Onosma visianii were investigated. By using different chromatographic techniques and appropriate spectroscopic methods, the seven naphthoquinones were described: deoxyshikonin (1), isobutyrylshikonin (2), α-methylbutyrylshikonin (3), acetylshikonin (4), β-hydroxyisovalerylshikonin (5), 5,8-O-dimethyl isobutyrylshikonin (6) and 5,8-O-dimethyl deoxyshikonin (7). Among the tested compounds, 3 and 4 exhibited the highest antibacterial activities toward all tested bacterial species . Also, naphthoquinones 3 and 4 exhibited strong cytotoxic activity against MDA-MB-231 cells , while compounds 1, 3, 4 and 5 significantly decreased viability of HCT116 cells . Our results indicated that all tested naphthoquinone pigments are potential candidates for clinical uses as antibacterial and cytotoxic agents. In this study, the antibacterial and cytotoxic activities of isolated compounds from the roots of  The emergence of antibiotic resistance presents one of the most important challenges for the pharmaceutical industry and the healthcare sector both in the developing and developed countries. The inadequate and excessive use of antibiotics, significantly diminishes the efficacy of current drugs, at the same time demanding new drugs against multi-drug resistant bacteria  contains about 150 species, distributed mainly in the Mediterranean region and Central Asia .Onosma sericeum Willd., Onosma microcarpum DC., Onosma argentata Hub.-Mor. and Onosma armeniaca Klokov have been widely used in traditional medicine for the treatment of wounds, burns, dyspnea, hoarseness, hemorrhoids, abdominal aches and stomach ulcers . Onosma has led to the discovery of a series of shikonin and alkannin derivatives, as compounds which exhibit a wide spectrum of biological properties, like wound healing, antiinflammatory, antitumor, antimicrobial and antithrombotic properties . On theal., 1999; Damianaal., 1999) and pheal., 1999).Onosma visianii Clem (Boraginaceae) is a biennial to perennial plant which inhabits areas of Balkan Peninsula and Southeast Europe. The point of view of the chemical composition, O. visianii is little investigated. Except that it contains shikonin , other O. visianii. Also, antibacterial activity against ten bacterial species, as well as cytotoxic activity against MDA-MB-231 and HCT116 tumor cell lines was described.We herein report for the first time isolation and structural characterization of these naphthoquinones from the roots of 3), dimethyl sulfoxide (DMSO) and HPLC grade methanol were obtained from Sigma-Aldrich . Water was treated in a Milli-Q water purification system . Mueller Hinton agar was obtained from Merck (Germany). Nutrient liquid medium, Mueller Hinton broth was purchased from Biolife . 3--2,5-diphenyltetrazolium bromide (MTT) was obtained from SERVA . Annexin V-FITC/7-AAD Kit was purchased from Beckman Coulter (USA). Standard antibiotics, Cisplatin, Phosphate-buffered saline (PBS), Ribonuclease A (RNAse A), fetal bovine serum (FBS) and 7-Aminoactinomycin D (7-AAD) were obtained from Sigma-Aldrich . Petroleum ether (boiling point ranges: 40 °C-60 °C), methylene chloride, ethyl acetate, deuterochloroform (CDCl-1) and Perkin-Elmer FT-IR Spectrometer Spectrum One . The NMR spectra were recorded on a Varian Gemini 200 spectrometer (1H at 200 MHz and 13C at 50 MHz) and a BrukerAvance III 500 spectrometer (1H at 500.26 MHz and 13C at 125.80 MHz), solvent CDCl3, TMS internal standard. Chemical shifts were given in δ (ppm), J-coupling constants in Hertz (Hz), abbreviations: s-singlet, d-doublet, dd-doublet of doublet, t-triplet, m-multiplet, br s-broadend singlet. Low resolution mass spectra were recorded on an Agilent 5973 mass spectrometer . High resolution mass spectra were obtained on a Agilent 6210 Time of Flight (TOF) mass spectrometer  and linear-trap quadrupole (LTQ) orbitrap hybrid mass spectrometer equipped with a heated-electrospray ionization probe . [α]D values were measured in dichloromethane using a Perkin Elmer 341 polarimeter. UV/VIS spectra were recorded on an Agilent Cary 300 UV-VIS spectrophotometer . MALDI TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) Biotyper  was used for confirmation of test bacteria. Semi preparative high performance liquid chromatography was performed on Agilent 1100 Series liquid chromatograph  equipped with diode array detector , autosampler, and fraction collector; conditions: injection volume, 600 μL ; column Zorbax Eclipse XDB C18 ; mobile phase (6 mL/min), water (40 %) and methanol (60 %). Silica gel 60  was used for column chromatography. Preparative TLC was performed by using silica gel P/UV254 with CaSO4 . Analytical TLC was performed on silica gel . Microanalysis of carbon and hydrogen was carried out with a Carlo Erba 1106 microanalyser. Infrared spectra were run on Thermo Scientific Nicolet 6700 FT-IR spectrometer (4000-400 cmO. visianii Clem roots were collected (June 2015) in the region of mountain Rumija . A voucher herbarium specimen was deposited at the Department of Botany, Faculty of Biology, University of Belgrade, Serbia .O. visianii were powdered and extracted at room temperature three times with a  petroleum ether-methylene chloride mixture (1:1). The combined extracts were evaporated under reduced pressure to give concentrated extract as red-brown semi-viscous residue (6.5 g). Dried roots (135 g) of F1-11, F12-15, F16-20, F21-23 and F24-29) and further subjected to preparative TLC. Used mobile phases: for F1-11 and F12-15 petroleum ether:ethyl acetate (8:2), for F16-20 and F21-23 petroleum ether:ethyl acetate (7:3), for F24-29 petroleum ether:ethyl acetate (6:4). After TLC examinations, compounds 1 and 2 were obtained from F1-11, 2 and 3 were obtained from F12-15, from F16-203, 4 and 5, from F21-23 was isolated compound 5, while F24-29 provided compounds 6 and 7. In order to obtain high purity, all isolated compounds were subjected to semi preparative HPLC on Zorbax Eclipse XDB C18 reversed phase column with isocratic elution of mixture water and methanol (40:60). The petroleum ether-methylene chloride extract (6 g) was subjected to silica gel column chromatography. The column was eluted with petroleum ether (100 %), followed by an increasing gradient of ethyl acetate starting from 1 % up to 30 %. Twenty-nine collected fractions were analyzed on TLC plates using petroleum ether:methylene chloride (97:3 and 95:5) and petroleum ether:ethyl acetate (95:5) solvent systems. Similar fractions were combined (max (ε) 552 (3.33), 515 (3.41), 486 (3.34), 277 (3.80), 216 (4.47); IR  3650-3160 (OH), 3055 (=CH), 2923 (CH), 1621, 1573 and 1457 (C=C), 1222 and 1102 (C-O); 1H NMR  δ 1.60 , 1.70 , 2.28 , 2.60 , 5.13 , 6.84 , 7.21 , 12.48  and 12.64 ; 13C NMR  δ 17.9 (C-16), 25.7 (C-15), 26.4 (C-12), 29.7 (C-11), 111.6 (C-9), 111.8 (C-10), 122.3 (C-13), 130.7 (C-6), 131.0 (C-7), 133.5 (C-14), 134.5 (C-3), 151.5 (C-2), 161.9 (C-5 or C-8), 162.6 (C-8 or C-5), 183.2 (C-1 or C-4) and 183.3 (C-4 or C-1); HRESIMS m/z [M-H]-=271.0956 .naphthalene-1,4-dione). Red purple solid. UV/VIS  λD25  +321. UV/VIS  λmax (ε) 556 (3.21), 518 (3.48), 489 (3.49), 275 (3.81), 217 (4.73); IR  3680-3630 (OH), 3074 and 3048 (=CH), 2965, 2925 and 2853 (CH), 1735 (C=O from COOCH(CH3)2), 1607, 1569 and 1457 (C=C), 1238, 1213, 1146 and 1116 (C-O); 1H NMR  δ 1.20 , 1.22 , 1.58 , 1.69 , 2.47 , 2.62 , 2.64 , 5.12 , 6.02 , 6.97 , 7.18 , 12.42  and 12.58 ; 13C NMR  17.9 (C-16), 18.9 (C-3' and C-4'), 25.7 (C-15), 32.8 (C-12), 33.9 (C-2'), 68.8 (C-11), 111.4 (C-9), 111.8 (C-10), 117.7 (C-13), 131.3 (C-3), 132.5 (C-6), 132.8 (C-7), 135.9 (C-14), 148.5 (C-2), 166.6 (C-5 or C-8), 167.3 (C-8 or C-5), 175.7 (C-1'), 176.7 (C-1 or C-4) and 178.3 (C-4 or C-1); HRESIMS m/z [M-H]-=357.1320 .[(+)-1--4-methylpent-3-enyl isobutyrate]. Red purple solid. [α]D25  +221. UV/VIS  λmax (ε) 557 (3.20), 518 (3.47), 489 (3.38), 275 (3.87), 216 (4.53); IR  3660-3622 (OH), 3041 (=CH), 2970, 2932 and 2878 (CH), 1742 (C=O from COOCH(CH3)CH2CH3), 1610, 1571 and 1455 (C=C), 1232, 1204, 1148 and 1113 (C-O); 1H NMR  δ 0.93 , 1.17 , 1.53 , 1.59 , 1.69 , 2.27 , 2.47 , 2.61 , 5.13 , 6.04 , 6.98 , 7.18 , 12.42  and 12.58 ; 13C NMR  δ 11.4 (C-4'), 17.9 (C-16), 18.8 (C-5'), 25.7 (C-15), 26.7 (C-3'), 33.0 (C-12), 41.0 (C-2'), 69.0 (C-11), 111.6 (C-9), 111.8 (C-10), 117.8 (C-13), 131.3 (C-2), 132.6 (C-6), 132. 8 (C-7), 135.9 (C-14), 148.6 (C-3), 166.9 (C-5 or C-8), 167.4 (C-8 or C-5), 175.3 (C-1'), 176.7 (C-1 or C-4), and 178.3 (C-4 or C-1); HRESIMS m/z [M-H]-=371.1473 .[(+)-1--4-methylpent-3-enyl 2-methylbutanoate]. Red purple solid. [α]D25  +692. UV/VIS  λmax (ε) 556 (3.21), 518 (3.48), 490 (3.41), 273 (3.89), 215 (4.53); IR  3650-3620 (OH), 3065 (=CH), 2921 and 2859 (CH), 1732 (C=O from COOCH3), 1604, 1575 and 1454 (C=C), 1236, 1209, 1114 and 1049 (C-O); 1H NMR  δ 1.58 , 1.69 , 2.14 , 2.46 , 2.61 , 5.12 , 6.02 , 7.00 , 7.18 , 12.43  and 12.58 ; 13C NMR  δ 17.9 (C-16), 20.9 (C-2'), 25.7 (C-15), 32.8 (C-12), 69.5 (C-11), 111.5 (C-9), 111.8 (C-10), 117.7 (C-13), 131.4 (C-2), 132.7 (C-6), 132.9 (C-7), 136.1 (C-14), 148.1 (C-3), 166.9 (C-5 or C-8), 167.4 (C-8 or C-5), 169.6 (C-1'), 176.8 (C-1 or C-4) and 178.2 (C-4 or C-1); HRESIMS m/z [M-H]-=329.1004 .[(+)-1--4-methylpent-3-enyl acetate]. Red purple solid. [α]D25  +91. UV/VIS  λmax (ε) 556 (3.19), 518 (3.48), 489 (3.40), 276 (3.97), 217 (4.54). IR  3680-3620 (OH), 3043 (=CH), 2974, 2927 and 2853 (CH), 1737 (C=O from COOCH2C(CH3)2OH), 1612, 1571 and 1455 (C=C), 1264, 1204, 1111 and 1154 (C-O); 1H NMR  δ 1.31 , 1.59 , 1.69 , 2.50 , 2.59 , 2.63 , 3.26 , 5.12 , 6.10 , 7.03 , 7.18 , 12.41  and 12.60 ; 13C NMR  δ 18.3 (C-16), 25.7 (C-15), 29.2 (C-4'), 29.5 (C-5'), 33.1 (C-12), 46.5 (C-2'), 69.1 (C-3'), 69.8 (C-11), 111.5 (C-9), 111.8 (C-10), 117.7 (C-13), 131.3 (C-2), 133.1 (C-6), 133.3 (C-7), 136.4 (C-14), 147.5 (C-3), 168.2 (C-5 or C-8), 168.7 (C-8 or C-5), 171.6 (C-1'), 175.3 (C-1 or C-4) and 176.9 (C-4 or C-1); HRESIMS m/z [M-H]-=387.1419 .[(+)-1--4-methylpent-3-enyl 3-hydroxy-3-methylbutanoate]. Red purple solid. [α]. [α]D25  +289. UV/VIS  λmax (ε) 452 (3.45), 259 (4.11), 215 (4.79); IR  3044 (=CH), 2919 and 2847 (CH), 1732 (C=O from COOCH(CH3)2), 1650 and 1457 (C=C), 1278, 1155 and 1058 (C-O); 1H NMR  δ 1.18 , 1.19 , 1.57 , 1.66 , 2.44 , 2.60 , 3.96 , 5.12 , 5.90 , 6.65 , 7.31 ; 13C NMR  δ 17.9 (C-16), 18.9 (C-3' and C-4'), 20.9 (C-2'), 25.7, (C-15), 32.8 (C-12), 56.8 (C-5-OCH3 or C-8-OCH3), 56.9 (C-8-OCH3 or C-5-OCH3), 69.7 (C-11), 118.1 (C-13), 120.1 (C-7), 120.6 , 131.3 (C-3), 133.3 (C-6), 135.6 (C-14), 148.3 (C-2), 153.0 (C-5 or C-8), 154.0 (C-8 or C-5), 169.5 (C-1'), 183.3 (C-1 or C-4), 184.5 (C-4 or C-1); HRESIMS m/z [M+H]+=387.1824 .