Design, synthesis and docking study of Vortioxetine derivatives as a SARS-CoV-2 main protease inhibitor

Designing of different analogues and synthesis for exploring structure-activity relationship (SAR)

Commercially available analytical grade solvents and reagents were purchased from commercial suppliers and were used without any further purification unless otherwise mentioned. Thin-layer chromatography (TLC) was performed for monitoring progress of reaction using commercially available Merck 60 F254 silica gel plate and visualized under UV light, and/or by spraying with freshly prepared phosphomolybdic acid (PMA) in methanol, followed by charring at high temperature. For purification of the crude compounds, column chromatography was performed on silica gel (100–200 mesh) or by using combiflash. All the 1H NMR and 13C NMR spectra were recorded at 25 °C using chloroform-d(CDCl3) or DMSO-D6 as deuterated solvents with tetramethylsilane (TMS) as an internal standard. The multiplicity of the reported peaks singlet, broad singlet, doublet, triplet, quadruplet and multiplet (or unwell-resolved signals) are denoted by s, br. s, d, t, q, and m respectively. All chemical shifts are reported in ppm (δ) and coupling constants (J) are in hertz (Hz). Mass Spectrometry (MS) data was recorded for unknown compounds 23–32 on Qtof-micro quadruple mass spectrophotometer. Elemental microanalyses were performed on elemental analyzer model flash 2000 thermo fisher for all new compounds.

General procedure for the synthesis of 23–32

New vortioxetine derivatives were synthesized according to following procedures. The synthetic procedure was modified for the synthesis of the vortioxetine derivatives. To a stirred solution of vortioxetine (1) (1 eq) in dry tetrahydrofuran (THF) (10 mL) was added sodium hydride (2 eq) at 0 °C. Contents were stirred at same temperature for 20 min, and then added alkyl/aryl/acyl/sulfonyl halide (1.5 eq) drop wise. The reaction mixture was stirred at room temperature until the reaction completes. The reactions were quenched using cold water and extracted with ethyl acetate (3 × 10 mL). The combined organic layer was washed with brine, dried over anhydrous sodium sulphate and evaporated under reduced pressure. Crude product was purified by silica gel column chromatography to afford pure compound 2325, 30–32.

1-(2-((2,4-dimethylphenyl)thio)phenyl)-4-vinylpiperazine, (23)

Yield: 52%; Off white solid, Rf: 0.8, AcOEt: Hexane (1:9), 1H-NMR (CDCl3, 300 MHz) δ 7.29 (d, J = 7.8 Hz, 1H), 7.16–6.73 (m, 5H), 6.39 (d, J = 7.5 Hz, 1H), 5.88–5.79 (m, 1H), 5.17–5.08 (m, 2H), 3.02–3.00 (m, 4H), 2.59–2.56 (m, 4H), 2.26 (s, 3H), 2.22 (s, 3H); 13C-NMR (CDCl3, 75 MHz) δ: 149.15, 142.39, 139.09, 136.19, 134.87, 134.56, 131.58, 127.70, 126.02, 125.36, 124.24, 119.77, 118.13, 61.79, 53.40, 51.49, 21.11, 20.51; MS (m/z): 325.18 [M + H]+. Anal. Calcd for C20H24N2S: C, 74.03; H, 7.46; N, 8.63; Found C, 74.21; H, 7.27; N, 8.48.

1-(2-((2,4-dimethylphenyl)thio)phenyl)-4-(prop-2-yn-1-yl)piperazine, (24)

Yield: 48%; Off White solid, Rf: 0.75, AcOEt: Hexane (1:9), 1H-NMR (CDCl3, 300 MHz) δ 7.29 (d, J = 7.8 Hz, 1H), 7.05–6.74 (m, 5H), 6.41 ( d, J = 7.8 Hz, 1H), 3.28 (s, 2H), 3.06–3.04 (m, 4H), 2.71–2.69 (m, 4H), 2.26 (s, 3H), 2.23 (s, 3H), 2.20 (m, 1H); 13C-NMR (CDCl3, 75 MHz) δ: 149.07, 142.47, 139.19, 136.25, 134.62, 131.69, 128.02, 127.81, 126.19, 125.48, 124.42, 119.88, 78.93, 73.34, 52.44, 51.43, 46.96, 21.22, 20.63; MS (m/z): 337.18 [M + H]+. Anal. Calcd for C21H24N2S: C, 74.96; H, 7.19; N, 8.33; Found C, 74.77; H, 7.25; N, 8.18.

