H)-quinazolinone and 2-methyl-1-substituted phenyl-4(1H)-quinazolinone derivatives and evaluate their activity against HCV in HCV subgenomic replicon assays. The biological data revealed that compound 11a showed the highest activity against HCV GT1b at a micromolar concentration (EC50 = 0.984 µM) followed by compound 11b (EC50 = 1.38 µM). Both compounds 11a and 11b had high selectivity indices (SI = CC50/EC50), 160.71 and 71.75, respectively, which make them very interesting candidates for further development of more potent and selective anti-HCV agents.Chronic hepatitis C virus (HCV) infections are a worldwide medical problem responsible for diverse types of liver diseases. The NS5B polymerase enzyme has become a very interesting target for the development of anti-HCV drugs owing to its fundamental role in viral replication. Here we report the synthesis of a novel series of 1-substituted phenyl-4(1 HCV is the main causative agent of chronic hepatitis ,2,3,4,5.Studying these crystal structures revealed that most of the above NNIs have the same conformational patterns and binding interactions with the NS5B TP-2 allosteric site. Two direct or water-mediated hydrogen bonds between the inhibitor molecules and backbone NHs of Ser476 and Tyr477 were observed. Furthermore, the inhibitor’s hydrophobic moieties bind with two hydrophobic pockets of the enzyme via stacking with the side chain of Leu419, Met423, Leu497, and Trp528 ,35,36. RI, II, IV, V, and VI), thiazolone , and quinazolinone  moieties. Aryl substituents in blue  +; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Yellow powder, yield 80%, mp 188–190 °C; IR (KBr) νmax/cmH)-one .General procedure for the synthesis of 6-nitro-1-(substitutedphenyl)quinazolin-4(13a or 3b (5 mmol) and triethyl orthoformate (10 mL) in ice path, sulfuric acid (2.5 mL) was added dropwise over 15 min. Then, the mixture was refluxed for 8 h (monitored by TLC). After the reaction’s completion, it was poured into ice water. The product was separated. The precipitated solid was filtrated off, washed with hot hexane, and dried in vacuo.To a stirring mixture of compound H)-one (4a).6-Nitro-1-phenylquinazolin-4(1−1: 3059 (CH-Ar), 2989 (CH-sp3), 1699 (C=O), 1550, 1334 (NO2); 1H NMR : δppm = 7.11 , 7.71–7.79 , 8.48 , 8.66 , 8.82 ; 13C NMR  δ 108.1, 119.3, 119.6, 123.7, 128.3 (2C), 128.7, 130.9, 131.0 (2C), 137.8, 145.1, 155.3, 167.9 (CO); MS m/z (%): 267.08 , 77 (100); Anal. Calcd. for C14H9N3O3 (267.08): C, 62.92%; H, 3.39%; N, 15.72%; Found: C, 63.15%; H, 3.61%; N, 15.92%. Greenish yellow powder, yield 85%, mp 246–248 °C; IR (KBr) νmax/cmm-tolyl)quinazolin-4(1H)-one (4b).6-Nitro-1-(−1: 3055 (CH-Ar), 2989 (CH-sp3), 1670 (C=O), 1527, 1334 (NO2); 1H NMR : δppm = 2.44 , 7.14 , 7.51–7.79 , 8.48 , 8.64 , 8.81 ; 13C NMR  δ 21.3 (CH3), 119.4, 119.50, 123.7, 125.3, 128.6, 128.7, 130.7, 131.2, 137.7, 140.8, 145.0, 145.1, 155.3, 167.9 (CO); C15H11N3O3; LC-ESI-MS (m/z): 282 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Green powder, yield 85%, mp 185 °C; IR (KBr) νmax/cmH)-one .General procedure for the synthesis of 6-amino-1-(substituted phenyl)quinazolin-4(14a or 4b (5 mmol), stannous chloride dihydrate , and conc. HCl (5 mL) in 15 mL ethyl acetate was stirred at room temperature for 2 h. The reaction was monitored with TLC. After the reaction was complete, the reaction mixture was diluted with a mixture of ethyl acetate and methanol (3:1) (200 mL) and the pH was adjusted to 7 through the addition of sodium carbonate powder. The solution mixture was filtered, and the organic filtrate was dried over sodium sulphate. Filtration of sodium sulphate was carried out, and the filtrate was evaporated under a vacuum to afford the crude products. The obtained residue was purified by silica gel chromatography using a hexane/ethyl acetate mixture (2:1) to give the desired compounds. A mixture of compound H)-one (5a).6-Amino-1-phenylquinazolin-4, 3055 (CH-Ar), 2885 (CH-sp3), 1635 (C=O); 1H NMR  δ 5.61 , 6.66 , 7.00 , 7.26 , 7.55–7.65 , 8.24 ; 13C NMR  δ 108.1, 118.2, 121.3, 122.2, 128.0 (2C), 130.4, 130.7 (2C), 131.9, 138.1, 148.2, 151.0, 169.0 (CO); C14H11N3O; LC-ESI-MS (m/z): 238 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, 67%, mp 293–295 °C; IR (KBr) νmax/cmm-tolyl)quinazolin-4(1H)-one (5b).6-Amino-1-, 3059 (CH-Ar), 2981 (CH-sp3), 1631 (C=O); 1H NMR  δ 2.41 , 5.60 , 6.67–6.72 , 6.98 , 7.62 , 7.23– 7.68 , 8.22 ; 13C NMR  δ 21.2, 108.0, 118.2, 121.3, 122.1, 127.3, 128.3, 130.4, 130.8, 130.9, 131.9, 138.1, 148.2, 150.9, 169.0(CO); C15H13N3O; LC-ESI-MS (m/z): 252 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, 65%, mp 288–290 °C; IR (KBr) νmax/cmN-acetamide .General procedure for the synthesis of 2-chloro-5a or 5b (4.2 mmol) and K2CO3  in CH2Cl2 (10 mL). The reaction mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, the solvents were evaporated, and ice water (100 mL) was added to the residue. The precipitate was filtered, washed with water, and left to dry in vacuo.Chloroacetyl chloride  was slowly added to a magnetically stirred mixture of compound N-acetamide (6a).2-Chloro-−1: 3275 (NH), 3070 (CH-Ar), 2958 (CH-sp3), 1689 (C=O); 1H NMR  δ 4.28 , 6.94 , 7.62–7.70 , 7.84 , 8.44–8.45 , 10.69 ; 13C NMR  δ 43.8, 116.8, 118.3, 120.3, 126.5, 128.1 (2C), 130.6 (2C), 130.8, 137.1, 137.2, 137.7, 153.0, 165.6 (CO); C16H12ClN3O2; LC-ESI-MS (m/z): 314 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Reddish white powder, 82%, mp < 300 °C; IR (KBr) νmax/cmN-(4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (6b).2-Chloro-−1: 3294 (NH), 3070 (CH-Ar), 2954 (CH-sp3), 1693 (C=O); 1H NMR  δ 2.40 , 4.31 , 6.94–7.00 , 7.38–7.74 , 7.86 , 8.42 , 8.47 , 10.85 ; 13C NMR  δ 20.1 (CH3), 43.8 (CH2), 116.7, 118.5, 120.3, 126.7, 127.2, 128.3, 130.6, 131.0, 131.4, 137.4, 137.5, 138.5, 152.8, 165.8 (CO), 167.5 (CO); C17H14ClN3O2; LC-ESI-MS (m/z): 328 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Reddish white powder, 84%, mp 290 °C; IR (KBr) νmax/cm-N-(4-oxo-1-(substituted phenyl)-1,4-dihydro quinazolin-6-yl)acetamide (7a–f).General procedure for the synthesis of 2-substituted phenoxy2CO3 (2.4 mmol), and compound 6a or 6b (1.6 mmol) in CH3CN (50 mL) was refluxed for 8 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated and a 4% NaOH solution (60 mL) was added to the residue to remove any remaining excess phenol as water-soluble sodium phenoxide salt. The product was separated and the precipitate was filtered, washed with water, and recrystallized to form methanol.A mixture of substituted phenol (2.4 mmol), KN--2-(p-tolyloxy)acetamide (7a).−1: 3271 (NH), 3032 (CH-Ar), 2920 (CH-sp3), 1643 (CO); 1H NMR  δ 2.25 , 4.69 , 6.92 , 7.11 , 7.27–7.66 , 7.96 , 8.43 , 8.51 , 10.44 ; 13C NMR  δ 20.6 (CH3), 67.8 (CH2), 112.0, 115.1 (2C), 117.2, 117.9, 126.6, 128.3, 130.3 (2C), 130.4, 130.8 (2C), 137.1, 138.1, 146.5, 147.9, 153.1, 156.2, 167.6 (CO); C23H19N3O3; LC-ESI-MS (m/z): 386 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Off-white powder, yield 80%; mp 220 °C; IR (KBr) νmax/cmN-acetamide (7b).2-(4-Chlorophenoxy)-−1: 3278 (NH), 3066 (CH-Ar), 2843 (CH-sp3), 1705 (CO), 1643 (CO); 1H NMR  δ 4.76 , 6.95 , 7.05 , 7.37 , 7.65 , 7.97 , 8.43 , 8.51 , 10.41 ; 13C NMR  δ 67.7 (CH2), 117.0 (2C), 117.2, 117.9, 120.2, 125.5, 126.7, 128.2 (2C), 129.7 (2C), 130.5, 130.8 (2C), 137.0, 137.2, 137.9, 153.1, 157.0, 167.2 (CO), 168.7 (CO); C22H16ClN3O3; LC-ESI-MS (m/z): 406 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Off-white powder, yield 80%, mp 262 °C; IR (KBr) νmax/cmN-acetamide (7c).2-(4-Chloro-3-methylphenoxy)-−1: 3275 (NH), 3066 (CH-Ar), 2978 (CH-sp3), 1685 (CO), 1643 (CO); 1H NMR  δ 2.31 , 4.73 , 6.89 , 6.93 , 7.05 , 7.33 , 7.63–7.74 , 7.94 , 8.45 , 8.50 , 10.42 ; 13C NMR  δ 20.2 (CH3), 67.6 (CH2), 114.2, 117.2, 118.0, 120.2, 125.8, 126.7, 128.2 (2C), 130.0, 130.5, 130.8 (2C), 137.0, 137.2, 137.9, 153.1, 156.9, 167.3 (CO), 168.7 (CO); C23H18ClN3O3; LC-ESI-MS (m/z): 420 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 85%, mp 255 °C; IR (KBr) νmax/cmN-(4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)-2-(p-tolyloxy)acetamide (7d).−1: 3282 (NH), 3062 (CH-Ar), 2958 (CH-sp3), 1705 (C=O); 1H NMR  δ 2.23 , 2.42 , 4.7 , 6.91 , 6.95–6.99 , 7.11 , 7.41–7.73 , 7.96 , 8.43 , 8.52 , 10.64 ; 13C NMR  δ 20.1 (CH3), 20.5 (CH3), 67.8 (CH2), 115.0 (2C), 117.1, 118.0, 120.2, 126.6, 127.5, 128.6, 130.3 (2C), 130.5, 131.0 (2C), 135.2, 136.8, 137.1, 138.3, 153.0, 156.1, 167.6(CO), 168.5 (CO); C24H21N3O3; LC-ESI-MS (m/z): 400 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS. Brown powder, 82%, mp 206 °C; IR (KBr) νmax/cmN-(4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (7e).2-(4-Chlorophenoxy)-−1: 3228 (NH), 3070 (CH-Ar), 2927 (CH-sp3), 1701 (C=O), 1643 (CO); 1H NMR  δ 2.42 , 4.80 , 6.94 , 7.04 , 7.35 , 7.41–7.74 , 7.97 , 8.42 , 8.52 , 10.86 ; 13C NMR  δ 21.3 (CH3), 67.7 (CH2), 117.0, 117.1, 118.0, 120.2, 125.2, 125.4, 126.6, 127.5, 128.6, 129.7, 130.5, 131.0, 135.2, 136.8, 137.0, 137.1, 138.3, 152.9, 157.1, 167.1 (CO), 168.5 (CO); C23H18ClN3O3; LC-ESI-MS (m/z): 420 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, 79%, mp 255 °C; IR (KBr) νmax/cmN-(4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (7f).2-(4-Chloro-3-methylphenoxy)-−1: 3278(NH), 3066 (CH-Ar), 2970 (CH-sp3), 1705 (CO), 1643 (CO); 1H NMR  δ 2.32 , 2.42 , 4.73 , 6.89 , 6.97 , 7.05 , 7.33 , 7.42–7.73 , 7.95 , 8.44 , 8.49 , 10.43 ; 13C NMR  δ 20.2 (CH3), 21.2 (CH3), 67.6 (CH2), 114.2, 117.2, 118.0, 118.1, 120.1, 125.1, 125.8, 126.8, 127.4, 128.4, 130.0, 130.6, 131.2, 136.9, 137.1, 137.8, 140.7, 153.0, 156.8, 167.4 (CO), 168.8 (CO); C24H20ClN3O3; LC-ESI-MS (m/z): 434 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Pale yellow powder, yield 85%, mp = 269 °C; IR (KBr) νmax/cmH)-one .General procedure for the synthesis of 2-methyl-6-nitro-1-(substituted phenyl)quinazolin-4(13a or 3b (5 mmol), acetic anhydride (10 mL) and sulfuric acid (1mL) was refluxed for 8 h (monitored by TLC). After the reaction was complete, it was poured into ice water. The product was separated out. The precipitated solid was filtrated off, washed with hot hexane, and dried in vacuo.A mixture of compound H)-one (8a).2-Methyl-6-nitro-1-phenylquinazolin-4(1−1: 3062 (CH-Ar), 1651 (CO), 1543, 1330 (NO2); 1H NMR : δppm = 2.18 , 6.77 , 7.66–7.82 , 8.40 , 8.80 ; 13C NMR  δ 24.9 (CH3), 118.9, 119.2, 122.6, 123.3, 128.5, 128.9, 129.9, 131.0, 131.4, 137.9, 144.6, 146.6, 163.5, 167.55 (CO); C15H11N3O3; LC-ESI-MS (m/z): 282 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Greenish yellow powder, yield 80%, mp 260–262 °C; IR (KBr) νmax/cmm-tolyl)quinazolin-4(1H)-one (8b).2-Methyl-6-nitro-1-(−1: 3059 (CH-Ar), 2993 (CH-sp3), 1658 (C=O), 1543, 1334 (NO2); 1H NMR  δ 2.18 , 2.44 , 6.82 , 7.45–7.82 , 8.35–8.40 , 8.71 ; 13C NMR  δ 21.3(CH3), 24.9(CH3), 118.7, 119.3, 123.2, 125.8, 128.1, 128.4, 129.1, 131.1, 131.6, 137.8, 141.3, 144.5, 146.5, 163.6 (CO); C16H13N3O3, LC-ESI-MS (m/z): 296 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Greenish yellow powder, 83%, mp 258–260 °C IR (KBr) νmax/cmH)-one .General procedure for the synthesis of 6-amino-2-methyl-1-(substituted phenyl)quinazolin-4(18a or 8b (5 mmol), stannous chloride dihydrate , and conc. HCl (5 mL) in 15 mL ethyl acetate was stirred at room temperature for 2 h. The reaction was monitored with TLC. After the reaction was complete, the reaction mixture was diluted with a mixture of ethyl acetate and methanol (3:1) (200 mL) and pH was adjusted to 7 through the addition of sodium carbonate powder. The solution mixture was filtered, and the organic filtrate was dried over sodium sulphate. The filtration of sodium sulphate and the evaporation of the solvent under a vacuum afforded the crude