N-substituted benzisoselenazol-3(2H)-ones and their non-selenium-containing analogues have been synthesized and tested against selected viruses  to determine the extent to which selenium plays a role in antiviral activity. The data presented here show that the presence of selenium is crucial for the antiviral properties of benzisoselenazol-3(2H)-ones since their isostructural analogues having different groups but lacking selenium either did not show any antiviral activity or their activity was substantially lower. The open-chain analogues of benzisoselenazol-3(2H)-ones—diselenides also exhibited high antiviral activity while selenides and disulfides were completely inactive towards model viruses.Various  Received: 29 October 2010; in revised form: 5 November 2010 / Accepted: 9 November 2010 / Published: 12 November 2010H)-ones and diselenides have been successfully developed -selenophenes, which are more stable forms of benzo[b]selenophen-3(2H)-ones 5 -ones 5  9,10]. ,10. N-suN-substituted benzisothiazol-3(2H)-ones 2 the analogous reaction of 2-(chlorosulfanyl)benzoyl chloride (12), obtained in a two-step synthesis starting from 2,2’-dithiodibenzoic acid (14) thiophen-3(2H)-ones or/and 3-hydroxybenzo[b]thiophenes selenophenes (more stable forms of benzo[b]selenophen-3(2H)-ones 5) have been found to be inactive towards tested viruses (MIC >1000 μg/mL) while the corresponding benzisoselenazol-3(2H)-ones  exhibited high activity against HHV-1 and EMCV (MIC in a range of 4.0-10.0 μg/mL) ) and low activity against VSV (MIC = 600 μg/mL). Thus, it can be supposed that antiviral activity similarly to other biological activities of benzisoselenazol-3(2H)-ones is related to the presence of the Se-N bond which can undergo cleavage.From compared . 3-Hydro6 exhibited virucidal activity towards HHV-1 and EMCV in the same range that corresponding benzisoselenazol-3(2H)-ones 1 (MIC in a range of 2.0-10.0 μg/mL). Only two diselenides 6f, 6g have been found inactive against EMCV. Like the benzisoselenazol-3(2H)-ones, none of tested diselenides exbibited antiviral activity against VSV. The replacement of the Se-Se bond in diselenides by the S-S one resulted in complete loss of antiviral activity. The corresponding selenides 8 have been found completely inactive against all tested viruses as well  or a 600 MHz Bruker AVII spectrometer (77Se-NMR). Chemical shifts are reported in ppm relative to TMS or dimethyl selenide. Reaction progress was monitored by a thin layer chromatography (TLC) on silica gel 60F254 coated aluminium TLC plates from Merck.All reagents and solvents were purchased from Sigma-Aldrich or Fluka. Melting points were determined in open glass capillaries with an Electrothermal IA 9100 digital melting point apparatus. IR spectra were measured on a Perkin Elmer 2000 FT-IR spectrophotometer in KBr pellets or in thin films and peaks are reported in cm1a–g ,,1a–g 1a–g 2d–j. 2-n 3a 3a ,,6a–g was,,6a–g wa [6f [6g  were preBis[2-(N-ethylcarbamoyl)phenyl] diselenide (6c). 79% yield, white powder, m.p. 210-212 °C, 1H-NMR (DMSO-d6) δ: 1.17 , 3.30-3.38 , 7.29-7.40 , 7.69 , 7.78 , 8.71 ; 77Se-NMR (DMSO-d6) δ: 455; Anal. Calcd for C18H20N2O2Se2: C, 47.59; H, 4.44; N, 6.17. Found: C, 47.86; H, 4.47; N, 6.14.Bis[2-(N-n-propylcarbamoyl)phenyl] diselenide (6d). 79% yield, white powder, m.p. 223-224 °C, 1H-NMR (DMSO-d6) δ: 0.93 , 1.52-1.64 , 3.23-3.30 , 7.29-7.40 , 7.69 , 7.78 , 8.71 ; 77Se-NMR (DMSO-d6) δ: 455; Anal. Calcd for C20H24N2O2Se2: C, 49.80; H, 5.02; N, 5.81. Found: C, 49.93; H, 4.98; N, 5.83.Bis[2-(N-n-butylcarbamoyl)phenyl] diselenide (6e). 89% yield, white powder, m.p. 181-183 °C, 1H-NMR (DMSO-d6) δ: 0.93 , 1.31-1.43 22CHCH3), 1.50-1.60 , 3.26-3.33 2CH3), 7.29-7.39 , 7.69 , 7.77 , 8.69 ; 77Se-NMR (DMSO-d6) δ: 455; Anal. Calcd for C22H28N2O2Se2: C, 51.77; H, 5.53; N, 5.49. Found: C, 51.84; H, 5.52; N, 5.46. 