Investigation of embelin synthetic hybrids as potential COVID-19 and COX inhibitors: Synthesis, spectral analysis, DFT calculations and molecular docking studies

Embelin (2, 5-dihydroxy-3-undecyl-1,4-benzoquinone), a benzoquinone isolated from fruits of Embelia ribes has miscellaneous biological potentials including; anticancer, anti-inflammation, antibiotic, and anti-hyperglycemic activities. Also, embelin down-regulates the overexpression of inflammatory pathways like NF-kB, TACE, TNF-α, and other cytokines. Furthermore, embelin fascinated synthetic interest as a pharmacologically active compound. The present article involves the design, synthesis, DFT calculations, and molecular docking studies of embelin derivatives as cyclooxygenase inhibitors. The structure of these derivatives is confirmed by the various spectral analyses such as IR, NMR, and Mass. The DFT calculations were carried out for the molecules (1-8) using CAM-B3LYP hybrid functional with a 6-31+g(d) all-electron basis set using the Gaussian 09 package. Second-order harmonic vibrational calculations are used to check the minimum nature of the geometry. Further, HOMO and LUMO analyses were used for the charge transfer interface between the structures. Based on our previous work and structural activity relationship study, foresaid embelin derivatives were evaluated for in vitro COX-1 and COX-2 inhibitory activity. The compounds 3, 4, 7, and 8 demonstrated excellent COX inhibitions with IC50 values of 1.65, 1.54, 1.56, and 1.23 μM compared to standard drugs Celecoxib and Ibuprofen. Finally, the molecular docking studies carried out with Covid-19 and cyclooxygenase with all the newly synthesized embelin derivatives.


Introduction
The various non-steroidal anti-inflammatory drugs (NSAIDs) are available in the market cures the inflammation, pain and fever ( Fig. 1 ) [1] .

Instrumentation
On a Perkin-Elmer FT-IR spectrophotometer (Spectrum ONE), ( v max in per centimetre, KBr) were documented, 1 H (400 MHz) and 13 C NMR (100 MHz) spectra were documented on a Bruker AMX spectrophotometer using DMSO-d 6 as solvent and TMS as an internal standard ESI-mass was recorded on a mass spectrometer equipped with an electrospray ionization source having mass a range of 40 0 0 amu in quadruple and 20,0 0 0 amu in Tof. The purity of the compounds was checked using TLC (silica gel 60G F254 plates) and iodine vapors were utilized as visualizing agents. The elemental analysis was carried out using the elemental analyzer Flash EA1112 series.

Experimental
The chemicals used in the experiments were procured from certified suppliers and used without further purification.

Isolation, purification and characterization of embelin (1)
Isolation of embelin involves slight modification of earlier reported method [2] . Coarsely powdered fruits of Embelia ribes (200 g) were exhaustively extracted initially with hexane and then with chloroform by cold extraction method (500 ml). After 48 h, the solvent was decanted and evaporated using a Rota evaporator. The obtained extract was concentrated and subjected to column chromatography over silica gel (10 0-20 0 mesh). Elute column with chloroform first then with chloroform and methanol. Collect the fractions of chloroform: methanol (9:1) and evaporating under reduced pressure using Rota evaporator yielded an orange color powder which on crystallization with ether afforded orange plates of embelin (Yield: 1.2 gm). The purity of the compound was monitored by TLC. Isolated embelin was further characterized by spectral data such as 1 H, 13

Drug like profile
The physiochemical characteristics, ADMETand drug-likeness for molecules are critical in their fundamental unique proof as a synthetic lead and serve as a baseline against which integrated compounds are tested during lead development. The http://www. swissadme.ch/index.php was used to screen the stages of absorption, distribution, metabolism, and excretion of the ligand molecules. Numerous properties like molecular weight, numberof heavy atoms, number of aromatic heavy atoms, number ofrotatable bonds, molar refractivity, topological polar surfacearea, solubility, gastrointestinal absorption, blood-brain,barrier penetration, Lipinski's rule of five, Ghose rule, Veberrule, bioavailability score, and synthetic susceptibility werepredicted ( Table 1 ) [28] .The main goal of this study is to estimate the pharmacokinetic characteristics of the substances under investigation.

In vitro anti-inflammatory activity
Inflammation is a multi-step progression driven by intensely produced arachidonic acid and its metabolic products like prostaglandins. The COX-1 and COX-2 are two cyclooxygenases (COX) isozymes involved in prostaglandin synthesis. In continuation of our previous reported research articles on the antiinflammatory activity of heterocycles [29][30][31] , herein we report in vitro anti-inflammatory of synthesized compounds via COX-1 and COX-2 inhibition by enzyme immune assay.

