Discovery of novel thioquinazoline-N-aryl-acetamide/N-arylacetohydrazide hybrids as anti-SARS-CoV-2 agents: Synthesis, in vitro biological evaluation, and molecular docking studies

In the current investigation, two novel series of (tetrahydro)thioquinazoline-N-arylacetamides and (tetrahydro)thioquinazoline-N-arylacetohydrazides were designed, synthesized and investigated for their antiviral activity against SARS-CoV-2. The thioquinazoline-N-arylacetamide 17g as well as the tetrahydrothioquinazoline-N-arylacetohydrazides 18c and 18f showed potent antiviral activity with IC50 of 21.4, 38.45 and 26.4 µM, respectively. In addition, 18c and 18f demonstrated potential selectivity toward the SARS-CoV-2 over the host cells with SI of 10.67 and 16.04, respectively. Further evaluation of the mechanism of action of the three derivatives 17g, 18c, and 18f displayed that they can inhibit the virus at the adsorption as well as at the replication stages, in addition to their virucidal properties. In addition, 17g, 18c, and 18f demonstrated satisfactory physicochemical properties as well as drug-likeness properties to be further optimized for the discovery of novel antiviral agents. The docking simulation on Mpro binding site predicted the binding pattern of the target compounds rationalizing their differential activity based on their hydrophobic interaction and fitting in the hydrophobic S2 subsite of the binding site


Introduction
Recently, coronavirus disease 2019 (COVID -19) has been identified as a global pandemic disease that affects the survival of population all over the world [1] . COVID-19 is a respiratory disease that causes upper and lower respiratory tract infection which can be further progressed further into respiratory failure by complex mechanisms and may end up with premature mortality [ 1 , 2 ]. It was reported that COVID-19 is caused by a novel zoonotic member of betacoronaviruses called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a single stranded RNA virus of the Coronaviridae family [ 3 , 4 ].
The huge problem, which is currently facing the world, is the remarkable ability of SARS-CoV-2 to mutate over a short period of time [5] . Hence, a plethora of studies have been performed to get some insightful knowledge about SARS-CoV-2 virus [6] . Successfully, some information has been reported including its molecular structure, life cycle, and its interactions with the host cells. This enabled the development various vaccines with different mechanisms of action to be used by humans [7] .
Up till now, the effective and specific antiviral agents for the treatment of SARS-CoV-2 infection are limited or rare [8] . Hence, numerous investigations have been carried out to identify new targets to control this pathogen without affecting the host cells. Recently, some structural elements that can act as potential therapeutic targets have been recognized. Spike glycoprotein was identified as a promising target that is present on the virus surface, and it is responsible for the virus binding to the host cell [9] . RNAdependent RNA polymerase (RdRp) is another attractive target participating in the replication of RNA from an RNA template [10] . Meanwhile, the 3-C-like protease (3CL pro or M pro ) and papain-like protease (PL pro ) were pointed out to be the most important targets for the design of promising antiviral agents against SARS-CoV-2 as they play a key role in the life cycle of SARS-CoV-2 virus with no homologues proteins in human cells [11][12][13][14] . Hence, inhibition of M pro and/or PL pro can result in a selective antiviral activity without side effects on humans [12] .
In this context, quinazolines are regarded as one of the most interesting scaffolds for designing antiviral agents [ 15 , 16 ]. Quinazolines were reported to possess potential antiviral activity against diverse types of RNA viruses including respiratory syncytial virus (RSV) [17] , influenza A virus (IAV) [18] as well as hepatitis C virus (HCV) [19] . For instance, Zhang and co-workers [18] reported the design of a series of 2,4-disubstituted quinazoline derivatives incorporating S -acetamide and NH -acetamide moieties at position 4. Compound I, a representative of this series, was found to have a potent antiviral activity against IAV. Moreover, Hwu et al., [19] described the potent antiviral activity of a class of quinazolinecoumarin conjugates, for example compound II showed potent antiviral activity against hepatitis C virus and chikungunya virus. Meanwhile, Lee et al. [20] reported the synthesis of a series of 4-anilino-6-aminoquinazolines as anti-MERS-CoV inhibitors, compound III , a representative example of the synthesized series, showed IC 50 = 0.157 μM, CC 50 = 3.59 μM and SI = 25. Recently, some studies reported the activity of quinazoline derivatives against SARS-CoV-2 [ 21 , 22 ]. For example, Zhao et al. [21] reported the promising activity of a series of quinolone and quinazoline derivatives in inhibiting RNA synthesis driven by SARS-CoV-2 RdRp. For instance, compounds IV and V revealed 58.43% and 58.83% inhibition on SARS-CoV-2 RdRp at 10 μM concentration. Additionally, Rothan and Teoh [22] reported the expected interesting potential of quinazoline derivatives, for example compound VI , in inhibiting SARS-CoV-2 Main Protease (M pro ) in a high throughput virtual screening campaign ( Fig. 1 ).
On the other hand , several studies reported the promising antiviral properties of various heterocycles substituted with N -aryl-2-(thio)acetamide moieties. For instance, RDEA806 ( VII ) was reported to possess a potential HIV-1 reverse transcriptase (RT) inhibitory activity against wild-type (WT) as well as some non-nucleoside reverse transcriptase (NNRT) resistant viruses [23] . Moreover, Zhan and co-workers [24] reported the synthesis and the interesting antiviral properties of novel 1,2,4-triazin-6-yl-thioacetamide derivatives as potent HIV-1 NNRTIs. Compound VIII was an example of this series displaying nanomolar activity against HIV-1(WT) as well as a moderate potency against the double mutant strain RES056. Furthermore, Zhang et al. [25] reported the discovery of some indol-3-yl-thio-N -phenyl-acetamides with potent antiviral activity. For instance, compound IX revealed a dual potency against RSV and IAV [25] . Moreover, Yu et al. [26] reported the potent antiinfluenza activity of different pyrimidines substituted at 2 position with a N -aryl-2-(thio)acetamide moiety. For example, compound X demonstrated a broad activity against IAV and IBV. In addition, Zhan and co-workers [27] reported the anti-influenza properties of some thiazolyl-N -aryl-2-(thio)acetamides, for example, the derivative XI displayed a potent activity on influenza A/H1N1 virus ( Fig. 2 ).

