The new compound was identified as (20S)-protopanaxadiol-3-O--β-D-glucopyranoside-20-O-β-D-xylopyranosyl(1→3)-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside on the basis of extensive 1D and 2D NMR as well as HRESI-MS spectroscopic data analysis.A new saponin, malonylginsenoside Ra Panax ginseng C.A. Meyer has been used in China for thousands of years as a traditional medicine and proved to exhibit wide pharmacological properties, such as anti-fatigue, anti-diabetes, as well as activity in the prevention of cancer and the ageing process -, 1281 [M-CO2]-, 1239 [M-COCH2COOH]-, 1107 [M-COCH2COOH-xyl]-, 945 [M-COCH2COOH-glc-xyl]-, 783 [M-COCH2COOH-xyl-2glc-H]-, 621 [M-COCH2COOH-xyl-3glc-H]-, 459 [M-COCH2COOH-xyl-4glc-H]-, in the negative ESI-MS, indicating its molecular weight to be 1326. The molecular formula was determined as C62H102O30 based on HRESI-MS [M+Na]+: m/z 1349.6348 [M+Na]+ . IR (KBr) νmax/cm-1: 3423 cm-1 (OH), 1732 cm-1 (C=O), 1608 (C=C) and 1386 cm-1 (-CH3). Since compound 1 can’t be dissolved in pyridine-d5, we added 0.1 mL of D2O in 0.5 mL of pyridine-d5 as NMR solvent. Ginsenoside m-Rb1 (compound 2) and the alkaline hydrolysis product of 1, ginsenoside Ra3 (compound 1a) were also dissolved in the same mixture solvent for NMR measurement. Analysis of the 13C-NMR spectrum . The malonyl group was assigned to C3-glc-C-6’’ position by HMBC experiment .The periment , which t3-glc-C-6’’ (δC 66.1) than seen in ginsenoside-Ra3. Alkaline hydrolysis of compound 1 yield compound 1a, which showed the structure identical to ginsenoside Ra3 by 1D NMR analysis -protopanaxadiol 3-O--β-D-glucopyranoside)-20-O-β-D-xylopyranosyl(1→3)-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside, which we have named malonylginsenoside Ra3.Malonyl group connection also caused a 2.7 ppm lower-field shift for Canalysis . The abs1H- and 13C-NMR spectra were measured on a Bruker Avance DRX 500 NMR spectrometer, using TMS as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm), with the coupling constants (J) reported in Hertz (Hz). The ESI-MS spectra were recorded on a triple quadrupole mass spectrometer Quattro  and the HRESI-MS spectra on a Bruker FT-ICRMS spectrometer. Column chromatographies were carried out with silica gel 60 M (200–300 mesh), Lichrospher RP-18 (20 μm); TLC was performed with silica gel plates , with spots detected by UV254 and H2SO4 (10%). HPLC were carried out with a Agilent 1100 system.The Panax ginseng was collected from Fu-Song, Jilin, China, in August 2003, and identified by one of the authors, Prof. Yi-Nan Zheng. A voucher specimen (ZYC-RS-03-08) has been deposited in College of Chinese Medicinal Material, Jilin Agricultural University. The fresh root of Panax ginseng (10 kg) was extracted five times with MeOH-H2O (4:1), and the extract was concentrated under reduced pressure at 40 °C. The residue (~2 kg) obtained was suspended in water and subjected to D-101 resin column chromatography, using MeOH-H2O  as eluted solvent to give total-ginsenoside (~300 g). The total-ginsenoside was applied to silica gel column and eluted with CHCl3-MeOH-H2O (6:4:1) to yield three fractions (F1-F3). Fraction F1 was further chromatographed on preparative HPLC eluted with gradient CH3CN-H2O (20% to 50%) to give the known saponins: malonylginsenoside-Rb1 , malonylginsenoside-Rb2 , malonylginsenoside-Rc , malonylginsenoside-Rd  and the new saponin malonylginsenoside Ra3 . Compound 1: 1H-NMR : δ 0.73 , 0.87 , 0.95 , 0.98 , 1.17 , 1.63 , 1.65 , 1.69 , 5.29 , 5.11 , 4.93  4.85 , 4.81 , 5.19 ; 13C-NMR data, see The root of 1 (20 mg) in MeOH (3 mL) was treated with 5% KOH-MeOH (0.1 mL) and the whole mixture was stirred at room temperature (22 °C) for 30 min , IR(KBr), MS and 13C NMR spectral comparisons. Compound 1a: IR (KBr) νmax / cm-1: 3432, 1728, 1605, 1385, 1078; ESI-MS [-]: m/z = 1239 [M-H]-, 1107 [M-xyl]-, 945 [M-glc-xyl]-, 783 [M-xyl-2glc-H]-, 621 [M-xyl-3glc-H]-, 459 [M-xyl-4glc-H]-; 1H-NMR : δ 0.70 , 0.84 , 0.86 , 0.96 , 1.17 , 1.49 , 1.55 , 1.58 , 5.20 , 5.04 , 4.4.96  4.83 , 4.80 , 5.26 ; 13C-NMR data, see A solution of r 30 min . The reac sample  by TLC c1 (2.0 mg) was refluxed with 6 N HCl (5 mL) at 100 °C for 2 h [3 to afford the aglycone, and the aqueous layer was neutralized with Na2CO3 and filtered. The aqueous layer was dried under vacuum and the residue was re-dissolved in H2O for sugar analysis by TLC with n-BuOH-AcOH-H2O (4:1:2) as the solvent. The sample spots were detected by spraying aniline hydrogen phthalate reagent  and heating at 120 °C. D-Glucose and D-xylose were used as authentic standards. The absolute configuration of glucose was further determined by chiral GC analysis using a SatoChrom GC and a 0.25 mm × 25 m Hydrodexb-6-TBDM chiral capillary column . β-D-Glucose and β-D-xylose were used as an authentic GC standard. The aqueous layer residues mentioned above were re-suspended in dichloromethane (1 mL), and trifluoroacetic anhydride (50 µL) was added. The mixtures were allowed to react at room temperature overnight and dried under a stream of nitrogen at room temperature. The sugar derivatives were separated using the following temperature program: inlet temperature was set at 240 °C, with hydrogen carrier gas and a 1/20 split, using nitrogen makeup gas. Column temperatures started at 120 °C, ramped to 220 °C at 50 °C·min–1 and were maintained for 12 min.To determine the stereochemistry of sugar moiety, compound  for 2 h ,15. The Panax ginseng led to the isolation of a new saponin (20S)-protopanaxadiol 3-O--β-D-glucopyranoside)-20-O-β-D-xylopyranosyl(1→3)-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (1) along with four known ginsenosides (2–5).A phytochemical investigation on the fresh root of


The new azo compounds I(a-c) and II(a-c) were prepared by the reaction of I and II with three different aromatic amines, namely aniline, p-aminophenol and p-toluidine. The structures of these compounds were confirmed by CHN, FT-IR, 1H-NMR, 13C-NMR, mass spectrum and UV/Vis spectroscopy.Maleic anhydride was reacted with  N,N-dimethylaminopyridine, ammonium chloride, hydroxylamine hydrochloride and sodium acetate to minimize the temperature and time of the reaction have been published [p-aminophenol and p-toluidine, respectively, in the presence of diphosphorus pentoxide (P2O5) as a catalyst, which decreased the temperature needed for ring closure from 150–300 °C to 20–70 °C.Small molecules and macromolecules containing imide groups exhibit great electrical properties, good solubility in polar media, resistance to hydrolysis and high thermal stability ,5,6,7,8.ublished ,16,17,18I, II, I(a-c) and II(a-c) is shown in 1H-NMR, 13C-NMR, mass spectrum and UV/Vis spectroscopy. The preparation of compounds I and II showed the presence of C=O absorbances at 1,702 cm−1, alkene group (HC=CH) ones at 3,119 cm−1 and the presence of aromatic rings indicated by bands at 1,589, 1,600 and 1,512 cm−1. In addition a hydroxyl group at 3,481 cm−1 and a methyl group at 1,316 cm−1 were seen for compounds I and II, respectively.The FT-IR spectra of compounds I and II were produced. The FT-IR data for compounds I(a-c) and II(a-c) showed the same characteristic bands of the coupling agents I and II, namely imide, methyl, hydroxyl group, alkene, and p-substituted band while the presence of the azo (N=N) group band in 1,630–1,575 cm−1 range confirmed the success of the synthesis. Besides, the o-substituted benzene ring absorbtion at 750–775 cm−1 proved that the azo group was attached to the ortho position of the benzene rings. The FT-IR spectrum of compound Ia as a typical example is shown in All these peaks clearly proved that compounds 1H-NMR and 13C-NMR for azo compounds I and Ia have been chosen as typical examples and the corresponding spectra are shown in 1H-NMR spectrum, the protons of the alkene group (HC=CH) and the protons of aromatic ring appeared at 6.62–6.52 ppm and 6.75–7.39 ppm, respectively. The new peak appeared at 6.93 ppm was assigned to the ortho position and that proved the reaction between compound I and aniline has occurred. The broad peak at 9.45–9.75 ppm was assigned to the free O-H proton. The 13C-NMR spectrum, the following signals are the characteristic of the structure; 167.43 ppm (C=O), 155.44 ppm , 134.87 ppm  and 122.56, 115.42 ppm . In the Ia has been successfully prepared. The mass spectrum (70 eV) of Ia (The addition of other aromatic carbons proved that the pure azo compound V) of Ia  shows thI(a-c) would exhibited higher λmax than compounds II(a-c) due to the presence of an auxochrome group such as hydroxyl group in the compounds [−1 in the 13C-NMR spectrum (I(a-c) and II(a-c) are given in the Experimental section.In the UV/Visible spectra, the azo group (N=N) usually gives an absorption in the 350–370 nm range . We expeompounds . Howeverompounds . The appspectrum  suggestsp-aminophenol , aniline , p-toluidine , sulfuric acid 98% , hydrochloric acid 37% , sodium hydroxide , N,N-dimethylformamide , diphosphorus pentoxide , sodium nitrite , 2-propanol  and glacial acetic acid . All the chemicals were used as received without further purification except for aniline, was distilled before use.Maleic anhydride , 1H-NMR and 13C-NMR spectra were obtained using a Bruker 400 MHz NMR spectrophotometer with tetramethylsilane (TMS) as the internal reference. CHN microanalyses were performed using Perkin Elmer 2400 Series II Combustion Analyzer. The MS were recorded on a Perkin Elmer Clarus 500 Gas Chromatography-Mass Spectrometry system (GC-MS). UV/Vis Spectroscopy were determined using a Shimadzu UV-1601 PC instrument. The entire sample was weighed and dissolved in methanol.FT-IR spectra were measured at room temperature using a Perkin-Elmer 2000 FT-IR equipped with a high-purity dried potassium bromide (KBr) beam splitter. The P-aminophenol  and maleic anhydride  were dissolved separately in DMF (50 mL) to yield solutions A and B, respectively. Solution B was added dropwise into solution A to give solution C. Solution C was stirred for 2 hours at 20 °C in a water bath. P2O5 (12 g) was dissolved in H2SO4 (10 mL) and DMF (70 mL). This mixture was added dropwise into solution C and was stirred for 2 hours at 70 °C. The mixture was kept chilled in the ice bath and poured into cold water. A precipitate formed that was filtered, washed with distilled water and finally recrystallized from 2-propanol and dried in a vacuum oven at 65 °C for 24 hours. Yield was 84%; m.p. 182–184 °C; color: yellow; FT-IR (KBr disc): 3,481 cm−1 (O-H), 3,108 cm−1 (HC=HC), 1,705 cm−1 (C=O), 1,601 cm−1 (aromatic ring) and 828 cm−1 ; 1H-NMR (CD3OD): 6.78–7.48 (aromatic), 6.90–7.15 , 6.31–6.58 (HC=CH) ppm; 13C-NMR (CD3OD): 170.79 (C=O), 157.44, 134.37 , 128.23, 112.00  ppm. II was prepared by following the procedure of the preparation of I except that p-toluidine was substituted for p-aminophenol. Yield was 50% with a melting point of 148–150 °C; color: yellow; FT-IR (KBr disc): 3,088 cm−1 (HC=CH), 1,708 cm−1 (C=O), 1,632 cm−1 (aromatic ring), 1,316 cm−1 (CH3) and 