# Isolation andIn Silico Anti-COVID-19 Main Protease (Mpro) Activities of Flavonoids and a Sesquiterpene Lactone from Artemisia sublessingiana

Authors: Roza I. Jalmakhanbetova; Yerlan M. Suleimen; Masayoshi Oyama; Eslam B. Elkaeed; Ibrahim. H. Eissa; Raigul N. Suleimen; Ahmed M. Metwaly; Margarita Yu. Ishmuratova
Journal: Journal of Chemistry
 (2021)

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## Abstract

The emergence of the COVID-19 pandemic declared the huge need of humanity for new and effective antiviral drugs. The reported antimicrobial activities ofArtemisia sublessingiana encouraged us to investigate the ethanol extract of its aerial parts which led to the isolation of six flavonoids and a sesquiterpenoid. The structures of the isolated compounds were elucidated by EI-MS, 1D, and 2D NMR spectroscopic methods to be (1) eupatilin, (2) 3′,4′-dimethoxyluteolin, (3) 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone, (4) hispidulin, (5) apigenin, (6) velutin, and (7) sesquiterpene lactone 8α,14-dihydroxy-11,13-dihydromelampolide. The isolated compounds were in silico examined against the COVID-19 main protease (Mpro) enzyme. Compounds 1–6 exhibited promising binding modes showing free energies ranging from −6.39 to −6.81 (kcal/mol). The best binding energy was for compound 2. The obtained results give hope of finding a treatment for the COVID-19 pandemic.


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## Body

## 1. Introduction

COVID-19 is the pandemic caused by the new coronavirus strain SARS-CoV-2 (severe acute respiratory syndrome corona virus-2). The pandemic started in Wuhan, China, at the end of 2019 and spread all over the world [1]. By December 2020, COVID-19 infected more than 35 million patients and caused more than a million deaths according to the WHO [2]. Unfortunately, there is no accessible treatment for COVID-19 till now. The available treatment for infected patients is just symptomatic treatment by using anticoagulants, oxygen therapy, analgesics, and some research drugs [3]. Coronaviruses have caused serious diseases to humans before, such as Middle East respiratory syndrome (MERS-CoV) which appeared in 2012 and severe acute respiratory syndrome (SARS-CoV) in 2003 [4, 5].The proteases, especially main protease (Mpro), play a vital role in the life cycle of coronaviruses [6]. Mpro is a cysteine protease that is enrolled in the maturation cleavage events within the polyprotein’s precursors [7, 8]. This enzyme is crucial for the processing and translation of polyproteins from the viral RNA [9]. Accordingly, Mpro is a very essential element for the replication and transcription of coronaviruses, and the inhibiting of its activity would block viral replication [10]. Consequently, Mpro could be an interesting target to explore the efficacy of new drugs against the coronavirus.Now, there is an urgent need to find an effective drug against COVID-19. Since computational chemistry is a rapid and reliable screening method for bioactive compounds, there were many efforts to explore the effect of different ligands against COVID-19 [11–13].Natural secondary metabolites are a significant source for anti-infective agents since dawn of history. These metabolites are found in many sources in nature such as plants [14, 15], marines [16, 17], and microbes [18–20], and they could be assorted following their chemical structure into countless classes such as saponins [21, 22], diterpenes [23], pyrones [24], isochromenes [25], flavonoids [26, 27], and alkaloids [28].Artemisia L. (Asteraceae) species have been used in traditional medicine to treat various diseases and have shown several interesting pharmacological activities such as antimalarial, hepatoprotective, antioxidant, antibacterial, and cytotoxic activities [29–31]. Some flavonoids such as isorhamnetin-3-O-rutinoside and 5, 7, 4′-trihydroxy-6, 3′-dimethoxyflavone [32] have been isolated from Artemisia sublessingiana before. Furthermore, some sesquiterpene lactones like the eudesmanolide arsubin were also found in this species [33]. Secondary metabolites, such as flavonoids and sesquiterpenoids were reported to have diverse vital biological activities, such as antitumor [34], cytotoxic [35], antitrypanosomal [36], antibacterial, and anti-inflammatory activities [37].In this study, the main bioactive contents of the aerial parts ofA. sublessingiana (Krasch. ex Poljak.) Poljak. (Synonium Seriphidium sublessingianum (Krasch ex Poljakov)) have been investigated. This paper reports the isolation, structural determination, and in silico anti-COVID-19 main protease (Mpro) activities of six flavonoids and one sesquiterpene lactone from A. sublessingiana. Their structures were determined by spectrum analysis of 1D, 2D NMR, and ESI-MS data.