COX-1 and COX-2 inhibition by colorimetric assay
The ability of the test compounds listed in Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC 50 value, μM) was determined using an enzyme immuno assay (EIA) kit according to the reported method [32] .

Molecular docking studies
The primary protein structure of SARS-CoV-2 Omicron virus variant (PDB ID:7T9L) and cyclooxygenase-2 (PDB ID:6COX) were obtained from the Protein Data Bank. The 3D structures of a few ligands were obtained from PubChem. The software Chemdraw 12 was used to draw the newly synthesized compounds. 3D structures were loaded into the PyRX 0.9 and energy minimized using the Universal Force Field (UFF) available under the OpenBabel [33] . A molecular docking study of the synthesized compounds and celecoxib and ibuprofen taken as reference was performed using PyRX-0.9.2 software. The ligands were blindly docked using the AutoDock Vina program to identify the compound with the best binding properties. The grid box for 7T9L was set to the XYZ coordinates as 214.866, 163.360, and 254.26 respectively with the centres of XYZ in the positions of 86.869, 84.09, and 122.748, respectively. The docking of the cocrystallized ligand S58 (1-Phenylsulfonamide-3-trifluoromethyl-5parabromophenylpyrazole) bound to the COX-2 protein was redocked and the interaction of the ligand with the protein is validated along with the docking procedure. S58 formed three hydrogen bonds along with nine hydrophobic interactions which matches with the PDBSum results for the S58-6COX complex.The grid box for the COX protein was set to the XYZ coordinates as 47.82, 24.69, and 36.44 respectively with the centres of XYZ in the Table 2 Computed bonding, non-bonding distances, bond angles and dihedrals for the molecules 1-8.     positions of 108.49, 77.37 and 77.47, respectively. The nine conformations of each compound along with the Vina score and RMSD value were used to screen for the best binding interaction. The 2D interaction diagrams generated from LigPlot + were used to analyze the ligand interactions and the 3D interactions were done using PyMOL [34][35][36][37] .

Chemistry
The literature suggests Embelia Ribes and their chemical constituent's embelin is known to inhibit NF-κB activity and COX-2 expression, which are related to various inflammation diseases like chronic inflammation, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and asthma. In light of this , embelin (2, 5-dihydroxy-3-undecyl-1,4-benzoquinone) subjected to react with different amines in acetic acid obtain compounds ( 3 and 4) in moderate to good yield.
The compound (  . The formation of molecular ion peak at m/z 407 clearly indicated the formation of compound 4 from embelin. Further, the compounds ( 5-6 ) were synthesized by using the known method by the reaction of embelin and primary amines in ethanol [2] . Also, cyclization followed by aromatization with 2-amino-3-hydroxy pyridine gives the target compound ( 7 ) having 73% yield. This cyclization was confirmed by the presence of four aromatic protons at δ7.517- 7.562 and mass spectra showing a molecular peak at 368. Finally, the reaction of embelin with acetic anhydride furnished diacetyl embelin ( 8 ) [11] . The physical data of the newly synthesized compound 8 is agreement with reported one ( Scheme-1 ).

DFT calculations
The DFT calculations were carried out for the molecules 1-8 using CAM-B3LYP hybrid functional [  proposed as a measure of lowering energy due to maximal electron flow between donor and acceptor. Finally, HOMO-LUMO ( ) gap establishes the correlation between chemical structure and biological activity.
Vibrational spectral assignments are done at B3LYP/6-31 + G(d,p). The structure shows the presence of a single N-H…O and O-H…O intra-molecular H-bonds. In all structures, the aromatic ring is attached to the long alkane chain, which increases the ring C-C bond distance, as shown in Fig. 3 and Table-1. The computed bond distances and stretching frequencies are in good corroboration with the experimental parameters that validates the method adopted here ( Table 3 ).The HOMO and LUMO energy values show the electron-donating and accepting ability of the molecules, respectively Fig. 4 ). Computed HOMO and LUMO Eigen functions indicate the MOs localized at the aromatic centre, which does not have any contribution from the longer alkane chain. Calculated HOMO and LUMO values range from -7.6 to -7.9 eV and -1.7 to -2.3 eV. The molecular electrostatic potential in Fig.  5 shows the charge separation between the two ends within the molecule. The relatively more minor HOMO-LUMO gaps, hardness values, and more considerable chemical reactivity make molecule c more reactive. The molecules' larger electronegativity ( χ) and Table 3 Computed and experimentally measure vibrational data for molecules 1-8.