823 cm−1 (p-substituted Ar); 1H-NMR (CD3OD): 7.17–7.57 (aromatic), 6.98–7.21 , 6.32–6.55 (HC=CH), 2.35 (CH3) ppm; 13C-NMR (CD3OD): 164.99 (C=O), 134.48 , 128.11, 126.36, 125.81, 120.71 , 19.96 (CH3) ppm.Compounda, 0.93 g, 0.01 mol) with concentrated HCl (3 mL) and water (3 mL) and cooling at 5 °C in an ice bath. NaNO2  was dissolved in water (10 mL) at 5 °C to obtain solution B. Then solution A was added dropwise to solution B at 5 °C with stirring. The mixture was then slowly added into the solution of compound I , which was dissolved in 10% NaOH (20 mL) at 5 °C. The mixture was keep chilled in the ice bath and stirred continuously for 10 min. The precipitate formed was filtered and recrystallized from glacial acetic acid, and washed with methanol and finally dried in a vacuum oven at 65 °C for 24 hours. The procedure was repeated by substituted I with II, where was substituted by p-aminophenol (b) and p-toluidine (c).Solution A was prepared by mixing pure aniline (Phenylazo-3-N-(4-hydroxyphenyl)maleimide (Ia). Color: pale yellow; yield: 85%; melting point: 199–200 °C; FT-IR (KBr disc): 3,290 cm−1 (O-H), 3,116 cm−1 (HC=CH), 1,703 cm−1 (C=O), 1,618 cm−1 (aromatic ring), 1,547 cm−1 (N=N), 836 cm−1 and 770 cm−1; 1H-NMR (CD3OD): 7.20–7.43 (aromatic), 6.93–7.25 , 6.26–6.52 (HC=CH) ppm; 13C-NMR (CD3OD): 167.43 (C=O), 165.01 (C=O), 155.44, 134.87 , 122.56, 115.42  ppm; elemental analysis: found: C, 62.76; H, 7.65; N, 13.86, (C16H23N3O3), calc.: C, 62.92; H, 7.60; N, 13.77; UV/Vis λmax (nm): 327.00 (N=N); 302.50 (C=O); 230.00 (Ar-CN); 208.50 (Ar-OH).4-Hydroxyphenylazo-3-N-(4-hydroxyphenyl)maleimide (Ib). Color: yellow; yield: 81%; melting point: 210–212 °C; FT-IR (KBr disc): 3,302 cm−1 (O-H), 3,166 cm−1 (HC=CH), 1,701 cm−1 (C=O), 1,618 cm−1 (aromatic ring) and 1,580 cm−1 (N=N), 835 cm−1 and 716 cm−1; 1H-NMR (CD3OD): 7.17–7.57 (aromatic), 6.96–7.21 , 6.30–6.51 (HC=CH) ppm; 13C-NMR (CD3OD): 170.95 (C=O), 155.71, 155.36, 134.77 , 129.93, 122.84, 112.00  ppm; elemental analysis: found: C, 60.12; H, 6.98; N, 13.10, (C16H23N3O4), calc.: C, 60.01; H, 6.93; N, 13.17; UV/Vis λmax (nm): 326.50 (N=N); 302.50 (C=O); 230.50 (Ar-CN); 210.00 (Ar-OH).4-Methylphenylazo-3-N-(4-hydroxyphenyl)maleimide (Ic). Color: yellow; yield: 80%; melting point: 203–204 °C; FT-IR (KBr disc): 3,302 cm−1 (O-H), 3,202 cm−1 (HC=CH), 1,703 cm−1 (C=O), 1,618 cm−1 (aromatic ring), 1,511 cm−1 (N=N), 835 cm−1 and 719 cm−1; 1H-NMR (CD3OD): 7.17–7.52 (aromatic), 6.90–7.20 , 6.29–6.53 (HC=CH), 2.35 (CH3) ppm; 13C-NMR (CD3OD): 170.11 (C=O), 155.20, 134.48 , 133.66, 129.74, 127.00, 125.00, 122.76, 112.00 , 20.01 (CH3) ppm; elemental analysis: found: C, 64.09; H, 7.84; N, 13.18, (C17H25N3O3), calc.: C, 63.91; H, 7.89; N, 13.16; UV/Vis λmax (nm): 330.00 (N=N); 302.50 (C=O); 231.50 (Ar-CN); 210.00 (Ar-CH3).Phenylazo-3-N-(4-methylphenyl)maleimide (IIa). Color: yellow; yield: 82%; melting point: 185–186 °C; FT-IR (KBr disc): 3,092 cm−1 (HC=CH), 1,703 cm−1 (C=O), 1,630 cm−1 (aromatic ring), 1,540 cm−1 (N=N), 758 cm−1 and 1,314 cm−1 (CH3); 1H-NMR (CD3OD): 7.17–7.53 (aromatic), 6.96–7.19 , 6.32–6.59 (HC=CH), 2.35 (CH3) ppm; 13C-NMR (CD3OD): 164.99 (C=O), 134.75 , 135.29, 133.55, 129.43, 128.11, 126.36, 125.81, 120.71 , 19.96 (CH3) ppm; elemental analysis: found: C, 67.46; H, 8.14; N, 13.91, (C17H25N3O2), calc.: C, 67.28; H, 8.31; N, 13.86; UV/Vis λmax (nm): 340.50 (N=N); 225.50 (Ar-CN); 205.50 (phenyl).4-Hydroxyphenylazo-3-N-(4-methylphenyl)maleimide (IIb). Color: yellow; yield: 83%; melting point: 190–191 °C; FT-IR (KBr disc): 3,210 cm−1 (O-H), 3,093 cm−1 (HC=CH), 1,703 cm−1 (C=O), 1,630 cm−1 (aromatic ring), 1,541 cm−1 (N=N), 758 cm−1 and 1,312 cm−1 (CH3); 1H-NMR (CD3OD): 7.19–7.57 (aromatic), 6.95–7.20 , 6.32–6.48 (HC=CH), 2.35 and 2.40 (CH3) ppm; 13C-NMR (CD3OD): 167.12 (C=O), 155.68, 143.19, 136.46, 136.01, 134.78 , 129.11, 128.26, 127.43, 112.01 , 19.93 (CH3) ppm; elemental analysis: found: C, 63.83; H, 7.89; N, 13.12, (C17H25N3O3), calc.: C, 63.91; H, 7.89; N, 13.16; UV/Vis λmax (nm): 366.50 (N=N); 225.50 (Ar-CN); 207.00 (Ar-OH).4-Methylphenylazo-3-N-(4-methylphenyl)maleimide (IIc). Color: pale yellow; yield: 85%; melting point: 186–187 °C; FT-IR (KBr disc): 3,091 cm−1 (HC=CH), 1,703 cm−1 (C=O), 1,630 cm−1 (aromatic ring), 1,540 cm−1 (N=N), 778 cm−1 and 1,315 cm−1 (CH3); 1H-NMR (CD3OD): 7.20–7.59 (aromatic), 6.90–7.19 , 6.32–6.55 (HC=CH), 2.39 (CH3) ppm; 13C-NMR (CD3OD): 165.36 (C=O), 144.33, 134.80 , 128.08, 127.11, 126.47, 112.33 , 20.10, 20.03, 19.96 (CH3) ppm; elemental analysis: found: C, 68.01; H, 8.72; N, 13.19, (C18H27N3O2); calc.: C, 68.14; H, 8.58; N, 13.24; UV/Vis λmax (nm): 352.00 (N=N); 225.50 (Ar-CN); 206.00 (Ar-CH3).2O5 as catalyst has minimized the reaction temperature from 150–300 °C to 20–70 °C as well as giving high yields.Six new azo compounds based on N-(4-hydroxyphenyl)maleimide and N-(4-methylphenyl)maleimide have been successfully synthesized and characterized. The use of P


Z-4-2 was isolated from the fermentation broth of Streptomyces djakartensis NW35, together with the known compound N-acetyltryptamine (Z-9-2) by bioassay-guided fractionation. Its chemical structure was elucidated as (E)-2-methoxy-1,4 naphthoquinone-1-oxime (Z-4-2) mainly by NMR analyses and MS spectral data. Their antibacterial activities against bacteria were evaluated by the filter paper method. The results of indicated that these compounds possess significant antibacterial activities.The new compound  Streptomyces djakartensis which was isolated from a pesticide-polluted soil sample has been investigated. In this paper, we report the antibacterial composition of the fermentation broth of S. djakartensis NW35.Soil actinomycetes are still important sources of novel antibiotics. The vast majority (70%) of the known antibiotics were first isolated from actinomycetes ,2. In thZ-4-2 . The IR spectrum of Z-4-2 showed absorptions of hydroxyl (3546 cm−1) and conjugated carbonyl (1640 cm−1) moieties. The 1H-NMR spectrum of Z-4-2 revealed the presence of four aromatic protons at δH 7.20 , 7.59 , 7.24 , 8.10  and one hydroxyl proton at δH 9.75 , one olefinic proton at δH 6.16 , one methoxy proton at δH 3.89 . The 13C-NMR spectrum of Z-4-2 exhibited 11 carbon signals, which were resolved through a DEPT experiment into one methyl, five methine, and five quaternary carbons  (E)-2-Methoxy-1,4 naphthoquinone1-oxime (Z-4-2) is a new compound.HMBC correlations from the conjugated carbonyl position . Based o was (E) . (E)-2-MZ-9-2 , 7.06 , 6.99 , 7.31  and one indole proton at δH 7.05 , one methyl proton at δH 1.89 , two methylene proton at δH 2.92 , 3.45 . The 13C-NMR spectrum of Z-9-2 exhibited 12 carbon signals, which were resolved through a DEPT experiment into one methyl δC 22.6, two methylenes δC 41.6, 22.6, five methines δC 112.2, 119.2, 119.6, 122.3, 123.3, and four quaternary carbons δC 173.2, 138.2, 128.8, 113.3. The above spectral data agreed with the literature values of N-acetyltryptamine [Compound Z-9-2  was obtaZ-4-2 and Z-9-2 showed significant antibacterial activities  in CDCl3 with TMS as internal standard. HR-ESI-MS data were obtained by Bruker Daltonics APEX II 49e (ESI) mass spectrophotometer. Silica gel  was used for chromatographic separations.Melting points (uncorrected) were taken on a Fisher-Johns melting point apparatus. IR spectra were acquired using a Bruker TENSOR2 spectrophotometer. The NMR data were recorded on a Bruker Avance 500 instrument , K2HPO4 (0.2%), KNO3 (0.2%), NaCl (0.2%), Casein (0.03%), MgSO4 (0.005%), CaCO3 (0.002%), FeSO4 (0.001%) and agar (1.5%). Fermentation was performed in two stages: seed growth and antibiotics production. The spores of S.djakartensis NW35 grown on starch casein agar were used to inoculate a 250 mL flask containing 60 mL of a sterile seed medium consisting of glucose (1.0%), millet steep liquor (1.0%), peptone (0.5%), (NH4)2SO4 (0.1%), NaCl (0.25%),and CaCO3 (0.05%); pH 7.2. The flask was shaken on a shaker at 180 rpm for 18 h at 28 °C. Six mL of the seed culture were transferred to 250 mL flasks containing 60 mL of a sterile production medium consisting of glucose (1.0%), millet steep liquor (1.0%), peptone (0.3%), (NH4)2SO4 (0.1%), NaCl (0.25%) and CaCO3 (0.1%); pH 7.2. Fermentation was carried out at 180 rpm for 4 days at 28 °C on a rotary shaker.2O and MeOH in sequence. The MeOH fraction was evaporated in vacuum. The concentrate was subjected to column chromatography and eluted with EtOAc and MeOH in sequence. The antimicrobial fraction was concentrated under vacuum, and further purified on a Waters 600E HPLC apparatus  equipped with a Hypersil ODS-BP  reverse phase column, using methanol-water as the mobile phase, flow rate of 3.0 mL/min, monitored by UV detector at 230 nm to afford two compounds Z-4-2 (20 mg) and Z-9-2 (10 mg).The culture of 90 L was filtered through cheesecloth to separate the medium and culture liquid at 25 °C, pH 7.0. The filtrate was absorbed onto HPD100 macroporous resin , and then eluted with HBacillus cereus (1.1846), Bacillus subtilis (1.88), Staphylococcus aureus (1.89), Escherichia coil (1.1574), and Pseudomonas aeruginosa (1.2031) were obtained from China General Microbiological Culture Collection Center. Pseuomonas syringae pv. Actinidiae, Erwinia carotovora were obtained from College of Plant Protection, Northwest A & F University. A clinical isolate of MRSA was obtained from Nanjing Medical University. Ampicillin  was used as positive control. The antibacterial activities of compounds against eight strains of bacteria were evaluated by the filter paper method [8 cfu·mL−1. Next, the mixture was poured on 9 cm Petri dishes. The tested compounds were dissolved in acetone at the concentration of 1,000 ppm, The filter paper (5 mm in diameter) were impregnated with 10 μL/disc of each compound, then were absolutely dried and placed on the inoculated agar. The inoculated plates were incubated at 37 °C for 24 h. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organism. Experiments were run in triplicate. The results indicated that Z-4-2 and Z-9-2 could effectively inhibit Gram-positive bacteria, such as Bacillus cereus, B. subtilis, whereas they were inactive towards Gram-negative bacteria -2-methoxy-1,4 naphthoquinone-1-oxime (Z-4-2) and N-acetyltryptamine (Z-9-2) were isolated from the extract of fermented broth of Streptomyces djakartensis NW35 by bioassay-guided fractionation. (E)-2-Methoxy-1,4 naphthoquinone-1-oxime (Z-4-2) is a new natural product. These two compounds both showed significant antibacterial activities against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coil, Pseuomonas syringae pv. Actinidiae and MRSA.Two compounds (