## 2. Materials and Methods

### 2.1. General Experimental Procedures

Column chromatography separations (CC) were performed on glass columns packed with silica gel (ASTM, 230–400 mesh, Merck, LTD, Japan). Thin-layer chromatography (analytical and preparative thin-layer chromatography (TLC) was performed on silica gel 60 F 254 glass plates (Merck, LTD, Japan). Spots were visualized under UV light (254 and 366 nm) and by spraying with alcoholic 10% H2SO4 reagent followed by heating. Isolated compounds were identified by 1D and 2D NMR analysis (1H 500 MHz, 13C 125 MHz), acquired on a Jeol Delta 2 NMR spectrometer at 297 K. Internal standards: TMS. Solvents: chloroform-d, acetone-d6, and DMSO-d6. Coupling constants are given in Hertz. The chemical shifts were expressed in δ ppm. Mass spectra (EIMS) were recorded on an IT-TOF-MS spectrometer.


### 2.2. Plant Material

The aerial parts ofA. sublessingiana were collected 90 km from Kyzylorda city, Kazakhstan (Kyzylkum sand desert). The material was authenticated by Professor M. Ishmuratova, Department of Botany, E.A. Buketov Karaganda University, Republic of Kazakhstan. A sample was deposited in the herbarium of the Faculty of Biology and Geography.


### 2.3. Extraction and Isolation

Air-dried powered aboveground parts ofA. sublessingiana (1.0 kg) were grounded and extracted with EtOH for 1 day. The extract was filtered, and the extraction process was repeated twice. The combined extracts were evaporated under reduced pressure to yield a crude extract of 93 g. The total crude extract was subjected to column chromatography over silica gel eluting with hexane and gradually increasing the polarity with acetone (up to 100%) and then MeOH. The fractions were studied on TLC and combined into twenty-three fractions (1F–23F). Compound (1) (216 mg) was separated from fraction 17F. Further chromatography of fraction 18F (2.5 g) on a column of silica gel with chloroform-acetone (in a manner of increasing polarity) and further chromatography of the obtained fractions 18F2 (0.1 g) with chloroform-methanol (in a manner of increasing polarity) gave compound (2) (5 mg). Fraction 18F4 (0.04 g) was dissolved in a solvent and repeatedly washed to give (3) (28 mg). Fraction 19F (2.91 g) was further fractionated on a silica gel column (60 g) eluting with chloroform-methanol (in a manner of increasing polarity) to obtain compounds (4) (19 mg) and (5) (15 mg). Fraction 14 F (0.65 g) was fractionated on a silica gel column eluting with chloroform-acetone (in a manner of increasing polarity) to give fraction (14F10). Fraction 14F10 (0.041 g) was further subjected to PTLC in system chloroform-methanol to obtain compound (6) (4 mg). Fraction 17F (2.93 g) was further purified by column chromatography on silica gel with chloroform-methanol (in a manner of increasing polarity) to give (7) (68.8 mg).


