Cucumis sativus L.) stems and identified as -2-[(2'R)-2-hydroxytetra-cosanoylamino]-1,3,4-octadecanetriol-10-ene (1), 1-O-β-D-glucopyranosyl-2-[(2'R)-2-hydroxy-tetracosanoylamino]-1,3,4-octadecanetriol-10-ene (2) and soya-cerebroside I (3) by their physicochemical properties and spectroscopic analysis. They were evaluated to show antifungal and antibacterial activity on test microorganisms including four fungal and three bacterial species. Among them, compound 1, a relatively low polarity aglycone, exhibited stronger antimicrobial activity than its corresponding glycoside 2. The results indicated that sphingolipids could be the main antimicrobial compounds in the crude methanol extract of cucumber stems.Three antimicrobial sphingolipids were separated by bioassay-guided isolation from the chloroform fraction of the crude methanol extract of cucumber ( Plants are capable of synthesizing a diverse array of secondary metabolites. These may be produced constitutively  or in response to pathogen or herbivore attack or stress  . There hCucumis sativus L.), which belongs to the Cucurbitaceae, is now widely planted in the temperate and tropical zones, including all the districts in China [Cucumber -2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octa- decanetriol-10-ene (1), 1-O-β-D-glucopyranosyl--2-[(2'R)-2-hydroxytetracosan-oylamino]-1,3,4-octadecanetriol-10-ene (2) and soya-cerebroside I (3), whose structures were shown in Three sphingolipids were isolated from the chloroform fraction of the crude methanol extract from cucumber stems based on bioassay-guided fractionation. After comparing their physicochemical and spectrometric data with those reported in the literature ,11,12,132S,3S,4R,10E)-2-[(2'R)-2-Hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (1) was first isolated from the medicinal fungus Engleromyces goetzei [O-β-D-Glucopyranosyl--2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (2) was first isolated from the roots of Incarvillea arguta (Bignoniaceae) -1,3,4-octadecanetriol-10-ene (1). White amorphous powder (MeOH); m.p. 138-139 °C; C42H83NO5; FAB-MS (negative) m/z, 680 ([M-H]-); 1H-NMR (pyridine-5d) δ (ppm), 8.57 , 5.52 , 5.50 , 5.10 , 4.61 , 4.51 , 4.42 , 4.33 , 4.27 , 1.25-1.30 , 0.85 ; 13C-NMR (pyridine-5d) δ (ppm), 62.0 (C-1), 52.9 (C-2), 76.9 (C-3), 73.0 (C-4), 33.3 (C-5), 26.8 (C-6), 32.2 (C-9), 130.8 (C-10), 130.7 (C-11), 33.0 (C-12), 175.3 (C-1'), 72.5 (C-2'), 35.7 (C-3'), 25.8 (C-4'), 29.6-30.3 (C-13-16 and C-5'-22'), 22.9 (C-17 and C-23'), 14.3 (C-18 and C-24'). The structure was confirmed by comparison with literature data [1-O-β-D-glucopyranosyl--2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecane-triol-10-ene (2). White amorphous powder (MeOH); m.p. 203-205 °C; C48H93NO10; FAB-MS (negative) m/z, 842 [M-H]-, 680; 1H-NMR (pyridine-5d) δ (ppm), 8.54 , 5.50 , 5.49 , 5.27 , 4.93 , 1.24-1.31 , 0.85 ; 13C-NMR (pyridine-5d) δ (ppm), 70.4 (C-1), 51.9 (C-2), 75.9 (C-3), 72.5 (C-4), 33.9 (C-5), 33.0 (C-6), 32.9 (C-7), 32.8 (C-8), 32.2 (C-9), 130.9 (C-10), 130.7 (C-11), 33.3 (C-12), 175.7 (C-1'), 72.5 (C-2'), 35.6 (C-3'), 25.9 (C-4'), 29.6-30.0 (C-13-16 and C-5'-22'), 23.0 (C-17 and C-23'), 14.3 (C-18 and C-24'); Glc: 105.5 (C-1"), 75.2 (C-2"), 78.5 (C-3"), 71.6 (C-4"), 78.6 (C-5"), 62.7 (C-6"). The structure was confirmed by comparison with literature data -, 550; 1H-NMR (pyridine-5d) δ (ppm), 8.38 , 5.77 , 5.49 , 4.92 , 4.51 , 4.38 , 4.25 , 4.21 , 4.05 , 3.92 , 2.14 , 1.99 , 1.71 , 1.37 , 1.25-1.35 , 0.86 ; 13C-NMR (pyridine-5d) δ (ppm), 70.2 (C-1), 54.6 (C-2), 71.5 (C-3), 131.1 (C-4), 132.1 (C-5), 32.2 (C-6), 32.1 (C-7), 130.0 (C-8), 132.1 (C-9), 32.9 (C-10), 175.7 (C-1'), 72.3 (C-2'), 35.7 (C-3'), 25.9 (C-4'), 29.6-30.0 (C-11-16 and C-5'-14'), 23.0 (C-17 and C-15'), 14.3 (C-18 and C-16'); Glc: 105.7 (C-1″), 75.2 (C-2″), 78.5 (C-3″), 71.5 (C-4″), 78.6 (C-5″), 62.7 (C-6″). The structure was confirmed by comparison with literature data  × 100, where cD is the average diameter increase of fungal colony with the negative control, and tD is the average diameter increase of a fungal colony with the treatment. To evaluate antifungal activity of the three sphingolipids, the test method was the same as that described above, only the concentration of the compound in medium was different.Mycelial radial growth inhibition assay was used to evaluate antifungal activity of the extract and fractions ,17. BrieXanthomonas vesicatoria ATCC 11633, Pseudomonas lachrymans ATCC 11921 and a Gram-positive bacterium, Bacillus subtilis ATCC 11562. X. vesicatoria is a pathogen of tomato bacterial scab, P. lachrymans is a pathogen of cucumber bacterial spot and B. subtilis is widely distributed in plants. All bacterial species were obtained from the microbial culture stock in the Department of Plant Pathology, China Agricultural University and maintained in LB medium at 28 ± 1 °C for antibacterial tests.The bacterial species included two Gram-negative bacteria, 6 CFU/mL, 150 µL) was thoroughly mixed with LB agar medium (15 mL) at 50 ºC and was immediately poured onto the solidified water agar layer. The inoculated Petri-dishes were placed uncovered in an incubator at 28 ± 1 °C for 20 min to evaporate the liquid medium on the surface completely. Equidistant wells of 5 mm in diameter were then poured into the solid medium with a sterile cork borer and each was filled with the test solutions (50 µL) at the desired concentration (50 mg/mL). The positive control was streptomycin sulfate with quantity in the well being 8 µg. The culture dishes were kept at 4 ºC for 12 h and then incubated for 24 h at 28 ± 1 °C. The diameter  of bacteria-free zone surrounding each well remaining after the incubation period was measured as the antibacterial activity.To evaluate antibacterial activity of the extract or fractions from the plant material, agar-well diffusion assay was employed . Briefly50) of the three sphingolipids against bacteria, a colorimetric assay by using chromogenic reagent 3--2,5-diphenyl tetrazolium bromide (MTT) was employed  × 100, where Ac was an average of six replicates of light absorption values at wavelength 510 nm of the negative controls, and At was an average of six replicates of light absorption values at wavelength 510 nm of the samples. The IC50 value was calculated using the linear relation between the inhibitory probability and concentration logarithm according to the method of Sakuma [To further evaluate the median effective inhibitory concentration -2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (1), 1-O-β-D-glucopyranosyl--2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene (2) and soya-cerebroside I (3). They may be contributors to the antimicrobial activity of the extract and fractions. In addition to the chloroform fraction, the n-butanol fraction also showed consistent antimicrobial activity, in some cases being similar to that of the chloroform fraction. It may contain other antimicrobial compounds that deserve isolation and examination of their bioactivity. The results from the present study provided the chemical basis for the efficacy of cucumber stems against plant bacterial and fungal pathogens including those affecting cucumber itself.In this work, we reported for the first time the isolation of three antimicrobial sphingolipids from cucumber stems by bioassay-guided fractionation. These compounds mainly existed in the chloroform fraction of the crude methanol extract with medium polarity, and were identified as (


The newly synthesized compounds were characterized by means of different spectroscopic methods such as IR, NMR, mass spectrometry and by elemental analyses. The new compounds displayed significant activity against both Gram-positive and Gram negative bacteria, in addition to fungi. Molecular docking and POM analyses show the crucial role and impact of substituents on bioactivity and indicate the unfavorable structural parameters in actual drug design: more substitution doesn’t guaranty more efficiency in bioactivity.Several series of novel substituted thienothiophene derivatives were synthesized by reacting the synthone  In addition, the bioactivity of the newly prepared compounds will be presented.On the other hand, addition of the enaminone group to the thienothiophene nucleus has added new activities to these compounds; one derivative that contains both of these groups is used as a drug to treat epilepsy ,22. In v1–7 were synthesized as shown in 1–7 were characterized by elemental analyses, MS and NMR spectral data. These data, detailed in the experimental part, are consistent with the suggested structures. Thus, the mass spectra display the correct molecular ion peaks for which the measured high resolution (HRMS) data are in good agreement with the calculated values. DEPT and 2D  experiments showed correlations that helped in the 1H- and 13C-signal assignments to the different carbons and their attached, and/or neighboring hydrogens.Compounds 1, a synthon required in this study, was prepared by following a procedure outlined by Mabkhot, et al. + (19%) at m/z 694 corresponding to the molecular formula C38H46O8S2 in addition to fragments at 98 (100%) and 57 (89%).The IR spectrum of compound 6 was prepared by refluxing a mixture of compound 1 and 3-amino-1H-1,2,4-triazole in methanol/DMF  and 8.96 (J = 12.5 Hz) attributed to the two CH protons of the pyrimidine ring and to a singlet at 8.67 of the triazole hydrogen. Moreover, the 13C-NMR spectrum of the prepared compound is in agreement with the proposed structure and showed the following signals pertaining to the different carbon atoms: δ 115.4 (CH), 127.0, 129.1, 130.0 (Ph), 125.0, 130.2, 132.8, 135.8 (thiophene), 152.8, 154.4, 160.0, and 162.9 (Ar-C). Further support of the structure came from mass spectral data; mass spectrum of compound 6 displayed the correct molecular ion [M]+ at m/z = 530 corresponding to the molecular formula C28H18N8S2. Fragments at m/z 81 (86%), 67 (100%), and 55 (88%), in addition to other small ones have also appeared. Other prominent fragments at 368 (100%), 165 (98%), and 105 (40%) also appeared.Its 7 was prepared by refluxing a mixture of 1 and 2-aminobenzimidazole in EtOH/DMF for 7 h (−1 (C=C) in addition to the disappearance of the C=O absorption band of compound 1. 1H-NMR spectrum in DMSO-d6 displayed two doublets corresponding to the pyrimidine CH protons at δ 8.16 (J = 12.5 Hz) and 8.80 (J = 12.5 Hz) in addition to a multiplet corresponding to the phenyl and to the benzimidazole rings in the range δ 7.42–7.52. 13C-NMR spectrum is also in agreement with the suggested structure; it showed signals corresponding to the different carbon atoms in the compound as follows: δ 112.1, 115.1, 122.5, 127.1, 128.1, 129.9, 131.3 ,136.0, 139.1, 142.0, 148.1, 148.4, 156.0 (Ar-C). Similarly, the mass spectrum showed the correct molecular ion [M]+ (25%) at m/z = 648 corresponding to the molecular formula C38H24N6S2 in addition to other related fragments at m/z = 439 (55%), 183 (100%) and at 57 (64%).Finally, compound  for 7 h . The IR Pseudomoas aeruginosa (RCMB 010043) and Escherichia coli (RCMB 010052) for Gram-negative bacteria and Staphylococcus pneumonia (RCMB 010010), Bacillis subtilis (RCMB 010067) for Gram-positive, and Aspergillus fumigates (RCMB 02568) and Candida albicans (RCMB 05036) for fungi. Results of the biological activity are displayed in To investigate the biological activity of the newly prepared compounds, the Cup-plate agar diffusion method was adopted by using discs of disinfected filter papers (6 mm in diameter). Tested compounds were dissolved in DMSO and were charged on the discs with concentrations of 5 mg/mL. The discs were then placed in Petri dishes and were loaded with different Gram-positive and Gram negative bacterial strains: 2a has exhibited remarkable activity against both Gram-positive and negative bacteria while compounds 4a, 5 and 6 showed good activity against Gram-positive bacteria. On the other hand, compound 2a was the most active against tested fungus. Results also reveal that compound 2a was the most potent; it has antibacterial and antifungal activities due to the presence of electron-rich functional groups. These groups could bind with the microorganisms. In addition, the presence of acrylides bonded to the thionothiophene nucleus and the effect of thionothiophene itself against bacteria and fungi may explain the potency of these compounds; the tautomeric forms of these compounds penetrate the cell wall.Results shown in b]thiophene derivatives, nine different target proteins i.e., dihydrofolate reductase (DHFR) (PDB ID 4HOF), secreted aspartic protease (PDB ID 3Q70), and N-myristoyl transferase (PDB ID 1IYL) from C. albicans were selected as fungal targets, while for bacterial targets dihydrofolate reductase (PDB ID 3FYV), gyrase B (PDB ID 4URM) and sortase A (PDB ID 2MLM) from S. aureus and rhomboid protease (PDB ID 3ZMI), methionine peptidase (PDB ID 4PNC) and undecaprenyl diphosphate synthase (PDB ID 4H2M) from E. coli were fetched from the Protein Data Bank thiophene derivatives unveiled their potency as anti-fungal and anti-bacterial inhibitors. All newly synthesized compounds contain large aromatic systems, exhibiting noticeable hydrophobic and Van der Waals (VDW) interactions along with few additional hydrogen bonding with the crucial residues i.e., Ile9, Glu32, Phe36, Ile112, and Tyr118 of 4HOF  thiophene-2,5-diyl)diethanone , DMF-DMA  was refluxed in m-xylene (15 mL) for 7 h. After cooling, the resulting solid product was collected by filtration and recrystallized by using DMF/EtOH to give the desired product 1. Yield: 73%; m.p. 250 °C; IR (νmax): 1622 (C=O), 1539, 1457 cm−1; 1H-NMR (DMSO-d6) δ (ppm): 2.99 , 4.53 , 5.38 , 7.41–7.65 ; 13C-NMR (DMSO-d6) δ (ppm): 192.2, 153.2, 147.7, 145.8, 141.8, 138.8, 134.8, 129.8, 129.6, 129.2, 125.3, 30.6; MS m/z (%): 488 , 386 (100), 368 (47), 213 (73), 43 (46); Anal. calcd. for C28H26N2O2S2: C, 69.11; H, 5.39; N, 5.76; Found: C, 69.15; H, 5.41; N, 5.78.A mixture of 1,1'- was added to hydrazine hydrate (2 mL), phenylhydrazine (1 mL), or 4-chlorophenyl hydrazine (0.5 g) . The mixture of compound 1 and the appropriate hydrazine was refluxed until compound 1 is completely dissolved, followed by addition of the mixed solvent EtOH/DMF. The reaction mixture was then refluxed for 6–7 h and the product was filtered after cooling. The desired product was obtained in a pure form by recrystallization from EtOH/DMF. Using the general procedure, the following compounds were prepared.Compounds −1): 3192 (NH); 1508 (C=N). 