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

Abstract

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.

Graphical Abstract

1. Introduction

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

Fig. 1.

Various NSAIDs drugs available in the market.

The plant Embelia ribes commonly known as a false black pepper used for the treatment to fever, inflammation, and gastrointestinal disorders. Further, a bioactive chemical constituent, embelin, has been known for anti-cancer, antimalarial, antimicrobial, and anti-inflammatory activity [2], [3], [4], [5] (Fig. 2 ).

Fig. 2.

Embelin and its important analogs reported in the literature.

This cell-permeable molecule reduces oxidative stress and inflammation by blocking NF-κB pathways compared to standard anti-inflammatory agents [6], [7], [8]. Knowing the importance of the anti-inflammatory potential of embelin, in 2014, Mahendran et al. reported the synthesis of embelin analogs as anti-inflammatory agents [9]. Later, Bezu et al. reported phenazine derivative of embelin as an analgesic and anti-inflammatory agent [10]. Further, Vault et al. reported the synthesis and anti-inflammatory activity of mono, di-acetyl embelin prepared in one step. Their result suggests that these compounds exhibited significant X-linked inhibitors of apoptosis protein (which plays a critical role in cell death and inflammation) inhibition and apoptosis in various cancer cell lines, which is probably independent of inhibition [11,12]. In view of these findings and with the continuation of our research work on biologically active molecules [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], we herein were reporting the synthesis of embelin synthetic hybrids as cyclooxygenase inhibitors and molecular docking studies with SARS-CoV-2 omicron protease & cyclooxygenase enzyme. Initially, embelin was isolated from Embelia ribes fruit by our early reported method. After purification, embelin on treatment with different aliphatic and heterocyclic amines in acidic condition produce targeted compounds (compounds 2, 3, 4, and 8) in good yield (Scheme-1 ). The compounds 5, 6, and 8 were prepared using reported methods [2,11]. All compounds were purified by column chromatography and characterized by spectral techniques such as IR, NMR, and Mass spectra.

Scheme 1.

Synthesis of embelin derivatives.

2. Materials and methods

2.1. Instrumentation

On a Perkin-Elmer FT-IR spectrophotometer (Spectrum ONE), (v _max in per centimetre, KBr) were documented, ¹H (400 MHz) and ¹³C NMR (100 MHz) spectra were documented on a Bruker AMX spectrophotometer using DMSO-d₆ 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 4000 amu in quadruple and 20,000 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.

2.2. Experimental

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

2.2.1. 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 (100-200 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 ¹H,¹³C NMR and ESI-MS.

2.2.2. Spectral data of embelin (1)

Orange crystals, m. p. 144-145°C, IR (KBr) cm⁻¹: 3320 (OH), 1660 (C=O); ¹H NMR (400 MHz, CDCl₃) d: 5.221 (s, 1H, H-6), 2.293 (t, 2H, H- 1′), 1.22-1.30 (m, 18H, H-2′, H-3′ to 10′), 0.83 (t, 3H, H-11′). ¹³C NMR (100 MHz, CDCl₃) 14.15 (C-11′), 22.04 to 40.16 (C-1′ to C10′), 99.23 (C-6), 110.01 (C-3), 178.81 (C-2), 181.35 (C-5), 206.37 (C=O). ESI-MS: m/z 295 [M+H]⁺.

2.2.3. General procedure for synthesis of embelin amine derivatives(2-4)

Embelin (0.4 mmol) in acetic acid (15 ml) and substituted amines (0.5 mmol) refluxed for 4 hr. Pour the reaction mixture in ice cold water, separated solid was filtered and dried to get the product. The crude product was purified through column chromatography using mobile phase hexane: ethyl acetate (8:2).