### 2.4. Compound Identification

5,7-Dihydroxy-6,3′,4′-trimethoxyflavone (eupatilin) (1): yellow crystals, C18H16O7. 1H NMR (DMSO, 500 MHz) δ (ppm): 13.05 (s, 5-OH), 10.73 (s, 7-OH), 6.98 (s, H-3), 6.65 (s, H-8), 7.57 (d, J = 2.5 Hz, H-2′), 7.12 (d, J = 8.5 Hz, H-5′), 7.68 (dd, J = 2.0, 8.5 Hz, H-6′), 3.76 (s, 6-OCH3), 3.88 (s, 3′-OCH3), 3.86 (s, 4′-OCH3); 1H NMR (CDCl3, 500 MHz) δ (ppm): 13.08 (s, 5-OH), 6.52 (s, 7-OH), 6.61 (s, H-3), 6.59 (s, H-8), 7.34 (brs, H-2′), 6.98 (d, J = 8.5 Hz, H-5′), 7.52 (brd, J = 8.5 Hz, H-6′), 4.05 (s, 6-OCH3), 3.99 (s, 3′-OCH3), 3.97 (s, 4′-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.38 (C-2), 103.37 (C-3), 182.21 (C-4), 152.74 (C-5), 131.35 (C-6), 157.32 (C-7), 94.38 (C-8), 152.43 (C-9), 104.13 (C-10), 122.92 (C-1′), 109.40 (C-2′), 149.00 (C-3′), 152.11 (C-4′), 111.67 (C-5′), 120.02 (C-6′), 59.97 (6-OCH3), 55.73 (3′-OCH3), 55.85 (4′-OCH3). HRESIMS: m/z 367.0780 [M +Na]+, C18H16O7Na, calc. 367.0788.5,7-Dihydroxy-3′,4′-dimethoxyflavone(3′,4′-dimethoxyluteolin) (2): yellow amorphous powder, C17H14O6. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.93 (s, 5-OH), 10.85 (s, 7-OH), 6.98 (s, H-3), 6.21 (d, J = 2.5 Hz, H-6), 6.54 (d, J = 1.8 Hz, H-8), 7.57 (d, J = 1.8 Hz, H-2′), 7.13 (d, J = 8.6 Hz, H-5′), 7.69 (dd, J = 1.8, 8.7 Hz, H-6′), 3.88 (s, 3′-OCH3), 3.91 (s, 4′-OCH3). HRESIMS: m/z 337.0686 [M + Na]+, C17H14O6Na, calc. 337.0683.5,7,3′-Trihydroxy-6,4′,5′-trimethoxyflavone (3): yellow powder, C18H16O8. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.99 (s, 5-OH), 10.70 (s, 7-OH), 9.60 (s, 3′-OH), 6.92 (s, H-3), 6.60 (s, H-8), 7.17 (dd, J = 1.5, 4.5 Hz, H-2′), 7.17 (dd, J = 1.5, 4.5 Hz, H-6′), 3.70 (s, 6-OCH3), 3.70 (s, 4′-OCH3), 3.80 (s, 5′-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.25 (C-2), 104.22 (C-3), 182.23 (C-4), 152.46 (C-5), 131.41 (C-6), 153.57 (C-7), 94.28 (C-8), 152.78 (C-9), 104.22 (C-10), 125.88 (C-1′), 102.12 (C-2′), 157.46 (C-3′), 139.63 (C-4′), 150.91 (C-5′), 107.68 (C-6′), 59.98 (6-OCH3), 60.08 (4′-OCH3), 56.15 (5′-OCH3). HRESIMS: m/z 383.0740 [M + Na]+, C18H16O8Na, calc. 383.0737.5,7,4′-Trihydroxy-6-methoxyflavone (hispidulin) (4): yellow powder, C16H12O6. 1H NMR (DMSO, 500 MHz) δ (ppm): 13.09 (s, 5-OH), 10.70 (brs, 7-OH), 10.40 (brs, 4′-OH), 6.79 (s, H-3), 6.60 (s, H-8), 7.93 (d, J = 8.3 Hz, H-2′), 6.93 (d, J = 8.3 Hz, H-3′), 6.93 (d, J = 8.3 Hz, H-5′), 7.93 (d, J = 8.3 Hz, H-6′), 3.74 (s, 6-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.80 (C-2), 152.40 (C-3), 182.14 (C-4), 152.78 (C-5), 131.34 (C-6), 157.26 (C-7), 94.23 (C-8), 152.40 (C-9), 104.07 (C-10), 121.21 (C-1′), 128.47 (C-2′), 115.96 (C-3′), 161.17 (C-4′), 115.96 (C-5′), 128.47 (C-6′), 59.95 (6-OCH3).5,7,4′-Trihydroxyflavone (apigenin) (5): yellow powder, C15H10O5. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.98 (s, 5-OH), 6.79 (s, H-3), 6.20 (d, J = 2.0 Hz, H-6), 6.49 (d, J = 2.5 Hz, H-8), 7.94 (d, J = 8.5 Hz, H-2′), 6.93 (d, J = 9.5 Hz, H-3′), 6.93 (d, J = 9.5 Hz, H-5′),7.94 (d, J = 8.5 Hz, H-6′). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.23 (C-2), 103.34 (C-3), 182.28 (C-4), 161.96 (C-5), 99.35 (C-6), 164.65 (C-7), 94.48 (C-8), 157.82 (C-9), 104.20 (C-10), 121.68 (C-1′), 129.02 (C-2′), 116.46 (C-3′), 161.69 (C-4′), 116.46 (C-5′), 129.02 (C-6′).5,4′-Dihydroxy-7,3′-dimethoxyflavone, luteolin 7,3′-dimethyl etherluteolin (velutin) (6): yellow powder, C17H14O6. 1H NMR (CDCl3, 500 MHz) δ (ppm): 12.81 (s, 5-OH), 6.04 (brs, 4′-OH), 6.58 (s, H-3), 6.38 (d, J = 2.0 Hz, H-6), 6.50 (d, J = 2.0 Hz, H-8), 7.34 (d, J = 2.0 Hz, H-2′), 7.04 (d, J = 8.3 Hz, H-5′), 7.50 (dd, J = 8.3, 2.0 Hz, H-6′), 3.89 (s, 7-OCH3), 4.01 (s, 3′-OCH3).8α,14-Dihydroxygermacra-1(10)E,4E-dien-6β,7α,11βH-12,6-olide (8α,14-dihydroxy-11,13-dihydromelampolide) (7): colorless crystals, C15H23O4. 1H NMR (CDCl, 500 MHz) δ (ppm): 5.49 (m, 2H, J = 7.0, 9.0, 16.0 Hz, H-1), 2.14 (m, H-2a), 1.89 (m, H-2b), 2.16 (m, H-3a), 1.91 (m, H-3b), 5.01 (brd, J = 10.0 Hz, H-5), 4.53 (t, J = 10.0 Hz, H-6), 2.12 (m, H-7), 3.90 (brs, H-8), 2.32 (brd, J = 15.0 Hz, H-9a), 2.25 (dd, J = 3.0, 15.0 Hz, H-9b), 2.57 (m, H-11), 1,43 (d, J = 6.0 Hz, H-13), 4.33 (brd, J = 12.0 Hz, H-14a), 4.13 (brd, J = 12.0 Hz, H-14b), 1.83 (brs, H-15), 2.84, 3.90 (brs, each, OH-3, 4). 13C NMR (CDCl, 500 MHz) δ (ppm): 128.73 (C-1), 25.22 (C-2), 38.05 (C-3), 138.58 (C-4), 124.60 (C-5), 77.26 (C-6), 55.17 (C-7), 73.75 (C-8), 35.15 (C-9), 139.02 (C-10), 41.73 (C-11), 179.45 (C-12), 16.41 (C-13), 69.22 (C-14), 17.24 (C-15).