[(+)-1--4-methylpent-3-enyl isobutyrate]. Orange oil. UV/VIS  λmax (ε) 443 (3.33), 259 (4.21), 216 (4.75); IR  3054 (=CH), 2923 and 2823 (CH), 1644 and 1448 (C=C), 1278, 1207, 1046 and 1018 (C-O); 1H NMR  δ 1.58 , 1.67 , 2.24 , 2.52 , 3.95 , 5.12 , 6.60 , 7.30 ; 13C NMR  δ 17.9 (C-16), 25.8, (C-15), 32.8 (C-12), 56.8 (C-5-OCH3 or C-8-OCH3), 56.9 (C-8-OCH3 or C-5-OCH3), 69.7 (C-11), 118.1 (C-13), 120.1 (C-7), 120.6 , 133.3 (C-6), 134.5 (C-3), 135.6 (C-14), 151.4 (C-2), 153.0 (C-5 or C-8), 154.0 (C-8 or C-5), 183.3 (C-1 or C-4), 184.5 (C-4 or C-1); HRESIMS m/z [M+H]+= 301.1450 .naphthalene-1,4-dione). Dark orange oilBacillus megaterium, Enterococcus faecalis, Microbacterium arborescens, Micrococcus luteus and Staphylococcus epidermidis) and five gram negative bacteria . The tested bacteria  were provided from a collection of Department of Microbiology, Faculty of Biotechnology and Food Sciences, Nitra, Slovakia.Antibacterial activity was tested against five gram positive bacteria  for 24 h at 36 °C. Regulation of initial suspension turbidity was conducted by comparison with 0.5 McFarland's standard  were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute document  in MuelDifferences in absorbance between the measurements before and after the analysis were expressed as a set of binary values. These values were assigned to exact concentrations. The following formula was created for this specific experiment: value 1 (inhibitory effect) was assigned to absorbance values lower than 0.05, while value 0 (no effect or stimulant effect) was assigned to absorbance values higher than 0.05. For this statistical evaluation the probit analysis in Statgraphics software was used. Tested isolated naphthoquinones were diluted in DMSO at the concentration of 200 μg/mL, filtered through a 0.22 μm Millipore filter before use, and diluted by a cell culture medium to diverse working concentrations, so that the final concentration of DMSO in cell culture medium never exceeded 0.5 % (v/v).Human breast carcinoma cell line MDA-MB-231 and colon cancer cell line HCT116 were purchased from the American Type Culture Collection  and cultured as suggested by the supplier. Both cell lines were sustained in culture medium supplemented with 10 % heat-inactivated FBS. 4/mL and allowed to adhere overnight. Cells were then treated with tested substances at indicated concentrations, or with media alone (control). The plates were incubated for 24, 48 and 72 h at 37 °C in an atmosphere of 5 % CO2 and absolute humidity. The media was removed and 100 μL of MTT (0.5 mg/mL PBS) was added to each well. After 4h incubation at 37 °C, MTT solution was removed and 150 μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured with a multiplate reader  at 595 nm  for linear regression downloaded from http://www.sciencegateway.org/protocols/cellbio/drug/data/.MDA-MB-231 and HCT116 cells were plated onto microtiter plates at density of 5 x 101-5 and 500 μg/mL of compounds 6 and 7. After 48 h at 37 °C in an atmosphere of 5 % CO2 and absolute humidity, both attached and detached cells were collected, washed in PBS and fixed with 70 % ethanol at 4 °C. Fixed cells were washed in PBS and finally resuspended in 1mL PBS containing RNAse A (500 μg/mL). After 30 min incubation at 37 °C, cells were stained with 5 μL PI (10 mg/mL PBS), incubated for 15 min in the dark and immediately analyzed by flow cytometer Cytomics FC500 . Distribution of cells with different DNA content was determined with Flowing Software (http://www.flowingsoftware.com/) and presented by histograms. MDA-MB-231 and HCT116 cells were incubated with media alone (control) or with 100 μg/mL of compounds 1-5 and 500 μg/mL of compounds 6 and 7. After 48 h incubation at 37 °C in the atmosphere containing 5 % CO2, both attached and detached cells were collected. After washing with PBS, cells were resuspended in 500 μL of ice cold binding buffer. Ten μL of Annexin V-FITC and 20 μL of 7-AAD were added and after 15 min incubation in the dark, 400 μL of binding buffer was added to each tube. Samples were analyzed by flow cytometer Cytomics FC500 . Data were analyzed using Flowing Software and presented by dot plots. Apoptosis was determined using Annexin V-FITC/7-AAD Kit. MDA-MB-231 and HCT116 cells were incubated with media alone (control) or with 100 μg/mL of compounds O. visianii using open column chromatography, preparative thin layer chromatography, as well as semipreparative HPLC, resulted in isolation of seven pure naphthoquinone derivatives: deoxyshikonin (1) (19 mg), isobutyrylshikonin (2) (38 mg), α-methylbutyrylshikonin (3) (63 mg), acetylshikonin (4) (166 mg), β-hydroxyisovalerylshikonin (5) (131 mg), 5,8-O-dimethyl isobutyrylshikonin (6) (18 mg) and 5,8-O-dimethyl deoxyshikonin (7) (32 mg)  was found in nature for the first time.Separation of petroleum ether-methylene chloride root extract of  Figure 1. Structu1-5 showed IR absorptions in the region of 3680-3160 cm-1 which indicated presence of OH groups attached to naphthoquinone moiety, as well as tertiary OH group from hydroxyisovaleryl part of compound 5. Except in the cases of 1 and 7, observed stretching vibrations of compounds 2-6 in the region of 1742-1732 cm-1 belong to the aliphatic ester C=O groups. Also, for all isolated naphthoquinones (1-7), strong bands from C-O groups were noted (at 1278-1018 cm-1). The 1H NMR spectra of 1-5 showed two deshielded protons from hydroxyl groups attached to C-5 and C-8 carbons from naphthoquinone part (12.41 ppm-12.64 ppm). Also, compound 5 contains one additional broadened singlet (at 3.26 ppm) from hydroxyl group linked to C-3'. Instead of singlets of OH protons, resonances from C-5-OCH3 and C-8-OCH3 groups  were observed in the 1H NMR spectra of 6 and 7 (as dimethoxy derivatives). In contrast to other isolated compounds , deoxyshikonin 1 and it 5,8-dimethoxy derivative 7 showed signals of C-11-H protons at lower fields . All derivatives contain characteristic triplets from vinyl C-13-H protons (at 5.12 ppm-5.13 ppm), singlets from C-3-H protons , as well as singlets from C-H-6 and C-H-7 protons . In the 13C NMR spectra of 2-6, signals from C-1' carbons (at 169.5 ppm-175.7 ppm) were recorded, indicating the presence of aliphatic ester C=O groups. These signals were not observed in cases of compounds 1 and 7, as deoxyshikonin and its dimethoxy derivative. Also, in contrast to isolated compounds 2 and 1, 5,8-dimethoxy derivatives 6 and 7 showed two additional signals from C-5-OCH3 and C-8-OCH3 carbons . Finally, the positive values of measured optical rotations ([α]D25) confirmed that isolated naphthoquinones 2-6 from the roots of O. visianii are shikonin derivatives.The compounds 50 and MIC90 in μg/mL, respectively) were showed in Table 250 and MIC90 values of isolated naphthoquinones are in the range of 4.27 μg/mL-68.27 μg/mL and 4.77 μg/mL-76.20 μg/mL, respectively. In accordance with literature , with the fact of remarkable differences of antibacterial potency due to different approaches of measuring MIC values , compoual., 1999; AndujarE. faecalis (MIC50 6.40 μg/mL and MIC90 6.82 μg/mL), as well as against S. maltophilia (MIC50 4.27 μg/mL and MIC90 4.77 µg/mL), while compound 4 was very active against M. arborescens (MIC50 6.40 μg/mL and MIC90 6.82 μg/mL) and P. proteolytica (MIC50 4.27 μg/mL and MIC90 4.7 μg/mL). In contrast to previous report that alkylation of the phenolic groups leads to lower activity .Among the isolated compounds, α-methylbutyrylshikonin 3 and acetylshikonin 4 exhibited the highest antibacterial activities toward all tested bacterial species. Observed MIC50 and MIC90 values for gram positive bacteria are in the range of 6.40 µg/mL-12.79 μg/mL and 6.82 μ g/mL-13.60 μg/mL, respectively, while for gram negative bacteria are in the range of 4.27 μg/mL-8.53 μg/mL and 4.77 μg/mL-9.54 μg/mL, respectively. The shikonin derivative 3 had strong potency against al., 1999), we herFrom the above mentioned results, in cases of developed resistance (or in cases of preventing resistance) examined naphthoquinones may substitute clinical antibiotics.1-7 against two cancer cell lines was performed by conducting MTT cell viability assays. Generally, tested compounds decreased viability of both tumor cell lines in a dose and time dependent manner. Yet, the extent of cytotoxicity on the designated cell lines was diverse, being more potent on HCT116 cell line . Also, all examined compounds, except compounds 6 and 7, decreased viability of MDA-MB-231 cells after 48 hours of incubation. However, HCT116 cells were generally more sensitive to the effects of isolated naphthoquinones. Compound 3 showed the strongest cytotoxic effect against HCT116 cells with IC50 value of 15.2 μg/mL. Also, compounds 1, 4 and 5 significantly decreased viability of HCT116 cells and these compounds exhibited IC50 values of 97.8 μg/mL, 24.6 μg/mL and 30.9 μg/mL, respectively. The results presented in Table 3in vivo activity against W256 in rats and P388 lymphoid leukemia in mice, as well as significant in vitro potency toward KB cells and CCRF-CEM leukemia cells . To det4  . Compounds 1 and 2 showed no changes in cell cycle profile of MDA-MB-231 cells  to 38.9 %-49.6 %, with most pronounced arrest when treated with compound 3 (p < 0.05). Contrary, treatment with compounds 2 and 7 showed an increasing trend in the number of the cells arrested at G0/G1 phase: quantitatively it was 24.3 % and 14.7 % respectively over the control values. Compound 1 showed no changes in cell cycle profile of HCT116 cells  or S phase arrest in MDA-MB-231 (compounds 6 and 7) point to blockade of DNA replication and inhibition of topoisomerase II and I as a possible mechanism that triggers apoptosis . Our real., 2003), whereaal., 2003) may be O. visianii are a rich source of bioactive naphthoquinone derivatives which possess strong activity toward selected resistant bacterial strains, as well as high cytotoxic activity against MDA-MB-231 and HCT116 tumor cell lines. According to our results, α-methylbutyrylshikonin 3 and acetylshikonin 4 showed notable activities toward all tested gram positive and gram negative bacterial species. Also, compounds 3 and 4 exhibited strong cytotoxic potency against MDA-MB-231 cells, while compounds 3, 4 and 5 showed the strongest cytotoxic effect toward HCT116 cells. Overall, this research provided a strong indication of the potential use of O. visianii naphthoquinone pigments as antibacterial and cytotoxic agents. In this study, for the first time we presents that roots of This work was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under Grants III41010, OI 172053, and co-funded by VEGA project No. 1/0611/14.