F5 exhibited higher fungicidal activity against Rhizoctonia solani(53.35%) than commercial antiviral agent validamycin (36.4%); compound F16 exhibited high fungicidal activity against Oospora citriaurantii ex Persoon(43.28%). Some of the alkaloids and their derivatives (compounds F4 and F25) exhibited broad-spectrum fungicidal activity. Specifically, compound F4 exhibited excellent high broad-spectrum fungicidal activity in vitro, and the curative and protection activities against P. litchi in vivo reached 92.59% and 59.26%, respectively. The new derivative, F4, with optimized physicochemical properties, obviously exhibited higher activities both in vitro and in vivo; therefore, F4 may be used as a new lead structure for the development of fungicidal drugs.A series of β-Carboline derivatives were designed, synthesized, and evaluated for their fungicidal activities in this study. Several derivatives electively exhibited fungicidal activities against some fungi. Especially, compound  Plant pathogenic microorganisms can infect crops, causing local or whole plant disease and leading to significant economic losses. How to control them in modern agriculture is still a big challenge. Many kinds of fungicides are used to prevent and cure the diseases caused by fungi; however, these chemical agents cannot fully protect the crops or completely cure the crops’ tissues from fungal infection under field conditions. Therefore, novel and more practical fungicidal reagents are urgently needed.b] indole ring structure  of tetrahydro-β-carboline compounds. Since the reaction temperature was moderate and the by-product generated was less, the product could be obtained with a purity of more than 90% by suction filtration and washing. Then the product could be used directly in the next reaction after drying. The carboxyl on the 3-position of tetrahydro-β-carboline must be protected by esterification, due to the fact that a carboxyl with high reaction activity could be easily decarboxylated, thereby losing carbonyl in the potassium permanganate conditions. Using KMnO4 to oxidize tetrahydro-β-carboline derivatives produced a lower yield, but the reagent was relatively inexpensive, and the reaction was easy to operate in the laboratory. The acylation reaction of amide synthesis used acid halide with ammonia or amine. Step 1: β-carboline-3-carboxylic acid reacted with an excess of thionyl chloride to become the corresponding acid chloride in the situation of catalyzer MDF of 1‰. The excess of thionyl chloride was both reactant and reaction solvent in the reaction, which may also remove water to reduce moisture in the system requirements of dry operation. HCl gas and SO2 gas were generated by the reaction; we utilized lye to absorb them. Step 2: acid chloride reacted with the corresponding amine. The reaction requires adding week base to neutralize the HCl so as to avoid amine reacting with HCl to generate the amine hydrochloride, which does not participate in the reaction. Because the reaction is intense, the product of the first step should be dissolved firstly in methylene chloride, and added slowly dropwise to the mixture of amine and triethylamine that was placed on an ice bath, and then the mixture stirred at room temperature for half an hour.Compounds harmine, harmane, harmaline, and harmalol were obtained from Sigma-Aldrich, St. Louis, MI, USA. Their chemical structures are shown in eactions  with a vFungicide Screening. Compounds F1–30 were evaluated in a series of in vitro fungicidal tests, against a range of phytopathogenic species. The resulting data , R. solani, P. litchii, P. nicotianae, and O. citriaurantii ex Persoon. When the R1 was phenyl, the compounds exhibited higher activities than those with 4-nitrophenyl, 4-methoxyphenyl,3,4,5-trimethoxyphenyl,4-trifluoromethylphenyl, or 4-chlorophenyl as the R1. When the skeleton was β-carboline, the types of the substituents on the R2 and R3 had a significant influence on the fungicidal activities. For instance, compound F4 (N-(2-pyridyl)-1-phenyl-9H-pyridoindole-3-formamide) exhibited excellent fungicidal activity against most of the tested fungi, whereas compound F2  exhibited only moderate activity; compound F5, containing a 4-trifluoromethylphenyl group, exhibited higher fungicidal activity than the compound F1 and the group containing 4-chlorophenyl. Several of these compounds selectively exhibited fungicidal activities against some fungi. For example, the activities of harmine (against F. oxysporum f.sp.cubense and R. solani), compound F16 (against O. citriaurantii ex Persoon), and compound F25 (against C. gloeosporioides (Penz.)) were much higher than that against other fungi and were higher than the other compounds against some fungi, as shown in ing data  revealedF1–30 were evaluated in in vivo fungicidal tests (litchi leaf-piece assays), against the phytopathogenic of P. litchii. The resulting data  and the isopropyl group  displayed higher curative activity than preventative, and compound F11 had the highest curative activity against P. litchi on litchi leaf-piece assay. Comparatively, the 2-pyridyl group  and the 2-chlorophenyl group  exhibited higher preventative activity, and compound F4 not only showed excellent preventative activity (92.6%) much better than the activity of metalaxyl (59.3%) against P. litchi on litchi leaf-piece assay, but also had a good curative activity (59.3%) against P. litchi. All the alkaloids and their derivatives had no phytotoxicity to the leaf pieces on the assay.The alkaloids and their derivatives compounds ing data  revealedAll anhydrous solvents were dried and purified by using standard techniques. The synthetic routes are given in N-ethyl-1-phenyl-9H-pyridoindole-3-formamide (F1). l-Tryptamine  was added in three neck flasks, and 100 mL of acetic acid was added with stirring and dissolved by heating. Press material quality 1:1 ratio dropping weighed benzaldehyde , the reaction was heated to reflux, and the reaction progress was followed by thin layer chromatography (TLC). When TLC showed the point of material starting to disappear, we stopped the reaction. The resulting solution was cooled by suction filtration, and washed with water as eluent to remove acetic acid and give1-phenyl-1,2,3,4-tetrahydro-9H-pyridoindole-3-carboxylic acid indole-3-carboxylic acid 11.68 g was added to the reaction mixture, then warmed slowly to reflux and the reaction was refluxed for 4 h, using TLC to track the progress of the reaction. When the reaction was completed, the reaction solution was stirred into 500 mL of ice water after it was cooled, and the pH was adjusted with 30% NaOH to neutral; at this time, the solution appeared as a heavy white precipitate. The precipitate was washed with water and filtered, then dried in a vacuum to give 1-phenyl-1,2,3,4-tetrahydro-9H-pyridoindole-3-carboxylic acid methyl ester as a white powdery solid .Four milliliters of SOClH-pyridoindole-3-carboxylic acid methyl ester, of which 6.84 g was added in three neck flasks that were assembled in the ice salt bath; DMF was used as menstruum and KMnO4 was added according to the molar ratio of 1:1.2 ratio, reacting for 0.5 h in the ice bath. The reaction progress was followed by TLC. The mixture was cooled to room temperature after the reaction was finished, and then filtered in a vacuum after stirring for 12 h. The filtrate was added to water and stirred sufficiently to give the precipitated solid, the solution was filtered by suction, then the solid was washed with water to give1-phenyl-9H-pyridoindole-3-carboxylic acid methyl ester as a pale yellow solid .The product of the previous step was 1-phenyl-1,2,3,4-tetrahydro-9H-pyridoindole-3-carboxylic acid methyl ester (6.13 g) and ethanol (25.0 mL) were added into a 100 mL flask, then the flask was heated in an oil bath with stirring, and then 1 mL aqueous solution containing 0.5 g of sodium hydroxide was added dropwise in the pot. The, reaction was heated for 2 h and followed by TLC. Dilute hydrochloric acid was added dropwise until pH = 5–6 after completion of the reaction; the mixture was cooled in a refrigerator at 4 °C overnight, then filtered, washed with water, and dried to give 1-phenyl-9H-pyridoindole-3-carboxylic acid as a yellow solid .1-Phenyl-9H-pyridoindole-3-carboxylic acid (5.76 g) was added to a 50 mL flask that was installed in a reflux condenser tube, drier, and exhaust absorption device, then 20 mL of thionyl chloride was added to the flask with stirring and the mixture was heated at reflux for 1 h. The condensed reflux into recovery device after the reaction was completed, and then the excess thionyl chloride was distilled off to give1-phenyl-9H-pyridoindole-3-carbonyl chloride as an off-white solid powder .1-phenyl-9F1 as a yellow solid.The solid that was obtained at the previous step was dissolved with dichloromethane, then the solution, under an ice bath with stirring, was added dropwise slowly to a 50-mL single neck flask that had 1.5 g 30% ethylamine solution mixed with 2.0 g of alcohol of triethylamine. The mixture was stirred at room temperature for 30 min, and evaporated to dryness by rotary evaporation of dichloromethane, then dissolved in an appropriate amount of methanol. After that the solution was poured into water and cooled with stirring to sufficiently precipitate solid, and then the mixture was filtered. The resulting residue was washed with the amount of 5% sodium hydroxide solution and water until neutral, and finally dried to give F2–30 were prepared by following the same procedure as for compound F1. The corresponding starting materials were all commercially available.The target compounds N-Ethyl-1-phenyl-9H-pyridoindole-3-formamide  (F1): yield, 79.54%; m.p. 210–227 °C. IR (KBr) cm−1: 3430 (νN-H), 2924, 2850 (νC-H), 1647 (νC=O), 1023 (νC-N); 1H-NMR  δ 11.82 H, c), 8.82 H, c), 8.70 , 8.43–8.39 H, c), 8.15 H), 8.03–8.01 H, c), 7.65 H, c, Ph(4)H), 7.60–7.57 H), 7.34–7.31 H, c), 3.44–3.40 , 1.18 .N,N-Diethyl-1-phenyl-9H-pyridoindole-3-formamide  (F2): yield, 57.1%; a pale yellow solid. m.p. 210–227 °C. IR (KBr) cm−1: 3449 (νN-H), 2918, 2851 (νC-H), 1623 (νC=O), 1022 (νC-N); 1H-NMR  δ 11.71 H, c), 8.42 H, c), 8.35 H, c), 8.02 H), 7.67 H, c), 7.63 H), 7.56 H, c, Ph(4)H), 7.29 H, c), 3.50 , 1.23–1.18 .N-Isopropyl-1-phenyl-9H-pyridoindole-3-formamide  (F3): yield, 47.27%; a pale yellow solid. m.p. 188–191 °C. IR (KBr) cm−1: 3430 (νN-H), 2924, 2850 (νC-H), 1647 (νC=O), 1252 (νC-N); 1H-NMR  δ 11.82 H, c), 8.82 H, c), 8.41 , 8.28 H, c), 8.12 H), 7.67 H, c, PhH), 7.59 H, c, Ph(4)H), 7.35–7.23 H, c), 4.20 , 1.26 .N-(2-Pyridyl)-1-phenyl-9H-pyridoindole-3-formamide  (F4): yield, 81.49%; a yellow-brown solid. m.p. 182–183 °C. IR (KBr) cm−1: 3447, 3423 (νN-H), 1623 (νC=O), 1245 (νC-N), 1493 (νC=C); 1H-NMR  δ 12.05 H, c), 10.66 , 9.05 H, c), 8.88 H, c), 8.49 H), 8.39 H), 8.35 H, c), 8.13 H), 7.72 H, c), 7.60 H), 7.35 H, C(6)H, c), 7.31 H), 7.20 H).N-(2-Chlorophenyl)-1-phenyl-9H-pyridoindole-3-formamide  (F5): yield, 56.98%; a brown-yellow solid. m.p. 206–207 °C. IR (KBr) cm−1: 3424 (νN-H), 1657 (νC=O), 1497 (νC=C), 742 (νC-Cl); 1H-NMR  δ 12.07 H, c), 11.06 , 9.02 H, c), 8.63–8.60 H, c), 8.49 H), 8.19 H), 7.73 H, c), 7.70 H), 7.67–7.61 H, C(7)H, c), 7.61–7.60 H), 7.45 H), 7.36 H, c), 7.20–7.17 H).N-Ethyl-1-(4-nitrophenyl)-9H-pyridoindole-3-formamide  (F6): yield, 77.82%; a bright yellow solid. m.p. 269–272 °C. IR (KBr) cm−1: 3435 (νN-H), 2921, 2848 (νC-H), 1624 (νC=O), 1249 (νC-N), 1520, 1347 (νNO2); 1H-NMR  δ 12.20 H, c), 9.02 H, c), 8.48 H), 8.47–8.46 H), 8.46–8.44 H, c), 8.32 , 7.72 H, c), 7.63 H, c), 7.37–7.34 H, c), 2.80 , 1.14 .N,N-Diethyl-1-(4-nitrophenyl)-9H-pyridoindole-3-formamide  (F7): yield, 66.08%; a yellow-brown solid. m.p. 252–254 °C. IR (KBr) cm−1: 3446 (νN-H), 2925, 2851 (νC-H), 1608 (νC=O), 1105 (νC-N), 1515, 1342 (νNO2); 1H-NMR  δ 11.97 H, c), 8.53 , c), 8.46 H), 8.39 H, c), 8.31 H), 7.69 H, c), 7.61 H, c), 7.32 H, c), 3.53–3.46 , 1.24–1.19 .N-Isopropyl-1-(4-nitrophenyl)-9H-pyridoindole-3-formamide  (F8): yield, 84.58%; a pale yellow solid. m.p. 260–262 °C. IR (KBr) cm−1: 3426 (νN-H), 2918, 2850 (νC-H), 1652 (νC=O), 1249 (νC-N), 1519, 1345 (νNO2); 1H-NMR  δ 12.02 H, c), 8.91 H, c), 8.47 H), 8.43 H), 8.34 H, c), 8.31 , 7.69 H, c), 7.66–7.63 H, c), 7.35 H, c), 4.22 , 1.26 .N-(2-Pyrido)-1-(4-nitrophenyl)-9H-pyridoindole-3-formamide  (F9): yield, 62.69%; a yellow-brown solid. m.p. 253–255 °C. IR (KBr) cm−1: 3427 (νN-H), 1625 (νC=O), 1321 (νC-N), 1601, 1495 (νC=C), 1519, 1348 (νNO2); 1H-NMR  δ 12.15 H, c), 10.61 , 9.02 H, c), 8.53 H, c), 8.47 H), 8.31 H), 8.17 H), 7.84 H, c), 7.72 H), 7.68–7.65 H, c), 7.61–7.57 H, c), 7.37 H), 7.35–7.29 H).N-(2-Chlorophenyl)-1-(4-nitrophenyl)-9H-pyridoindole-3-formamide  (F10): yield, 43.6%; a yellow-brown solid. m.p. 277–278 °C. IR (KBr) cm−1: 3439 (νN-H), 1629 (νC=O), 1495 (νC=C), 737 (νC-Cl), 1519, 1348 (νNO2); 1H-NMR  δ 12.15 H, c), 10.61 , 9.02 H, c), 8.53 H, c), 8.47 H), 8.43 H), 8.39–8.37 H), 8.31 H), 8.17 H, c), 7.84 H, c), 7.72 H), 7.68–7.65 H, c), 7.37 H).N-Ethyl-1-(4-methoxyphenyl)-9H-pyridoindole-3-formamide  (F11): yield, 41.48%; a pale yellow solid. m.p. 171–173 °C. IR (KBr) cm−1: 3429 (νN-H), 2923, 2853 (νC-H), 1653 (νC=O), 1249 (νC-N), 1249, 1119 (νC-O-C); 1H-NMR  δ 11.80 H, c), 8.76 H, c), 8.69 , 8.39 H, c), 8.13 H), 7.69 H, c), 7.58 H, c), 7.31 H, c), 7.19 H), 3.89 , 3.45–3.38 , 1.23–1.14 .N,N-Diethyl-1-(4-methoxyphenyl)-9H-pyridoindole-3-formamide  (F12): yield, 36.66%; a pale yellow solid. m.p. 225–226 °C. IR (KBr) cm−1: 3427(νN-H), 2921, 2851 (νC-H), 1609 (νC=O), 1243 (νC-N), 1243, 1113 (νC-O-C); 1H-NMR  δ 11.67 H, c), 8.36 H, c), 8.35–8.32 H, c), 7.99 ), 7.67 H, c), 7.57 H, c), 7.28 H, c), 7.19 H), 3.88 , 3.49 , 1.23–1.18 .N-Isopropyl-1-(4-methoxyphenyl)-9H-pyridoindole-3-formamide  (F13): yield, 23.92%; a yellow solid. m.p. 188–189 °C. IR (KBr) cm−1: 3430 (νN-H), 2924, 2850 (νC-H), 1648 (νC=O), 1176 (νC-N), 1248, 1176 (νC-O-C); 1H-NMR  δ 11.78 H, c), 8.77 H, c), 8.39 H, c), 8.28 , 8.09 H), 7.68 H, c), 7.60−7.58 H, c), 7.37−7.26 H, c), 7.22−7.20 H), 4.21−4.18 , 3.89 , 1.26 .N-(2-Pyridyl)-1-(4-methoxyphenyl)-9H-pyridoindole-3-formamide  (F14): yield, 40.36%; a brown solid. m.p. 244–245 °C. IR (KBr) cm−1: 3414, 3340 (νN-H), 1686 (νC=O), 1300 (νC-N), 1608, 1512 (νC=C), 1252, 1176 (νC-O-C); 1H-NMR  δ 12.14 H, c), 10.66 , 9.05 H, c), 8.40 H, C(5)H, c), 8.09 H), 7.94–7.91 H, C(8), c), 7.73–7.71 H, c), 7.64–7.62 H, c), 7.28–7.25 H), 6.98 H), 6.88–6.83 H), 3.91 .N-(2-Chlorophenyl)-1-(4-methoxyphenyl)-9H-pyridoindole-3-formamide  (F15): yield, 7.66%; a yellow-brown solid. m.p. 250–251 °C. IR (KBr) cm−1: 3422 (νN-H), 1665 (νC=O), 1510 (νC=C), 746 (νC-Cl), 1249, 1114 (νC-O-C); 1H-NMR  δ 12.02 H, c), 11.08 , 8.98 H, c), 8.62 H, c), 8.47 H), 8.15 H), 7.74–7.71 H, c), 7.62 H, c, Ph2(3)H), 7.46 H, c), 7.35 H), 7.25 H), 7.19 H), 3.91 .N-Ethyl-1--9H-pyridoindole-3-formamide  (F16): yield, 54.13%; a pale yellow solid. m.p. 241–243 °C. IR (KBr) cm−1: 3429 (νN-H), 2925, 2850 (νC-H), 1653 (νC=O), 1250 (νC-N), 1250, 1104 (νC-O-C); 1H-NMR  δ 11.73 H, c), 8.92 H, c), 8.41 , 7.58 H, C(8)H, c), 7.30 H, c, C(6)H, c), 4.02 OCH3), 3.93 OCH3), 3.17 , 1.15 .N,N-Diethyl-1--9H-pyridoindole-3-formamide  (F17): yield, 14.84%; a pale yellow solid. m.p. 268–270 °C. IR (KBr) cm−1: 3454 (νN-H), 2929, 2850 (νC-H), 1623 (νC=O), 1238 (νC-N), 1238, 1127 (νC-O-C); 1H-NMR  δ 11.72 H, c), 8.90 H, c), 8.42 H, c), 8.13 H, c), 7.64–7.54 H, c, PhH), 7.31 H, c), 4.03 OCH3), 3.93 OCH3), 3.31–3.03 , 1.21 .N-Isopropyl-1--9H-pyridoindole-3-formamide  (F18): yield, 22.55%; a yellow solid. m.p. 311–317 °C. IR (KBr) cm−1: 3433 (νN-H), 2925, 2855 (νC-H), 1651 (νC=O), 1052 (νC-N), 1252, 1116 (νC-O-C); 1H-NMR  δ 11.72 H, c), 9.03–8.72 , 8.44–8.41 H, c), 8.35 H, c), 7.67 , c), 7.61–7.56 H, c), 7.29–7.27 H, c), 7.23 H), 3.93–3.90 OCH3), 3.78 OCH3), 3.50 , 1.23 .N-(2-Pyridyl)-1--9H-pyridoindole-3-formamide  (F19): yield, 41.63%; a khaki solid. m.p. 279–281 °C. IR (KBr) cm−1: 3433 (νN-H), 1651 (νC=O), 1252 (νC-N), 1623, 1525 (νC=C), 1252, 1113 (νC-O-C); 1H-NMR  δ 11.89 H, c), 10.58 , 9.09 H, c), 8.50–8.48 H, c), 8.37 H), .94 H, c, Py(5)H), 7.63 H, c, C(6), c), 7.35 H), 7.22–7.19 H), 3.95–3.92 OCH3), 3.87 OCH3).N-(2-Chlorophenyl)-1--9H-pyridoindole-3-formamide  (F20): yield, 10.09%; a bright yellow solid. m.p. 275–278 °C. IR (KBr) cm−1: 3429 (νN-H), 1624 (νC=O), 1484 (νC=C), 749 (νC-Cl), 1250, 1109 (νC-O-C); 1H-NMR  δ 12.11 H, c), 11.07 , 9.54–9.53 H, c), 9.06 H), 9.01 H, c), 8.51–8.47 , c, Ph2(6)H), 7.64 , c, C(6)H, c), 7.41 H), 7.26 H), 7.19–7.18 H), 3.97–3.92 OCH3), 3.80 OCH3).N-Ethyl-1-(4-trifluoromethylphenyl)-9H-pyridoindole-3-formamide  (F21): yield,78.1%; a beige solid. m.p. 261–263 °C. IR (KBr) cm−1: 3428 (νN-H), 2926, 2848 (νC-H), 1625 (νC=O), 1250 (νC-N), 1119 (νCF3); 1H-NMR  δ 11.95 H, c), 8.87 , 8.76 H, c), 8.40 H), 7.99 H), 7.68 H, c), 7.65–7.58 H, c), 7.43–7.27 H, c), 3.38 , 1.18 .N,N-Diethyl-1-(4-trifluoromethylphenyl)-9H-pyridoindole-3-formamide  (F22): yield, 83.4%; a beige solid. m.p. 238–239 °C. IR (KBr) cm−1: 3435 (νN-H), 2927, 2851 (νC-H), 1615 (νC=O), 1323 (νC-N), 1122 (νCF3); 1H-NMR  δ 11.83 H, c), 8.49 H, c), 8.38 H, c), 8.22 H), 7.99 H), 7.66 H, c), 7.60 H, c), 7.31 H, c), 3.53–3.47 , 1.20 .N-Isopropyl-1-(4-trifluoromethylphenyl)-9H-pyridoindole-3-formamide  (F23): yield, 69.3%; a beige solid. m.p. 245–247 °C. IR (KBr) cm−1: 3437 (νN-H), 2925, 2854 (νC-H), 1646 (νC=O), 1325 (νC-N), 1122 (νCF3); 1H-NMR  δ 11.95 H, c), 8.88 H, c), 8.43 H, c), 8.33 , C(8)H, c), 8.00 H), 7.68 , 7.64–7.58 H, c), 7.33 H, c), 4.21 , 1.25 .N-(2-Pyridyl)-1-(4-trifluoromethylphenyl)-9H-pyridoindole-3-formamide  (F24): yield, 57.7%; a beige solid. m.p. 269–271 °C. IR (KBr) cm−1: 3438 (νN-H), 1627 (νC=O), 1322 (νC-N), 1524 (νC=C), 1121 (νCF3); 1H-NMR  δ 12.15 H, c), 10.61 , 9.10 H, c), 8.51 H, c), 8.38 , C(8)H, c), 8.34 H), 8.06 H), 7.70 H, c), 7.62–7.57 H, c), 7.41–7.35 H), 7.26–7.14 H).N-(2-Chlorophenyl)-1-(4-trifluoromethylphenyl)-9H-pyridoindole-3-formam-ide  (F25): yield, 95.6%; a beige solid. m.p. 295–297 °C.IR (KBr) cm−1: 3442 (νN-H), 1641 (νC=O), 1539 (νC=C), 744 (νC-Cl), 1116 (νCF3); 1H-NMR  δ 12.17 H, c), 10.96 , 9.05 H, c), 8.56 H, c), 8.49 H), 8.37 H), 8.04 H), 7.72 H, c), 7.67–7.63 H), 7.59 H, c), 7.50–7.42 H), 7.41–7.33 H, c), 7.18 H).N-Ethyl-1-(4-chlorophenyl)-9H-pyridoindole-3-formamide  (F26): yield, 49.84%; a gray-yellow solid. m.p. 234–236 °C. IR (KBr) cm−1: 3440 (νN-H), 2926, 2855 (νC-H), 1644 (νC=O), 1091 (νC-N), 746 (νC-Cl); 1H-NMR  δ 12.06 H, c), 8.93 H), 8.43 , 8.07–8.05 H, c), 7.71–7.69 H, c, Ph1H), 7.63–7.60 H, c), 7.34 H, c), 3.93 , 1.36–1.05 .N,N-Diethyl-1-(4-chlorophenyl)-9H-pyridoindole-3-formamide  (F27): yield, 25.64%; a pale yellow solid. m.p. 247–248 °C. IR (KBr) cm−1: 3436 (νN-H), 2925, 2855 (νC-H), 1626 (νC=O), 1093 (νC-N), 747 (νC-Cl); 1H-NMR  δ 11.88 H, c), 8.82 H, c), 8.41 H, c), 8.21–8.19 H), 7.70–7.68 H, c, Ph1H), 7.62–7.59 H, c), 7.32 H, c), 3.16 , 1.16 .N-Isopropyl-1-(4-chlorophenyl)-9H-pyridoindole-3-formamide  (F28): yield, 60.15%; a pale yellow solid. m.p. 257–259 °C. IR (KBr) cm−1: 3439 (νN-H), 2969, 2927 (νC-H), 1648 (νC=O), 1092 (νC-N), 746 (νC-Cl); 1H-NMR  δ 11.87 H, c), 8.85 H, c), 8.45–8.28 H), 8.22–8.07 H, c, CO-NH), 7.70 H, c, Ph1H), 7.64–7.56 H, c), 7.37–7.28 H, c), 4.24–4.13 , 1.25 .N-(2-Pyridyl)-1-(4-chlorophenyl)-9H-pyridoindole-3-formamide  (F29): yield, 7.29%; a pale yellow solid. m.p. 263–265 °C. IR (KBr) cm−1: 3436 (νN-H), 1628 (νC=O), 1455 (νC=C), 1094 (νC-N), 747 (νC-Cl); 1H-NMR  δ 12.03 H, c), 8.93 H, c), 8.43 H), 8.10 H, c, Py(3)H), 7.70 H, c, Ph1H, Ph2(5)H), 7.62 H, c), 7.32 H).N-(2-Chlorophenyl)-1-(4-chlorophenyl)-9H-pyridoindole-3-formamide  (F30): yield, 35.84%; a pale yellow solid. m.p. 271–273 °C. IR (KBr) cm−1: 3449 (νN-H), 1649 (νC=O), 1594, 1497 (νC=C), 1094 (νC-N), 741 (νC-Cl); 1H-NMR  δ 12.16 H, c), 11.00 , 9.02 H, c), 8.58 H, c), 8.49 H), 8.24–8.17 H), 7.78–7.72 H, Ph2(3)H), 7.64 H, c), 7.60 H, c), 7.47–7.43 H), 7.40–7.33 H, c), 7.22–7.16 H).Fusarium oxysporum f.sp.cubense,Colletotrichum gloeosporioides (Penz.), R. solani, Peronophthora litchii, Phytophthora nicotianae, and O. citriaurantii ex Persoon, at the concentration of 100 μg/mL. Each assay contained three replicates for each concentration. The plates were stored in controlled environment cabinets for between two and seven days, depending on the assay, after which mycelia growth was assessed. Treatments were scored as percentage inhibition of mycelial growth relative to untreated controls. The biological data presented in The compounds were screened on a broader range of fungal pathogens in mycelial growth tests in artificial media against P. litchi on young leaves of litchi. The formulated chemicals were applied to leaf pieces prior to inoculation with spores of the pathogen, and also after they had been inoculated with pathogen spores. Each assay contained three replicates for each concentration. Assessments were made three days after inoculation, depending on the assay, after which disease inhibition was assessed. Treatments were scored as percentage inhibition of disease development relative to untreated controls. The biological data presented in The compounds were also evaluated in leaf-piece assays, at the concentration of 100 μg/mL for in vitro and in vivo. The results showed that most of these compounds exhibited good fungicidal activities, and these compounds had certain selectivity toward fungi species. Of all the new compounds synthesized, F4 (N-(2-pyridyl)-1-phenyl-9H-pyridoindole-3-formamide) showed activity almost at the same level as the control compound metalaxyl against P. litchii and P. nicotianae in vitro. Very interestingly, this compound exhibited better activity than the control compound validamycin against R. solani in vitro, and also had excellent preventative activity against P. litchi in vivo. Further studies on the activity mechanism of these compounds are underway in our laboratory.In summary, a series of β-carbolines derivatives were designed, synthesized, and first assayed for their fungicidal activities