15 has been obtained according to the procedure described in references cited  disulfide (7b), bis[2-(N-ethylcarbamoyl)phenyl] disulfide (7c), bis[2-(N-n-propylcarbamoyl)phenyl] disulfide (7d), bis[2-(N-n-butylcarbamoyl)phenyl] disulfide (7e), bis[2-(N-phenylcarbamoyl)phenyl] disulfide (7f), bis[2-(N-benzylcarbamoyl)phenyl] disulfide (7g). General procedure: To a stirred solution of corresponding amine (8.80 mmol) in dry dichloromethane (25 mL), a solution of chloride 15  in dry dichloromethane (15 mL) was added dropwise over 30 min in room temperature. After the reaction has finished the solvent was evaporated in vacuo and the crystalline residue was treated with water (70 mL) and stirred for 4 h. The insoluble solid was filtered off, washed with water and dried in the air. Crude products were recrystalized from dioxane , ethanol (7c–e) or ethyl acetate (7g). Yields for 7a–g: 87%, 59%, 61%, 73%, 85%, 87%, 63% respectively. The compounds 7a 15 has be7b 15 has be7e  selenide (8b). 82% yield, colourless needles, m.p. 239.5-242 °C, 1H-NMR (DMSO-d6) δ: 2.84 , 7.22-7.36 , 7.57 , 7.93 ; 77Se-NMR (DMSO-d6) δ: 440; νmax (KBr): 3360, 3319 (NH), 1650, 1626 (CO); Anal. Calcd for C16H16N2O2Se: C, 55.34; H, 4.64; N, 8.07. Found: C, 54.13; H, 4.38; N, 7.84.Bis[2-(N-n-propylcarbamoyl)phenyl] selenide (8d). 90% yield, white powder, m.p. 180-182 °C (decomp.), 1H-NMR (DMSO-d6) δ: 0.88 , 1.43-1.55 , 3.13-3.19 , 7.21-7.36 , 7.53 , 8.42 ; 77Se-NMR (DMSO-d6) δ: 438; νmax (KBr): 3350, 3261 (NH), 1653, 1627 (CO); Anal. Calcd for C20H24N2O2Se: C, 59.55; H, 6.00; N, 6.94. Found: C, 59.41; H, 6.01; N, 6.86.Bis[2-(N-phenylcarbamoyl)phenyl] selenide (8f). 98% yield, white powder, m.p. 238-240 °C (decomp.), 1H-NMR (DMSO-d6) δ: 7.09 , 7.31-7.35 , 7.38-7.40 , 7.42-7.45 , 7.69-7.72 , 10.43 ; 77Se-NMR (DMSO-d6) δ: 434; νmax (KBr): 3310 (NH), 1664 (CO); Anal. Calcd for C26H20N2O2Se: C, 66.24; H, 4.28; N, 5.94. Found: C, 65.70; H, 3.95; N, 5.85.2 in air. The cells were plated at a density of 2 × 105 cells/mL on 96-well plates (Costar-Corning) in 100 µL of fresh media the day before treatment with compounds.Human lung adenocarcinoma A549 (ATCC 185) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutammine, 5% heat-inactivated foetal bovine serum (FBS), 100 U/mL penicilin and 100 µg/mL streptomycin  at 37 ºC in the atmosphere of 5% CO1-8 at various concentrations  were incubated with following viruses: HHV-1 , EMCV  and VSV . Viruses EMCV and VSV were used at the dose of 107 TCID50/mL and HHV-1 at the dose of 105 TCID50/mL. After 1 hour incubation at room temperature, the virus titer was measured in human cell line A549. Concentration of compound causing 1000 times decrease of virus titer was taken as minimal inhibitory concentration MIC (µg/mL). The compounds 2 in air. Then, the cultures were observed for cytotoxic effects. The minimal concentration of compound which was toxic to approximately 50% of cells was taken as TCCD50 (tissue culture cytotoxic dose). Final concentrations of compounds used in virucidal activity assay were below TCCD50 value that means they were not toxic for the cells.The cytotoxicity of compounds was carried out with human lung adenocarcinoma cell line A549 (ATCC 185). The cells were incubated with serially diluted compounds for 48 hours at 37 ºC in the atmosphere of 5% COH)-one ring of benzisoselenazol-3(2H)-ones 1 allowed us identify the fragment which is necessary for the antiviral activity of these compounds. The comparison of antiviral properties of benzisoselenazol-3(2H)-ones 1 and their isostructural analogues that do not contain selenium 2–4 clearly indicates the crucial role of this element for antiviral activity. The replacement of selenium by other atoms or functional groups drastically diminished the antiviral activity. While benzisoselenazol-3(2H)-ones 1 exhibited high antiviral activity directed towards HHV-1 and EMCV their non-selenium-containing analogues were completely inactive 3, 4 or their activity was substantially lower (compounds 