Thiofluorenone, a relatively stable and highly reactive aryl thioketone, yields upon treatment with TMS-CHN2 at −60 °C the corresponding 1,3,4-thiadiazoline. This unstable cycloadduct undergoes decomposition at ca. −45 °C and the silylated thiocarbonyl S-methanide generated thereby is trapped with complete regioselectivity by aryl or hetaryl thioketones forming also sterically crowded 2-trimethylsilyl-1,3-dithiolanes. The obtained 1,3-dithiolanes, by treatment with an equimolar amount of TBAF in a one-pot procedure, are converted in high yields into hetaryl/aryl-substituted ethenes or dibenzofulvenes, respectively, via a cycloreversion reaction of the intermediate 1,3-dithiolane carbanion. The presented protocol offers a new, highly efficient approach to tetrasubstituted ethenes and dibenzofulvenes bearing aryl and/or hetaryl substituents.Trimethylsilyldiazomethane (TMS-CHN Th. Th2N2, 2 with 1a and some dihetaryl thioketones, e.g., 1d and 1e, were reported -cycloadduct 8, which only at ca. −40 °C extruded N2 -cycloaddition of the in situ generated (at ca. −45 °C) silylated thiocarbonyl S-methanide 10 with thioketones 1a,c,d,h and i. The obtained products of type 6, without isolation, were desilylated at rt to give the expected ethenes 9g–k with a fluorenylidene moiety .The unexpected formation of tetrasubstituted ethenes lane 13c . In the  13c , are superior substrates for the preparation of tetraarylethenes. Another group of 2-trimethylsilylated 1,3-dithiolanes, obtained through the [3 + 2]-cycloaddition of trimethylsilylated thiofluorenone S-methanide with aryl and hetaryl thioketones can also be used for this transformation. The described protocol opens a new, straightforward access to a series of new dibenzofulvenes dithiophene (9i): Yield: 132 mg (77%); chromatographic purification (petroleum ether/CHCl3 7:3). Orange crystals; mp 175–177 °C; IR (KBr) ν: 3094 (w), 3066(w), 1556 (w), 1445 (m), 1416 (m), 1252 (w), 1220 (w), 837 (w), 777 (m), 713 (s) cm−1; 1H NMR  δ 6.68–6.94 , 7.02–7.04 , 7.14–7.18 , 7.42–7.43 , 7.59–7.61  ppm; 13C NMR  δ 119.3, 124.8, 126.6, 127.4, 128.1, 128.5, 129.2 (14 CHarom), 137.7, 138.4, 140.6, 144.6  ppm; HRMS–ESI+ (TOF): [M]+ calcd for C22H14S2, 342.0537; found, 342.0527.Preparation of 1,3-dithiolanes 13a,b: Both compounds were prepared from commercial methyl (trimethylsilyl) ketone or phenyl (trimethylsilyl) ketone [3·Et2O following a literature protocol [) ketone , respectprotocol .2-Phenyl-2-(trimethyl)silyl-1,3-dithiolane (13b): Yield: 191 mg (75%); colourless solid; mp 52–53 °C; IR (KBr) ν: 2952 (m), 2920 (m), 1591 (m), 1496 (m), 1480 (m), 1435 (m), 1245 (vs), 1078 (m), 1036 (m), 922 (m), 840 (vs), 736 (s), 698 (vs), 609 (m), 504 (s) cm–1; 1H NMR  δ 0.10 , 2.99–3.05 , 3.20–3.25 , 7.12–7.15 , 7.23–7.26 , 7.65–7.68  ppm; 13C NMR  δ 2.1 (3 CH3), 38.9 (2 CH2), 58.7 (C(2)), 125.7, 127.4, 127.8 (5 CHarom), 144.6 (1 Carom) ppm; anal. calcd for C12H18S2Si (254.49): C, 56.63; H, 7.13; S, 25.20; found: C, 56.64; H, 7.37; S, 25.31.Desilylation of 1,3-dithiolanes 13a,b. General procedure: The corresponding 1,3-dithiolane (0.5 mmol) was dissolved in THF (2 mL) and the solution was cooled to 0 °C (ice bath). Then, a solution of TBAF  was added portion-wise. The progress of the desilylation was controlled by TLC (petroleum ether/CH2Cl2 7:3). After removal of the solvent under vacuum, the residue was subjected to 1H NMR analysis in CDCl3 solution. The crude products were purified by column chromatography .File 19, 13, 15 and copies of the original 1H and 13C NMR spectra.Experimental data for compounds


Penicilliumdecumbens as biocatalyst was investigated and selective hydroxylation of 1 was observed. The products were separated by silica gel column chromatography, and the structure determination was performed by MS, NMR, and X-ray crystallography. Biotransformation of 1 afforded 7β-hydroxy-16α, 17α-epoxyprogesterone (2). and 7β,11α-dihydroxy-16α,17α- epoxyprogesterone (3). The two novel metabolic products 2 and 3 were reported for the first time. Moreover, the identified C7β- and C11-αhydroxylation is a novel reaction of microbial transformation of steroids by P.decumbens. Microbial transformation has been successfully applied in the production of steroid intermediates with therapeutic use and commercial value in pharmaceutical industry due to its high regio- and stereo-selectivity. As such, it is still important to screen microbial strains with novel activity or more efficient abilities in the development of the commercial steroid industry. Biotransformation of steroid: 16α, 17α-epoxyprogesterone (1). using  Steroid, an important class of bioactive compounds, has been widely used as anti-inflammatory, anti-microbial, anti-diabetic, anti-allergenic, and anti-cancer drugs in clinical therapy -3. It isAlthough the biotransformation of  has alrePenicillium decumbens was observed to efficiently transform 16α, 17-epoxyprogesterone during the screening of fungal strains and two new hydroxylated steroid derivatives were obtained. Since P. decumbensis already known to have activity in the reduction of double bonds and hydroxylation of steroids . With acetate ester/petroleum ester . 1H-NMR spectra were recorded on a JEOL JNM GSX 400M spectrometer in DMSO. 13C-NMR spectrawere measured at 100 MHz in DMSO. Mass spectra were performed on Waters 3100/2767with electron impact ionization (EI) at 70 eV. X-ray crystallography were carried out with Mo Karadiation (k = 0.07 nm) using a bruker APEX CCD diffractometer at 293 K.As asolvent. Spots were detected through UV-light (254 nm). The CCDC numbers of the crystal of 2 and 3were obtained after their crystallographic data wasdeposited with the Cambridge Crystallographic Data Centre. Further details ofthe crystallographic parameters canbe obtained for free on application to CCDC assupplementary publication, 12 UnionRoad, Cambridge CB2 1EZ, UKMicroorganism cultivation and substrate biotransformationP.decumbens TCCC 41604, stored in our laboratory,was cultured on potato-dextrose-agar medium ina test tube at 28 °C for 4-5 d. 8 mL of sterilized water was added subsequently and 1ml of the suspension was inoculated into a50 mL medium containing 20 g L-1 glucose, 20 g L-1peptone and 10g L-1 yeast extract. After 24 h. cultivation on a rotary shaker (180 rmin-1) at 28 °C, 50 mg of 16α, 17α-epoxyprogesterone (1) was added to the flask and the biotransformation was conducted for 24 h. under the same condition. The culture medium was analyzed by TLC.Separation and purification of the productsThe products were extracted from the medium by equal volume of ethyl acetatefor three times. After the evaporation of ethyl acetate in vacuum, white powder was obtained. After purification through silica gel column chromatography, two white products were obtained. Re-crystallization of products was performed and crystals were obtained, respectively 7β-hydroxy-16α, 17α-epoxyprogesterone (2): m/z 345.35 [M]+. 1H NMR  δ (ppm)): 5.64 ; 4.70 ; 3.90 ; 3.15-3.13 ; 2.45-2.38 ; 2.35-2.12 ; 2.01-1.89 ; 1.63-1.54 ; 1.53-1.42 ; 1.41-1.25 ; 1.22-1.00 ; 0.89-0.88 . 13C NMR (DMSO): 205.5 (C(20)); 198.5 (C(3)); 168.7 (C(5)); 124.2 (C (4)); 73.5 (C(17)); 70.1 (C(7)); 61.1 (C(16)); 50.4 (C(6)); 44.4 (C(10)); 39.8 (C(2)); 39.6 (C(13)); 39.4 (C(8)); 38.1 (C(9)); 35.3 (C(1)); 34.1(C(12)); 31.4 (C(14)); 30.3(C(15)); 26.2 (C(11)); 20.4 (C(19)); 17.2 (C(21)); 15.3 (C(18)).Whitecrystal.MS: Calcd. for C21H28O47β,11α-dihydroxy-16α,17α-epoxyprogesterone (3): m/z 361.39 [M]+. 1H NMR  δ (ppm): 5.62 ; 4.71 ; 4.40 ; 3.91 ; 3.87-3.84 ; 3.17-3.14 ; 2.50-2.33 ; 2.19-2.15 ; 2.01-1.96 ; 1.64-1.55 ; 1.26-1.17 ; 1.04-0.97 . 13C NMR (DMSO): 204.3 (C=O(20)); 198.0 (C(3)); 167.8 (C(5)); 123.5 (C(4)); 72.1 (C(17)); 68.8 (C(7)); 66.1 (C(11)); 60.3 (C(16)); 54.9 (C(9)); 42.5 (C(6))); 41.0 (C(2)); 39.1 (C(12)); 38.9 (C(10)); 38.7 (C(13)); 38.5 (C(8)); 35.8 (C(1)); 33.3 (C(14)); 29.2 (C(15)); 25.1 (C(19)); 17.5 (C(21)); 15.2 (C(18)).White crystal. MS: Calcd. for C21H28O5J =6.4Hz) and 3.15-3.13 implied the 7β-hydroxylation of (1). In case of compound (3) the occurrence of new H signals at ppm values of 4.71 . 3.17-3.14 . 4.40  and 3.87-3.84  suggested the 7β, 11α-dihydroxylation of (1). Moreover, other chemical shifts of protons and carbons in methylene groups, methine groups, methyl groups, C=CH units and aryl rings were in reasonable ranges, and the data reflected the crystal structure of (2) and (3). Further MS data was in good accordance with (2) and (3). TLC was used to analyze the extracts from the culture of fungus. The biotransformation process of 16α, 17α-epoxyprogesterone (1) using P. decumbens in 24 h of incubation was also investigated. As shown in P. decumbens for the first time. Their structures were characterized by 1H NMR, 13C NMR, X-ray crystallography and mass spectra. The biological activity of two new steroids will be studied in the near future.Two new steroidal compounds were prepared from16α, 17α-epoxyprogesterone by