Compounds
E HOMO E LUMO ɳ χ ω a The concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme, the result is the mean of two value obtained by assay of enzyme. b The in vitro COX-2 selectivity index (COX-1/COX-2).  Table 4 .

In Vitro cyclooxygenase inhibition assay
The in vitro COX-1/COX-2 enzyme inhibitory activities determine the aptitude of examined embelin hybrids to inhibit ovine COX-1 and human recombinant COX-2 using an enzyme im-  munoassay (EIA). The consequences were (

Molecular docking studies
The conformation that had the least binding affinity and most stability based on docking analysis was identified.

Docking study withSARS-CoV-2 Omicron virus variant (PDB ID:7T9L)
All the compounds 1-8 were taken for molecular docking study with SARS Cov-2 omicron variant and the standard drugs Celecoxib and Ibuprofen were used as reference.The conformation that had   10. The brief SAR study for synthesized embelin derivatives the least binding affinity and most stability based on docking analysis was identified. In silico analysis revealed that the compounds 4 and 8 showed the best binding interactions with SARS-CoV-2 Omicron virus variant. Compound 4 formed two hydrogen bonds with Tyr196 and Gly205 amino acids ( Fig. 6) . Compound 8 formed four hydrogen bonds with Ser43, Ser44 and Asp350 amino acids ( Fig. 7 ). Molecular docking results of the structures considered for docking with SARS-CoV-2 Omicron virus variant are depicted in the Table-S1 .

Docking study with cyclooxygenase-2 (COX2) (PDB ID:6COX)
All the compounds 1-8 were taken for molecular docking study with SARS Cov-2 omicron variant and the standard drugs Celecoxib and Ibuprofen were used as reference. Bioinformatics anal-ysis revealed that compounds 4 and 8 showed the best binding interactions with cyclooxygenase-2 ( PDB ID: 6COX ). Ibuprofen was observed to be having hydrophobic interactions with the -heme edge of cyclooxygenase-2.The compound 4 with a Vina score of -7.9kcal/mol and with forms two hydrogen bonds with ASN39, and GLN461 amino acids. Further, compound 8 showed strong interaction with the COX compared to the reference drug ibuprofen with a Vina score of -8.2kcal/mol and 4 exhibited four hydrogen bonds with ASN34, PRO154, ASN39 and CYS47. It can be noted that all the ligands are having interaction with both the proteins. Based on the vina score and number of hydrogen bonds and hydrophobic interactions 2D and 3D interaction diagrams for compound 4 and compound 8 is generated and shown in the Fig. 8 and 9 respectively ( Table-S2 ).

Structure activity relationship studies
Embelin, a bioactive molecule, a combination of the hydrophilic-OH group, keto group, and hydrophobic aliphatic long chain, makes this molecule a perfect scaffold to develop drug candidates. In our previous study, we reported that the presence of OH, C = O, and an alkyl group in embelin is responsible for its strong binding to histone acetyl transferases, an enzyme involved in the post-translation modification referred to as histone acetylation. Replacement of the hydroxyl group by substituted amines and acetylation of the -OH function, the activity increases. Further, by the introduction of a heterocyclic system, activity increases. The brief SAR for synthesized compounds highlighted in Fig. 10 .

Conclusion
In conclusion, we have lucratively intended and produced embelin hybrids in single-step reactions. The probable pharmacokinetic and pharmacodynamic properties of all the synthesized compounds were performed. The DFT calculations are used to compare the structure of the synthesized molecules. The antiinflammatory activity studies of all the compounds ( 1-8 ) were carried out. In vitro, cyclooxygenase inhibition testing found that compounds 3 , 4 , 7 , and 8 are more effective inhibitors of COX-1 and COX-2 enzymes. Molecular docking study accessible the possible interactions between the synthesized compounds at the active situate of cyclooxygenase. The outcome is that some of the synthesized compounds bind with an enzyme in a very similar way to standard drugs. The imminent results in the current investigation could be obliged for the rising novel loom in developing embelincontaining anti-inflammatory drugs. Finally, from the SAR study, it has been suggested that acetyl and amine derivatives of embelin may be an excellent lead for further COX inhibition studies.

Author credit statement
All the authors have contributed and extended their support for making this manuscript to communicate for publications.

Declaration of Competing Interest
The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Data will be made available on request.