products. The obtained residue was purified by silica gel chromatography using a hexane/ethyl acetate mixture (2:1) to produce the desired compounds. A mixture of compound H)-one (9a).6-Amino-2-methyl-1-phenylquinazolin-4, 1708 (C=O); 1H NMR  δ 2.07 , 5.51, 6.28 , 6.89 , 7.22 , 7.52 –7.73 ; 13C NMR  δ 24.0 (CH3), 107.8, 118.1, 120.4, 121.9, 128.8, 130.4, 131.1, 134.0, 138.3, 147.5, 158.8, 159.5, 168.8, 175.4 (CO); C15H13N3O; LC-ESI-MS (m/z): 252 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, 69%, mp 268–270 °C; IR (KBr) νmax/cmm-tolyl)quinazolin-4(1H)-one (9b).6-Amino-2-methyl-1-, 3066 (CH-Ar), 2978 (CH-sp3), 1635 (C=O); 1H NMR  δ 2.08 , 2.40 , 5.52 , 6.35 , 6.91 , 7.21 , 7.38 , 7.54 , 7.00 ; 13C NMR  δ 20.1 (CH3), 24.1 (CH3), 107.9, 118.1, 120.5, 121.8, 128.1, 131.3, 133.8, 135.2, 137.2, 138.7, 147.5, 158.6, 168.6, 175.9 (C=O); C16H15N3O; LC-ESI-MS (m/z): 266 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Yellow powder, 67%, mp 270–273 °C IR (KBr) νmax/cmN-(2-methyl-4-oxo-1-(substituted phenyl)-1,4-dihydroquinazolin-6-yl)acetamide derivatives .General procedure for the synthesis of 2-chloro-9a or 9b (4.2 mmol) and K2CO3  in CH2Cl2 (10 mL). The reaction mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, the solvents were evaporated, and ice water (100 mL) was added to the residue. The product was separated out. The precipitate was filtered, washed with water, and left to dry in vacuo.Chloroacetyl chloride  was slowly added to a magnetically stirred solution of compound N-acetamide (10a).2-Chloro-−1: 3271(NH), 3074 (CH-Ar), 2962 (CH-sp3), 1701 (C=O); 1H NMR  δ 2.14 , 4.28 , 6.6 , 7.56–7.76 , 8.44 , 10.67 ; 13C NMR  δ 23.7 (CH3), 43.8 (CH2), 116.4, 118.6, 119.6, 126.5, 128.6, 130.8, 130.9 (2C), 131.3, 136.9, 137.5, 138.7, 161.4, 165.7 (CO), 166.8 (CO); C17H14ClN3O2; LC-ESI-MS (m/z): 328 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Yellow powder, 83%, mp 285 °C; IR (KBr) νmax/cmN-(2-methyl-4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (10b).2-Chloro-−1: 3271 (NH), 3073 (CH-Ar), 2958 (CH-sp3), 1701 (C=O); 1H NMR  δ 2.14 , 2.39 , 4.30 , 6.63 , 7.33–7.78 , 8.42 , 10.85 ; 13C NMR  δ 20.1 (CH3), 24.3 (CH3), 43.8 (CH2), 116.5, 118.2, 119.3, 126.3, 126.7, 128.0, 131.3, 131.4, 135.5, 136.5, 136.6, 139.0, 161.1, 165.6 (CO), 168.4 (CO); C18H16ClN3O2; LC-ESI-MS (m/z): 342 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.Reddish white powder, 88%, mp 280 °C; IR (KBr) νmax/cmN-acetamide (11a–f).General procedure for the synthesis of 2-substituted phenoxy-2CO3 , and compound 10a or 10b (1.6 mmol) in CH3CN (50 mL) was refluxed for 8 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated, and a 4% NaOH solution (60 mL) was added to the residue to remove any remaining excess phenol as water-soluble sodium phenoxide salt. The product was separated out. The precipitate was filtered, washed with water, and recrystallized to form methanol.A mixture of substituted phenol (2.4 mmol), KN--2-(p-tolyloxy)acetamide (11a).−1: 3271(NH), 3066 (CH-Ar), 2970 (CH-sp3), 1705 (CO); 1H NMR  δ 2.13 , 2.23 , 4.69 , 6.55 , 6.90 , 7.11 , 7.57–7.78 , 7.85 , 8.46 , 10.47 ; 13C NMR  δ 20.5 (CH3), 24.4 (CH3), 67.6 (CH2), 115.0 (3C), 116.9, 117.9, 119.4, 126.5, 128.9 (2C), 130.3 (2C), 130.6, 131.2 (2C), 136.3, 138.1, 139.1 156.0, 161.1, 167.6 (CO), 168.5 (CO); C24H21N3O3; LC-ESI-MS (m/z): 400 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 85%, mp 268 °C; IR (KBr) νmax/cmN-acetamide (11b).2-(4-Chlorophenoxy)-−1: 3267 (NH), 3070 (CH-Ar), 2974 (CH-sp3), 1705 (CO); 1H NMR  δ 2.12 , 4.76 , 6.56 , 7.05 , 7.36 , 7.57–7.78 , 7.84 , 8.47 , 10.52 ; 13C NMR  δ 24.4 (CH3), 67.6 (CH2), 117.0 (2C), 117.9, 119.4, 125.5, 126.5, 128.9 (2C), 129.7 (2C), 130.6, 131.2 (2C), 136.3, 138.1, 139.0, 157.0, 161.2, 167.2, 168.4 (CO), 169.6 (CO); C23H18ClN3O3; LC-ESI-MS (m/z): 420 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 87%, mp 254 °C; IR (KBr) νmax/cmN-acetamide (11c).2-(4-Chloro-3-methylphenoxy)-−1: 3267 (NH), 3074 (CH-Ar), 2927 (CH-sp3), 1701 (CO); 1H NMR  δ 2.13 , 2.30 , 4.71 , 6.53 , 6.89 , 7.04 , 7.34 , 7.60–7.69 , 7.82 , 8.48 , 10.32 ; 13C NMR  δ 20.3 (CH3), 24.5 (CH3), 67.7 (CH2), 114.3, 116.9, 117.9, 118.0, 119.4, 125.8, 126.4, 128.9 (2C), 130.0, 130.6, 131.2 (2C), 136.3, 137.0, 138.2, 139.0, 157.0, 160.9, 167.2(CO), 168.3(CO); C24H20ClN3O3; LC-ESI-MS (m/z): 434 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 80%, mp 271 °C; IR (KBr) νmax/cmN-(2-Methyl-4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)-2-(p-tolyloxy)acetamide (11d).−1: 3221 (NH), 3074 (CH-Ar), 2962 (CH-sp3), 1701 (CO); 1H NMR  δ 2.14 , 2.23 , 2.41 , 4.66 , 6.62 , 6.91 , 7.11 , 7.47–7.77 , 7.85 , 8.46 , 10.38 ; 13C NMR  δ 20.2 (CH3), 20.5 (CH3), 24.5 (CH3), 67.8 (CH2), 115.0 (2C), 116.9, 117.9, 119.4, 126.4, 128.2, 130.3 (2C), 130.4, 131.4, 131.6, 135.4, 136.5, 137.0, 138.8, 156.2, 160.7, 167.5 (CO), 168.2 (CO); C25H23N3O3; LC-ESI-MS (m/z): 414 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 85%, mp 270 °C; IR (KBr) νmax/cmN-(2-methyl-4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (11e).2-(4-Chlorophenoxy)-−1: 3221 (NH), 3074 (CH-Ar), 2927 (CH-sp3), 1701 (CO); 1H NMR  δ 2.14 , 2.42 , 4.73 , 6.64 , 7.04 , 7.36 , 7.46–7.75 , 7.82 , 8.45 , 10.40 ; 13C NMR  δ 20.2, 24.5, 67.8, 116.9(2C), 117.0, 118.0, 119.4, 121.8, 125.4, 126.3, 128.3, 129.7(2C), 131.4, 131.7, 135.4, 137.00, 138.8, 138.9, 157.1, 160.7, 167.0 (CO), 168.2(CO); C24H20ClN3O3; LC-ESI-MS (m/z): 434 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 85%, mp 283 °C; IR (KBr) νmax/cmN-(2-methyl-4-oxo-1-(m-tolyl)-1,4-dihydroquinazolin-6-yl)acetamide (11f).2-(4-Chloro-3-methylphenoxy)-−1: 3221 (NH), 3074 (CH-Ar), 2927 (CH-sp3), 1701 (CO), 1643 (CO); 1H NMR  δ 2.14 , 2.30 , 2.42 , 4.71 , 6.63 , 6.87 , 7.03 , 7.32 , 7.39–7.75 , 7.83 , 8.45 , 10.37 ; 13C NMR  δ 20.2 (CH3), 21.3 (CH3), 24.4 (CH3), 67.7 (CH2), 114.3, 116.4, 116.9, 118.0, 119.4, 125.8, 126.0, 128.2, 130.0, 131.6, 135.4, 136.4, 136.9, 137.0, 138.9, 138.9, 157.0, 160.9, 165.4, 167.2 (CO), 168.2 (CO); C25H22ClN3O3; LC-ESI-MS (m/z): 448 [M+H]+; purity was confirmed by HPLC-UV (254 nm)-ESI-MS.White powder, yield 87%, mp 290 °C; IR (KBr) νmax/cmv/v) fetal calf serum (referred to as complete DMEM). Complete DMEM was supplemented with G418 at 500 μg/mL for Huh5-2, 750 μg/mL for Huh7.5-3a, and 350 μg/mL for Huh7.5-4a, or puromycin at 0.5 μg/mL for Huh7-D2.The Huh5-2 stable cell line, containing the HCV subgenomic reporter replicon of GT 1b I389luc-ubi-neo/NS3-3′/Con1/5.1 (strain Con1), has been previously described  and was ® reduction bioassay, which is based on alamarBlue® reduction by cellular enzymes involved in the oxidation-reduction reaction. Specifically, 104 replicon cells were seeded in 96-well flat-bottom plates in a total volume of 100 μL of complete DMEM per well. After 24 h, cells were incubated with the compounds for 3 days at 37 °C (5% CO2). Cells treated with the solvent DMSO were used as the control. Ten microliters of alamarBlue® were added to each well, and cells were further incubated at 37 °C for 3 h. Absorbance was read at 570 nM (reduced) and 600 nm (oxidized) using a plate reader. The percent difference in dye reduction between treated and control cells was calculated and used for the determination of the compound concentration causing 50% cell death (CC50). CC50 values were determined by nonlinear regression analysis after converting the drug concentrations into log-X using the Prism 5.0 software .Cytotoxicity of the compounds was determined by the alamarBlue4 replicon cells per well in a 96-well flat bottom plate. The medium was exchanged with serial dilutions of the test compounds in complete DMEM. Compounds were diluted in DMSO, and the final concentration of DMSO in complete DMEM was 0.2%. Cells were cultured at 37 °C (5% CO2) and lysed after 3 days. Viral replication levels were determined by measuring Firefly luciferase (F-Luc) activity in cell lysates by using the Luciferase Assay System (Promega), as recommended by the manufacturer. Measurements were performed with a GloMax 20/20 single-tube luminometer (Promega) for 10 s. F-Luc activity was normalized to the total intracellular protein amount as determined by the Bradford assay (Pierce). Relative luminescence units (RLUs) were calculated as the percentage of the respective values from DMSO-treated control cells. The median effective concentration (EC50) was defined as the concentration of the compound that reduced the luciferase signal by 50%. EC50 values were determined by nonlinear regression analysis after converting the drug concentrations into log-X using the Prism 5.0 software (GraphPad Software Inc.). Daclatasvir used in the drug combination experiments was kindly provided by Dr. Marc Windisch (Institute Pasteur Korea). The coefficient of drug interaction in these experiments was calculated as follows: CDI = AB/(A × B) where AB is the ratio of the viral replication (luciferase activity levels) resulting from cells treated by the 2-drug combination, normalized to the control group (cells treated with the solvent DMSO), which is set to 1, and A or B is the ratio of the viral replication resulting from the single drug, normalized to the control group.An antiviral assay was performed by seeding 10Total RNA was extracted from replicon cells seeded in 12-well plates using the NucleoZOL reagent (Macherey-Nagel) based on the manufacturer’s recommendation and subjected to reverse-transcription (RT) for cDNA synthesis. cDNA was then used as a template for the quantitative real-time polymerase chain reaction (qPCR). RT was performed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega) and reverse primers specific for Con1 IRES (5’-GGATTCGTGCTCATGGTGCA-3’) and the housekeeping gene YWHAZ (5’-GGATGTGTTGGTTGCATTTCCT-3’). For the qPCR reactions, Con1 IRES-specific primers (forward: 5’-GGCCTTGTGGTACTGCCTGATA-3’ and reverse: 5’-GGATTCGTGCTCATGGTGCA-3’) and the Luna Universal qPCR Master Mix (NEB) were used. YWHAZ mRNA was used for normalization (forward: 5’-GCTGGTGATGACAAGAAAGG-3’ and reverse: 5’-GGATGTGTTGGTTGCATTTCCT-3’). Denaturing SDS-polyacrylamide gel electrophoresis and Western blotting were performed as previously described . The folt-test with p ≤ 0.05 were considered statistically significant and presented. Statistical calculations were performed with Excel Microsoft Office®.In the diagrams, bars represent the mean values of three independent experiments in triplicate. Error bars represent standard deviation. Only results subjected to statistical analysis using Student’s 11a or 11b was added to 1mL aliquot of deionized water. A stirrer magnet was placed, the tube was capped and rapped with aluminum foil then placed on a stirring plate at an ambient temperature. The suspension was agitated at an ambient temperature for 24 h after which it was centrifuged at 5000 rpm for 5 min. The clear supernatant was pipetted and filtered through a 0.2 µm syringe filter disc and then transferred to an HPLC vial. A 20 µL aliquot was injected into an HPLC system equipped with a UV detector set at 254nm. Elution was achieved isochratically on an RP C18 column (250 × 4.5 mm) with 90% methanol in water as a mobile phase. The column temperature was thermostatically controlled at 40 °C and the mobile phase flow rate was 1 mL/min. Linear regression analysis of integrated peak areas versus the concentration of a series of standard solutions of each compound in methanol (1–500 µg/mL) was used to prepare the calibration curve from which the concentrations of the solubility assessment samples were estimated. In a capped 5 mL polypropylene tube, an excess quantity of compounds All protein and ligand preparation, docking, and free energy calculations, as