Among them, three compounds, methyl 3,6-di-O-galloyl-α-d-glucopyranoside (9), ethyl 2,3-di-O-galloyl-α-d-glucopyranoside (11) and ethyl 2,3-di-O-galloyl-β-d-glucopyranoside (12), are new compounds and other six, 1,6-di-O-galloyl-β-d-glucopyranose (1), 1,4,6-tri-O-galloyl-β-d-glucopyranose (2), 1,2-di-O-galloyl-β-d-glucopyranose (3), 1,3-di-O-galloyl-β-d-glucopyranose (4), 1,2,3-tri-O-galloyl-α-d-glucopyranose (6) and methyl 3,4,6-tri-O-galloyl-α-d-glucopyranoside (10), were synthesized for the first time in the present study. In in vitro MTT assay, 1–12 inhibited human cancer K562, HL-60 and HeLa cells with inhibition rates ranging from 64.2% to 92.9% at 100 μg/mL, and their IC50 values were determined to be varied in 17.2–124.7 μM on the tested three human cancer cell lines. In addition, compounds 1–12 inhibited murine sarcoma S180 cells with inhibition rates ranging from 38.7% to 52.8% at 100 μg/mL in the in vitro MTT assay, and in vivo antitumor activity of 1 and 2 was also detected in murine sarcoma S180 tumor-bearing Kunming mice using taxol as positive control.Twelve galloyl glucosides  O-galloyl-β-d-glucopyranose (PGG), can be obtained in large amounts by acidic methanolysis of tannic acid from plants   1, 3, 4,  1, 3, 4, or 5% FeCl3 reagent (5 g of FeCl3 dissolved in 100 mL of 95% aqueous EtOH). Polyamide  and Sephadex™ LH-20  were used for column chromatography. HPLC was carried out on a Waters HPLC system  equipped with Waters 600 controller, Waters 600 pump, Waters 2996  or 2998 (for preparative) photodiode array detector, and Waters Empower™ software. The Capcell Pak C18 columns  were used in analytical and preparative HPLC, respectively.Precoated silica gel GFd-glucose (lot No. 20110514) were purchased form Sinopharm Chemical Reagent Co., Ltd. . All other chemicals were purchased as the reagent grade and used without purification unless otherwise noted. Benzaldehyde was distilled and pyridine was dried by KOH for 24 h, before use in reactions. The petroleum ether with boiling point 60–90 °C was used throughout the experiments and was abbreviated as Pet.Et2O in the following descriptions.The human chronic myelogenous leukemia K562 cell line was provided by Prof. Dr. Lili Wang . Human acute promyelocytic leukemia HL-60 and human cervical cancer HeLa cell lines were provided by Prof. Dr. Wenxia Zhou (Beijing Institute of Pharmacology and Toxicology). Fetal bovine serum was purchased from Tianjin Hao Yang Biological Manufacture Co., Ltd. . The RPMI-1640 medium (lot No. 1403238) was purchased from Gibco  and MTT (lot No. 0793) from Amresco . Streptomycin (lot No. 121104) and penicillin (lot No. X1303302) were purchased from North China Pharmaceutical Group Corporation . The 5-fluorouracil , docetaxol  and taxol (lot No.20110427) were purchased from Aladdin Chemistry Co. Ltd. . Gallic acid (lot No. F20070926) and 15  in aqueous 2 N HCl (640 mL) was refluxed at 80 °C for 16 h to hydrolyze 15. The reaction mixture was then extracted with EtOAc (300 mL × 4) to give an EtOAc extract. The EtOAc extract was separated by column chromatography on silica gel  to obtain hydrolyzed product 18 and quite a larger amount of the 1-OCH3 unhydrolyzed materials, methyl 2,3-di-O-benzyl-α-d-glucopyranoside. The latter was further subjected, without weighing, to the same hydrolysis and separation steps to obtain additional amounts of 18, and the recovered raw materials in this round of experiments were also hydrolyzed and then separated once again to obtain further additional amount of 18. Eventually, 18  was obtained from 17.4 g (0.0376 mol) of 15 as an amorphous powder in CH2Cl2-MeOH. Totally over 10 g of 18 were prepared from 15 by additionally repeated hydrolyses. 2,3-Di-O-benzyl-d-glucopyranose (18): An amorphous powder (CH2Cl2-MeOH), positive ion ESI-MS m/z: 361 [M+H]+, 383 [M+Na]+; negative ion ESI-MS m/z: 405 [M+HCO2]−. 1H-NMR  δ for α-isomer: 7.39–7.21 , 5.21 , 4.85 and 4.84 , 4.69 and 4.65 , 3.81–3.75 , 3.79 , 3.77 , 3.68 , 3.45 , 3.42 ; δ for β-isomer: 7.39–7.21 , 4.94 and 4.80 , 4.86 and 4.68 , 4.64 , 3.86 , 3.65 , 3.46 , 3.43 , 3.34–3.29 , 3.23 . 13C-NMR  δ for α-isomer: 140.7, 140.0, 129.5–128.7 (10C), all Ph-C6 in PhCH2 × 2; 92.1 (C-1), 83.0 (C-3), 81.5 (C-2), 76.7 (PhCH2), 73.9 (PhCH2), 73.2 (C-5), 71.9 (C-4), 62.7 (C-6); δ for β-isomer: 140.5, 140.3 129.5–128.7 (10C), all Ph-C6 in PhCH2 × 2; 98.8 (C-1), 86.2 (C-3), 84.6 (C-2), 78.1 (C-5), 76.1 (PhCH2), 75.9 (PhCH2), 72.0 (C-4), 63.0 (C-6).A solution of 18  and 13  in 30 mL of anhydrous pyridine was stirred at 60 °C for 48 h and then evaporated under reduced pressure to obtain a reaction mixture. The reaction mixture was separated by silica gel column chromatography  to afford crude 19 , which did not give pseudo-molecular ion peaks in both positive and negative ESI-MS measurements. Crude 19  was dissolved in 200 mL of THF-95% EtOH (15:5) solution, transferred into a 0.5 L high-pressure reactor, and then reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (0.8 g) as catalyst. The reaction mixture was filtered to filter out insoluble materials and evaporated under reduced pressure to give a reaction product. This product was separated by repeated Sephadex LH-20 column chromatography eluted with H2O-MeOH (75:25→25:75) to afford crude 1. The crude 1 was recrystallized in 30% MeOH to obtain pure 1 . Needles (MeOH-H2O), m.p. 180–182 °C;c 0.52, MeOH); positive ion ESI-MS m/z: 507 [M+Na]+; negative ion ESI-MS m/z: 483 [M−H]−. 1H- and 13C-NMR: A mixed solution of 18  and 13  in anhydrous pyridine (170 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to obtain a reaction mixture. The mixture was subjected to silica gel column chromatography eluted with CH2Cl2-EtOAc (100:1) to afford crude 20 , which did not give the corresponding pseudo-molecular ion peaks in positive and negative ESI-MS measurements. The crude 20  was dissolved in THF-95% EtOH , transferred into a 1 L high-pressure reactor, and reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (1.0 g) as catalyst. The reaction mixture was filtered to filter out insoluble materials and evaporated to give a reaction product. This product was separated by repeated Sephadex LH-20 column chromatography eluted with H2O-EtOH (45:55) to afford crude 2, which was recrystallized in 40% MeOH to give pure 2 . Needles (MeOH-H2O), m.p. 173–175 °C;c 0.51, MeOH); positive ion ESI-MS m/z: 656 [M+Na]+; negative ion ESI-MS m/z: 635 [M−H]−, 671 [M+Cl]−. 1H- and 13C-NMR: A mixed solution of 16  and 13  in anhydrous pyridine (40 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to obtain a reaction mixture that was separated by silica gel column chromatography  using CH2Cl2 as eluting solvent to afford a mixture of three esterified products 21 (5.8 g). The mixture was dissolved in THF-95% EtOH , transferred into a 0.5 L high-pressure reactor, and then reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd–C (0.7 g) as catalyst. The reaction mixture was filtered to filter out the undissolved materials and evaporated under reduced pressure to obtain a reaction product. This reaction product was separated by Sephadex LH-20 column  to afford 4  and fractions Fr-2 and Fr-3. Further separation of Fr-2 by preparative HPLC  gave 5  and 6 , while HPLC separation of Fr-3 at the same conditions, except for the use of 10% methanol as mobile phase, afford 3 . d-glucopyranose1,2-Di-O-galloyl-β- (3): A crystalline powder (MeOH), m.p. 150–153 °C;c 0.44, MeOH); positive ion ESI-MS m/z: 502 [M+NH4]+, 507 [M+Na]+; negative ion ESI-MS m/z: 483 [M−H]−, 519 [M+Cl]−. d-glucopyranose1,3-Di-O-galloyl-β- (4): A crystalline powder (MeOH), m.p. 161–163 °C;c 0.44, MeOH); positive ion ESI-MS m/z: 507 [M+Na]+; negative ion ESI-MS m/z: 483 [M−H]−, 519 [M+Cl]−, 529 [M+HCO2]−. d-glucopyranose1,2,3-Tri-O-galloyl-β- (5): A crystalline powder (MeOH), m.p. 176–178 °C;c 0.48, MeOH); positive ion ESI-MS m/z: 654 [M+NH4]+ 659 [M+Na]+; negative ion ESI-MS m/z: 635 [M−H]−, 671 [M+Cl]−, 681 [M+HCO2]−. d-glucopyranose1,2,3-Tri-O-galloyl-α- (6): A crystalline powder (MeOH), m.p. 1165–167 °C;c 0.49, MeOH); positive ion ESI-MS m/z: 654 [M+NH4]+, 659 [M+Na]+; negative ion ESI-MS m/z: 635 [M−H]−, 671 [M+Cl]−. 1H- and 13C-NMR data for 3–6: A mixed solution of 14  and 13  in anhydrous pyridine (50 mL) was stirred at 60 °C for 48 h and evaporated under reduced pressure to give a reaction mixture. The reaction mixture was subjected to column chromatography on silica gel (bed: 5.0 × 10.0 cm), eluted by CH2Cl2, to afford 22 . A portion of 22  was dissolved in 150 mL of THF-95% EtOH (13:2), transferred into a 0.5 L high-pressure reactor, and then reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (1.5 g) as catalyst. The reaction mixture was filtered to filter out insoluble substances and evaporated to give a reaction product containing 23  as major component, which was further hydrolyzed without purification in 40 mL of aqueous 1 N HCl at 55 °C for 6 h. The hydrolysate was separated by column chromatography on polyamide  and then Sephadex LH-20  to afford 7 . A crystalline powder (MeOH), m.p. 160–162 °C;c 0.52, MeOH); positive ion ESI-MS m/z: 499 [M+H]+, 521 [M+Na]+; negative ion ESI-MS m/z: 497 [M−H]−, 533 [M+Cl]−. 1H- and 13C-NMR: A mixed solution of 15  in 50% aqueous AcOH (200 mL) was refluxed at 55 °C for 24 h and then evaporated under reduced pressure to give a reaction mixture. The reaction mixture was separated by column chromatography on silica gel (bed: 4.0 × 10.0 cm), eluted by gradient Pet.Et2O-CH2Cl2 (3:1)→CH2Cl2-MeOH (20:1), to afford methyl d-glucopyranoside2,3-di-O-benzyl-α- (24), 6.0 g, 0.0160 mol, 74.1% yield from (15) as a crystalline powder (CH2Cl2-MeOH), m.p. 81–83 °C,c 1.00, CH2Cl2); positive ion ESI-MS m/z: 397 [M+Na]+; negative ion ESI-MS m/z: 373 [M−H]−, 419 [M+HCO2]−. 1H-NMR  δ: 7.39–7.28 , 5.02 and 4.71 , 4.77 and 4.66 , 4.59 , 3.83–3.70 , 3.61 , 3.52 , 3.49 , 3.38 . 13C-NMR  δ: 138.7, 138.0, 128.8 (2C), 128.6 (2C), 128.3 (2C), 128.2 (2C), 128.1, 128.0, all Ph-C6 in PhCH2 × 2; 98.3 (C-1), 81.4 (C-3), 79.8 (C-2), 75.5 (PhCH2), 73.3 (PhCH2), 70.7 (C-5), 70.4 (C-4), 62.5 (C-6), 55.4 (OCH3).A solution of 24  and 13  in anhydrous pyridine (30 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to obtain a reaction mixture that was separated by silica gel column chromatography  to afford a crude 25 . The crude 25  was dissolved without further purification in THF-95% EtOH , transferred into a 0.5 L high-pressure reactor, and then reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (2.0 g) as catalyst. The reaction mixture was filtered to filter out insoluble substances and evaporated under reduced pressure to give a reaction product. This product was separated by Sephadex LH-20 column  to give 8 . A crystalline powder (MeOH), m.p. 163–165 °C;c 0.47, MeOH); positive ion ESI-MS m/z: 521 [M+Na]+, 537 [M+K]+; negative ion ESI-MS m/z: 497 [M−H]−. 