and binding free energy evaluation serves as a promising alternative for identifying potential drug candidates to combat COVID-19 pandemic.

9-Bromopentane (Aldrich), 4-bromo-2-hydroxybenzaldehyde (Aldrich), 1,2-dibromoethane (Avra), Sodium azide (Aldrich), Propargyl Bromide (80% in toluene, Aldrich), potassium carbonate (Avra), dibromoethane (Aldrich), NaN 3 (Aldrich), bromotris (triphenylphosphine) copper (I) (Sigma Aldrich) were used as received. Solvent were purified and dried before use. 1 H and 13 C NMR spectra were recorded on a JEOL (AL 400 MHz) spectrometer using CDCl 3 as an internal reference and chemical shifts were reported relative to TMS, values are given in Hz. The FT-IR spectra were recorded on a Thermo Scientific NICOLET IS50 spectrophotometer. Melting points were measured in a Mel Temp II device using sealed capillaries and were uncorrected

Single crystals of compound 6 suitable for X-ray diff raction were obtained by slow evaporation from a saturated chloroform solution at room temperature. A suitable crystal was carefully selected under polarizing microscope in order to perform its structural analysis by X-ray diffraction. Diffraction data were collected on a Super Nova, Single source at offset/far, HyPix3000 diffractometer. The crystal was kept at 298 (2) calculations. The final R 1 was 0.0972 (I > 2σ (I) and wR 2 was 0.3035 (all data). The goodness-of-fit was 1.000. CCDC 2094938 contains the supplementary crystallographic data for compound 6. Crystallographic parameters and basic information pertaining to data collection and structure refinement for the compound are summarized in Table 1 .

Initially 3-bromopentane (1) was made to react with Zinc and lithium chloride in minimum amount of THF at 50 ˚C for 2 hours followed by addition of 4-bromo-2hydroxybenzaldehyde (2) and Pd(PPh 3 ) 4 and continued to stir for 10 hours (scheme 1). The reaction was monitored by TLC and on complete consumption of starting materials, the resulting reaction mixture was allowed to cool and filtered to get 2-hydroxy-4-(pentan-3yl)benzaldehyde (3). Further, compound 3 was dissolved in minimum amount of DMF along with 1.5 equiv of anhydrous K 2 CO 3 and allowed to react with propargyl bromide, which was added slowly and allowed to stir at room temperature for 16 hours to get 2-hydroxy-4-(pentan-3-yl)benzaldehyde (4) . The progress of the reaction was monitored by TLC. Finally, to get the desired product 6, compound 4 was allowed to react with diazidoethane (5) in presence of copper catalyst (10 mol%) using THF:TEA (1:1) as solvent. The crude product was further purified by column chromatography to get pure compound 6 and thoroughly characterised by NMR and IR. 1,2-diazidoethane (5) 190.97, 190.92, 162.84, 160.62, 142.07, 131,65, 129.82, 124.98, 115.51, 114.54, 61.39, 55.74, 49.03, 39.42, 39.25 . Experimental characterization of compound 6 is reported in supplementary information.

Required PDB file for the crystal structure of COVID-19 main protease (PDB ID: 6LU7) downloaded from Protein Data Bank (https://www.rcsb.org) and the Ligand (compound 6) for screening was synthesized according to scheme 1. The ligand was drawn in the Avogadro molecule editor, and then their stable structure obtained by energy minimization with the MMFF94 Force Field and converted into PDB format with the help of 3D-chemdraw. The 

A three step synthetic route, shown in scheme 1, was employed to synthesize compound 6.

Firstly, 3-bromopentane (1) was made to undergo Negishi coupling with 4-bromo-2hydroxybenzaldehyde (2) in THF to yield 2-hydroxy-4-(pentan-3-yl)benzaldehyde (3). The obtained compound 3 was then stirred at room temperature with propargyl bromide in DMF using potassium carbonate as base to give 2-hydroxy-4-(pentan-3-yl)benzaldehyde (4).

Finally, desired product 6 was synthesized by reaction between compound 4 and 1,2diazidoethane (5) 

Single crystal X-ray diffraction studies disclose that the compound 6 crystallized in monoclinic crystal system containing eight molecules per unit cell with P2 1 /c space group and has comparable structure, as is evident from the bond angles and bond parameters ( Table   S2) . Crystallographic data and structure determination parameters are summarized in Table 1 .