Cordia exaltata Lam. (Boraginaceae) led to the isolation, through chromatographic techniques, of nineteen secondary metabolites: 8,8'dimethyl-3,4,3',4'-dimethylenedioxy-7-oxo-2,7'cyclolignan (1), 8,8'-dimethyl-4,5-dimethoxy-3',4'-methylenodioxy-7-oxo-2,7'cyclolignan (2), sitosterol (3a), stigmasterol (3b), sitosterol-3-O-β-d-glucopyranoside (4a), stigmasterol-3-O-β-d-glucopyranoside (4b), phaeophytin A (5), 132-hydroxyphaeophytin A (6), 173-ethoxypheophorbide A (7), 132-hydroxy-173-ethoxypheophorbide A (8), m-methoxy-p-hydroxybenzaldehyde (9), (E)-7--7-propenoic acid (10), 1-benzopyran-2-one (11), 7-hydroxy-1-benzopyran-2-one (12), 2,5-bis--3,4-dimethyltetrahydrofuran (13), 3,4,5,3',5'-pentamethoxy-1'-allyl-8.O.4'-neolignan (14), 3,5,7,3',4'-pentahydroxyflavonol (15),5,7-dihydroxy-4'-methoxyflavone (16), 5,8-dihydroxy-7,4’-dimethoxyflavone (17), kaempherol 3-O-β-d-glucosyl-6''-α-L-ramnopyranoside (18) and kaempherol 3,7-di-O-α-l-ramnopyranoside (19). Their structures were identified by 1H and 13C-NMR using one and two-dimensional techniques. In addition, the antimicrobial activity of compounds 1, 2, 13 and 14 against bacteria and fungi are reported here for the first time.The phytochemical study of  Cordia, one of the most representative in the Boraginaceae family [Cordia leucocephala, popularly known as “Maria Preta”, are used as an infusion for the treatment of dysmenorrhea [Cordia report antiviral effects against herpes type I, cytotoxic effects on tumor cells CA-9KB [C. alliadora and commonly found in other species of this genus, was identified as a larvicide for Aedes aegypti [C. goetzei and C. corymbosa, are mentioned in the literature for their fungicidal effects against phytopatogens [C. exaltata in order to associate them to the observed pharmacological properties. We now report the isolation and identification of four neolignans 1, 2, 13 and 14, two mixtures of steroids 3a, 3b and 4a, 4b, four porphyrins 5-8, two phenolic acids 9 and 10, two coumarins 11 and 12 and five flavonoids 15-19. Besides we report for the first time the antimicrobial activity of compounds 1, 2, 13 and 14 against bacteria and fungi.The Boraginaceae family has 148 genera and 2,740 species distributed in the temperate and tropical zones of Europe, Asia, Australia and America [e family . Its speenorrhea . Biologis CA-9KB , anti-in aegypti . Some supatogens ,12. The 1 was obtained as colorless crystals. Its IR spectrum exhibited the characteristic absortion of a carbonyl group at 1,678 cm−1 and an aromatic nucleus at 1,475 cm−1 [1H- and 13C-NMR spectra exhibited the general absortions of a neolignan, with two methyl and two methylenedioxy groups on aromatic rings [1H-NMR spectrum of 1 exhibited a singlet at δH 3.80, a multiplet at δH 2.41 and another multiplet at δH 1.93, which were attributed to H-7', H-8 and H-8', respectively, while signals at δH 1.09 and 0.90 were assigned to two methyl groups (H-9 and H-9'). In the aromatic region, an ABX-type system was observed at δH 6.80 , 6.62  and 6.60 , attributed to hydrogens of a 1',3',4'-trisubstituted aryl group. Two additional aromatic hydrogens resonate at δH 7.55  and δH 6.95 . The 13C-NMR spectrum showed 20 carbon resonances, which were assigned by the DPT spectrum as two methyl carbons, three methine carbons, five phenyl methine carbons, two alcoholic methylene carbons, seven phenyl quaternary carbons and a carbonyl carbon. The long-range 1H-13C-correlations of the HMBC spectrum showing 3J between C-4 (δC 151.41), C-3 (δC 144.90) with δH 5.93 , C-4' (δC 145.50), C-3' (δC 147.00) with δH 5.83  and δH 5.78  (OCH2O-C), allowed the location of the two methylenedioxy groups to be established and also the assignments of the two methyl positions by way of correlations between C-8 (δC 42.60) and C-8' (δC 46.48) with δH 2.41  and δH 1.09 , respectively, 2J and 3J. Additionally, a carbonyl group was located at C-7 by the correlation between the resonances at δC 197.48 (C-7) and δH 2.41  and δH 7.55 . Thus, compound 1 was identified as 8,8'-dimethyl-3,4,3',4''-dimethylenedioxy-7-oxo-2,7'-cyclolignan, isolated before from another source [Cordia.Compound 475 cm−1 . The 1H-ic rings . The 1H-n C-4 δC 1.41, C-32 formed white needle-like crystals. Spectral analysis revealed similarities with compound 1, thus suggesting a neolignan structure for 2 as well. The main difference consisted of two methoxyl groups instead of a methylenedioxy on ring A. This could be inferred by the IR spectrum, which exhibited a C-H absortion from a methoxyl group at 2,850 cm−1. The HMBC spectrum showed 3J correlations between resonances at δC 149.10 (C-5) and δH 3.83 , and also δC 155.06 (C-4) and δH 3.58 , allowing the assignments of the methoxyl groups’ positions. Thus, through a complete spectral analysis, it was possible to identify compound 2as 8,8'-dimethyl-4,5-dimethoxy-3',4'-methylenodioxy-7-oxo-2,7'-cyclolignan [1 and 2 are shown in Compound lolignan , a compo13 showed in its 1H-NMR spectrum absorptions in the aromatic region, which corresponded to two ABX-systems: at δH 6.86 , at δH 6.78  and another doublet at δH 6.94 , suggesting a symmetrical structure. This information was supported by a strong singlet at δH 5.94, attributed to four hydrogens of two methylenedioxy groups, which is consistent with a neolignan structure. Also, a tetrahydrofuran ring could be inferred fromy two multiplets at δH 2.24 (2H) and δH 4.40 (2H), related to H-3/4 and H-2/5, respectively. Two symmetric methyl groups were revealed at δH 0.99 . The 13C-NMR spectrum of 13 showed 10 strong peaks, which allowed the identification of two methyl carbons, four methine carbons, two alcoholic methylene carbons, six phenyl methine carbons and six phenyl quaternary carbons, corresponding to 20 chemically equivalent carbons. The HMBC spectrum showed long-range 1H-13C-correlations between H-2/H-5 (δH 4.40) and the aromatic carbons at C-6'/C-6' (δC 120.10) through 3J and also C-1'/C-1 (δC 136.16) through 2J, allowing the location of the two aromatic rings at C-2/C-5. This spectrum could also establish the locations of the two methyl groups, showing 2J interactions between H-3/H-4  and CH3-3/CH3-4 (δC 12.97). The 1H-1H-NOESY spectrum reinforced these positions by showing spatial interactions between H-2/H-5 (δH 4.40), H-3/H-4 (δH 2.24) and CH3-3/CH3-4 (δH 0.99). Therefore, compound 13 was identified as 2,5-bis--3,4-dimethyltetrahydrofuran [Compound drofuran , describ14 appeared as yellow oil. Its 1H-NMR spectrum showed resonances for five methoxyl groups, being two peaks at δH 3.81  and δH 3.77 , which correspond to two pair of symmetric aromatic methoxyls and another group at δH 3.80 . An allyl group was shown at δH 5.95  (H-8'), δH 5.09 , δH 5.05  and δH 3.31 . Additionally, two singlets were observed at δH 6.44 (2H) and δH 6.38 (2H), referring to two pairs of symmetric phenyl hydrogens, H-2/H-6 and H-2'/H-6', respectively. From these absorptions it was possible to suggest an aryloxyarylpropane structure for compound 14, consistent with a neolignan skeleton. Through HMBC analysis, the methoxyl groups positions could be assigned by 3J1H-13C correlations showed between resonances at δH 3.81 (H3CO-3/5) with δC 153.64 (C-3/5) and δH 3.77 (H3CO-3'/5') with δC 152.85 (C-3'/5'). Also, hydrogen H-7'  showed 3J and 2J correlations with C-2'/6' (δC 105.94) and C-9' (δC 115.94), respectively, leading to the location of the allyl group at C-1'. Therefore, compound 14 was identified as 3,4,5,3',5'-pentamethoxy-1'-allyl-8.O.4'-neolignan [Compound 1H/13C-NMR data by means of one and two-dimensional techniques was also performed for the other compounds, which made possible their identification as sitosterol (3a), stigmasterol (3b), sitosterol-3-O-β-d-glucopyranoside [4a), stigmasterol-3-O-β-d-glucopyranoside (4b) [5) [2-hydroxyphaeophytin A (6) [3-ethoxypheophorbide A (7) [2-hydroxy-173-ethoxypheophorbide A (8) [m-methoxy-p-hydroxy-benzaldehyde (9) [10) [11) [12) [15) [16) [17) [O-β-d-glucosyl-6''-α-L-ramnopyranoside (18) [O-α-l-ramnopyranoside (19) [The spectral analysis using IR and ranoside  (4a), stide (4b) , phaeoph(4b) [5) , 132-hydin A (6) , 173-ethde A (7) , 132-hydde A (8) , m-methohyde (9) , (E)-7-((9) [10) , 1-benzo10) [11) , 7-hydro11) [12) , 3,5,7,312) [15) , 5,7-dih15) [16) , 5,8-dih16) [17) , kaempfeide (18) , and kaeide (19)  .The MIC values of compound 2 are also summarized in 2 to the highest concentration was 40%, therefore higher than that found with the compound 1 (10%). As for the antibiotics tested, they showed inhibitory effect on 60% of the strains, similar to compound 2.The MIC data of the compound 1 had the strongest antimicrobial effect, which could be explained by the lower MIC values against microorganisms [13 and 14 were also tested against the same strains and following the same protocol described for 1 and 2, but did not show any significant activity.Thus, one can conclude that both the tested compounds have detectable antimicrobial activity against a variety of pathogens, including bacteria and yeasts, in infectious processes relevant from a clinical point of view. It also may be noted the compound rganisms ,32,33,34C. exaltata. TLC was used to analyse and compare the fractions obtained from chromatographic column procedures. Solvents used in TLC were basically mixtures of the same solvents of the chromatographic experiments. For pure compounds analysed by TLC, three different systems of solvents were tested, to make sure that these compounds were free of impurities. The melting point of the constituents was recorded on a MQAPF-302 apparatus. IR spectra were recorded on a FT-IR-1750 Perkin-Elmer spectrometer. 1H and 13C-NMR spectra were recorded on a Varian Oxford 200 NMR spectrometer (200/50 MHz) and on a Varian 500 NMR spectrometer (500/125 MHz).Silica gel 60 (Merck) 7734 , flash silica  and Sephadex LH-20 were used for the fractionation and isolation of the secondary metabolites from Cordia exaltata Lam, were collected near the city of Porto Grande–AP in July 2006 and were identified by Prof. Dr. Rosangela do Socorro Ferreira Rodrigues Sarquis-IEPA . A voucher specimen is deposited in the Herbarium Amapaense/AP/BR (HAMAP), under the code 2528.The leaves, stems, fruits and stem bark of Cordia exaltata Lam were dehydrated in an oven at 40 °C for 72 h and ground separately in a mechanical mill to yield 847.5 g, 1000.5 g, 281.0 g and 2.310.0 g of a powder, respectively. Each powdered part of the plant was submitted to maceration with methanol  for three consecutive days at room temperature and this process was repeated until the maximum amount of chemical constituents had been extracted. The obtained methanol extract solutions were concentrated in a rotatory evaporator, yielding 150.0 g, 150.0 g, 11.0 g and 211.0 g of the respective crude extracts.Leaves, stems, fruits and stem bark of 1 and 2, respectively.An amount of the crude methanolic extract from fruits (12.0 g) was subjected to filtration under reduced pressure using silica gel 60 as stationary phase and eluted with hexane (hex.), ethyl acetate (EtOAc) and methanol (MeOH) alone or in binary mixtures following an increasing gradient polarity. After that, the extracts were concentrated under pressure, leading to the respective phases. The hexane phase (1695.0 mg) yielded 847.8 mg of a precipitate, which was chromatographed on a silica gel column and eluted with hex., EtOAc and MeOH. This procedure led to 28 fractions, analysed by TLC , from