Convallaria majalis (Liliaceae) whole plants yielded 15 steroidal glycosides (1–15), including nine new compounds  with a lycotetrose unit. The structures of the new compounds were determined using two-dimensional Nuclear magnetic resonance (NMR) analyses and chemical methods. The isolated compounds were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and HSC-4 and HSC-2 human oral squamous cell carcinoma cell lines. Of these, (25S)-spirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (1) exhibited cytotoxic activity against HL-60, A549, HSC-4, and HSC-2 cells with IC50 values ranging from 0.96 to 3.15 μM. The corresponding furostanol glycoside of 1, (25S)-26-[(β-d-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (8), was cytotoxic to the adherent cell lines of A549, HSC-4, and HSC-2 cells with IC50 values of 2.97, 11.04, and 8.25 μM, respectively. The spirostanol lycotetroside (1) caused necrotic cell death in A549 cells in a dose-dependent manner. Alternatively, the furostanol lycotetroside (8) induced apoptotic cell death in A549 cells in a time-dependent manner, as was evident by morphological observations and flow cytometry analyses.Phytochemical examination of  Convallaria majalis L. (Liliaceae), commonly called lily of the valley, is a popular ornamental garden plant -O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranose (lycotetrose) unit  and six known compounds  were isolated. The structures of the new steroidal glycosides were determined based on spectroscopic analysis, including two-dimensional Nuclear magnetic resonance (NMR) data, and chemical methods. The isolated compounds were evaluated for their cytotoxicity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and HSC-4 and HSC-2 human oral squamous cell carcinoma cells. Apoptosis-inducing activity in A549 cells was also examined.en plant . It has en plant . Previousaponins ,4. HowevConvallaria majalis  whole plants were extracted with hot MeOH. The MeOH extract was passed through a porous-polymer polystyrene resin  column. The MeOH-eluted and MeOH-H2O (6:4)-eluted fractions were then subjected to silica gel and octadecylsilanized (ODS) silica gel column chromatography (CC) and reversed-phase preparative high-performance liquid chromatography (HPLC) to obtain compounds 1–15 (1–3 and 7–9 were identified as (25S)-spirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (1) [S)-14α-hydroxyspirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (2) [R)-spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→4)-β-d-glucopyranoside (3) [S)-26-[(β-d-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-ol (7) [S)-26-[(β-d-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (8) [S)-26-[(β-d-glucopyranosyl)oxy]-14α,22α-hydroxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (9) [nds 1–15 . The strMeOH-H2O :4-elutedranoside  [8], and4 was obtained as an amorphous solid, and its molecular formula was identified as C50H78O24, based on data from high-resolution electrospray ionization time-of-flight mass spectrometry  and 13C NMR  spectrum. The 1H and 13C NMR spectral features of 4 were closely related to those of 2, showing the following signals: four steroid methyl groups at δH 1.21 , 1.07 , 1.04 , and 0.99 , in addition to δC 20.4 (C-18), 17.1 (C-19), 16.3 (C-27), and 15.2 (C-21); an olefinic group at δH 5.75  and δC 166.6 (C-5) and 126.9 (C-6); oxygenated protons and carbons at δH 3.86  and δC 78.1 (C-3), δH 5.06  and δC 82.2 (C-16), and δH 4.02  and 3.31  and δC 65.0 (C-26); an oxygenated quaternary carbon at δC 110.0 (C-22); and four anomeric protons and carbons at δH 5.59 , 5.25 , 5.19 , and 4.83  and δC 105.2, 105.0, 104.9, and 102.8   correlations were found between the signals of H-8 at δH 2.74 and Me-18 at δH 1.04/Me-19 at δH 0.99, between the signals of H-12ax at δH 1.41 and H-9 at δH 2.41/H-17 at δH 2.72, between the signals of H-17 and Me-21, between the signals of H-20 at δH 2.02 and Me-18/H-23ax at δH 1.93, and between the signals of Me-27 at δH 1.07 and H-23ax at δH 1.93. These correlations in the NOE spectroscopy (NOESY) spectrum of 4 were consistent with the B/C trans, C/D trans, and D/E cis ring fusions, as well as the 14α, 20α, 22α, and 25S configurations. The linkage of lycotetrose, O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranose, at C-3 of the aglycone, was ascertained by acid hydrolysis of 4 with 0.5 M HCl, obtaining d-galactose, d-glucose, and d-xylose. Additionally, long-range correlations from the anomeric proton (H-1′′′) of Glc (II) at δH 5.59 to C-2′′ of Glc (I) at δC 81.3, from H-1′′′′ of Xyl at δH 5.25 to C-3′′ of Glc (I) at δC 86.7, from H-1′′ of Glc (I) at δH 5.19 to C-4′ of Gal at δC 79.8, and from H-1′ of Gal at δH 4.83 to C-3 of the aglycone at δC 78.1 in the HMBC spectrum of 4 were also observed. Thus, 4 was labeled (25S)-3β-[(O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4) β-d-galactopyranosyl)oxy]-14α-hydroxyspirost-5-en-7-one.Compound nd 102.8 . In addiC 200.6) . In the 1H and 13C NMR spectral data of 5 (C56H90O29) suggest that 5 is a spirostanol glycoside resembling 2 in structure, with a lycotetrose unit at C-3 and a hydroxy group at C-14. However, the molecular formula of 5 was in excess of 2 by C6H10O6, which corresponded to a hexyloxy group. When the 1H and 13C NMR spectra of 5 were compared with those of 2, the signals for the C-24 methylene protons at δH 2.15 and 1.37  and carbon at δC 26.2 were displaced by an oxymethine proton at δH 4.83  and carbon at δC 72.8. Acid hydrolysis of 5 gave d-galactose, d-glucose, and d-xylose as the sugar moieties. Analysis of the 1H-1H correlation spectroscopy (COSY) and heteronuclear multiple quantum coherence (HMQC) spectra allowed the hexosyl unit attached to C-24 to be assigned β-d-glucopyranosyl, and an HMBC correlation was observed from the anomeric proton at δH 5.04  to C-24 carbon at δC 72.8. In the NOESY spectrum of 5, NOE correlations between the signals of H-24 at δH 4.83 and H-25 at δH 2.24/H-26ax at δH 3.89, and J values of 3JH-24, H-23ax = 10.9 Hz, 3JH-24, H-23eq = 5.5 Hz, and 3JH-24, H-25 = 5.5 Hz provided evidence for the 24S and 25R configurations. Thus, the structure of 5 was determined to be -24-[(β-d-glucopyranosyl)oxy]-14α-hydroxyspirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The 6 (C56H90O29) was the same as that of 5. The 1H and 13C NMR spectra of 6 showed close similarity with those of 5, suggestive of a stereoisomer with respect to the C-25 configuration. In the NOESY spectrum of 6, the NOE correlations between the signals of H-23ax at δH 2.00 and H-20 at δH 2.07/H-25 at δH 1.91, between the signals of H-26ax at δH 3.58 and H-16 at δH 5.05/H-24 at δH 4.06/Me-27 at δH 1.13, between the signals of Me-27 and H-24, and J values of 3JH-24, H-23ax = 12.4 Hz, 3JH-24, H-23eq = 5.3 Hz, and 3JH-25, H-26ax = 11.8 Hz confirmed the 24S and 25S configurations. The structure of 6 was characterized as -24-[(β-d-glucopyranosyl)oxy]-14α-hydroxyspirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The molecular formula of C 110.6 and the positive color reaction in Ehrlich’s test suggest that 10 (C56H92O29) may be a 22-hydroxyfurostanol glycoside. The 1H and 13C NMR spectra of 10 were similar to those of 8; however, the molecular formula of 10 had one extra oxygen atom compared to that of 8, and a significant difference was observed in the 13C NMR signals from the B-ring portion. Enzymatic hydrolysis of 10 with β-d-glucosidase yielded the corresponding spirostanol saponin 10a. In the 1H-1H COSY spectrum of 10a, a proton signal of a hydroxy group at δH 5.73 , which disappeared following the addition of HCl vapor, showed a correlation peak with the C-7 oxymethine proton at δH 4.01 (m), indicating the presence of a hydroxy group at C-7. The NOE correlations between the signals of H-7 at δH 4.00 and H-9 at δH 1.05/H-12ax at δH 1.68/H-14 at δH 1.32 in the NOESY spectrum of 10a confirmed that the C-7 hydroxy group was β-oriented. Furthermore, the NOE correlations between the signals of Me-27 at δH 1.07 and H-23ax at δH 1.90 and between the signals of H-25 at δH 1.57 and H-24ax at δH 2.14 confirmed the 25S configuration. Thus, 10a was assigned as (25S)-7β-hydroxyspirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside. The linkage of a β-d-glucopyranosyl group (Glc (III)) to the C-26 hydroxy group of the aglycone of 10 was ascertained by an HMBC correlation from H-1′′′′′ of Glc (III) at δH 4.80  to C-26 of the aglycone at δC 75.4. The NOE correlations between the signals of H-20 at δH 2.24 and H2-23 at δH 2.08 and 1.96 confirmed the C-22α configuration. Therefore, 10 was identified as (25S)-26-[(β-d-glucopyranosyl)oxy]-7β,22α-dihydroxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The hemi-acetal carbon signal at δ1H and 13C NMR spectra of 11 (C56H90O28) showed similar features with those of 9; however, the molecular formula of 11 was smaller than that of 9 by H2O, and the signals assignable to H-17 and Me-21 were observed at δH 3.36 as a doublet (J = 9.7 Hz) and δH 1.67 as a singlet, respectively. Furthermore, signals for a pair of olefinic carbon were detected at δC 152.2 and 103.9, in addition to those attributable to C-5 and C-6. These data implied that 11 was the corresponding Δ20(22)-pseudo-furostanol glycoside of 9. This was confirmed by the fact that the peracetate (11a) of 11 was the same as the product obtained by treating 9 with Ac2O in pyridine at 110 °C for 3 h. Enzymatic hydrolysis of 11 with β-d-glucosidase gave the corresponding spirostanol saponin 2 and d-glucose. Accordingly, the structure of 11 was assigned (25S)-26-[(β-d-glucopyranosyl)oxy]-14α-hydroxyfurosta-5,20(22)-dien-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The 12 (C56H90O27) appeared to be the corresponding Δ20(22)-pseudo-furostanol glycoside of 8 (C56H92O28), based on the