2.2.4. 2-Hydroxy-5-(methylamino)-3-undecylcyclohexa-2,5-diene-1,4-dione (2)

Yield: 53%.Brown color solid. m. p.110-111°C_. IR (KBr) cm⁻¹: 3330 (NH), 1675 (C=O).¹H NMR (400 MHz, DMSO): 7.8 (s, 1H, -OH), 5.2 (s, 1H, H-6), 2.74 (s, 3H, NCH₃), 2.25 (t, 2H, H- 1′), 2.17 (s, 1H, NH), 1.32 (m, 2H, H-2′), 1.226-1.15 (m, 16H, H-3′ to 10′), 0.831 (t, 3H, H-11′); MS (MALDI-ToF, CHCA): m/z 308.09 [M+H]⁺, calc. 307.27) [2].

2.2.5. 5-(2-(Dimethylamino)ethylamino)-2-hydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione (3)

Yield: 71%. Brown crystals, m. p. 91-92°C, IR (KBr) cm⁻¹: 3321 (NH), 1694 (C=O); ¹H NMR (400 MHz, DMSO): 6.99 (s, 1H, -OH), 5.33 (s, 1H, H-6), 3.21 (t, 2H, -NHCH₂), 2.63 (t, 2H, -CH₂N(CH₃)₂), 2.40 (t, 2H, H-1′), 2.30 (s, 6H, -N(CH₃)₂), 2.18 (s, 1H, NH), 1.44-1.25 (m, 16H, H-2′ to 10′), 0.85 (t, 3H, H-11′).¹³C NMR (100 MHzCDCl₃) 14.18 (C-11′), 22.93 to 40.78 (C-1′ to C10′), 44.32 (NCH2), 47.67 (N(CH₃)₂), 62.47 (N(CH3)₂ CH2), 102.33 (C-6), 116.21 (C-3), 176.91 (C-2), 181.35 (C-5), 204.81 (C=O). ESI MS: m/z 365 [M+1].Elemental analysis: Calcd for C₂₁H₃₆N₂O3: C, 69.19: H, 9.95; N, 7.68. Found: C, 69.14: H, 9.93; N, 7.65.

2.2.6. 2-Hydroxy-5-(4-(2-aminoethyl)morpholino)-3-undecylcyclohexa-2,5-diene-1,4-dione (4)

Yield: 69%. Brownish solid, m. p. 120-121°C, IR (KBr) cm⁻¹: 3390 (OH), 1678 (C=O); ¹H NMR (400 MHz, DMSO): 7.40 (s, 1H, -OH), 5.29 (s, 1H, H-6), 4.03 (t, 4H, OCH₂), 2.54 (t, 2H, ring NCH₂), 2.49 (t, 2H, NCH₂), 2.31 (t, 2H, NCH₂), 2.29 (t, 2H, H-1′), 2.17 (s, 1H, NH), 1.38-1.15 (m, 18H, H-2′, H-3′ to 10′), 0.832 (t, 3H, H-11′);¹³C NMR (100 MHz, CDCl₃) 14.1 (C-11′), 22.74 to 40.38 (C-1′ to C10′), 44.90 (NCH₂ {attached to morpholine}), 55.62 (NCH₂{attached to quinone}), 56.82 (ring NCH₂), 67.72 (ring OCH₂), 103.23 (C-6), 117.01 (C-3), 174.91 (C-2), 180.35 (C-5), 205.31 (C=O). MS (MALDI-ToF, CHCA): m/z 407.21 [M+H]⁺.Elemental analysis: Calcd for C₂₃H₃₈N₂O₄: C, 67.95: H, 9.42; N, 6.89. Found: C, 67.93, H, 9.40; N, 6.86.

2.2.7. Synthesis of (3E, 6E)-3,6-bis(alkylimino)-2-undecylcyclohexa-1,4-diene-1,4-diol (5-6)[2]

To a mixture of embelin (0.34 mmol)) and amines (0.68 mmol) added 10 ml ethanol refluxed for 2 hr. Evaporation of excess ethanol gives crude residue, which is further purified by column purification to obtain the desired compound using mobile phase hexane and ethyl acetate(7:3).