which the combined fractions 5/14 (200.0 mg) and 17/20 (170.0 mg) gave compounds C. exaltata Lam. (100.0 g) was suspended in methanol-H2O (9:1) and successively partitioned with hex., chloroform (CHCl3), EtOAc and n-butanol, increasing the gradient polarity. The hexane phase (10.0 g) was chromatographed under the same conditions as described previously for the crude extract of fruits, giving 80 fractions, which were combined by TLC . Fractions 1/19, after purification, led to 100.0 mg of a mixture of compounds 3a and 3b. Fraction 20/27 was chromatographed on a gel silica column, from which 84 fractions were obtained and analysed by TLC. From this chromatography, fractions 01/08, 09/18 and 28/36 were purified and this led to the isolation of compounds 4 (40 mg), 5 (100.0 mg) and 6 (100.0 mg), respectively. The CHCl3 extract (8.18 g) was also chromatographed following the described methodology, which allowed the isolation and purification of 30.0 mg of compound 11, 35.0 mg of compound 12 and 17.0 mg of 16. The EtOAc (3.7 g) and n-butanol (1.4 g) phases were submitted to chromatography on Sephadex LH-20, affording compound 15 (40.0 mg) and 19 (15.0 mg), respectively.The methanolic crude extract from the leaves of 2O (7:3) and successively partitioned under vacuum using silica gel with hex., CHCl3, EtOAc and n-butanol as described in the previous procedures. The hexane phase (5.0 g) was submitted to column chromatography with silica gel using hexane (hex.), ethyl acetate (EtOAc) and methanol (MeOH) alone or in binary mixtures with increasing polarity, leading to 93 fractions, which were further combined according to TLC . Using this technique fractions 16/24, 37/47 and 57/70 were pure and gave 25.0 mg of compound 8, 18.0 mg of compound 9 and 40.0 mg of compound 10. Through column chromatography on Sephadex LH-20, the EtOAc (2.9 g) and n-butanol (2.6) phases yielded compound 17 (15.0 mg) and compound 18 (30.0 mg), respectively.The methanolic crude extract from the stem bark (100 g) was solubilized in methanol-HC. exaltata Lam (7.5 g) was chromatographed on silica gel under reduced pressure with the following solvents: hex., hex-EtOAc (9:1), hex.-EtOAc (6:4), hex.-EtOAc (1:1), Hex.-EtOAc (4:6), AcOEt, EtOAc-MeOH (9:1) and EtOAc-MeOH (1:1). Fraction Hex.:AcOEt (6:4) (175.0 mg) was fractionated by column using gel silica and an increasing polarity system of solvents, such as Hex., EtOAc and MeOH, which result in 30 fractions. These fractions were analysed by TLC, using Hex.-EtOAc in three different mixtures of polarity. Fractions 02/04 and 08/11 were purified and led to the isolation of compound 13 (10.0 mg) and compound 14 (12.0 mg), respectively. The fraction eluted with EtOAc-MeOH (9:1) yield a supernatant and a precipitate. The latter was separated from the former, purified and analysed by TLC using EtOH-MeOH (9:1) as eluting solvent, which resulted in a mixture of compounds 4a and 4b (30 mg).The bark extract of 8.8'-Dimethyl-3,4,3', 4'-dimethylenedioxy-7-oxo-2,7'-cyclolignan (1). Colorless crystals; mp 176 °C; IR (KBr) vmáx (cm−1): 3081, 2963, 1678 (C=O), 1475; 1H-NMR : δ 7.55 , 6.95 , 6.80 , 6.62 , 6.60 , 5.93 , 5.83 , 3,80 , 2.41 , 1.09 , 1.93 , 0.90 ; 13C- NMR : δ 197.48 (C-7), 151.41(C-4), 147.00 (C-3'), 145.50 (C-4), 144.90 (C-3), 137.50 (C-1'), 126.84 (C-2), 122.05 (C-6), 121.83 (C-6'), 108.67 (C-2'), 107.82 (C-5'), 107.46 (C-5), 100.72 (OCH2O-A), 101.54 (OCH2O-C), 47.97 (C-7'), 46.48 (C-8'), 42.60 (C-8), 17.11 (C-9), 12.45 (C-9').8,8'-Dimethyl-4,5-dimethoxy-3',4'-methylenedioxy-7-oxo-2,7'cyclelignan (2). Fine white crystals; mp 131 °C; IR (KBr) vmáx (cm−1): 3081, 2963, 2850 (OCH3), 1678 (C=O), 1475; 1H-NMR : δH 7.47 , 6.24 , 6.81 , 6.70 , 6.59 , 5.94 , 3.83 , 3.74 , 3.58 , 2.39 , 2.04 , 1.26 , 0.93 ; 13C-NMR : δC 200.79 (C-7), 155.06 (C-4), 149.49 (C-3'), 149.10 (C-5), 143.51 (C-2), 138.89 (C-1'), 126.68 (C-1), 124.20 (C-5'), 112.57 (C-3), 110.14 (C-2'), 109.42 (C-6), 109.10 (C-6'), 102.40 (OCH2O-C), 56.17 (OCH3-4), 54.28 (C-7'), 49.52 (C-8), 44.98 (C-8'), 18.17 (C-9'), 12.87 (C-9).Sitosterol (3a) and stigmasterol (3b). 1H-NMR and 13C-NMR data were consistent with the literature values [e values .Sitosterol-3-O-β-d-glucopyranoside (4a) and stigmasterol-3-O-β-d-glucopyranoside (4b). 1H-NMR and 13C-NMR data were in agreement with the literature [terature .Phaeophytin A (5). IR, 1H-NMR and 13C-NMR data were in agreement with the literature [terature .2-Hydroxyphaeophytin A13 (6) (without phytyl ester). Green powder. IR (KBr) vmax: 3494, 2926, 2854, 1739, 1706, 1620, 1377 cm−1. 1H-NMR and 13C-NMR data were in agreement with the literature [terature .3-Ethoxyphaeophorbide A17 (7). 1H-NMR and 13C-NMR data were in agreement with the literature [terature .2-Hydroxy-173-ethoxyphaeophorbide A13 (8). Bluish green powder with a metallic luster. 1H-NMR and 13C-NMR data were in agreement with the literature [terature .m-Methoxy-p-hydroxybenzaldehyde (9). White crystals; mp 81 °C–82 °C. 1H-NMR and 13C-NMR data were in agreement with the literature [terature .Kaempferol 3,7-di-O-α-L-ramnopyranoside (19). Yellow crystals; mp 201 °C–203 °C. 1H-NMR and 13C-NMR data were in agreement with the literature [terature .(E)-7--7-propenoic acid (10). White solid; mp 220–221 °C. 1H-NMR : δH7.40 , 6.08 , 6.91 , 6.79 , 6.64 ; 13C-NMR : δC127.71 (C-1), 115.43 (C-2), 116.45 (C-5), 122.88 (C-6), 149.35(C-4), 146.66 (C-3), 171.12 (C-9), 114.98 (C-8), 147.04 (C-7).1-Benzopyran-2-one (11). White solid, mp 69–70 °C. 1H-NMR : δH 7.52 , 6.22 , 7.12 , 7.06 , 7.35  7.30 ; 13C-NMR : δC 160.89 (C-2), 116.96 (C-3), 116.76 (C-8), 118.90 (C-4a), 154.10 (C-8a), 143.57 (C-4), 131.93 (C-7), 127,97 (C-5), 124.53 (C-6).7-Hydroxy-1-benzopiran-2-one (12). White solid; mp 230–231 °C.1H-NMR : δH 7.87 , 6.22 , 7.48 , 6.83 , 6.75 ; 13C-NMR : δC 163.66 (C-2), 112.34 (C-3), 103.41 (C-8), 113.49 (C-4a), 157.21 (C-8a), 145.99 (C-4), 163.09 (C-7), 130,62 (C-5), 114.49 (C-6).2,5-bis--3,4-dimethyltetrahydrofurane (13). 1H-NMR : δH 6.94 , 6.86 , 6.78 , 5.94 , 4.40 , 2.24  and 0.99 , 13C-NMR : δC 147.92 (C-3' and 3''), 147.14 (C-4' and 4''), 136. 16 (C-1' and 1''), 120.10 (C-6' and C-6''), 108.15 (C-5' and 5''), 106.91 (C-2' and 2''), 101.09 (-OCH2O-), 87.59 (C-2 and 5), 44.80 (C-3 and C-4), 12.97 (C-CH3-3 and CH3-4).3,4,5,3',5'-Pentamethoxy-1'-allyl-8.O.4'-neolignan (14). 1H-NMR : δH 6.44 , 6.38 , 5.95 , 5'.09  5,05 , 4.33 , 3.81 , 3.80 , 3.77  3.31 , 3.10 , 2.72 , 1.20 , 0.93 ; 13C-NMR : δC 153.64 (C-3 and 5), 152.85 (C-1'), 137.24 (C-8'), 136.33 (C-4), 135.49 (C-4'), 134.89 (C-1), 134.35 (C-1'), 115.94 (C-9'), 106.58 (C-2 and 6), 105.94 (C-2' and 6'), 79.66 (C-8), 60.82 (C- H3CO-4), 56.08 (OCH2O-C), 56.17 (C- H3CO-3 and 5), 56.01 (C- H3CO-3' and 5'), 43.66 (C-7), 40.51 (C-7') and 19,76 (C- CH3-9). 3,5,7,3',4'-Pentahydroxyflavone (15). Yellow crystals, mp 313–314 °C. 1H-NMR  δH: 6.38 , 6.17 , 7.62 , 6.88 , 7.72 . 13C-NMR  δC: 148.01 (C-2), 137.23 (C-3), 177.36 (C-4), 162.50 (C-5), 99.27 (C-6), 165.62 (C-7), 94.43 (C-8), 158.24 (C-9), 104.51 (C-10), 124.16 (C-1'), 116.23 (C-2'), 146.21 (C-3'), 148.76 (C-4), 116.01 (C-5'), 121.68 (C-6').5,7-Dihydroxy-4’-methoxyflavone (16). Yellow crystals, m.p. 261–262 °C. 1H-NMR  δH: 6.86 , 6.20 , 6.50 , 8.03 , 7.10 , 3.85 . 13C-NMR  δC: 164.34 (C-2), 103.38 (C-3), 181.86 (C-4), 161.52 (C-5), 98.99 (C-6), 163.38 (C-7), 94.13 (C-8), 157.42 (C-9), 103.81 (C-10), 122.87 (C-1'), 128.40 (C-2'/C-6'), 114.67 (C-3'/C-5'), 162.38 (C-4), 55.64 (OCH3 C-4').5,8-Dihydroxy-7,4'-dimethoxyflavone (17). Yellow powder. mp 265–266 °C. 1H-NMR  δH: 6.84 , 6.53 , 8.09 , 7.12 , 3.88 , 3.85  and 12.41 . 13C-NMR  δC: 163.56 (C-2), 103.03 (C-3), 182.41 (C-4), 153.08 (C-5), 95.73 (C-6), 154.39 (C-7), 126.26 (C-8), 144.49 (C-9), 103.90 (C-10), 123.00 (C-1'), 128.51 (C-2'/C-6'), 114.59 (C-3'/C-5'), 162.41 (C-4'), 56.37 (OCH3 C-7) and 55.58 (OCH3 C-4').Kaempferold-glucosyl-6''-α-L-ramnopyranoside3-O-β- (18). Yellow powder; mp 220–221 °C. 1H-NMR : δH 8.08 , 6.92 , 6.42 , 6.23 , 5.15 , 3.30 , 3.84  3.47 , 3.68 , 3.55 , 3.36 , 3.29 , 1.16 . 13C-NMR : δC 159.36 (C-2), 135.51 (C-3), 179.35 (C-4), 162.92 (C-5), 100.02 (C-6), 166.15 (C-7), 94.96 (C-8), 158.24 (C-9), 105.59 (C-10), 122.73 (C-1'), 132.35 , 116.12 , 161.44 (C-4'), 104.64 (C-1''), 71.43 , 75,76 (C-4''), 69.71 , 72.07 (C-2'''), 72.30 (C-3'''), 77.18 (C-4'''), 73.90 (C-5'''), 17.90 (C-6''').S. aureus (ATCC 6538), S. aureus (ATCC 25923), S. epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633), P. aeruginosa (ATCC 25853), P. aeruginosa (ATCC 9027), E. coli , E. coli (ATCC 18739), E. coli (ATCC 8733), Shigella flexineri (LM 412), C. albicans (ATCC 90028), C. albicans , C. albicans (LM 142V), C. albicans (JCB 12), C. tropicalis , C. tropicalis (LM 028), C. krusei (ATCC 6258), C. krusei (12 LM), C guilliermondii (LM 2101) and C. guilliermondii (LM 011). The strains were acquired from the collection of the Mycology Laboratory (LM), Institute of Biomedical Sciences, University of São Paulo (ICB-USP/SP) and the Oswaldo Cruz Foundation-FIOCRUZ (Rio de Janeiro). The stock bacterial strains were maintained in Muller Hinton agar (AMH) and the yeast on Sabouraud dextrose agar (SDA) under refrigeration (8 °C). The inoculum of microorganisms was prepared and standardized in sterilized saline (0.85%), containing Tween 80 (1%). Turbidity was visually compared and adjusted to the range of 0.5 tube McFarland, which corresponds to an inoculum of approximately 106 CFU/mL (CFU/mL) [®) for the bacteria and ketoconazole  for yeasts.The microorganisms used througout the tests were: (CFU/mL) ,38,39. T(CFU/mL) . In each®) recognized as an indicator of oxidation-reduction colorimetric for bacteria. And in parallel, 20 μL of triphenyltetrazolium chloride (TTC) 1% (Sigma-Aldrich®), a colorimetric indicator for the redox yeast [The assay was performed in duplicate and incubated at 35 °C/24 h. After the incubation time was added 20 µL of resazurin sodium 0.01% (w/v) (Sigma-Aldrichox yeast ,42,43. TBiological properties of the compounds were considered either active or non-active according to the criteria reported ,32,33,34C. exaltata, resulted in nineteen compounds: two mixtures of steroids 3a, 3b, 4a and 4b, four neolignans 1, 2, 13 and 14, four porphyrins 5-8, two phenolic acids 9 and 10, two coumarins 11 and 12 and five flavonoids 15–19. For the first time compounds 4a/4b, 7, 11, 12, 13, 14, 16, 19 were isolated in Boraginaceae family, compounds 1, 2, 5, 6, 8, 9 and17 in genus Cordia and compounds 10, 15 and 18 in species C. exaltata. Compounds 1 and 2 showed significant antimicrobial activity against bacteria and yeasts, whereas compounds 13 and 14 did not show a relevant effect.The chemical study of the crude extracts from