characteristic proton signals of the H-17 doublet at δH 2.44 (J = 9.9 Hz) and Me-21 singlet at δH 1.62, as well as the olefinic carbon signals at δC 152.4 and 103.5. Enzymatic hydrolysis of 12 with β-d-glucosidase gave the corresponding spirostanol saponin 1 and D-glucose. Thus, the structure of 12 was characterized as (25S)-26-[(β-d-glucopyranosyl)oxy]-furosta-5,20(22)-dien-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.Compound 1H and 13C NMR spectral data for 13 (C56H90O28) suggest that it is a furostanol glycoside, having a structure similar to that of 8. However, significant differences were recognized in the signals from the ring E and the side chain moiety, where a tertiary hydroxy group [δC 76.7 (C)] and an oxygen-bearing trisubstituted olefinic group  were supposed to be located. The deshielded methyl singlet signal at δH 1.73 assignable to Me-21 and the olefinic proton at δH 4.53 exhibited long-range correlations with the quaternary carbon at δC 76.7 and the oxygen-bearing olefinic carbon at δC 163.0 in the HMBC spectrum of 13. The olefinic proton at δH 4.53 was shown to be coupled with the methylene protons at δH 2.52 and 2.15  attributable to H2-24 in the 1H-1H COSY spectrum. These correlations allowed the tertiary hydroxy group and trisubstituted olefinic group to be placed at C-20 and between C-22 and C-23, respectively. The other correlations supporting the partial structure are depicted in H 1.73 and H-23 at δH 4.53. Enzymatic hydrolysis of 13 with β-d-glucosidase yielded the spirostanol saponin 13a and D-glucose. The NOE correlations between the signals of Me-21 at δH 1.73 and Me-18 at δH 1.19/H-23ax at δH 1.79, between the signals of H-23eq at δH 2.37 and H-16 at δH 5.06, and between the signals of H-25 at δH 1.69 and H-23ax/Me-27 at δH 0.75 in the NOESY spectrum of 13a, in addition to the large J value of the H-26ax proton , were indicative of the 20S, 22S, and 25S configurations -20-hydroxyspirost-5-en-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside. An HMBC correlation from H-1′′′′′ of Glc (III) at δH 4.85  to C-26 of the aglycone at δC 75.3 in the HMBC spectrum of 13 confirmed that a β-d-glucopyranosyl group was attached to C-26. Therefore, the structure of 13 was elucidated to be -26-[(β-d-glucopyranosyl)oxy]-20-hydroxyfurosta-5,22-dien-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The urations . Thus, s1H and 13C NMR data for 14 (C56H90O29) were essentially analogous to those of 12, except for the lack of signals for the C-20(22)-tetrasubstituted olefinic group at δC 103.5 and 152.4. Instead, signals for a keto carbonyl carbon at δC 205.5 and an ester carbonyl carbon at δC 173.2 were newly observed in the 13C NMR spectrum of 14. All other signals appeared at almost the same positions between the two glycosides. In the HMBC spectrum of 14, the H-17 at δH 2.48  and Me-21 protons at δH 2.13 (s) showed long-range correlations with the keto carbonyl carbon at δC 205.5, which was assigned to C-20. Long-range correlations from the H-16 at δH 5.66 (m) and H2-23 protons at δH 1.84 (m) and 1.47 (m) to the ester carbonyl carbon at δC 173.2 resulted in the assignment of the ester carbonyl carbon to C-22. These data suggest that 14 was formed from 12 through the oxidative cleavage of the C-20(22) double bond. This was confirmed by the fact that the peracetate (14a) of 14 was identical to the product obtained by treating 12 with Ac2O in pyridine at room temperature for 12 h and then with CrO3 in AcOH. Accordingly, the structure of 14 was determined to be 16β-[[(4S)-5-(β-d-glucopyranosyloxy)-4-methyl-1-oxo-pentyl]oxy]-3β-[(O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl)oxy]-pregn-5-en-20-one.The 15 (C44H68O21) is a pregnane glycoside. Its 1H NMR spectrum showed signals for two angular methyl groups at δH 0.90  and 0.86 , a methyl group of an acetyl moiety at δH 2.22 , two olefinic protons at δH 6.57  and 5.29 , and four anomeric protons at δH 5.58 , 5.23 , 5.18 , and 4.88 . The existence of an α,β-unsaturated carbonyl group was verified by the IR (1660 cm−1), UV [239 nm (logε 3.81)], and 13C NMR  spectra. These spectral data and comparison with those of previously reported compounds identified the aglycone of 15 as 3β-hydroxypregna-5,16-dien-20-one -O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl)oxy]-pregna-5,16-dien-20-one.The data suggest that n-20-one . Acid hy1–15 were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, HSC-4, and HSC-2 human oral squamous cell carcinoma cells. Etoposide, cisplatin, and doxorubicin were used as positive controls. Compound 1 showed cytotoxic activity against the four tumor cell lines with IC50 values ranging from 0.96 ± 0.01 to 3.15 ± 0.43 μM. Compound 8, which is the corresponding furostanol glycoside of 1, was only cytotoxic to the adherent cell lines of A549, HSC-4, and HSC-2 cells, with IC50 values of 2.97 ± 0.06 μM, 11.04 ± 0.25 μM, and 8.25 ± 0.20 μM, respectively . Treatment of the cells with 1 led to necrotic cell death in the four cell lines, whereas the cells exposed to 8 morphologically displayed nuclear chromatin condensation and nuclear disassembly, implying that 8 induced apoptosis in the adherent cells , which stains the nuclear DNA in permeabilized cells D25 –50.4 . HR-ESI-TOF-MS m/z: 1085.4773 [M + Na]+ . IR νmax (film) cm−1: 3152 (OH), 2948 (CH), 1650 (C=O). UV λmax (MeOH) nm (log ε): 237 (3.86). For 1H and 13C NMR spectral data, see 1H and 13C NMR spectral data of the sugar moiety, see 13C-NMR spectral data of the aglycone moiety, see Compound 5: An amorphous solid. [α]D25 –64.6 . HR-ESI-TOF-MS m/z: 1249.5472 [M + Na]+ . IR νmax (film) cm−1: 3420 (OH), 2930 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 6: An amorphous solid. [α]D25 –38.2 . HR-ESI-TOF-MS m/z: 1249.5477 [M + Na]+ . IR νmax (film) cm−1: 3365 (OH), 2929 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 10: An amorphous solid. [α]D25 –49.4 . HR-ESI-TOF-MS m/z: 1251.5625 [M + Na]+ . IR νmax (film) cm−1: 3398 (OH), 2874 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 11: An amorphous solid. [α]D25 –29.1 . HR-ESI-TOF-MS m/z: 1233.5516 [M + Na]+ . IR νmax (film) cm−1: 3397 (OH), 2924 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 12: An amorphous solid. [α]D25 –33.3 . HR-ESI-TOF-MS m/z: 1195.5739 [M + H]+ . IR νmax (film) cm−1: 3397 (OH), 2935 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 13: An amorphous solid. [α]D25 –38.1 . HR-ESI-TOF-MS m/z: 1233.5531 [M + Na]+ . IR νmax (film) cm−1: 3366 (OH), 2931 and 2872 (CH). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 14: An amorphous solid. [α]D25 –37.9 . HR-ESI-TOF-MS m/z: 1249.5466 [M + Na]+ . IR νmax (film) cm−1: 3447, 3423, and 3400 (OH), 2923 and 2883 (CH), 1731 and 1707 (C=O). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 15: An amorphous solid. [α]D25 –32.8 . HR-ESI-TOF-MS m/z: 955.4127 [M + Na]+ . IR νmax (film) cm−1: 3481 (OH), 2921 and 2889 (CH), 1660 (C=O). UV λmax (MeOH) nm (log ε): 239 (3.81). For 1H and 13C NMR spectral data, see 13C-NMR spectral data of the aglycone moiety, see Compound 4 (3.0 mg) in 0.5 M HCl and dioxane-H2O  was heated at 95 °C for 2 h under an Ar atmosphere. The reaction mixture was neturalized by passing it through an Amberlite IRA-96SB column . The mixture was then eluted through a Diaion HP-20 column  with MeOH-H2O (4:6) and EtOH-Me2CO (1:1). The sugar fraction was analyzed using HPLC under the following conditions: Capcell Pak NH2 UG80 column ; mobile phase of MeCN-H2O (85:15); detection by refractive index and optical rotation; and a flow rate of 1.0 mL/min. d-Glucose, d-galacose, and d-xylose were identified by comparing their retention times (tR) and optical rotation with those of authentic samples: d-galactose , d-glucose , and d-xylose . Compounds 5 (5.0 mg), 6 (3.5 mg), 10a (3.5 mg), 13a (3.0 mg), and 15 (2.0 mg) were independently subjected to acid hydrolysis as described for 4. HPLC analysis of the sugar fractions under the same conditions as in the case of 4 indicated the presence of d-galacose, d-glucose, and d-xylose.A solution of 10 (10.0 mg) was treated with β-d-glucosidase  in HOAc/NaOAc buffer  at room temperature for 12 h. The reaction mixture was purified using CC on silica gel  eluted with CHCl3-MeOH-H2O (20:10:1) to obtain gitogenin  and a sugar fraction. HPLC analysis of the sugar fraction under the same conditions as those used for 4 indicated the presence of d-glucose . Compounds 11 (5.0 mg), 12 (5.0 mg), and 13 (7.0 mg) were independently subjected to enzymatic hydrolysis as described for 10 to obtain 2 , 1 , 13a , and sugar fractions. HPLC analysis of the sugar fractions under the same conditions as those used for 4 indicated the presence of d-glucose .Compound 11 (2.0 mg) and 14 (3.6 mg) were independently acetylated with Ac2O (1.0 mL) in pyridine (1.0 mL) at room temperature for 18 h. Each crude acetate was purified using preparative TLC with hexane-Me2CO (1:1) to yield 11a (1.9 mg) and 14a (2.8 mg).Compounds 9 (15.0 mg) and Ac2O (5.0 mL) in pyridine (3.0 mL) was stirred at 110 °C for 3 h. After the excess Ac2O was decomposed by H2O (10 mL), the reaction mixture was purified using preparative TLC with hexane-Me2CO (7:5) to yield 11a (4.5 mg).A mixture of 12 (5.0 mg) was treated with Ac2O (2.0 mL) in pyridine (4.0 mL) at room temperature for 12 h. After the excess Ac2O was decomposed by H2O (10 mL), the reaction mixture was evaporated to dryness. The crude product was dissolved in 95% AcOH (aq.) (5.0 mL), to which the CrO3 (15.0 mg) solution in 95% AcOH (aq) (1.0 mL) was added, and the mixture was stirred at room temperature for 3 h. After the excess CrO3 was decomposed by MeOH (3.0 mL), the crude product was diluted with H2O (20.0 mL) and extracted with Et2O (20.0 mL × 3). The Et2O extract was purified using ODS Si CC  eluted with MeCN-H2O (3:1) to obtain 14a (8.2 mg).Compound 10a: An amorphous solid. [α]D25 –21.7 . HR-ESI-TOF-MS m/z: 1071.4994 [M + Na]+ . IR νmax (film) cm−1: 3335 (OH), 2928 (CH). For 1H and 13C NMR spectral data, see Compound 11a: An amorphous solid. [α]D25 –14.3 . HR-ESI-TOF-MS m/z: 1925.7460 [M + H]+ . IR νmax (film) cm−1: 2924 and 2854 (CH), 1748 (C=O). 