2.2.8. (3E, 6E)-3,6-bis(Methylimino)-2-undecylcyclohexa-1,4-diene-1,4-diol (5)

Yield: 83%.Violet solid, m. p. 89-90°C, IR (KBr) cm⁻¹: 3310 (OH). ¹H NMR (400 MHz, DMSO): 8.9 (s, 2H, -OH), 5.23 (s, 1H, H-6), 2.98 (s, 3H, NCH₃), 2.95 (s, 3H, NCH₃), 2.21 (t, 2H, H-1′), 1.26-1.21 (m, 18 H, H-2′, H-3′ to 10′), 0.835 (t, 3H, H-11′); MS (MALDI-ToF, CHCA): m/z 321.14 [M+H]⁺).

2.2.9. (3E, 6E)-3,6-bis(Ethylimino)-2-undecylcyclohexa-1,4-diene-1,4-diol (6)

Yield: 81%. Purple crystals, m. p. 78-79°C, ¹H NMR (400 MHz, DMSO): 7.8 (s, 2H, OH), 5.2 (s, 1H, H-6), 2.74 (q, 3H, NCH₂C), 2.71 (t, 3H, NCCH₃), 2.20 (t, 2H, H-1′), 1.33 (m, 2H, H-2′), 1.28-1.12 (m, 16H, H-3′ to 10′), 0.82 (t, 3H, H-11′); MS (MALDI-ToF, CHCA): m/z 349.09 [M+H]⁺.Elemental analysis: Calcd for C₂₁H₃₆N₂O₂: C, 72.37, H, 10.41; N, 8.04. Found: C, 72.35, H, 10.36; N, 8.01.

2.2.10. Procedure for synthesis of 8-hydroxy-6-undecyl-7H-benzo[b]pyrido[2,3-e][1,4]oxazin-7-one oxazine (7)

Embelin (0.68 mmol) and 2-amino-3-hydroxypyridine (0.68 mmol) in acetic acid (10 ml) refluxed for 4 hr. Pour the reaction mixture in to ice water. Solid separated was dried and purified with column chromatography using hexane and ethyl acetate as a mobile phase (8:2).

Brown solid,Yield-73 %, m. p. 132-134°C, IR (KBr) cm⁻¹: 3390(OH), 1678 (C=O); ¹H-NMR (400 MHz, DMSO): 7.51-7.56 (m, 4H, pyridine-CH, OH), 6.62 (s, 1H, H6), 5.328 (s, 1H, H-6), 2.67 (t, 2H, H- 1′), 1.48-1.15 (m, 16H, H-2′ to 10′), 0.83 (t, 3H, H-11′).¹³C NMR (100 MHz, CDCl₃) 14.21 (C-11′), 22.34 to 40.11 (C-1′ to C10′), 103.55 (C-6), 115.12 (C-3), 121.4, 134.05, 140.05 (Pyridine-C), 149.3 (C=N oxazine), 159.5 (C=N Pyridine), 173.95 (C-2), 181.11 (C-5), 206.81 (C=O).MS (MALDI-ToF): m/z -368, 369[M+H]⁺, 390 [M+Na]⁺.Elemental analysis: Calcd for C₂₂H2₈N₂O₃: C, 71.71; H, 7.66; N, 7.60. Found: C, 71.69, H, 7.62; N, 7.56.

2.2.11. 3, 6-Dioxo-2-undecylcyclohexa-1,4-diene-1,4-diyl diacetate (8)

A solution of embelin (0.34 mmol) and acetic anhydride (10 ml) refluxed for 1 hour. Cool the reaction mixture, pour in to ice water. Solid separated was dried and recrystallize from ethanol to obtain diacetyl embelin [11].Yield: 72%. Pale yellow solid, m. p. 90-91°C, IR (KBr) cm⁻¹: 1659 (C=O); 1171 (C-O-C).

2.3. 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.

Table 2.

Computed bonding, non-bonding distances, bond angles and dihedrals for the molecules 1-8.

