Synthesis, spectroscopic characterization of novel phthalimides derivatives bearing a 1,2,3-triazole unit and examination as potential SARS-CoV-2 inhibitors via in silico studies

In the present study, novel phthalimide derivatives 8(a-f) and 9(a-f) bearing a 1,2,3-triazole subunit were synthesized via CuAAC reactions and characterized by 1H, 13C NMR, HR-MS, and FT-IR analyses. To support the fight against SARS-CoV-2, in silico molecular docking studies were carried out to examine their interactions with the proteins of SARS-CoV-2 (Mpro and PLpro) and the protein-protein interactions (PPI) between the ACE2-spike (S1) in comparison with various inhibitors reported to be active by in vitro experiments. The ligand-protein stabilities of compounds 8a-Mpro, 8b-PLpro, and 9a-‘ACE2-S1’ showing the best binding energy and predicted inhibition constant values (Ki) were examined by molecular dynamics simulation studies. Finally, in silico ADMET properties of the target compounds were investigated using the Swiss ADME and ProTox-II web tools. According to in silico results, all phthalimide analogs may block the PPI between S1 and ACE2. The compounds may also inhibit the progression of the Mpro, and PLpro proteins of SARS-CoV-2. Additionally, it has been estimated that the compounds are suitable for oral administration and exhibit low levels of toxicity.


Introduction
Since a new type of coronavirus disease, COVID-19 caused by SARS-CoV-2 virus, emerged in December 2019 in China [1] , scientists have conducted many studies on the mechanism of action of the SARS-CoV-2 in the human body. The spike (S) protein of SARS-CoV-2 has been reported to promote virus entry into the host cell by binding with the human ACE2 protein [2] . The S protein comprises two subunits: S1 and S2. The S1 facilities the binding to ACE2 of the virus and it consists of the receptor-binding domain (RBD), the N-terminal domain (NTD), and the other several subdomains, while the S2 mediates virus-cell membrane fusion and initiates the infection [3][4][5] . The S1 binds to ACE2 via its RBD part [ 6 , 7 ]. Blocking of the protein-protein interactions (PPI) between the S1 and ACE2 can impair infection efficiency [8] , therefore, this blockade has been considered as a possible drug target [9] . After the virus enters the host cell, viral replication and transcription are initiated by the other proteins of SARS-CoV-2 [ 10 , 11 ]. The main protease protein (Mpro or 3CLpro) of the virus is considered an attractive drug target because it is required in the processing and maturation of viral polyproteins [12] . The papain-like protease E-mail address: a.tan@alparslan.edu.tr protein (PLpro), which is another viral protease of the virus, plays an important role in both the cleavage for the proliferation cycle of viral polyproteins and the disruption of host responses [ 13 , 14 ]. Therefore, the PLpro has been reported as an attractive drug target [15] , as well. In silico approaches have been used to discover potential inhibitors of SARS-CoV-2 by scientists. The interactions of both commercially available antiviral drugs and some known bioactive compounds with the proteins (especially, Mpro and PLpro) of SARS-CoV-2 have been examined via in silico studies [15][16][17][18] . Recently, the synthesis of some new compounds containing various bioactive moieties such as 1,2,3-triazole, Schiff base, piperidine, and norcantharimides have been reported by scientists and some of these compounds have been suggested as possible therapeutic agents for the COVID-19 via in silico studies [19][20][21] . In addition to in silico studies, it has been reported in vitro antiviral studies against SARS-CoV-2 [22] .
In the light of these studies, in the present study, novel hybrid phthalimide derivatives 8 ( a -f ) and 9 ( a-f ) bearing 1,2,3-triazole subunit having three active components (the phthalimide ring, various amines, and 1,2,3-triazole ring) were synthesized. To support the fight against SARS-CoV-2, in silico molecular modeling studies were carried out to examine their interactions with the PPI of ACE2-S1 and the proteins of SARS-CoV-2 (Mpro and PLpro) by comparison with various inhibitors reported to be active in vitro experiments.
On the other hand, corresponding organic azido compounds 6 ( a -f ) [ 42 , 43 , 47 ] and 7 ( a -f ) [48][49][50][51] , which are known in the literature, were synthesized by the reaction steps given in Scheme 1 . For this, in the first step, 4 ( a -f ) and 5 ( a -f ) were synthesized from the reaction of various amines (secondary amines 2 ( a -f ) and primary amines 3 ( a -f )) with chloroacetyl chloride in the presence of NaHCO 3 in acetonitrile at 0 °C. In the second step, 2-chloro-Nsubstituted acetamide derivatives 4 ( a -f ) and 5 ( a -f ) were treated with NaN 3 in dimethyl sulfoxide at room temperature and azido compounds 6 ( a -f ) and 7 ( a -f ) were obtained in good yields. Finally, compound 1 was treated with azido compounds 6 ( a -f ) or 7 ( a -f ) in the presence of CuSO 4 •5H 2 O [45] and sodium L -ascorbate in a methanol:water solvent system at room temperature, and the target compounds ( 8 ( a -f ) and 9 ( a -f )) were obtained in good yields ( Scheme 1 ).