1-(but-2-en-1-yl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine, (25)

Yield: 29%; Light brown sticky liquid, Rf: 0.7, AcOEt: Hexane (1:9), 1H-NMR (CDCl3, 300 MHz) δ 7.28 (d, J = 7.8 Hz, 1H), 7.05–6.72 (m, 5H), 6.39 (d, J = 7.5 Hz, 1H), 5.60–5.44 (m, 2H), 3.03–2.93 (m, 6H), 2.59–2.56 (m,4H), 2.26 (s, 3H), 2.22 (s, 3H), 1.60 (d, 3H); 13C-NMR (CDCl3, 75 MHz) δ: 149.30, 142.43, 139.13, 136.21, 134.61, 131.64, 129.52, 128.14, 127.76, 126.53, 126.17, 125.46, 124.30, 119.87, 60.90, 53.49, 53.38, 21.16, 20.56, 17.81; MS (m/z): 353.55 [M + H]+; Anal. Calcd. for C22H28N2S: C, 74.95; H, 8.01; N, 7.95; Found: C, 74.77; H, 7.88; N, 7.91.

Benzyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1-carboxylate, (30)

Yield: 69%; White solid, Rf: 0.9, AcOEt: Hexane (1:9), 1H-NMR (CDCl3, 300 MHz) δ 7.30–7.24 (m, 6H), 7.16–6.77 (m, 5H), 6.44 (d, J = 7.8 Hz, 1H), 5.09 (s, 2H), 3.63–3.60 (m, 4H), 2.95–2.93 (m, 4H), 2.27 (s, 3H), 2.23 (s, 3H) 13C-NMR (CDCl3, 75 MHz) δ;155.34, 148.80, 142.23, 139.18, 136.71, 136.01, 134.59, 131.65, 128.46, 127.96, 127.85, 127.75, 126.33, 125.49, 124.65, 119.85, 67.12, 51.48, 44.32, 21.12, 20.52; MS (m/z): 433.19 [M + H]+; Anal. Calcd. for C26H28N2O2S: C, 72.19; H, 6.52; N 6.48; Found: C, 72.01; H, 6.60; N 6.37.

1-(4-bromobenzyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine, (31)

Yield: 51%; White solid, Rf: 0.3, AcOEt: Hexane (1:9), 1H-NMR (DMSO-d6, 400 MHz) δ 7.53 (dd, J = 8.4 Hz, 2H), 7.32–7.29 (m, 3H), 7.21 (s, 1H), 7.13–7.06 (m, 3H), 6.90–6.88 (m, 1H), 6.38–6.36 (dd, J = 8.0 Hz, 1H), 3.52 (s, 2H) 2.97 (m, 4H), 2.54–5.52 (m, 4H), 2.32 (s, 3H), 2.22 (s, 3H); 13C-NMR (CDCl3, 100 MHz) δ; 149.18, 142.46, 139.20, 137.14, 136.25, 134.60, 131.66, 131.38, 130.99, 127.99, 127.79, 126.11, 125.44, 124.32, 120.96, 119.82, 62.42, 53.51, 51.55, 21.20, 20.60; MS: (m/z) 469.00 [M + H]+.

1-(4-bromo-2-fluorobenzyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine, (32)

Yield: 55%; White solid, Rf: 0.4, AcOEt: Hexane (1:9), 1H-NMR (DMSO-d6, 400 MHz) δ 7.52 (dd, J = 8.8 Hz, 1H), 7.51–7.40 (m, 2H), 7.29 (dd, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.11–7.06 (m, 3H), 6.88 (m, 1H), 6.39–6.37 (dd, J = 8.0 Hz, 1H), 3.58 (s, 2H), 2.97 (m, 4H), 2.54–2.52 (m, 4H), 2.31 (s, 3H), 2.21 (s, 3H) 13C-NMR (CDCl3, 100 MHz) δ; 149.18, 142.39, 139.15, 136.16, 134.58, 132.81, 132.76, 131.65, 128.01, 127.78, 127.28, 127.24, 126.20, 125.46, 124.33, 124.03, 123.88, 121.12, 121.03, 119.85, 119.10, 118.84, 54.89, 53.24, 51.52, 21.19, 20.59; MS (m/z): 484.95 [M + H]+, Anal. Calcd for C25H26BrFN2S: C, 61.85; H, 5.40; N, 5.77; Found: C, 61.85; H, 5.40; N, 5.77.