	1		2	3	4	5	6	7	8
Bonding and Non-bonding distances (indicated in*), Å
C1-C2 C1-C1′ C1′- H1′	1.45 1.50 1.09		1.45 1.50 1.09	1.45 1.50 1.09	1.45 1.50 1.09	1.35 1.50 1.01	1.48 1.50 1.91	1.35 1.49 1.09	1.48 1.49 1.09
C2-C3 C2-O2′ O2′-H3′* C2-N2′ N2′-C2′ C2-O2a O2a- C2b C2b-C2c C2c-H2c' C2c-C2d C2d-H2d' C2d-C2e C2e-H2h' C2e-N2f N2f-C2g C2g- N2h N2h-C3	1.50 1.22 1.99 - - - - - - - - - - - - - -		1.51 1.21 2.15 - - - - - - - - - - - - - -	1.51 1.21 2.14 - - - - - - - - - - - - - -	1.51 1.21 2.13 - - - - - - - - - - - - - -	1.48 - - 1.35 1.45 - - - - - - - - - - - -	1.48 - - 1.27 1.45 - - - - - - - - - - - -	1.48 - - - - 1.35 1.36 1.38 1.08 1.38 1.08 1.39 1.08 1.32 1.33 1.38 1.29	1.49 1.20 - - - - - - – - - - - - - -
C3-C4 C3-O3′ O3′- H3′* C3-N3′ N3′-H3′* N3′-C3′ O3′- C3′ C3′- O3′'	1.34 1.32 0.97 - - - - -		1.36 - - 1.34 1.01 1.44 - -	1.35 - - 1.34 1.01 1.44 - -	1.35 - - 1.34 1.01 1.44 - -	1.34 1.35 0.96 - - - - -	1.33 1.36 0.96 - - - - -	1.44 - - - - - - -	1.32 1.36 - - - - 1.37 1.19
C4-C5 C4-H4′	1.45 1.08		1.44 1.08	1.44 1.08	1.44 1.08	1.45 1.08	1.46 1.08	1.34 1.08	1.47 1.08
C5-C6 C5-O5′ C5-N5′ N5′-C5′ C5-O5′ O5′-H5′	1.51 1.21 - - - -		1.52 1.21 - - - -	1.51 1.21 - - - -	1.51 1.21 - - - -	1.48 - 1.27 1.45 - -	1.48 - 1.27 1.45 - -	1.49 - - - 1.33 0.97	1.49 1.21 - - - -
C6-O6′ O6′-H6′ O6′-C6′ C6′- O6′'	1.33 0.96 - -		1.33 0.96 - -	1.33 0.96 - -	1.33 0.96 - -	1.34 0.96 - -	1.36 0.96 - -	1.22 - - -	1.37 1.37 1.19 -
BondAngles in º
C1C2C3 C2C3C4 C3C4C5 C4C5C6 C5C6C1 C6C1C2	119.66 121.90 119.88 117.54 123.16 117.84		119.11 120.81 121.09 117.18 123.24 118.56	119.11 120.78 121.10 117.18 123.25 118.55	119.17 120.77 121.07 117.20 123.23 118.53	115.74 122.11 122.43 115.74 122.81 121.11	115.51 121.62 123.71 114.70 122.79 121.55	123.93 117.99 119.63 121.96 119.02 117.42	118.16 122.19 120.24 117.33 123.59 118.29
C3O3′H3′ C6O6H6′ C3N3′H3′ C3N3′H3′ C2N2′C2′ C5O5′C5′ C4C3N2h O6′C6C1 C1C2O2a C4C5O5′ C3O3′C3′ C6O6C6′	106.31 110.27 - - - - - - - - - -		- 110.07 115.29 - - - - - - - - -	- 110.04 115.13 - - - - - - - - -	114.95 110.08 - - - - - - - - - -	109.64 109.28 - - - 126.11 119.84 - - - - -	109.29 109.42 126.18 125.65 - - - - - - - -	- - - - - - 119.37 124.02 118.56 123.99 - -	- - - - - - - - - - 116.98 116.95
Dihedrals in º
C1′C1C2O2′ C1′C1C6O6′ C1C6O6′H6′ C2C3O3′H3′ C2C3N3′H3′ C1′C1C2N2′ C1′C1C6O6′ C1C6O6′H6′ C2C3O3′H3′ O6′C6C5N5′ C1′C1C202a C2C3N2HC2 O6′ C6 C5O5′ C1C6O6′C6′ C2C3O3′C3′		1.13 -1.04 -0.70 -0.09 - - - - - - - -	0.78 -0.92 -0.81 - -0.26 - - - - - - - - - -	0.41 -0.97 -0.94 - -1.37 - - - - - - - - - -	1.65 -1.07 -0.76 - 3.12 - - - - - - - - - -	- - - - - -0.67 -0.56 -0.81 -179.8 -0.30 – - - - -	0.09 -0.42 -0.42 179.89 -1.06 - - - - - - - - - -	- - - - - - 0.81 1.09 0.03 0.16 - - - - -	6.38 2.69 - - - - - - - - - - - 117.34 65.81