Characterization
The structures of phthalimide-1,2,3-triazole hybrid compounds 8 ( a -f ) and 9 ( a -f ) are similar. Among these compounds, 8a and 9a were chosen as the model compounds. The 1 H NMR spectrum of the compound 8a was recorded in CDCl 3 and the 13

Molecular docking studies
Molecular docking studies play an important role in explaining the protein-ligand relationship [52][53][54] and prevent both in vitro and in vivo studies of molecules that are impossible to be effective, thus the studies provide great financial savings. In the present studies, the interactions of the target compounds with SARS-CoV-2 proteins were examined and their estimated Ki values were calculated via molecular docking studies. For this purpose. The interactions of the target compounds with the PPI of ACE2-S1, Mpro, and PLpro proteins were investigated. PDB files: 6M0J for the ACE2-S1, 7K40 for the Mpro, and 7CMD for the PLpro were used. Bojadzic et al . [9] have reported that methylene blue (MB) (IC 50 : 3.03 μM) inhibits the PPI of between the S1 and ACE2. Therefore, MB was used as a reference inhibitor/ligand. The target compounds and MB were docked on the PPI of ACE2-S1. According to the docking results, all target compounds showed better interactions than MB with the PPI. The binding energy values vary between −6.40 and −9.70 kcal/mol, while the estimated Ki values vary between 0.077 and 20.35 μM. The best binding energy and estimated Ki values of MB were −6.18 kcal/mol and 29.32 μM. Compound 9a in the target compounds exhibited the best binding energy ( −9.70 kcal/mol) and estimated Ki value (0.077 μM) ( Table 1 ). According to the docking results, MB interacted with the RDB of ACE2, while compound 9a interacted with both the RDB of ACE2 and S1. The phthalimide moiety of 9a formed a conventional hydrogen bond with Lys353 residue (3.00 Å ) of the ACE2, its triazole ring formed a πanion with Asp38 residue (3.92 Å ) of the ACE2 protein. The ph- Table 1 The docking results of the target compounds and the inhibitors with the PPI of ACE2-S1, Mpro, and PLpro.

