General: All the chemicals were acquired from S. D. fine chemicals, Spectrochem, Sigma-Aldrich, or Avra chemicals. Pre-coated silica gel TLC plates were used for reaction monitoring. All the mentioned yields are from un-optimized processes. Melting points were determined either using melting point apparatus or by differential scanning calorimetry (DSC) and are uncorrected. Using Perkin-Elmer FT-IR / Bruker spectrophotometer all IR spectra were recorded. The 1H-NMR spectra of final lead molecules were recorded on a Bruker Advance-II 500 MHz spectrometer using DMSO-d6 solvents and corresponding chemical shifts (δ) are expressed in parts per million (ppm). LC-MS-, Shimadzu (EI) was used to record the mass of compounds.
4.1 Chemistry:
2-Bromo-1-(substitutedphenyl)ethanone (2a – 2d)
Bromine was added drop-wise to a stirred solution of an acetophenone (1 mM) (1a – 1d) in ethanol (30 ml) and the solution was stirred at room temperature for 1 h and then poured into water to form a precipitate. This was re-crystallized from ethanol to give pure bromoacetophenone derivatives (2a – 2d) with an 85-95% yield.
2-bromo-1-phenylethan-1-one (2a): yield 87%; m.p. 53-56 ˚C; IR (KBr, cm-1): 3098, 3072, 2939, 1686, 1599, 1572, 748, 687.
2-bromo-1-(p-tolyl)ethan-1-one (2b): yield 92%; m.p. 51-52 ˚C; IR (KBr, cm-1): 3085, 3038, 2999, 2955, 1691, 1591, 725, 665.
2-bromo-1-(4-chlorophenyl)ethan-1-one (2c): yield 95%; m.p. 94-97 ˚C; IR (KBr, cm-1): 3083, 3019, 2966, 2940, 1676, 1592, 798, 758.
2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (2d): yield 85%; m.p. 32-34 ˚C; IR (KBr, cm-1): 3082, 3010, 2965, 2940, 1681, 1585, 868, 797, 769.
3,4-Dimethoxybenzonitrile (4a, 4b)
Added a solution of benzaldehyde / 3,4-dimethoxybenzaldehyde (3a, 3b) (1 equiv.) in 10 mL dimethylsulfoxide (DMSO) along with hydroxylamine hydrochloride (1.2 equiv.). The reaction mixture was stirred at 110 °C for 10 hours. After the reaction completion, the reaction mixture was poured into the ice-cooled water. Filtered the obtained precipitate, washed it with water and dried it under vacuum to get 4a, 4b.
Benzonitrile (4a): yield 83%; b.p. 188-190 ˚C; IR (KBr, cm-1): 3078, 3016, 2922, 2223, 1618, 1604, 1488, 812.
3,4-Dimethoxybenzonitrile (4b): yield 88%; m.p. 68-70 ˚C; IR (KBr, cm-1): 3122, 3085, 2962, 2840, 2223, 1596, 1582, 1466, 818.
3,4-Dimethoxybenzimidamide (5a, 5b)
To a solution of benzonitrile / 3,4-dimethoxybenzonitrile (1 equiv.) in ethanol, dry HCl gas was purged to saturation. The resulted solution was stirred for 10 hrs. Excess HCl gas was removed and ammonium carbonate (3 equivalent) was added to it. The resulted solution was stirred further for 10 hrs and concentrated on rotary evaporator to obtain the product 5a, 5b.
Benzimidamide (5a): yield 87%; m.p. 78-80 ˚C; IR (KBr, cm-1): 3333 (broad), 3053, 2968, 2903, 2842, 1615, 1598, 852, 808.
3,4-Dimethoxybenzimidamide (5b): yield 85%; m.p. 110-112 ˚C; IR (KBr, cm-1): 3305 (broad), 3210, 3089, 2996, 2835, 1644, 1606, 1591, 814.