### 2.5. Docking Studies Experiment

The crystal structure of the target enzymes COVID-19 main protease (Mpro) (PDB ID: 6lu7, resolution: 2.16 Å) was downloaded from Protein Data Bank (http://www.pdb.org). Molecular operating environment (MOE) was used for the docking analysis [38]. In these studies, the free energies and binding modes of the examined molecules against Mpro were determined. At first, the water molecules were removed from the crystal structure of Mpro, retaining only one chain which is essential for binding. The cocrystallized ligand (PRD-002214) was used as a reference ligand. Then, the protein structure was protonated, and the hydrogen atoms were hidden. Next, the energy was minimized, and the binding pocket of the protein was defined [39].The structures of the examined compounds and the cocrystallized ligand were drawn using ChemBioDraw Ultra 14.0 and saved in SDF format. Then, the saved file was opened using MOE software, and 3D structures were protonated. Next, the energy of the molecules was minimized. The validation process was performed for the target receptor by running the docking process for only the cocrystallized ligand. Low RMSD values between docked and crystal conformations indicate valid performance [40, 41]. The docking procedures were carried out utilizing a default protocol. In each case, 30 docked structures were generated using genetic algorithm searches. The output from MOE software was further analyzed and visualized using Discovery Studio 4.0 software [42, 43].


## 2.1. General Experimental Procedures

Column chromatography separations (CC) were performed on glass columns packed with silica gel (ASTM, 230–400 mesh, Merck, LTD, Japan). Thin-layer chromatography (analytical and preparative thin-layer chromatography (TLC) was performed on silica gel 60 F 254 glass plates (Merck, LTD, Japan). Spots were visualized under UV light (254 and 366 nm) and by spraying with alcoholic 10% H2SO4 reagent followed by heating. Isolated compounds were identified by 1D and 2D NMR analysis (1H 500 MHz, 13C 125 MHz), acquired on a Jeol Delta 2 NMR spectrometer at 297 K. Internal standards: TMS. Solvents: chloroform-d, acetone-d6, and DMSO-d6. Coupling constants are given in Hertz. The chemical shifts were expressed in δ ppm. Mass spectra (EIMS) were recorded on an IT-TOF-MS spectrometer.


## 2.2. Plant Material

The aerial parts ofA. sublessingiana were collected 90 km from Kyzylorda city, Kazakhstan (Kyzylkum sand desert). The material was authenticated by Professor M. Ishmuratova, Department of Botany, E.A. Buketov Karaganda University, Republic of Kazakhstan. A sample was deposited in the herbarium of the Faculty of Biology and Geography.