Compounds
ACE2-S1 Mpro PLpro thalimide moiety of 9a also formed a π -cation interaction with Arg403 (3.91 Å ) and a carbon-hydrogen bond with Tyr495 residue (2.97 Å ) of the S1 protein. At the same time, while the carbonyl group of amide moiety of 9a formed a conventional hydrogen bond with Ser494 residue (2.07 Å ) of the S1 protein, its benzyl group formed a π -alkyl interaction with Leu452 (5.28 Å ) of the S1. 9a also formed van der Waals interactions with both the ACE2 and S1 proteins ( Figs. 3 and 6 ). The Mpro protein of SARS-CoV-2 are necessary for maturation, viral polyproteins processing [12] , and it has been recognized as an important drug target. Therefore, all target compounds were docked on the active site of the Mpro protein (PDB ID: 7K40) containing boceprevir as a co-crystallized ligand. The docking mode of 7K40 was determined according to the location of boceprevir using a directed docking procedure. The validation study was carried out for boceprevir and the root mean square deviation (RMSD) value was 1.630 Å (see SI, Figure S49-a). This value shows to be valid for the docking procedure. Boceprevir was also used as a reference inhibitor/ligand, it has been reported to inhibit the Mpro protein (IC 50 : 8.0 μM) [12] . For all target compounds, the binding energy values vary between −7.03 and −8.76 kcal/mol, while the estimated Ki values vary between 7.03 and 0.379 μM. The best binding energy and estimated Ki values of boceprevir were −10.04 kcal/mol and 0.043 μM, respectively. 8a in the target compounds showed the best binding energy ( −8.76 kcal/mol) with the active site of the Mpro. The estimated Ki value of 8a was 0.379 μ ( Table 1 ). The other target compounds were predicted to show acceptable inhibition constants. When the interactions of 8a with the Mpro, which showed the best binding energy and inhibition constant values, were examined, it was observed that the amide carbonyl group of 8a formed two conventional hydrogen bonds with His41 (2.65 Å ) and Cys145 (3.69 Å ) residues of the Mpro. The phthalimide moiety of 8a formed a π -sigma interaction with Leu167   a directed docking procedure. The RMSD value was 1.021 Å (see SI, Figure S49-b) for GRL0617. This value is valid for validation. At the same time, GRL0617 was a strong inhibitor against PLpro (IC 50 : 2.4 μM) [55] and was evaluated as a reference inhibitor/ligand. According to the docking results, the binding energy values of the target compounds vary between −7.31 and −8.87 kcal/mol, while its estimated Ki values vary between 4.30 and 0.315 μM. The best binding energy and estimated Ki values of GRL0617 were −9.83 kcal/mol and 0.061 μM, respectively. Compound 8b exhibited the best binding ( −8.87 kcal/mol) and estimated Ki value (0.315 μM) in the target compounds ( Table 1 ). When the results were examined in detail, the triazole ring of 8b formed a conventional hydrogen bond with Gln269 (2.02 Å ), a ππ stacked with Tyr264 (3.69 Å ). The triazole ring also formed π -anion and carbonhydrogen bond interactions with Asp164 (4.25 Å and 3.77 Å , respectively). The phthalimide moiety formed two amide-π -stacked interactions with Gly163 (3.67 Å and 4.55 Å ), π -anion with Glu167 (4.94 Å ), π -alkyl with Leu162 (5.42 Å ), and π -sigma interaction with Gln269 (3.64 Å ). Its pyrrolidine ring formed an alkyl interaction with Arg166 (4.57 Å ) ( Figs. 5 and 6 ).
In this study, the interactions of the respective inhibitors ( Fig. 7 ) with all proteins were investigated at the same time. Both the interactions of MB with the Mpro and PLpro proteins and the interactions of boceprevir with the PPI of ACE2-S1 complex are weaker than the target compounds. Regarding GRL0617, while it showed weaker than the target compounds in the interactions with the PPI of the ACE2-S1, it showed the binding energy value close to the target compounds in the interactions with Mpro. Multi-targeting inhibitors can be beneficial for solving limited efficiencies, resistant profiles of an individual target. Therefore, the target compounds can be considered as effective inhibitors than the respective inhibitors against all three proteins ( Table 1 ).

Molecular dynamics simulation
Molecular docking studies were supported by molecular dynamics (MD) simulations for the complexes 8a -Mpro, 8b -PLpro, and 9a -the PPI of ACE2-S1 to check their stability and dynamic properties. Their Desmond simulation trajectories were analyzed. The RMSD, root mean square fluctuation (RMSF), and proteinligand contacts were calculated from MD trajectory analysis. The RMSD plots are shown in Fig. 8 . The RMSD plot of the complex 8a -Mpro indicates that the complex reaches stability at 5 ns. From then, an average RMSD value of 0.8 Å for 8a -Mpro persists up to 50 ns. After that, the changes in the RMSD values remain within 0.5 Å during the simulation period, which is quite acceptable ( Fig. 8 a). The ligand fit to protein RMSD values fluctuates within an average of 1.0 Å after being stable. There was a decrease in the RMSD of ligand due to flip. It increases at 40 ns and became stable for the rest of the time. The RMSD plot of the complex 8b -PLpro indicates that the complex reaches stability at 10 ns. From then, an average RMSD value of 1.0 Å for 8b -PLpro persists up to 50 ns. The ligand fit to protein RMSD values fluctuates within 1.0 Å after being stable ( Fig. 8 b). The RMSD plot of the complex 9a -'ACE2-S1' indicates that the complex reaches stability at 5 ns. From then, an average RMSD value of 1.0 Å for 9a -'ACE2-S1' persists up to 50 ns. The ligand fit to protein RMSD values fluctuates within 1.0 Å after being stable. There was a slight increase in the ligand's RMSD at 15 ns but became stable again. These indicate that the ligand remains stably bound to the binding site of the receptor during the simulation period ( Fig. 8 c). Fig. 9 shows the residue-wise RMSF value of the protein bound to the ligand. The residues showing higher peaks correspond to loop regions, as identified from MD trajectories ( Fig. 10 )      tions of ligand-proteins determined with MD are hydrogen bonds and hydrophobic interactions. Gln189, Thr190, and Gln192 are the most important ones in terms of H-bonds for 8a -Mpro ( Fig. 11 a  and 11 b). Tyr273 is the most important one in terms of H-bonds for 8b -PLpro ( Fig. 11 c and 11 d). A: His34, A: Glu35, A: Asp38, A: Lys353, E: Ser494, and E: Gly496 are the most important ones in terms of H-bonds for 9a -'ACE2-S1' ( Fig. 11 e and 11 f).