1H-NMR (DMSO-d6) δ (ppm): 6.45 , 7.40–7.53 , 7.81 , 13.00 . 13C-NMR δ: 103.0 (CH), 127.3 (CH), 128.6 (CH), 129.0 (C), 129.1 (CH), 130.0 (C), 130.4 (C), 131 (N-CH), 131.3 (C), 135.0 (C), 136.4 (C), 145.8 (C), 147.3 (Ar-C). MS m/z (70 eV): 580, M+ (4%); 279, [M−301]+ (100%); 189, [M−391]+ (46%); 104, [M−476]+ (33%). Anal. Calcd for C24H16N4S2: C, 67.90; H, 3.80; N, 13.20; S, 15.10. Found: C, 67.78; H, 4.21; N, 13.00; S, 15.19.Yellow crystals; yield: 89%; mp: 133–135 °C. IR (cm−1): 1610. 1H-NMR (DMSO-d6): δ, 6.48 , 6.53–7.20 , 7.55 . 13C-NMR: δ, 102.0 (CH), 127.3 (CH), 128.5 (CH), 132.0 (N-CH), 129.2 (CH), 129.9 (C), 130.4 (C), 130.5, 136.4 (C), 145.8 (C), 147.3 (C), 152.8 (C). MS m/z (70 eV): 646, [M−301]+ (100%); 189, [M−391]+ (46%); 104, [M−476]+ (33%). Anal. Calcd for C36H22Cl2N4S2: C, C, 66.97; H, 3.43; Cl, 10.98; N, 8.68; S, 9.93. Found: C, 66.99; H, 3.47; Cl, 11.00; N, 8.55; S, 9.78.Red powder; yield: 67%; mp: 339–341 °C. IR (cm−1): 1618. 1H-NMR (DMSO-d6): δ, 6.50 , 6.53–7.20 , 7.57 . 13C-NMR: δ, 102.1 (CH), 132.0 (N-CH), 128.4, 190.4, 130.5, 131.5, 132.0, 132.9, 133.50, 141.4, 153.3, 160.8 (Ar-C). MS m/z (70 eV): 576, M+ (694%); Anal. Calcd for C36H24N4S2 C, 74.97; H, 4.19; N, 9.71; S, 11.12. Found: C, 74.87; H, 4.00; N, 9.83; S, 10.92.Brown powder; yield: 64%; mp: 194–196 °C. IR  was dissolved in glacial acetic acid (10 mL) containing ammonium acetate (1.0 g) in a 100-mL round bottomed flask connected to a condenser. The mixture was refluxed for 5 h and after cooling, the precipitate was filtered and recrystallized from DMF/EtOH to afford the desired product as yellow powder. Yield: 75%; mp: 298–300 °C. IR (cm−1): 1618. The product was insoluble in most organic solvents, which prevented us from recording its 1H- and 13C-NMR spectra. MS m/z (70 eV): 821, M+ (2%); 262, [M−559]+ (27%); 67, [M−754]+ (100%). Anal. Calcd for C52H39N3O3S4: C, 70.80; H, 4.46; N, 4.76; S, 14.54; Found: C, 70.81; H, 4.46; N, 4.78; S, 14.50.Compound 4a and 4b were prepared according to the following general procedure: a mixture of compound 1 , acetyl acetone , and ammonium acetate (1.0 g) in glacial acetic acid (10 mL) was refluxed for 6 h. The solid product was filtered and recrystallized from DMF/EtOH to afford the desired product in pure form.Compounds −1): 1573 (C=N), 1671 (C=O). 1H-NMR (DMSO-d6) δ (ppm): 2.47 , 2.62 , 7.29–7.59 , 7.95 , 8.26 . 13C-NMR (DMSO-d6) δ (ppm): 29.4 (CH3), 30.6 (COCH3), 119.0 (CH), 129.2 (CH), 129.5 (CH), 129.9 (CH), 134.8 (CH), 138.8 (C), 141.8 (C), 147.7 (C), 151.2 (C), 166.1 (C), 194.1 . MS m/z (70 eV): 560, M+ (3%); 522, [M−38]+ (25%); 431, [M−1291]+ (100%); 326, [M−234]+ (94%). Anal. Calcd for C34H26N2O2S2: C, 73.09; H, 4.69; N, 5.01; S, 11.48; Found: C, 73.20; H, 4.74; N, 4.91; S, 11.40.Obtained as a deep yellow powder. Yield 77%; mp: >360 °C. IR (cm−1): 1568, 11718. 1H-NMR (DMSO-d6) δ (ppm): 1.29 , 2.57 , 4.21 , 7.29–7.59 , 8.20 , 8.26 . 13C-NMR (DMSO-d6) δ (ppm): 29.4 (CH3), 30.6 (COCH3), 62.17 (CH2), 119.1 (CH), 129.2 (CH), 129.6 (CH), 129.9 (CH), 134.8 (CH), 138.8 (C), 141.8 (C), 147.7 (C), 151.2 (C), 166.1 (C), 194.1 . MS data: m/z (70 eV) 580, M+ (4%); 279, [M−301]+ (100%); 189, [M−391]+ (46%); 104, [M−476]+ (33%). Anal. Calcd for C36H30N2O4S2: C, 69.88; H, 4.89; N, 4.53; S, 10.36; Found: C, 69.89; H, 5.09; N, 4.70; S, 10.17.Obtained as a brown powder; yield: 74%; mp: 245–247 °C. IR  and triethyl orthoformate  in presence of ZnCl2 (0.5 g) as a catalyst. The mixture was stirred for 4 h followed by addition of DMF/EtOH. The solid product was collected by filtration and recrystallized from DMF/EtOH to afford compound 5 as a brown solid. Yield: 56%; mp: >360 °C. IR (cm−1): 1626. 1H-NMR (DMSO-d6): δ (ppm): 1.05 , 2.95, 5.69 , 6.53 , 7.42–7.52 . 13C-NMR (DMSO-d6) δ (ppm): δ, 22.4 (CH3), 44.0 (OCH2), 127.8 (C) 129.3 (CH), 129.9 (CH), 130.5 (CH), 131.5 (C), 133.3 (C), 136.5 (C), 142.0 (C), 151.0 (C), 182.4 (C=O). MS data: m/z (70 eV) 694, M+ (19%); 480, [M−205]+ (32%); 98, [M−587]+ (100%). Anal. Calcd for C38H44O8S2: C, C, 65.87; H, 6.40; S, 9.26; Found: C, 65.89; H, 6.59; S, 9.40.The title compound was prepared by mixing compound 6 was prepared according to the following procedure: compound 1  was dissolved in DMF/EtOH  followed by addition of 3-amino-1H-1,2,4-triazole . The mixture was then refluxed for 6 h and the solid that formed was filtered while hot to afford the desired compound which was recrystallized from DMF/EtOH to afford the title compound in a pure form as pale brown crystals; yield: 68%; mp: 349–351 °C. IR (cm−1): 1588. 1H-NMR (DMSO-d6) δ (ppm): 7.41–7.66 , 8.14 , 8.67 , 8.96 . 13C-NMR (DMSO-d6) δ (ppm): 115.4 (CH), 125.0 (C), 127.0 (C), 127.3 (CH), 129.1 (CH), 129.9 (CH), 130.2 (C), 132.8 (C), 135.8 (C), 152.8 (C), 154.4 (CH), 159.9 (CH), 162.9 (C). MS data: m/z (70 eV) 530, M+ (1%); 368, [M−162]+ (100%); 165, [M−165]+ (98%); 105, [M−425]+ (40%). Anal. Calcd for C28H16N8S2: C, 63.62; H, 3.05; N, 21.20; S, 12.13; Found: C, 63.63; H, 5.31; N, 21.30; S, 12.18.Compound 1  was dissolved in ethanol (15 mL) followed by addition of 2-aminobenzimidazole  in DMF/EtOH. The mixture was heated under reflux for 7 h and the solid product was filtered while hot to afford the desired product in a pure form as a white powder. Yield: 70%; mp: 248–350 °C. IR (cm−1): 1626. 1H-NMR (DMSO-d6): δ (ppm): 7.42–7.52 , 8.16 , 8.80 . 13C-NMR (DMSO-d6) δ (ppm): 112.1 (CH), 115.1 (CH), 122.6 (CH), 125 (C), 127.0 (C), 127.1 (CH), 128.1 (CH), 129.9 (CH), 131.3(C), 136.0 (C), 139.1 (C), 142.0, 148.2 (C), 148.4 (CH), 156 (C). MS data: m/z (70 eV) 622, M+ (25%); 567, [M−45]+ (56%); 439, [M−183]+ (55%); 183, [M−439]+ (100%). Anal. Calcd for C38H22N6S2: C, C, 72.82; H, 3.54; N, 13.41; S, 10.23. Found: C, 72.82; H, 3.54; N, 13.41; S, 10.23.The title compound was prepared by the following procedure: Compound 2a has remarkable activity against both Gram-positive and negative bacteria and against the tested fungi, while compounds 4a, 5 and 6 showed good activity against Gram-positive bacteria and against fungi. However, because of these promising results of quantitative structure-activity relationship studies, an extensive synthesis of the analogue series of compounds 1–7 are under way in order to revise the structural errors of pharmacophoric sites and to carry out more efficient drugs on the basis of a POM better understanding of the relationship between the physicochemical properties and biological activity observed for these compounds.In summary, we have succeeded in the synthesis of a number of novel substituted thienothiophenes through known chemical routes. The structures of the new compounds were confirmed by means of different spectroscopic methods and by elemental analyses. On the basis of preliminary screening data for these new compounds, the antibacterial and antifungal activity was evaluated. Results revealed that compound


Ganoderma triterpenes (GTs) are the major secondary metabolites of Ganoderma lucidum, which is a popularly used traditional Chinese medicine for complementary cancer therapy. In the present study, systematic isolation, and in silico pharmacological prediction are implemented to discover potential anti-cancer active GTs from G. lucidum. Nineteen GTs, three steroids, one cerebroside, and one thymidine were isolated from G. lucidum. Six GTs were first isolated from the fruiting bodies of G. lucidum, including 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (1), 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (2), 3β,7β,15α,28-tetrahydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (3), ganotropic acid (4), 26-nor-11,23-dioxo-5α-lanost-8-en-3β,7β,15α,25-tetrol (5) and -dihydroxy-lanosta-8,24-dien- 11-one (6). -N-d-2′-hydroxypalmitoyl-l-O-β-d-glucopyranosyl-9-methyl-4,8-spingodienine (7), and stigmasta-7,22-dien-3β,5α,6α-triol (8) were first reported from the genus Ganodema. By using reverse pharmacophoric profiling of the six GTs, thirty potential anti-cancer therapeutic targets were identified and utilized to construct their ingredient-target interaction network. Then nineteen high frequency targets of GTs were selected from thirty potential targets to construct a protein interaction network (PIN). In order to cluster the pharmacological activity of GTs, twelve function modules were identified by molecular complex detection (MCODE) and gene ontology (GO) enrichment analysis. The results indicated that anti-cancer effect of GTs might be related to histone acetylation and interphase of mitotic cell cycle by regulating general control non-derepressible 5 (GCN5) and cyclin-dependent kinase-2 (CDK2), respectively. This research mode of extraction, isolation, pharmacological prediction, and PIN analysis might be beneficial to rapidly predict and discover pharmacological activities of novel compounds. Ganoderma lucidum (Leyss. ex Fr.) Karstis is one of the most highly used medicinal fungi in the world. Its fruiting body, called Lingzhi or Reishi, has been widely used in traditional Chinese medicine (TCM) as a dietary supplement and medicinal herb in China and other eastern countries for health promotion. Modern medical research has indicated that G. lucidum had comprehensive biological activities, such as anti-cancer, anti-inflammatory, immune-modulating, anti-oxidant, anti-microbial, anti-HIV-1, and so on, between which the most attractive is anti-cancer activity [activity ,6,7,8,9.G. lucidum. Wherein, more than 150 compounds belonged to GTs which were regarded as the main medicinal components, such as ganoderic acid A (GA-A), GA-C2, GA-D, GA-DM, lactone, ganoderiol F, ganodermanotriol, and so on [G. lucidum are still unknown.To date, over 400 compounds were isolated and identified from nd so on ,13,14,15nd so on ,17,18. HVirtual screening is an effective method to predict the biological activity of compounds . Pharmacin silico anti-cancer pharmacological prediction and profiling of GTs had been attempted. According to the docking studies of anti-cancer targets IKK1 and IKK2, Balraj [Several , Balraj  discover, Balraj . GA-D waG. lucidum, studies on chemical constituents of G. lucidum and their biological activity prediction were carried out deeply and systematically. Solvent extraction and silica gel column chromatography were utilized to isolate the novel active constituents from the fruiting bodies of G. lucidum, including GTs, sterides and cerebrosides. Then, reverse target identification of novel isolated GTs was implemented by pharmacophore database and hit targets were used to construct ingredient-target network. Thereafter, high frequency targets from ingredient-target network were selected and their target-related proteins were utilized to construct PIN. According to the results of module analysis of PIN, anti-cancer modules were recognized and pharmacological activity of GTs was explained by gene ontology (GO) enrichment analysis.In order to search for more active GTs from 1H- and 13C-NMR spectra. 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (1) [2) [3) [4) [5) [6) [G. lucidum. Meanwhile, -N-d-2′-hydroxypalmitoyl-l-O-β-d-glucopyranosyl-9-methyl-4,8-spingodienine (7) [8) [Ganodema, together with 4,4,14α-trimethyl-3-oxo-5α-pregna-7,9(11)-dien-20S-carboxylate (9), lucidenic acid C (10), lucidone F (11), lucidone D (12), 7-oxo-ganoderic acid Z (13), ganosineniol A (14), ganoderic acid XL1 (15), ganoderic acid C2 (16), methyl ganoderate C (17), ganolucidic acid γa (18), methyl ganoderate C2 (19), lucidenic lactone (20), ganodericacid C (21), ganodermacetal (22) and ergosterol (23), and ergosta-7,22-dien-3-one (24).The structures of the isolated compounds were elucidated from the data obtained from ster (1) , 3β,7β,1 (1) [2) , 3β,7β,1 [2) [3) , ganotro [3) [4) , 26-nor- [4) [5) ,  [5) [6)  were firnine (7)  and stig (7) [8)  were fir1 was obtained as a yellow oily solid, yielded a positive reaction to 10% H2SO4–EtOH reagent. 