well as the analysis of the poses, were conducted using the Schrödinger platform, LLC, New York, NY, USA, release 2019-1. Ligand preparation was performed with the default protocol of LigPrep using Epik ,58 to ge3a,b, 2-substituted phenoxy-N-(4-oxo-1-(substituted phenyl)-1,4-dihydro quinazolin-6-yl)acetamide derivatives 7a–f, and 2-substituted phenoxy-N-(2-methyl-4-oxo-1-(substitutedphenyl)-1,4-dihydroquinazolin-6-yl)acetamide 11a–f are illustrated in 1a  and 11b  exhibited the highest anti-HCV potency. Similarly, compounds 11d and 11e demonstrated good anti-HCV activity but, unfortunately, with cytotoxic activity . We believe that the presence of a mono-substituted phenoxy ring in the para-position together with methyl substitution at C-2 of the quinazoline ring is essential for this potent anti-HCV activity.The anti-HCV activity of the analogues was also tested against other genotypes. Specifically, we measured their effect against viral replication-driven F-Luc activity in Huh7.5-3a and Huh7.5-4a stable cell lines containing the HCV GT 3a (strain S52) and 4a (strain ED43) replicons, respectively. Compounds = 8.4 μM . Neverth11a measured by the luciferase assay in HCV GT 1b was confirmed by determining viral RNA and NS5A protein levels. Serial dilutions of the compound, or its solvent DMSO (control), were used to treat Huh5-2 replicon cells. Viral RNA was quantified by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) and NS5A was evaluated by Western blotting. We found that compound 11a caused a reduction in HCV RNA replication  (To examine the activity of compound ir (DCV) . The two11a is 2.7 µgm/mL and for compound 11b is 3.5 µgm/mLThe observed water solubility of compound 11a–f compounds. The crystal structure of HCV NS5B polymerase with Protein Data Bank code 4JU1  was employed [IX  or extracted from crystal structures . The reserved crystal water forms a hydrogen bond with Leu497 but not with the ligands’ ether O, mostly due to the discrepancy in the conserved orientation of the water hydrogen since the latter is in very close proximity to it (aqueous O-ether O: 2.97 Å). Hence, in a less static environment for the crystal water, the water bridge between 11c-Leu497 is expected to take place. Moreover, a cation–π interaction between the protonated guanidine of Arg490 is observed  without forming hydrogen bonds. They both form a water bridge between the ether O and the Leu497 backbone and a sandwich π–π stacking with His475  and surprisingly even closer to the amide carbonyl O (3.07 Å) (Compound gand cf. . It form(3.07 Å) .3 substitution. In addition, its proximity to Trp528, His475, and the guanidine group of Arg501 open a possibility that the phenyl might form π–π interactions  and cation–π interactions (Arg501) in a dynamic environment. Furthermore, it is interesting that between the two pairs with identical binding modes, i.e., 11a, 11b, and 11d, 11e, the only difference is the exchange of a p-CH3 to a p-Cl, but this nonetheless results in differences in the respective EC50. We have shown that this structural characteristic also results in a difference in water solubility for 11a and 11b. Furthermore, since a cation–π interaction with the guanidine of Arg490 has been illustrated by our model for compounds 11c and 11f, which bear both groups, it cannot be excluded that such an interaction may take place during the binding process of the other ligands. In that case, the methyl substitution would enhance a possible cation–π interaction by enriching the electron density of the ring compared to the chloro-analogue—a feature difficult to simulate accurately in the MM environment.As observed in all docking poses, the phenyl group of the phenylquinazolinone core can be well accommodated within the newly revealed cleft, even with an m-CH11a–f displays a variety of SARs; however, not all of them are of equal importance  the co-crystallized ligands VIII and IX have the lowest ΔGbind; (b) the ligands 11a and 11b are next, although at a higher energy level; (c) the rest of the ligands follow at an even higher energy level. A trend can be clearly seen between the values of ΔGbind and the EC50 values. This profiling highlights that the formation of the two hydrogen bonds between Ser476 and Tyr477 and the quinazolinone core, or at least the optimal positioning of the latter towards the critical amino acids via other interactions , is of the highest importance, similarly to the lead compound binding mode. Ligands lacking these interactions and drifting away from this vicinity resulted in higher ΔGbind and EC50 values. Furthermore, it is notable that compounds bearing a 3-methyl group on the quinazolinone phenyl groups receive an energy penalty due to rotation hindrance and clash with the protein cleft.As presented above, the series of portance . In addi7a–f and 11a–f have lower LogP values (3.52–4.79) than the reported lead compound VII (LogP = 8.09) and the ligand compounds VIII and IX . Moreover, the target compounds 7a–f and 11a–f have better LogS values (−5.67 ~ −4.72) than compound VII (Log S = −6.02) and the ligand compounds VIII and IX . The ClogP and Log S values for all compounds were illustrated in H)-quinazolinone, and 2-methyl-1-substituted phenyl-4(1H)-quinazolinone derivatives have been prepared and tested for their antiviral potency against various HCV GTs. Compound 11a was the most potent against HCV GT 1b (EC50 = 0.98 µM) and showed a synergistic effect in combination with an approved NS5A inhibitor daclatasvir (DCV). The SAR study of the synthesized compounds revealed that the presence of both the mono-substituted phenoxy ring in the para-position and the methyl group at C-2 of the quinazolinone ring, as seen in compounds 11a, 11b, 11d, and 11e, is essential for potent antiviral activity. Due to the proximity of the C-2 methyl group to the phenyl group of the quinazolinone scaffold, it could be hypothesized that its presence may facilitate the opening of the cleft, rendering it better approachable by the rigid phenyl ring. Our computational investigations show that the phenyl analogues, which by a rigid approach were deemed not to fit in the binding site, can reach a lipophilic cleft within the pocket where they can be accommodated. As exhibited in the docking poses of 11a–f, there are possible SARs that might take place between the phenyl group and the newly revealed cleft in a dynamic environment. However, as shown both in the computational modelling and the pharmacologic data, these prospective SARs would be of less significance than the ones of the benzyl analogues, since neither our model showed any clear solid interactions forming in this cleft nor did our pharmacological evaluation result in higher affinity than the lead compounds. Nonetheless, it is important to exhibit that by minimal movement of lipophilic side chains, the known binding pocket can be enlarged to accommodate more rigid ligands, but also that this new cleft presents opportunities for SAR, which can be exploited in future drug design. Furthermore, the hydrogen bonds from the quinazolinone core were highlighted as the most important SARs. These findings may play an important role in further structure optimization aimed at the development and production of more potent anti-HCV agents.In conclusion, a novel series of anthranilic acid, 1-substituted phenyl-4(1


Conjugate addition proceeded upon treatment of the ortho-hydroxy-β-nitrostyrene with an amine; however, subsequent C–C bond cleavage readily occurred to afford the corresponding imine. Moreover, conversion of the trans-isomer to a cis-isomer did not occur efficiently, even when UV light was irradiated. We studied these unusual behaviors of β-nitrostyrene, focusing on the role of the ortho-hydroxy group.Nitrostyrene derivatives are widely used in organic syntheses as a substrate for Michael addition, photoisomerization and cycloaddition. In contrast,  A strongly electron-withdrawing nitro group activates the scaffold to undergo versatile reactions, which are used as building blocks for diverse purposes . In the 1a using a primary amine and N-chlorosuccinimide (NCS) . Th. Th1a usposition . Accordiynthesis ,20,21, i1a was subjected to reaction with 3 equiv. propylamine at room temperature in THF, conjugate addition occurred and was completed within 5 min to afford the corresponding adduct 2a in an 86% yield .When nitrostyrene 6% yield , Entry 11b. Using dry solvent did not effectively suppress C–C bond cleavage, which rules out the possibility that water contained in the solvent is involved. Hence, an ortho-hydroxy group might facilitate C–C bond cleavage by intramolecular hydrogen bonds . When the solution of the latter reaction mixture was left at room temperature for 2 h, the formation of N-chlorinated product 3b, imine 4b and nitrostyrene 1b was observed (Entry 3). The imine 4b was formed by C–C bond cleavage, and nitrostyrene 1b was a product of the E2 elimination of chloramine from 3b. Only small amounts of 3b were obtained, even though 2 equiv. of NCS was used and reaction time was prolonged (Entries 4 and 5). It is considered that the low reactivity of 2b is caused by the ortho-hydroxy group. The intramolecular hydrogen bond between the hydroxy and amino groups decreases the nucleophilicity of the amino group to suppress the N-chlorination, resulting in the low conversion of 2b. Even though 3b is produced, the hydroxy-supported formation of 4b might occur predominantly  in CDCl3 was subjected to the measurement of 1H-NMR, new signals appeared in the lower field. A couple of doublet splits with a 9.0 Hz coupling constant indicated that isomerization to cis-form  occurred, and half the amount of E-1c was consumed after 5 h under ambient conditions. While this isomerization was not influenced by the bulkiness of the O-acyl group and the addition of acids such as acetic acid, methanesulfonic acid and trifluoroborane–ether complex, the isomerization was accelerated under sunlight, which indicates that photoisomerization proceeded. On the contrary, E-1b revealed considerably lower reactivity for the photoisomerization in comparison with E-1a and E-1c.When a solution of E-1a–c, UV-Vis. spectra were measured  for 2.5 h in CDCl3  photoisomerization of E-1c to Z-1c, (ii) cycloaddition of Z-1c with nitrone 6 and (iii) hydrolysis of the acetate moiety of the cycloadduct, which afforded the formal adduct of Z-1b with nitrone 6 , four kinds of cycloadducts resulted in an 86% total yield. The stereochemistry for each isomer was determined by 1H-NssMR by comparing coupling constants with those of the literature : 188.0318, found: 188.0319.To a solution of ammonium acetate  in acetic acid (4 mL) were added nitromethane  and salicylaldehyde , and the resultant mixture was heated under reflux for 6 h. After cooling down to room temperature, water (40 mL) was added and the mixture was extracted with dichloromethane (30 mL × 3). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to afford ene (1b) ,29  in dichloromethane (8 mL) were added triethylamine  and acetyl chloride  at 0 °C, then the mixture was warmed to room temperature. After the mixture was stirred at room temperature for 14 h, water (50 mL) was added and was extracted with dichloromethane (30 mL × 3). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to afford E-2-(2-acetoxyphenyl)-1-nitroethene (1c) : 230.0424, found: 230.0423.To a solution of omethane  mL × 3. 