1H- and 13C-NMR: A mixed solution of 14  and NaH (25 g) in DMF (1200 mL) was stirred for 40 min, during which time the solution became hot while stirring and was thus cooled down to room temperature. To the mixture under stirring benzyl bromide (200 mL) was added dropwise and reacted at room temperature for 6 h. The reaction mixture solution was poured into distilled water (2000 mL) and extracted with CHCl3 (2000 mL × 3) to give a CHCl3 solution. Without washing with water and drying by anhydrous MgSO4 to remove remaining NaOH (from NaH) in CHCl3, the CHCl3 solution was directly evaporated under reduced pressure, at the lower temperature at first to remove CHCl3 and further at 90 °C to remove remaining DMF, to obtain a reaction mixture that was separated by silica gel column chromatography  to afford 26 . d-glucopyranosideMethyl 2-O-benzyl-α- (26): A crystalline powder (MeOH), m.p. 113–115 °C,c 1.00, MeOH); positive ion ESI-MS m/z: 302 [M+NH4]+, 307 [M+Na]+, 323 [M+K]+; negative ion ESI-MS m/z: 283 [M−H]−, 319 [M+Cl]−, 329 [M+HCO2]−. 1H-NMR  δ: 7.44–7.26 , 4.76 and 4.64 , 4.64 , 3.79 , 3.74 , 3.65 , 3.49 , 3.34 , 3.33–3.26 . 13C-NMR  δ: 139.8, 129.4 (2C), 129.2 (2C), 128.9, all Ph-C6 in PhCH2; 99.2 (C-1), 81.0 (C-2), 74.3 (C-3), 74.0 (PhCH2), 73.3 (C-5), 71.8 (C-4), 62.6 (C-6), 55.4 (OCH3).A mixed solution of 26  and 13  in anhydrous pyridine (50 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to give a reaction mixture that was separated by silica gel column chromatography  to afford crude 27 , eluted by Pet. Et2O-EtOAc (2:1), and 28 , eluted by Pet. Et2O-EtOAc (4:1). The crude 28  was dissolved in THF-95% EtOH , transferred into a 0.5 L high-pressure reactor, and reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd–C (0.6 g) as catalyst. The reaction mixture was filtered to filter out the insoluble substances and evaporated under reduced pressure to give a reaction product that was separated by column chromatography on Sephadex LH-20  to afford pure d-glucopyranosidemethyl 3,6-di-O-galloyl-α-  in the 20.4% yield. A crystalline powder (MeOH), m.p. 154–156 °C;c 0.51, MeOH); positive ion ESI-MS m/z: 521 [M+Na]+, 537 [M+K]+; negative ion ESI-MS m/z: 497 [M−H]−. 1H- and 13C-NMR: 27 , followed by separation of reduction product by Sephadex LH-20 column  afforded additional amount of 8  for a 37.0% yield.A mixed solution of 26  and 13  in anhydrous pyridine (30 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to give a reaction mixture which was separated by silica gel column chromatography  to give crude 29 . A portion of the crude 29  was dissolved in THF-95% EtOH , transferred into a 0.3 L high-pressure reactor, and reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (0.4 g) as catalyst. The reaction mixture was filtered to filter out the insoluble substances and evaporated under reduced pressure to give a reduction product. This product was separated by Sephadex LH-20 column chromatography  to afford d-glucopyranosidemethyl 3,6-di-O-galloyl-α- . A crystalline powder (MeOH), m.p. 171–173 °C;c 0.49, MeOH); positive ion ESI-MS m/z: 651 [M+H]+, 668 [M+NH4]+, 673 [M+Na]+; negative ion ESI-MS m/z: 649 [M−H]−, 685 [M+Cl]−. 1H- and 13C-NMR: A mixed solution of 17  and 13  in anhydrous pyridine (40 mL) was stirred at 60 °C for 48 h and then evaporated under reduced pressure to give a reaction mixture that was separated by silica gel column chromatography  to afford crude 30 . The crude 30 without further purification was dissolved in THF-95% EtOH , transferred into a 0.5 L high-pressure reactor, and reduced by H2 under 10 atmospheric pressure at 40 °C for 12 h using 10% Pd-C (1.5 g) as catalyst. The reaction mixture was filtered to filter out the insoluble substances and evaporated under reduced pressure to give a reduction product which was then separated by repeated Sephadex LH-20 column chromatography  to afford a mixture of crude 11 and 12 . This mixture was further separated by preparative HPLC on a Capcell Pak C18 column , eluted by 30% MeOH, to obtain 11  and 12  in yields of 22.7% and 17.2%, respectively. d-glucopyranosideEthyl 2,3-di-O-galloyl-α- (11): A crystalline powder (MeOH), m.p. 157–159 °C;c 0.44, MeOH); positive ion ESI-MS m/z: 513 [M+H]+, 535 [M+Na]+; negative ion ESI-MS m/z: 511 [M−H]−, 547 [M−Cl]−. UV λmax nm (log ε) in MeOH: 223.5 (4.17), 276.4 (4.06). IR νmax cm−1 : 3427, 2988, 2941, 1700, 1613, 1534, 1450, 1338, 1224. 1H- and 13C-NMR: d-glucopyranosideEthyl 2,3-di-O-galloyl-β- (12): A crystalline powder (MeOH), m.p. 160–162 °C;c 0.51, MeOH); positive ion ESI-MS m/z: 513 [M+H]+, 535 [M+Na]+; negative ion ESI-MS m/z: 511 [M−H]−, 547 [M+Cl]−. UV λmax nm (log ε) in MeOH: 220.1 (4.14), 276.4 (3.91). IR νmax cm−1 : 3400, 2986, 2948, 1704, 1613, 1532, 1381, 1325, 1241. 1H- and 13C-NMR: A mixed solution of 2CO3 (200 g) in DMSO (500 mL) was added dried gallic acid  under stirring and heated to 140 °C. Then, benzyl chloride (300 mL) was added dropwise over 1.5 h under nitrogen atmosphere and then reacted at 140 °C for 8 h. After cooling down the reaction solution to the room temperature, the reaction mixture was diluted with cooled-distilled water (2000 mL) and extracted with CH2Cl2 (2000 mL × 4). The CH2Cl2 solution was combined and evaporated under reduced pressure to give a solid product. This product was suspended in MeOH (1800 mL), harvested by filtration, and washed with a suitable amount of methanol in the same filter to give a white powder. The powder was dried in vacuo and dissolved in CH2Cl2 (1000 mL). To the solution, MeOH (approximately 1000 mL) was added in portions, and when a lot of fine crystal species appeared, held at room temperature for two hours. Then, the crystals formed in the solution were filtered and then dried in vacuo to obtain benzyl tri-O-benzylgallate . Fine needles (from CH2Cl2-MeOH), m.p. 91–93 °C; positive ESI-MS m/z: 548 [M+NH4]+. 1H-NMR  δ: 7.37–7.12 , 5.24 , 5.03 . 13C-NMR  δ: 166.1 (CO), 152.6 , 142.5 (C-4); 137.5, 136.7 (2C), 136.1, 128.7–127.6 (20C), all Ph-C6 in PhCH2 × 4; 125.2 (C-1), 109.2 ; 75.2, 71.3 (2C), 66.9, all PhCH2 × 4.To a solution of anhydrous KO-benzylgallate  and MeOH (700 mL), in turn, and the mixture was refluxed at 90 °C for 5 h to hydrolyze the ester bond in the benzyl tri-O-benzylgallate by the base-catalyzed reaction. The solution was poured, after cooling to room temperature, into aqueous 1.2 N HCl (2500 mL) to neutralize the NaOH and stirred at room temperature for 30 min to precipitate the hydrolyzed product. The precipitate was filtered, washed with distilled water, and dried in vacuo. The precipitate was suspended in CH2Cl2-MeOH  and refluxed to dissolve the precipitate in full, and the solution was kept at 4 °C overnight for crystallization. Then, the formed crystals in the solution were filtered, washed with MeOH, and dried in vacuo to obtain pure tri-O-benzyl gallic acid . Fine needles (CH2Cl2-MeOH), m.p. 183–185 °C; positive ESI-MS m/z: 441 [M+H]+, 463 [M+Na]+. 1H-NMR  δ: 12.9 , 7.23–7.53 , 5.18 , 5.05 . 13C-NMR  δ: 166.8 (COOH), 152.0 , 141.0 (C-4); 137.4, 136.8 (2C), 127.6–128.4 (15C), all Ph-C6 in PhCH2 × 3; 126.0 (C-1), 108.2 ; 74.2, 70.2 (2C), all PhCH2 × 3.To a solution of NaOH (200 g) in distilled water (500 mL) were added the benzyl tri-O-benzyl gallic acid  was dissolved in SOCl2 (400 mL) and refluxed at 90 °C for 9 h. The solution was evaporated under reduced pressure to give a reaction mixture that was dried in vacuo and then dissolved in cyclohexane (600 mL) by refluxing. The solution was kept at room temperature overnight, and the crystals formed in the solution were collected by vacuum filtration, washed with cyclohexane, and dried in vacuo to obtain tri-O-benzylgalloyl chloride . Needles (from cyclohexane), m.p. 113–115 °C; positive ESI-MS m/z: 459 [M+H]+. 1H-NMR  δ: 7.22–7.56 , 5.18 , 5.04 . 13C-NMR  δ: 166.9 (CO), 152.0 , 141.0 (C-4); 137.4, 136.8 (2C), 127.6–128.4 (15C), all Ph-C6 in PhCH2 × 3; 126.1 (C-1), 108.2 ; 74.3, 70.2 (2C), all PhCH2 × 3.The tri-d-glucose , and the solution was refluxed for 72 h and then evaporated under reduced pressure to give a reaction mixture that was separated by silica gel column chromatography , followed by further purification through recrystallization in EtOH to obtain pure d-glucopyranosidemethyl α- . Needles (from EtOH), m.p. 163–165 °C; positive ESI-MS m/z: 212 [M+NH4]+, 217 [M+Na]+; negative ESI-MS m/z: 193 [M−H]−. 1H-NMR  δ: 4.67 , 4.61 , 3.81 , 3.67 , 3.52 , 3.39 , 3.41 , 3.28 . 13C-NMR  δ: 101.2 (C-1), 75.1 (C-3), 73.5 , 71.7 (C-4), 62.6 (C-6), 55.5 (OCH3).To stirring MeOH (700 mL) acetyl chloride (32 mL) was added, followed in turn by dried α-d-glucopyranoside , freshly distilled benzaldehyde (60 mL), triethyl orthoformate (20 mL), p-toluenesulfonic acid monohydrate (1.0 g) and THF (200 mL) was refluxed at 85 °C for 16 h. After cooling the reaction mixture to room temperature, K2CO3 (1.0 g) was added and the mixture was stirred at room temperature for 30 min. The mixture was filtered, and the filtrate was suspended in distilled water (400 mL) and a suitable amount of 95% EtOH was added to dissolve the suspended materials in full. Then, the solution was evaporated under reduced pressure, until a fine crystalline species appeared, and kept at 4 °C for 12 h. The crystals formed in the solution were filtered, washed with water (100 mL ×2) and cyclohexane (100 mL ×3), and dried in vacuo to give pure d-glucopyranosidemethyl 4,6-O-benzylidene-α- . Needles (EtOH-H2O), m.p. 163–165 °C; positive ion ESI-MS m/z: 283 [M+H]+, 300 [M+NH4]+, 305 [M+Na]+; negative ESI-MS m/z: 327 [M+HCO2]−. 1H-NMR  δ: 7.52–7.46 , 7.40–7.34 , 5.52 , 4.76 , 4.28 , 3.91 , 3.79 , 3.73 , 3.60 , 3.47 , 3.44 . 13C-NMR  δ: 137.1, 129.4, 128.5 (2C), 126.4 (2C), all Ph-C6 in PhCH; 102.1 (PhCH), 99.9 (C-1), 81.0 (C-4), 72.9 (C-2), 71.8 (C-3), 69.0 (C-6), 62.4 (C-5), 55.7 (OCH3).A mixture of methyl-14  and NaH (5 g) in DMF (300 mL) was stirred for 30 min, during which it became hot and was thus cooled down to room temperature. To the mixture benzyl bromide (45 mL) was added dropwise with stirring and reacted at room temperature for 4 h. To the reaction mixture was added distilled water (200 mL), and then it was extracted with CHCl3 (500 mL). The CHCl3 solution was washed with water (200 mL × 3), then dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure, at a lower temperature at first to remove CHCl3 and further at 90 °C to remove the remaining DMF, to obtain a yellow-colored product. This product was separated by silica gel column chromatography , eluted with Pet. Et2O–EtOAc (10:1), to obtain a fraction containing the target product 15. Recrystallization of the fraction in Pet. Et2O gave 15  in 87.1% yield. Fine needles (Pet.Et2O), m.p. 91–93 °C; positive ion ESI-MS m/z: 463 [M+H]+, 480 [M+NH4]+, 485 [M+Na]+. 1H-NMR  δ: 7.54–7.24 , 5.47 , 4.93 and 4.76 , 4.78 and 4.62 , 4.61 , 4.28 , 4.07 , 3.85 , 3.72 , 3.62 , 3.57 , 3.42 . 13C-NMR  δ: 138.8, 138.2, 137.5, 129.0, 128.6 (2C), 128.4 (2C), 128.3 (2C), 128.2 (2C), 128.1 (2C), 128.0, 127.7, 126.1 (2C), all Ph-C6 in PhCH2 × 2 and PhCH × 1; 101.3 (PhCH), 99.3 (C-1), 82.2 (C-4), 79.2 (C-2), 78.7 (C-3), 75.5 (PhCH2), 73.9 (PhCH2), 69.2 (C-6), 62.4 (C-5), 55.5 (OCH3).A mixed solution of d-glucose , newly distilled benzaldehyde (20 mL), p-toluenesulfonic acid monohydrate (1.0 g) and DMF (100 mL) was refluxed at 45 °C for 16 h. After the reaction mixture was cooled to room temperature, K2CO3 (1.0 g) was added into the mixture, stirred for 30 min, and filtrated to obtain a filtrate. To the filtrate was added distilled water (300 mL), and then the mixture was concentrated under reduced pressure to give a slimy product, which was separated by silica gel column chromatography , eluted by a gradient of CH2Cl2→CH2Cl2-MeOH (15:1), to afford 16 . A crystalline powder (CH2Cl2-MeOH), m.p. 161–163 °C; positive ion ESI-MS m/z: 269 [M+H]+, 291 [M+Na]+, 559 [2M+Na]+; negative ion ESI-MS m/z: 267 [M−H]−, 303 [M+Cl]−, 313 [M+HCO2]−. 