Table. 1.

Physiochemical parameters of compounds 1-8 predicted by SwissADME.

Parameters*	1	2	3	4	5	6	7	8
MW	294.39	307.43	364.52	406.56	320.47	348.52	368.47	378.46
NHA	21	22	26	29	23	25	27	27
NAHA	0	0	0	0	0	0	14	0
NRB	10	11	14	14	10	12	10	14
NHBA	4	3	4	5	4	4	5	6
NHBD	2	2	2	2	2	2	1	0
MR	84.31	90.35	107.67	120.17	100.9	110.51	109.75	102.97
TPSA	74.6	66.4	69.64	78.87	65.18	65.18	76.22	86.74
iLOGp	3.28	3.59	4.31	4.59	4.33	4.75	4.04	4.23
Log S (ESOL)	-4.42	-4.65	-4.83	-4.85	-4.81	-5.23	-5.8	-4.82
MLOGP	1.21	1.44	1.3	0.92	1.68	2.13	2.36	1.94
GI	High	High	High	High	High	High	High	High
BBBP	Yes	Yes	Yes	Yes	No	No	No	No
vLROF	0	0	0	0	0	0	0	0
vGR	0	0	0	0	0	1	0	0
vVR	0	1	1	1	0	1	0	1
BS	0.85	0.85	0.55	0.55	0.55	0.55	0.55	0.56
SA	3.66	3.88	4.3	4.42	4.36	4.67	4.02	4.11

^⁎MW: Molecular weight; NHA: Num. heavy atoms; NAHA: Number of aromatic heavy atoms; NRB: Num. rotatable bonds; NHBA: Num. H-bond acceptors; NHBD: Num. H-bond donors; MR: Molar Refractivity; TPSA:Topological Polar Surface Area; Log S: Solubility class; MLOGP: Moriguchi octanol-water partition coefficient: GI: Gastrointestinal absorption; BBBP: Blood Brain Barrier Penetration; vLROF: Violation ofLipinski's rule of five; vGR: Violation of Ghose rule; vVR: Violation of Veber rule; BS: BioavailabilityScore; SA: Synthetic accessibility.

2.4. 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 anti-inflammatory 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.

2.4.1. 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 (IC₅₀ value, μM) was determined using an enzyme immuno assay (EIA) kit according to the reported method [32].

2.5. 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 co-crystallized ligand S58 (1-Phenylsulfonamide-3-trifluoromethyl-5-parabromophenylpyrazole) 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 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].

3. Results and discussion

3.1. 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 (3) formation was confirmed by the new peaks in 1H NMR at δ 2.637-2.364 (t, CH₂, NCH₂), 2.402 (t, 2H, H- 1′), 2.303 (s, 6H, N(CH₃)₂), and 2.170 (s, 1H, NH). Further, supported by the presence of a molecular peak of 365 (M+1) in its mass spectra. Compound (4) confirmed by ¹H NMR showing peaks at δ 4.032 (t, 2H OCH₂), 3.582 (t, 2H OCH₂), 3.163 (t, 2H, ring NCH₂), 2.54 (t, 2H, ring NCH₂), 2.49 (t, 2H, NCH₂), and 2.312 (t, 2H, NCH₂). 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).