1a  in THF (3 mL), propylamine  was added and the resultant mixture was stirred at room temperature for 5 min. The solvent was removed under reduced pressure to afford adduct 2a  as a yellow oil. When 1b and 1c were used, the reaction was conducted in the same way; however, adduct 2b could not be isolated upon treatment with column chromatography on silica gel, presumably due to the decomposition of the product. So, assignment of 1H-NMR was performed using a reaction mixture.To a solution of nitrostyrene 1-Nitro-2-phenyl-2-(propylamino)ethane (2a): Yellow oil. 1H-NMR  δ 0.87 , 1.41–1.51 , 2.45 , 4.40 , 4.49 , 4.57 , 7.30–7.40 ; 13C-NMR  δ 11.7 (CH3), 23.2 (CH2), 49.3 (CH2), 61.1 (CH), 81.1 (CH2), 127.2 (CH), 128.5 (CH), 129.1 (CH), 138.8 (C); IR (KBr/cm−1) 3400–3280 (br), 1634, 1602, 1553, 1521, 1380, 1343; HR-MS (ESI/TOF) calcd for C11H16N2O2 [(M + H)+]: 209.1277, found: 209.1285.2-(Hydroxyphenyl)-1-nitro-2-(propylamino)ethane (2b): 1H-NMR  δ 0.93 , 1.57 , 2.61 , 4.50 , 4.53 , 4.77 , 6.83 , 6.84 , 7.02 , 7.22 . N-H and O-H signals were not observed.2-(Acetoxyphenyl)-1-nitro-2-(propylamino)ethane (2c): 1H-NMR  δ 0.86 , 1.36–1.63 , 2.12 , 3.14–3.33  4.90 , 5.43 , 5.90 , 6.85 , 6.95 , 7.06 , 7.26 .1a  in acetonitrile (3 mL) was irradiated by UV light (310 nm) in the dark at room temperature for 17 h. After removal of the solvent under reduced pressure, the isomeric ratio was determined by 1H-NMR of the residue. The cis-isomer could be isolated by column chromatography on silica gel (eluted with hexane/ethyl acetate = 90/10) and the structure was confirmed by comparison with NMR data of authentic sample : 381.1001, found: 381.1000. Crystallographic data: empirical formula C21H17ClN2O3; formula weight 380.82; temperature (K) 93(2); crystal system orthorhombic; space group Pbca; unit cell dimensions a = 14.3835(8) Å, b = 10.4902(4) Å, c = 23.8298(12) Å; volume (Å) 3595.6(3); Z 8; ρcalc (g·cm–3) 1.407; absorption coefficient (mm–1) 0.237; θ range (°) 2.220–25.496; reflections collected 3345; independent reflections 2579; completeness to θ 99.9; goodness of fit 1.082; final R indices [I > 2σ(I)] R1 = 0.0499, wR2 = 0.1319; R indices  R1 = 0.0642, wR2 = 0.1426; largest diff. peak (e Å) 0.391; largest diff. hole (e Å) -0.357; CCDC 2,178,609.3,4-cis-4,5-cis-2-(4-Chlorophenyl)-3,5-diphenyl-4-nitroisoxazolidine (8′a): 1H-NMR  δ 5.31 , 5.59 , 5.95 , 6.94 , 7.23 , 7.34–7.58 .3,4-trans-4,5-trans-2-(4-Chlorophenyl)-3,5-diphenyl-4-nitroisoxazolidine (9a): 1H-NMR  δ 5.32 , 5.52 , 5.75 , 7.03 , 7.26 , 7.34–7.57 .3,4-cis-4,5-trans-2-(4-Chlorophenyl)-3,5-diphenyl-4-nitroisoxazolidine (9′a): 1H-NMR  δ 4.93 , 5.46 , 6.05 , 6.96 , 7.16 , 7.35–7.56 .1c and nitrone 7 was conducted in the same way as 1a. Only cis-isomer 8c could be separated by column chromatography on silica gel (eluted with hexane/ethyl acetate = 95/5), but other isomers were obtained as a mixture.The reaction of 3,4-trans-4,5-cis-5-(2-Acetoxyphenyl)-2-(4-chlorophenyl)-4-nitro-3-phenylisoxazolidine (8c): 1H-NMR  δ 2.25 , 5.21 , 5.44 , 5.77 , 7.01 , 7.21 , 7.24 , 7.28 , 7.42 , 7.59 , 7.42–7.54 .3,4-cis-4,5-cis-5-(2-Acetoxyphenyl)-2-(4-chlorophenyl)-4-nitro-3-phenylisoxazolidine (8′c): 1H-NMR  δ 2.35 , 5.34 , 5.53 , 5.98 , 6.93 , 7.18 7.24 , 7.31 , 7.35–7.41 , 7.40 , 7.56 , 7.78 .3,4-trans-4,5-trans-5-(2-Acetoxyphenyl)-2-(4-chlorophenyl)-4-nitro-3-phenylisoxazolidine (9c): Yellow oil, 1H-NMR  δ 2.21 , 5.20 , 5.33 , 6.02 , 7.02 , 7.15 , 7.25 , 7.33 , 7.67 .3,4-cis-4,5-trans-5-(2-Acetoxyphenyl)-2-(4-chlorophenyl)-4-nitro-3-phenylisoxazolidine (9′c): Signals of 9′c were too small to be analyzed, except for signals of the acetyl group and ring protons of isoxazolidine. 1H-NMR  δ 2.40 , 4.87 , 5.50 , 6.21 .8c  in a mixed solvent , 1 M hydrochloric acid  was added, and the resultant mixture was heated in a sealed tube at 100 °C for 3 h. After cooling to room temperature, water (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL × 3). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to afford a brown oil as a residue. The formation of the hydrolyzed product was confirmed via observation of a newly formed isoxazolidine ring; however, other aromatic protons could not be analyzed because of overlap with those by-products.To a solution of isoxazolidine 3,4-trans-4,5-cis-2-(4-Chlorophenyl)-5-(2-hydroxyphenyl)-4-nitro-3-phenylisoxazolidine (8b): 1H-NMR  δ 5.19 , 5.61 , 5.96 .


Z)-N-(adamantan-1-yl)-3,4-diarylthiazol-2(3H)-imines (5a-r) was synthesized via condensation of 1-(adamantan-1-yl)-3-arylthioureas (3a-c) with various aryl bromomethyl ketones (4a-f). The structures of the synthesized compounds were characterized by 1H NMR, 13C NMR and by X-ray crystallography. The in vitro inhibitory activities of the synthesized compounds were assessed against a panel of Gram-positive and Gram-negative bacteria, and pathogenic fungi. Compounds 5c, 5g, 5l, 5m, and 5q displayed potent broad-spectrum antibacterial activity, while compounds 5a and 5o showed activity against the tested Gram-positive bacteria. Compounds 5b, 5l and 5q displayed potent antifungal activity against Candida albicans. In addition, the synthesized compounds were evaluated for anti-proliferative activity towards five human tumor cell lines. The optimal anti-proliferative activity was attained by compounds 5e and 5k which showed potent inhibitory activity against all the tested cell lines. Molecular docking analysis reveals that compounds 5e and 5k can occupy the positions of NAD cofactor and the histone deacetylase inhibitor EX527 at the active site of SIRT1 enzyme.A series of ( The chemotherapeutic effectiveness of adamantane-based compounds was early explored after the discovery of amantadine6 and rimantadine7 as efficient medications for the control of Influenza A viral infections. Tromantadine was also developed as potent antiviral drug for the treatment of skin infection caused by herpes simplex virus8. Adamantane cage constitutes an important moiety of currently employed anticancer drugs. Adaphostin is a tyrosine kinase inhibitor that displays anti-proliferative activity in leukemia, non-small cell lung-cancer, and prostate cancer9. Adarotene, a IκB kinase-β inhibitor, was also developed as potent anticancer drug for the treatment of lymphoma, leukemia and prostate cancer10. The adamantane-based synthetic retinoid CD437 was also reported as a promising anticancer agent acting by inhibition of DNA polymerase11, while Opaganib is a recently approved adamantane-based anticancer drug for the treatment of advanced solid tumors12. Opaganib induces its anticancer activity via inhibition of sphingosine kinase (SK). Recently, opaganib proved to be a promising candidate for the treatment of severe COVID-19 pneumonia13. Other adamantane-based derivatives have demonstrated potent activity against pathogenic bacteria, mycobacteria and fungi. The adamantane-diamine hybrid derivative SQ109 was discovered for treatment of drug-resistant tuberculosis14, while the structurally-related dipiperidine analogue SQ609 was developed as a lead compound with potent action against Mycobacterium tuberculosis15 29. Dasatinib30, dabrafenib31 and alpelisib32 are currently-used drugs for the treatment of acute lymphoblastic leukemia, advanced melanoma and breast cancer, respectively -3-arylthiourea derivatives 3a-c in good yields41. The thiourea derivatives 3a-c were subsequently reacted with different aryl bromomethyl ketones 4a-f via prolonged heating in ethanol followed by addition of sodium acetate to yield the corresponding (Z)-N-(adamantan-1-yl)-3,4-diarylthiazol-2(3H)-imine derivatives 5a-r rather than the isomeric (Z)-3-(adamantan-1-yl)-4,N-diarylthiazol-2(3H)-imines 6a-r. The structures of the diarylthiazol-2(3H)-imines 5a-r were assigned based on 1H NMR, 13C NMR and elemental analysis. Single crystal X-ray analysis of compounds 5d and 5f. was also performed to assign their configuration and to exclude the formation of compounds 6a-r  as three singlets (condensed multiplets) at δ 1.65–1.76 (6H), 1.84–1.99 (6H) and 2.06–2.17 (3H) ppm. The thiazole CH protons were shown as distinguished singlets at δ 5.78–6.04 ppm. Meanwhile, the 13C NMR spectra of compounds 5a-r showed the adamantane carbons as four characteristic peaks at δ 29.64–30.12, 36.60–37.08, 40.63–41.17 and 53.49–54.19 ppm. In addition, the C5, C4 and C2 carbons of the thiazole ring were shown at δ 96.11–99.09, 148.47–150.28 and 158.89–160.08 ppm, respectively. The aromatic protons and carbons and their substituents were properly shown in their 1H NMR and 13C NMR spectra , symmetry element: 1-x, 1-y, 1-z). In addition, a weak C–H…S interaction  links the neighboring molecules into a chain that runs parallel to the crystallographic a axis as shown in Fig. The six-membered rings of the adamantane cage exhibits chair conformation as observed in related structures. In the crystalline state, the molecules pack in a columnar fashion Fig. b. Figure5a-r was assessed against a panel of standard pathogenic strains of the Institute of Fermentation of Osaka (IFO) namely; Staphylococcus aureus IFO 3060 (SA), Bacillus subtilis IFO 3007 (BS), Micrococcus luteus IFO 3232 (ML) (Gram-positive bacteria), Escherichia coli IFO 3301 (EC), Pseudomonas aeruginosa IFO 3448 (PA) (Gram-negative bacteria ) and the pathogenic fungi Candida albicans IFO 0583 (CA), Aspergillus oryzae IFO 4177 (AO) and Aspergillus niger IFO 4414 (AN). The initial screening was carried out using the semi-quantitative agar disc-diffusion method using Müller-Hinton agar medium42.The in vitro antibacterial and antifungal activity of newly designed compounds 5a-r (200 µg/disc); the antibacterial antibiotics Ampicillin trihydrate, Ciprofloxacin (100 µg/disc) and the antifungal drug Fluconazole (100 µg/disc) are shown in Table 5a, 5c, 5e, 5g, 5h, 6j, 5l, 5m, 5n, 5o, 5p and 5q, which produced growth inhibition zones ≥ 18 mm against one or more of the tested bacterial strains. Meanwhile, compounds 5b, 5d, 5f., 5i, 5k and 5r displayed moderate antibacterial activity (growth inhibition zones 14–17 mm). In addition, the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and to a lesser extent Micrococcus luteus are considered the most sensitive among the tested bacterial strains. The inhibitory activity against the tested Gram-negative bacteria was generally lower than the Gram-positive bacteria; compounds 5c, 5g, 5l, 5m and 5q showed potent inhibitory activity against Escherichia coli, while only compounds 5c, 5l and 5m were strongly active against Pseudomonas aeruginosa. The optimum antibacterial activity was attained by compounds 5c, 5g, 5l, 5m and 5q which displayed potent broad spectrum antibacterial activity.The results of the initial antimicrobial screening of compounds 5a-r was generally lower than their antibacterial activity. Compounds 5b, 5l and 5q displayed high activity against the yeast-like pathogenic fungus Candida albicans, while compounds 5j and 5k were moderately active and compounds 5c and 5h exhibited marginal activity (growth inhibition zones 10–13 mm). Compounds 5g, 5h and 5q were almost weakly against Aspergillus oryzae, and all the compounds were totally inactive against Aspergillus niger.The antifungal activity of the compounds 5a-f, the 3,4-diphenyl analogue 5a displayed potent antibacterial activity against the tested Gram-positive bacteria and lacked activity against the tested Gram-negative bacteria and pathogenic fungi. Replacement of the 4-phenyl group with 4-(4-chlorophenyl) substituent (compound 5b) deteriorated the antibacterial activity and greatly enhanced the activity against Candida albicans. Meanwhile, the 4-(p-tolyl) 5d and 4-(2-naphthyl) 5f. analogues retained moderate activity against the tested Gram-positive bacteria, with only marginal activity against the tested Gram-negative bacteria, and no antifungal activity. In addition, the 4-(4-methoxyphenyl) derivative 5e displayed good activity against Staphylococcus aureus