1H-NMR  δ for α-isomer: 7.52–7.46 , 7.40–7.32 , 5.58 , 5.18 , 4.14 , 3.96 , 3.87 , 3.70 , 3.48 , 3.43 ; δ for β-isomer: 7.52–7.46 , 7.40–7.32 , 5.58 , 4.65 , 4.20 , 3.72 , 3.46 , 3.41 , 3.28 . 13C-NMR  δ for α-isomer: 139.3, 129.4 (2C), 128.7, 127.3 (2C), all Ph-C6 in PhCH; 102.2 (PhCH), 94.3 (C-1), 82.8 (C-4), 74.3 (C-2), 71.7 (C-3), 69.7 (C-6), 63.2 (C-5); δ for β-isomer: 139.2, 129.4 (2C), 128.7, 127.3 (2C), all Ph-C6 in PhCH; 102.0 (PhCH), 98.8 (C-1), 82.1 (C-4), 77.2 (C-2), 74.4 (C-3), 69.3 (C-6), 67.2 (C-5).A mixture of d-glucose , newly distilled benzaldehyde (45 mL), triethyl orthoformate (20 mL), p-toluenesulfonic acid monohydrate (1.0 g) and THF (200 mL) was refluxed at 85 °C for 16 h. After the reaction mixture cooled down to room temperature, K2CO3 (1.0 g) was added to the mixture, which was stirred for 30 min, and filtered to obtain a filtrate. The filtrate was concentrated under reduced pressure and then separated by column chromatography on silica gel , using a gradient of CH2Cl2→CH2Cl2-EtOH (20:1) as eluting solvent, to afford 17  as a mixture of α,β-isomers. An amorphous powder (CH2Cl2-MeOH), positive ion ESI-MS m/z: 297 [M+H]+, 314 [M+NH4]+, 319 [M+Na]+; negative ion ESI-MS m/z: 341 [M+HCO2]−. A portion of 17 (approximate 250 mg) was separated by preparative HPLC  to obtain α-isomer 17a  and β-isomer 17b . d-glucopyranosideEthyl 4,6-O-benzylidene-α- (17a): positive ion ESI-MS m/z: 297 [M+H]+, 314 [M+NH4]+; negative ion ESI-MS m/z: 341 [M+HCO2]−. 1H-NMR  δ: 7.53–7.46 , 7.39–7.31 , 5.57 , 4.84 , 4.20 , 3.88–3.70 , 3.84 , 3.64–3.41 , 3.51 , 3.45 , 1.27 . 13C-NMR  δ: 129.9, 129.0 (2C), 127.5 (2C), 139.2, Ph-C6 in PhCH; 103.3 (PhCH), 100.7 (C-1), 83.0 (C-4), 74.0 (C-2), 72.0 (C-3), 70.0 (C-6), 64.9 (OCH2CH3), 64.0 (C-5), 15.4 (OCH2CH3). d-glucopyranosideEthyl 4,6-O-benzylidene-β- (17b): positive ion ESI-MS m/z: 297 [M+H]+, 314 [M+NH4]+, 615 [2M+Na]+. 1H-NMR  δ: 7.54–7.47 , 7.39–7.31 , 5.57 , 4.40 , 4.28 , 3.91 , 3.76 , 3.68–3.59 , 3.50–3.39 , 3.48 , 3.27 , 1.24 . 13C-NMR  δ: 129.9, 129.0 (2C), 127.5 (2C), 139.1, Ph-C6 in PhCH; 104.8 (C-1), 102.9 (PhCH), 82.3 (C-4), 75.9 (C-2), 74.6 (C-3), 69.7 (C-6), 67.6 (C-5), 66.6 (OCH2CH3), 15.5 (OCH2CH3).A mixed solution of 1–12, 5-FU, DOC and taxol were dissolved in DMSO to prepare 10.0 mg/mL stock solutions, respectively, and serial dilutions were made for the MTT assay. 5-FU, DOC and taxol were used as positive control, and DMSO was used as blank control. The MTT assay was performed according to our previous procedure [control − ODsample)/ODcontrol × 100%, and the IC50 value for a sample was obtained from its IR% values at different concentrations.Compounds rocedure ,42,43,441 and 2 (30 mg of each) and taxol (20 mg) was dissolved in DMSO (1 mL) and mixed with oxidized castor oil (1 mL) to prepare stock solutions of 1 and 2 at 15 mg/mL and taxol at 10 mg/mL, respectively. Stock solutions were repeatedly prepared according to the need. Before administration, each 0.4 mL of the stock solution was diluted with 3.6 mL of 5% glucose aqueous solution to obtain diluted solutions of 1 and 2 at 1.5 mg/mL and taxol at 1.0 mg/mL for each round of administrations. A blank solvent without test samples was also prepared in the same manner and used for the model group as blank control.Compounds in vivo antitumor activity of 1 and 2 was tested by a procedure similar to that described in [7 cells/mL) into the armpit, were randomly divided into six groups: the model (blank solvent) group with 11 mice, positive control taxol (20 mg/kg) group with eight mice, and four test groups with 11 mice in each group for 1 and 2 at both 15 and 30 mg/kg, respectively. The drug administration was performed by intravenous injection via the mouse tail vein from the next day of tumor cell injection. The four test groups were injected every day continuously 6 days, while the taxol group was injected every other day for 6 days . The model group was continuously injected the same volume of blank solvent as that of 30 mg/kg test sample solutions every day for 6 days. On the third day of the last administration, the mice were sacrificed, the body and tumor weights were weighed, and the inhibition rate of 1–2 and taxol on the tumor growth was calculated as described in the main text. Statistical analysis for the test and positive control taxol groups was performed by the Student’s t test using body and tumor weights in mean ± S.D. values in comparison with the model group.The ribed in ,53. Sixt1–12 with two or three galloyl groups were synthesized from d-glucose and gallic acid. Three of them, 9, 11 and 12, were new compounds and six others, 1–4, 6 and 10, were synthesized for the first time. In an in vitro MTT assay, 1–12 inhibited human cancer K562, HL-60 and HeLa cells with inhibition rates ranging from 64.2% to 92.9% at 100 μg/mL and their IC50 values were determined to vary between 17.2–124.7 μM. In addition, 1–12 also inhibited murine sarcoma S180 cells with inhibition rates ranging from 38.7% to 52.8% at 100 μg/mL in an in vitro MTT assay, and the in vivo antitumor activity of 1 and 2 was also detected on murine sarcoma S180 tumor-bearing Kunming mice using taxol as positive control.Twelve galloyl glucosides


H-benzimidazol-2-yl)-benzene-1,3-diols, modified in both rings, have been synthesized and their efficacies as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors have been determined. The modified Ellman’s spectrophotometric method was applied for the biological evaluation. The compounds showed strong (IC50 80–90 nM) AChE and moderate (IC50 5–0.2 µM) BuChE inhibition in vitro. Some compounds were effective toward AChE/BuChE, exhibiting high selectivity ratios versus BuChE, while the other compounds were active against both enzymes. The structure–activity relationships were discussed. The compounds inhibited also in vitro self-induced Aβ(1–42) aggregation and exhibited antioxidant properties. The docking simulations showed that the benzimidazoles under consideration interact mainly with the catalytic site of AChE and mimic the binding mode of tacrine.In the present study, new 4-(1 The therapy of neurodegenerative diseases, including Alzheimer’s disease (AD), is a great challenge in the modern world . Both acN-substituted 2-aminobenzimidazoles were described as good inhibitors of AChE and BuChE. The molecular modeling studies revealed that the heterocyclic ring of the active molecule creates face-to-face π–π stacking interactions in the form of a “sandwich” with the indole moiety of the appropriate amino acids  (4-(1H-benzimidazol-2-yl)-6-chlorobenzene-1,3-diol (1). Yield: 84%; m.p.: 340–341 °C; EI MS : 260 , 231 (6), 197 (17), 169 (25), 143 (5), 116 (6), 92 (5), 69 (4), 65 (7), 63 (5), 52 (3), 36 (17); 1H NMR  δ: 14.12 , 11.46 –OH), 10.44 –OH), 8.27 –H), 7.80 –H), 7.49 –H), 7.03 –H) ppm; 13C NMR  δ: 161.1, 157.3, 152.3, 140.3 (2C), 129.3, 123.2 (2C), 114.8 (2C), 112.6, 111.9, 103.1 ppm; IR : 3281 (OH), 3051 (C–H), 1608 (C=N), 1572 (C=N), 1518 (C=C), 1503 (C=C), 1460, 1403, 1390, 1344, 1308, 1285, 1243, 1229 (C–OH), 1215, 1153, 1098, 1041, 1003, 951, 895, 849, 816, 779, 757, 737, 704. Anal. Calc. for C13H9ClN2O2 (260.68): C, 59.90; H, 3.48; N, 10.75. Found: C, 59.94; H, 3.51; N, 10.68%.4--6-chlorobenzene-1,3-diol (2). Yield: 88%; m.p.: 354–355 °C; EI MS : 329 , 296 (18), 256 (14), 201 (50), 187 (21), 169 (14), 146 (6), 141 (8), 114 (7), 95 (2), 86 (3), 73 (9), 69 (8), 63 (15), 51 (5), 45 (3), 40 (9); 1H NMR  δ: 11.20 –OH), 10.78 –OH), 8.11 –H), 7.52 –H), 6.72 –H) ppm; IR : 3496 (OH), 3354 (NH), 3071 (OH), 1640 (C=N), 1594 (C=N), 1512 (C=C), 1471, 1422, 1399, 1299, 1271, 1211 (C–OH), 1197, 1161, 1089, 1062, 967, 875, 846, 832, 803, 774, 748, 731. Anal. Calc. for C13H7Cl3N2O2 (329.57): C, 47.38; H, 1.14; N, 8.50. Found: C, 47.29; H, 2.17; N, 8.57%.4-[6-(Trifluoromethyl)-1H-benzimidazol-2-yl]-2-methylbenzene-1,3-diol (3). Yield: 72%; m.p.: 263–264 °C; EI MS : 308 , 289 (7), 279 (37), 263 (12), 251 (9), 237 (7), 211 (4), 167 (61), 154 (7), 140 (5), 65 (5), 39 (4); 1H NMR  δ: 13.07 –OH), 10.54 –OH), 8.17 –H), 7.85 –H), 7.71 –H), 6.65 –H), 6.59 –H), 2.00  ppm; IR : 3378 , 2924 (CH), 1611 (C=N), 1573 (C=C), 1504 (C=C), 1455, 1377, 1336, 1305, 1267 (C–OH); 1194, 1131, 1111, 1077, 1037, 930, 856, 832, 822, 793, 756, 705. Anal. Calc. for C15H11F3N2O2 (308.26): C, 58.45; H, 3.60; N, 9.09. Found: C, 58.52; H, 3.58; N, 9.16%.4-(7-Chloro-5-(trifluoromethyl)-1H-benzimidazol-2-yl)-6-ethylbenzene-1,3-diol (4). Yield: 81%; m.p.: 284–286 °C; EI MS : 357 , 356 , 341 , 313 (4), 287 (9), 271 (8), 212 (5), 181 (46), 164 (16), 149 (3), 77 (5), 69 (11), 45 (2), 39 (3); 1H NMR  δ: 11.30 , 10.75 –OH), 10.04 –OH), 7.75 –H), 7.58 –H), 7.43 –H), 6.44 –H)), 2.46 , 1.13  ppm; IR : 3456 (OH), 3402 (OH), 3356 (NH), 2971 (CH), 2939 (CH), 2874 (CH), 1624 (C=N), 1594 (C=C), 1508 (C=C), 1488, 1467, 1346, 1291, 1248 (C–OH), 1146, 1094, 1071, 987, 916, 887, 844, 768, 748. Anal. Calc. for C16H12ClF3N2O2 (356.73): C, 53.87; H, 3.39; N, 7.58. Found: C, 53.94; H, 3.37; N, 7.62%.4-(6-Methoxy-1H-benzimidazol-2-yl)-2-methylbenzene-1,3-diol (5). Yield: 76%; m.p.: 228–229 °C; EI MS : 270 , 255 (57), 241 (11), 227 (13), 198 (3), 167 (6), 151 (4), 135 (8), 79 (3), 77 (4), 65 (2), 44 (7), 39 (3), 33 (5); 1H NMR  δ: 13.37 –OH), 10.42 –OH), 8.15 –H), 7.38 –H), 7.03 –H), 6.98 –H), 6.57 –H), 3.30 , 2.00  ppm; 13C NMR  δ: 161.1, 159.2, 156.9, 156.0, 154.6, 139.5, 138.1, 126.2, 114.8, 111.2, 108.3, 108.8, 102.9, 59.8, 8.2 ppm; IR : 3382 (OH), 2937 (CH), 1611 (C=N), 1571 (C=C),1502 (C=C), 1457, 1335, 1319, 1252, 1236 (C–OH), 1183, 1159, 1108, 1091, 1036, 955, 916, 857, 815, 777, 758, 738, 700. Anal. Calc. for C15H14N2O3 (270.16): C, 66.66; H, 5.22; N, 10.36. Found: C, 66.74; H, 5.24; N, 10.29%.4-(6-Methoxy-1H-benzimidazol-2-yl)-6-chlorobenzene-1,3-diol (6). Yield: 73%; m.p.: 219–222 °C; EI MS : 290 , 275 (82), 261 (5), 247 (20), 241 (4), 187 (6), 145 (6), 80 (4), 78 (3), 69 (3), 51 (3), 33 (6); 1H NMR  δ: 12.77 –OH), 11.40 –OH), 8.19 –H), 7.41 –H), 7.06 –H), 7.02 –H), 6.56 –H), 3.27  ppm; 13C NMR  δ: 161.3, 157.1, 156.2, 154.9, 139.1, 134.3, 116.2, 114.7, 111.9, 111.5, 108.2, 104.2, 102.9, 59.8 ppm; IR : 3403 (NH), 3198 (OH), 2951 (CH), 1633 (C=N), 1512 (C=C), 1476, 1328, 1228 (C–OH), 1187, 1137, 1109, 1090, 974, 883, 784, 756, 727. Anal. Calc. for C14H11ClN2O3 (290.70): C, 57.84; H, 3.81; N, 9.64. Found: C, 57.92; H, 3.78; N, 9.70%.4-(6-Methoxy-1H-benzimidazol-2-yl)benzene-1,2,3-triol (7) [riol (7) .Ethyl 2--1H-benzimidazole-5-carboxylate (8). Yield: 73%; m.p.: 213–214 °C; EI MS : 312 , 284 (31), 267 (15), 255 (13), 239 (11), 167 (5), 119 (5), 90 (3), 63 (3); 1H NMR  δ: 12.21 , 10.91 –OH), 10.06 –OH), 7.66 –H), 7.62 –H), 7.56 –H), 6.79 –H), 6.42 –H), 3.75 , 1.82 , 1.38  ppm; IR : 3419 (OH), 3328 (NH), 3204 (OH), 2920 (CH), 1673 (C=O), 1627 (C=N), 1602 (C=C), 1493, 1459, 1394, 1369, 1294, 1263, 1242 (C–OH), 1202, 1155, 1088, 1015, 966, 857, 821, 796, 765, 726. Anal. Calc. for C17H16N2O4 (312.32): C, 65.38; H, 5.16; N, 8.97. Found: C, 65.31; H, 5.19; N, 8.90%.Ethyl 2--1H-benzimidazole-5-carboxylate (9). Yield: 74%; m.p.: 316–317 °C; EI MS : 326 , 311 , 297 (10), 283 (48), 238 (10), 169 (3), 133 (12), 123 (9), 91 (4), 44 (4), 36 (5); 1H NMR  δ: 10.71 –OH), 8.33 –H), 8.03 –H), 7.84 –H), 7.45 –H), 6.74 –H), 4.38 , 2.50 , 1.37 , 1.21  ppm; IR : 3425 (NH), 3163 (OH), 2968 (CH),1677 (C=O), 1618 (C=N), 1569 (C=C), 1511 (C=C), 1466, 1408, 1373, 1296, 1245 (C–OH), 1149, 1094, 1014, 951, 837, 765, 742. Anal. Calc. for C18H18N2O4 (326.35): C, 66.25; H, 5.56; N, 8.58. Found: C, 66.31; H, 5.55; N, 8.51%.Ethyl 2--1H-benzimidazole-5-carboxylate (10). Yield: 63%; m.p.: 300–301 °C; EI MS : 314 , 286 (9), 269 (9), 250 (10), 230 (21), 222 (22), 206 (6), 194 (13), 185 (6), 177 (18), 149 (8), 126 (100), 111 (7), 97 (8), 90 (5), 85 (22), 80 (13), 69 (23), 63 (10), 52 (12), 44 (27), 39 (10), 36 (7); 1H NMR  δ: heteroatoms protons are illegible; 8.02 , 7.65 , 5.91 , 5.89 , 4.35 , 1.35  ppm; IR : 3435 , 1667 (C=O), 1615 (C=N), 1517 (C=C), 1478, 1390, 1366, 1289, 1244 (C–OH), 1189, 1156, 1028, 934, 823, 766, 729. Anal. Calc. for C16H14N2O5 (314.29): C, 61.14; H, 4.49; N, 8.91. Found: C, 61.08; H, 4.52; N, 8.85%.2--1H-benzimidazole-6-carbonitrile (11). Yield: 76%; m.p.: 338–339 °C; EI MS : 265 , 236 (32), 220 (10), 208 (10), 194 (6), 181 (3), 168 (3), 117 (4), 90 (4), 36 (4); 1H NMR  δ: 10.07 –OH), 8.11 –H), 7.75 , 7.65 –H), 6.57 –H), 2.07  ppm; IR : 3369 (NH), 3134 (OH), 2837 (CH), 1659 (C=N), 1605 (C=C), 1570 (C=C), 1474, 1413, 1369, 1317, 1273, 1235 (C–OH), 1213, 1087, 981, 874, 819, 756, 710. Anal. Calc. for C15H11N3O2 (265.27): C, 67.92; H, 4.18; N, 15.84. Found: C, 68.01; H, 4.20; N, 15.77%.2--1H-benzimidazole-6-carbonitrile (12). Yield: 84%; m.p.: 284–285 °C; EI MS : 279 , 264 ; 236 (5), 207 (4), 194 (7), 181 (3), 117 (3), 90 (3), 40 (2), 36 (3); 1H NMR  δ: heteroatoms protons are illegible; 8.18 –H), 7.89 –H), 7.84 –H), 7.77 –H), 6.66 –H), 2.45 , 1.20  ppm. Anal. Calc. for C16H13N3O2 (279.29): C, 68.81; H, 4.69; N, 15.05. Found: C, 68.72; H, 4.71; N, 14.98%.4-(6-Nitro-1H-benzimidazol-2-yl)benzene-1,3-diol (13) [iol (13) . 2-Methyl-4-(6-nitro-1H-benzimidazol-2-yl)benzene-1,3-diol (14). Yield: 74%; m.p.: 213–215 °C; EI MS : 285 , 256 (14), 239 (55), 210 (16), 167 (13), 151 (11), 119 (5), 90 (8), 77 (6), 63 (11), 52 (5), 39 (5), 33 (3); 1H NMR  δ: 12.15 , 10.99 –OH), 10.15 –OH), 7.98 –H), 7.92 –H), 7.65 –H), 6.82 –H), 6.47 –H), 2.02  ppm; 13C NMR  δ: 195.1, 158.3, 151.3, 135.3, 133.2, 126.2, 124.8, 123.4, 122.6, 117.1, 111.3, 110.1, 107.4, 8.4 ppm; IR : 3426 (NH), 3340 (OH), 3222 (OH), 2855 (CH), 1641 (C=N), 1604 (C=C), 1514 (C=C), 1499, 1462, 1428, 1382, 1382, 1307, 1262 (C–OH), 1203, 1153, 1080, 887. Anal. Calc. for C14H11N3O4 (285.25): C, 58.95; H, 3.89; N, 14.73. Found: C, 59.03; H, 3.91; N, 14.68%.3·min−1 at room temperature. Measurements were made at 292 nm. The retention time of an unretained solute (t0) was determined by the injection of a small amount of acetone dissolved in water. All compounds showed the regular retention behavior in the function of methanol concentration (v/v). This is described by the Soczewiński–Wachtmeister dependence:w is the retention factor of a solute with pure water as the mobile phase (intercept) and S is the regression curve (slope) [R − t0)/t0, where tR is the retention time of a solute and t0 is the retention time of an unretained solute. The Eurosil Bioselect C18  column was used as the stationary phase. The mobile phase consisted of different volume mixtures of methanol and 20 mM acetate buffer as the aqueous phase to give pH 4.0. The organic modifier concentration ranged from 0.30 to 0.75, depending on the structure of the compound at 0.05 (0.1) intervals. The flow rate was 0.5 cm (slope) ,33. The Acetylcholinesterase , butyrylcholinesterase , acetylthiocholine iodide (ATCh), butylthiocholine iodide (BTCh), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), donepezil, and neostigmine bromide were purchased from Sigma-Aldrich . The inhibitory activities against AChE and BuChE of the prepared compounds were performed by means of the method previously developed by Ellman et al. using neostigmine and donepezil as the reference compound . The det1 inhibitor:  and 5  and without it. The tested medium contained DTNB solution (0.4 mg/mL), AChE solution (2 U/mL) or BuChE solution (4 U/mL), solution of the examined compound, and different concentrations of the ATC iodide substrate solution. Absorbance was detected at 412 nm at room temperature after 7 min. The obtained data were calculated and presented on the Lineweaver–Burk plot. The determined the Michaelis-Menten constant (KM) and the maximum velocity of the enzyme (υmax) values were used to define the type of enzyme inhibition.The kinetic analysis was evaluated according to the Ellman’s method . Plots oI and IF0 correspond to the fluorescence intensities, in the presence and absence of the test compounds, respectively, minus the fluorescence intensities due to the blanks. The reported values were obtained as the mean ± SD of triplicate of three independent experiments. Amyloid β-peptide, (1–42) (Aβ42), was purchased from Aldrich. Aβ42 was diluted in the phosphate buffer (pH 8.0) to obtain a 125.8 µM solution. The compounds under study were first dissolved in a small amount of methanol, and then further diluted in the phosphate buffer to the final concentration of 5–160 μM. To study the Aβ42 aggregation inhibition, the fluorescence emission of thioflavin T (ThT) was followed with curcumin as the reference compound ,36. At f0) is the absorbance of the blank sample and (A1) is the absorbance of the tested compound (t = 30 min). Concentration of each compound required for scavenging 50% of DPPH (IC50) was also determined [Antioxidant activity of each compound and standards (rutin) was assessed based on the radical scavenging effect of stable DPPH free radical . An amoutermined .The partition coefficient (log P) values were predicted using Crippen′s fragmentation by the CS ChemProp module from ChemDraw Ultra 12  according to the fragmentation method introduced by Crippen . The pol1, 5, and 7 were created by Corina online (Molecular Networks) and later prepared by Sybyl 8.0 (Tripos). All atom and bond types were checked, necessary hydrogen atoms were added, and finally, the Gasteiger–Marsili charges were assigned. The pKa values for the compounds and the ionization states corresponding to the physiological conditions (pH 7.4) were assessed by Marvin (ChemAxon). The compounds were docked to acetylcholinesterase from the 1ACJ crystal structure. Before docking with GoldSuite (CCDC), the enzyme structure was prepared. All histidine residues were protonated at Nε, the hydrogen atoms were added, and some water molecules  were retained. The binding site was defined as all amino acid residues within the radius of 12 Å from the reference compound, tacrine. A standard set of genetic algorithms was applied. The population size was equal to 100, and the number of operations 100,000. As a result, 10 conformations for each compound were obtained. The GoldScore function was used for the selection of the best poses. The results were visualized by PyMOL 0.99 .The spatial structures of compounds STB) or its analogs substituted by -Me, -Et, -Cl, or -OH on the resorcinol moiety in the reaction. These compounds are the agents cyclizing and introducing resorcinol (or modified one) into final products. They were obtained according to the previously described way [The target benzimidazole derivatives  have beeibed way . The syn1H NMR, and 13C NMR, as well as by IR spectroscopy. The obtained data are consistent with those for the other derivatives [The structures of the compounds were confirmed by mass spectrometry, ivatives ,42.50 [All obtained compounds have been assessed as the AChE and BuChE inhibitors. Their potency was determined using the modified Ellman’s method and expressed as the half of maximal inhibitory concentration, IC50 . Neostig50 values toward AChE and BuChE ranged from 76 to 98 nM or from 0.117 to 18.711 µM, respectively, for the most active derivatives. All considered compounds showed greater activity toward AChE than BuChE. Some of them were highly selective for AChE with respect to BuChE (compounds 4 and 6), whereas compound 5 was a strong inhibitor of both cholineserases (IC50 = 86 nM and 117 nM). Differences in the inhibitory potency toward these enzymes result from some structural differences between the hydrophobic gorges of the active center of both enzymes [The results show that the potency of compounds is varied and some of them are characterized by good to moderate inhibitory activities. The IC enzymes . The studied phenol–benzimidazole conjugates with the additional substituent including the modified amine group on the phenolic moiety showed a lower activity compared with the considered polyphenols and the highest activity was found for 5-nitroderivative ,7. This 1 and 5 as the most valuable compounds. The Lineweaver–Burk plots present 1/velocity versus 1/substrate concentrations in the range of 100–10 µM for compounds 5  and 1  and without inhibitors (0 µM)  . Based otrations , Table 2 of AChE , Table 31, 5, 6), thioflavin-T (ThT) assay was performed with curcumin as the reference compound. Interestingly, all the synthesized compounds presented moderate to good potencies on the self-induced Aβ(1–42) aggregation. The data in 6, followed by 5 and 1; their IC50 values were 27.04 ± 2.29, 33.07 ± 2.07, and 48.00 ± 3.43 µM, respectively . On the contrary, in compound 8 (IC50(Aβ(1–42)) = 0.125 ± 0.06 µM), the radical scavenging properties were at a level similar to the used standard.Reactive oxygen species are a causal factor in the process of aging and the cumulative damage has been associated with AD . In this1, 5) while one was inactive (7). The crystal structure of AChE from the electric ray in a complex with the reference inhibitor, tacrine, was used for docking (PDB code: 1ACJ). The docking procedure was fully validated and described previously [Three benzimidazole derivatives were taken for the molecular docking studies to present their binding mode within the enzyme and to explain the differences in the activity. Two of them were active (eviously .5) was not engaged in any specific interactions and it seems that it was not necessary for the enzyme inhibition. The substituted phenyl moiety created π–π interactions with Trp84.All compounds presented a similar arrangement within the active site of AChE, interacting mainly with the catalytic site . They mi7, log P = 1.57) led to a decrease of lipophilic properties in comparison with the compounds containing an extra methyl group ). The detailed binding mode of compound 5 is presented in The hydroxyl group in position 4 was always bound to the carboxyl group of Glu199 by the hydrogen bond, whereas the hydroxyl group in position 2 usually created the H bond with the water