Rationale design of thioquinazoline-N -aryl-acetamide/ Narylacetohydrazide hybrids
Encouraged by the previous findings, and since most of the antiviral drugs in the clinical use were not designed specifically for SARS-CoV-2, we were interested in the current study in designing a new series of anti-SARS-CoV-2 through the application of the molecular hybridization strategy between the quinazoline ring and the N -aryl-2-(thio)acetamide moiety to design a novel series of (tetrahydro)thioquinazoline-N -arylacetamide hybrids XII ( Fig. 3 ). For further structure-activity relationship investigation, further elongation of the N -aryl-2-(thio)acetamido linkage was carried out by its replacement with a 2-(thio)acetohydrazide linker to afford scaffold XIII ( Fig. 3 ). Derivatives from the designed scaffolds were synthesized utilizing the conventional methods of organic chemistry. Subsequently, the synthesized compounds were assayed for their antiviral activity against SARS-CoV-2. Promising hits were further examined for their expected mode of antiviral activity. Additionally, their physicochemical as well as pharmacoki-  netic properties were predicted using the SwissADME free webtool. Concurrently, in silico molecular docking studies were performed in the SARS-CoV-2 Main Protease (M pro ) binding site to study their binding mode in order to rationalize their promising antiviral activity.

Chemistry
Initially, the starting quinazoline 3 was synthesized by the reaction of 1 with CS 2 ( 2 ) in the presence of KOH at 30 o C [ 28 , 29 ]. Meanwhile, the starting material 6 was synthesized by the condensation of 4 with thiourea ( 5 ) under basic conditions [ 30 , 31 ] ( Scheme 1 ).
For the synthesis of the target compounds 11a-c and 12ac ; 2-aminobenzoic acid ( 7a ), 3-aminobenzoic acid ( 7b ), and 4aminobenozic acid ( 7c ) were reacted with chloroacetyl chloride ( 8 ) in DMF to afford the intermediates 9a-c , respectively, which were subsequently reacted with the starting materials 3 and 6 under basic conditions to afford the target compounds 11a-c and 12a-c , respectively, in excellent yields ( Scheme 2 ).
For the synthesis of the target quinazolines 17 and 18 , different benzoic acid derivatives 13 were first esterified to give the corresponding esters 14 which were subsequently reacted with hydrazine hydrate to afford the corresponding acid hydrazides 15 ( Scheme 3 ). Further reaction of 15 with chloroacetyl chloride ( 8 ) was performed to give the intermediates 16 which were reacted with the thioquinazolines 3 and 6 to yield the target compounds 17 and 18 , respectively, in excellent yields.

Biological evaluation
All the target compounds 11a-c , 12a-c , 17a-g and 18a-g were evaluated for their antiviral activity against NRC-03-nhCoV as well as for their cytotoxic activity on Vero-E6 cells employing MTT assay [32] . The CC 50 (concentration necessary for 50% growth inhibition of normal cell line compared to the control experiment) of the target compounds on Vero-E6 cells and IC 50 (concentration necessary for 50% reduction of virus-induced cytopathic effect (CPE) compared to the virus control experiment) of the target compounds against NRC-03-nhCoV virus in Vero-E6 cells are presented in Table 1 . The selectivity indices which are the ratio of CC 50 relative to IC 50 of the tested compounds were calculated and depicted in Table 1 . In addition, Fig. 4 presents the inhibition curves of the most potent compounds 17g , 18c , and 18f (For the rest of the inhibition curves see the SI).