1H-NMR  δ: 3.57 , 2.91 , 4.96 , 5.49 , 5.44 , 2.78 , 1.61 , 0.95 , 1.96 , 3.12 , 3.02 , 2.50 , 2.54 , 2.40 , 2.73 , 1.25 , 1.22 , 1.02 , 1.11 , 1.03 , 1.01 , 1.02 ; 13C-NMR  δ: 34.77 (C-1), 28.65 (C-2), 77.03 (C-3), 39.24 (C-4), 49.52 (C-5), 28.59 (C-6), 69.32 (C-7), 161.39 (C-8), 141.47 (C-9), 39.20 (C-10), 199.79 (C-11), 46.65 (C-12), 51.56 (C-13), 56.48 (C-14), 77.43 (C-15), 123.89 (C-16), 154.22 (C-17), 22.46 (C-18), 19.62 (C-19), 27.78 (C-20), 20.22 (C-21), 48.54 (C-22), 207.57 (C-23), 48.29 (C-24), 35.27 (C-25), 176.17 (C-26), 16.99 (C-27), 28.83 (C-28), 16.65 (C-29), 23.46 (C-30), 52.10 (C-O-Me).Compound 2 was obtained as a yellow oily solid, yielded a positive reaction to 10% H2SO4–EtOH reagent. The structure was the same as that of compound 1, except for the substituent of C-26, with a carboxy group on it. 1H-NMR  δ: 3.11 , 4.47 , 5.38 , 5.21 ; 13C-NMR  δ: 78.82 (C-3), 70.10 (C-7), 161.91 (C-8), 142.65 (C-9), 201.68 (C-11), 77.96 (C-15), 125.61 (C-16), 155.77 (C-17), 209.82 (C-23), 179.6 (C-26).Compound 3 was obtained as a yellow oily solid, yielded a positive reaction to 10% H2SO4–EtOH reagent. The structure was the same as that of compound 1, except for its more than a hydroxyl of C-28 and opposite stereoconfiguration of chiral C-15. 1H-NMR  δ: 4.29 , 5.09 , 5.96 , 5.59 , 4.22 , 3.76 ; 13C-NMR  δ: 71.9 (C-3), 69.3 (C-7), 161.7 (C-8), 141.6 (C-9), 200.0 (C-11), 77.3 (C-15), 125.8 (C-16), 154.6 (C-17), 210.3 (C-23), 183.2 (C-26), 66.6 (C-28).Compound 4 was obtained as a white amorphous power, yielded a positive reaction to 10% H2SO4–EtOH reagent. The structure was possessed two oxygenic five-membered rings system in the side chain of lanostane skeleton. 1H-NMR  δ: 3.20 , 4.37 , 5.30 , 2.28 , 1.85 , 2.83 , 2.62 , 2.08 , 2.87 , 1.21 ; 13C-NMR  δ: 77.43 (C-3), 69.09 (C-7), 161.12 (C-8), 141.77 (C-9), 200.24 (C-11), 72.83 (C-15), 45.54 (C-16), 95.21 (C-17), 44.05 (C-20), 17.97 (C-21), 44.50 (C-22), 113.29 (C-23), 44.57 (C-24), 35.66 (C-25), 178.64 (C-26), 15.01 (C-27).Compound 5 was obtained as a white solid, yielded a positive reaction to 10% H2SO4–EtOH reagent. The structure was the same as that of compound 1, except for its the degradation of the carboxyl group and the attachment of a hydroxyl group to C-25 and opposite stereoconfiguration of chiral C-15, with its more than two H atoms of C-16 and C-17. 1H-NMR  δ: 3.20 , 4.42 , 4.68 , 4.12 , 1.81 ; 13C-NMR  δ: 78.07 (C-3), 67.39 (C-7), 160.72 (C-8), 140.89 (C-9), 199.45 (C-11), 70.5 (C-15), 36.57 (C-16), 47.89 (C-17), 213.84 (C-23), 76.39 (C-25).Compound 6 was obtained as a white amorphous powder, yielded a positive reaction to 10% H2SO4–EtOH reagent. The structure was most likely a lanostane triterpenoid with the presence of a trisubstituted C=C bond at C-24. 1H-NMR  δ: 3.46 , 3.48 , 5.23 , 2.97 , 1.67 , 1.73 , 1.87 , 1.73 , 2.65 , 2.56 , 2.00 , 1.88 , 1.62 , 1.51 ; 13C-NMR  δ: 77.48 (C-3), 69.35 (C-7), 160.42 (C-8), 141.78 (C-9), 199.93 (C-11), 29.98 (C-15), 27.84 (C-16), 50.10 (C-17), 23.50 (C-23), 123.88 (C-24), 135.10 (C-25), 23.50 (C-26), 17.42 (C-27). Compound 7 was obtained as a white amorphous powder, yielded a positive reaction to 10% H2SO4–EtOH reagent. 1H-NMR  δ: 8.32 , 6.02 , 3.83 , 4.86 , 4.50 , 5.28 , 3.97 , 1.59 , 6.00 , 0.84 ; 13C-NMR  δ: 14.23 (C-1), 30.27, 29.99, 29.99, 29.97, 29.97, 29.89, 29.89 (C-2-8), 32.08 (C-9), 124.06 (C-10), 131.8 (C-11), 35.56 (C-12), 33.00 (C-13), 132.33 (C-14), 135.58 (C-15), 72.2 (C-16), 54.45 (C-17), 70.04 (C-18), 32.08 (C-19), 14.23 (C-1′), 16.01 (C-2′), 29.86, 29.86, 29.83, 29.78, 29.58, 29.30, 28.26, 28.11, 25.82, 22.89, 22.77 (C-3′-13′), 39.93 (C-14′), 72.39 (C-15′), 175.66 (C-16′), 105.54 (C-1′′), 75.00 (C-2′′), 78.31 (C-3′′), 71.36 (C-4′′), 78.44 (C-5′′), 62.50 (C-6′′). Compound 8 was obtained as a white solid, which when sprayed with 10% H2SO4 ethanol solution followed by heating showed a blue purple color. 1H-NMR  δ: 5.07 , 5.23 , 5.16 , 4.21 , 3.75 , 3.58 , 4.48 , 3.36 , 2.02–1.82 , 1.82–1.76 , 1.68–1.62 , 1.60–1.20 , 0.53 , 0.88 , 0.98 , 0.79 , 0.87 , 0.80 ; 13C-NMR  δ: 21.29 (C-1), 40.17 (C-2), 65.92 (C-3), 31.15 (C-4), 74.43 (C-5), 72.09 (C-6), 119.41 (C-7), 139.62 (C-8), 42.23 (C-9), 36.61 (C-10), 32.42 , 38.93 (C-12), 42.95 (C-13), 54.13 (C-14), 22.55 (C-15), 27.67 (C-16), 55.28 (C-17), 12.02 (C-18), 17.65 (C-19), 40.00 (C-20), 20.94 (C-21), 135.34 (C-22), 131.34 (C-23), 41.96 (C-24), 19.70 (C-26), 19.42 (C-27), 17.24 (C-28).Compound 1, 2, 3, and 5 and two positive GTs could match with CDK2 pharmacophore model (PDB: 1KE5) with fit value more than 0.5, which indicated they might have better biological action to CDK2. The superior mapping result of compound 3 and the best model was showed in 1, 2, 3 and 5 are more similar to that of GA-A and GA-D, because the branch chain of substituent of C-17 is a straight chain, including carboxyl, hydroxyl, or methyl ester, which might be the common structural basis of CDK2 activities, while, the branch chain of compound 4 had two oxygenated quaternary carbons (C-23 and C-17) instead of the carbonyl (C-23) and methyl (C-17) in compound 3, and the branch chain of compound 6 had also no carbonyl or hydroxyl (C-23), which may be the cause of poor biological action to CDK2. Reverse pharmacophoric profiling of six isolated GTs and two positive GTs (GT-A and GT-D) was implemented to explore the pharmacological activity of GTs. In total, the reverse profiling results of eight GTs contained 30 human targets, and targets from the other creatures were eliminated because of less correlation with cancer. Top 20 interaction information of GTs and targets were showed in In order to observe the biological effect of GTs visually, ingredient-target network of GTs was constructed in In this study, 19 high frequency targets were used to construct PIN  for furtHistone acetylation (HAT) of module 1 was closely related to cancer, including KAT2A, TAF10, TAF9, TADA2A, TADA3, and so on. KAT2A is the gene name of GCN5, which is a typical transcriptional cofactor and requirement for HAT . GCN5 coModule 3 showed anti-cancer activity including CCNE1, CCNB1, RBL1, RB1 and CDK2. Cyclin-dependent kinase-2 (CDK2) is an important member in protein kinase family, which could regulate division cycle in eukaryotic cell . PreviouModule 6 and module 7 indicated that GTs might play an important role in anti-tumor effect by regulating receptor tyrosine kinase signaling pathway, especially fibroblast growth factor receptor (FGFR) signaling pathway. Module 11 showed that GTs might inhibit the negative regulation of apoptosis by activating the serine/threonine protein kinase (Pim1), which could promote the apoptosis of tumor cells.G. lucidum were collected from Hainan in February 2013. The samples were authenticated by Professor Chun-Sheng Liu . A voucher specimen (No.130201) has been deposited at the 513 lab of School of Chinese Pharmacy, Beijing University of Chinese medicine.The fruiting bodies of G. lucidum (30 kg) were milled and soaked into 240 L 70% ethanol in the ratio of 1:8 (w/v). The mixtures were left for 12 h at room temperature, and then refluxed for three times, each time for two hours. The extract was filtered through a Whatman number 3 filter paper and concentrated with the rotary evaporator  at 55 °C to obtain the crude 70% ethanol extract of G. lucidum. The crude extract was dissolved in water (12 L), and extracted with ethyl acetate (12 L) and n-butyl alcohol (12 L) for three times, respectively. The extract was filtered and concentrated, yielded an ethyl acetate fraction  and an n-butyl alcohol fraction .The dried fruiting bodies of 3:EtOAc (4:1) as the eluent, yielded 40 fractions. The 40 fractions were then combined to five groups (F1 to F5) according to the thin layer chromatography (TLC) .The ethyl acetate fraction was subjected to silica gel  dry column chromatography using CHCl11 to F14) with petroleum ether: EtOAc  according to the TLC. F11 and F12 were applied to precipitation crystallization, filtering, recrystallization to obtain compounds 23 and 24. F13 was subjected to CHCl3: EtOAc (2:1) as the eluent, then applied to preparation TLC  with petroleum ether: chloroform: methanol (5:5:1) as mobile phase to obtain compound 9. F13 was subjected to a silica gel column chromatography using CHCl3:MeOH (5:1) as mobile phase to obtain compound 10.The F1 fraction (20.0 g) was fractionated into four groups  according to the TLC. F21 was rechromatographed over silica gel column using petroleum ether: EtOAc (1:1) as mobile phase to obtain compound 6. F22 was also subjected to a silica gel column using CH2Cl2: MeOH (1:1) as the eluent, and further applied to Sephadex LH-20 column , ODS-A  and MCI-gel CHP-20-P  with a step gradient of MeOH/H2O to yield compounds 11, 12, 13. The F2 fraction (78 g) was fractioned into three groups with CHCl2Cl2/MeOH  to yield seven groups (F31–F37). F31was submitted to column chromatography using CH2Cl2:MeOH (3:1) as mobile phase to yield compound 4. F33 and F34 were further subjected with Sephadex LH-20, ODS-A and MCI-gel CHP-20-P, and eluted with a step gradient of MeOH:H2O to obtain compounds 1, 2, 5, 8. F33, F34, and F35 were subjected to a silica gel chromatography eluted with a CH2Cl2:MeOH:H2O (30:1:0–8:2:0.2), and further reapplied to Sephadex LH-20, ODS-A, and MCI-gel CHP-20-P column, and using MeOH as eluting solvents to yield compounds 14, 16, 17, 18, 19, 15. The F34 fraction (10 g) was subjected to Sephadex LH-20 and MCI-gel CHP-20-P open column using MeOH as eluting solvents to yield compound 3.Fraction F3 (55.0 g) was subjected to a silica gel column  and eluted with a step gradient of CH2Cl2/MeOH/H2O (40:1:0–7:3:0.5) as mobile phase, and further submitted to Sephadex LH-20, ODS-A, and MCI-gel CHP-20-P, and eluted with MeOH/H2O to yield compounds 7, 20, 21, 22. The structures of compounds 1–24 are shown in A 160 g sample of n-butyl alcohol extract was fractionated into three groups according to the TLC with EtOAc: MeOH in the following ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 1:1. Fraction 1 and 3 were applied to silica gel column using a step gradient of CHLigand profile module was utilized to reversely identify targets for six isolated and two known positive GTs in Discovery Studio 4.0 . Previous researches indicated GA-A and GA-D have the clear anti-cancer activity ,41. Therin silico profiled by structure-based pharmacophore (SBP) models database called pharmaDB in DS [Eight GTs were DB in DS ,43,44. IDB in DS . Fit valDB in DS . AccordiDB in DS . The higThe PPI information of high-frequency targets was derived from the online database of String 9.1  which cop-value was utilized to represent the probability that a group of genes was from the same gene ontology, and less p-value indicated more probability.A functional module is the structural unit of PIN, which can represent cellular functional organization and extract meaningful information from the complex PIN. In this paper, functional modules with more than four sides were identified by the MCODE methods . Based oG. lucidum is one of the most commonly used TCM for thousands of years. The discovery of therapeutic basis and action mechanism of G. lucidum was essential to clinical use of drugs from G. lucidum. In this paper, an innovated research mode of extraction, isolation, pharmacological prediction, and PIN analysis was presented to discover the novel compounds and predict the anti-cancer activity of G. lucidum. Twenty-four compounds were isolated and identified from the 70% ethanol extract of the fruiting bodies of G. lucidum, including nineteen triterpenes, three steroids, one cerebroside, and one thymidine. Six GTs (Compounds 1–6) were first reported from the species of G. lucidum. One cerebroside and one steroid were first isolated from the genus Ganodema. Then, pharmacophoric profiling of these six GTs was implemented to analyze their potential therapeutic targets. Nineteen high-frequency targets were obtained and mainly related to cancer and metabolic syndrome, especially CDK2 and PPAR γ. PIN and modules analysis of the 19 high frequency targets indicated that the cell division cycle regulating and protein acetylation might be the main anti-cancer mechanism of these six GTs. In this paper, molecular simulation and PIN analysis were directly utilized to research the potential activity of isolated compounds from the source of extraction of TCM. Actually, the results also need to be validated by wet experiments. However, compared to biochemistry and animals experiment, it might be a rapid, economical, and efficient method for discovering potential pharmacological activity of new isolated compounds.