## 2.3. Extraction and Isolation

Air-dried powered aboveground parts ofA. sublessingiana (1.0 kg) were grounded and extracted with EtOH for 1 day. The extract was filtered, and the extraction process was repeated twice. The combined extracts were evaporated under reduced pressure to yield a crude extract of 93 g. The total crude extract was subjected to column chromatography over silica gel eluting with hexane and gradually increasing the polarity with acetone (up to 100%) and then MeOH. The fractions were studied on TLC and combined into twenty-three fractions (1F–23F). Compound (1) (216 mg) was separated from fraction 17F. Further chromatography of fraction 18F (2.5 g) on a column of silica gel with chloroform-acetone (in a manner of increasing polarity) and further chromatography of the obtained fractions 18F2 (0.1 g) with chloroform-methanol (in a manner of increasing polarity) gave compound (2) (5 mg). Fraction 18F4 (0.04 g) was dissolved in a solvent and repeatedly washed to give (3) (28 mg). Fraction 19F (2.91 g) was further fractionated on a silica gel column (60 g) eluting with chloroform-methanol (in a manner of increasing polarity) to obtain compounds (4) (19 mg) and (5) (15 mg). Fraction 14 F (0.65 g) was fractionated on a silica gel column eluting with chloroform-acetone (in a manner of increasing polarity) to give fraction (14F10). Fraction 14F10 (0.041 g) was further subjected to PTLC in system chloroform-methanol to obtain compound (6) (4 mg). Fraction 17F (2.93 g) was further purified by column chromatography on silica gel with chloroform-methanol (in a manner of increasing polarity) to give (7) (68.8 mg).