The presented study showed promising activity in comparison to the results reported in the literature [ 20 , 21 ]. From the IC 50 results presented in Table 1 , it is obvious that the synthesized thioquinazolines and tetrahydrothioquinazolines displayed moderate to potent antiviral activity against NRC-03-nhCoV. Compounds 17g, 18c and 18f showed the most potent inhibitory activity with IC 50 of 21.4, 38.45 and 26.4 μM, respectively, on NRC-03-nhCoV. Close investigation of the antiviral results revealed that the thioquinazoline-N -aryl-acetamide hybrids 11a-c demonstrated moderate antiviral properties with an IC 50 range of 79.2 to 104 μM. Compounds 11b and 11c incorporating 3 and 4 carboxylic groups, respectively, showed higher potencies in comparison to the 2-carboxylic acid derivative 11a with IC 50 of 79.7, 79.2, and 104 μM, respectively, as well as higher SI s. Replacing the thioquinazoline moiety in series 11a-c with a tertahydrothioquinazoline moiety in series 12a-c decreased the antiviral activity (IC 50 range of 173.6 to 531.3 μM). Compound 12a , with a carboxylic acid moiety at the 2 position, showed an IC 50 of 173.6 μM and a 2.5-fold higher selectivity toward the virus over the host cells (CC 50 = 431.4 μM). Shifting the COOH moiety to the 3 and 4 positions in 12b and 12c , respectively, resulted in more than a 2-fold decrease in the antiviral potency with no preferential selectivity ( SI = 0.98 and 0.63, respectively).
Replacing the thio-N -aryl-acetamide moiety of series 11a-c with thioacetohydrazide moiety in series 17a-g , resulted in a different pattern of potency as well as specificity. The thioquinazoline-Naryl-acetohydrazide derivative 17a with an unsubstituted terminal phenyl moiety displayed a weak antiviral activity (IC 50 = 317.5 μM) and high cytotoxic activity (CC 50  Hydrogenation of the fused phenyl group of the thioquinazoline ring in series 17a-g to afford the tetrahydrothioquinazoline series 18a-g increased the antiviral activity in all cases with the exception of compounds 18e and 18g . Compound 18a with unsubstituted phenyl group displayed a moderate antiviral activity (IC 50 = 140.2 μM) with a low SI of 1.68 (CC 50 = 235 μM). Introduction of 4-NO 2 and 4-Cl groups at the terminal phenyl moiety to yield 18c and 18f , respectively, resulted in a more than three-fold increase in potency in comparison to 18a with IC 50 of 38.45 and 26.4 μM, respectively, in addition to a more than 10-fold higher selectivity toward the virus over the host cell ( SI = 10.67 and 16.04, respectively). On the other hand, introduction of 4-Me, 2-OMe, 2-Br groups at the terminal phenyl moiety to give 18b, 18d , and 18g showed a decrease in the antiviral activity with IC 50 = 93.40 to 113.7 μM. Slight decrease in the antiviral activity was found for 18e (IC 50 = 459.8 μM) in comparison to 17e (IC 50 = 457.8 μM) and more than four-fold reduction was obsereved for 2-bromo derivative 18g (IC 50 = 93.40 μM) in copmarison to 17g (IC 50 = 21.4 μM).
In summary, although compound 17g displayed the highest antiviral activity with IC 50 of 21.4 μM, it was less favored because of its cytotoxic properties (CC 50 = 184.9 μM and SI = 8.64). On the contrary, 18f and 18c were regarded to be the most promising derivatives in terms of their potency (IC 50 = 26.4 and 38.45 μM) as well as their selectively and safety ( SI = 16.04 and 10.67).