Compound 1 exhibited melanogenesis inhibitory activities comparable with those of arbutin. In cytotoxicity assays, compounds 1 and 3 exhibited weak cytotoxicity, with IC50 values of 34.5–93.7 μM against HL-60 and P388 cells.Three new multiflorane-type triterpene esters,  Cucurbita moschata, C. pepo, and C. maxima, are gourd squashes of the genus Cucurbita and the family Cucurbitaceae. Cucurbita moschata seeds have been used as an anthelmintic + ion in the HREIMS data at m/z 586.4019 compatible with the molecular formula C39H54O4 , therefore it was suggested that the molecular formula of 1 is C39H56O5. The IR spectrum showed the presence of a hydroxy group (νmax 3437 cm−1) and ester carbonyl groups . The 1H and 13C-NMR spectra ], an oxymethylene , two oxymethines , a tetrasubstituted olefin , an acetoxy group , and a benzoyl group . The 1H- and 13C-NMR spectra are similar to those of 3-O-p-aminobenzoyl-29-O-benzoylmultiflor-8-ene-3α,7α,29-triol  to C-3 [δc 77.4 (d)], C-4, C-5, and C-24; Me-24 [δH 0.91 (s)] to C-3, C-4, C-5, and Me-23; Me-25 [δH 0.92 (s)] to C-1, C-5, C-9 [δC 140.1 (s)], and C-10; Me-26 [δH 1.03 (s)] to C-8 [δC 136.4 (s)], C-13, C-14, and C-15; Me-27 [δH 1.06 (s)] to C-12, C-13, C-14, and C-18; Me-28 [δH 1.12 (s)] to C-16, C-17, C-18, and C-22; H2-29 [δH 4.14 ] to C-19, C-20, C-21, C-30, and 29-OCO [δC 166.7 (s)]; Me-30 [δH 1.10 (s)] to C-19, C-20, C-21, and C-29; H-3 [δH 4.69 (t)] to 3-OCO [δC 170.9 (s)]; H-5 and H-6α to C-7 [δC 64.3 (d)]; H-6α and H2-11 to C-8 [δC 136.4 (s)]; H-11 and H-12δ to C-9 [δC 140.1 (s)]. In the 1H-1H COSY experiment, H-7 [δH 4.16 (brs)] correlated with H2-6  ] and NOEs between H-3 and Me-24. Therefore, the structure of 1 was determined to be as shown in Compound  spectra  displaye0, 1.74] . The folβ, H-21β . In addiβ, H-21β . The con2 exhibited a [M]+ ion in the HREIMS data at m/z 680.4447 compatible with the molecular formula C45H60O5 . The IR spectrum showed absorption indicating ester carbonyl groups . The 1H- and 13C-NMR spectra ], an oxymethylene , two oxymethines , a tetrasubstituted olefin , and two benzoyl groups . The above data suggested that the structure of 2 is similar to that of 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate  to 29-OCO [δC 166.6 (s)]; H-3 [δH 4.95 (t)] to 3-OCO [δC 166.3 (s)]; H-5 and H-6α to C-7 [δC 73.8 (d)]; H-6α, H-7β, and Me-26 to C-8 [δC 135.3 (s)]; H-7β, H2-11 and Me-25 to C-9 [δC 139.7 (s)]. In the 1H-1H COSY experiment, H-7 [δH 3.54 (brs)] correlated with H2-6  ]. Therefore, 2 was established as shown in Compound  spectra  displaye4, 1.95] . Additio4, 1.95] . The con3 exhibited a [M]+ ion in the HREIMS data at m/z 680.4446 compatible with the molecular formula C45H60O5 . The IR spectrum showed absorption indicating ester carbonyl groups . The 1H- and 13C-NMR spectra ], an oxymethylene , two oxymethines , a tetrasubstituted olefin , and two benzoyl group . The 1H- and 13C-NMR spectra of 3 were very similar to those of 2, except for the H-7 signal . The coupling constants of H-7, and the NOE correlations of H-7/H-5α, and H-15α; 7-OMe/Me-26 suggested that the methoxy group at C-7 is in the β -orientation ]. The above data established that 3 was a 7β-methoxy epimer of 2 . In the melanogenesis inhibitory assay, compound 1 reduced melanin content (88.5%) at a non-toxic concentration, 10 μM. The melanogenesis inhibitory activity of compound 1 was comparable with that of the positive control, arbutin (melanin content 88.9% at 10 μM), which has been recognized as a useful depigmentation compound for skin whitening in the cosmetic industry  at 25 °C with n-hexane/EtOAc , and on ODS column  at 25 °C with Me2CO:H2O .Chemicals and reagents were purchased as follows: fetal bovine serum (FBS) from Invitrogen , 3--2,5-diphenyl-2Cucurbita maxima, produced in India, were purchased from Takada Seeds Co., Ltd.  in 2011. A voucher specimen was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.The seeds of Cucurbita maxima (10 kg) produced in India, were subjected to extraction with MeOH under reflux . The MeOH extract (310 g) was then partitioned between Et2O and H2O. The Et2O-soluble fraction (150 g) was subjected to SiO2 column chromatography (CC)  resulting in 9 fractions (Fr. A–I). Fr. B, eluted with CHCl3, was subjected to SiO2 CC to yield 18 fractions, B1–B18. Preparative HPLC of B7 (42.5 mg) (HPLC system II), eluted with hexane/EtOAc (10:1), gave 4 . Fr. C, eluted with CHCl3, was subjected to SiO2 CC to yield 24 fractions, C1–C24. Preparative HPLC of C5 (67.9 mg)(HPLC system II), eluted with hexane/EtOAc (10:1), gave Fr. C5-4 , and then re-preparative HPLC gave 2 (HPLC system I). Preparative HPLC (HPLC system II) of C6 (64.0 mg), eluted with hexane/EtOAc (10:1), gave 15 fractions; C6-1–C6-15, and preparative HPLC (HPLC system II) of C7 (15.4 mg), eluted with hexane/EtOAc (10:1), gave 15 fractions; C7-1–C7-15. Preparative HPLC of C6-6  and C7-6 (0.4 mg) combination gave 3 (HPLC system IV). Fr. E, eluted with CHCl3, was fractionated with SiO2 CC to E1–E11. Preparative HPLC (HPLC system III) of E8 (119.8 mg), eluted with hexane/EtOAc (5:1), gave Fr. E8-10 , and then re-preparative HPLC (HPLC system V) gave 1 .The seeds of 7α-Hydroxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (1): Colorless crystal (MeOH); mp 66–68 °C;  −85.8 ; UV (EtOH) λmax (logε) 205.0 (3.80), 220.0 (3.94), 232.5 (3.98), 270.5 (3.41), 280.0 (3.32), 321.0 (2.87) nm; IR (KBr) νmax 3437, 2938, 2876, 1718, 1272, 1244, 1110, 750, 729, 710 cm−1; 1H and 13C-NMR data see m/z 586 [M–H2O]+ (26), 540 (33), 527 (40), 511 (100), 389 (12), 387 (9), 253 (15), 225 (16); HREIMS m/z 586.4019 .7α-Methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (2): Colorless crystal (MeOH); mp 88–90 °C;  −20.7 ; UV (EtOH) λmax (logε) 238.5 (3.70), 271.5 (3.21), 278.5 (3.11) nm; IR (KBr) νmax:2948, 2883, 1717, 1456, 1367, 1314, 1274, 1113, 1069, 716 cm−1;1H and 13C-NMR data see m/z 680 (6) [M]+, 648 [M–MeOH]+ (12), 526 (25), 511 (100), 389 (8), 355 (10), 324 (8); HREIMS m/z 680.4447 .7β-Methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (3).Colorless crystal (MeOH); mp 67–68 °C;  −43.7 ; UV (EtOH) λmax (logε) 206.5 (3.86), 220.0 (3.97), 235.0 (4.00), 271.0 (3.29), 280.0 (3.16) nm; IR (KBr) νmax:3437, 1717, 1272, 1110, 1028, 975, 711 cm−1; 1H and 13C-NMR data see +, 665 (38), 648 [M–MeOH]+ (14), 526 (29), 511 (100), 393 (18), 381 (18), 354 (28); HREIMS m/z 680.4446 [M]+ .2 humidified incubator.The cell lines HL-60 (human leukemia) and P388 (murine leukemia) were grown in RPMI 1640 medium, while B16 4A5 cells were grown in D-MEM. The medium was supplemented with 10% FBS and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). The cells were incubated at 37 °C in a 5% COThe assay of B16 4A5 cells proliferation was examined according to a method reported previously . The assay of melanin content was performed as described previously .The cytotoxicity assay was determined previously .i.e. 7α-hydroxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (1), 7α-methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (2), and 7β-methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (3), from pumpkin seeds. Isolated compounds were evaluated for melanogenesis inhibitory and cytotoxic activities. In the melanogenesis inhibitory assay, we revealed that compound 1 possessed melanogenesis inhibitory activities comparable with arbutin at a non-toxic concentration. In a cytotoxicity assay against cancer cell lines, none of the compounds showed remarkable cytotoxic activities. We will continue to explore other biological activities of multiflorane-type triterpenes.In this study, we isolated three new multiflorane-type triterpene esters,


Laurencia johnstonii collected in Baja California Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed us to identify the amoebicidal diastereoisomers α-bromocuparane (4) and α-isobromocuparane (5). Furthermore, bromination of the inactive laurinterol (1) and isolaurinterol (2) yielded four halogenated derivatives, (6)–(9), which improved the activity of the natural sesquiterpenes. Among them, the most active compound was 3α-bromojohnstane (7), a sesquiterpene derivative which possesses a novel carbon skeleton johnstane.Focused on our interest to develop novel antiparasistic agents, the present study was aimed to evaluate the biological activity of an extract of  Acanthamoeba genus + at m/z 371.9711/373.9702/375.9677 , indicative of two bromine atoms and six degrees of unsaturation. The structure of (6) was determined by comparison of its NMR data with those for aplysin (3) (1H NMR spectra were the absence of one of the two aromatic singlets of (3), H-8 (δH 6.59), and a deshielded aromatic methyl group H3-15 (δH 2.49) for (6) compared with that of 3 H3-15 (δH 2.30). These values agree with the presence of a bromine atom at C-8 in the aromatic ring as well as with the information provided by mass spectrometry.Compound (ysin (3) . The mai3) was easily obtained after treatment of laurinterol (1) with HBr (1) ([α]3 ([α]1): [α]3: [α]3-12, H3-13, H3-14 and H-3 between 6 and aplysin (3)  , the absby GC-MS . Later,  species  refers 67 was obtained as a colorless oil. The HREIMS analysis provided four molecular ions [M]+ at m/z 449.8840/451.8800/453.8798/455.8758  suggesting a molecular formula C15H17OBr3 and six degrees of unsaturation. The structure of 7 was established based on the analysis of its spectroscopic data and comparison with those of 6. 1D and 2D NMR spectra evidenced three methyl, three methylenes, and two methine groups (one on sp2 carbon and one bearing heteroatom), in addition to five sp2 and two sp3 quaternary carbons. Similar to 6, the pentasubstituted phenyl ring was confirmed by the presence of a singlet at δH 7.14 (H-12) and the methyl group at δH 2.53 (H3-15) in the 1H NMR spectrum  and H3-14  to a quaternary carbon at δC 92.3 (C-2), and from H3-14 to C-7 (δC 136.3) confirming the presence of the same dihydrofuran ring found in 6. The main difference between both compounds was a COSY spin system from the diasterotopic protons H2-3 (δH 2.42/1.99), sequentially coupled with the bromomethine H-4 (δH 3.84), methylene H2-5 (δH 2.18/1.76), and methylene H2-6 (δH 2.18/1.54). HMBC correlations from H3-14 (δH 1.04) to C-6 (δC 32.8) and H3-13 (δH 2.54) to C-3 (δC 47.6) led to the connection of this substructure within the molecule and thus established the planar structure of the rearranged sesquiterpene 7 as shown in Compound spectrum , and the3-13 to H3-14 and H-4, and from H-4 to the diastereotopic proton H-3β located all these protons on the same face of the molecule. Since the absolute configuration of laurinterol (1) has been confirmed and configuration at C-1 is conserved in 7, it is possible to establish the absolute configuration of its chiral centers as 1S, 2S, and 4S.NOE correlations observed from H3) and other related sesquiterpenes − , 295.0518 [M − H]−  1H NMR  δ 0.55 , 0.58 , 1.15 , 1.28 , 1.32 , 1.41 , 1.66 , 1.95 , 2.09 , 2.29 , 5.26 , 6,61 , 7.61 ; 13C NMR  δ 16.2 (C-12), 18.6 (C-13), 22.2 (C-14), 23.5 (C-15), 24.4 (C-3), 25.3 (C-4), 29.6 (C-2), 35.9 (C-5), 114.9 (C-10), 118.8 (C-8), 132.3 (C-11), 134.0 (C-6), 135.9 (C-9), 153.3 (C-7).White crystal; [α]c 0.14, CH2Cl2); HRESIMS m/z 293.0536 [M − H]− , 295.0528 [M − H]−  1H NMR  δ 1.21 , 1.42 , 1.46 , 1.59 , 2.05 , 2.20 , 2.31 , 2.85 , 4.94 , 5.11 , 5.56 , 6.73 , 7.45 ; 13C NMR  δ 21.2 (C-14), 22.2 (C-15), 27.8 (C-12), 31.2 (C-4), 37.6 (C-3), 39.1 (C-5), 49.8 (C-1), 106.9 (C-13), 115.5 (C-10), 120.4 (C-8), 131.2 (C-11), 132.7 (C-6), 137.2 (C-9), 153.0 (C-7), 165.4 (C-2).Colorless amorphous solid; [α]c 0.19, CH2Cl2); HRESIMS m/z 293.0551 [M − H]− , 295.0529 [M − H]− . 