N′-hydroxyindazolecarboximidamides. Among the compounds synthesized, compound 8a inhibited both tryptophan depletion and kynurenine production through the IDO1 enzyme. Molecular docking studies revealed that 8a binds to IDO1 with the same binding mode as the analog, epacadostat (INCB24360). Here, we report the synthesis and biological evaluation of these hydroxyindazolecarboximidamides and present the molecular docking study of 8a with IDO1.Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that is highly overexpressed in various cancer cells and antigen-presenting cells. It has emerged as an attractive therapeutic target for cancer immunotherapy, which has prompted high interest in the development of small-molecule inhibitors. To discover novel IDO1 inhibitors, we designed and synthesized a series of  T. T8a forAll materials were obtained from commercial suppliers and used without further purification. Solvents in this study were dried using an aluminum oxide column. Thin-layer chromatography was performed on pre-coated silica gel 60 F254 plates. Purification of intermediates was carried out by normal phase column chromatography . NMR spectra were recorded on a Avance 300 MHz . LC/MS data were obtained using a Waters 2695 LC and Micromass ZQ spectrometer . HR-MS was detected on JMS-700 . The identity of the final compounds was confirmed by proton NMR and mass spectrometry.2 atmosphere. The mixture was filtered through a pad of Celite and washed with EtOH. The filtrate was concentrated to give compound 3 .1H-NMR  δ 8.09 , 7.24 , 7.10–6.96 , 6.68 , 4.12 .A mixture of 7-nitroindazole ) and 10% Pd/C (0.6 g) in EtOH (10 mL) was stirred at rt under H3  in c-HCl (0.56 mL) and 98% H2SO4 (2 mL) was added dropwise a solution of NaNO2  in H2O (2 mL). The mixture was stirred at 0 °C for 30 min. Then, a solution of KI  in H2O (2 mL) was added dropwise and the mixture was stirred at rt for 2 h. The mixture was diluted with EA, washed with sat. NaHCO3, water, and brine. The organic layer was dried over MgSO4 and purified by column chromatography (MPLC) to give compound 4 . 1H-NMR  δ 10.14 , 8.26 , 7.82–7.71 , 7.04–6.92 .To a solution of 4 , dppf , Pd2(dba)3 , Zn , Zn(CN)2  in DMA  was heated at 120 °C for 5 h. The mixture was concentrated and diluted with EA. The organic layer was washed with 2 N NH4OH and brine. The organic layer was dried over MgSO4 and concentrated to obtain the crude mixture which was purified by column chromatography (MPLC) to give compound 5 . 1H-NMR  δ 11.85 , 8.41 , 8.09 , 7.81 , 7.36–7.30 .A solution of 5  in H2O (1.5 mL)/EtOH (3 mL) were added hydroxylamine hydrogen chloride  and sodium bicarbonate  and heated at 85 °C for 8 h. The reaction mixture was diluted with EA, washed with water and brine. The organic layer was dried over MgSO4 and concentrated to give compound 6  1H-NMR  δ 12.22 , 9.82 , 8.12 , 7.76 , 7.17 , 6.07 .To a solution of 2  in water (1 mL) was added dropwise while maintaining the temperature below 0 °C. After complete adding stirring was continued in the ice bath for 1.5 h and then the mixture was allowed to warm to 15 °C. The precipitate was collected by filtration and washed with cold water to give the desired compound 7  1H-NMR  δ 12.74 , 12.47 , 8.23 , 7.94 , 7.82 , 7.29 .To a suspension of 6  in water (1 mL) were added acetic acid (0.5 mL) and 6 N HCl  and this suspension was stirred at 45 °C until complete solution was achieved. Sodium chloride  was added to the reaction mixture and stirred in an ice/water/methanol bath. A solution of NaNOZ)-N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8a). To a solution of 7  in THF (1 mL) at 60 °C was 3-bromo-4-fluoroaniline  was added and stirred for 10 min. A solution of NaHCO3  in water (1 mL) was added dropwise and stirred at 60 °C for 3 h. The mixture was extracted with EA and washed with brine. The organic layer was dried over MgSO4 and concentrated to obtain the crude mixture which was purified by column chromatography (MPLC) to give compound 8a. 1H-NMR  δ 12.01 , 8.14 , 7.73 , 7.48 , 7.20 , 7.01 , 6.95 , 6.78 , 6.58–6.43 ; LC/MS (ESI) m/z 349 [M + H]+; HRMS (EI) m/z calcd. for C14H10BrFN4O [M+] 348.0022, found 348.0019.(Z)-N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8b).1H-NMR  δ 11.96 , 8.15 , 7.74 , 7.44 , 7.22 , 7.06–6.94 , 6.85–6.75 , 6.53–6.39 ; LC/MS (ESI) m/z 305 [M + H]+ ; HRMS (EI) m/z calcd. for C14H10ClFN4O [M+] 304.0527, found 304.0519.(Z)-N-(3-Chlorophenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8c).1H-NMR  δ 11.91 , 8.16 , 7.74 , 7.43 , 7.34–7.25 , 7.08–6.94 , 6.90 , 6.77 , 6.51 ; LC/MS (ESI) m/z 287 [M + H]+; HRMS (EI) m/z calcd. for C14H11ClN4O [M+] 286.0621, found 286.0627.(Z)-N-(4-Fluorophenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8d).1H-NMR  δ 11.98 , 8.15 , 7.71 , 7.43 , 7.21 , 7.02–6.95 , 6.82–6.74 , 6.72–6.63 ; LC/MS (ESI) m/z 271 [M + H]+.(Z)-N--N′-hydroxy-1H-indazole-7-carboximidamide (8e).1H-NMR  δ 8.21 , 7.83 , 7.46–7.39 , 7.18 , 7.17–7.10 , 7.10–6.99 , 6.66–6.53 , 6.36–6.24 ; LC/MS (ESI) m/z 289 [M + H]+.(Z)-N-(3-Acetylphenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8f).1H-NMR  δ 12.80 , 10.79 , 8.64 , 8.09 , 7.78 , 7.35–7.27 , 7.16–7.04 , 6.88 , 2.31 ; LC/MS (ESI) m/z 295 [M + H]+.(Z)-N′-Hydroxy-N-(m-tolyl)-1H-indazole-7-carboximidamide (8g).1H-NMR  δ 11.80 , 8.17 , 7.74 , 7.37–7.27 , 7.11–6.95 , 6.79 , 6.66 , 6.54 , 2.21 ; LC/MS (ESI) m/z 267 [M + H]+.(Z)-N-(4-Chloro-3-methylphenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8h).1H-NMR  δ 12.77 , 10.75 , 8.44 , 8.10 , 7.78 , 7.26 , 7.12–7.03 , 6.99 , 6.74 , 6.39 , 2.10 ; LC/MS (ESI) m/z 301 [M + H]+.(Z)-N-(3-Chloro-4-methoxyphenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8i).1H-NMR  δ 12.0 , 8.13 , 7.70 , 7.40 , 7.20 , 6.98 , 6.86 , 6.58 , 6.49 , 3.78 ; LC/MS (ESI) m/z 317 [M + H]+.(Z)-N′-Hydroxy-N-phenyl-1H-indazole-7-carboximidamide (8j).1H-NMR  δ 11.59 , 8.18 , 7.76 , 7.34 , 7.16 , 7.08–6.95 , 6.81 ; LC/MS (ESI) m/z 253 [M + H]+.(Z)-N-(4-Chlorophenyl)-N′-hydroxy-1H-indazole-7-carboximidamide (8k).1H-NMR  δ 11.71 , 8.18 , 7.77 , 7.37–7.25 , 7.10 , 7.06 , 6.71 ; LC/MS (ESI) m/z 287 [M + H]+.(Z)-N′-Hydroxy-N-(4-methoxyphenyl)-1H-indazole-7-carboximidamide (8l).1H-NMR  δ 11.95 , 8.14 , 7.70 , 7.37 , 7.23 , 6.97 , 6.72 , 6.65 , 3.72 ; LC/MS (ESI) m/z 283 [M + H]+.(Z)-N′-Hydroxy-N-(p-tolyl)-1H-indazole-7-carboximidamide (8m).1H-NMR  δ 11.53 , 8.17 , 7.75 , 7.32 , 7.27 , 7.07–6.99 , 6.97 , 6.73 , 2.27 ; LC/MS (ESI) m/z 267 [M + H]+., NCS  in DMF was stirred at rt for 6 h. The mixture was diluted with EA and washed with water and brine. The organic layer was dried over MgSO4 and concentrated to give the crude product 11. 1H-NMR  δ 10.11 , 8.08 , 7.84 , 7.58 , 7.24–7.19 , 4.15 .A solution of 1-methyl-1H-indazole-7-carbaldehyde oxime Z)-N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-1-methyl-1H-indazole-7-carboximidamide (12).1H-NMR  δ 8.02 , 7.82 , 7.60 , 7.35 , 7.20–7.11 , 6.82–6.75 , 6.73–6.64 , 6.38–6.29 , 4.09 ; LC/MS (ESI) m/z 363 [M + H]+.(Z)-N-(3-Bromo-4-fluorophenyl)-N′,2-dihydroxybenzimidamide (16).1H-NMR  δ 7.27–7.18 , 7.13–7.07 , 7.07–6.98 , 6.98–6.89 , 6.76–6.65 ; LC/MS (ESI) m/z 325 [M + H]+. was inserted into pcDNA5/FRT/TO expression vector  and transfected into Flp-In-Rex- HEK293 cells (Invitrogen). After transfection, homogenous IDO1 expressing HEK293 cells were selected using hygromycin and confirmed for their IDO1 expression using Western blot.18 column  with a Security Guard C18 guard column  maintained at 25 °C. Isocratic mode was used to achieve separation of the analytes using mobile phase (acetonitrile with 0.1 formic acid and water with 0.1% formic acid (20:80 v/v) at a flow rate of 300 μL/min for 3 min. The multiple reaction monitoring (MRM) transition was m/z 204.94 → 188.1 for tryptophan and m/z 208.97 → 192.00 for kynurenine under positive-ionization. Optimized values for de-clustering potential, collision energy, entrance potential, and collision-exit potential were 26 V, 15 eV, 9.5 V and 8 V and 21 V, 13 eV, 10 V and 8 V for tryptophan and kynurenine, respectively. The optimized instrument conditions were as follows: ion spray voltage, 5500 V; source temperature, 450 °C; curtain gas, 25 psi; nebulizing gas (GS1), 30 psi; heating gas (GS2), 30 psi. Data analysis was performed using Analyst Software version 1.4.2. .To analyze anti-IDO1 activity of compounds, human IDO1 expressing HEK293 recombinant cells were seeded in 100 μL of complete MEM media containing 10% FBS in a 96-well plate. After incubation for 48 hours, various concentrations of compounds with 0.2 ng/mL doxycyclin  was treated and incubated for 24 h. Culture supernatants were collected and prepared for analysis of tryptophan and kynurenine using LC-MS. For preparation of samples for LC-MS analysis, 30 μL of culture supernatant was mixed with 2 μM caffeine containing 270 μL methanol. After centrifugation at 12,000 rpm for 10 min, 100 μL of supernatant was transferred into new 1.5 mL tubes. Distilled water (900 μL) was added into 100 μL of supernatant and vortexed. The concentrations of tryptophan and kynurenine were determined by an LC-MS/MS system consisting of an Agilent 1200 series HPLC system  with an API 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer  equipped with a turbo electrospray interface. Chromatographic separation of tryptophan and kynurenine was achieved on a Unison UK-Cλ = 320 to 325 nm.To analyze the hIDO1 inhibitory activities of compounds, we used IDO1 inhibitor screening assay kit and followed the protocol provided from BPS Bioscience . Briefly, 5 μL of IDO solution was aliquoted into a 384-well plate and treated with 5 μL of compound in buffer at room temperature for 3 h. The data was measured at 8a against IDO1 was performed using the Schrödinger Suite 2017-2  [8a was performed using the Standard Precision method in Glide v.7.5 with positional constraints for covalent bonding with heme iron. The docking model of 8a was visualized using Discovery Studio 2016  [Molecular modeling study of NY, USA) . The X-rNY, USA) . The IDOCA, USA) .N′-hydroxyindazolecarboximidamides for inhibition of IDO1. Among the compounds synthesized, compound 8a exhibited moderate IDO1 inhibitory activity in both tryptophan depletion and kynurenine production assays. In addition, the molecular docking study with compound 8a suggested a structural basis for its IDO1 inhibition. Although IDO1 inhibitory activities are relatively lower than those of known inhibitors, an indazole scaffold might be a useful novel structure for further optimization study as an IDO1 inhibitor.In conclusion, we designed and synthesized a series of