and moderate activity against Bacillus subtilis and Micrococcus luteus with no activity against the tested Gram-negative bacteria and fungi. The optimum antibacterial activity was attained in the 4-bromophenyl analogue 5c which displayed excellent broad-spectrum antibacterial activity.In general, the antibacterial and antifungal potency and spectrum are greatly influenced by the nature of the aryl groups at positions 3 and 4 of the core thiazole nucleus. In the 3-phenyl series 5g-l, the 4-phenyl, 4-(4-chlorophenyl) and 4-(p-tolyl) derivatives 5g, 5h and 5j showed marked broad-spectrum antibacterial activity and weak antifungal activity against Candida albicans and/or Aspergillus oryzae. Unlike the antimicrobial activity in the 3-phenyl derivatives 5a-f, the antibacterial activity of the 4-bromophenyl analogue 5i was found inferior compared to the majority of the 3-(4-fluorophenyl) derivatives 5g-l, and with no antifungal activity. Meanwhile, the 4-(p-tolyl) 5j and 4-(4-methoxyphenyl) 5k analogues showed almost moderate or weak antibacterial activity and moderate activity against Candida albicans. In contrary to the antimicrobial activity of the 3-phenyl derivatives 5a-f, the 4-(2-naphthyl) analogue 5l displayed excellent broad-spectrum antibacterial activity in addition to potent antifungal activity against Candida albicans.In the 3-(4-fluorophenyl) series 5m-r, the optimum antibacterial activity was shown by the 4-phenyl 5m and 4-(4-methoxyphenyl) 5q derivatives which almost displayed potent broad-spectrum antibacterial activity, while the 4-(4-bromophenyl) derivative 5o retained potent and almost weak activity against the tested Gram-positive and Gram-negative bacteria, respectively. The antibacterial activity of the 4-(4-chlorophenyl) 5n and 4-(p-tolyl) 5p derivatives was found similar, but to lower extent, to the activity of the 4-(4-bromophenyl) derivative 5o. The 4-(2-naphthyl) analogue 5r only retained moderate or marginal activity against the tested Gram-positive bacteria. Potent and moderate antifungal activity against Candida albicans and Aspergillus oryzae, respectively, was displayed by the 4-(4-methoxyphenyl) 5q derivative.In the 3-(4-chlorophenyl) series 5c, 5l, 5m and 5q. The same results were clearly demonstrated by the antifungal activity of compounds 5b, 5l and 5q. Upon comparing the antibacterial activity of the two isomeric structures 5b and 5m, it has been discovered that the halogen substitution pattern is more beneficial at the N-3 position than the C-4 position.Taken together, the broad spectrum antibacterial activity was exhibited by monohalogenated derivatives rather than the dihalogenated ones. This is illustrated by the activity 5a, 5c, 5g, 5l, 5m, 5o and 5q) against the same microorganisms used in the primary screening were carried out using the micro-dilution susceptibility method in Müller-Hinton Broth45. The MIC and MBC values of compounds 5a, 5c, 5g, 5l, 5m, 5o, 5q, Ampicillin trihydrate, Ciprofloxacin and Fluconazole are depicted in Table 46. The MBC/MIC ratio for the tested compounds (Table Z)-N-(adamantan-1-yl)-3,4-diarylthiazol-2(3H)-imine derivative 5a, 5c, 5g, 5l, 5m, 5o and 5q could be considered as potential antibacterial candidates for further studies. Though these compounds are of inferior activity compared to the reference drugs, they have displayed MBC/MIC ratio equivalent to that of the references.The minimal inhibitory concentrations (MIC) and the minimal biocidal concentrations  for seven of the most active compounds  of compounds 5a, 5c, 5l, 5o and the broad-spectrum antibiotic Erythromycin towards the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus are shown in Table 50 (the lowest concentrations of tested compounds that exhibited 50% inhibition on the biofilm formation) ranged from 1.6 to 2.5 and 3.4 to 7.4 μg/mL against Staphylococcus aureus and Micrococcus luteus, respectively.The adamantane-linked thiazoles 5a-r was assessed against five human tumor cell lines namely; PC-3 (human prostate cancer), HCT-116 , HepG-2 (human hepatocellular cancer), HeLa  and MCF-7 (human breast cancer) using the 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay50. The obtained results, together with that of the anticancer drug doxorubicin, are shown in Table The in vitro anti-proliferative activity of the adamantane-linked thiazoles 5a-r displayed variable degrees of inhibitory activity against the tested cancer cell lines. In general, compounds 5e, 5g, 5h, 5k, 5o and 5q displayed potent anti-proliferative activity (IC50 < 25 μg/mL) against at least three of the tested cancer cell lines, compounds 5a, 5d and 5n showed moderate activity (IC50 > 25 to 50 μg/mL), compounds 5b, 5c, 5j and 5r showed weak activity (IC50 > 50 to 75 μg/mL) while compounds 5f., 5i and 5l were inactive (IC50 > 75 μg/mL). In addition, the optimal anti-proliferative activity was attained by compounds 5e and 5k which showed excellent activity against all the tested cell lines.Compounds N-(adamantan-1-yl)-thiazol-2(3H)-imine core. Within the 3-phenyl derivatives 5a-f, the 3,4-diphenyl analogue 5a exhibited moderate activity against HepG-2 and HeLa cell lines with weak activity against PC-3, HCT-116 and MCF-7 cell lines. Replacement of the 4-phenyl group with 4-(4-chlorophenyl), 4-(4-bromophenyl) or 4-(p-tolyl) substituents greatly deteriorated the anti-proliferative activity against PC-3 and HCT-116 cell lines with almost weak or marginal activity against HepG-2, HeLa and MCF-7 cell lines. The 4-(4-methoxyphenyl) substituent (compound 5e) is optimistic for anti-proliferative activity against all the tested cell lines. In contrary, a 4-(2-naphthyl) substituent (compound 5f.) dramatically declined the anti-proliferative activity.The anti-proliferative potency was found to be mainly dependent on the nature of the aryl groups at positions 3 and 4 of the 5g-l, the 4-phenyl analogue 5g displayed potent activity against PC-3, HepG-2 and MCF-7 cell lines and moderate activity against HCT-116 and HeLa cell lines, while the 4-(4-chlorophenyl) analogue 5h exhibited high activity against PC-3, HepG-2 and HeLa cell lines and moderate activity against HCT-116 and MCF-7 cell lines. In addition, the 4-(4-bromophenyl), 4-(p-tolyl) and 4-(2-naphthyl) derivatives 5i, 5j and 5l were almost inactive against all the tested cell lines. The 4-(4-methoxyphenyl) analogue 5k was found to the most active among the 3-(4-fluorophenyl) derivatives 5g-l which resembles the anti-proliferative activity of Doxorubicin.In the 3-(4-fluorophenyl) derivatives 5m-r was rather lower than the 3-(4-fluorophenyl) derivatives 5g-l. the 4-phenyl analogue 5m displayed potent activity against HeLa cell lines and moderate activity against PC-3, HCT-116, HepG-2 and MCF-7 cell lines, while the 4-(4-chlorophenyl) analogue 5n showed moderate activity against all the tested cell lines. In contrary to the activity of the 3-phenyl and 3-(4-fluorophenyl) derivatives (5c and 5i), the 4-(4-bromophenyl) analogue 5o showed potent activity against HCT-116, HeLa and MCF-7 cell lines, moderate activity against HepG-2 and week activity against PC-3 cell lines. As in case of the 3-phenyl and 3-(4-fluorophenyl) derivatives (5e and 5k). the anti-proliferative activity of the 4-(4-methoxyphenyl) analogue 5q was found to the most active among the 3-(4-chlorophenyl) derivatives 5m-r as it in displayed potent activity against HCT-116, HeLa and MCF-7 cell lines, moderate activity against HepG-2 and week activity against PC-3 cell lines.The anti-proliferative activity of the 3-(4-chlorophenyl) derivatives In view of the available results, it can be concluded that the presence of a 4-methoxyphenyl group at C-4 of the thiazole ring is optimal for the anti-proliferative activity, regardless of the nature of the C-3 substitution. Other C-4 substitutions diminish such activity, especially the 2-naphthyl group that impaired activity against all tested cell lines.51 was utilized to predict a suitable target of the designed (Z)-N-(adamantan-1-yl)-3,4-diarylthiazol-2(3H)-imine compounds 5a-r. The PASS analysis results indicated that the predicted highest probability of biological activity (Pa) was for histone deacetylase (HDAC) SIRT1 enzyme (Table 54 and thiazole59 derivatives.The online PASS (Prediction of Activity Spectra) structure–activity relationship toolme Table . The PASN-acetyl group from acetylated lysine residues in histones or some non-histone proteins. The overexpression of HDACs has been linked to a variety of solid and hematological malignancies, neurological disorders, inflammatory diseases and metabolic disorders62.HDACs are capable of catalyzing the removal of 5e and 5k showed excellent anti-proliferative activity against all the tested cell lines. In order to understand the bioactivity of these compounds, we carried out molecular docking against the SIRT1 enzyme. Before docking of 5e and 5k with SIRT1, we carried out a positive control docking simulation for the NAD cofactor and EX527 inhibitor63 (PDB ID: 4I5) with SIRT1. The result showed that there is an excellent agreement between experimental conformations and the predicted poses of NAD cofactor and EX527 .As shown in Table 5e and 5k bound in a similar manner at the active site of the SIRT1. Moreover, the adamantane cage of these compounds occupies the position of the cycloheptanecarboxamide moiety of the nanomolar inhibitor EX52764. The five-membered ring overlaps with the position of the 5-chlorophenyl of EX527. The methoxyphenyl and the substituted phenyl moieties of 5e and 5k are closer to the position of the nicotinamide and pentose sugar moieties of the NAD cofactor. The predicted pose of 5e and 5k clearly suggest that they could act as potential inhibitors.As can be seen from Fig. 5e and 5k because the binding poses of 5e and 5k are similar. The adamantane moiety establishes hydrophobic interactions with Ala 262, Ile 270, Asn 346, Ile 347 and Asp 348. The substituted phenyl also participated in the hydrophobic interaction with Gln 345 and Val 445. It is interesting to note that His 363 establishes π-stacking interaction with the substituted phenyl ring and the methoxy phenyl ring in compounds 5e and 5k. The active site residue Phe 414 has participated in a hydrophobic interaction with the methoxy phenyl ring.As shown in Fig. 1H NMR, 13C NMR data in addition to single crystal X-ray crystallography. The in vitro antibacterial, antifungal activities of the synthesized compounds were assessed against a panel of pathogenic Gram-positive, Gram-negative bacteria and fungi. Compounds 5c, 5g, 5l, 5m, and 5q displayed potent broad-spectrum antibacterial activity and compounds 5a and 5o showed potent activity against the tested Gram-positive bacteria, while compounds 5b, 5l and 5q displayed potent antifungal activity against Candida albicans. The anti-proliferative activity of the synthesized compounds was evaluated towards five human tumor cell lines. Compounds 5e and 5k showed potent inhibitory activity against all the tested cell lines. Molecular docking simulation analysis demonstrated that the potent compounds 5e and 5k bind to the active site of the SIRT1 enzyme, occupying the positions of the NAD cofactor and the known histone deacetylase inhibitor EX527. The adamantane moiety of these compounds establishes extensive hydrophobic interactions with the active site residues in addition to other interactions.Eighteen new adamantane-linked thiazole derivatives were synthesized and their structures were confirmed by 1H and 100.63 MHz for 13C; and Jeol ECA 500 III at 500.16 MHz for 1H and 125.77 MHz for 13C using CDCl3 as solvent. The chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) as