4-(Substitutedphenyl)-2-(3,4-dimethoxyphenyl)-1H-imidazole (6-10)
A solution of benzimidamide (5a) / 3,4-dimethoxybenzimidamide (5b) (3 mmol), potassium bicarbonate (12 mmol) in THF (16 ml) and water (4 ml) was heated vigorously at reflux. Various bromoketone (2a-2d) (3 mmol) in THF (4 ml) was added over period of 30 mins and reflux further maintained for 2 hrs. THF was then recovered, and product was washed with water and recrystallized with ethanol to get pure products 6-10 respectively.
2,4-diphenyl-1H-imidazole (6): yield 67%; m.p. 168-170 ˚C; IR (KBr, cm-1): 3065, 3032, 1607, 1583, 1459, 714; 1H NMR (DMSO-d6): δ 12.63 (s, 1H, -NH), 8.02-8.01 (d, 2H, ArH), 7.86-7.85 (d, 2H, ArH), 7.72 (s, 1H, ArH), 7.49-7.46 (t, 2H, -SCH2), 7.40-7.35 (m, 3H, ArH), 7.24-7.21 (t, 1H, ArH); MS (m/z): 221.20 (M+H)+.
2-(3,4-dimethoxyphenyl)-4-phenyl-1H-imidazole (7): yield 71%; m.p. 120-122 ˚C; IR (KBr, cm-1): 3455, 3078, 3002, 2938, 2840, 1606, 1504, 765; 1H NMR (DMSO-d6): δ 12.48 (s, 1H, -NH), 7.86-7.85 (d, 2H, ArH), 7.72 (s, 1H, ArH), 7.61 (s, 1H, ArH), 7.56-7.54 (d, 1H, ArH), 7.38-7.35 (t, 2H, ArH), 7.21-7.18 (t, 1H, ArH), 7.06-7.04 (d, 1H, ArH), 3.86 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3); MS (m/z): 281.40 (M+H)+.
2-(3,4-dimethoxyphenyl)-4-(p-tolyl)-1H-imidazole (8): yield 68%; m.p. 105-108 ˚C; IR (KBr, cm-1): 3460, 3060, 3002, 2915, 2835, 1606, 1505, 764; 1H NMR (DMSO-d6): δ 12.42 (s, 1H, -NH), 7.73 (br, 2H, ArH), 7.61 (s, 1H, ArH), 7.57-7.56 (d, 2H, ArH), 7.20-7.19 (d, 2H, ArH), 7.05-7.04 (d, 1H, ArH), 3.85 (s, 3H, -OCH3), 3.80 (s, 3H, -OCH3), 2.31 (s, 3H, -CH3); MS (m/z): 295.20 (M+H)+.
4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)-1H-imidazole (9): yield 70%; m.p. 107-110 ˚C; IR (KBr, cm-1): 3457, 3000, 2920, 2835, 1590, 1495, 765; 1H NMR (DMSO-d6): δ 12.53 (s, 1H, -NH), 7.87-7.86 (d, 2H, ArH), 7.77 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.56-7.54 (d, 1H, ArH), 7.43-7.42 (d, 2H, ArH), 7.06-7.04 (d, 1H, ArH), 3.85 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3); MS (m/z): 315.20 (M+H)+, 317.20 (M+H+2)+.
4-(2,4-dichlorophenyl)-2-(3,4-dimethoxyphenyl)-1H-imidazole (10): yield 73%; m.p. 189-190 ˚C; IR (KBr, cm-1): 3459, 3035, 3002, 2957, 2836, 1590, 1498, 765; 1H NMR (DMSO-d6): δ 12.71 (s, 1H, -NH), 8.27-8.25 (d, 1H, ArH), 7.87 (s, 1H, ArH), 7.62 (s, 2H, ArH), 7.59-7.57 (d, 1H, ArH), 7.49-7.47 (d, 1H, ArH), 7.07-7.06 (d, 1H, ArH), 3.85 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3); MS (m/z): 349.25 (M)+, 351.20 (M+2)+, 353.25 (M+4)+.
4-(4-substitutedphenyl-1H-imidazol-2-yl)benzene-1,2-diol (11-14)
To obtain products 11-14, A solution of compound 6-10 respectively (1 equiv.) in dry DCM (36 mL) at 0 °C under N2 was treated drop wise with BBr3 (1M in DCM, 4 equiv.). The resulting mixture was allowed to attain the RT and stirred overnight, then it was drop wise poured to a stirring ice water (50 mL). The mixture was stirred for 30 min at RT then filtered and dried to obtain the products 11-14 as a yellow solid.