The known ellagitannins were identified based on spectroscopic data. The structure of davicratinic acid A (5), a monomeric ellagitannin possessing a unique, skew-boat glucopyranose core, was established based on spectroscopic data. Additionally, we examined the effects of several tannins with good yields from this plant on drug-resistant bacteria and human oral squamous cell carcinomas, and found that davidiin (1) exhibited the most potent antibacterial and antitumor properties among the tannins examined.We isolated a new ellagitannin, davicratinic acid A ( Davidia involucrata Baill., known as the dove tree or handkerchief tree, is the sole species of the genus Davidia of the family Davidiaceae (as per the Engler system) + ion peak corresponding to the molecular formula C41H31O28. The 1H-NMR spectrum of 5 showed that the signals were due to a galloyl group  and a 4,4′,5,5′,6,6′-hexahydroxydiphenoyl (HHDP) group . In addition, the spectrum exhibited sugar signals at δ 3.93 , 4.04 , 4.26 , 4.60 , 5.13 , 5.37 , and 5.88 , which were assigned to the glucose H-4, H-6a, H-5, H-6b, H-2, H-3, and H-1 signals based on 1H-1H-COSY. The appearance of the H-4 signal in the higher field indicated that the C-4 hydroxyl group was unacylated. According to the coupling constants of these glucose proton signals, the glucopyranose ring in 5 was assigned a skew-boat–type conformation. The HMBC spectrum revealed that glucose O-3 was acylated by the galloyl group and the O-1 and O-6 positions of glucose were bridged via the HHDP group  and 5.49 (H-3′) with a carboxyl carbon signal at δ 165.5 (C-7′) of the remaining acyl group. Because this carboxyl carbon signal was further correlated with the glucose H-2 signal, the acyl group containing these protons and carbons is connected to glucose O-2. The HMBC spectrum also showed correlations with the methine proton signals at δ 5.15 (H-2′) and 5.49 (H-3′) with a carboxyl carbon signal at δ 171.4 (C-1′), and those of two methine proton signals (δ 5.15 (H-2′), 7.10 (H-3″)) with a carboxyl carbon signal (δ 166.8 (C-7″)). These spectral observations led us to formulate a dehydrochebuloyl group for the acyl group at glucose O-2. H-5′ showed an ROE correlation between H-2′ and H-3′, indicating that the geometrical isomerism of the double bond was Z  -HHDP-2-O--(4′-dehydrochebuloyl)-3-O-galloyl-β-d-glucose  a 23], w, w5) wasDavidia tannins, we examined the major tannin 1 and related tannins 2 and 4, obtained in high yield from D. involucrata, for antibacterial effects on MRSA (S. aureus OM481 and OM584) and VRE  by the liquid dilution method c = 1.0, MeOH); UV λmax (MeOH) nm (ε): 219 (0.621), 275 (0.252); ECD (MeOH) [θ] (nm) −2.7 × 104 (200), +2.8 × 104 (213), −2.7 × 104 (231), +2.3 × 104 (244), −2.5 × 104 (261), +4.4 × 104 (284), −1.0 × 104 (319); 1H-NMR (acetone-d6/D2O = 9/1) δ: 3.93 , 4.04 , 4.26 , 4.60 , 5.13 , 5.15 , 5.37 , 5.49 , 5.88 , 6.74 , 6.83 , 6.89 , 7.03 , 7.10 . 13C-NMR (acetone-d6/D2O = 9/1) δ: 34.7 (C-3′), 65.1 (C-6), 68.9 (C-4), 72.0 (C-2), 72.2 (C-3), 78.2 (C-5), 78.8 (C-2′), 95.2 (C-1), 108.8 (HHDP-3′), 108.9 (C-3″), 109.3 (HHDP-3), 110.0 , 115.0 (C-1″), 115.9 (HHDP-1), 116.1 (HHDP-1′), 116.9 (C-2″), 120.2 , 124.8 (HHDP-2), 125.4 (HHDP-2′), 136.4 (HHDP-5), 136.6 (HHDP-5′), 137.3 (C-4′), 137.5 (C-5′), 139.3 , 139.5 (C-5″), 144.0 (C-6″), 144.3 , 144.6 (HHDP-6), 144.9 (HHDP-6′), 145.3 (C-4″), 145.7 , 165.5 (C-7′), 166.8 (C-7″), 166.9 , 168.8 , 171.4 (C-1′). HR-ESI-MS m/z: 971.0952 [M + H]+ .Enterococcus faecium FN-1 and E. faecalis NCTC 12201 used in this study were provided by Prof. Y. Ike, Gunma University. The bacterial cells were pre-cultured in Mueller-Hinton broth at 37 °C under aerobic conditions. They were incubated in the presence of compounds with final concentrations 16, 32, 64, and 128 µg/mL, obtained by two-fold serial dilution at 37 °C without shaking, in the same broth, for 24 h on 96-well plates, as shown in a previous study /[CC50 (Ca9-22) + CC50 (HSC-2) + CC50 (HSC-3) + CC50 (HSC-4)] × 4/3. We have previously confirmed that the TS value determined by this method reflects the antitumor potential of test samples, although these normal oral cells and OSCC cell lines are classified as mesenchymal or epithelial cells [Human normal oral cells  and OSCC  were cultured at 37 °C in DMEM supplemented with 10% heat-inactivated FBS, 100 units/mL of penicillin G, and 100 µg/mL of streptomycin sulfate under a humidified 5% COT method ,34. In bD. involucrata is an important tannin-rich resource. In particular, ellagitannin 1, which possesses the unique skew-boat–type glucopyranose core, is abundant in this plant. In this study, we investigated new ellagitannins of this plant, and characterized the new tannin 5, which also has a skew-boat glucopyranose core. We consider that 5 is metabolized from 4 via oxidation of the DHHDP moiety [1 is a prominent antibacterial compound. The anti-MRSA activity of ellagitannins has been previously reported [1 is a possible candidate for a lead compound for antitumor agents. Further studies will be needed to isolate other ellagitannins related to 1 from D. involucrata, and will be important in understanding the tannin metabolism in plants; examination of the structure-activity relationship of these tannins on antibacterial and antitumor effects will also be important.The findings obtained in the present study indicate that P moiety , and havreported ; to the


TPA-TPD, TPA-PT-TPD and TPA-TT-TPD with donor-acceptor structure and their use in organic photovoltaic cells are reported. For the synthesis, 5-(2-ethylhexyl)-4H-thienopyrrole-4,6(5H)-dione was used as electron withdrawing fragment while the triphenylamine was used as electron donating fragment. Molecular electronic geometry and electronic distribution density were established by density functional theory (DFT) calculations and confirmed by optical and chemical characterization. These molecules were employed as electron-donors in the active layer for manufacturing bulk heterojunction organic solar cells, where -phenyl C71 butyric acid methyl ester (PC71BM) was used as electron-acceptor. As cathode, Field′s metal (FM), an eutectic alloy  with a melting point above 62 °C, was easily deposited by drop casting under vacuum-free process and at air atmosphere. Prepared devices based on TPA-TPD:PC71BM (1:4 w/w ratio) presented a large VOC = 0.97 V, with JSC = 7.9 mA/cm2, a FF = 0.34, then, a power conversion efficiency (PCE) of 2.6%. In this work, microwave synthesis, chemical, optical and electrochemical characterization of three small organic molecules,  The synthesis and study of organic semiconducting molecules have increased considerably in recent years due to their electronic and optoelectronic properties ,2,3,4,5;Particularly, OSCs have attracted great attention because they are a source of renewable energy and their manufacture is inexpensive, they are thin, lightweight as well as semi-transparent; further, because of their potential flexibility, these devices can be used almost on any surface ,18,19,20OC) values in photovoltaic devices pyrrole-4,6(5H)-dione (TPD), which is attractive because it is symmetric, compact and rigid pyrrole-4,6(5H)-dione (TPA-TPD) synthesis by direct arylation under conventional heating at 110 °C for 48 h using Pd(OAc)2 as catalyst, reaching yields of 51%. In this work, the synthesis of TPA-TPD is reported through Suzuki reaction by using Pd(PPh3)4 as catalyst and microwave (MW) heating at 110 °C for 1 h, getting yields of 70%. For the synthesis of -3,3′--4,6-dioxo-5,6-dihydro-4H-thieno-pyrrole-1,3-diyl)bis)bis(2-(4′-(diphenylamino)--4-yl)acrylonitrile) (TPA-PT-TPD) and -2,2′--4,6-dioxo-5,6-dihydro-4H-thienopyrrole-1,3-diyl)bis)bis(3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)acrylonitrile) (TPA-TT-TPD), firstly the synthesis of (Z)-2-(4-bromophenyl)-3-(thiophen-2-yl)acrylonitrile (PT) was carried out by Knoevenagel condensation between 4-Bromophenylacetonitrile and 2-thiophenecarboxaldehyde, while the synthesis of (E)-3-(5-bromothiophen-2-yl)-2-(thiophen-2-yl)acrylonitrile (TT) was carried out by Knoevenagel reaction between 2-thienylacetonitrile and 5-Bromo-2-thiophenecarboxaldehyde. Synthetic route of the conjugated organic small molecules (SMs) are depicted in N,N-diphenylamine)-1-phenylboronic acid with PT or TT precursors, using Pd(PPh3)4 as catalyst and microwave heating, (Z)-2-(4′-(diphenylamino)--4-yl)-3-(thiophen-2-yl)acrylonitrile (TPA-PT) and (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-2-(thiophen-2-yl)acrylonitrile (TPA-TT) were obtained; finally TPA-PT-TPD and TPA-TT-TPD were synthesized through direct arylation reaction between TPA-PT and TPA-TT with 1,3-dibromo-5-(2-ethylhexyl)-4H-thienopyrrole-4,6(5H)-dione (TPD), using Pd(OAc)2 as catalyst and microwave heating by 1 h.Subsequently, through Suzuki reaction between 4- calculated for these SMs are also shown in Geometrical structure and density electronic properties of the reported SMs were studied by theoretical calculation using density functional theory (DFT) with Gaussian 09 package . GeometrTPA-TPD and TPA-TT-TPD. It is observed that electronic density in the HOMO orbital is delocalized along the π-conjugated system for these molecules, whereas for TPA-PT-TPD it is located in the TPA fragment. It is because the geometry of TPA-TPD and TPA-TT-TPD is flat, which allows the delocalization of the electrons. The geometry of TPA-PT-TPD is not flat, so the orbitals are not aligned for an electronic delocalization. On the other hand, in the LUMO orbital, the electronic density in the three molecules is located on the TPD fragment. It is because TPA groups act as donor groups of the electronic density, while TPD molecules act as an acceptor of the electronic density, so we conclude that electronic architecture for the three SMs is D-A-D.gcal is TPA-TPD with 3.02 eV, while TPA-PT-TPD has the lowest one: 2.08 eV; this fact agrees with the size of the π-conjugated system. However, the value of Egcal for TPA-TT-TPD (2.20 eV) is reversed in comparison with the experimental data (see below). This fact could be because, in solid state, the crystal packing plays a very important role in the optical properties due to stronger intermolecular chemical interactions  eV, where oxE is the recorded onset oxidation potentials of SMs. 4.4 eV is the energy level of ferrocene/ferrocenium (Fc/Fc+) below the vacuum level and 0.22 eV is the correction by the reference used electrode pyrrole-4,6(5H)-dione, were purchased from Sigma-Aldrich . The other reagents and solvents were purchased from Karal . All reagents and chemicals were used without further purification unless stated otherwise. The potassium pivalate (PIVOK) was synthesized according to the literature + Found: 289.9634 for C13H8BrNS; [M + H]+ Calcd.: 289.9640.(E)-3-(5-Bromothiophen-2-yl)-2-(thiophen-2-yl)acrylonitrile (TT). Yellow solid, Yield: >98%, m.p. 116–118 °C. FTIR , 3100 (C–H), 2214 (CN), 1585 (C=C), 1433 (C–S). 1H-NMR : δ 7.24–7.34 , 7.0–7.1 , 13C-NMR  δ 138.4, 103.4, 132.7, 139.2, 118.2, 128.34, 131.2, 127.4, 126.4, 130.7, 116.9 (CN). HRMS–TOF : [M + H]+ Found: 295.9197 for C11H6BrNS2; [M + H]+ Calcd.: 295.9204.3)4. The mixture was placed under nitrogen, then 5 mL of dimethylacetamide (DMA) is added, the mixture was stirred for 2 min and then heated up in a microwave oven at 110 °C with a power of 100 W, after 1 h the reaction was cooled down to 50 °C and 50 mL of cold EtOH are added, a solid precipitate is observed, which is filtered and washed first with 50 mL of EtOH and then with 50 mL of hexane. The solid was recovered and recrystallized using a mixture of ketone/EtOH 40:60.In a flask of 20 mL are placed the diboronic acid and the corresponding aryl halides; subsequently, 3 mol equivalents of PIVOK is added as well as 0.05% mol of the catalyst tetrakistriphenylphosphine palladium Pdphenyl)-5-(2-ethylhexyl)-4H-thienopyrrole-4,6(5H)-dione (TPA-TPD). Orange solid, Yield: 70%, m.p. 178–179 °C. FTIR , 3068 (C-H), 1687 (C=O), 1494 (C–S). 1H-NMR  δ 7.95–8.05 , 7.27–7.40 , 7.00–7.23 , 3.54 , 1.75–1.91 , 1.25–1.40 , 0.82–0.98 ; 13C-NMR  δ 163.4, 149.3, 146.8, 144.1, 129.4, 129.0, 128.6, 125.4, 124.0, 123.7, 121.4, 42.4, 38.2, 30.6, 28.6, 23.9, 23.0, 14.1, 10.5. HRMS–TOF : [M + H]+ Found: 752.3307 for C50H45N3O2S; [M + H]+ Calcd.: 752.3311. (Z)-2-(4′-(Diphenylamino)--4-yl)-3-(thiophen-2-yl)acrylonitrile (TPA-PT). Orange solid, Yield: 84%, m.p. 174–175 °C, FTIR , 2928 (C–H), 2207 (CN), 1586 (C=C), 1485 (C–S). 1H-NMR  δ 7.6–7.7 , 7.50 , 7.5 , 7.23–7.33 , 7.10–7.20 , 7.01–7.09 ; 13C-NMR  δ 147.8, 147.6, 141.3, 138.2, 133.5, 132.3, 132.3, 130.0, 129.4, 127.9, 127.7, 127.2, 126.2, 124.7, 124.7, 123.6, 123.3, 118.2, 108.1. HRMS–TOF : [M + H]+ Found: 455.1573 for C31H22N2S; [M + H]+ Calcd.: 455.1565. (E)-3-(5-(4-(Diphenylamino)phenyl)thiophen-2-yl)-2-(thiophen-2-yl)acrylonitrile (TPA-TT). Brown solid, Yield: 78%, m.p. 157–158 °C. FTIR , 3100 (C–H), 2211 (CN), 1587 (C=C), 1433 (C–S). 