Isolation and In Silico Anti-COVID-19 Main Protease (M pro ) Activities of Flavonoids and a Sesquiterpene Lactone from Artemisia sublessingiana

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 8 α ,14-dihydroxy-11,13-dihydromelampolide. The isolated compounds were in silico examined against the COVID-19 main protease (M pro ) 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.


Introduction
COVID-19 is the pandemic caused by the new coronavirus strain SARS-CoV-2 (severe acute respiratory syndrome corona virus-2). e 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. e 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]. e proteases, especially main protease (M pro ), play a vital role in the life cycle of coronaviruses [6]. M pro is a cysteine protease that is enrolled in the maturation cleavage events within the polyprotein's precursors [7,8]. is enzyme is crucial for the processing and translation of polyproteins from the viral RNA [9]. Accordingly, M pro is a very essential element for the replication and transcription of coronaviruses, and the inhibiting of its activity would block viral replication [10]. Consequently, M pro 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][12][13].
In this study, the main bioactive contents of the aerial parts of A. sublessingiana (Krasch. ex Poljak.) Poljak. (Synonium Seriphidium sublessingianum (Krasch ex Poljakov)) have been investigated. is paper reports the isolation, structural determination, and in silico anti-COVID-19 main protease (M pro ) activities of six flavonoids and one sesquiterpene lactone from A. sublessingiana. eir structures were determined by spectrum analysis of 1D, 2D NMR, and ESI-MS data. in-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% H 2 SO 4 reagent followed by heating. Isolated compounds were identified by 1D and 2D NMR analysis ( 1 H 500 MHz, 13 C 125 MHz), acquired on a Jeol Delta 2 NMR spectrometer at 297 K. Internal standards: TMS. Solvents: chloroform-d, acetone-d 6 , and DMSO-d 6 .

Materials and Methods
Coupling constants are given in Hertz. e chemical shifts were expressed in δ ppm. Mass spectra (EIMS) were recorded on an IT-TOF-MS spectrometer.

Plant Material.
e aerial parts of A. sublessingiana were collected 90 km from Kyzylorda city, Kazakhstan (Kyzylkum sand desert). e 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.

Extraction and Isolation.
Air-dried powered aboveground parts of A. sublessingiana (1.0 kg) were grounded and extracted with EtOH for 1 day. e extract was filtered, and the extraction process was repeated twice. e combined extracts were evaporated under reduced pressure to yield a crude extract of 93 g. e 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. e 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 chloroformmethanol 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).

Docking Studies Experiment.
e crystal structure of the target enzymes COVID-19 main protease (M pro ) (PDB ID: 6lu7, resolution: 2.16Å) 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 M pro were determined. At first, the water molecules were removed from the crystal structure of M pro , retaining only one chain which is essential for binding. e cocrystallized ligand (PRD-002214) was used as a reference ligand. en, 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]. e structures of the examined compounds and the cocrystallized ligand were drawn using ChemBioDraw Ultra 14.0 and saved in SDF format. en, the saved file was opened using MOE software, and 3D structures were protonated. Next, the energy of the molecules was minimized. e 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]. e docking procedures were carried out utilizing a default protocol. In each case, 30 docked structures were generated using genetic algorithm searches. e output from MOE software was further analyzed and visualized using Discovery Studio 4.0 software [42,43].

Docking Studies.
Docking studies were carried out for compounds (1-7) against the COVID-19 main protease (M pro ) (PDB ID: 6lu7, resolution: 2.16Å) to examine the mode of binding with the proposed target. e cocrystallized ligand (PRD-002214) was used as a reference molecule. e 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). e crystallized ligand (PRD-002214) showed binding energy of −7.83 kcal/mol. e 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 M pro . 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).
Compound (2) showed the best binding mode and highest binding energy of −6.81 kcal/mol. e 7-hydroxy-6methoxy-4H-chromen-4-one moiety occupied the first pocket of M pro , 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 M pro forming two hydrophobic interactions with Met165 and His41 (Figures 5-7).

Conclusions
is study focused on the phytochemical and the in silico biological investigation against the COVID-19 main protease (M pro ) 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 (M pro ). e 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.

Data Availability
NMR data of the isolated compounds are available.

Conflicts of Interest
e authors declare that they have no conflicts of interest.