4-(4-phenyl-1H-imidazol-2-yl)benzene-1,2-diol (11): yield 73%; m.p. 98-100 ˚C; IR (KBr, cm-1): 3426, 3369, 3035, 2799, 1643, 1607, 1518, 686; 1H NMR (DMSO-d6): δ 14.33 (s, 1H, -NH), 10.13 (s, 1H, broad, variable –OH proton peaks), 9.53 (s, 1H, broad, variable –OH proton peaks), 8.20-8.18 (s, 1H, ArH), 7.91-7.90 (d, 2H, ArH), 7.58-7.39 (m, 5H, ArH), 7.02-7.00 (d, 1H, ArH); MS (m/z): 253.25 (M+H)+.
4-(4-(p-tolyl)-1H-imidazol-2-yl)benzene-1,2-diol (12): yield 69%; m.p. 118-120 ˚C; IR (KBr, cm-1): 3366, 3167, 2967, 1642, 1601, 1520, 715; 1H NMR (DMSO-d6): δ 14.15 (s, 1H, -NH), 10.12 (s, 1H, broad, variable –OH proton peaks), 9.51 (s, 1H, broad, variable –OH proton peaks), 8.13 (s, 1H, ArH), 7.80-7.78 (d, 2H, ArH), 7.48 (s, 1H, ArH), 7.44-7.42 (d, 1H, ArH), 7.36-7.35 (d, 2H, ArH), 7.01-6.99 (d, 1H, ArH), 2.37 (s, 3H, -CH3); MS (m/z): 267.25 (M+H)+.
4-(4-(4-chlorophenyl)-1H-imidazol-2-yl)benzene-1,2-diol (13): yield 72%; m.p. 305-307 ˚C; IR (KBr, cm-1): 3301, 3276, 3135, 2972, 2888, 1639, 1601, 1510, 808, 704, 647; 1H NMR (DMSO-d6): δ 14.35 (s, 1H, -NH), 10.13 (s, 1H, broad, variable –OH proton peaks), 9.53 (s, 1H, broad, variable –OH proton peaks), 8.23 (s, 1H, ArH), 7.95-7.93 (d, 2H, ArH), 7.65-7.63 (d, 2H, ArH), 7.49-7.48 (d, 1H, ArH), 7.45-7.43 (d, 1H, ArH), 7.01-6.99 (d, 1H, ArH); MS (m/z): 287.20 (M+H)+, 289.20 (M+H+2)+.
4-(4-(2,4-dichlorophenyl)-1H-imidazol-2-yl)benzene-1,2-diol (14): yield 70%; m.p. 269-271 ˚C; IR (KBr, cm-1): 3377, 3140, 2987, 1635, 1603, 849, 808, 705; 1H NMR (DMSO-d6): δ 14.55 (s, 1H, -NH), 10.07 (s, 1H, broad, variable –OH proton peaks), 9.51 (s, 1H, broad, variable –OH proton peaks), 8.04 (s, 1H, ArH), 7.89-7.88 (d, 1H, ArH), 7.85-7.83 (d, 1H, ArH), 7.69-7.67 ((d)d, 1H, ArH), 7.46-7.45 (d, 1H, ArH), 7.41-7.39 ((d)d, 1H, ArH), 6.99-6.97 (s, 1H, ArH); MS (m/z): 321.15 (M)+, 323.15 (M+2)+, 325.15 (M+4)+.