1H-NMR  δ 7.48–7.53 , 7.42 , 7.18–7.33 , 7.23–7.33 , 7.03–7.09 ; 13C-NMR  δ 149.9, 148.3, 147.0, 139.0, 135.0, 134.0, 132.1, 129.9, 129.3, 128.1, 126.7, 126.4, 125.5, 124.8, 123.4, 122.8, 122.6, 117.2, 101.5. HRMS–TOF : [M + H]+ Found: 461.1125 for C19H20N2S2; [M + H]+ Calcd.: 461.1146. H-thienopyrrole-4,6(5H)-dione (TPD) and then 0.2 mmol of TPA-PT or TPA-TT are added; subsequently 3 molar equivalents of PIVOK are added as well as 0.05 mol % of catalyst palladium (II) acetate Pd(OAc)2, the mixture was placed under nitrogen atmosphere, then 5 mL of DMA are added. The mixture was stirred for 2 min and then heated up in a microwave oven at 110 °C with a power of 100 W. After 1 h, the reaction was cooled down to 50 °C and 50 mL of cold EtOH was added and a solid precipitate is observed, which is filtered and washed first with 50 mL of EtOH and then with 50 mL of hexane. The solid was recovered and recrystallized using a mixture of acetone/EtOH 40:60.In a flask of 20 mL are placed 0.1 mmol of 1,3-dibromo-5-(2-ethylhexyl)-4-3,3′--4,6-dioxo-5,6-dihydro-4H-thienopyrrole-1,3-diyl)bis)bis(2-(4′-(diphenylamino)--4-yl)acrylonitrile) (TPA-PT-TPD). Dark solid, Yield: 60%, m.p. 173–175 °C. FTIR , 3010 (C–H), 2219 (CN), 1696 (C=O), 1588 (C=C), 1432 (C–S). 1H-NMR  δ 7.7–7.81 , 7.51–7.62, 7.23–7.33 , 7.10–7.20 , 7.01–7.09 , 3.57 , 1.81–1.93 , 1.23–1.47 , 0.81-1.02 . 13C-NMR  δ 159.5, 145.2, 144.8, 138.6, 133.2, 132.7, 130.2, 130.5, 129.9, 128.7, 128.2, 127.6, 126.9, 124.2, 123.4, 122.3, 120.5, 120.8, 106.7, 106.5, 40.3, 35.7, 28.2, 26.2, 21.4, 20.7, 11.7, 8.0, 159.7, 115.0 (CN). HRMS–TOF : [M + H]+ Found: 1169.7796 for C76H59N5O2S3; [M + H]+ Calcd.: 1169.7783. -2,2′--4,6-dioxo-5,6-dihydro-4H-thienopyrrole-1,3-diyl)bis)bis(3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)acrylonitrile) (TPA-TT-TPD). Dark solid, Yield: 70%, m.p. 179–182 °C. FTIR , 2926 (C–H), 2211 (CN), 1699 (C=O), 1587 (C=C), 1493 (C–S). 1H-NMR  δ 7.68 , 7.16–7.60 , 6.79–7.16 , 3.57 , 1.81–1.93 , 1.23–1.47 , 0.81–1.02 . 13C-NMR  δ 150, 148.4, 147.1, 147.0, 142.8, 135.5, 135.4, 132.7, 132.0, 130.5, 129.5, 128.9, 126.9, 126.2, 125.1, 123.8, 122.6, 122.5, 122.3, 116.5, 42.9, 38.3, 30.7, 28.7, 23.9, 23.3, 14.3, 10.6, 162.4 (C=O), 100 (CN). [M + H]+ Found: 1128.4051 for C72H55N5O2S5; [M + H]+ Calcd.: 1181.2959.TPA-TPD, TPA-PT-TPD and TPA-TT-TPD molecules were synthesized by using microwave assistance and thus decreasing the reaction time from 48 to 1 h. It could help to reduce costs in the synthesis of semiconductor materials. These small molecules were used in solution processed OSCs fabrication for the first time; the largest VOC was almost of 1 V for those cells based on TPA-TPD:PC71BM; while JSC value was 7.9 mA/cm2 and thus PCE = 2.6%. Through AFM measurements, it was observed that one of the factors involved in the low photovoltaic parameters of OSCs based on TPA-PT-TPD:PC71BM and TPA-TT-TPD:PC71BM is an irregular morphology of the formed films. They showed a roughness of 22 and 28 nm, respectively . Although power energy efficiencies of our OSCs based on the described SMs containing TPD are still lower than those for OSCs based on other SMs containing this same fragment, our SMs synthesis procedure is easier and faster than in other reports. Furthermore, our OSCs were fabricated and tested under regular atmosphere and by using as cathode the eutectic alloy Field’s metal, which is easily free vacuum deposited. However, it is necessary to improve film morphology; probably it could be through evaporation or by improving solubility of the molecules by adding alkyl chains.


H-indol-2-yl)methylene)-1,3-diethyl-2-thioxodihydro-pyrimidine-4,6-dione π-bridge (D–A–D) donor–acceptor–donor compound. Its exo-isomer structure has been proven by XRD-single-crystal analysis for the first time. The IR, UV–Vis., MS, CHN-, 1H and 13C NMR analysis were also carried out. The DFT-optimized structural-parameters were matched with the XRD-crystallographic data. The experimental-XRD-interactions in the lattice were compared to the computed Hirshfeld analysis (HSA), MEP map and Mulliken charge population. The DFT/6-311G(d) calculations like IR/B3LYP, TD-SCF, HOMO–LUMO, GRD and GIAO-NMR have been compared to their corresponding experimental parameters. Non-linear optical (NLO) crystal theoretical-analysis was carried out then compared to urea reference. The compound thermal activity was evaluated in an open-atmosphere by TG/DTG analysis.This work deals with the synthesis and characterization of the novel 5-((5-chloro-1 Barbituric acid and thiobarbituric acid and their derivatives as hypnotic-compounds containing the active methylene are considered being as a good starting material to prepare specific class of heterocyclic molecules via Knoevenagel mild condensation condition –4. CombiMany polar-organic crystalline molecules with non-centrosymmetric crystal structures reflected a very high second-order non-linear optical (NLO) properties , 19. SevH-indol-2-yl)methylene)-1,3-diethyl-2-thioxodihydro-pyrimidine-4,6 -dione compound has been prepared through one pot condensation reaction in a good yield, the structure of exo-isomer was confirmed by XRD-single crystal and spectrally characterized. Several experimental spectral measurements were compared with their corresponding theoretical parameters. Initially, exo–endo isomerization reaction was DFT-computed and its T.S was detected under QTS2 level of calculation.In this study, 5-((5-chloro-1H-indol-2-yl)methylene)-1,3-diethyl-2-thioxodihydro-pyrimidine-4,6-dione derivative was prepared through one pot Knoevenagel dehydration reaction, as in Scheme H,5H)-dione with 5-chloro-1H-indole-2-carbaldehyde under reflux conditions revealed the formation of the desired thiobarbiturate in a very good yield. The synthetic methodology here reflected high yield without side products, moreover, short refluxed time was required compared to recent synthetic methodologies  m/z = 361.3 . 1H NMR : (ppm) 1.2 , 4.5 , 7.0–9.0 , 9.2 , 13.1 . 13C-NMR : (ppm) 13.5 , 43.1 , 110.8, 116.1, 117.7, 121.8, 123.4, 126.1, 131.5, 128.8, 130.7, 139.8, 145.6, 146.9 , 160.8 , 173.8 . FT-IR main vibrations, VN–H = 3319 cm−1, VC–HAr = 3176 cm−1, VC–H aliph = 2979 cm−1, VC=O = 1669 cm−1, VC=S = 1365 cm−1, VC=C = 1286 cm−1.The yellow powder product, with a m.p = 360 °C, was collected; molecular formula CHirshfeld surface analysis (HSA) been performed using the CRYSTAL EXPLORER 3.1 program . All Com


The planar structures and relative configurations of the new compounds were determined by extensive analysis of the NMR and HRESIMS data. The absolute configurations were established by comparison of the experimental and calculated ECD (electronic circular dichroism) spectra or OR  value. Compound 9 exhibited potent anti-food allergic activity with IC50 value of 28.7 μM, while 4 showed weak cytotoxicity against ECA-109 tumor cells (IC50 = 28.7 μM).Four new (penigrisacids A–D,  Aspergillus terreus + in its HRESIMS spectrum, indicating five degrees of unsaturation. The 1H NMR spectrum  and one oxymethine . The 13C NMR spectrum, in association with the DEPT and HSQC spectra, indicated 15 carbon signals , five methylenes , one oxygenated methine , and six non-protonated carbons, including one carboxyl , two olefinic , two oxygenated ; and one aliphatic  carbons. Since one carboxyl and two olefinic carbons accounted for two degrees of unsaturation, 1 was assumed to be a tricyclic molecule. The COSY correlations showed three isolated spin systems from H-2 (δH 4.48) to H2-1 , H2-4  to H2-5 , and H2-8  through H2-9 (δH 2.38) to Me-12 and Me-13. The HMBC spectrum displayed correlations from Me-15 to C-1/C-6/C-7/C-8, H-2 to C-3/C-4/C-6/C-14, and H2-5 to C-6/C-7/C-10. Taking together the COSY and HMBC correlations, the planar structure of 1 was established  to Me-15, H-1b (δH 2.01) to H-2, Me-15 to H-5a (δH 1.55), suggesting the configurations of C-2 and C-6 at the epoxy moiety were opposite to that of Me-15. Accordingly, the relative structure of 1 was assigned as 10,11-dehydro-3α-hydroxy-2α,6α-epoxycarotan-14-oic acid.Compound spectrum  showed t signals , includiablished , which wS,3R,6S,7R-1 (1a) and 2R,3S,6R,7S-1 (1b) were obtained by time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+G level in ACN (acetonitrile). As shown in 1a fits well with the experimental one. From the above-mentioned evidences, the structure of 1 was then established as -10,11-dehydro-2,6-epoxy-3-hydroxy-14-carotanoic acid, and was named penigrisacid A.To determine the absolute configuration, a theoretical calculation on its ECD spectrum was performed. The calculated ECD spectra of 22 was also obtained as a colorless oil. The molecular formula C15H24O4 was established according to its positive HRESIMS spectrum at m/z 291.1563 , indicating four degrees of unsaturation. The 1H NMR spectrum exhibited two methyl singlets , one oxygenated methylene , and one olefinic proton . The 13C NMR spectrum displayed 15 carbons signals which, in combination with DEPT and HSQC spectra, could be categorized as two methyls , one oxymethylene , five sp3 methylene , two methines , one protonated sp2 , and four non-protonated carbons, including one carboxyl , one olefinic  and one oxygenated  carbons. Since one carboxyl and two olefinic carbons accounted for two degrees of unsaturation, 2 was deduced to be a bicyclic molecule. The COSY spectrum exhibited correlations from H-2 (δH 7.07) to H2-1  and the overlapped proton at δH 1.93; H-5a  to H-4a  and H-6 ; H2-9  to H2-8  and the overlapped proton at δH 1.93. The HMBC spectrum showed correlations from Me-12 to C-10, C-11, C-13, from Me-15 to C-1, C-6, C-7, C-8, from H2-4 to C-2, C-3, C-14, and from H2-9 to C-6, C-10, C-11. Taking together the molecular formula, the COSY, and HMBC spectra, the planar structure of 2 was then established. The NOESY spectrum showed correlations from Me-15 to H-1a (δH 2.41)/H-5a (δH 1.25)/H-9a (δH 1.78), from H-1b (δH 2.14) to H-6, and from H2-13 to H-5b (δH 1.93)/H-6/H-9b (δH 1.68)/H-10 (2-13 were on the same face which was opposite to that of Me-15.Compound 68)/H-10 . Therefo2, a theoretical calculation of its ECD spectrum in ACN was performed. As shown in S,7R,10S,11S)-2 (2a) fit well with that of the experimental one. Accordingly, 2 was elucidated as -2,3-dehydro-11,13-dihydroxy-14-carotanoic acid, and was named penigrisacid B.To determine the absolute configuration of the stereogenic carbons of 3 showed a molecular formula C15H24O5, as established by its positive HRESIMS spectrum at m/z 307.1523 , indicating four degrees of unsaturation. The 13C NMR spectrum showed 15 carbon signals which, according to DEPT and HSQC spectra, could be classified as two methyls , five methylenes , one oxymethylene , two methines , one oxymethine , and four non-protonated carbons including one carboxyl group (δC 175.8), two oxyquaternary carbons (δC 77.6 and 71.5). The general aspect of the 13C NMR spectrum of 3 was similar to that of aspterric acid, except that the signals of the olefinic carbons (C-10/C-11) in aspterric acid were replaced by the signals of one methine  and one oxyquaternary carbon  in 3, respectively. This was confirmed by the HMBC correlations from Me-12 and Me-13 to C-10 and C-11. The NOESY spectrum showed a correlation from H-10  to H-15a , indicating H-10 is in the β position. Further by comparison of the experimental OR -3 (+ .Penigrisacid A (2): colorless oil; + .Penigrisacid B (3): colorless oil; + .Penigrisacid C (4): colorless oil; + .Penigrisacid D  and five known compounds , craterellone D, 3,4-dihydro-3,4,8-trihydroxy-1(2H)-naphthalenone) were obtained. 3,4-Dihydro-3,4,8-trihydroxy-1(2H)-naphthalenone, showed potent anti-food allergic activity (IC50 = 28.7 μM), whereas penigrisacid D showed weak cytotoxicity against esophageal cancer cell line ECA-109 tumor cells (IC50 = 28.7 μM).From the deep-sea-derived fungus