molecule. Comparing two active compounds with an inactive one, it could be noticed that they differed significantly from one another as far as lipophilicity is concerned. Introduction of the third hydroxyl group in position 3  resulted in reduction of hydrophobic and aromatic interactions, especially those of the phenyl moiety, and as a consequence, a lack of the activity. The other compound  to AChE . Structu to AChE ,22.w obtained by RP-HPLC and calculated in silico log P, the topological polar surface area (tPSA), and the molar refractivity (MR)  . The logcriptors ,49,50.H-benzimidazol-2-yl)-benzene -1,3-diol  as a parent compound was included in the biological screening (7 and 10) or -NO2 , particularly -CN  on the benzimidazole ring decreases significantly the inhibitory potency of the compounds against both enzymes. Introduction of the hydrophobic substituent  as well as -OMe on the heterocyclic ring or -Cl in the aryl moiety enhances biological activity of analogs in comparison to the unsubstituted bib-1,3-diol. The activity of compounds with the ethyl ester group is moderate (8–10) and it deteriorates with the addition of the hydroxyl substituent.To obtain better results, the previously described compound 4-.On the other hand, taking into account the values of the calculated descriptors , the low1 and 5 are the strongest inhibitors in the series . At the same time, compound 5 is highly active against BuChE (IC50 = 117 nM). The kinetic studies suggest the mixed type of inhibition for both enzymes. Besides the ability to inhibit AChE and BuChE, the most active derivative also inhibited in vitro self-induced Aβ(1–42) aggregation and exhibited antioxidant properties. The compounds under consideration have the properties expected for potential drugs used in AD therapy.In summary, a series of new benzimidazoles functionalized by 2,4-dihydroxyphenyl moiety has been obtained and evaluated as AChE and BuChE inhibitors. All compounds were more active against AChE than BuChE. Analogs 1 and 5 create a lot of interactions with the catalytic site of AChE and they mimic the binding mode of tacrine, which confirms their high inhibitory potency. The force of binding with the enzyme and the activity depend on the lipophilicity of the compounds, molar refractivity, and topological polar surface area. Introduction of the third hydroxyl group into the phenyl ring leads to reduction of lipophilic properties (increase of tPSA) as well as of hydrophobic and aromatic interactions of enzymes, particularly those of the phenyl moiety, and as a consequence, a lack of activity is observed. The presence of the additional lipophilic substituent of both rings enhances the anticholinesterase potency of analogs compared with the parent compound.The docking simulation exhibited that modeled compounds


The F-derivative of the –SOMe substituted furan-3(2H)-ones exhibited the best activity  of 54% (dose 0.01 mmol/kg), and MCF-7 and HSC-3 cytotoxicity with IC50 values of 10 μM and 7.5 μM, respectively). A cytotoxic effect related to the COX-1 inhibitory action was observed and a synergistic effect with the anti-neoplastic drugs gefitinib and 5-fluorouracil was found. A phenanthrene derivative exhibited the best synergistic effect with gefitinib. Apart from their anti-inflammatory action, COX inhibitors have gathered the interest of many scientists due to their potential use for the treatment and prevention of cancer. It has been shown that cyclooxygenase inhibitors restrict cancer cell growth and are able to interact with known antitumor drugs, enhancing their in vitro and in vivo cytotoxicity. The permutation of hydrophilic and hydrophobic aryl groups in COX inhibitors leads to cardinal changes in the biological activity of the compounds. In the present study, thirteen heterocyclic coxib-like 4,5-diarylfuran-3( Prostaglandins produced during inflammation are responsible for most of the undesirable effects of the inflammatory process and cyclooxygenase isoenzymes—COX-1 and COX-2—involved in their biosynthesis are the main targets of most anti-inflammatory drugs. As COX-2 is the isoenzyme induced during inflammation, COX-2-specific inhibitors have been developed during the last decades. Since 1999, when the first selective cyclooxygenase-2 (COX-2) inhibitors rofecoxib , an inteCurrently, COX inhibitors including even rofecoxib  are of rCancer cells present cellular and genomic heterogeneity that contribute to mechanisms leading to drug resistance development and to therapy failure in clinical practice ,6. This Among the two COX isoenzymes, COX-2 was mostly related with tumor development. The antiapoptotic role of COX-2 activity in chemoresistant cancer cells was noted in a study concerning drug resistance in patients with acute myeloid leukemia during standard treatment . COX-2 iMost interestingly, COX inhibitors may act synergistically with known antitumor drugs, enhancing their in vitro and in vivo cytotoxicity ,22,23,242H)-one derivatives -one and phenanthrofuran-3-one derivatives -ones and their annelated derivatives involved 5–7 stages furanones  was carrfragment . Despitep-Cl, and, especially, for m-F-substituted compounds, for which only the ratio of products after 1,2-aryl migration was established. The main difficulties in the synthesis arose for 1o with the higher predicted COX-2 activity as well as the other two most selective compounds, 1h and 1k (predicted selectivity: 100–125), of the –SO2CH3 group were tested. The less selective compound 1c of the same group was also tested for comparison. Furthermore, the most selective, 1g, and the less selective, 1j, compounds of – SOCH3 group as well as the two most selective compounds of the The COX-1/2 inhibitory action, mainly of the most active and COX-2 selective inhibitors according to the prediction results, was evaluated in vitro using ovine COX-1 and human recombinant COX-2 isoenzymes included in the COX inhibition assay kit of Cayman as described at the experimental part. Specifically, compound 2NH2 group were also tested. The results are shown in SO1v which showed a slightly lower IC50 value for COX-2 compared to COX-1 isoenzyme (20 μΜ vs. 28 μΜ). Compound 1v, the p–Cl derivative of the –SO2NH2 substituted compounds was the most active COX-2 inhibitor (IC50 = 20 μΜ) followed by the m–Cl derivative 1o (IC50 = 71 μΜ) of the –SO2 CH3 substituted compounds.Despite the coxib like structure and the prediction results which indicated a preference in COX-2 inhibition, most of the compounds exhibited mainly COX-1 inhibitory action with the exception of compound 1g, the p–F derivative of the –SOMe substituted group was the best COX-1 inhibitor with an IC50 value of 2.8 μΜ followed by two compounds of the –SO2 CH3 Me substituted group, 1o and 1k with IC50 values 22 μΜ and 29 μΜ respectively and two compounds of the –SO2NH2 substituted group 1v and 1w with IC50 values of 28 μΜ and 70 μΜ. Polarity of the substituents of both aryl moieties influences the activity as shown by the comparison between the IC50 values of the p–F derivatives 1g and 1w where the less polar –SO CH3 Me group appears to favor inhibitory activity compared to the –SO2NH2 group. On the other hand, introduction of the less electronegative Cl atom at the p-position of the phenyl ring in the –SO2NH2 derivatives (1v) resulted in enhanced inhibition of both isoenzymes compared to the strongly electronegative F-substituted 1w. It was found that none of the substituents favor COX-2 selective inhibition. However, compound 1v appears to be a better COX-2 inhibitor than is the reference drug naproxen. In contrast, compound x-1 was expected to be one of the best COX-2 isoenzyme inhibitors.It should be mentioned that in general, the docking analysis failed to predict the inhibitory action of the compounds especially in case of COX-1 as well as their selectivity. This may mean that the human protein 3D structures used for the prediction are not appropriate. Docking analysis using other COX-1 protein structures such as the ovine 1EQH and 2AYL structures co-crystalized in complex with flurbiprofen and the ovine 4O1Z in complex with meloxicam did not show better correlation with the in vitro activity. COX-2 inhibition was better predicted in all cases. Docking analysis results using the human COX-2 structure 5IKT derived from its complex with tolfenamic acid are presented in Interestingly, docking analysis indicate that all studied compounds may form stable complexes with the human COX-2 or the ovine COX-1 structures which were used. However, they are not placed deep inside the active site pocket as observed with most known inhibitors but are preferably oriented near the entrance of the active site . Much hi1v are formed while Tyr115 also participates in polar interactions with the O atom of the furanone ring. Similar interactions are observed in the case of all compounds stabilizing their orientation at the entrance of the active site cleft, including the more rigid phenanthrene derivatives. On the other hand, a negative effect leading to the formation of a high energy complex  is observed when the compound is oriented deep inside the active site, mainly because of the hydrophobic interactions between Leu352 -ones, including annelated derivatives are very similar.For the evaluation of anti-inflammatory activity of the tested compounds in vivo carrageen-induced mouse paw edema assay was used, with celecoxib (64.6%) and indomethacin (47%) as reference compounds. In general compounds in concentration of 0.1 mmol/g exhibited inhibition of inflammation varying between 38–57.7%. From the data of Thus, the study of structure–activity relationship revealed that anti-inflammatory activity of the compounds tested is almost independent of the nature and position of substituents. It should be noted that COX-1/COX-2 inhibition does not correlate with anti-inflammatory activity, as has been observed in anti-inflammatory results of other researches. Furthermore, since other mechanisms are involved in the first steps of edema formation, linear correlation between in vivo and in vitro results cannot be expected to be found. The anticancer treatments traditionally were based on the inhibition of DNA synthesis and function. Nowadays, many researchers turn their interest to selective inhibition of signaling pathways involved in proliferation, as another approach for anticancer therapy. Epidermal growth factor receptor (EGFR), which is overexpressed in major number of human tumors, plays a significant role in growth signaling. One representative of potent and selective EGER inhibitors is gefitinib, which is a competitive inhibitor of the ATP binding site of EGFR tyrosine kinase.Gefitinib is an antineoplastic drug well known for its inhibitory effect in cell proliferation and induction of cell death in various cancer cell lines, including breast cancer. It inhibits the catalytic activity of numerous tyrosine kinases and may also induce cell cycle arrest and inhibit angiogenesis ,46.p–F derivative of the SO CH3 group (1g) with the best COX inhibitory action (COX-1 IC50:2.8 μM), the p–F derivative of SO2CH3 group (1h) with low COX inhibitory action and one compound from the phenanthren group (x-1) with moderate predicted inhibitory action on both COX-1/2 isoenzymes and the best predicted selectivity over COX-2 were chosen. Three compounds were selected for the evaluation of their effect on cancer cell growth in the human epithelial breast adenocarcinoma cell line (MCF-7 cells) and in human tongue squamous cell carcinoma HSC-3 cell line. The 50 values of each compound after 48 h exposure of the cells were determined.The cells were grown in the presence of each of the selected compounds as well as of the two well-known anti-neoplastic drugs gefitinib and 5-fluorouracil. Celecoxib, a selective COX-2 inhibitor was used as a control. The IC50 value of 24 µM for 1h and 10 µM for 1g and x-1, while the IC50 values of the control compounds were 70 µM for gefitinib, 0.03 µM for 5-fluorouracil, and 29.2 µM for celecoxib (50:70 μΜ), with the less active COX inhibitor, 1h, to exhibit similar cytotoxicity under the culture conditions (IC50:24 μΜ). As far as the HSC-3 cell line is concerned, compound 1g exhibited remarkable growth inhibition of this cell line (64%) at the concentration of 10 μΜ with an IC50 value of 7.5 μΜ. The two other compounds exhibited much lower cytotoxicity at the HSC-3 cells. The inhibition potency of celecoxib and 5-fluouracil is comparable with that presented in the literature -5-phenylfuran-3phenyl]-5-phenylfuran-3(2H)-one (1b racemic mixture) Yield: 270 mg 86% colorless oily substance.1H NMR , δ, ppm: 1.58 , 2.74 , 7.38 , 7.49–7.51 , 7.63 . 