internal standard; coupling constants (J) are pressed in Hz. The splitting patterns were designated as: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Monitoring the reactions and checking the purity of the final products were carried out by thin layer chromatography (TLC) using silica gel pre-coated aluminum sheets , petroleum ether/ethyl acetate (8:2) as eluent and visualization with ultraviolet light (UV) at 365 and 254 nm. Compounds 3a-c were prepared via condensation of adamantan-1-amine 1 with the corresponding aryl isothiocyanate according to the reported procedures41. Elemental analyses  were in agreement with the proposed structures within ± 0.3% of the theoretical values (Table 50 are presented in the supplementary data. The reference drugs Ampicillin trihydrate (CAS 7177-48-2), Ciprofloxacin (CAS: 85721-33-1), Fluconazole (CAS: 86386-73-4), Erythromycin (CAS: 114-07-8) and Doxorubicin (CAS 23214-92-8) were purchased from Sigma-Aldrich Chemie GmbH, Germany.Melting points  were measured in open glass capillaries using Stuart SMP30 electro-thermal melting point apparatus. NMR spectra were obtained on a Bruker 400 Avance III at 400.20 MHz for es Table . The sta3a-c (2.0 mmol) and the appropriate aryl bromomethyl ketone 4a-f (2.0 mmol), in ethanol (10 mL), was heated under reflux for 24 h. On cooling, sodium acetate (1.0 g) and crushed ice (100 g) were added and the mixture was stirred for 30 min. and allowed to stand at room temperature for 3 h. The precipitated crude product was filtered, washed with water, dried and crystallized from EtOH/CHCl3 or EtOH to yield the target compounds (5a-r) as fine colorless crystals.A mixture of the appropriate 1-(adamantan-1-yl)-3-arylthiourea (Z)-N-(Adamantan-1-yl)-3,4-diphenylthiazol-2(3H)-imine5a. Yield: 78%, m.p.: 198–200 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H26N2S (386.56). 1H NMR (500.16 MHz): δ 1.68 , 1.89 , 2.09 , 5.88 , 7.05–7.07 , 7.11–7.23 . 13C NMR (125.77 MHz): δ 29.64, 36.60, 40.63, 53.49 (Adamantane-C), 96.97 (Thiazole C-5), 126.02, 127.60, 127.77, 127.90, 129.05, 132.52, 138.84, 139.08 (Ar–C), 148.98 (Thiazole C-4), 160.02 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-chlorophenyl)-3-phenylthiazol-2(3H)-imine5b. Yield: 62%, m.p.: 176–178 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H25ClN2S (421.0). 1H NMR (400.20 MHz): δ 1.68 , 1.88 , 2.08 , 5.89 , 6.98 , 7.10–7.18 , 7.22–7.28 . 13C NMR (100.63 MHz): δ 29.83, 36.77, 40.82, 53.90 (Adamantane-C), 98.14 (Thiazole C-5), 123.99, 124.59, 126.65, 128.28, 128.45, 129.21, 133.82, 137.98 (Ar–C), 149.30 (Thiazole C-4), 159.34 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-bromophenyl)-3-phenylthiazol-2(3H)-imine5c. Yield: 55%, m.p.: 179–181 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H25BrN2S (465.45). 1H NMR (400.20 MHz): δ 1.68 , 1.88 , 2.08 , 5.89 , 6.93 , 7.10–7.18 , 7.22–7.30 . 13C NMR (100.63 MHz): δ 29.78, 36.65, 40.75, 54.19 (Adamantane-C), 99.09 (Thiazole C-5), 120.69, 122.28, 127.12, 128.57, 129.12, 129.58, 131.44, 138.22 (Ar–C), 148.90 (Thiazole C-4), 159.97 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-phenyl-4-(p-tolyl)thiazol-2(3H)-imine5d. Yield: 88%, m.p.: 158–160 °C (EtOH), Mol. Formula (Mol. Wt.): C26H28N2S (400.58). 1H NMR (400.20 MHz): δ 1.70 , 1.92 , 2.11 , 2.28 , 5.85 , 6.97–6.99 , 7.14–7.17 , 7.22–7.28 . 13C NMR (100.63 MHz): δ 21.29 (CH3), 29.89, 36.83, 40.86, 53.78 (Adamantane-C), 96.56 (Thiazole C-5), 123.90, 126.27, 127.98, 128.89, 129.36, 130.0, 137.78, 139.19 (Ar–C), 149.42 (Thiazole C-4), 160.08 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-methoxyphenyl)-3-phenylthiazol-2(3H)-imine5e. Yield: 85%, m.p.: 156–158 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C26H28N2OS (416.58). 1H NMR (400.20 MHz): δ 1.67 , 1.89 , 2.08 , 3.73 , 5.78 , 6.67 , 6.97 , 7.11–7.15 , 7.20–7.26 . 13C NMR (100.63 MHz): δ 29.87, 36.82, 40.86, 53.80 (Adamantane-C), 55.22 (OCH3), 96.13 (Thiazole C-5), 113.56, 125.12, 126.38, 128.10, 129.39, 138.88, 139.25, 157.71 (Ar–C), 149.77 (Thiazole C-4), 159.23 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4--3-phenylthiazol-2(3H)-imine5f. Yield: 80%, m.p.: 177–179 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C29H28N2S (436.62). 1H NMR (400.20 MHz): δ 1.76 , 1.99 , 2.17 , 6.04 , 7.08–7.26 , 7.47- 7.50 , 7.59 , 7.74- 7.78 . 13C NMR (100.63 MHz): δ 29.85, 36.78, 40.84, 53.94 (Adamantane-C), 98.21 (Thiazole C-5), 125.55, 126.38, 126.42, 126.51, 127.15, 127.56, 127.64, 128.08, 128.19, 129.21, 132.66, 133.02, 139.03, 139.18 (Ar–C), 150.02 (Thiazole C-4), 159.02 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-fluorophenyl)-4-phenylthiazol-2(3H)-imine5g. Yield: 90%, m.p.: 193–195 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H25FN2S (404.55). 1H NMR (500.16 MHz): δ 1.65 , 1.85 , 2.06 , 5.85 , 6.86–6.89 , 7.03–7.08 , 7.15–7.17 . 13C NMR (125.77 MHz): δ 29.88, 36.84, 40.93, 53.77 (Adamantane-C), 97.24 (Thiazole C-5), 114.86, 128.02, 128.29, 130.76, 132.54, 135.25, 138.90, 161.73 (Ar–C), 149.27 (Thiazole C-4), 159.78 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-chlorophenyl)-3-(4-fluorophenyl)thiazol-2(3H)-imine5h. Yield: 92%, m.p.: 194–196 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H24ClFN2S (438.99). 1H NMR (500.16 MHz): δ 1.66 , 1.85 , 2.07 , 5.87 , 6.89–6.93 , 6.96–6.99 , 7.04–7.07 , 7.14–7.16 . 13C NMR (125.77 MHz): δ 29.84, 36.76, 40.88, 53.99 (Adamantane-C), 98.20 (Thiazole C-5), 115.19, 128.62, 128.80, 129.11, 130.74, 134.09, 137.87, 161.95 (Ar–C), 149.76 (Thiazole C-4), 159.99 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-bromophenyl)-3-(4-fluorophenyl)thiazol-2(3H)-imine5i. Yield: 84%, m.p.: 221–223 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H24BrFN2S (483.44). 1H NMR (400.20 MHz): δ 1.66 , 1.85 , 2.07 , 5.88 , 6.89–6.95 , 7.04–7.08 , 7.29 . 13C NMR (100.63 MHz): δ 29.85, 36.81, 40.93, 53.80 (Adamantane-C), 97.90 (Thiazole C-5), 115.03, 122.14, 129.46, 130.71, 131.44, 135.04, 137.73, 162.04 (Ar–C), 148.72 (Thiazole C-4), 159.60 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-fluorophenyl)-4-(p-tolyl)thiazol-2(3H)-imine5j. Yield: 64%, m.p.: 144–146 °C (EtOH), Mol. Formula (Mol. Wt.): C26H27FN2S (418.57). 1H NMR (400.20 MHz): δ 1.69 , 1.89 , 2.10 , 2.29 , 5.84 , 6.90–7.01 , 7.09–7.12 . 13C NMR (100.63 MHz): δ 21.23 (CH3), 29.82, 36.76, 40.85, 53.78 (Adamantane-C), 96.68 (Thiazole C-5), 114.84, 127.95, 128.94, 130.77, 135.16, 137.94, 138.95, 161.96 (Ar–C), 149.56 (Thiazole C-4), 159.52 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-fluorophenyl)-4-(4-methoxyphenyl)thiazol-2(3H)-imine5k. Yield: 94%, m.p.: 179–181 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C26H27FN2OS (434.57). 1H NMR (400.20 MHz): δ 1.68 , 1.87 , 2.08 , 3.74 , 5.78 , 6.69 , 6.88–6.98 , 7.06–7.10 . 13C NMR (100.63 MHz): δ 29.86, 36.80, 40.90, 53.80 , 96.11 (Thiazole C-5), 113.67, 114.88, 124.85, 129.44, 130.85, 135.15, 138.68, 161.99 (Ar–C), 149.76 (Thiazole C-4), 159.55 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-fluorophenyl)-4-thiazol-2(3H)-imine5l. Yield: 60%, m.p.: 155–157 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C29H27FN2S (454.61). 1H NMR (500.16 MHz): δ 1.68–1.69 , 1.89–1.00 , 2.09 , 5.98 , 6.85–6.89 , 7.01–7.02 , 7.11–7.15 , 7.43–7.46 , 7.57 , 7.68 , 7.71–7.75 . 13C NMR (125.77 MHz): δ 29.86, 36.78, 40.85, 54.04 (Adamantane-C), 98.18 (Thiazole C-5), 115.10, 125.58, 126.58, 127.37, 127.74, 128.14, 129.79, 130.77, 130.84, 132.77, 133.06, 134.91, 139.13, 161.88 (Ar–C), 150.28 (Thiazole C-4), 159.93 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-chlorophenyl)-4-phenylthiazol-2(3H)-imine5m. Yield: 76%, m.p.: 190–192 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H25ClN2S (421.0). 1H NMR (500.16 MHz): δ 1.66–1.68 , 1.86–1.87 , 2.08 , 5.87 , 7.04–7.07 , 7.15–7.20 . 13C NMR (125.77 MHz): δ 30.12, 37.08, 41.17, 54.08 (Adamantane-C), 97.97 (Thiazole C-5), 128.25, 128.34, 128.44, 128.64, 130.67, 132.74, 138.09, 138.87 (Ar–C), 149.10 (Thiazole C-4), 159.09 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3,4-bis(4-chlorophenyl)thiazol-2(3H)-imine5n. Yield: 77%, m.p.: 197–199 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H24Cl2N2S (455.44). 1H NMR (400.20 MHz): δ 1.66 , 1.85 , 2.06 , 5.88 , 6.97 , 7.03 , 7.15–7.20 . 13C NMR (100.63 MHz): δ 29.81, 36.74, 40.86, 53.99 (Adamantane-C), 98.48 (Thiazole C-5), 128.45, 128.68, 129.18, 130.34, 134.08, 137.54 (Ar–C), 148.55 (Thiazole C-4), 159.02 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-4-(4-bromophenyl)-3-(4-chlorophenyl)thiazol-2(3H)-imine5o. Yield: 82%, m.p.: 210–212 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C25H24BrClN2S (499.90). 1H NMR (400.20 MHz): δ 1.66 , 1.84 , 2.07 , 5.89 , 6.91 , 7.03 , 7.18 , 7.30 . 13C NMR (100.63 MHz): δ 29.81, 36.75, 40.88, 53.93 (Adamantane-C), 98.46 (Thiazole C-5), 122.24, 128.42, 129.40, 130.33, 131.61, 137.53 (Ar–C), 148.47 (Thiazole C-4), 159.07 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-chlorophenyl)-4-(p-tolyl)thiazol-2(3H)-imine5p. Yield: 90%, m.p.: 188–190 °C (EtOH), Mol. Formula (Mol. Wt.): C26H27ClN2S (435.03). 1H NMR (500.16 MHz): δ 1.65–1.67 , 1.85–1.86 , 2.07 , 2.28 , 5.82 , 6.92 , 6.98 , 7.04–7.06 , 7.15–7.17 . 13C NMR (125.77 MHz): δ 21.33 (CH3), 29.87, 36.83, 40.91, 53.80 (Adamantane-C), 97.03 (Thiazole C-5), 127.90, 128.18, 129.09, 129.50, 130.47, 131.25, 138.02, 138.69 (Ar–C), 149.0 (Thiazole C-4), 158.89 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-chlorophenyl)-4-(4-methoxyphenyl)thiazol-2(3H)-imine5q. Yield: 82%, m.p.: 160–162 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C26H27ClN2OS (451.03). 1H NMR (500.16 MHz): δ 1.65–1.67 , 1.85–1.86 , 2.07 , 3.75 , 5.78 , 6.70 , 6.95 , 7.04 , 7.16 . 13C NMR (125.77 MHz): δ 29.85, 36.80, 40.86, 53.84 (Adamantane-C), 55.30 (OCH3), 96.47 (Thiazole C-5), 113.76, 124.84, 128.23, 129.39, 130.53, 131.71, 137.78, 138.44 (Ar–C), 149.43 (Thiazole C-4), 159.36 (Thiazole C-2).(Z)-N-(Adamantan-1-yl)-3-(4-chlorophenyl)-4-thiazol-2(3H)-imine5r. Yield: 89%, m.p.: 177–179 °C (EtOH/CHCl3), Mol. Formula (Mol. Wt.): C29H27ClN2S (471.06). 1H NMR (500.16 MHz): δ 1.68 , 1.88–1.89 , 2.09 , 5.99 , 6.99–7.02 , 7.10–7.15 , 7.44–7.48 , 7.58 , 7.69 , 7.71–7.75 . 13C NMR (125.77 MHz): δ 29.87, 36.83, 40.93, 53.90 (Adamantane-C), 98.34 (Thiazole C-5), 125.49, 126.59, 127.11, 127.76, 127.89, 128.13, 128.28, 130.05, 130.37, 131.72, 132.77, 133.12, 137.79, 138.71 (Ar–C), 148.94 (Thiazole C-4), 158.96 (Thiazole C-2).5d and 5f. (50 mg) were dissolved in 15mL cyclohexane/CHCl3 and EtOH/CHCl3 , respectively. Slow evaporation of the solutions at room temperature yielded the single crystals as colorless prisms.Analytically-pure samples of compounds 5e and 5k were drawn using ChemSketch Freeware (www.acdlabs.com) and these structures were subsequently optimized with the B3LYP/6-31G level of theory using the Gaussian 09 program65. The optimized structures were used for molecular docking simulations. The crystal structure of SIRT1 catalytic domain bound to NAD and an EX527 analog was retrieved from the protein data bank (PDB ID: 4I5I). The molecular docking simulation against SIRT1 was performed using the AutoDock Vina program66 available in PyRX-0.8 virtual tools67. The chain A of 4I5I and the ligands 5e and 5k were prepared and used for docking calculation. The active site grid was constructed based on the position of NAD ligand. To check the predictive power of the AutoDock Vina program, we also performed docking for the cocrystallized ligand NAD. The experimental and predicted poses were very similar. The top ranked binding poses were considered for protein–ligand interaction analysis using PLIP web server68.The structures of Supplementary Information.