Mechanism of Anti-SARS-CoV-2 Activity
For further investigation of the mechanism of virus inhibition of the most promising derivatives 17g, 18c and 18f , a plaque infectivity reduction assay was performed [34] . This assay studied whether the promising derivatives affected the virus at the adsorption stage and/or replication stage and/or due to their direct virucidal effect. The inhibition results are depicted in Table 2 . Interestingly, the three derivatives were found to have multiple inhibitory effects to different extents in the three stages. Nevertheless, the replication stage was found to be the most affected following treatment with the tested compounds (40-50% viral inhibition at 125 μM and 38.9-40% viral inhibition at 62.5 μM).
As can be seen in Table 2 , the thioquinazoline-N -aryl acetamide derivative 17g showed at 125 μM a moderate inhibition of the virus at the adsorption stage 29.6%, whereas it showed a potent inhibition at the replication stage (50%) as well as a potent virucidal effect (47.8%). The tetrahydrothioquinazoline-N -arylacetohydrazide 18c displayed moderate inhibitory activity on the three tested mechanisms. At 125 μM, it revealed inhibition% of 44.4 and 43.3% by applying adsorption and replication mechanisms, respectively, and a 38.9% virucidal effect was noticed. Moreover, at 125 μM, compound 18f was found to have 53% virucidal activity as well as a 40% inhibitory effect on virus replication and only 20% inhibition of the viral adsorption mechanism.