In silico ADMET studies
In silico ADME studies of compounds 8 ( a-f ) and 9 ( a-f ) were performed to predict their physicochemical descriptors using the Swiss ADME website [56] . Some of the ADME values of the compounds are given in Table 2 and the bioavailability radar images are shown in Figure S48 (see SI). The colored zone in the bioavailability radar image is the convenient physicochemical region for oral bioavailability [29] (the colored zone; Lipo (Lipophilicity): −0.7 < XLOGP3 < + 5.0, size: 150 < MW < 500 g/mol, polar (polarity): 20 Å 2 < TPSA < 130 Å 2 , insolu (Insolubility): 0 < log S(ESOL) < 6, insatu (insaturation): 0.25 < Fraction Csp 3 < 1, flex (flexibility): 0.25 < num. rotatable bonds < 9). According to this, the bioavailability radar images show that all compounds, except 8f and 9a , have acceptable physicochemical properties. These results mean that the compounds are suitable for oral bioavailability. Log S (ESOL) value is defined as in the water solubility and classes of solubility are listed as follows insoluble < −10 < poorly < −6 < moderately < −4 < soluble < −2 < very < 0 < highly. According to this, while compounds 8c, 8d, 9b are very soluble in the water, the other compounds are soluble in the water. All compounds have high GI absorption and do not cross the blood-brain barrier (BBB). The BBB diffusion is a crucial parameter due to toxicity, therefore the target compounds may be considered as good drug candidates. While compounds 8 ( a -c ), 9a, 9c , and 9e act as P-glycoprotein substrates, compounds 8d, 8e, 8f, 9b , and 9e do not act as P-glycoprotein substrates. All compounds obey the Lipinski, Ghose, Veber, Egan, and Muegge rules. In silico toxicity predictions of the target compounds were evaluated through the ProTox-II [57][58][59] , which is a free website. The oral rat acute toxicity (LD 50 ) values of the target compounds were predicted as 500 mg/kg, and the predicted toxicity classes of all were 4 ( Table 2 ). These predicted results mean that the target compounds show low toxicity profiles.

Materials and methods
All reagents and anhydrous solvents were purchased from Sigma-Aldrich, Alpha, Acros, and Merck and used without further purification. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker (Avance III) spectra were recorded on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS. Compounds 1, 4 ( a -f ), 5 ( a -f ), 6 ( a -f ), and 7 ( a -f ) were synthesized as mentioned in previous studies [ 42 , 43 , 46-51 ].