## 2.4. Compound Identification

5,7-Dihydroxy-6,3′,4′-trimethoxyflavone (eupatilin) (1): yellow crystals, C18H16O7. 1H NMR (DMSO, 500 MHz) δ (ppm): 13.05 (s, 5-OH), 10.73 (s, 7-OH), 6.98 (s, H-3), 6.65 (s, H-8), 7.57 (d, J = 2.5 Hz, H-2′), 7.12 (d, J = 8.5 Hz, H-5′), 7.68 (dd, J = 2.0, 8.5 Hz, H-6′), 3.76 (s, 6-OCH3), 3.88 (s, 3′-OCH3), 3.86 (s, 4′-OCH3); 1H NMR (CDCl3, 500 MHz) δ (ppm): 13.08 (s, 5-OH), 6.52 (s, 7-OH), 6.61 (s, H-3), 6.59 (s, H-8), 7.34 (brs, H-2′), 6.98 (d, J = 8.5 Hz, H-5′), 7.52 (brd, J = 8.5 Hz, H-6′), 4.05 (s, 6-OCH3), 3.99 (s, 3′-OCH3), 3.97 (s, 4′-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.38 (C-2), 103.37 (C-3), 182.21 (C-4), 152.74 (C-5), 131.35 (C-6), 157.32 (C-7), 94.38 (C-8), 152.43 (C-9), 104.13 (C-10), 122.92 (C-1′), 109.40 (C-2′), 149.00 (C-3′), 152.11 (C-4′), 111.67 (C-5′), 120.02 (C-6′), 59.97 (6-OCH3), 55.73 (3′-OCH3), 55.85 (4′-OCH3). HRESIMS: m/z 367.0780 [M +Na]+, C18H16O7Na, calc. 367.0788.5,7-Dihydroxy-3′,4′-dimethoxyflavone(3′,4′-dimethoxyluteolin) (2): yellow amorphous powder, C17H14O6. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.93 (s, 5-OH), 10.85 (s, 7-OH), 6.98 (s, H-3), 6.21 (d, J = 2.5 Hz, H-6), 6.54 (d, J = 1.8 Hz, H-8), 7.57 (d, J = 1.8 Hz, H-2′), 7.13 (d, J = 8.6 Hz, H-5′), 7.69 (dd, J = 1.8, 8.7 Hz, H-6′), 3.88 (s, 3′-OCH3), 3.91 (s, 4′-OCH3). HRESIMS: m/z 337.0686 [M + Na]+, C17H14O6Na, calc. 337.0683.5,7,3′-Trihydroxy-6,4′,5′-trimethoxyflavone (3): yellow powder, C18H16O8. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.99 (s, 5-OH), 10.70 (s, 7-OH), 9.60 (s, 3′-OH), 6.92 (s, H-3), 6.60 (s, H-8), 7.17 (dd, J = 1.5, 4.5 Hz, H-2′), 7.17 (dd, J = 1.5, 4.5 Hz, H-6′), 3.70 (s, 6-OCH3), 3.70 (s, 4′-OCH3), 3.80 (s, 5′-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.25 (C-2), 104.22 (C-3), 182.23 (C-4), 152.46 (C-5), 131.41 (C-6), 153.57 (C-7), 94.28 (C-8), 152.78 (C-9), 104.22 (C-10), 125.88 (C-1′), 102.12 (C-2′), 157.46 (C-3′), 139.63 (C-4′), 150.91 (C-5′), 107.68 (C-6′), 59.98 (6-OCH3), 60.08 (4′-OCH3), 56.15 (5′-OCH3). HRESIMS: m/z 383.0740 [M + Na]+, C18H16O8Na, calc. 383.0737.5,7,4′-Trihydroxy-6-methoxyflavone (hispidulin) (4): yellow powder, C16H12O6. 1H NMR (DMSO, 500 MHz) δ (ppm): 13.09 (s, 5-OH), 10.70 (brs, 7-OH), 10.40 (brs, 4′-OH), 6.79 (s, H-3), 6.60 (s, H-8), 7.93 (d, J = 8.3 Hz, H-2′), 6.93 (d, J = 8.3 Hz, H-3′), 6.93 (d, J = 8.3 Hz, H-5′), 7.93 (d, J = 8.3 Hz, H-6′), 3.74 (s, 6-OCH3). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.80 (C-2), 152.40 (C-3), 182.14 (C-4), 152.78 (C-5), 131.34 (C-6), 157.26 (C-7), 94.23 (C-8), 152.40 (C-9), 104.07 (C-10), 121.21 (C-1′), 128.47 (C-2′), 115.96 (C-3′), 161.17 (C-4′), 115.96 (C-5′), 128.47 (C-6′), 59.95 (6-OCH3).5,7,4′-Trihydroxyflavone (apigenin) (5): yellow powder, C15H10O5. 1H NMR (DMSO, 500 MHz) δ (ppm): 12.98 (s, 5-OH), 6.79 (s, H-3), 6.20 (d, J = 2.0 Hz, H-6), 6.49 (d, J = 2.5 Hz, H-8), 7.94 (d, J = 8.5 Hz, H-2′), 6.93 (d, J = 9.5 Hz, H-3′), 6.93 (d, J = 9.5 Hz, H-5′),7.94 (d, J = 8.5 Hz, H-6′). 13C NMR (DMSO, 500 MHz) δ (ppm): 163.23 (C-2), 103.34 (C-3), 182.28 (C-4), 161.96 (C-5), 99.35 (C-6), 164.65 (C-7), 94.48 (C-8), 157.82 (C-9), 104.20 (C-10), 121.68 (C-1′), 129.02 (C-2′), 116.46 (C-3′), 161.69 (C-4′), 116.46 (C-5′), 129.02 (C-6′).5,4′-Dihydroxy-7,3′-dimethoxyflavone, luteolin 7,3′-dimethyl etherluteolin (velutin) (6): yellow powder, C17H14O6. 1H NMR (CDCl3, 500 MHz) δ (ppm): 12.81 (s, 5-OH), 6.04 (brs, 4′-OH), 6.58 (s, H-3), 6.38 (d, J = 2.0 Hz, H-6), 6.50 (d, J = 2.0 Hz, H-8), 7.34 (d, J = 2.0 Hz, H-2′), 7.04 (d, J = 8.3 Hz, H-5′), 7.50 (dd, J = 8.3, 2.0 Hz, H-6′), 3.89 (s, 7-OCH3), 4.01 (s, 3′-OCH3).8α,14-Dihydroxygermacra-1(10)E,4E-dien-6β,7α,11βH-12,6-olide (8α,14-dihydroxy-11,13-dihydromelampolide) (7): colorless crystals, C15H23O4. 1H NMR (CDCl, 500 MHz) δ (ppm): 5.49 (m, 2H, J = 7.0, 9.0, 16.0 Hz, H-1), 2.14 (m, H-2a), 1.89 (m, H-2b), 2.16 (m, H-3a), 1.91 (m, H-3b), 5.01 (brd, J = 10.0 Hz, H-5), 4.53 (t, J = 10.0 Hz, H-6), 2.12 (m, H-7), 3.90 (brs, H-8), 2.32 (brd, J = 15.0 Hz, H-9a), 2.25 (dd, J = 3.0, 15.0 Hz, H-9b), 2.57 (m, H-11), 1,43 (d, J = 6.0 Hz, H-13), 4.33 (brd, J = 12.0 Hz, H-14a), 4.13 (brd, J = 12.0 Hz, H-14b), 1.83 (brs, H-15), 2.84, 3.90 (brs, each, OH-3, 4). 13C NMR (CDCl, 500 MHz) δ (ppm): 128.73 (C-1), 25.22 (C-2), 38.05 (C-3), 138.58 (C-4), 124.60 (C-5), 77.26 (C-6), 55.17 (C-7), 73.75 (C-8), 35.15 (C-9), 139.02 (C-10), 41.73 (C-11), 179.45 (C-12), 16.41 (C-13), 69.22 (C-14), 17.24 (C-15).