1H NMR : δH 5.65 , 3.97 , 3.70 , 3.45 , 2.38, 2.34, 2.32, 2.16 × 2, 2.14, 2.13 × 2, 2.07, 2.04 × 2, 2.03, 2.01, 1.97 × 2, 1.96 , 1.69 , 1.15 , 0.98 , 0.97 .Compound 13a: An amorphous solid. [α]D25 –22.5 . HR-ESI-TOF-MS m/z: 1071.4935 [M + Na]+ . IR νmax (film) cm−1: 3328 (OH), 2926 (CH). For 1H and 13C NMR spectral data, see Compound 14a: An amorphous solid. [α]D25 –21.2 . HR-ESI-TOF-MS m/z: 1921.7156 [M + Na]+ . IR νmax (film) cm−1: 2936 (CH), 1750 (C=O). 1H NMR : δH 3.72 , 3.40 , 2.39, 2.35, 2.33 , 2.17―2.13 (Ac × 5), 2.07 × 2, 2.05, 2.03, 2.02, 1.98, 1.97, 1.96 , 1.23 , 1.04 , 0.90 .Compound v/v) FBS supplemented with L-glutamine, penicillin G sodium salt (100 units/mL), and streptomycin sulfate (100 μg/mL). HL-60 (4 × 104 cells/mL), A549 (1 × 104 cells/mL), HSC-4 (2 × 104 cells/mL), HSC-2 (1 × 104 cells/mL), and TIG-3 (5 × 104 cells/mL) cells were continuously treated with each compound for 72 h, and cell growth was measured using an MTT reduction assay as previously described [50) was calculated from the dose response curve. The time courses for 0, 3, 6, 16, 24, 48, and 72 h of the antiproliferative effects of 1 and 8 at 1.0, 10, and 20 μM on HL-60, A549, HSC-4, and HSC-2 cells were also examined using the MTT assay.HL-60 cells were maintained in an RPMI-1640 medium, A549 and TIG-3 cells were maintained in MEM, and HSC-4 and HSC-2 cells were maintained in DMEM. The cell media contained heat-inactivated 10% (escribed . Data ar4 cells/well), A549 (1 × 104 cells/well), HSC-4 (1 × 104 cells/well) and HSC-2 (4 × 103 cells/well) were independently plated on coverslips in 96-well plates. After 24 h, the cells were treated with either 1 (20 μM) for 3 h, 8 (20 μM) for 24 h, etoposide (15 μM), or cisplatin (33 μM). The cells were fixed with 1% glutaraldehyde for 30 min at room temperature and before staining with DAPI (0.5 μg/mL). They were observed using a CKX41 fluorescence microscope . A549 cells (1 × 105 cells/well) were seeded in 96-well plates. After 24 h, the A549 cells were treated with either 1 (20 μM) or 8 (20 μM) and stained with caspase-3/7 assay reagent (1 μM)  or YOYO-1 . Cells images were obtained after 3, 6, and 30 h, respectively, using the IncuCyte® ZOOM  in 5% CO2/air at 37 °C.HL-60 (2 × 105 cells/ml) were treated with 8 (20 μM) for 30 h, in separate experiments, according to a previously reported method [Cell cycle distribution was evaluated using a flow cytometer . A549 cells  whole plants yielded 15 steroidal glycosides (1–15), including nine new compounds  with a lycotetrose unit. The structures of the new compounds were determined using two-dimensional NMR analyses and chemical methods. The isolated compounds were evaluated for their cytotoxicity against HL-60, A549, HSC-4, and HSC-2 cells. Of these, the spirostanol lycotetroside (1) exhibited cytotoxic activity against HL-60, A549, HSC-4, and HSC-2 cells with IC50 values ranging from 0.96 to 3.15 μM. Alternatively, the corresponding furostanol lycotetroside (8) was cytotoxic to the adherent cell lines of A549, HSC-4, and HSC-2 cells with IC50 values of 2.97, 11.04, and 8.25 μM, respectively. Compound 1 caused necrotic cell death in A549 cells in a dose-dependent manner. In contrast, the corresponding furostanol lycotetroside (8) induced apoptotic cell death in A549 cells in a time-dependent manner, as was evident by morphological observations and flow cytometry analyses. This study demonstrated that 8 has induced apoptosis against A549 cells through caspase-3/7 and had the potential for anti-lung cancer agent. Further investigation of the pathway of apoptosis induced by 8 should be addressed.Chromatographic separation of the MeOH extract of


Three isomeric methylated 2-(thiophen-2-yl)acetohydrazides show little consistency in the pattern of weak  interactions in their crystal structures. E)-N-methyl-N′-(nitrobenzylidene)-2-(thiophen-2-yl)acetohydrazides (formula C14H13N3O3S) are described, with the nitro group in ortho, meta and para positions in the benzene ring. In each crystal structure, molecules are linked by various weak interactions , leading to three-dimensional networks in each case, but with little similarity between them.The crystal structures of three isomeric ( R,R′NCOCH2)-thiophene -thiophene -N-methyl-N′-arylidene-2-(thiophen-2-yl)acetohydrazides. The anti-TB activities of these compounds will be reported elsewhere: here, we present the crystal structures of three isomeric derivatives in this family bearing a nitro group on the aromatic ring, viz. (E)-N-methyl-N′-(2-nitronitrobenzylidene)-2-(thiophen-2-yl)acetohydrazide, (I)E)-N-methyl-N′-(3-nitronitrobenzylidene)-2-(thiophen-2-yl)acetohydrazide, (II)E)-N-methyl-N′-(4-nitronitrobenzylidene)-2-(thiophen-2-yl)acetohydrazide, (III)Our ongoing interest in the biological activities and structural chemistry of heterocyclic compounds have led us to investigate compounds containing a thiophene ring system. We have reported the syntheses and anti-TB activities of acetamido derivatives, 2-°: this substantial twist can in part be ascribed to steric reasons. The central CH=N—N(CH3)—C(=O)—CH2 fragment in (I)The molecular structure of (I)meta-N3/O1/O2 nitro group is almost coplanar with its attached benzene ring [dihedral angle = 1.96 (2)°]. The almost planar central methylidene–acetohydrazide grouping in (II)The molecular structure of (II)A’, thiophene ring ‘B’ and CH=N—N(CH3)—C(=O)—CH2 fragment ‘C’ (r.m.s. deviation = 0.034 Å), are A/B = 79.36 (6), A/C = 12.75 (12) and B/C = 69.60 (6)°. Equivalent dihedral-angle data for the S2 molecule are 88.23 (6), 15.51 (13) and 82.51 (6)°, respectively. The para-nitro group is twisted from its attached ring by 9.2 (3) (S1 molecule) and 8.8 (3)° (S2 molecule). The dihedral angles are broadly similar but even so, the two molecules have different conformations ns Fig. 4 as indicCg1 (where Cg1 is the centroid of the thiophene ring) bonds; this second inversion dimer (about The packing in (I)s Table 1 generatens Fig. 6 in whichC(7) chains propagating in [010]. The two C8 (methyl-group) bonds lead to (101) sheets. Taken together, these interactions lead to a three-dimensional network of molecules in the crystal. There are no C—H⋯π or π–π stacking interactions in (II)The packing in (II)The packing for (III)et al., 20163)—C(=O)—CH2– fragment of the title compounds revealed just three matches, viz. FOTMUX -N-Methyl-N′-(2-nitrophenylmethylidene)-2-(thiophen-2-yl)acetohydrazide, (I)1H NMR : δ 8.21 , 8.12 , 8.04 , 7.83–7.80 , 7.69–7.67 , 7.37  6.96–6.94 , 4.34 , 3.32 . 13C NMR : δ 171.0 (C=O), 148.3 (C-7′), 136.8 (N=CH), 136.1 (C-2), 133.4 (C-10′), 130.4 (C-9′), 128.8 (C-11′), 128.3 (C-6′), 126.8 (C-3), 126.5 (C-4), 125.2 (C-5), 124.5 (C-8′), 33.9 (N-CH3), 28.1 (CH2). MS/ESI: [M + Na]: 326. IR νmax : 1681 (C=O); 3088 (N-CH3).(E)-N-Methyl-N′-(3-nitrophenylmethylidene)-2-(thiophen-2-yl)acetohydrazide, (II)s; N=CH), 8.29–8.25 , 8.17 , 7.79–7.75 , 7.37–7.35 , 7.00–6.99  6.96–6.94 , 4.40 , 3.35 . 13C NMR  δ: 170.9 (C=O), 148.2 (C-8′), 138.6 (N=CH), 136.9 (C-2), 136.5 (C-6′), 132.8 (C-11′), 130.4 (C-10′), 126.7 (C-9′), 126.6 (C-3), 125.2 (C-4), 123.9 (C-5), 121.6 (C-7′), 34.3 (N-CH3), 28.2 (CH2). MS/ESI: [M + Na]: 326. IR νmax : 1668 (C=O); 2962 (N—CH3).(E)-N-Methyl-N′-(4-nitrophenylmethylidene)-2-(thiophen-2-yl)acetohydrazide, (III)1H NMR  δ: 8.32 , 8.13 , 8.07 , 7.36 , 7.00-6.99 , 6.96-6.94 , 4.41 , 3.36 . 13C NMR  δ: 171.0 (C=O), 147.6 (C-9′), 140.9 (N=CH), 138.4 (C-6′), 136.8 (C-2), 128.0 (C-3), 126.8 (C-4), 126.5 (C-5), 125.2 (C-7′ and C-11′), 124.0 (C-C-8′ and C-10′), 34.2 (N-CH3), 28.3 (CH2). MS/ESI: [M + Na]: 326. IR νmax : 1678 (C=O); 3101 (N-CH3).