General procedure for the preparation of 8a-f and 9a-f
To a stirred solution of compound 1 (1 mmol) in 20 mL of methanol were added 6 ( a-f ) or 7 ( a-f ) (1 mmol) and then, added solutions of sodium L -ascorbate (0.1 mmol) in 5 mL of H 2 O and CuSO 4 •5H 2 O (0.1 mmol) in 5 mL of H 2 O, consecutively, at room temperature. Until the consumption of compound 1 , the mixture was stirred at room temperature for about 5 h. Then, to the mixture was added 20 mL of ethyl acetate, and extraction was performed with H 2 O (3 × 10 mL). The organic layer was dried over Na 2 SO 4 . The crude product was purified by washing with diethyl ether. ((1-(2-(4-methylpiperidin-1-yl) −2-oxoethyl) −1H-1,2,3-triazol-4yl)methyl)isoindoline-1,3-dione (8a).  compounds 8 ( a-f ) and 9 ( a -f ) [61] . 3D crystallographic structures of the ACE2-S1 complex, Mpro, and PLpro proteins were downloaded from the Protein Data Bank ( www.rcsb.org ) [62] (PDB ID:6M0J for the ACE2-S1 complex, PDB ID:7K40 for the Mpro, PDB ID:7CMD for the PLpro). All co-crystallized ligands and waters were deleted using Discovery Studio Visualizer 2021 [63] . The 2D structures of the compounds were drawn on ChemDraw Professional 16.0. The 2D structures of boceprevir, GRL0617, and MB were retrieved from the PubChem website [64] . The 2D structures of all compounds were minimized using the MMFF94s force field via Avogadro software [65] . In the docking analysis, the compounds were flexible, and the proteins were rigid. The grid boxes were created with the AutoDock 4.

Molecular dynamics simulation
Molecular dynamics simulations were performed for 50 ns using Desmond [67] , a Package of Schrödinger LLC (Schrödinger, LLC, New York, NY, 2014). The initial stage of protein and ligand complexes for molecular dynamics simulation were obtained from docking studies. Molecular Docking Studies provide a prediction of ligand binding status in static conditions. Simulations were carried out to predict the ligand binding status in the physiological environment. The protein-ligand complexes were preprocessed using Protein Preparation Wizard in Maestro, which also included optimization and minimization of the complexes. All systems were prepared by the System Builder tool. Solvent Model with an orthorhombic box was selected as TIP3P (Transferable Intermolecular Interaction Potential 3 Points). The OPLS_2005 force field [ 68 , 69 ] was used in the simulation. The models were made neutral by adding counter ions where needed. To mimic the physiological conditions, 0.15 M salt (NaCl) was added. The NPT [ 70 , 71 ] ensemble with 300 K temperature and 1 atm pressure were selected for complete simulation. The models were relaxed before the simulation. The trajectories were saved after every 100 ps for analysis, and the stability of simulations was evaluated by calculating the RMSD of the protein and ligand over time.

Conclusion
In summary, novel phthalimide analogs 8 ( a-f ) and 9 ( a-f ) bearing a 1,2,3-triazole framework were synthesized via CuAAC in good yields and characterized by various spectroscopic methods. The interactions with the proteins of SARS-CoV-2 (Mpro and PLpro) and the PPI between ACE2-S1 of the target compounds were examined via the molecular docking studies by comparison with MB, boceprevir, and GRL0617 inhibitors. All phthalimide analogs showed better interactions than MB in the interactions with the PPI of ACE2-S1 and compound 9a in the target compounds exhibited the best binding energy value ( −9.70 kcal/mol) and estimated Ki value (0.077 μM). In the interactions with the Mpro protein, compound 8a showed the best binding energy ( −8.76 kcal/mol) and estimated Ki value (0.379 μM). Regarding the interactions with the PLpro, compound 8b exhibited the best binding energy ( −8.87 kcal/mol) and estimated Ki value (0.315 μM) in the target compounds. The ligand-protein stabilities of compounds 8a -Mpro, 8b -PLpro, and 9a -'ACE2-S1' showing the best binding energy and predicted Ki were investigated via the molecular dynamics simulation studies. The stabilities of 8a -Mpro, 8b -PLpro, and 9a -'ACE2-S1' support the docking results. Finally, in silico ADMET studies were carried out. According to the predicted results, the target compounds have acceptable pharmacokinetic profiles and are suitable for oral administration. The compounds were estimated to display low toxicity levels. According to the in silico results, the target compounds may prevent the entry of SARS-CoV-2 into the host-cell, and the progression of the Mpro and PLpro proteins. Therefore, the compounds can be evaluated in vitro and in vivo studies as potential inhibitors of SARS-CoV-2.

Declaration of Competing Interest
The author declares no competing interests.