## 2.5. Docking Studies Experiment

The crystal structure of the target enzymes COVID-19 main protease (Mpro) (PDB ID: 6lu7, resolution: 2.16 Å) was downloaded from Protein Data Bank (http://www.pdb.org). Molecular operating environment (MOE) was used for the docking analysis [38]. In these studies, the free energies and binding modes of the examined molecules against Mpro were determined. At first, the water molecules were removed from the crystal structure of Mpro, retaining only one chain which is essential for binding. The cocrystallized ligand (PRD-002214) was used as a reference ligand. Then, the protein structure was protonated, and the hydrogen atoms were hidden. Next, the energy was minimized, and the binding pocket of the protein was defined [39].The structures of the examined compounds and the cocrystallized ligand were drawn using ChemBioDraw Ultra 14.0 and saved in SDF format. Then, the saved file was opened using MOE software, and 3D structures were protonated. Next, the energy of the molecules was minimized. The validation process was performed for the target receptor by running the docking process for only the cocrystallized ligand. Low RMSD values between docked and crystal conformations indicate valid performance [40, 41]. The docking procedures were carried out utilizing a default protocol. In each case, 30 docked structures were generated using genetic algorithm searches. The output from MOE software was further analyzed and visualized using Discovery Studio 4.0 software [42, 43].


## 3. Results and Discussion

### 3.1. Compounds Isolation

Using different chromatographic techniques, seven compounds have been isolated from the ethanol extracts of the aerial parts ofA. sublessingiana. The obtained compounds were identified using different 1D and 2D NMR spectroscopic methods to be (1) eupatilin [44, 45], (2) 3′,4′-dimethoxyluteolin [46–48], (3) 5, 7, 3′-trihydroxy-6,4′,5′-trimethoxyflavone [49], (4) hispidulin [50, 51], (5) apigenin [52, 53], (6) velutin [54], and (7) sesquiterpene lactone 8α,14-dihydroxy-11,13- dihydromelampolide [55–57]. The chemical structures of compounds (1–7) were confirmed by comparison of the reported spectral data in the literature (Figure 1).Figure 1
Chemical structures of the isolated compounds.
(a)(b)(c)(d)(e)(f)(g)


### 3.2. Docking Studies

Docking studies were carried out for compounds (1–7) against the COVID-19 main protease (Mpro) (PDB ID: 6lu7, resolution: 2.16 Å) to examine the mode of binding with the proposed target. The cocrystallized ligand (PRD-002214) was used as a reference molecule. The results of docking studies revealed that the docked compounds have good binding affinities against COVID-19 main protease with binding free energies ranging from −4.94 to −6.81 kcal/mol (Table 1).Table 1
The docking binding free energies of seven compounds, simeprevir, and the cocrystallized ligand (PRD-002214) against COVID-19 main protease.
CompoundBinding free energy (kcal/mol)1−6.532−6.813−6.514−6.445−6.396−6.557−4,94Cocrystallized ligand (PRD-002214)−7.83The crystallized ligand (PRD-002214) showed binding energy of −7.83 kcal/mol. The detailed binding mode of the crystallized ligand was as follows: making three hydrogen bonds with Phe140, His163, and Glu166, the 2-oxopyrrolidin-3-yl moiety occupied the first pocket of the enzyme. Additionally, tert-butyl carbamate moiety occupied the second pocket of Mpro. Furthermore, the phenyl ring of phenylalanine moiety occupied the third pocket of the receptor, forming hydrophobic interaction with His41. Finally, ethyl propionate moiety was incorporated in the fourth pocket (Figures 2–4).Figure 2
Cocrystallized ligand (PRD-002214) docked into the active site of the COVID-19 main protease. The hydrogen bonds are represented in green dashed lines, and the hydrophobic interactions are represented in orange dashed lines.Figure 3
Mapping surface showing the cocrystallized ligand (PRD-002214) occupying the active pocket of the COVID-19 main protease.Figure 4
2D interaction of the cocrystallized ligand (PRD-002214) in the active site of the COVID-19 main protease.Compound (2) showed the best binding mode and highest binding energy of −6.81 kcal/mol. The 7-hydroxy-6-methoxy-4H-chromen-4-one moiety occupied the first pocket of Mpro, forming three hydrogen bonds with Phe140 and His163. Also, it formed one hydrophobic interaction with His163. Additionally, 1,2-dimethoxybenzene moiety occupied the second pocket of Mpro forming two hydrophobic interactions with Met165 and His41 (Figures 5–7).Figure 5
Compound (2) docked into the active site of the COVID-19 main protease. The hydrogen bonds are represented in green dashed lines, and the hydrophobic interactions are represented in orange dashed lines.Figure 6
Mapping surface showing compound (2) occupying the active pocket of the COVID-19 main protease.Figure 7
2D interaction of compound (2) in the active site of the COVID-19 main protease.