(Uiso(H) = 1.2Ueq(carrier) or 1.5Ueq(methyl) was applied in all cases. The methyl group was allowed to rotate, but not to tip, to best fit the electron density (AFIX 137 instruction). In each case, this group rotated from its initial orientation to minimize steric interaction with atom H7; the final orientation leads to a short C8—H⋯O1 intramolecular contact but we do not regard this as a bond. The thiophene rings in (I)Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989016016856/hg5478sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989016016856/hg5478Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016016856/hg5478IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989016016856/hg5478IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989016016856/hg5478Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016016856/hg5478IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016016856/hg5478IIIsup7.cmlSupporting information file. DOI: 1510866, 1510865, 1510864CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S-myricanol (2), myricanone 5-O- -D-glucopyranoside (4), and (+)-S-myricanol 5-O- -D-glucopyranoside (5) were isolated from the EtOAc fraction of Alnus japonica Steud (family: Betulaceae) stem bark. Their structures were established by different spectroscopic analyses, as well as optical rotation measurement. Compounds 1, 2, 4, and 5 are isolated for the first time from A. japonica. The antioxidant and anti-inflammatory activities of compounds (1-5) were assessed using DPPH assay and carrageenin induced rat paw edema model, respectively. They displayed significant antioxidant activity in relation to propyl gallate (standard antioxidant) at concentration 50 µM. Compound 2 demonstrated anti-inflammatory effect at a dose 10 mg/kg compared with indomethacin (positive control).A new cyclic diarylheptanoid namely alnuheptanoid B (3), along with four known cyclic diarylheptanoids: myricanone (1), (+)- Acer (Aceraceae), Platycarya (Juglandaceae), Myrica (Myricaceae), Centrolobium (Leguminosae), Alpinia, Curcuma, and Zingiber (Zingiberaceae), and Alnus and Betula (Betulaceae)  is widely distributed in the low mountains  , 2. The ompounds . The latl ethers . In addil ethers . They shl ethers , 5-15. Iountains , 13. Pre tannins -22. Contptanoids  and 2. TGeneral 18 column . A linear gradient (H2O:MeOH 80:20 % to MeOH 100 % over 45 min) was applied. Chromatographic separation was achieved using RP18 (0.04-0.063 mm) and silica gel (0.04-0.063 mm) 60 . The TLC analysis was performed using the following systems: MeOH:CHCl3 , MeOH:CHCl3 , and n-BuOH:H2O:HOAc . Pre-coated silica gel 60 F254 TLC plates  was used for TLC. Propyl gallate (PG), 2,2-diphenyl-1-picrylhydrazyl (DPPH), carrageenin, and indomethacin were provided by Sigma-Aldrich Co. . Melting points were determined by Electrothermal Digital Melting Point 9100 instrument (England). Shimadzu 1601 UV/VIS and Shimadzu Infrared-400 (Japan) spectrophotometers were used to measure the UV and IR spectra, respectively. Optical rotation was measured by Perkin-Elmer Polarimeter 341 LC Model (USA). LRMS spectra were assessed by a MATTSQ7000 Finnigan spectrometer. A Micromass Qtof 2 spectrometer was used for HRESIMS spectra measurements. Bruker Avance DRX 400  was used to record NMR spectra. HPLC separations were performed on a HPLC system consisting of a UV L-7400 detector (280 nm) and a Lachrom-Merck L-7100 Hitachi pump using a CPlant material The plant sample was obtained from the Heinrich-Heine University`s botanical garden  in March 2005. The plant was taxonomically authenticated and identified by Peter Westhoff, Prof. of Plants Molecular Biology and Development . A specimen (Registration code AJB-2005) was kept at the Faculty of Pharmacy, Department of Pharmacognosy, Al-Azhar University, Egypt. Extraction and isolation 3:n-hexane (500 mL x 4) and EtOAc (500 mL x 4) to obtain 2.6 and 4.1 g, respectively. The VLC of the EtOAc (4.1 g) fraction using CHCl3:MeOH gradient elution afforded 6 sub-fractions (A-F). Sub-fractions B-E were previously investigated by authors (2 column (2 cm 50 cm 50 g) of sub-fraction A  with CHCl3:MeOH gradient, followed by semi-preparative HPLC yielded 1  and 2 . RP-18 column (2 cm 50 cm 60 g) of sub-fraction F  using MeOH:H2O gradient elution followed by HPLC gave 3 (13.7 mg), 4 (15.3 mg), and 5 (18.9 mg). The powdered stem bark (200 g) was extracted exhaustively with EtOH . The EtOH extract was concentrated to afford 12 g brown residue. The residue was chromatographed over vacuum liquid chromatography (VLC) using 50 % CHCl authors . SiO2 coMyricanone (1)max: 215, 258, 295 nm; NMRdata: see m/z 357 [M + H]+.White needles (22.1 mg); m.p. 191-192 °C; UV (MeOH) S-Myricanol (2)D +38.5 ; UV (MeOH) max: 221, 259, 295 nm; NMR data: see m/z 359 [M + H]+.White needles (14.8 mg); m.p. 103-104 °C; [α]Alnuheptanoids B (3)max (log  ): 219 (4.71), 251 (4.36), 293 (3.95) nm; IR (KBr)  max: 3465, 1725, 1715, 1598 cm-1; NMR data: see m/z 561 [M + H]+, 398.9 [(M + H)-Glu]+, 357.2 [(M + H)-(Glu+Acetyl)]+; HRESIMS: m/z 561.2339 .White amorphous powder (13.7 mg); UV (MeOH) Myricanone 5-O--D-glucopyranoside (4)max: 220, 251, 294 nm; NMR data: see m/z 519 [M + H]+, 357 [(M + H)-Glu]+.White amorphous powder (15.3 mg); UV (MeOH)  (+)-S-Myricanol 5-O--D-glucopyranoside (5)D +82.3 ; UV (MeOH) max: 231, 253, 294 nm; NMR data: see m/z 521 [M + H]+, 359 [(M + H)-Glu]+. White amorphous powder (18.9 mg); [α]Antioxidant activityThe antioxidant effect of compounds 1-5 was evaluated by 2,2`-diphenylpicrylhydrazyl (DPPH) assay as previously outlined , 24. Carrageenin-induced rat paw edema methodThe anti-inflammatory activity was evaluated on adult male albino rats (120-150 g b. wt.) using the same procedures as previously described , 25 ,26.Statistical analysist test. ANOVA  was used for evaluation of statistical significance. The values were considered to be significantly different when P < 0.01.All data were expressed as mean ± standard error of mean using the student 29H36O11 was established from the HRESIMS quasi-molecular ion peak at m/z 561.2339 [M + H]+. The IR, UV, and NMR spectral data of 3 were in agreement with those of 4 except for the appearance of new signals at H 2.08 /C 20.8 (COCH3) and 171.7 (COCH3) characteristic for an acetyl group in 3. Its attachment at C-17 was confirmed by the 3J HMBC cross peak of H-16 to the carbonyl group of acetyl moiety at C 171.7 and further secured by the ESIMS ion peak at 357.2 [(M + H)-(Glu+Acetyl)]+. In addition, 3 was 42 mass units and one degree of unsaturation more than 4, confirming the presence of the acetyl moiety. The UV absorption maxima at 219, 251, and 293 nm indicated a diarylheptanoid structure of 3 (-1), ester carbonyl (1725 cm-1), ketone carbonyl (1715 cm-1), and benzene (1598 cm-1) functionalities (1H NMR spectrum showed two singlet methoxy groups at H 3.95 (4-OCH3) and 3.79 (3-OCH3) (C 61.9 (4-OCH3) and 61.7 (3-OCH3), respectively in HMQC spectrum. Their connectivity at C-4 and C-3 was proven by the HMBC cross peaks of 4-OCH3 to C-4 (C 145.0) and 3-OCH3 to C-3 (C 146.9). Four aromatic proton signals at H 7.06 , 6.87 , 6.69 , and 6.67 , which correlated with carbons at C 129.6 (C-15), 122.8 (C-16), 132.4 (C-18), and 129.1 (C-19) in HMQC spectrum, indicating the presence of 1,2,4-tri-substituted and 1,2,3,4,5-penta-substituted benzene moieties in 3 . They were established by the COSY cross peaks of H-15 to H-16 and H-18 and further secured by the HMBC correlations of H-15 to C-17 and C-18, H-16 to C-1 and C-14, H-18 to C-14 and C-17, and H-19 to C-3 and C-5  showed cross peak to the signal at C 105.0 (C-1`), indicating the presence of β-glucopyranose moiety (m/z 398.9 [(M + H)-Glu]+ and 357.2 [(M + H)-(Glu+Acetyl)]+. In the HMBC, the cross peak of H-1` to C-5 (C 148.7) established the placement of glucose at C-5. Furthermore, signals for six methylene groups at H 1.80-3.07 and ketone carbonyl at C 213.6 (C-11), characteristic for heptanoid moiety in 3 were observed. In the COSY spectrum, the spin system started from H-7 to H-10 and cross peak of H-12 to H-13 established this moiety. It was secured by the observed HMBC cross peaks of H-9/C-7 and C-11, H-10/C-8, H-12/C-13, and H-13/C-11. The attachment of heptanoid moiety at C6-C14 of the biphenyl moiety was secured by the HMBC cross peaks of H-8/C-6, H-19/C-7, and H-18/C-13. Consequently, 3 was concluded to be 17-O-acetyl myricanone 5-O-β-glucopyranoside and named alnuheptanoid B.Compound 3 was isolated as white amorphous powder. A molecular formula Cure of 3 . Its IR nalities . The 1H (3-OCH3) . They co and C-5 . The con8 to C-2 . Moreovee moiety . This waS-myricanol (O- -D-glucopyranoside (S-myricanol 5-O- -D-glucopyranoside (Compounds 1-5 were identified to be myricanone (1) , 30, (+)yricanol  30,31),,S-myricaranoside  32), an, anS-myrranoside  by the iµM. The results showed that, 1 and 2 had significant antioxidant activity. While, 3-5 showed moderate activity in comparison with propyl gallate (a known antioxidant) (The antioxidant activity of the isolated cyclic diarylheptanoids -5 was deoxidant) . Their aoxidant) .Compounds 1-5 were evaluated for their anti-inflammatory effects using carrageenin induced paw edema model. Compound 2 showed the highest activity comparable to indomethacin (10 mg/kg) . Also, 1The observed activity of these compounds might be through the inhibition of the inflammatory prostanoids , 37. In A. japonica for the first time. Their chemical structures were established by different spectroscopic analyses. Compounds 1 and 2 showed significant antioxidant activity. Compound 2 exhibited potent anti-inflammatory activity.A new cyclic diarylheptanoid and four known compounds were isolated from


The compound that was used in our article was N′-(3-bromo-6-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and not N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide. All experiments described in the paper were done using BHBM [N′-(3-bromo-6-hydroxybenzylidene)-2-methylbenzohydrazide], and the structure noted in Volume 6, no. 3, e00647-15, 2015, mBio studies was confirmed by proton nuclear magnetic resonance (NMR), which indicated the following chemical shifts: 1H NMR  δ 2.38 , 6.90 , 7.28 to 7.32 , 7.37 to 7.43 , 7.47 , 7.78 , 8.47 , 11.19 , 12.05 .The structure of BHBM used in the All findings illustrated in the paper are correct.