13CNMR , δ, ppm: 23.3 (CH3), 23.4 (CH3), 43.9 (SOCH3), 87.6 (CH3)2), 112.4 (C4), 123.9, 127.9 (2C), 128.5 (2C), 128.7(2C), 129.5, 130.3(2C), 132.2, 144.6 , 179.2 (C5), 204.9 (C=O). IR (in CCl4), cm−1: 955 w., 1051 m., 1090 w., 1383 s., 1616 m., 1701 s. (C=O), 2931 w., 2982 w. HRMS, m/z, calculated for C19H18NaO3S 349.0869, found: 349.0876 [M + Na]+.5-(4′-Fluorophenyl) 2,2-dimethyl-4-[4-(methylsulfinyl)phenyl]-furan-3(2H)-one (1g racemic mixture) Yield: 27%. Slightly yellow oily substance. 1H NMR , δ, ppm: 1.54 , 2.72 , 7.04 , 7.45–7.47 , 7.60–7.65 . 13C NMR , δ, ppm: 23.4 (2CH3), 43.9 (SOCH3), 87.9 (H3)2), 116.7 , 124.8 (2C), 127.1 , 129.0, 131.0 (2C), 131.8 , 134.0, 145.9, 165.5 , 178.3 (C5), 204.5 (C=O). 19F NMR  δ, ppm: −105.4. IR (in CCl4), cm−1: 842 m, 902 w, 1049 m (S=O), 1161 m, 1242 s., 1381 s, 1515 m, 1616 s, 1703 s (C=O), 2980 w. (C-H). LCMS, m/z, calculated for C19H18FO3S+: 345.0956, found 345.1015 [M + H]+.5-(4′-Chlorophenyl)-2,2-dimethyl-4-[4-methylsulfinyl)phenyl]-furan-3(2H)-one (1j racemic mixture) Yiled: 8%. Slightly yellow oily substance. 1H NMR , δ, ppm: 1.54 , 2.74 , 7.32, 7.45 , 7.58 , 7.73 . 13C NMR , δ, ppm: 23.2 (2CH3), 43.6 (SOCH3), 88.0 (CH3)2), 112.7 (C4), 125.2 (2C), 127.3 (2C), 128.4, 128.7 (2C), 129.0 (2C), 133.2, 134.5, 144.2, 177.5 (C5), 204.6 (C=O). LCMS, m/z calculated for C19H18ClO3S+: 361.0660, found 361.0740 [M + H]+. 5-(3′-Chlorophenyl)-2,2-dimethyl-4-[4′-(methylsulfinyl)phenyl]-furan-3(2H)-one (1n racemic mixture) Yield: 33%. Slightly yellow oily substance. 1H NMR , δ, ppm: 1.53 , 2.79 , 7.23–7.27 , 7.37–7.50 , 7.60–7.70 . 13C NMR , δ, ppm: 23.3 (2CH3), 44.2 (SOCH3), 88.2 (CH3)2), 114.1 (C4), 124.6 (2C), 127.8, 128.7, 131.0 (2C), 131.3, 132.6, 132.8, 133.5, 135.0, 147.1, 177.6 (C5), 204.7 (C=O). IR (in CCl4), cm-1: 1051 m. (S=O), 1145 m, 1238 w, 1384 s, 1618 m, 1706 s, (C=O), 2983 w (C-H). LCMS, m/z, calculated for C19H18ClO3S+: 361.0660, found 361.0740 [M + H]+. 2H)-one  in acetic acid  a mixture of 35% hydrogen peroxide , 98% H2SO4 (0.05 mL), and acetic acid (3 mL) was added to the reaction dropwise with vigorous stirring for 1 h. The reaction mixture was stirred for 30 minutes at 75–85 °C (control by TLC). After completion of the reaction, the mixture was poured into water, extracted with ethyl acetate (4 × 20 mL), organic phase was washed by NaHCO3/water  were used. To a solution of 2,2-dimethyl-4-[4′-(methylthio)phenyl]-5-phenylfuran-3phenyl]-5-phenylfuran-3(2H)-one (1c) Yield: 94%. 1H NMR , δ, ppm: 1.58 , 3.04 , 7.38 , 7.50–7.54 , 7.61 , 7.91 . 13C NMR , δ, ppm: 23.3 (2CH3), 43.5 (SO2CH3), 87.9 (CH3)2), 111.6 (C4), 127.5, 128.4, 128.6, 129.2, 130.1, 132.4, 136.1, 139.0 , 179.8 (C5), 204.4 (C=O). IR (in CCl4), cm−1: 955 m, 1051 m., 1157 s, 1327 m., 1383 m., 1614 m., 1701 s. (C=O), 2932 w., 2982 w. HRMS, m/z, calculated for C19H18NaO4S+: 365.0818, found 365.0823 [M + Na]+.1o)5-(3′-Chlorophenyl)-2,2-dimethyl-4-[4′-(methylsulfonyl)phenyl]-furan-3(2H)-one , δ, ppm: 1.56 , 3.04 , 7.29 , 7.38 , 7.47 , 7.51 , 7.67 , 7.91 . 13C NMR , δ, ppm: 23.2 (2CH3), 44.4 (SO2CH3), 88.1 (CH3)2), 112.4, 126.7, 127.6 (2C), 128.0, 130.0, 130.1 (2C), 131.0, 132.3, 135.0, 135.6, 139.3, 177.8 (C5), 204.2 (C=O). IR (in CCl4), cm−1: 957 m., 1157 s. (SO2), 1330 s. (SO2), 1384 m., 1616 m., 1705 s. (C=O), 2931 w., 2982 w. (C-H). LCMS, m/z, calculated for C19H18ClO4S+: 377.0609, found 377.0667 [M + H]+. 5-(4′-Chlorophenyl)-2,2-dimethyl-4-[4′-(methylsulfonyl)phenyl]-furan-3(2H)-one (1k) Yield: 9%. Colorless crystals, m.p. 223–225 °C. 1H NMR , δ, ppm: 1.57 , 3.06 , 7.37 , 7.51-7.56 , 7.53 , 7.92 . 13C NMR , δ, ppm: 23.3 (2CH3), 44.5 (SO2CH3), 88.1 (CH3)2), 112.0, 127.7 (2C), 129.2 (2C), 129.7 (2C), 130.2 (2C), 130.6, 135.9, 138.8, 139.3, 178.3 (C5), 204.2 (C=O). IR (in CCl4), cm−1: 956 m., 1095 m., 1158 s. (SO2), 1329 s. (SO2), 1384 s., 1616 m., 1703 s. (C=O), 2931 w., 2982 w.(C-H). LCMS, m/z calculated for C19H18ClO4S+: 377.0609, found 377.0679 [M + H]+.5-(4′-Fluorophenyl)-2,2-dimethyl-4-[4′-(methylsulfonyl)phenyl]-furan-3(2H)-one (1h) Yield: 23%. Colorless crystals, m.p. 191–192 °C. 1H NMR , δ, ppm: 1.57 , 3.06 , 7.09 , 7.52 , 7.63 , 7.92 . 13C NMR , δ, ppm: 23.3 (2CH3), 44.5 (SO2CH3), 88.0 (CH3)2), 116.2 d , 126.3 (1C), 127.8 (2C), 130.2 (2C), 130.9 d , 136.1, 139.3, 165.1 d , 178.4 (C5), 204.2 (C=O). 19F NMR  δ, ppm: -104.8. IR (in CCl4), cm−1: 955 m., 1157 s. (SO2) 1243 m., 1327 s. (SO2), 1383 m., 1613 m., 1704 s. (C=O), 2933 w. (C-H). LCMS, m/z, calculated for C19H18FO4S+: 361.0905, found 361.0953 [M + H]+.3 (5mL) 2,2-dimethyl-4-[4′-(chlorosulfonyl)phenyl]-5-(3′-chlorophenyl)furan-3(2H)-one (200 mg) in THF (5 mL) was added and mixture was stirred at room temperature for 24 h. Then, water, ammonia and tetrahydrofuran were removed in vacuo on a rotary evaporator, the solid residue was dissolved in ethanol, then silica gel (5 g) was added, and ethanol was removed in vacuo. The silica gel with the product was washed on a Schott filter 3 times with dichloromethane (5 mL each), after which the compound was washed off the filter by ethanol after removing the alcohol and vacuum drying . For isolation and purification of compounds the mixtures of n-hexane/ CH2Cl2/ethyl acetate (from 10:2:1 to pure ethyl acetate) were applied.To an aqueous solution of 25% NH5-(3′-Chlorophenyl)-2,2-dimethyl-4-[4′-(aminosulfonyl)phenyl]-furan-3(2H)-one (1u) Yield: 150 mg, 79%. Colorless crystals, m.p. 198–200 °C. 1H NMR 2CO, reference: 2.05 ppm), δ, ppm: 1.54 , 6.63 , 7.45–7.62 , 7.49 , 7.68–7.71 , 7.89 . 13CNMR 2CO, reference: 206.26 ppm), δ, ppm: 204.54, 177.90, 144.07, 135.09, 134.77, 132.77, 132.70, 131.37, 130.61, 128.67, 127.86, 127.07, 113.86, 88.33, 23.30. HRMS, m/z, calculated for C18H17ClNO4S+: 378.0562, found: 378.0563 [M + H]+.5-(4′-Phenyl)-2,2-dimethyl-4-[4′-(aminosulfonyl)phenyl]-furan-3(2H)-one (1q) Yield: 23%.Colorless crystals, m.p. 158–160 °C. 1H NMR , δ, ppm: 1.53 , 6.60 , 7.44-7.49 , 7.58 , 7.65 , 7.87 . 13CNMR , δ, ppm: 23.4 (2C), 88.0 (C2), 113.0 (C4), 127.0 (2C), 129.2 (2C), 129.6 (2C), 130.6 (2C), 130.7, 133.0, 135.3, 143.7, 179.7 (C5), 204.5 (C=O). IR (in KBr), cm−1: 3346 br. (-SO2NH2), 3234 br. (-SO2NH2), 2988 w. , 2938 w. , 1662 v.s., 1607 s., 1568 s., 1485 m., 1386 s., 1342 s., 1238 m., 1170 s., 1058 m., 904 m., 746 m., 658 m., 597 m., 553 m. HRMS, m/z, calculated for C18H18NO4S+: 344.0952, found: 344.1006 [M + H]+. 5-(4′-Chlorophenyl)-2,2-dimethyl-4-[4′-(aminosulfonyl)phenyl]-furan-3(2H)-one (1v) Yield 29%. Colorless crystals, m.p. 224–225 °C. 1H NMR , δ, ppm: 1.53 , 4.82 , 7.44-7.49 , 7.64 , 7.86 . 13C NMR , δ, ppm: 204.51, 178.16, 140.80, 138.76, 134.76, 129.98 (2C), 129.76, 129.14 (2C), 127.68 (2C), 126.85 (2C), 112.16, 88.03, 23.31. HRMS, m/z, calculated for C18H17ClNO4S+: 378.0562, found: 378.0578 [M + H]+. 5-(4′-Fluorophenyl)-2,2-dimethyl-4-[4′-(aminosulfonyl)phenyl]-furan-3(2H)-one (1w) Yield: 36%. Colorless crystals, m.p. 167–169 °C. 1H NMR , δ, ppm: 1.55 , 5.42 , 7.06 , 7.41 , 7.60–7.65 ), 7.86 . 13CNMR , δ, ppm: 204.72, 178.32, 164.96 d (J = 255.2 Hz), 141.07, 134.64, 130.94, 130.83, 129.93, 128.46, 128.32, 126.72, 125.43, 116.04 , 111.80, 87.93, 23.24. HRMS, m/z, calculated for C18H17FNO4S+:362.0857, found: 362.0861 [M + H]+.2H)-one and 2,2-dimethyl-4-[4′-chlorophenyl]-5-(phenyl)furan-3(2H)-one was dissolved in triethylamine (10 mL), presaturated by CH3NH2, and obtained by heating a 60% solution of methylamine in water and passing gas through a layer of CaCl2.The resulting solution was stirred at 45 ° C for 24 h. Then the volatile products were removed on a rotary evaporator. The solid residue was applied to silica gel (3 g) and washed 3 times with a mixture of hexane-methylene chloride  to remove nonpolar impurities. After that, desired product 1vMe was washed off from silica gel by ethanol. Finally, 160 mg (67%) of the product 1vMe was obtained after removing the solvent. For the purification of compound the mixtures of n-hexane/ CH2Cl2/ethanol (from 10:1:0.1 to 1:1:1) were used.A mixture (450 mg) of 2,2-dimethyl-4-[4′-(chlorosulfonyl)phenyl]-5-(4′-chlorophenyl)furan-3 , δ, ppm: 1.52 , 2.57 , 4.82 , 7.48-7.52  7.66 , 7.83 . ESMS, calculated for C19H19ClNO4S+: m/z = 392.0718, found: 392.0745.Colorless crystals, m.p. 204–205 °C. 2H)-one  in dichloromethane (5 mL) placed in a quartz photochemical reactor with water cooling and a volume of 50 mL and irradiated with a mercury UV lamp of 150 W during 4 h in the presence of oxygen. After completion of the reaction, the solvent was distilled off on a rotary evaporator, the mixture was applied onto a preparative chromatography plate and divided into fractions (eluent: hexane–ethyl acetate–dichloromethane). The yield of 2′,2′-dimethyl-6-(methylthio)phenanthrofuran-3′(2′H)-one was 70 mg (14%), colorless crystals, violet fluorescence (254 nm). The degree of conversion of the starting 3(2H)furanone was 90%.A solution of 2,2-dimethyl-4-[4′-(methylthio)phenyl]-5-phenylfuran-3(2′H)-one may be prepared by oxidation of corresponding sulfide as described above. Yield: 6%. Colorless crystals, m.p. 222–224 °C. 1H NMR , δ, ppm: 1.68 , 3.06 , 7.72 , 7.80 , 8.10 , 8.32 , 8.50 , 8.79 , 9.30 . LCMS, m/z, calculated for C19H18O4S+: 342.0843, found: 342.0826 [M + H]+.2′,2′-Dimethyl-6-(methylsulfonyl)-phenanthrofuran-3′, δ, ppm: 1.62 , 2.64 , 7.60 , 7.70 , 7.90–7.84 , 8.33 , 8.45 , 8.63 , 8.76 . LCMS, m/z, calculated for: C19H16NaO2S+: 331.0764, found: 331.0769 [M + Na]+.Slightly yellow crystals, m.p. 178–180 °C. 2′H)-one (X-2) may be prepared by oxidation of corresponding sulfide as described above.3-Fluoro-2′, 2′-dimethyl-6-(methylsulfonyl)-phenanthrofuran-3′(2′H)-one.Here we present synthesis of intermediate 3-fluoro-2′, 2′-dimethyl-6-(methylthio)-phenanthro-furan-3′-phenanthrofuran-3′(2′H)-one.2H)-one  (305 mg) dissolved in boiling n-hexane (30 mL) was placed in a quartz reactor and irradiated by a low-pressure UV mercury lamp  during 2 h. After chromatography 27 mg (9%) of the product was obtained. The conversion rate was 34% and 200 mg of starting 3(2H)furanone was isolated. 