Kadsura coccinea (Lem.) A. C. Smith, which was used for food and as a healthy supplement. Their structures were elucidated by comprehensive analyses of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic data. To search healthy components, an acetylcholinesterase (AChE) inhibitory activity test by Ellman’s Method was conducted, kadsuricoccins A and B showed activity with the AChE inhibit index (AII) up to 68.96% ± 0.19% and 57.8% ± 0.11% at 94 nM , respectively. Two new triterpenoids, named kadsuricoccins A and B, together with three known ones, were isolated from the Li folk herb Heilaohu, the stems of  Kadsura coccinea (Lem.) A. C. Smith, Schisandraceae] fruit is a new taste, and its stems with their full aroma and sedative effect is used for pork and beef stew by the Li folk people + , indicative of seven degrees of unsaturation. The IR spectrum revealed the presence of carbonyl  and double bonds  absorptions. The 1H-NMR spectra ], four methyls , and three olefinic protons , 4.84  and 6.65 . The 13C-NMR and DEPT spectroscopic data (1 was similar to those of 16-oxo-mansumbin-3(28),13(17)-dien-3-oic-acid methyl ester /H-12, H-15[δH 1.99  and 1.54 (1.54)]/H-16 [δH 2.46  and 2.28 ], and the HMBC correlations from H-12 to C-17 (δC 27.3 t) and C-10 (δC 37.7 s), and from H-15 and H-16 to C-17 verified this hypothesis. The other 1H-1HCOSY and HMBC correlations (1 was determined to be likely to the 16-oxo-mansumbin-3(28),13(17)-dien-3-oic-acid methyl ester ]/H-5 [δH 2.18 ], H-19/H-9 [δH 2.25 ], H-9/H-22, H-22/H-7β [δH 2.08 ], and H-7α [δH 1.17 ]/H-18. Thus, the structure of compound 1 was assigned as shown, and named kadsuricoccins A (1).Kadsuricoccins A ’s molecular formula of C30H44O3 was determined by the positive HRESIMS ion at m/z 475.3183 [M + Na]+  with nine degrees of unsaturation. The IR spectrum showed the presence of carbonyl (1705 cm−1) and double bonds (1457 and 1380 cm−1) absorptions. The 13C-NMR and DEPT spectroscopic data , 46.9 , 121.5 , 126.6 , 140.3 , 138.8  and 19.0 , instead of δC 135.7 , 134.2 , 33.3 , 29.3 , 143.8 , 134.6  and 21.5  in kadindutic acid, indicating compound 2 was generated from kadindutic acid for the double bond moved from C-8-C-9 to C-11-C-12, and the methyl group 30-Me moved from C-12 to C-8. Furthermore, the determined molecular formula C15H22O4 and the key 1H-1H COSY correlations with H-9 [δH 2.40 ]/H-11 [δH 6.27 ], H-11/H-12 [δH 5.59 ] and the HMBC correlations from H-30 [δH 0.94 ] to C-8 (δC 37.5 s), C-9 (δC 46.9 d) and C-14 (δC 55.1 s), from H-11to C-9 and C-10 (δC 35.6 s) and from H-12 to C-13 (δC 140.3 s) confirmed this hypothesis. The other 1H-1HCOSY and HMBC correlations ]/H-30 [δH 0.95 ], H-9/H-18 [δH 1.02 ]. Thus, the structure of compound 1 was assigned as shown and named kadsuricoccins B.Kadsuricoccins B ’s 1–5 were all tested for their AchE-inhibitory activity by Ellman’s Method as described before  were collected in Changjiang Hainan Province , People′s Republic of China, in May 2017. The voucher specimen (HUANG00012) identified by Prof. Dr. Jun Wang  was deposited at the Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou, People’s Republic of China. The stems of Heilaohu + 381(50); HRESIMS m/z [M + Na]+ 381.2503 .Colorless, amorphous powder; + 475 (40); HRESIMS m/z [M + Na]+ 475.3208 .Colorless oil; [αThe AchE-inhibitory activity of the compounds was tested as described by Ellman’s Method in the literature ,14. The E − S)/E × 100 . The acetylthiocholine hydrolysis reaction was monitored at 405 nm after 30 min later. All the reactions were performed in triplicate. The AChE inhibit index (AII) was calculated according to the equation: %inhibition = (Schisandraceae family makes the phytochemisty and organic synthesis chemistry of this family of great interest to our research field [Schisandraceae plant, whilst other compounds isolated from Kadsura coccinea indicated the probability of biosynthesis as other Kadsura plants [The skeleton diversity of triterpenoids in the ch field ,15. An oa plants . Furthera plants , while ta plants  and myasa plants . With th


Psephellus pyrrhoblepharus (Boiss.) Wagenitz (syn. Centaurea pyrrhoblephara Boiss.) resulted in the isolation of six guaianolid-type sesquiterpenes, chlorojanerin (1), 19-deoxychlorojanerin (2), 15-hydroxyjanerin (3), aguerin B (4), cynaropicrin (5), eleganin (6); three flavonoids, apigenin, 6-methoxyluteolin and jaceosidine; two glycosides, benzyl-1-O-β-d-glucoside and 3(Z)-hexenyl-1-O-β-d-glucoside; and the coumarin scopoletin. The structures were established by the interpretation of their ESI-MS and 1D and 2D NMR data including 1H-NMR, JMOD, 1H,1H-COSY, HSQC, HMBC, and NOESY experiments. All compounds were isolated for the first time from P. pyrrhoblepharus. Compounds 1–6, the isolated flavonoids and scopoletin were evaluated for their antiproliferative activities on human gynecological cancer cell lines  using the MTT -2,5-diphenyltetrazolium bromide) assay. Chlorojanerin (1), 19-deoxychlorojanerin (2), aguerin B (4), cynaropicrin (5), eleganin (6) were shown to have noteworthy effects on all of the tested cell lines, while apigenin, jaceosidine, and 6-methoxyluteolin were moderately active on HeLa cells. The highest activities were demonstrated by the chlorine-containing derivatives chlorojanerin (1) and 19-deoxychlorojanerin (2) with IC50 values of 2.21 and 2.88 µM, respectively, against the triple negative breast cancer model MDA-MB-231 cells.Multistep chromatographic separations of the chloroform extract of the Turkish endemic plant  Psephellus Cass., belonging to the Asteraceae family, tribe Cynareae, includes approximately 80 species. In Turkey, this genus is represented by 32 species. Some of these are new members of the genus as some sections of the genus Centaurea have been transferred into the genus Psephellus in recent years. Psephellus pyrrhoblepharus (Boiss.) Wagenitz (syn. Centaurea pyrrhoblephara Boiss.) is an endemic plant of Turkey, and grows in the Middle and East Black Sea Region, Upper Red River Area, and Upper Fırat Area of the country +, 399 [M + H]+, 297 [M + H − C4H6O3]+, 279 [M + H − C4H6O3 − H2O]+; 1H-NMR  see 13C-NMR  see 19-Deoxychlorojanerin (2): colorless amorphous solid; 1H-NMR  see 13C-NMR  see 15-Hydroxyjanerin (3): colorless amorphous solid; ESI-MS (positive): m/z 398 [M + H + NH3]+, 381 [M + H]+, 279 [M + H − C4H6O3]+, 261 [M + H − C4H6O3 − H2O]+, 243 [M + H − C4H6O3 − 2 × H2O]+; 1H-NMR  see 13C-NMR  see Aguerin B (4): colorless oil; 1H-NMR  see Cynaropicrin (5): colorless oil; 1H-NMR  see 13C-NMR  see Eleganin (6): colorless oil; 1H-NMR  see 13C-NMR of 1–3, 5 and 6  see Jaceosidine: yellow powder; 1H-NMR  6.88 , 6.60 , 7.55 , 6.93 , 3.75 , 3.89 ; 13C-NMR  163.6 (C-2), 102.7 (C-3), 182.1 (C-4), 152.7 (C-5), 131.4 (C-6), 157.7 and 152.5 , 94.4 (C-8), 103.9 (C-10), 121.6 (C-1′), 110.2 (C-2′), 148.0 (C-3′), 150.7 (C-4′), 115.8 (C-5′), 120.3 (C-6′), 56.0 (3′-OCH3).6-Methoxyluteolin: yellow powder; 1H-NMR and 13C-NMR  data are identical with the published data ) assay on a panel of human adherent cancer cells of gynecological origin. HeLa (HPV 18+ cervix carcinoma) and MDA-MB-231 (triple-negative breast cancer) cell lines were purchased from the European Collection of Cell Cultures  while SiHa (HPV 16+ cervix carcinoma) was obtained from the American Tissue Culture Collection . The cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, and 1% penicillin-streptomycin. All media and supplements for these experiments were obtained from Lonza Group Ltd. . The cells were maintained at 37 °C in a humidified atmosphere containing 5% COeviously . BrieflyPsephellus species and accumulation of antitumor compounds in genera Centaurea, Arctium, Cirsium, and Psephellus belonging to tribe Cynareae initiated the present phytochemical investigation that resulted in the isolation and structural elucidation of 12 secondary metabolites. The isolated compounds were subjected to antiproliferative screening using a set of human gynecological cancer cell lines, and several sesquiterpenes exhibited significant antiproliferative activities against SiHa, HeLa, and MDA-MB-231 cells with IC50 values ranging from 2.21 to 19.62 μM. 19-Deoxychlorojanerin was first investigated in our experiment for antiproliferative activity. Chlorojanerin, hydroxyjanerin, and eleganine were previously tested only against other tumor cell lines, melanoma , epidermoid (KB), ductal (BT-549) and ovarian carcinomas (SK-OV-3), non-small lung cancer (A549), breast (MCF-7), prostate (PC-3), and colorectal (HCT-15) adenocarcinomas [P. pyrrhoblepharus are the chlorinated sesquiterpenes chlorojanerin (1) (IC50 from 2.21 to 11.37 μM) and 19-deoxychlorojanerin (2) (IC50 from 2.88 to 5.54 μM) with higher activities than that of cynaropicrin; these compounds are attractive substrates for further studies to explore their mechanism of action, selectivity, and safety profile.Ethnopharmacological relevance of rcinomas ,26,31. Hrcinomas . Cynaroprcinomas ,22,23,31


Their structures were elucidated using extensive interpretation of 1D and 2D-NMR data, as well as HRESIMS. The aglycon of all compounds is a rare 30-norlonastane and the sugar residues were identified after acid hydrolysis and GC analyses. Cytotoxicities of the isolated compounds were evaluated against Jurkat and CHO cell lines by a MTT in vitro assay as well as a hemolysis assay. Unexpectedly, all these saponin derivatives showed very low activity in our bioassays.Three new triterpene glycosides, named ulososide F ( Secondary metabolites play critical roles in the chemical defense systems of many marine organisms and they are frequently involved in the organization of benthic communities. Marine sponges are already well known as producers of such secondary metabolites with ecological roles, especially in the Caribbean region, where they can dominate some ecosystems . The higEctyoplasia ferox   was selected for a full chemical study due to its high abundance in the Urabá Gulf, our region of interest. This species has also been described at sites in the Northern part of the Caribbean, like Florida or even the Bahamas, and our chemical study will therefore also shed light on some possible differences in the secondary metabolome induced by geographical and/or environmental factors. The place of E. ferox in the order Poecilosclerida has been demonstrated, but the absence of guanidinic alkaloids was intriguing, as they are usually recognized as chemotaxonomic markers of this group +) and 13C-NMR data. The saponin nature of this compound was evidenced in the 1H-NMR spectrum of 1 by the presence of characteristic terpenoid methyl singlets (δH between 0.6 and 1.5) together with sugar residues (δH between 3.1 and 5.0). The portion of the 1H-NMR spectrum corresponding to the aglycon evidenced seven methyl signals that were reminiscent of a 29-functionalized 24-methyl-30-norlanostane at δH 0.67 , 0.85 , 0.93 , 0.95 , 0.96 , 1.01 , 1.34  , 3.68 , 1.56  and δC 73.4 , 72.2 , 41.0 . The structure was then very similar to the previously isolated ulososides isolated from an Ulosa sp. sponge collected in Madagascar . Th. Th1 was01 s, H3-, 1.34 , and 105.1  and protons at δH 4.56  and 4.49 , with coupling constants at J = 8.1 and 7.8 Hz suggesting a β anomeric configuration  and 3.89  which was reminiscent of a methylene oxy at C-4  and the key H-22a, H-22b, H-24/C-23 HMBC correlations. Interestingly, this side-chain was identical to the one of some pandarosides, steroidal saponins found in another Poecilosclerida sponge of the genus Pandaros + in HRESIMS and 13C-NMR data indicated an empirical molecular formula of