4.2. Enzyme inhibition (3CLpro) assay
The synthesized compounds were screened as inhibitors of SARS-CoV-2 3CLPro at 20 µM using an in vitro quenched fluorescence resonance energy transfer (FRET) assay using a fluorogenic substrate to measure the residual activity. For the study, the MBP-tagged 3CL Protease (SARS-CoV-2) Assay Kit (BPS Bioscience, San Diego, CA, USA) was used according to the manufacturer’s instructions [13,14]. For determination of the 3CLPro activity, 10 µL of the compounds was pre-incubated with 30µl of the 3CLPro for 30 min. Subsequently, the fluorogenic substrate was added to a final concentration of 50 µM and the reaction was incubated for 4 h in the dark in the presence of 1 mM 1,4-dithio-D, L-threitol (DTT). The fluorescence intensity was recorded at 460 nm / 360 nm. A positive control was included to measure the maximum activity of the protease in the absence of potential inhibitors. Moreover, an inhibition control GC 376 at 20 µM was included in the study. All the compounds were tested initially at 20 µM to establish the enzyme inhibition and antiviral activity. The hit molecules identified from the antiviral screening against SARS-CoV-2 (ancestral Wuhan and Delta) were subjected to IC50 determination against SARS-CoV-2 variants by plaque assay and 3CLpro inhibition by FRET assay.
4.3 Cell lines and viruses
The African green monkey kidney epithelial cell (Vero E6) was cultured in a humidified CO2 (5%) incubator at 370C, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 µg/mL) and Amphotericin B (0.25 µg/mL).
SARS-CoV-2 isolate USA-WA1/2020 (ancestral Wuhan strain) and B.1.617.2 (Delta) were obtained from bei Resources, USA. The virus stocks were prepared by propagating in Vero E6 cells by following the standard protocol [15,16]. The virus stocks were quantified by the gold standard plaque assay (Case, et. al., 2020). The SARS-CoV-2 infection study was carried out in high containment (BSL-3) facility.
4.4 Screening and determination of IC50 value through dose response curve generation
The compounds were solubilized in dimethyl sulfoxide (DMSO) and screened against SARS-CoV-2 (ancestral Wuhan and Delta) by plaque assay. For the initial antiviral screening, Vero E6 cells were seeded at a density of ~30000 cells per well, in 96 well flat bottom tissue culture plate in 200 µL of complete DMEM. The plate was incubated for 18-24 h, at 370 C in a humidified CO2 (5%) incubator. Next day, the medium was removed from the wells and the test compounds were added in duplicate to respective wells at a final concentration of 20 µM, followed by infection with SARS-CoV-2 isolates at approximately 30 plaque forming units per well. The plate was incubated at 370 C for 1 h, in a humidified CO2 (5%) incubator for adsorption of virus. The DMEM supplemented with 2.5% FBS (infection medium) was used to dilute the test compounds and virus. The final volume of infection medium containing test compounds and virus was maintained at 40µL per well to maximize the virus adsorption. After 1 h of incubation, the infection medium was removed from the wells and overlayed with DMEM-CMC and incubated for 72 h and then the plates were processed to score the plaques. The controls including virus only wells (with infection and without test compound) and cell only wells (without infection and test compound) were maintained as positive and negative controls, respectively. The percentage reduction of virus in test compound treated wells were calculated in comparison to positive control (virus only well).
The hit molecules identified in the initial screen were subjected to six-point dose response curve (DRC) generation (20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM, and 0.625 µM) and IC50 determination in Vero E6 cells. The IC50 of the test compounds was calculated by non-regression analysis using GraphPad prism version 9.2.0.
4.5 Molecular Docking:
The interactions of molecules under study with 3CLpro enzyme (PDB Code: 6LU7) were analyzed using ‘flexible docking’ protocol within BIOVIA Discovery Studio software [17]. To perform the flexible docking, residues (THR25, LEU27, HIS41, VAL42, CYS44, SER46, MET49, LEU50, TYR54, PHE140, LEU141, ASN142, SER144, CYS145, HIS163, HIS164, MET165, GLU166, LEU167, HIS172, ALA173, PHE181, VAL186, ASP187, ARG188, GLN189, THR190 and GLN192) were considered flexible in study. In the study, these residues were considered in creating flexible protein conformations using ChiFlex and side-chain refinement in the presence of the ligand using ChiRotor. In the process, generating protein confirmation was kept true with maximum number to 100. Ligand conformation generation was allowed using BEST method with maximum 255 conformations and energy threshold value to 20 for each ligand under study. Docking protocol was run with 100 number of hotspots and docking refinement was carried using simulated annealing with 2000 heating and 5000 cooling steps. The x,y,z coordinates for docking was set as -11.859595 13.885757 69.446622 respectively.