The ORTEP diagram and atomic labeling for empirical formula C 32 H 38 N 6 O 4 (6) is shown in 

Inter-molecular hydrogen bonding 

M pro also termed 3CL protease which is a potential drug target for corona virus infections due to its essential role in processing the poly-proteins that are translated from the viral RNA and mediates the maturation of functional polypeptides involved in the assembly of replicationtranscription machinery 24 . M pro digests the poly-protein at no. less than 11 conserved sites, starting with the autolytic cleavage of this enzyme itself from pp1a and pp1ab. In addition, M pro has no human homolog and is highly conserved among all corona-virus 25 . These above features make it an attractive drug target against corona viruses. Therefore, in order to find out the binding conformation of synthesized compound 6 within the active sites of main protease of COVID-19 and to obtain additional validations for experimental results, molecular docking was performed. All single bonds were considered as rotatable and fitness function was given as the score and all the calculations were made by using standard default settings. After Docking several pose was saved for compound 6 out of them best are shown in Figure 

On the contrary, all interaction between compound 8 and the protein residues summarized in Table 2a and The binding energy (EB) of compound 6 was −6.43 kcal/mol with rmsd value, 0.00. The more negative binding energy (EB) and smaller value of inhibition constant (Ki) implies best docking score. Hydrogen bonds are primary contributing factor in supporting the binding affinity of drugs with the receptor. Strong hydrogen bonding interaction represents a high binding capability between ligand and protein which points toward the strong inhibitory activity of synthesized compound 6 against the main protease of corona virus.

In summary, we have successfully synthesized a new bis-triazolyl probe via click chemistry.

The synthesis is very simple, high yielding and employs commercially available starting materials. The molecular structure of the compound 6 is authenticated by single crystal X-ray diffraction analysis showing intra and intermolecular hydrogen bonding which has great importance in many biological properties of the compound in medicinal chemistry. Docking analysis showed interactions of synthesized compound with M pro of COVID-19 with binding affinity of −6.43 kcal/mol and concludes that tested compound can serve as a potential M pro inhibitor. In brief, the present work opens a promising approach to design new drug and will probably be useful in molecular modeling, drug designing and wider scope in medicinal chemistry.

Supporting information contains 1 H NMR, 13 C NMR and single X-Ray structure data, bond parameters and CCDC number (2094938) of compound 6 crystal.

The epidemiology and pathogenesis of corona virus disease (COVID-19) outbreak

World Health Organization declares global emergency: a review of the 2019 Novel Coronavirus (COVID-19)

Emergence of novel coronavirus 2019 nCoV: need for rapid vaccine and biologics development

An overview of the synthetic routes to the best selling drugs containing 6-membered heterocycles

An overview of N-heterocyclic carbenes

A mini review of pyrimidine and fuse pyrimidine marketed drugs

Synthesis and biological evaluation of 3,4,6-triaryl-2-pyranones as a potential new class of anti-breast cancer agents

An efficient and improved synthesis of 1, 5-diketones: versatile conjugate addition of nucleophiles to a,b-unsaturated enones and alkynones

Privileged scaffolds for library design and drug discovery

Pharmacological significance of synthetic heterocycles scaffold: a review

Structural requirements for the binding of modified proteins to the scavenger receptor of macrophage

Synthesis and biological evaluation of indolyl bisphosphonates as anti-bone resorptive and anti-leishmanial agents

Synthesis and structure guided evaluation of estrogen agonist and antagonist activities of some new tetrazolyl indole derivatives

Synthesis, HIV-RT inhibitory activity and SAR of 1-benzyl-1H-1,2,3-triazole derivatives of carbohydrates

A copper(I)-catalyzed 1,2,3-triazole azide-alkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1 protease variant

Broad-spectrum antihuman immunodeficiency virus (HIV) potential of a peptide HIV type 1 entry inhibitor

The Hsp90 inhibitor 17-allylamide-17-demethoxygeldanamycin induces apoptosis and differentiation of Kasumi-1 harboring the Asn822Lys KIT mutation and down-regulates KIT protein level

Clicktophycin-52": a bioactive cryptophycin-52 triazole analogue

Synthesis and structure activity relationship of 1-and 2-substituted-1,2,3-triazole letrozole-based analogues as aromatase inhibitors

Synthesis of some 1,2,3-triazoles derivatives and evaluation of their antimicrobial activity

Synthesis and biological activity of some new 1,2,3-triazole hydrazone derivatives

Azide-alkyne cycloaddition en route to 1H-1,2,3-triazole-tethered 7-chloroquinoline-isatin chimeras: Synthesis and antimalarial evaluation

triazol-4-yl)methyl)piperazin-1-yl)phenanthridine analogues as antimycobacterial agents

Discovery of 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole, a novel CRF1 receptor antagonist

OLEX2: a complete structure solution, refinement and analysis program

Crystal structure refinement with SHELXL

AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility

Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

The authors are thankful to CSIR (01(2950)/18/EMR-11, New Delhi, DST-PURSE, DST-SERB (SB/FT/CS-132/2014) for providing financial support and University Grant Commission (UGC), New Delhi, for junior research fellowship.There is no conflict of interest.