## 3.1. Compounds Isolation

Using different chromatographic techniques, seven compounds have been isolated from the ethanol extracts of the aerial parts ofA. sublessingiana. The obtained compounds were identified using different 1D and 2D NMR spectroscopic methods to be (1) eupatilin [44, 45], (2) 3′,4′-dimethoxyluteolin [46–48], (3) 5, 7, 3′-trihydroxy-6,4′,5′-trimethoxyflavone [49], (4) hispidulin [50, 51], (5) apigenin [52, 53], (6) velutin [54], and (7) sesquiterpene lactone 8α,14-dihydroxy-11,13- dihydromelampolide [55–57]. The chemical structures of compounds (1–7) were confirmed by comparison of the reported spectral data in the literature (Figure 1).Figure 1
Chemical structures of the isolated compounds.
(a)(b)(c)(d)(e)(f)(g)


## 3.2. Docking Studies

Docking studies were carried out for compounds (1–7) against the COVID-19 main protease (Mpro) (PDB ID: 6lu7, resolution: 2.16 Å) to examine the mode of binding with the proposed target. The cocrystallized ligand (PRD-002214) was used as a reference molecule. The results of docking studies revealed that the docked compounds have good binding affinities against COVID-19 main protease with binding free energies ranging from −4.94 to −6.81 kcal/mol (Table 1).Table 1
The docking binding free energies of seven compounds, simeprevir, and the cocrystallized ligand (PRD-002214) against COVID-19 main protease.
CompoundBinding free energy (kcal/mol)1−6.532−6.813−6.514−6.445−6.396−6.557−4,94Cocrystallized ligand (PRD-002214)−7.83The crystallized ligand (PRD-002214) showed binding energy of −7.83 kcal/mol. The detailed binding mode of the crystallized ligand was as follows: making three hydrogen bonds with Phe140, His163, and Glu166, the 2-oxopyrrolidin-3-yl moiety occupied the first pocket of the enzyme. Additionally, tert-butyl carbamate moiety occupied the second pocket of Mpro. Furthermore, the phenyl ring of phenylalanine moiety occupied the third pocket of the receptor, forming hydrophobic interaction with His41. Finally, ethyl propionate moiety was incorporated in the fourth pocket (Figures 2–4).Figure 2
Cocrystallized ligand (PRD-002214) docked into the active site of the COVID-19 main protease. The hydrogen bonds are represented in green dashed lines, and the hydrophobic interactions are represented in orange dashed lines.Figure 3
Mapping surface showing the cocrystallized ligand (PRD-002214) occupying the active pocket of the COVID-19 main protease.Figure 4
2D interaction of the cocrystallized ligand (PRD-002214) in the active site of the COVID-19 main protease.Compound (2) showed the best binding mode and highest binding energy of −6.81 kcal/mol. The 7-hydroxy-6-methoxy-4H-chromen-4-one moiety occupied the first pocket of Mpro, forming three hydrogen bonds with Phe140 and His163. Also, it formed one hydrophobic interaction with His163. Additionally, 1,2-dimethoxybenzene moiety occupied the second pocket of Mpro forming two hydrophobic interactions with Met165 and His41 (Figures 5–7).Figure 5
Compound (2) docked into the active site of the COVID-19 main protease. The hydrogen bonds are represented in green dashed lines, and the hydrophobic interactions are represented in orange dashed lines.Figure 6
Mapping surface showing compound (2) occupying the active pocket of the COVID-19 main protease.Figure 7
2D interaction of compound (2) in the active site of the COVID-19 main protease.


## 4. Conclusions

This study focused on the phytochemical and thein silico biological investigation against the COVID-19 main protease (Mpro) of six flavonoids and one sesquiterpene lactone obtained from A. sublessingiana. Eupatilin, 3′, 4′-dimethoxyluteolin, 5, 7, 3′-trihydroxy-6, 4′,5′-trimethoxyflavone, velutin, and 8α,14-dihydroxy-11,13-dihydromelampolide were isolated from Artemisia species for the first time. Compound (2) exhibited the best binding mode with a binding energy of −6.81 kcal/mol against COVID-19 main protease (Mpro). The obtained results open a window of hope to find an effective cure to the pandemic of COVID-19. Further in vitro and clinical studies should be conducted on compound 2 to confirm its potential against the contagious virus SARS-CoV-2.



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*Source: 5547013-2021-05-27.xml*