Cesium carbonate (3 eq), and xantphos (0.05 eq) were added to a solution of vortioxetine (1) (1 eq), aryl bromide (1.5 eq) in 1,4 dioxane (8 mL) solvent. For 20 min, inert argon gas was purged through the reaction mixture, followed by catalytic amounts of Pd2(dba)3(10 mol%) being added. Then the reaction mixture was heated at 110 °C for 12 h. The crude reaction mixture was passed through celite bed and washed with ethyl acetate (50 mL). The organic layer was diluted with water (20 mL), washed with brine, separated, dried over anhydrous Na2SO4 and concentrated to get crude sticky liquid material. Column chromatography (30% ethyl acetate in hexanes) of the crude extract yielded the product as a white solid.

4-(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)quinoline (26)

Yield: 44%; Off-White Solid. Rf: 0.4 (AcOEt: Hexane (3:7), 1H-NMR (DMSO-d6, 400 MHz) δ 8.72 (d, J = 4.8 Hz, 1H); 8.13 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.25 (br.s, 1H), 7.18–7.08 (m, 3H), 6.97 (m, 1H), 6.42 (d, J = 7.6 Hz, 1H), 3.36 (m, 4H), 3.31 (m, 4H), 2.33 (s, 3H), 2.26 (s, 3H). 13C-NMR (CDCl3, 100 MHz): δ 157.14, 150.87, 149.55, 148.79, 142.46, 139.37, 136.26, 134.81, 131.76, 129.97, 129.11, 127.88, 127.74, 126.28, 125.56, 125.34, 124.75, 123.77, 123.60, 120.02, 109.01, 52.66, 51.69, 21.22, 20.64; MS (m/z): 425.90 [M + H]+. Anal. Calcd for C27H27N3S: C, 76.20; H, 6.39; N, 9.87; Found: C, 76.47; H, 6.51; N, 9.82.

1-(2-((2,4-dimethylphenyl)thio)phenyl)-4-(5-(trifluoromethyl)pyridin-2-yl) piperazine (27)

Yield: 49%; Off-White Solid. Rf:0.3, AcOEt: Hexane (4:6), 1H-NMR (DMSO-d6, 400 MHz) δ; 8.44 (s, 1H), 7.84 (dd, J = 8.8,2.4 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.24 (br.s, 1H), 7.17–7.09 (m, 3H), 7.04 (d, J = 9.2 Hz, 1H), 6.95–6.91 (m, 1H), 6.42 (d, J = 8.0 Hz, 1H), 3.80 (m, 4H), 3.08 (m, 4H), 2.32 (s, 3H), 2.26 (s, 3H).13C-NMR (CDCl3, 100 MHz) δ; 160.58, 148.78, 145.80, 145.76, 142.39, 139.34, 136.18, 134.65, 134.53, 134.50, 131.75, 127.87, 127.75, 126.34, 125.57, 124.73, 119.81, 114.98, 105.64, 51.43, 45.26, 21.22, 20.63. MS (m/z) 443.97 [M + H]+. Calculated for C24H24F3N3S: C, 64.99; H, 5.45; N, 9.47; Found: C, 64.78; H, 5.29; N, 9.24.

To a solution of vortioxetine (1) (1 eq) in dry DCM (4 mL) solvent, was added N,N-diisopropyl ethylamine(2 eq) at 0 °C, stirred for 5 min, followed by dropwise addition of isocyanate (2 eq). The contents were stirred at room temperature for 2 h and TLC was checked for the completion of reaction. The reaction mixture was quenched using ice water and extracted with DCM (20 mL). The extract was washed with brine, dried over anhydrous Na2SO4, filter and evaporated to get dark colored crude semi-solid material. Crude product was further purified by combiflash column chromatography to get pure solid compound.

N-(3-chloro-4-fluorophenyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1-carboxamide (28)

Yield: 55%; Light brown solid. Rf:0.3 (AcOEt: Hexane 5:5), 1H-NMR (DMSO-d6, 400 MHz) δ; 8.77 (s, 1H); 7.75 (dd, J = 7.2, 2.8 Hz, 1H), 7.45–7.42 (m, 1H), 7.35–7.24 (m, 3H), 7.17–7.09 (m, 3H), 6.93 (t, J = 8.0 Hz, 1H), 6.42 (d, J = 8.0 Hz, 1H), 3.61 (m, 4H), 3.00 (m, 4H), 2.32 (s, 3H), 2.25 (s, 3H). 13C-NMR (DMSO-d6, 100 MHz) δ; 155.12, 151.56, 149.20, 142.08, 139.61, 138.32, 136.17, 133.87, 132.18, 128.49, 127.70, 126.38, 126.19, 125.10, 121.15, 120.90, 120.02, 119.95, 116.99, 51.67, 44.63, 21.19, 20.58; MS (m/z): 470.10 [M + H]+. Anal. Calcd for C25H25ClFN3OS: C, 63.89; H, 5.36; N, 8.94; Found: C, 64.02; H, 5.39; N, 8.80;