Molecular modeling
Protease enzyme plays a critical role in viral protein maturation by cleaning proproteins after their translation into the host cell cytosol. As a result, viral proteases are considered potential antiviral drug targets [35] . The inhibition of a viral protease can reduce the assembly of mature viral particles. Therefore, SARS-CoV-2 main protease (M pro ) could be a plausible target for the newly synthesized compounds, especially with its reported pyrimidinedione inhibitors which are analogues to our designed antiviral derivatives [36] , thus, molecular docking simulations have been carried out to study the binding pattern of the target compounds 11a-c, 12a-c, 17a-g , and 18a-g in the active site of SARS-CoV-2 main protease (M pro ). For this end, the X-ray crystallographic structure of SARS-CoV-2 main protease (M pro ) co-crystalized with a pyrimidine-2,4dione inhibitor (YD1) (PDB ID: 7LTJ) was retrieved from the protein data bank ( https://www.rcsb.org/ ) [36] . The molecular docking protocol was first validated by self-docking of the co-crystallized ligand (YD1) in the vicinity of the enzyme active site. The selfdocking step reproduced the co-crystalized ligand pose efficiently with a docking score (S) of −13.26 kcal/mol and a root mean square deviation (RMSD) of 1.538 Å . Moreover, the docking protocol reproduced all the key interactions with the active site amino acids ( Figs. 5 and 6 ). Using the validated molecular docking protocol, the target compounds 11a-c, 12a-c, 17a-g , and 18a-g were docked in the SARS-CoV-2 M pro active site.
Generally, the target compounds showed a common binding pattern in the target enzyme (M pro ) active site accommodated in subsites S1 and S2 ( Figs. 7-9 ). The (tetrahydro)quinazolinone moiety occupies YD1 uracil binding subsite S1 driven by polar contacts interacting by its carbonyl group with the key amino acid His163 through hydrogen bonding. On the other side, the substituted phenyl group occupies the largely hydrophobic S2 subsite occupied by YD1 dichlorophenyl moiety. The target compounds' substituted phenyl group is stabilized through ππ stacking with the imidazole side chain of the key catalytic amino acid His41. The substituted phenyl group is sandwiched between the side chains of the amino acids His41 and Gln189. The linker in-between interacts through multiple H-bond interactions with the surrounding key amino acids Asn142, Gly143, Cys145, and His164. In compounds 11a-c and 12a-c , the peripheral carboxylate moiety on the distal phenyl group is involved in extra hydrogen bond interactions    with the amino acids His41, Cys44, Met49, Pro52, and/or Met165 (For further details see supporting materials). Table 3 shows the docking score of the target compounds and the co-crystalized ligand ( YD1 ). The newly synthesized compounds show a predicted docking score range of −13.99 to −11.73 kcal/mol, whereas the co-crystalized ligand YD1 showed a predicted docking score of −13.26 kcal/mol. As for the most promising compounds 17g, 18c , and 18f , compound 18c showed the most negative docking score −13.99 kcal/mol more negative than that of YD1 . Whereas compounds 17g and 18f exhibited comparable docking score ( −12.45 and −12.07 kcal/mol, respectively) which is less negative than that of the co-crystalized ligand (YD1) ( −13.26 kcal/mol) indicating their less predicted binding affinity than YD1 .  Compounds 17d, 18a, 17a , and 17e showed the least negative predicted docking scores ( −11.54, −11.47, −11.38, and −11.37 kcal/mol) which agree with their poor experimental activity (258.8, 140.2, 317.5, and 457.8 μM).
The predicted binding pattern of the target compounds could rationalize their differential activity based on their hydrophobic interaction and fitting in the hydrophobic S2 subsite of the bind- ing site. Compounds 11a-c and 12a-c showed a relatively less predicted binding affinity due to their ionic polar carboxylate substitution on their distal phenyl group which decreases the probable hydrophobic interactions with the hydrophobic S2 subsite, however, this loss of proper hydrophobic interactions is somehow compensated by the carboxylate involvement in multiple extra hydrogen bond interactions with the amino acids His41, Cys44, Met49, Pro52, and/or Met165 like in case of compound 11b . On the other hand, compounds 17a-g and 18a-g show better predicted binding affinity because of their hydrophobic substitutions on the distal phenyl group and the longer thioacetohydrazide linker which makes the (substituted)phenyl group better fitted in the hydrophobic S2 subsite. Compounds achieving higher hydrophobic interaction with S2 subsite and better fit of their (substituted)phenyl group show strong predicted binding affinity as reflected in their promising experimental activity e.g., compounds 17g, 18c , and 18f with o -bromo, p -nitro, and p -chloro substitution, respectively. Alternatively, compounds with unsubstituted distal phenyl ring 17a and 18a show less predicted binding affinity relative to their substituted congeners due to their less possible hydrophobic interactions with S2 subsite. Compounds with less hydrophobic substituents (OMe) e.g., compounds 17d or that are not well fitted in the S2 subsite e.g., compounds 17e and 18e show weaker predicted binding affinity which is reflected