Vaccinium bracteatum are well recorded in ancient Chinese medical books and were also demonstrated by modern researches. However, the relationship between its beneficial functions and specific chemical constituents has not been fully characterized. This study investigated the bioactive small-molecule constituents in the leaves of V. bracteatum, which afforded 32 compounds including ten new ones (1–9) and ten pairs of enantiomers (9–18). Their structures with absolute configurations were elucidated by spectroscopic methods, especially nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) analyses, with 1–4 bearing a novel revolving-door shaped scaffold. While half-compounds exhibited decent antioxidant activity by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, all except 19 and 20 exerted significant capturing activity against diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals. In addition, the new iridoids 1, 5, 6, and 7 exerted apparent neuroprotective activity toward PC12 cells, with 1 being comparable to the positive control, and selective compounds also displayed anti-diabetic and anti-inflammatory properties by inhibiting α-glucosidase and NO production, respectively. The current work revealed that the bioactive small-molecule constituents could be closely related to the functional food property of the title species.The health benefits of  Vaccinium Linn. represents a large group of shrubs or small arbors with about 450 species distributed worldwide +; HR-ESIMS m/z 399.1414 [M + Na]+ .Yellowish gum; -d4) see ; 1H NMR c 0.2, MeOH); UV (MeOH) λmax (log ε) 312 (4.61) nm; 1H and 13C NMR data (methanol-d4)  δH 7.60 , 7.55 , 6.80 , 6.38 , 4.79 , 4.70 , 3.96 , 4.08 , 3.97 , 3.32 , 3.33 , 2.07 , 2.06 , 1.90 , 1.59 , 1.72 ; 13C NMR  δC 166.6 (C-9′), 159.9 (C-4′), 144.8 (C-7′), 130.3 (C-2′/6′), 125.1 (C-1′), 115.8 (C-3′/5′), 114.2 (C-8′), 98.6 (C-1), 97.3 (C-3), 77.2 (C-8), 73.4 (C-6), 69.9 (C-10), 55.1 (1-OCH3), 54.7 (3-OCH3), 45.4 (C-9), 45.3 (C-7), 40.2 (C-5), 28.8 (C-4); ESIMS m/z 416.9 [M + Na]+; HR-ESIMS m/z 417.1517 [M + Na]+ .Yellowish gum; -d4) see ; 1H NMR λmax (log ε) 278 (4.16) nm; 1H NMR  δH 6.81 , 6.78 , 6.69 , 6.66 , 4.80 , 4.30 , 3.85 , 3.80 -OCH3), 3.78 , 3.77 , 3.54 , 2.59 , 1.79 ; 13C NMR  δC 151.7 (C-3′), 148.9 (C-3(5)), 147.2 (C-4′), 137.9 (C-1′), 135.9 (C-4), 133.3 (C-1), 121.8 (C-6′), 119.3 (C-5′), 113.9 (C-2′), 105.6 (C-2(6)), 86.4 (C-8), 74.3 (C-7), 62.3 (C-9), 62.2 (C-9′), 56.7 (3(5)-OCH3), 56.4 (3′-OCH3), 35.5 (C-8′), 32.7 (C-7′); ESIMS m/z 430.9 [M + Na]+; HR-ESIMS m/z 443.1481 [M + Cl]– .Colorless gum; data see ; UV ; UV (MeOH) λmax (log ε) 283 (4.17) nm; ECD  λ (Δε) 209 (–15.3) nm. 1H NMR  δH 6.63 , 6.58 , 6.56 , 6.52 , 6.47 , 3.82 , 3.68 , 3.76 , 3.73 , 3.51 , 3.40 , 2.99 , 2.87 , 2.53 , 1.74 ; 13C NMR  δC 148.7 (C-3′), 148.3 (C-5), 145.2 (C-4), 143.7 (C-4′), 133.72 (C-1), 133.70 (C-1′), 129.4 (C-5′), 122.6 (C-2), 122.0 (C-6′), 115.6 (C-3), 113.8 (C-6), 110.6 (C-2′), 65.8 (C-9), 62.2 (C-9′), 56.5 (3′-OCH3), 56.2 (5-OCH3), 45.6 (C-8), 37.5 (C-7), 35.8 (C-8′), 32.8 (C-7′); ESIMS m/z 385.2 [M + Na]+.White solid; 5 (8 mg) in 2.5 mL methanol was added excess 8% aqueous NaOH (0.2 mL), and the reaction mixture was stirred at 80 °C for 90 min till the finish of hydrolysis monitored by TLC. The reaction solvent was then evaporated, and the residue was purified by a silica gel column  to give the desired product 5r (4.0 mg) as a yellowish gum. 1H and 13C NMR data (CDCl3)  δH 4.95 , 4.76 , 4.10 , 3.47 , 3.46 , 3.46 , 3.45 , 2.17 , 2.15 , 2.06 , 1.98 , 1.77 , 1.54 ; 13C NMR  δC 99.5 (C-3), 99.4 (C-1), 81.7 (C-8), 77.1 (C-6), 69.6 (C-10), 56.0 (1-OCH3), 55.9 (3-OCH3), 47.4 (C-9), 46.2 (C-7), 44.5 (C-5), 32.5 (C-4).To a stirred solution of molecule Cl3) see . 1H NMR 8 (10 mg) was processed with the same procedure to afford 8r (7.7 mg). 1D NMR data (CDCl3)  δH 4.77 , 4.76 , 4.11 , 3.455 , 3.448 , 3.445 , 3.42 , 2.19 , 2.16 , 1.98 , 1.88 , 1.79 , 1.68 ; 13C NMR  δC 100.8 (C-1), 99.5 (C-1), 80.4 (C-8), 75.0 (C-6), 69.9 (C-10), 56.02 (1-OCH3), 55.96 (3-OCH3), 46.7 (C-9), 45.7 (C-7), 42.6 (C-5), 29.9 (C-4).Compound Cl3) see . 1H NMR 5 (2.0 mg) in pyridine (0.5 mL) was added (R)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (10 μL) and 4-dimethylaminopyridine . The reaction mixture was stirred at room temperature for 4.5 h, and the crude product acquired after removal of the solvent was further purified by HPLC to give the (S)-MTPA ester 5a (0.9 mg). The (R)-MTPA ester 5b, as well as the (S) and (R)-MTPA esters 8a and 8b of 8, was prepared via the same protocol.To a solution of compound See The antioxidant effect of the VBL constituents was assessed by besting their free radical scavenging activity with the ABTS  and DPPH  methods, according to the protocols described previously , and ascThe nitric oxide (NO) production inhibitory assay was conducted in murine RAW264.7 macrophages as we recorded in an early report , with quThe acetylcholinesterase (AChE) inhibitory assay was carried out according to a method reported formerly , and tacThe α-glucosidase inhibitory effect of all the molecules was tested by a previously described method , with acStaphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633, as well as the Gram– strains Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739, were screened using the liquid growth inhibition method as formerly documented . In addition, signals for two acetal methines , an oxygenated sp3 quaternary carbon (δC 82.2), an sp3 oxymethine , an sp3 oxymethylene , and a methoxy group , as well as non O-bonded aliphatic signals , were further observed. The aforementioned NMR features accounted for six indices of hydrogen deficiency, and the remaining three suggested a tricyclic backbone for 1. Further inspection of 2D NMR 1H–1H COSY and HMBC data -coumaroyloxy unit was linked to C-10 by the HMBC correlations from H2-10 to C-9′. Finally, considering the remaining undescribed ring, the HMBC signal from H-3 to C-8, and the chemical shifts for C-3 and C-8, an ether bridge between C-3 and C-8 was established. The relative configuration of 1 was determined by 3D conformational analysis and the examination of NOESY data as shown in 1 is very rigid and presents an interesting revolving-door shape , which leads to the only possibility for the relative configuration assignments at C-3, C-5, C-8, and C-9. Then, the NOESY correlations of H-1/H-10a and H-4b/H-6 assigned the C-1 and C-6 relative configurations as drawn.The molecular formula CNMR data  for 1 exMBC data  establis2 was given a molecular formula same as 1 based on the HR-ESIMS ion at m/z 385.1261 , being an isomer of the latter. The NMR data -coumaroyloxy group in the former, replacing the (E)-counterpart in the latter, which was evidenced by the resonances at δC 167.8, 160.2, 145.5, 133.7 (2C), 127.6, 116.1, 115.9 (2C), together with δH 7.64, 6.76 , 6.90, 5.82 . Subsequent examination of 2D NMR spectra , being 14 mass units less than that of 1 and indicative of a demethyl analogue of the latter. Analysis of the NMR data . The NMR data . In addition to the (E)-coumaroyloxy unit as in 1, the NMR data , 4.77 ], an oxygenated sp3 quaternary carbon (δC 80.0), an sp3 oxymethine , an sp3 oxymethylene , two methoxy groups , and a series of non-oxygenated aliphatic units , 2.04, 1.83, 1.56]. These NMR features were consistent with those of 1, except for the appearance of an extra methoxy unit (3-OMe). Further inspection of 1H–1H COSY data (2)CH2CH– (H-1/H-9/H-5/(H-6/H2-7)/H2-4/H-3), which was subsequently linked to other structural moieties and/or quaternary carbon(s) by the HMBC signals from 1-OCH3 and H-3 to C-1, 3-OCH3 to C-3, and H2-10 to C-7, C-8, C-9, and C-9′. The constitution structure of 5 was thus constructed, and the difference of 5 from 1 was that the epoxy bond linking C-3 and C-8 in the latter was cleaved and replaced by the 8-OH and 3-OMe groups in the former. The relative configuration of 5 was established to be identical with that of 1 by interpretation of NOESY and 1D-NOE data . Careful comparison of the NMR data -coumaroyloxy group in the latter by a (Z)-coumaroyloxy group in the former. As with the case of 1 and 2, compound 6 was identified to be the Z-isomer of 5, which was further confirmed by examination of full 2D NMR data , being 18 mass units less that of 5 and indicative of a dehydration analogue. Detailed inspection of the NMR data CH2– (C-6/C-7) moiety in 5 being replaced by those for a 1,2-disubstituted double bond  in 7, which was also supported by analyses of 1H–1H COSY and HMBC data , being isomeric with the latter. The NMR data  to that of 5r (4.9 Hz) in methanol-d4, established the remaining chiral centers as shown. Compound 8 was thus characterized as the 3-epimer of 5.Compound NMR data  for 8 wedata see  demonstrdata see . The key1–6 and 8 all had only one UV chromophore, which is not vicinal to any of the chiral centers, and thus they did not show discernible Cotton effects in the ECD measurements. Therefore, it is not possible to assign their absolute configurations via ECD-related methods , a 1,3,4,5-tetrasubstituted and symmetrical benzene unit , three methoxy groups , two sp3 oxymethines , two sp3 oxymethylenes , and two normal aliphatic methylenes .,23.9 waserythro-4,7,9,9′-tetrahydroxy-3,3′-dimethoxy-8,4′-oxyneolignane (10) ntiomers , lignansproducts . However reports ,41. Anot in 2006  and a pa in 2006  based on-workers , whereas9a/9b, the 8R and 8S absolute configurations for 10a and 10b were determined by the (–) and (+) Cotton effects at 239 nm, respectively [11a and 11b were assigned 8R and 8S absolute configurations based on their respective (–) and (+) Cotton effects at 236 nm. For the benzofuran-type lignans, molecule 12a was determined to have the 7S,8R-configuration on the basis of its (+) and (–) Cotton effects at 293 (1Lb band) and 227 (1La band) nm, respectively [12b thus had the 7R,8S-configuration. Compounds 13a/13b and 14a/14b showed highly consistent ECD curves with the corresponding enantiomers 12a/12b, and their absolute configurations were hence established as shown. Owing to the introduction of an additional methoxy group, the ECD spectra of 15a/15b exhibited some variations compared to those of 12–14, and their absolute configurations were assigned as drawn via the Cotton effects at 292 nm [16a and 16b were differentiated from each other and assigned the drawn absolute configurations by comparing their ECD curves with the computed ones -enantiomeric form with the trivial name icariol A2 [21 was separated, without acquisition of its enantiomer, from the mixture of 12a/12b via chiral HPLC separation, and only one previous report described the separation of both enantiomers but without determining the absolute configuration [R-configuration to 21 based on the TD-DFT-based ECD calculation , compounds 9a, 12a, 17a/17b, 18a/18b, and 21 performed particularly well, with IC50 values below 10 μM. Meanwhile, all the iridoid constituents (1–8) did not exhibit a scavenging effect toward DPPH radicals, whereas most lignans showed moderate activity.The antioxidant activities of extracts acquired through