4-(2-((2,4-dimethylphenyl)thio)phenyl)-N-(pyridin-2-yl)piperazine-1-carboxamide (29)

Yield: 50%; Off white solid. Rf:0.35 (AcOEt: Hexane 3:7), 1H-NMR (DMSO-d6, 400 MHz) δ; 8.77 (br s, 1H), 8.66 (d, J = 2.0 Hz,1H), 8.16 (d, J = 3.2 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.29–7.27 (m, 2H), 7.18–7.12 (m, 3H), 6.95 (m, 1H), 6.43 (d, J = 8.0 Hz, 1H), 3.16 (m, 4H), 3.01 (m, 4H), 2.33 (s, 3H), 2.25 (s, 3H). 13C-NMR (CDCl3, 100 MHz,) δ; 155.06, 148.39, 143.63, 142.24, 141.16, 139.28, 136.43, 136.04, 134.54, 131.68, 127.79, 127.68, 126.25, 125.52, 124.78, 123.72, 123.59, 119.85, 51.36, 44.58, 21.11, 20.53; MS (m/z) 419.00 [M + H]+; Anal. Calcd for C24H26N4OS: C, 68.87; H, 6.26; N, 13.39; Found: C, 68.66; H, 6.12; N, 13.54.

Molecular docking

The X-ray crystal structure of SARS-CoV-2 Mpro complexed with co-ligand (N3) PDB ID: 6LU7 (2.16 Å) [6], was retrieved from Research Collaboratory for Structural Bioinformatics RCSB Protein Data Bank (www.rcsb.org). The Mpro crystal structure was imported in AutoDock Tools 1.5.6 [34] and removed water molecules and hetero atoms, and then added polar hydrogen’s followed by computing Gasteiger and adding Kollman charge. Finally, the protein was saved in pdbqt format. The OpenBabel software was used to convert ligands into PDB format [35]. Furthermore, the ligands were prepared by detecting the torsion root, correcting the torsion angles, assigning charges, optimizing using UFF [36] and finally converted into pdbqt format.

In this study, docking was performed using ADV in PyRx virtual screening open-source software [37]. The protein and ligand molecules to be docked are selected under the vina wizard control. The grid was generated by selecting the co-crystallized ligand and grid size can be adjusted according to the active site residues. A grid box with the size 58 × 68 × 70 with coordinates of center_x = -10.883, y = 13.934, and z = 68.209. During docking the grid spacing and exhaustiveness were 0.375 Å and 50, respectively. The docking Lamarckian Genetic Algorithm (LGA) was used [38].

These compounds were again re-docked using AD [39] to eliminate false positive software considering identical receptor grid coordinates. The top docking pose of ADV output file was visualized using Discovery Studio 2020 Client (BIOVIA 2016) software. The virtual screening and ADME were performed using Windows 10 OS in a 64-bit machine, Core 2 Duo CPU microprocessor with 4 GB RAM.

In-silico ADME and drug-likeness prediction

In silico Absorption, Distribution, Metabolism, Elimination (ADME) prediction is one of the important as well as significant criteria to estimate drug-likeness of the selected hits. Conventionally, ADME properties of drug molecules were determined in the last stage of the drug discovery process. In modern drug discovery, ADME properties can be predicted using the in-silico method in the early stage. Due to poor ADME properties, 60% of drug molecules failed in the development process. Therefore, early prediction of these properties would lead to the reduction of drug discovery costs [40]. In the present study, the potential hits were subjected to ADME prediction using a publicly available online web server: SwissADME (http://www.swissadme.ch [41]. Several properties like molecular weight, number of heavy atoms, number of aromatic heavy atoms, number of rotatable bonds, molar refractivity, topological polar surface area, solubility, gastrointestinal absorption, blood–brain, barrier penetration, Lipinski's rule of five, Ghose rule, Veber rule, bioavailability score, and synthetic susceptibility were predicted.

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