5-(4′-fluorophenyl)-2,2-dimethyl-4-[4′-(methylthio)phenyl]-)furan-3, δ, ppm: 1.65 , 2.64 , 7.48 , 7.66 , 8.08 , 8.14 , 8.38 , 8.56 . LCMS, m/z, calculated for C19H16FO2S+: 327.0850, found: 327.0792 [M + H]+.Slightly yellow oily substance. H)-one The second step: Unreacted 5-(4′-fluorophenyl)-2,2-dimethyl-4-[4′-(methylthio)phenyl]-furan-3-(2n-hexane and the irradiation procedure was repeated. A unreacted starting 3(2H)furanone (143 mg) was isolated and 27 mg (13.5%) of desired phenanthrofuranone was obtained. Total yield: 54 mg (17.7%). (200 mg) dissolved in 25 mL of 1H NMR , δ, ppm: 1.65 , 3.16 , 7.50-7.55 , 8.17 , 8.37 , 8.43 , 9.02 d , 9.04 . LCMS, m/z, calculated for C19H16FO4S+: 359.0748, found: 359.0763 [M + H+]. Colorless crystals, m.p. 242–244 °C. The COX-1 and COX-2 activities of the compounds were measured using ovine COX-1 and human recombinant COX-2 enzymes included in the “COX Inhibitor Screening Assay” kit provided by Cayman  as reported previously ,61 The anti-inflammatory activity of compounds was performed using the carrageenan mice paw oedema assay. Animal of both sex (AKR), with exception of pregnant females were housed under standard conditions, receiving only water during the experimental period. The compounds (0.01 mmol/kg body weight) were administered intraperitoneally simultaneously with the carrageenin injection. The detailed description of the whole experiment reported previously . All theThe antitumor activity of the compounds was tested using two different cancer cell lines, breast cancer cell line, MCF-7, which mainly expresses the COX-1 isoenzyme  and the v/v) CO2) in Dulbecco’s Modified Eagle’s medium supplemented with 10% (v/v) fetal bovin serum (FBS), 10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin, and 25 μg/mL of amphotericin B. In order to allow the continuous logarithmic phase of cell growth in culture, cells were split accordingly before reaching 80% confluency (approximately every 48–72 h) with trypsin-EDTA (0.25% w/v). According to ATCC (American Tissue Culture Collection), the MCF-7 cell express the WNT7B oncogene, the estrogen receptor, and the insulin-like growth factor binding proteins (IGFBP) BP-2, BP-4, and BP-5; addition of 0.01 mg/mL human recombinant insulin in the cell culture medium is proposed. EGF Receptors are expressed when the other growth factors’ pathways are inhibited or after long term culture in the presence of Epidermal Growth Factor [Malignant MCF-7 cells  and HSC-3  cells were grown in culture  after every dilution. Cells were seeded in 24-well plates, at an initial density of 5 × 104 cells/mL. Cell cultures were left overnight at 37 °C to ensure appropriate cell attachment, before the compounds’ addition at the specified concentrations. Cells were allowed to grow for additional 48 h before being harvested by trypsinization and subsequently counted  using a hemocytometer (Neubauer counting chamber) and optical microscopy. Cell growth was expressed as a percentage relative to that for the untreated control culture . For the combination experiments, a Combination Index was measured using the equation CI = CA,x/Cx,A + CB,x/Cx,B where CA,x and CB,x stand for the concentrations of compounds A and B respectively in the combination mixture which results in x% inhibition of cell growth, while Cx,A and Cx,B are the concentrations of compounds A and B, respectively, which alone cause x% inhibition of cell growth. A CI of less than, equal to, and more than 1 indicates synergy, additivity, and antagonism, respectively [To estimate growth inhibition rate, we tested increasing concentrations (0.01–100 μM) of the compounds exposure to MCF-7 cells after appropriate serial dilutions of every compound stock solution. Each cell culture was treated appropriately, ensuring that DMSO concentration was kept below ≤ 0.2% (ectively .2H) ones were synthesized based on molecular docking for COX inhibitory action. Their anti-inflammatory and COX-1/COX-2 inhibitory activities were evaluated in vitro. Cytotoxicity of compounds on MCS-7 and HSC-3 cancer cell lines were performed.Thirteen new 4,5-diarylfuran- 3. Lower synergistic effect with 5-fluorouracil was shown by the less active compound 1h (CI = 0.533). A synergistic effect with gefitinib was found for compounds 1g (CI = 0.780) and X-1 (CI = 0.260), while the low activity compound 1h exhibited antagonistic effect. The most active COX-1 selective inhibitor, 1g with the known COX-2 selective inhibitor, celecoxib, was also observed (CI = 0.75), which may be indicative for the involvement of both cyclooxygenase isoenzymes in cancer cell development and the value of combined COX1/2 inhibition for best inhibitory effect.A synergistic effect of the most active COX-1 inhibitor


Kielmeyera variabilis (Clusiaceae) using DPPH•  and ABTS•+  colorimetric assays. The ethyl acetate and n-butanol fractions of the leaves of K. variabilis displayed the strongest activity . Chromatographic fractionation of the most potent fractions led to identification of three flavonols with previously described antioxidant activity, quercitrin (1), quercetin-3-O-β-glucoside (3), and quercetin-3-O-β-galactoside (4), and of one biflavone, podocarpusflavone A (2). This is the first time that the presence of these flavonoids in Kielmeyera variabilis has been reported.As part of our ongoing research on antioxidant agents from Brazilian flora, we screened the free radical scavenging activity of two extracts and eight fractions of  Clusiaceae is a tropical family of trees, shrubs, and herbs comprising approximately 50 genera and 1,200 species  radicals.The present study aimed to screen the extracts, fractions, and isolated compounds from the leaves and branches of •+ that can be reduced by antioxidants; whereas the DPPH assay is based on the reduction of the purple DPPH• to 1,1-diphenyl-2-picrylhydrazine . The ethanol extract of the leaves exhibited lower activity  than the medium-polarity leaves fractions, suggesting that the active substances were concentrated in the ethyl acetate and n-butanol fractions after partition.For the DPPH•+ radical, the ethanol extract of the branches of K. variabilis was the most active , whilst its ethyl acetate and n-butanol fractions gave IC50 values (μg mL−1) of 13.5 ± 1.0 and 3.2 ± 0.3, respectively. The ethanol extract obtained from the leaves of K. variabilis furnished IC50 (μg mL−1) of 7.0 ± 1.0, and its ethyl acetate and n-butanol fractions afforded IC50 (μg mL−1) of 6.6 ± 0.4 and 3.1 ± 0.1, respectively.For the ABTS1) from the ethyl acetate fraction of the leaves, which gave IC50 values (μg mL−1) of 9.1 ± 0.5 and 12.2 ± 0.7 for DPPH• and ABTS•+, respectively, and the biflavone podocarpusflavone A (2), inactive against both radicals.The phytochemical study of the most active fractions of the leaves identified four compounds belonging to the flavonoid class. We isolated one flavonol identified as quercitrin  and quercetin-3-O-β-galactoside (4), from the second most active fraction of the leaves of K. variabilis (n-butanol). The free radical scavenging activity of the 3 and 4 mixture was evaluated and we obtained an IC50 (μg mL−1) value of 4.1 ± 0.2 and 5.0 ± 0.4 for DPPH• and ABTS•+, respectively. This mixture was more active than the commonly employed antioxidant rutin . 1–4.We isolated In Nature, plants synthesize flavonoids to prevent damage by free radicals. The structures of this class of secondary metabolites contain a polyphenol structure with numerous double bonds and hydroxyl groups that can donate electrons through resonance and thus stabilize free radicals . The rad1, 3, and 4 are quercetin derivatives. Quercetin is the major representative of the flavonol subclass and has received considerable attention. Quercetin and its sugar-bound, or glycosylated, forms correspond to 60–75% of flavonoids intake − ; NMR 1H : δ 6.19 ; 6.38 ; 7.29 ; 6.86 ; 7.25 ; 5.25 ; 3.97 ; 3.50 ; 3.16 ; 3.20–3.23 ; 0.81 ; 12.6 ; NMR 13C : δ 157.2 (C-2); 134.2 (C-3); 177.7 (C-4); 161.3 (C-5); 98.7 (C-6); 164.2 (C-7); 93.6 (C-8); 156.2 (C-9); 104.0 (C-10); 120.7 (C-1'); 115.6 (C-2'); 145.2 (C-3'); 148.4 (C-4'); 115.4 (C-5'); 121.1 (C-6'); 101.8 (C-1''); 70.0 (C-2''); 70.5 (C-3''); 71.2 (C-4''); 70.3 (C-5''); 17.4 (C-6'').Podocarpusflavone A (2). ESI-MS m/z 551.0633 [M−H]− ; NMR 1H : δ 6.80 ; 6.17 ; 6.43 ; 7.97 ; 7.14 ; 8.0 ; 6.86 ; 6.40 ; 7.66 ; 6.91 ; 6.91 ; 7.66 ; 3.73 ; 12.95 ; 13.05 ; NMR 13C : δ 163.8 (C-2); 103.0 (C-3); 181.7 (C-4); 161.4 (C-5); 98.8 (C-6); 164.1 (C-7); 94.0 (C-8); 157.3 (C-9); 103.7 (C-10); 121.0 (C-1'); 131.3 (C-2'); 120.0 (C-3'); 159.6 (C-4'); 116.2 (C-5'); 127.8 (C-6'); 163.2 (C-2''); 103.2 (C-3''); 182.1 (C-4''); 160.5 (C-5''); 98.7 (C-6''); 161.0 (C-7''); 104.1 (C-8''); 154.5 (C-9''); 103.6 (C-10''); 123.0 (C-1'''); 128.0 (C-2'''); 114.5 (C-3'''); 162.2 (C-4'''); 114.5 (C-5'''); 128.0 (C-6'''); 55.5 (4'''-OCH3).Quercetin-3-O-β-glucoside (3). ESI-MS m/z 463.0510 [M−H]− ; NMR 1H : δ 6.21 ; 6.41 ; 7.55 ; 6.85 ; 7.66 ; 5.46 ; 3.24 ; 3.25 ; 3.11 ; 3.10 ; 3.57 ; 3.60 ; NMR 13C : δ 156.4 (C-2); 133.4 (C-3); 177.5 (C-4); 161.3 (C-5); 98.8 (C-6); 164.4 (C-7); 93.6 (C-8); 156.3 (C-9); 103.9 (C-10); 122.0 (C-1'); 116.0 (C-2'); 144.9 (C-3'); 148.5 (C-4'); 115.3 (C-5'); 122.0 (C-6'); 101.0 (C-1''); 74.1 (C-2''); 76.6 (C-3''); 70.0 (C-4''); 77.5 (C-5''); 61.0 (C-6'').Quercetin-3-O-β-galactoside (4). ESI-MS m/z 463.0510 [M−H]− ; NMR 1H : δ 6.21 ; 6.41 ; 7.59 ; 6.85 ; 7.57 ; 5.37 ; 3.58 ; 3.39 ; 3.67 ; 3.33 ; 3.34 ; 3.35 ; NMR 13C : δ 156.4 (C-2); 133.5 (C-3); 177.5 (C-4); 161.2 (C-5); 98.8 (C-6); 164.4 (C-7); 93.6 (C-8); 156.2 (C-9); 104.0 (C-10); 121.6 (C-1'); 116.3 (C-2'); 144.9 (C-3'); 148.6 (C-4'); 115.3 (C-5'); 121.6 (C-6'); 101.9 (C-1''); 71.3 (C-2''); 73.3 (C-3''); 68.0 (C-4''); 75.9 (C-5''); 60.2 (C-6'').1–4 were assayed in different concentrations, and their FRSA against DPPH• and ABTS•+ was investigated.The extracts, fractions, and the flavonoids −1 or the flavonoids 1–4 at concentrations of 33.3, 26.7, 20.0, 13.3, 6.70, and 1.70 µg mL−1) were added to 0.6 μM DPPH• in MeOH. The resulting mixture was incubated in the dark at 25 °C for 30 min, being the remaining DPPH• determined colorimetrically at 517 nm using MeOH as blank , where A is the absorbance without the tested sample  and Asample is the absorbance in the presence of the tested sample. The results are expressed as the mean IC50 values obtained from triplicates  was used the equation: Inhibition % = [1 − , podocarpusflavone A (2), quercetin-3-O-β-glucoside (3), and quercetin-3-O-β-galactoside (4) in the more electroactive fractions, which could be responsible, at least in part, for the observed free radical scavenging activity. In view of these findings, further chemical and pharmacological investigations to identify other secondary metabolites, and to evaluate the potential of this species as an antioxidant in vivo are recommended.