3.2. DFT calculations

The DFT calculations were carried out for the molecules 1-8 using CAM-B3LYP hybrid functional [27a]with 6-31+g(d) all-electron basis set using the Gaussian 09 package. Second-order harmonic vibrational calculations were performed to check the minimum nature of the geometry. The electronic properties chemical hardness [ɳ = (LUMO-HOMO)/2], electronegativity [χ =-(HOMO+LUMO)/2], chemical potential [μ = (H+L)/2], and electrophilicity index (ω = μ2/2ɳ) were calculated using the energies of Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO). Chemical hardness (ɳ) has been used to understand the molecular system's chemical reactivity [27b-f]. The concept of electronegativity (χ) explains the molecular electron-accepting ability. Electrophilicity (ω) has been proposed as a measure of lowering energy due to maximal electron flow between donor and acceptor. Finally, HOMO-LUMO (Δ) 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 electrophilicity index (ω) values suggest the binding mechanism towards their receptor. The molecule can bind with the receptor by donating the electron. The steric effect of longer chains is studied by recalculating the molecular geometries and electronic structures without the longer chain. The absence of a side-chain maintains a similar trend as synthesized molecules. The higher alkane chain can act as a hydrophobic unit and might help drug delivery.

Fig. 3.

Optimized geometries of molecules 1-8.The complete structural parameters are given in the table 2.

Table 3.

Computed and experimentally measure vibrational data for molecules 1-8.

Molecules	Stretching Modes	Experimental Stretching frequencies in cm−1	Computed Stretching frequencies in cm−1
1	νC=O νO-H	1660 3320	1646 3438
2	νC=O νO-H	1675 3330	1689 3441
3	νC=O νN-H	1694 3321	1685 3391
4	νC=O νO-H	1678 3390	1671 3339
7	νC-O νO-H	1678 3390	1672 3334
8	νC=O νC-O	1659 1171	1698 1192

Fig. 4.

Computed molecular orbital plots for synthesized molecules (1-8) with the counter value of 0.02 Å⁻³.

Fig. 5.

Molecular electrostatic charge distribution plots for synthesized molecules (1-8) with an isovalue of 0.004. Å⁻³.

The vibrational frequencies of experimental and theoretical values are analogous as illustrated in the Table 3. In compound 1 the C=O stretching vibrations are 1660 and 1646 cm⁻¹ respectively and O-H stretching vibrations are appeared at 3320 and 3438 cm⁻¹ respectively. In compound 2 these vibrations are appeared at 1675 & 1689 cm⁻¹ and 3330 & 3441 cm⁻¹ respectively for CO and OH functions. In the compound 3 the vibrational frequencies for CO and OH functions appeared at 1694 & 1685 cm⁻¹ and 3321 & 3391 cm⁻¹ experimental and computational values respectively. The peaks at 1678 and 1671cm⁻¹ for obtained and calculated vibrations due to CO stretching respectively and the peaks at 3390 and 3339 cm⁻¹ due to for –OH functions for experimental and calculated values for compound 4. In the compound 7 the vibrational frequencies for CO and OH functions appeared at 1678 & 1671 cm⁻¹ and 3390 & 3334 cm⁻¹ obtained and calculated values respectively. In the compound 8 the vibrational frequencies for COC and CO functions appeared at 1171 & 1192 cm⁻¹ and 1659 & 1698 cm⁻¹ experimental and computational values respectively.

The calculated values of E_HOMO, E_LUMO, HOMO-LUMO gap (∆), chemical hardness (ɳ), electronegativity (χ) and electrophilicity index (ω) in eV for all the synthesized molecules are shown in Table 4 .

Table 4.