N-Mannich bases are reported herein. In the crystals of both compounds, there are C—H⋯F hydrogen bonds present, forming inversion dimers in one and chains in the other.The syntheses and crystal structures of two novel adamantane-substituted  N-Mannich bases, 3-(adamantan-1-yl)-4-(4-fluorophenyl)-1-[(4-phenylpiperazin-1-yl)methyl]-4,5-dihydro-1H-1,2,4-triazole-5-thione (C29H34FN5S) (I), and 3-(adamantan-1-yl)-4-(4-fluorophenyl)-1-{[4-(2-methoxyphenyl)piperazin-1-yl]-methyl}-4,5-dihydro-1H-1,2,4-triazole-5-thione (C30H36FN5OS) (II), fluorophenyl, adamantane and piperazine moieties are linked to a planar triazole ring. There is an additional phenyl ring on the piperazine ring in (I) and a methoxyphenyl ring in (II). In compound (I), the fluorophenyl and phenyl rings are inclined to the triazole ring by 86.55 (13) and 60.52 (12)°, respectively, and the two aryl rings are inclined to one another by 66.37 (13)°. In compound (II), the corresponding dihedral angles are 83.35 (13), 71.38 (15) and 11.97 (16)°, respectively. The crystal structure of (I) shows pairs of C—H⋯F hydrogen bonds forming inversion dimers, while in the crystal of compound (II), in addition to the C—H⋯F hydrogen bonds that generate chains parallel to the b axis, there are C—H⋯π interactions present that link the chains to form layers parallel to the ab plane.In the title  The chains are linked by C—H⋯π interactions, forming layers parallel to the ab plane e Table 2.et al., 2016H)-thione moiety gave 14 hits. One compound, 3-(adamantan-1-yl)-4-phenyl-1-[(4-phenylpiperazin-1-yl)methyl]-1H-1,2,4-triazole-5(4H)-thione -1-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-4-methyl-1H-1,2,4-triazole-5(4H)-thione -thione -4-(4-fluoromethyl)-4H-1,2,4-triazole-5-thiol Compound (I): 1-Phenylpiperazine  and a 37% formaldehyde solution (1 ml) were added to a solution of 3-(adamantan-1-yl)-4-(4-fluoromethyl)-4H-1,2,4-triazole-5-thiol  in ethanol (10 ml), and the mixture was heated under reflux for 1 h then allowed to stand overnight. Cold water (3 ml) was slowly added and the mixture was stirred for 20 min. The precipitated crude product was filtered, washed with water, dried, and crystallized from ethanol to yield 846 mg (84%) of compound (I)1H NMR (700.17 MHz): δ 1.47–1.49 , 1.60–1.62 , 1.80 , 1.89 , 2.89–2.91 , 3.14–3.15 , 5.14 , 6.77–6.79 , 6.94 , 7.20–7.22 , 7.41–7.49 . 13C NMR (125.76 MHz): δ 27.61, 36.07, 39.62, 39.74 (adamantane-C), 48.73, 50.30 (piperazine-C), 69.06 (CH2), 116.0, 116.78, 119.41, 129.37, 132.80, 133.10, 151.48, 156.28 (Ar-C), 162.17 (triazole C-3), 170.95 (C=S).Compound (II): 1-(2-Methoxyphenyl)piperazine  and a 37% formaldehyde solution (1 ml) were added to a solution of 3-(adamantan-1-yl)-4-(4-fluoromethyl)-4H-1,2,4-triazole-5-thiol  in ethanol (10 ml), and the mixture was heated under reflux for 1 h then allowed to stand overnight. The precipitated crude product was filtered, washed with cold ethanol, dried, and crystallized from ethanol to yield 865 mg (81%) of compound (II)1H NMR (700.17 MHz): δ 1.49–1.50 , 1.61–1.63 , 1.83 , 1.90 , 2.89–2.90 , 2.96–2.98 , 3.78 , 5.11 , 6.88–6.96 , 7.42–7.52 . 13C NMR (125.76 MHz): δ 27.61, 35.77, 36.07, 39.61 (adamantane-C), 50.54, 50.58 (piperazine-C), 55.65 (OCH3), 69.39 (CH2), 112.08, 116.78, 118.51, 121.23, 123.02, 132.79, 133.13, 141.53, 152.36, 156.32 (Ar-C), 162.17 (triazole C-3), 171.0 (C=S).3:EtOH solutions  at room temperature.Suitable single crystals of compounds (I)Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017009756/su5368sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017009756/su5368Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017009756/su5368IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017009756/su5368Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017009756/su5368IIsup5.cmlSupporting information file. DOI: 1559732, 1559731CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


HPQJ-34. The structures of 1–4 were characterized using comprehensive spectroscopic analyses. The absolute configuration of 1 was determined by comparison of electronic circular dichroism (ECD) spectra with literature values used for the reported analogue, cyclopenicillone (5), which was not isolated in this research. Compound 1 was shown to scavenge 2,2-diphenyl-1-picrylhydrazyl free radicals, and decrease β-amyloid (Aβ) fibrillization in vitro. Moreover, 1 significantly reduced H2O2-induced neurotoxicity in SH-SY5Y cells. These findings suggested that compound 1, a newly discovered cyclopentenone, has moderate anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects, and might be a good free radical scavenger.A new cyclopentenone, 5-hydroxycyclopeni cillone ( Trichoderma sp. HPQJ-34, isolated from the marine sponge Hymeniacidon perleve, was found to have potent anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects during preliminary testing. Bioactivity-guided chromatographic separation of the EtOAc extract obtained from the fermentation broth of this strain resulted in the isolation of a new cyclopentenone, 5-hydroxycyclopenicillone (1), along with three known compounds: ar-turmerone (2) + . The 1H NMR spectrum of 1 in CD3OD showed two methyl doublets at δH 1.65  and 1.75 , one methyl singlet at δH 1.23 , two olefinic methines at δH 5.49  and 5.48 , two oxygenated methines at δH 4.49  and 4.72 , as well as signals with complex coupling patterns attributed to two methylenes between δH 1.76 and 2.12  spectra, indicated that the structure of 1 possesses one ring -2,5-dimethylcyclopent-2-en-1-one}.The use of ECD has been well established as a powerful tool for assigning the absolute configuration of natural products ,21,22. Id 209 nm  19]. In. In1, th1, three known metabolites were isolated during the course of this research. These were identified, by comparing their spectrometric and spectroscopic data with literature values, as ar-turmerone (2) c 0.1, MeOH); CD (MeOH) λmax (Δε) 321 (−4.1), 246 (+21.5), 214 (−8.7) nm; IR (film) νmax: 3369, 2926, 1710, 1634, 1379, 1055 cm–1; UV (MeOH) λmax (log ε) 320 (0.7), 238 (3.8) nm; HRESIMS: m/z 263.1247 [M + Na]+ ; The purity of compound 1 is 98%. 1H-NMR  δH 5.49 , 5.48 , 4.72 , 4.49 , 2.12 , , 1.75 , 1.64 , 1.23 ; 13C-NMR  δC 210.4 (C-1), 171.7 (C-3), 135.8 (C-2), 131.6 (C-4ʹ), 126.7 (C-5ʹ), 75.3 (C-4), 74.5 (C-5), 69.4 (C-1ʹ), 36.6 (C-2ʹ), 29.8 (C-3ʹ), 23.1 (C-7), 18.1 (C-6ʹ), 8.4 (C-6).5-hydroxycyclopenicillone (Ar-turmerone (2): White powder; UV (MeOH) λmax (log ε) 290 (3.84), 262 (4.03) nm; IR (film) νmax: 3363, 2925, 2854, 1726, 1660, 1615, 1381, 1287, 1121, 1074, 976, 543 cm−1; HRESIMS: m/z 239.1126 [M + Na]+ ; The purity of compound 2 is 97%. 1H-NMR  δH 7.07 , 7.07 , 7.07 , 7.07 , 6.12 , 3.21 , 2.66 , 2.27 , 2.04 , 1.86 , 1.21 ; 13C-NMR  δC 202.6 (C-9), 157.0 (C-11), 144.6 (C-1), 136.7 (C-4), 130.0 (C-3), 130.0 (C-5), 127.7 (C-2), 127.7 (C-6), 125.2 (C-10), 53.5 (C-8), 37.1 (C-7), 27.6 (C-13), 22.6 (C-14), 21.0 (C-15), 20.8 (C-12).3): Yellow oil; UV (MeOH) λmax (log ε) 323 (3.47), 293 (3.36) nm; IR (film) νmax: 3327, 1681, 1625, 1460, 1361, 1238, 1168, 1069, 968, 853, 695, 422, 408 cm–1; HRESIMS: 301.0539 [M + Na]+ ; The purity of compound 3 is 96%. 1H-NMR  δH 6.34 , 6.26 , 6.26 , 4.40 , 2.64 , 2.55 , 2.14 ; 13C-NMR  δC 209.8 (C-4ʹ), 167.8 (C-1), 167.3 (C-6), 164.8 (C-8), 155.6 (C-3), 141.1 (C-4a), 107.4 (C-4), 103.8 (C-8a), 102.7 (C-5), 99.9 (C-7), 66.4 (C-2ʹ), 51.1 (C-3ʹ), 41.9 (C-1ʹ), 30.7 (C-5ʹ).Citreoisocoumarin (O-methyl-citreoisocoumarin (4): Yellow oil; UV (MeOH) λmax (log ε) 330 (2.88), 290 (2.88) nm; IR (film) νmax: 3391, 2924, 1684, 1622, 1571, 1509, 1438, 1380, 1241, 1195, 1164, 1074, 1034, 978, 850, 799, 693, 575, 443, 433, 423 cm–1; HRESIMS: 315.0721 [M + Na]+ ; The purity of compound 4 is 96%. 1H-NMR  δH 6.50 , 6.50 , 6.47 , 4.46 , 3.88 , 2.70 , 2.62 , 2.19 ; 13C-NMR  δC 209.9 (C-4ʹ), 168.6 (C-6), 166.6 (C-1), 164.1 (C-8), 155.9 (C-3), 141.1 (C-4a), 107.4 (C-4), 102.2 (C-5), 101.6 (C-7), 100.9 (C-8a), 66.4 (C-2ʹ), 56.4 (C-6-O-Me), 51.1 (C-3ʹ), 41.9 (C-1ʹ), 30.7 (C-5ʹ).6-1) and cyclopenicillone (5) were generated using Spartan ′10 software . An equilibrium geometry calculation was performed for each molecule, at the ground state, using the Hartree–Fock method and the 6-31G* basis set, in a simulated vacuum. All computational operations were completed using an HP Elitebook 850 G1 laptop running 64-bit Windows 7 OS, containing 8 GB ram, and with an Intel i7-4600U CPU @ 2.1 GHz.The energy minimized structures of 5-hydroxycyclopenicillone (1) was determined by DPPH free radical scavenging assay, as previously reported  × 100.The anti-oxidant property of 5-hydroxycyclopenicillone  assay as previously reported . Briefly2 in a humidified environment. The medium was refreshed every two days. For the H2O2–induced neurotoxicity testing, SH-SY5Y cells in DMEM with low serum content (1% FBS) were seeded in 6-well or 96-well plates at a density of 1 × 105 cells/mL for 24 h before further experiments.SH-SY5Y cells were obtained from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences) and maintained in high glucose modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 μg/mL)/streptomycin (100 μg/mL) at 37 °C with 5% COCell viability was assessed by the 3--2.5-diphenyltetrazolium bromide (MTT) assay based on previous protocol ,32. HereViable cells were visualized by the fluorescein formed from FDA by esterase activity in viable cells. Non-viable cells were analyzed by PI staining, which only penetrates the membranes of dead cells. Then, the cells were examined after incubation with 10 μg/mL of FDA and 5 μg/mL of PI for 15 min. Images were acquired using UV light microscopy and these were compared with those taken under phase-contrast microscopy.p < 0.05 was considered as statistically significant.Results were expressed as mean ± SEM. Differences among groups were compared by analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s test. 1) was isolated together with three known compounds (2–4) from the sponge-derived fungus, Trichoderma sp. HPQJ-34. These molecules were obtained at purified yields of about 0.01%, 0.03%, 0.04%, and 0.06%, respectively, from the dry EtOAc extract. Compound 1 is a new structural analogue of cyclopenicillone (5), which has only been reported once previously in the literature, thus this investigation expands the research available on a rare group of natural products. Compound 1 was shown to exhibit moderate in vitro anti-oxidative, free radical scavenging and anti-Aβ fibrillization activities, along with neuroprotective effects. Accordingly, 5-hydroxycyclopenicillone and structurally related molecules may be of interest to neuropharmacology research and anti-AD drug discovery programs. However, further studies are necessary to validate these results in vivo and to examine the penetration of these molecules into brain tissue.A new cyclopentenone, 5-hydroxycyclopenicillone (