The structures of 1–4 were elucidated by using spectroscopic analyses. The relative stereoconfigurations of compounds 3 and 4 were determined by NOESY experiments. In the bioassay test, 1–4 showed no cytotoxicity against the Hela cell lines. Compound 4 also showed no inhibitory bioactivity on HCV protease and SecA ATPase and wasn’t active against VSVG/HIV-luc pseudotyping virus.Four new  Aegiceras corniculatum, Excoecaria agallocha, Kandelia candel, Bruguiera gymnorrhiza, Avicennia mariana, Heritiera littoralis, have antibacterial activities and 81.7% strains have antifungal activities  or the respective liquid medium at 22 ºC. For long-term preservation, cultures grown on agar plates supplemented with 5% glycerol were maintained in the vapor phase of liquid nitrogen. The strain isolate has been deposited in the strain collection of the Leibniz Institute for Natural Products Research and Infection Biology. The strain was isolated from the mangrove plant 2 from an agar plate of a well grown culture and cultivated as resting culture for 17 days at 22 ºC. The production culture was carried out in a 300 L fermentor filled with 200L of the above production medium; it was grown for 10 days at 22 ºC  and stirred at 125 rpm. The inoculum (2 L) was obtained after several steps of resting cultures with increasing cultivation volume in a modified malt extract medium .For screening purposes, the strain was grown in 250 mL Erlenmeyer flasks with 100 mL of the production culture medium, consisting of saccharose 20 g/L, soybean flour10 g/L, cornsteep 10 g/L, and KCl 8 g/L, adjusted to pH 6.5 prior to sterilization. It was inoculated with pieces (1 × 1 cmStreptomyces sp. (strain HK10552) was filtered by centrifugation and subjected to a XZD-161M resin column (20 × 20 cm) eluted with MeOH/H2O (gradient from 40:60 to 90:10 in 38 min). Seven fractions were collected and lyophilized. Fractions 5-6  was further purified on Sephadex LH-20 eluted with MeOH (900 mL) to give six subfractions. Subfraction 6 (900 mg) was separated on a RP-18 silica gel column eluted with MeOH/H2O  to give six fractions (T1-T6). Fraction T2 (88.6 mg) and T3 (30.2 mg) was purified by semi-preparative HPLC with MeOH–H2O (1:1) as a mobile phase to yield compound 1 (13.9 mg) and 2 (19.6 mg). T4 (119.8mg) and T5 (88.2mg) was followed by the same way as T2 with MeOH–H2O (7:3) as a mobile phase to obtain 3 (29.7 mg) and 4 (40.9 mg).Fermentation supernatant of (2E)-11-(4-Aminophenyl)-5,9-dihydroxy-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (1). Yellow oil; c 0.015, MeOH); UV maxλ  (MeOH) 318, 206 nm; IR (KBr) maxν 3,733, 3,357, 2,964, 2,931, 1,698, 1,650, 1,595, 1,379, 1,175, 826 cm-1; 1H- and 13C-NMR data, see m/z positive 364.4 [M + H]+; HRESIMS m/z 364.21266 [M + H]+ .9-(4-Aminophenyl)-3,7-dihydroxy-2,4,6-trimethyl-9-oxo-nonoic acid (2). Yellow oil; c 0.07, MeOH); UV λmax (MeOH) 319, 231, 195 nm; IR (KBr) νmax  3,733, 3,515, 3,446, 3,354, 2,961, 2,933, 2,869, 1,772, 1,651, 1,629, 1,179, 1,592, 830 cm-1; 1H- and 13C-NMR data, see m/z positive 338.5 [M + H]+; HRESIMS m/z 338.19701 [M + H]+ .(2E)-11-(4′-aminophenyl)-5,9-O-cyclo-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (3). Yellow oil; c 0.03, MeOH); UV λmax (MeOH) 314, 207 nm; IR (KBr) νmax 3,448, 3,356, 3,230, 2,964, 2,911, 1,700, 1,652, 1,596, 1,381, 1,025, 1,051, 828 cm-1; 1H- and 13C-NMR data, see m/z positive 346.6 [M + H]+; HRESIMS m/z 346.20109 [M + H]+ .9-(4′-aminophenyl)-3,7-O-cyclo-2,4,6-trimethyl-9-oxo-nonoic acid (4). Yellow oil; c 0.04, MeOH); UV λmax (MeOH) 316, 230,192 nm; IR (KBr) νmax 3,448, 3,358, 3,238, 2,923, 1,720, 1,636, 1,596, 1,379, 1,025, 828 cm-1; 1H- and 13C-NMR data, see m/z positive 320.4 [M + H]+; HRESIMS m/z 320.18587 [M + H]+ .p-aminoacetophenonic acids from an endophyte Streptomyces sp. (strain HK10552) of the mangrove plant A. corniculatum was reported. These compounds were identified as (2E)-11-(4′-aminophenyl)-5,9-dihydroxy-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (1), 9-(4′-amino-phenyl)-3,7-dihydroxy-2,4,6-trimethyl-9-oxo-nonoic acid (2), (2E)-11-(4′-aminophenyl)-5,9-O-cyclo-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (3) and 9-(4′-aminophenyl)-3,7-O-cyclo-2,4,6-trimethyl-9-oxo-nonoic acid (4). In the bioassay test, 1–4 showed no cytotoxicity against the Hela cell lines. Compound 4 also showed no inhibitory bioactivity on HCV protease and SecA ATPase and was not active against VSVG/HIV-luc pseudotyping virus. Further chemical and pharmaceutical investigation will be carried on in order to explore the rich source of bioactive secondary metabolites of mangrove endophytes.Isolation of four new


Trichilia casaretti C. DC. together with three known triterpenes—24-methylencycloart-3β,22-diol, 22,25-dihydrocycloart-23(E)-en-3β-ol, and 22(R)-hydroxycycloart-24-en-3-ol. These compounds were characterized on the basis of their spectral data, mainly 1D (1H and 13C) and 2D NMR , and mass spectra (EI-MS and HR-ESI-MS), also involving comparison with data from the literature.Two new triterpenes cycloartane-type, named 24-methylencycloartan-12-oxo-3β,22α-diol and trichiliol, were isolated from the leaves of  Trichilia genus (Meliaceae) comprises about 230 species distributed throughout tropical America, which are recognized for their significant economic importance and high commercial value. Phytochemical studies revealed that this genus is a potential source of terpenoids, including triterpenes, limonoids, steroids and other terpenes derivatives [T. casaretti collected in Espírito Santo State, Brazil, were reported by Vieira et al. in 2010 [The Meliaceae family has attracted such a great interest among phytochemists because of its relatively complex and diverse chemical structures and its biological activity, mainly against insects ,2,3,4. Tivatives ,6. Speciivatives ,7,8. TheT. casaretti specimen, which allowed the characterization of five triterpenes, including two novel cycloartane-type triterpenes named 24-methylencycloartane-12-oxo-3β,22α-diol (1) and trichiliol (2). The known triterpenes were identified as 24-methylencycloartane-3β,22-diol (3), cycloartane-22(E)-en-3β,22,25-triol (4), and 22(R)-hydroxycycloart-24-en-3-ol (5). Their structures  and trichiliol (2), along with three known triterpenes, cycloartane-type 24-methylencycloart-3β,22-diol (3) [E)-en-3β-ol (4) [R)-hydroxycycloart-24-en-3-ol (5) [1H and 13C-NMR spectral data, especially 2D NMR and mass spectral data, besides comparison with values described in the cited literature.Fractionation of the MeOH extract of the leaves from diol (3) ,10, 22,2β-ol (4) ,11 and 23-ol (5)  were cha1) was isolated as yellow oil, 3, c 0.02). The analysis of the {1H}- and 13C-APT NMR spectra  and one C=O group at δC 216.0 (C-12)), seven CH  and 70.3 (CH-22), nine methylene CH2 (including one sp2 at δC 109.8 (CH2-31)) and seven methyl groups CH3, allowing to deduce the expanded partial formula C6(C=O)CH5(OCH)2(CH2)10(CH3)7 = C31H48O3 or C6(C=O)CH5(HOCH)2(CH2)10(CH3)7 = C31H50O3 based in the presence of two hydroxyl groups.The new triterpene named 24-methylencycloartan-12-oxo-3β,22α-diol at m/z 471.3475, that in combination with 1H-NMR (1D and 2D) and comparative analysis of the {1H}- and 13C-APT NMR spectra (31H50O3 (seven degrees of unsaturation) compatible with a cycloartane-type 24-methylencycloart-3β,22-diol (3) skeleton [1 and 3 consists of a carbonyl group at C-12.The HR-ESI-MS spectrum of  spectra  enabled skeleton . The onlC 216.0 in the 13C-APT NMR spectrum suggested the presence of a carbonyl group in the triterpene cycloartane 1. The location of the carbonyl group at C-12 was supported in the HMBC spectrum by the correlation (3JHC) between C-12 (δC 216.0) and the hydrogens of the methyl group 3H-18 at δH 0.90. Thus, the new triterpene cycloartane-type was characterized as 24-methylidencycloartan-12-oxo-3β,22-diol (1).The signal at δ2 was isolated as a yellow oil, 3, c 0.001). The analysis of the {1H}- and 13C-NMR spectra (CH)(OCH)2(CH2)2(CH3)2 = C8H13O2 of the side chain, different of the cycloartane triterpene 1 with signals representing 31 carbon atoms including (C)(CH)2(HOCH)(CH2)2(CH3)3 = C9H17O of the side chain.The 13C-NMR spectrum involving the side chain of 2 revealed signals representing only two methyl groups when compared with those of 1 with three, one corresponding to CH3-21 at δC 19.5 correlated in the HSQC with the doublet (J = 6.3 Hz) signal at δH 1.22 (3H-21) and one attached to sp2 carbon , two sp2 carbon atoms at δC 147.8 (C-25) and 112.5 (CH2-26), featuring a methylidene group =CH2 confirmed by correlations (1JHC) between δC 112.48 (CH2-26) with two broad singlets at δH 5.32 (Ha-26) and 5.10 (Hb-26) observed in the HSQC spectrum.The 2JHC correlations between C-25 (δC 147.8) and 3H-27 (δH 2.01) and C-26 (δC 112.5) with 3H-27 (δH 2.01). The presence of a isopropenyl group (isopropyl in 1) attached to CH-24  was deduced by HSQC and HMBC correlations between CH-24 and 3H-27 . It was also possible to observe the interaction between CH-22  and 3H-21 . These data allowed to postulate the presence of oxygen atoms linked to carbon 22 (HC-O-22: δH 4.55\69.7) and 24 (HC-O-24 δH 5.32\86.6), in agreement with the 1H-1H-COSY spectrum which showed correlations between the methylene hydrogens 2H-23 (δH 2.10 and 1.90\δC 34.5) and both hydrogen atoms H-22 (δH 4.55) and H-24 at δH 5.32 . Other heteronuclear correlations observed in the HMBC spectrum of 2 were shown in The location of the methylidene group at the C-25 was confirmed through the 2 , named trichiliol.The relative stereochemistry indicated in 254 was used in thin layer chromatography analysis. 1H and 13C-NMR spectra were measured on a Bruker model DRX-500 and Jeol model Eclipse-400 spectrometers, equipped with inverse probes and field gradient, operating at 500 (1H) and 125 (13C) and 400 (1H) and 100 (13C) MHz. CDCl3 and pyridine-d5 were used as solvents and TMS as internal reference. Chemical shifts are given in the δ scale (ppm) and coupling constants J in Hz. One dimensional (1D) 1H and 13C-NMR spectra were acquired under standard conditions by using a direct detection 5 mm 1H/13C dual probe. Standard pulse sequences were used for two dimensional spectra by using a multinuclear inverse detection 5 mm probe with field gradient.Measures of optic rotation were obtained on a Perkin Elmer 343 digital polarimeter. Melting point was obtained on a Microquímica MQRPF. EI-MS (low resolution) mass spectra were obtained on Shimadzu QP5050A mass spectrometer. HRESI-MS (high resolution) mass spectra were obtained by using a ESI-IT-OF-MS SHIMADZU mass spectrometer, using the positive ion mode of analysis. Chromatographic purifications were carried out over silica gel 60 (70–230 mesh). Silica gel 60 FTrichilia casaretti C. DC. were collected at Vale Cia, Linhares City, Espirito Santo State, Brazil, and identified by Domingos Folly. A voucher specimen (CVRD-449) was deposited at the Vale Cia of Vale herbarium, Vale Cia, Linhares City, Espirito Santo State, Brazil.The leaves of Trichilia casaretti C. DC. were extracted with methanol at room temperature, yielding 15.0 g of crude methanol extract. The methanol extract was submitted to liquid-liquid partition . The CH2Cl2 fraction (10.1 g) was submitted to liquid-liquid partition . The methanol fraction (8.0 g) was chromatographed over a silica gel column with a gradient of ethyl acetate/hexane, affording eight fractions. Fraction 1 (19.3 mg) was similarly rechromatographed, yielding triterpene 5 (8.0 mg). Fraction 5 (518 mg) was analogously rechromatographed, affording eleven fractions. Fraction 5.7 (66.8 mg) was similarly chromatographed, affording triterpene 4 (4.0 mg). Fraction 7 (6.736 mg) was chromatographed affording fifteen fractions. The fraction 7.5 (1.300 mg) was rechromatographed affording fourteen fractions. The triterpene 2 (10 mg) was obtained from the fractions 7.5.3 (45 mg) and 7.5.4 (100 mg). The triterpene 1 (25 mg) was obtained from the fraction 7.6 (500 mg). The fraction 7.9 (163 mg) was chromatographed affording fifteen fractions. The fraction 7.9.2 (50.3 mg) yielded triterpene 3 (11 mg).Air dried and powdered leaves (920 g) from


Agastache rugosa flowering aerial parts against the root knot nematode, Meloidogyne incognita, and to isolate and identify any nematicidal constituents from the essential oil. The essential oil of A. rugosa aerial parts was obtained by hydrodistillation and analyzed by GC-FID and GC-MS. A total of 37 components of the essential oil were identified, with the principal compounds being methyleugenol (50.51%), estragole (8.55%), and eugenol (7.54%), followed by thymol (3.62%), pulegone (2.56%), limonene (2.49%) and caryophyllene (2.38%). Based on bioactivity-guided fractionation, the three active constituents were isolated from the essential oil and identified as methyleugenol, estragole and eugenol. The essential oil of A. rugosa exhibited strong nematicidal activity against M.incognita, with a LC50 value of 47.3 μg/mL. The components eugenol (LC50 = 66.6 μg/mL) and methyleugenol (LC50 = 89.4 μg/mL) exhibited stronger nematicidal activity against M.incognita (LC50 = 185.9 μg/mL). The results indicate that the essential oil of A. rugosa aerial parts and its constituent compounds have potential for development into natural nematicides for control of the root knot nematode.The aim of this research was to determine the chemical composition and nematicidal activity of essential oil of  Meloidogyne incognita (Kofoid and White) Chitwood is the most economically important and widely distributed nematode throughout China and it is responsible for considerable crop losses  instrument using CDCl3 as the solvent with TMS as internal standard. Electron impact mass spectra (EIMS) were determined on a Micromass VG7035 mass spectrometer at 70 eV (probe).The crude essential oil of Estragole  δ (ppm): 7.27 , 7.01 , 6.14 , 5.26 , 3.92 , 3.50 . 13C-NMR  δ (ppm): 158.22 (C-8), 138.08 (C-1), 132.16 (C-9), 129.61 (C-4), 115.52 , 113.99 , 55.18(C-10), 39.52 (C-7). EI-MS m/z (%): 149 (13), 148 (100), 147 (46), 133 (23), 121 (41), 117 (32), 105 (23), 91 (20), 77 (23). The spectral data matched that given in a previous report [agole 1, . A colorEugenol  δ (ppm): δ: 6.83 , 6.66 , 6.65 , 5.94 , 5.73 , 5.06 , 3.81 , 3.30 . 13C-NMR  δ (ppm): 146.60 (C-3), 144.03 (C-4), 137.91 (C-8), 131.94 (C-1), 115.49 (C-5), 114.46 (C-2), 111.26 (C-9), 55.84 (C-10), 39.92 (C-7). EI-MS m/z (%): 165 (11), 164 (100), 149 (29), 137 (15), 133 (15), 121 (14), 103 (19), 91 (14), 77 (21), 55 (18). The spectral data matched that given in a previous report [genol 2, . A colorMethyleugenol  δ (ppm): 6.81 , 6.74 , 6.71 , 5.96 , 5.07 , 3.86 , 3.87 , 3.34 . 13C-NMR  δ (ppm): 148.8 (C-4), 147.3 (C-3), 137.7 (C-8), 132.6 (C-6), 120.4 (C-1), 115.6 (C-9), 111.8 (C-2), 111.2 (C-5), 55.9(-OCH3), 55.7 (-OCH3), 39.8 (C-7). EI-MS m/z (%): 178 (100), 163 (33), 151 (15), 147 (32), 135 (18), 131 (11), 107 (28), 103 (39), 91 (50), 77 (26). The spectra data matched with previous report [genol 3, . A color(39), 91 , 77 (26)M. incognita obtained from tomato roots with aid of a stereomicroscope were maintained in Petri dishes during 24 h in distilled H2O for the juveniles to hatch. Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of A. rugosa essential oil (five concentrations) and pure compounds (five concentrations) was prepared in H2O solution with 2% DMSO. Aliquots of H2O (20 μL) containing ca. 100 juveniles (J2) were transferred to vials to which 980 μL of the solution containing essential oil or pure compounds was added. The vials were kept in a hood at 25 °C. The counting of the inactive nematodes was performed at every 24 h for 72 h. After the last count, the inactive juveniles were maintained in distilled H2O for 24 h to observe their revival. Six repetitions for each treatment were performed using H2O and a 2% DMSO in H2O solution as control. The experiments were repeated in three times. Results from all replicates for the pure compounds and essential oil were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC50  values and their 95% confidence intervals (CI 95%) [Egg masses of (CI 95%) .A. rugosa aerial parts and its main constituent compounds, eugenol, methyleugenol and estragole, have potential for development into natural nematicides for the control of root knot nematodes.The study indicates that the essential oil of