Estimation of physicochemical, pharmacokinetic and ADME properties
Encouraged by the promising antiviral properties of 17g, 18c and 18f , they were further selected to predict their physicochemical and ADME properties using SwissADME free web tool [37] . Table 4 presents some selected results of 17g, 18c and 18g .
Analysis of the obtained results ( Table 4 ) revealed that the target thioquinazoline-N -aryl acetamide 17g and tetrahydrothioquinazoline-N -aryl acetohydrazides 18c and 18f express acceptable levels of physicochemical properties. The molecular weight of 17g, 18c and 18f is spanning between 392.86 to 433.28 g/mol. They incorporate acceptable numbers of hydrogen bond donors (less than 5) and acceptors (less than 10). Additionally, the topological polar surface area (TPSA) for 17g and 18f is 129.25 Å , while it is slightly high in case of the 18c TPSA = 175.07 Å and the ilog P (octanol-water partition coefficient) is ranging from 1.60 to 2.62 [38] .
The target (tetrahydro)thioquinazoline-N -arylacetamides 17g and 18f are predicted to be well absorbed from the GIT while 18c is predicted to have low GIT absorption. The three target compounds 17g, 18c and 18f have no predicted ability to penetrate the blood brain barrier which decreases their probable adverse effect at the central level. Compounds 18c and 18f were found to be substrates for P-glycoprotein (P-gp), which plays a significant role in the removal of strange substances outside the cells. Meanwhile, 17g is not a substrate for P-glycoprotein (P-gp) [39] . Fig. 10 presents the bioavailability radar chart of compounds 17g, 18c and 18f provided by the SwissADME web tool [37] . Generally, the bioavailability radar presented by SwissADME demonstrates a pink-colored area that identifies the optimum space of six physicochemical parameters for oral bioavailability. These six properties are size, polarity, lipophilicity, solubility, flexibility, and sat- uration. Close investigation of the presented radar charts showed that compound 18f occupies the ideal space of the six physicochemical properties for oral bioavailability, whereas the derivatives 17g and 18c are nearly fully located in the pink area, only the degree of unsaturation slightly deviates from the optimum in 17g while the polarity is slightly deviating from the ideal for 18c ( Fig. 10 ).
Moreover, it is attractive that 17g and 18f follow all rules of drug-likeness, they do not violate Lipinski's rule [40] , Veber rule [41] , Ghose-filter [42] , Egan [43] or Muegge ś filter [44] . Because of the high TPSA of compound 18c , it satisfies only Lipinski's rule. In addition, it worth pointing out that the three derivatives do not incorporate in their structures Pan Assay Interference (PAINS) fragments [45] . Hence, in addition to the promising antiviral activity of the target compounds, their promising drug-likeness parameters suggests their potential to be subjected for future optimization for the discovery of chemotherapeutic agents.

Conclusion
The presented study involves the design, synthesis, and anti-SARS-CoV-2 activity evaluation of two novel series of (tetrahydro) thioquinazoline-N -arylacetamides and (tetrahydro)thioquinazoline-N -arylacetohydrazides. The thioquinazoline-N -arylacetamide 17g beside the tetrahydrothioquinazoline-N -arylacetohydrazides 18c and 18f showed potent antiviral activity with IC 50 = 21.4, 38.45, and 26.4 μM, respectively. The derivatives 18c and 18f exhibited SI = 10.67 and 16.04, respectively, towards the virus over the host cells. In addition, the three derivatives 17g, 18c , and 18f showed promising virucidal properties beside their ability to inhibit the virus at the adsorption as well as at the replication stages. Moreover, besides their promising antiviral activity, compounds 17g, 18c and 18f displayed acceptable physicochemical and pharmacokinetic properties for further optimization as antiviral agents.  Fig. 10. Bioavailability radar plot from SwissADME online web tool for 17g, 18c and 18f .