1H and 13C NMR data ; HREIMS [M − H]+m/z 279.0744 , 282.0723 , [M − Br]+m/z 201.1640 ; 1H NMR  δ 0.62 , 1.09 , 1.43 , 1.96 , 2.24 , 2.27 , 2.32 , 2.52 , 4.07 , 7.09 ; 13C NMR  δ 20.7 (C-15), 20.9 (C-13), 22.4 (C-12), 25.0 (C-14), 33.1 (C-4), 36.4 (C-5), 47.4 (C-2), 48.4 (C-1), 62.0 (C-3), 127.3 , 128.1 , 135.4 (C-9), 143.9 (C-6).White amorphous solid; [α]c 0.14, CH2Cl2); HREIMS [M]+m/z 280.0826 , 282.0801 , [M − Br]+m/z 201.1649 ; 1H NMR  δ 0.65 , 1.10 , 1.28 , 1.61 , 2.20 , 2.33 , 2.51 , 2.71 , 4.46 , 7.12 , 7.13 , 7.27 ; 13C NMR  δ 20.6 (C-15), 20.8 (C-13), 22.2 (C-12), 25.3 (C-14), 31.2 (C-4), 33.5 (C-5), 47.7 (C-2), 48.4 (C-1), 63.6 (C-3), 126.5 , 128.5 , 135.4 (C-9), 143.0 (C-6).Colorless oil; [α]1, 8.0 mg, 0.027 mmol) was dissolved in ethyl ether (5 mL) and bromine  was added. The reaction was left under magnetic stirring for 15 min at room temperature, after which the solvent was evaporated in vacuo. The reaction mixture was purified on a silica gel 0.2–0.5 mm  column (10 Ø × 50 mm) using a step gradient of n-Hex/EtOAc (100:0–95:5) to yield 8-bromoaplysin  and 7 . Similarly, isolaurinterol  was treated with bromine  in the same experimental conditions. After evaporation of the solvent, the reaction mixture was purified on a silica gel column ) to give compound 6 , 8  and 9 .Laurinterol  was dissolved in ethyl ether (5 mL) and HBr  was added. The reaction was left under magnetic stirring for 15 min at room temperature, after which the solvent was evaporated in vacuo. Quantitative conversion was observed to aplysin (3) which was purified by crystallization in n-hexane.Laurinterol ; IR υmax 3264, 1652, 1315, 1103, 1022, 904 cm−1; HREIMS [M]+m/z 371.9711 , 373.9702 , 375.9677 ; 1H and 13 C NMR data ; IR υmax 3259, 2362, 2158, 2031, 1975, 906, 800 cm−1; HREIMS [M]+m/z 449.8840 , 451.8800 , 453.8798 , 455.8758 ; 1H and 13 C NMR data ; HREIMS [M]+m/z 449.8836 , 451.8815 , 453.8783 , 455.8764 .Colorless oil; [α]c 0.08, CHCl3); HREIMS [M]+m/z 387.9680 , 389.9667 , 391.9639 .Colorless oil; [α]Acanthamoeba castellanii Neff (ATCC 30010) which was axenically grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 μg/mL of gentamicin .The amoeba strain used in this study was the type strain: 2 atmosphere, was used for the cytotoxicity assays.The murine macrophages J774A.1 (ATCC# TIB-67) cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C and 5% COAcanthamoeba assays were determined using the alamarBlue® method as previously described [Acanthamoeba strain was seeded in triplicate on a 96-well microtiter plates with 50 μL from a stock solution of 104 cells/mL. Amoebae were left to adhere to the bottom of the well for 15 min, which was checked using a Leika DMIL inverted microscope . After that, 50 μL of serial dilutions of the molecules were added to the wells (1% DMSO was used to dissolve the highest dose of the compounds with no effects on the parasites). As a control, we use chlorhexidine , which is a standard antiseptic belonging to the biguanide family of antiseptics that is commonly used in contact lens maintenance solutions; and voriconazole (Sigma) is an inhibitor of ergosterol synthesis that has been proven previously to be highly effective against clinical strains of Acanthamoeba [®  was placed into each well at an amount equal to 10% of the final volume. Then, plates containing alamarBlue® were incubated for 96 h at 26 °C with a slight agitation.To evaluate the biological activity of the fractions and molecules, the anti-escribed ,16. Firsthamoeba ,27. Fina® method as previously described [5 cells/mL. Macrophages were left to adhere to the bottom of the well for 15 min, which was checked using a Leika DMIL inverted microscope . After that, 50 μL of serial dilutions of the molecules were added to the wells. Finally, the alamarBlue Reagent®  was placed into each well at an amount equal to 10% of the final volume. Then, plates containing alamarBlue® were incubated for 24 h at 37 °C in presence of CO2 at 5%.The cytotoxicity assay was carried out using the alamarBlueescribed ,16. Firs® Multimode Plate Reader  using fluorescence, a test wavelength of 570 nm and a reference wavelength of 630 nm. The percentage of the growth inhibition, 50% inhibitory concentration (IC50 or CC50) was calculated by linear regression analysis with 95% confidence limits using Sigma Plot 12.0 statistical analysis software (Systat Software). All experiments were performed three times, and the mean values were also calculated. A paired two-tailed t-test was used for analysis of the data. Values of p < 0.05 were considered significant.Briefly, the test plates were analyzed, during an interval of time between 24 and 96 h, on an EnSpireAcantamoeba activity of the brominated sesquiterpenes α-bromocuparane (4) and α-isobromocuparane (5), isolated from Laurencia johnstonii, is reported. On the other hand, after chemical modification of the inactive metabolites laurinterol (1) and isolaurinterol (2), a set of brominated derivatives, 6–9, that substantially improve the activity of the natural sesquiterpenes have been obtained. The addition of a second bromine atom in the aromatic ring of 6–9 seems to be relevant to increase the antiamoeboid activity. Whereas all obtained derivatives possess a laurane-type framework, the most active compound, 3α-bromojohnstane (7), possesses a different rearranged carbon skeleton, johnstane, never found among natural sesquiterpene metabolites isolated from Laurencia species so far. Our results suggest that Laurencia-based brominated sesquiterpenes could be a potential source of novel therapeutic agents against Acanthamoeba in the near future.To the best of our knowledge, red algae have never been evaluated as a potential source of amoebicidal agents. Furthermore, this is the first time that the anti-


In this work, we describe a study on the biotransformation of seven natural occurring apocarotenoids by means of eleven selected fungal species. The substrates, namely ionone , 3,4-dehydroionone, damascone (α- and β-isomers) and theaspirane are relevant flavour and fragrances components. We found that most of the investigated biotransformation reactions afforded oxidized products such as hydroxy- keto- or epoxy-derivatives. On the contrary, the reduction of the keto groups or the reduction of the double bond functional groups were observed only for few substrates, where the reduced products are however formed in minor amount. When starting apocarotenoids are isomers of the same chemical compound  their biotransformation can give products very different from each other, depending both on the starting substrate and on the fungal species used. Since the majority of the starting apocarotenoids are often available in natural form and the described products are natural compounds, identified in flavours or fragrances, our biotransformation procedures can be regarded as prospective processes for the preparation of high value olfactory active compounds. GC-MS (EI): m/z (%) = 250 [M+] (<1), 235 [M+ − Me] (<1), 190 (28), 175 (29), 157 (14), 147 (100), 131 (32), 119 (20), 105 (39), 91 (27), 79 (15), 69 (14), 55 (8).3-Hydroxy-γ-ionone acetate  of compound 28 as a 5:1 mixture of trans/cis isomers:The biotransformation of 3,4-dehydro-β-ionone performed using trans-3,4-Dihydroxy-β-ionone diacetate (28) = -3,5,5-Trimethyl-4-((E)-3-oxobut-1-en-1-yl)cyclohex-3-ene-1,2-diyl diacetate. 1H-NMR: δ = 7.10 , 6.11 , 5.51 , 5.17–5.09 , 2.28 , 2.06 , 2.00 , 1.88–1.66 , 1.62 , 1.17 , 1.07 . 13C-NMR: δ = 197.8 (C), 170.7 (C), 170.4 (C), 141.0 (CH), 140.2 (C), 133.7 (CH), 128.5 (C), 74.7 (CH), 70.3 (CH), 41.4 (CH2), 36.4 (C), 29.7 (Me), 27.7 (Me), 27.5 (Me), 21.1 (Me), 20.8 (Me), 16.5 (Me). MS (ESI): 331.2 (M+ + Na).Curvularia lunata allowed isolating 95 mg (7% yield) of compound 35 and 65 mg (5% yield) of compound 36:The biotransformation of theaspirane performed using 2-ethyl acetate (35). 1H-NMR: δ = 5.13 , 4.05 , 2.46–2.37 , 2.06 , 1.92–1.79 , 1.74–1.52 , 1.66 , 1.44–1.33 , 1.08 , 1.00 . 13C-NMR: δ = 171.0 (C), 171.0 (C), 139.5 (C), 128.2 (C), 72.3 (CH), 63.3 (CH2), 35.1 (C), 34.6 (CH2), 28.2 (Me), 28.0 (CH2), 26.8 (Me), 25.3 (CH2), 21.3 (Me), 21.0 (Me), 16.8 (Me). GC-MS (EI): m/z (%) = 268 [M+] (<1), 226 (13), 208 [M+ − AcOH] (16), 166 (40), 148 (17), 133 (100), 120 (28), 110 (36), 91 (16), 79 (8).3,6,6-Trimethyl-1,4,5,6,7,8-hexahydro-3H-3,5a-epoxybenzo[c]oxepin-8-yl acetate (36). 1H-NMR: δ = 5.43–5.36 , 5.31 , 4.49 , 4.20 , 2.30–1.40 , 2.02 , 1.47 , 1.11 , 1.01 . 13C-NMR: δ = 170.5 (C), 138.7 (C), 116.3 (CH), 105.3 (C), 86.0 (C), 68.4 (CH), 63.7 (CH2), 40.7 (CH2), 35.3 (C), 34.3 (CH2), 30.5 (CH2), 24.6 (Me), 24.4 (Me), 23.3 (Me), 21.2 (Me). GC-MS (EI): m/z (%) = 266 [M+] (1), 236 (50), 224 (8), 207 [M+ − AcO] (59), 195 (36), 178 (27), 164 (84), 153 (100), 136 (61), 121 (59), 107 (62), 91 (53), 79 (29).13 apocarotenoids ionone, theaspirane and damascone. With respect to the eleven strains tested, we observed that the most common chemical transformations are oxidation reactions that afford oxygenated products such as hydroxy- keto- or epoxy-derivatives. On the contrary, the reduction of the keto groups or the reduction of the double bond functional groups are less relevant transformations, occurring for few substrates and yielding a minority amount of products.Our work provides some relevant findings. First, we demonstrated that fungi are able to perform different biotransformations on the isomeric forms of the CA very significant feature of our study concern the prospective applicability of the fungi-mediated biotransformation of apocarotenoids for the synthesis of high value natural flavours. Since some ionone, damascone and theaspirane isomers are available in natural form and the biotransformation of a natural precursor is considered a ‘natural method’ of synthesis, the flavours obtained by means of the fungi-mediated reactions described above possess the natural status and could be commercialized accordingly. 36) and we devised a new biocatalytic procedure for the synthesis of 2-hydroxy-γ-ionone, 3-hydroxy-γ-ionone, 3,4-dihydroxy-β-ionone and 2,6,10,10-tetramethyl-1-oxa-spiro[4.5]dec-6-ene-2,8-diol (identified as its monoacetate 34), which are natural apocarotenoids or their direct precursors. Finally, we would like to highlight that our microbial biotransformations allow the preparation of many derivatives whose synthesis, using the classical chemical reactions, is very difficult. For example, different fungal strains proved to be able to oxidize some inactivated positions of the ionone or theaspirane framework, such as the position 2 and 3 of the β- and γ-ionone and the methine carbon linked to the theaspirane oxygen atom. By means of these microbial capabilities, we isolated one new compound (compound