The E_HOMO, E_LUMO, HOMO-LUMO gap (∆), chemical hardness (ɳ), electronegativity (χ) and electrophilicity index (ω) in eV for all the synthesized molecules 1-8.

Compounds	EHOMO	ELUMO	∆	ɳ	χ	ω
1	-8.62	-2.27	6.35	3.17	5.44	5.01
2	-7.71	-1.77	5.93	2.97	9.45	3.87
3	-7.63	-1.73	5.89	2.95	4.68	3.71
4	-7.68	-1.79	5.89	2.95	4.74	3.81
5	-7.49	-1.23	6.26	3.13	4.36	3.04
6	-7.53	-1.14	6.39	3.19	3.76	2.94
7	-7.86	-2.32	5.54	2.77	5.09	4.68
8	-9.25	-2.47	6.77	3.38	5.86	5.08

3.3. 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 immunoassay (EIA). The consequences were (Table-5 ) exposed that the compounds 3, 4, 7 and 8 were showed admirable COX-1 inhibitory activity with IC₅₀ value 3.58, 3.18, 3.19 and 2.25μM respectively. While the compounds 3, 4, 7 and 8demonstrated excellent COX-2 inhibitory activities with IC₅₀ value 1.65, 1.54, 1.56, and 1.23μMrespectively. In the same condition standard drugs Celecoxib and Ibuprofen showed IC₅₀ 6.34 & 3.15μM for COX-1 and 0.56 μM & 1.28 μM for COX-2 respectively. Other compounds showed less to moderate cyclooxygenase inhibition activity as compared to standard drugs.

Table 5.

In vitro COX-1, COX-2 inhibition activity of embelin hybrids (1-8).

Compounds	COX-1 IC50 (μM)a	COX-2 IC50 (μM)a	COX-2 S. I.b
1	6.42	2.98	2.15
2	6.54	3.10	2.11
3	3.58	1.65	2.17
4	3.18	1.54	2.06
5	7.65	2.56	2.98
6	6.85	2.15	3.18
7	3.19	1.56	2.04
8	2.25	1.23	1.83
Celecoxib	6.34	0.56	11.32
Ibuprofen	3.15	1.28	2.46

^aThe 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. ^bThe in vitroCOX-2 selectivity index (COX-1/COX-2).

3.4. Molecular docking studies

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

3.4.1. 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 the least binding affinity and most stability based on docking analysis was identified. In silico analysis revealed that the compounds4 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.

Fig. 6.

Molecular docking interaction representation of ligand 4 with omicron. a) 3D representation ligand 4 (in stick) with omicron (molecular surface). b) 3D interaction of ligand 4 in stick and amino acids forming hydrogen bond and hydrophobic interaction in line representation. c) LigPlot 2D interaction representation of ligand 4 with protein.

Fig. 7.

Molecular docking interaction representation of ligand 8 with omicron. a) 3D representation ligand 8 (in stick) with omicron (molecular surface). b) 3D interaction of ligand 8 in stick and amino acids forming hydrogen bond and hydrophobic interaction in line representation. c) LigPlot 2D interaction representation of ligand 8 with protein.

3.4.2. 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 analysis revealed that compounds4 and 8showed 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).

Fig. 8.

Molecular docking interaction representation of ligand 4 with COX-2. a) 3D representation ligand 4 (in stick) with COX-2 (molecular surface). b) 3D interaction of ligand 4 in stick and amino acids forming hydrogen bond and hydrohphobic interaction in line representation. c) LigPlot 2D interaction representation of ligand 4 with protein.

Fig. 9.

Molecular docking interaction representation of ligand 8 with COX-2. a) 3D representation ligand 8 (in stick) with COX-2 (molecular surface). b) 3D interaction of ligand 8 in stick and amino acids forming hydrogen bond and hydrohphobic interaction in line representation. c) LigPlot 2D interaction representation of ligand 8 with protein.

4. 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 .

Fig. 10.

The brief SAR study for synthesized embelin derivatives

5. 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 anti-inflammatory 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 embelin-containing 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.

6. 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.