Table 3
Docking energy scores ( S ) in kcal/mol for the target compounds and the co-crystalized compound (YD1) in M pro active site (PDB ID: 7LTJ).

General remarks
Chemicals along with solvents used for chemical reaction were obtained from commercial companies. Follow up of the reactions were carried out using analytical thin layer chromatography (TLC). Uncorrected melting points were recorded on a Stuart SMP30 melting point apparatus. Elemental analyses of the synthesized hybrids were recorded in the micro analytical labs, National Research Centre, Cairo, Egypt. IR spectra (40 0 0-40 0 cm −1 ) were recorded on Jasco FT/IR 300 E Fourier transform infrared spectrophotometer. 1 H NMR as well as 13 C NMR spectra were measured in DMSO-d 6 as a solvent at 500 (125) MHz and 400 (100) MHz on Bruker instruments.

General procedure I for the synthesis of the target compounds
11a-c, 12a-c, 17a-g and 18a-g A mixture of 3 or 6 and anhydrous K 2 CO 3 was stirred for 30 min at room temperature then 3a-c or 16a-g was added and the mixture was heated under reflux for 2 h. The reaction mixture was then cooled to room temperature poured on ice and neutralized with few drops of 2 N HCl and the precipitated product was filtered, dried and purified by crystallization from MeOH/ DCM 1:1 mixture to give the corresponding target products 11a-c, 12a-c, 17a-g, 18a-g in analytical pure form.

Mechanism of action(s).
To investigate whether the most potent candidates affect the (a) viral adsorption, (b) viral replication, or (c) has a virucidal effect, the plaque infectivity reduction assay was performed according to the reported procedure [ 34 ].

Molecular modeling
Molecular docking studies were carried out using Molecular Operating Environment (MOE, 2020.0901) software. All minimizations were performed with MOE until an RMS gradient of 0.05 kcal •mol −1 Å −2 with MMFF94x force field and the partial charges were automatically calculated. The X-ray crystallographic structure of SARS-CoV-2 main protease (M pro ) co-crystalized with a pyrimidine-2,4-dione inhibitor (YD1) (PDB ID: 7LTJ) was downloaded from the protein data bank [36] . Water molecules and ligands which are not involved in the binding were first removed.
Next, the protein structure was prepared for the molecular docking study using QuickPrep protocol in MOE with the default options. YD1 exhibits several binding interactions with M pro active site with the amino acids Asn142, Gly143, Cys145, His163, Met165, Glu166, and Asp187 either directly or through water mediated interactions ( Fig. 5 ). To perform the molecular docking study, the cocrystalized ligand (YD1) was used to define the active site and Triangle Matcher placement method and London dG scoring function were used.
The molecular docking protocol was first validated by selfdocking of the co-crystallized ligand (YD1) in the vicinity of the enzyme active site. The self-docking step reproduced the cocrystalized ligand pose efficiently with docking score (S) of −13.26 kcal/mol and a root mean square deviation (RMSD) of 1.538 Å , moreover, the docking protocol reproduced all the key interactions with the active site amino acids indicating the suitability of the adopted molecular docking protocol for the intended molecular docking study ( Fig. 6 ).

Estimation of physicochemical, pharmacokinetic and ADME properties
The open SwissADME web tool available from the Swiss Institute of Bioinformatics (SIB) was used for the calculation of the physicochemical descriptors as well as to predict the ADME parameters, and pharmacokinetic properties of the most potent compounds [37] . The compounds' structures were drawn on the web user interface, converted to SMILES notations, then submitted to the online server for calculation.