C41H62O17. The signals corresponding in the NMR spectra of 3 were closely related with the signals of 2 indicating a close relationship between these two compounds. The sole difference appears for the signals corresponding to the positions C-3, C-4 and C-5 , thus confirming this assumption.Compound  and C-5 . The abs3 which were identified as a first glucuronic acid linked at C-3 to the aglycon due to the presence of a H-1′/C-3 HMBC correlation. Coupling constant values and a clear H-5′/C-6′ HMBC correlation confirmed the β-glucuronic acid. The second sugar residue was linked at C-2′ to this first residue on the basis of a H-1′′/C-2′ HMBC correlation. This second residue was found to be a galactose unit due to the coupling constant values of the signal at δH 3.76 . Furthermore, the value of J = 8.1 Hz for the coupling constant of H-1′′ ascertained a β linkage with the first residue. Using the same process used for 2 we confirmed the relative configurations and proposed a D absolute configuration for both residues.Two hexose residues were observed in 3 exhibited a low cytotoxicity, with an IC50 value of 100 µM. Other compounds were considered inactive, with IC50 > 100 µM, and with cytotoxicities below 30% up to 100 µM. Interestingly, compounds 1 to 4 did not induce hemolysis, which is the major known adverse effect of this kind of molecules and the major obstacle for clinical trials progress. Previous studies on the bioactivities of triterpene glycosides from E. ferox reported moderate cytotoxicity against J774 (murine monocyte-macrophage), WEHI164 (murine fibrosarcoma), and P388 (murine leukemia) cell lines at IC50 ranging from 8.5 to 19 μg/mL 20D −4.0 ; IR (thin film): γmax 3392, 2956, 2874, 1721, 1669 cm−1; 1H-NMR and 13C-NMR see m/z 892.46222 [M+Na]+ .D-Arabinopyranosyl-(1→2)-)-β-D-galactopyranosid]-3,28-dihydroxy-23-oxolanost-8(9)-en-29-oic acid3β-O-20D −330 ; IR (thin film): γmax3392, 2988,1625, 1072 cm−1; 1H-NMR and 13C-NMR see m/z 953.47327 [M+Na]+ .D-Galactopyranosyl-(1→2)-β-D-glucopyranosiduronic acid]-3-hydroxy-23-oxolanosta-8(9)-ene-28,29-dioic acid3β-O-20D−130 ; IR (thin film): γmax 3409, 2920, 1712, 1674 cm−1; 1H-NMR and 13C-NMR see m/z 849.38367 [M+Na]+ .D-Glucuronopyranosyl-(1→6)-β-D-glucopyranosyl)-3,22,23-trihydroxy-24-methyl-30-norlanost-8(9)-en-29-oic acid-3β-O-. White amorphous solid; [α]20D −9.5 ; IR (thin film): γmax 3425, 1715, 1648 cm−1; 1H-NMR  1.84 (m), 1.36 (m), 2.41 (m), 2.03 (m), 3.30 (m), 1.34 (m), 1.97 (m), 1.71 (m), 2.07 (m), 1.93 (m), 2.17 (m), 2.08 (m), 1.99 (m), 1.46 (m), 2.11 (m), 1.64 (m), 1.34 (m), 1.99 (m), 1.44 (m), 1.57 (m), 0.67 (s), 1.01 (s), 1.90 (m), 0.93 , 3.51 (m), 3.66 , 1.55 (m), 1.62 (m), 0.97 , 0.95 , 0.85 , 1.44 (s), 4.32 , 3.25 (m), 3.47 (m), 3.27 (m), 3.53 (m), 4.08 , 3.77 (m), 4.47 , 3.25 (m), 3.39 (m), 3.51 (m), 3.81 ; 13C-NMR  37.7 (C-1), 28.5 (C-2), 89.4 (C-3), 50.7 (C-4), 53.6 (C-5), 21.3 (C-6), 29.9 (C-7), 129.3 (C-8), 137.1 (C-9), 23.2 (C-11), 38.5 (C-12), 43.3 (C-13), 53.3 (C-14), 24.8 (C-15), 29.1 (C-16), 52.5 (C-17), 11.9 (C-18), 18.6 (C-19), 38.0 (C-20), 12.3 (C-21), 73.2 (C-22), 72.2 (C-23), 41.0 (C-24), 32.2 (C-25), 21.6 (C-26), 21.2 (C-27), 10.0 (C-28), 179.0 (C-29), 24.3 (C-30), 107.2 (C-1′), 75.3 (C-2′), 77.0 (C-3′), 71.7 (C-4′), 73.2 (C-5′), 70.2 (C-6′), 105.1 (C-1′′), 74.9 (C-2′′), 77.9 (C-3′′), 73.5 (C-4′′), 76.8 (C-5′′), 171.2 (C=O); HRESIMS (+): m/z 851.43524 [M + Na]+ .D- and L- determination was performed using combined gas chromatography/mass spectrometry (GC/MS) of the butylated derivatives of the monosaccharides produced from the compounds 1–4 by acidic hydrolysis. The sample materials were placed into individual test tubes. To this, aqueous 2 M TFA (400 µL) was added. The tubes were then placed at 121 °C for 1.5 h. Once cooled, the sample was dried under nitrogen. The dried samples were then re-N-acetylated with pyridine and acetic anhydride in methanol. After drying, the samples were butylated using S-(+)-2-butanol or R-(+)-2-butanol (Sigma) for 16 h at 80 °C. The samples were per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 °C (0.5 h). GC/MS analysis of the butylated derivatives was performed using a Supelco EC-1 fused silica capillary column (30m × 0.25 mm ID). Individual standards of all the detected residues were analyzed in parallel with each sample. Comparison of retention times allowed for the elucidation of the specific conformation of each monosaccharide in the compounds. All residues were of D configuration.2 atmosphere in 24 cm2 cell culture flasks in RPMI 1640 medium supplemented with 5% heat inactivated fetal bovine serum, 2 mM L-glutamine, free of antibiotics.Jurkat and CHO-K1 cells were cultured at 37 °C in a humidified 5% CO4/well for Jurkat cells and 8.0 × 103/well for CHO cells in RPMI 1640 medium with 5% of FBS) onto wells. Cells were treated with each sample for 24 h at 37 °C in 5% CO2. After treatment, the viability of cells was evaluated by the MTT assay. MTT reagent was added to each plate, and after 4 h of incubation, 100 µL of acidic isopropyl alcohol mixture  was added to dissolve the water-insoluble formazan salt for overnight. The OD570 nm absorbance was measured. Each concentration was tested by triplicated and unexposed cells were regarded as 100% viable [The purity of all tested compounds was found to be higher than 96% based on HPLC-ELSD analysis. Compounds at final concentrations of 1 and 10 μM was added to 100 μL of the CHO and Jurkat cells suspension  . As esta


Albiziae Cortex and their ability to ameliorate steatosis and promote proliferation and anti-oxidation in vitro. Together, five known lignan glycosides, -erythro-syringylglycerol-β–O-4′-sinapyl ether 9-O-β-D-glucopyranoside (1), (+)-lyoniresinol-9′-O-gluco-side (2), (−)-lyoniresinol-9′-O-glucoside (3), picraquassioside C (4), and icariside E5 (5), were isolated from the Albiziae Cortex. Their structures were elucidated by extensive NMR and high-resolution mass spectrometry analysis and compared with reported data. Oil Red O staining results revealed that compounds 1, 2, and 3 attenuated lipid accumulation and lipid metabolic disorders in FFAs -exposed HepG2 cells. The Cell Counting Kit 8 (CCK-8) assay results revealed that compounds 1 and 5 can significantly promote human umbilical vein endothelial cell (HUVEC) proliferation; meanwhile, these compounds did not exhibit significant cytotoxicity against HUVECs. In addition, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining results revealed that high glucose (HG)-induced reactive oxygen species (ROS) production was abolished by compounds 1, 2, and 3. This is the first report of the isolation of lignan skeletons from the genus Albizzia julibrissin with the ability to ameliorate steatosis and promote proliferation and anti-oxidation activities.This study describes the chemical constituents of  Lycium barbarum, which have been reported to prevent fatty liver −, indicating 10 degrees of unsaturation. The 13C-NMR spectrum showed eight non-protonated carbons and four methyl carbons, indicating the presence of one syringoylglycerol, one glucopyranose moiety, and one sinapyl alcohol  and δ H 3.83 . In addition, the 1H-NMR spectrum -erythro-syringylglycerol-β–O-4′-sinapyl ether 9-O-β-D-glucopyranoside, which had previously been reported − in combination with its extensive NMR. 1H-NMR  δ 6.76 , 6.74 , 6.56 , 6.34 , 4.81 , 4.65 , 4.29 , 4.24 , 3.96–3.88 , 3.85 , 3.83 , 3.77 , 3.73–3.63 , 3.60 , 3.56–3.39 , 3.22 . 13C-NMR  δ153.08 , 152.39 , 138.12 (C-4′), 134.99 (C-4), 134.13 (C-1′), 133.30 (C-1), 129.95 (C-7′), 128.51 (C-8′), 104.62 , 104.26 (C-1”), 103.51 , 85.66 (C-8), 76.94 (C-3”), 76.37 (C-5”), 74.32 (C-2”), 72.64 (C-7), 69.92 (C-4”), 62.16 (C-9), 61.17 (C-9′), 60.23 (C-6”), 55.61 , 55.28 .Compound 2, (+)-lyoniresinol-3α-O-β-D-glucopyranoside, was purified as a white powder and showed positive optical activity m/z 617.1981 [M + Cl]−, molecular formula of C28H38O13. 1H-NMR  δ 6.60 , 6.45 , 4.44 , 4.30 , 3.95–3.81 , 3.76 , 3.67 , 3.56 , 3.47 , 3.39 , 3.36 , 3.28–3.23 , 2.77–2.59 , 2.09 , 1.73 . 13C-NMR  δ 147.59, 147.24(C-5), 146.19(C-7), 137.95(C-1′), 137.51(C-6), 133.10(C-4′), 128.81(C-9), 125.03(C-10), 106.47(C-8), 105.55, 103.43(C-1”), 76.85(C-5”), 76.54(C-3”), 73.79(C-2”), 70.27(C-4”), 70.11(C-3α), 64.84(C-2α), 61.44(C-6”), 58.80(5-OMe), 55.48, 55.23(7-OMe), 45.30(C-3), 41.39(C-4), 39.22(C-2), 32.43(C-1).Compound 3, (−)-lyoniresinol-3α-O-β-D-glucopyranoside, was obtained as a pale yellow powder, showed negative optical activity 28H38O13 as evidenced by an ESI-MS peak at m/z 617.1890 [M + Cl]− in combination with its 1H-NMR, 13C-NMR, and a comparison with the literature − in combination with its 1H-NMR, 13C-NMR and comparison with the literature − in combination with its 1H-NMR, 13C-NMR, and a comparison with the literature. 1H-NMR  δ 6.94 , 6.59 , 6.50 , 6.32 , 4.69 , 4.24 , 4.03–3.94 , 3.92–3.85 , 3.84 , 3.82–3.74 , 3.72 , 3.71 , 3.67 , 3.62–3.35 , 3.19–3.11 , 2.99 , 2.74 . 13C-NMR  δ 152.05 (C-3), 147.02 (C-3′), 143.94 (C-4′), 143.61 (C-4), 137.53 (C-5), 134.01 (C-1), 131.82 (C-1′), 130.10 (C-8), 128.29 (C-7), 121.22 (C-6′), 117.76 (C-6), 114.29 (C-5′), 112.41 (C-2′), 107.75 (C-2), 103.97 (C-1”), 76.67 (C-5”), 76.46 (C-3”), 74.54 (C-2”), 69.85 (C-4”), 65.45 (C-9′), 62.27 (C-9), 61.06 (C-6”), 55.02 (3′-OCH3), 54.89 (5′-OCH3), 41.39 (C-8′), 37.76 (C-7′).Compound 2 at 37 °C. According to the literature reports, exposure of HepG2 cells to FFAs  can induce a steatosis model without influencing cell activities [5 per well in a 12-well plate and incubated at 37 °C for 12 h. Then, the cells were exposed to FFAs by supplementation with FFAs for 24 h, followed by treatment with compounds 1–5 for 24 h ; a positive control group was treated with EGCG for 24 h, and the control groups were cultured in normal DMEM medium. HUVECs were cultured in DMEM medium (low glucose) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator containing 5% CO2 at 37 °C. The sample compounds were dissolved in DMSO, then further diluted. Cells were plated at a density of 5 × 104 per well in a 12-well plate and incubated at 37 °C for 12 h. The HUVECs were pretreated with compounds 1, 2, 3, 4, and 5 (80 µM for 12 h) or NAC (500 µM) for 1 h before 35 mM HG incubation for another 24 h.HepG2 cells were cultured in DMEM containing 25 µM glucose, 10% FBS , 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator containing 5% COtivities . To induOil Red O staining was used to evaluate lipid accumulation . The Hep2 at 37 °C. The sample compounds were dissolved in DMSO, then further diluted. Cells were plated at a density of 3 × 103 per well in a 96-well microplate and incubated at 37 °C overnight. The cells were treated with various concentrations of test compounds  for 48 h. Then, cell viability was determined by CCK-8 assay [HUVECs were cultured in DMEM medium (low glucose) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator containing 5% CO-8 assay ,20.Intracellular reactive oxygen species (ROS) generation was detected by a fluorescence probe (DCFH-DA) as previously described . The HUVAlbiziae Cortex. The results revealed that three of the isolated compounds  can markedly alleviate the deposition of lipid droplets and eliminate HG-induced ROS production, while two of the isolated compounds (1 and 5) can effectively promote HUVEC proliferation. This is the first report of these lignans from Albiziae Cortex and their ability to ameliorate steatosis, promote proliferation, and provide anti-oxidation activities. These compounds exhibited no significant inhibitory activities against HUVECs. This study provides a new basis for further research into new drugs for the treatment of steatosis, atherosclerosis, and trauma.Five lignan glycosides were isolated and identified for the first time from