different methods from  reports , and sev reports . However reports . We thus1–8, and the promotion or inhibition on cell growth of these compounds was first excluded in the testing concentration range by the same assay. The 6-OHDA-induced cell injury was first evaluated in the range of 50–250 μM, and 150 μM with an inhibition ratio of 52.68% was chosen for the final experiment , 11b (IC50 = 61.80 ± 3.58 μM), 12a (IC50 = 69.37 ± 3.32 μM), and 18a (IC50 = 46.36 ± 7.92 μM) also exhibited significant inhibition against the enzyme, while compounds 9a/9b, 10a, 13a, 16a/16b, 17a/17b, and 18b were less active, with IC50 values ranging from 106.3 to 311.9 μM.The hypoglycemic effect, an important strategy to control diabetes, is the most well-known and investigated health benefit of tabolism . In the V. bracteatum, by evaluating their inhibition against cyclooxygenase-2 and NO production, are also documented in the literature [14a displayed mild activity (IC50 = 52.16 ± 6.17 μM).The anti-inflammatory properties of different extracts from S. aureus, B. subtilis, and E. coli with positive feedback [P. aeruginosa was further included in the present study’s antibacterial assays on all the molecules, but none of them displayed decent activity. Moreover, the inhibition of all these compounds against AChE, an important neurodegenerative disease target, was also screened, whereas they were all inactive.The preservative effect of VBL extract on food has long been known and applied by local residents , and thifeedback . In addiV. bracteatum afforded only several glycosides, and no free aglycones were reported [1–4 feature a novel revolving-door shaped scaffold. The reason of this constituent difference could be attributed to the extracting solvent and the research focus, as more polar solvent systems (mainly MeOH-H2O) were used in the most previous reports, and the separation work generally concentrated on the polar compounds. As for the lignan metabolites from V. bracteatum, there were only three members counted in the 2021 review [Iridoids represent a unique group of natural products of the monoterpene family, and they are mainly found in dicotyledonous plants . Former reported . Eight f1 review . In thisV. bracteatum is a rich source of natural antioxidants, and lignans and iridoids, in addition to the previously described flavonoids, could also make an important contribution to its antioxidation-relevant health benefits.Oxidative stress, a resultant physiological state from the imbalance of the redox system, is usually accompanied by the accumulation of destructive free radicals (or reactive species) and decreasing protection from antioxidant defense , and it In summary, both crude extracts and specific chemical constituents from VBL exhibit a variety of bioactivities related to its traditional health-beneficial applications . Althoug1–9) and ten pairs of enantiomers (9–18) from VBL, and their chemical structures and absolute configurations were determined by NMR, HR-ESIMS, and ECD analyses, as well as chemical derivatization and degradation. By screening these compounds in a panel of bioassays, the antioxidant activities of most isolates, along with the neuroprotective, α-glucosidase inhibitory, and NO release inhibitory effects of selective molecules, were validated. The current work expanded the knowledge on both the chemistry and bioactivity of the constituents from V. bracteatum and further consolidated our understanding toward this plant as a source of functional food. All in all, there is still a lot more to be explored and exploited regarding this fascinating herbal species.In the present study, we obtained 32 small molecule constituents including ten new ones (


Penicillium chrysogenum, located 15 km northwest of Jazan, KSA. The elucidation of the chemical structures of the isolated compounds was performed by analysis of their NMR, MS. Compounds 1–5 were tested for antibacterial activities against Gram-positive and Gram-negative bacteria. All of the compounds exhibited selective antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Bacillus licheniformis except compound 3. The chloro-dihydroisocoumarin derivative, compound 4, showed potential antimicrobial activities against all of the tested strains with the MIC value between 0.8–5.3 μg/mL followed by compound 5, which exhibited a moderate inhibitory effect. Molecular docking data showed good affinity with the isolated compounds to β-Lactamase enzymes of bacteria; NDM-1, CTX-M, OXA-48. This work provides an effective strategy for compounds to inhibit bacterial growth or overcome bacterial resistance.Antibiotic resistance is considered a major health concern globally. It is a fact that the clinical need for new antibiotics was not achieved until now. One of the most commonly prescribed classes of antibiotics is β-Lactam antibiotics. However, most bacteria have developed resistance against β-Lactams by producing enzymes β-Lactamase or penicillinase. The discovery of new β-Lactamase inhibitors as new antibiotics or antibiotic adjuvants is essential to avoid future catastrophic pandemics. In this study, five dihydroisocoumarin: 6-methoxy mellein (1); 5,6-dihydroxymellein (2); 6-hydroxymellein (3); 4-chloro-6-hydroxymellein (4) and 4-chloro-5,6-di-hydroxymellein (5) were isolated from Wadi Lajab sediment-derived fungus  Discovery of antibiotics and the broad use of antibiotics play a clinically significant role in the treatment of bacterial infections, prolonging human life and reducing mortality. However, the rise of antibacterial resistance has caused a severe loss in the effectiveness of antibiotics ,2. The pAcinetobacter spp., Escherichia coli and in Klebsiella pneumoniae S)-5,6-Dihydroxymellein 2(D25 + 60.0 (c = 0.06 in CHCl3), HREIMS m/z: 210.03, 1H-NMR : δH 1.45 , 2.40 , 2.98 , 4.54 , 6.20 , 11.0 (8-OH). 13C-NMR : δc 19.9 (CH3), 29.5 (C-4), 75.0 (C-3), 100.1 (C-9), 102.3 (C-7), 126.2 (C-5), 135.6 (C-10), 155.2 (C-6), 158.1 (C-8), 169.1 (C-1).White amorphous powder, [α]S)-6-Hydroxymellein 3(D25 − 51.0 (c = 0.10 in CH3OH), HREIMS m/z: 194.02, 1H-NMR : δH 1.39 , 2.82 , 2.92 , 4.68 , 6.20 , 6.24 , 11.14 (8-OH). 13C-NMR : δc 20.7 (CH3), 34.1 (C-4), 75.8 (C-3), 100.5 (C-9), 101.3 (C-7), 107.2 (C-5), 142.2 (C-10), 163.8 (C-6), 164.9 (C-8), 169.9 (C-1).White amorphous powder, [α]R)-4-Chloro-6-hydroxymellein 4(D25 − 74.5 (c = 1.05 in CHCl3), HREIMS m/z: 228.01, 1H-NMR : δH 1.41 , 4.86 , 4.60 , 6.35 , 6.55 , 11.05 (8-OH). 13C-NMR : δc 17.5 (CH3), 57.2 (C-4), 75.2 (C-3), 100.0 (C-9), 102.4 (C-7), 108.0 (C-5), 141.5 (C-10), 163.1 (C-6), 165.0 (C-8), 168.1 (C-1).White amorphous powder, [α]R)-4-Chloro-5,6-di-hydroxymellein 5(D25 − 5.8 (c = 0.4 in CHCl3), HREIMS m/z: 244.42, 1H-NMR : δH 1.44 , 4.85 , 4.65 , 6.43 , 11.21 (8-OH). 13C-NMR : δc 19.1 (CH3), 56.8 (C-4), 76.0 (C-3), 101.4 (C-9), 101.5 (C-7), 136.5 (C-5), 141.4 (C-10), 156.2 (C-6), 158.3 (C-8), 168.9 (C-1).White amorphous powder, [α]The antibacterial activity of the isolate was evaluated according to the reported method . Bacteri1–5 were determined by broth microdilution technique, as described by Ebrahim W., et al. -3-[4-dinitrophenyl]-5-phenyl-tetrazolium chloride (INT) and incubated at 37 °C for 30 min. Viable bacteria reduced the yellow dye to pink.The minimum inhibitory concentrations (MIC) of compounds , et al. . Compoun1–5) was performed using AutoDock4.2, as reported previously Morris et al. [1–5) were drawn in ChemSketch and saved as a mol2 file. The preprocessing of the ligands was performed by adding Gasteiger partial charges, merging non-polar H-atoms and defining rotatable bonds, as reported earlier [Molecular docking between the target proteins  and selected ligands  with the possible target proteins ; these compounds interacted with β-Lactamase enzymes. Thus, dihydroisocoumarins could be employed for antibacterial compound studies in the future.For many years, the media and the scientific community have published many fearful articles regarding the rise of antibiotic resistance, even citing treatment failure in multidrug-resistant bacteria (MDR)-infected patients which results in high mortality worldwide. This caused conjectures about the threat of multidrug-resistant bacteria toward the modern world, because of both a global rise in bacterial resistance and the lack of discovery of new compounds to treat MDR bacterial infections. Increased antibiotic resistance and an absence of new antibiotic development showed the need for finding new compounds. Despite the progress in new antibiotic development research, the main bottleneck in detecting potentially interesting compounds, which may be used to overcome bacterial resistance and the treatment of infections, was not yet solved. Interestingly, dihydroisocoumarin compounds isolated from the Wadi Lajab sediment fungus,


Our understanding of SARS-CoV-2 evolution is limited. Most estimates arise from analysis of global databases populated with unrelated sequences and is currently estimated at ∼27.7 substitutions/genome/year. SARS-CoV-2 polymerase contains a proofreading function encoded by NSP-14 limiting change. Additionally, virus evolution may be influenced by patient comorbidity. Intra-host mutational rate (MR) during infection remain poorly studied.To minimize effect of vaccination and/or natural immunity on MR analysis, paired samples from adults originating from the initial pandemic wave (3/17/2020 through 5/27/2020) were retrieved and analyzed at Cleveland Clinic. Viral genome analysis was performed using next generation sequencing, and mutations between paired samples were quantified at allele frequencies (AF) ≥ 0.1, ≥ 0.5 and ≥ 0.75 and compared. MR was calculated employing F81 and JC69 evolution models and compared between isolates with (Δ NSP-14) and without  non-synonymous mutations in NSP-14 and by comorbidity.A total of 40 patients (80 sample pairs) were identified. Median interval between paired tests was 15 days [range 5-32]. The estimated MR by F81 modeling was 317.2 (95%CI 312.0-322.3), 54.6 (95%CI 52.5-56.7) and 45.1 (95%CI 43.1-47.0) substitutions/genome/year at AF of ≥0.1, ≥0.5, ≥0.75 respectively. Rates in ΔNSP-14 (n=13) vs wt (n=27) groups were 557.7 (95%CI 537.0-578.2) vs 193.1 (95%CI 187.1-199.1) p-value 0.001, 50.8 (95%CI 44.3-57.3) vs 56.3 (95%CI 53.1-59.4) p-value 0.144, and 31.0 (95%CI 25.9-36.1) vs 51.3 (95%CI 48.3-54.3) p- value < 0.001 at AF ≥0.1, ≥0.5, ≥0.75 respectively. Patients with immune comorbidities (n=6) had significantly higher MR of 137.6 (95%CI 114.6-160.5) vs 40.5 (95%CI 38.4-42.7) p-value < 0.001, and 113.7 (95%CI 92.8-134.5) vs 33.4 (95%CI 31.5-35.4) p-value < 0.001 at AF ≥0.5 and ≥0.75 respectively. Similar results were obtained when using the JC69 model.Intra-host SARS-CoV-2 mutation rates are higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated MR at low AF. Immunosuppressed patients have elevated MR at higher AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.Frank Esper, M.D, Johnson and Johnson: Advisor/Consultant Daniel D. Rhoads, M.D. PhD, Luminex: Advisor/Consultant|Talis Biomedical: Advisor/Consultant|Thermo Fisher: Advisor/Consultant.