2). The possible mechanism of action probably requires the cleavage of the Se-N bond since compounds having the Se-C bond instead of it did not exhibit any antiviral activity.Structural modifications that have been done in the isoselenazol-3-ones 1 exhibit similar antiviral activity to the cyclic compounds, whereas the corresponding disulfides 7 and selenides 8 were completely inactive towards the model viruses. Our study has also demostrated that bis(2-carbamoylphenyl)diselenides N-substituted isoindolin-1-ones 3 has been elaborated. It has been found that N-acylation of primary amines by chlorocarbonyl group of 2-(chloromethyl)benzoyl chloride (13), followed by their N-alkylation in the presence of DBU resulted in the pyrrolidone ring closure. Based on these reactions the simple, general one-pot procedure for synthesis of N-substituted isoindolin-1-ones 3 has been established.During our studies on the influence of selenium on antiviral activity a new synthetic approach to


O-[(α-L-rhamnopyranosyl)(1→2)]-[β-D-glucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid (1) and a new indole alkaloid, 5-methoxy-2-oxoindolin-3-acetic acid methyl ester (5) were isolated from the leaves of Acanthopanax senticosus Harms along with six known compounds. The structures of the new compounds were determined by means of 2D-NMR experiments and chemical methods. All the isolated compounds were evaluated for their glycosidase inhibition activities and compound 6 showed significant α-glucosidase inhibition activity.A new triterpene glycoside, 3- Acanthopanax senticosus (Rupr. Maxim) Harms, a member of the Araliaceae family, is a shrub found mainly in the northeast of China, Korea and Japan. It usually grows up to 2 m in height and generally has prickly stems bearing five leaflets  and umbel-shaped flowers. The young leaves of A. senticosus are cultivated as a vegetable crop in Northeast China. As regards biological active constituents of A. senticosus, many of its triterpenoid saponins + signal at m/z 799.4605 by HR-ESI-MS . A close comparison of NMR data , 0.94 (3H), 0.97 (3H), 1.00 (3H), 1.07 (3H) and 1.29 (6H), one olefinic H-atom (br.s at δ 5.45) and a methoxy group at δ 3.71 (3H). The13C-NMR spectrum of 1 showed signals of a pair of olefinic C-atoms at δ 122.5, 144.8, two anomeric C-atoms at δ 102.5, 105.4, and two C=O groups at δ 180.2 and 170.5. The 1H- and 13C-NMR signals of 1 were assigned by the use of HMQC and HSQC-TOCSY. The full connectivity of 1 was deduced from the HMBC correlations (δ 5.64 (H-1′′) and the carbon signals at δ 83.4 (C-2′) suggested that α-L-rhamnose unit was linked to C-2′ of β-D-glucuronic acid. The cross-peak between the proton signals at δ 3.71 (3H) and the carbon signals at δ 170.5 suggested that a methoxy group was linked to C=O of D-glucuronic acid. The anomeric configuration of D-glucuronic acid was determined to be β-form on the basis of the JH-H value (7.6 Hz) in the 1H-NMR spectrum, and the configuration of L-rhamnose was determined to be α-form from examination of the chemical shifts  in the 13C-NMR spectrum of 1. Based on the above evidence, compound 1 was identified as 3-O-[(α-L-rhamnopyranosyl)(1→2)]-[β-D-glucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid.The rum of 1  showed selations . In part5 was obtained as a yellow oil. The molecular formula C12H13NO4 was defined by HR-ESI-MS . The 1H-NMR , 6.71  and 6.73 . The signals at δ 3.83  and 3.67  were assigned to proton signals of methoxy group. The 13C-NMR spectrum data (δ (C) 108.4, 110.0, 115.3, 131.6, 135.9 and 154.4 and two C=O groups at δ (C) 181.1 and 173.2. Compound e 1H-NMR  spectra rum data  showed 11H-1H COSY spectrum showed correlations between δ 3.05 , 2.78  and δ 0.70 , suggesting a –CHCH2– fragment. Moreover, in the HMBC spectrum, the long-range correlations between the proton signals at δ 3.70 (H-3) and the carbon signals at δ 110.0 (C-4), 131.6 (C-4a), 135.9 (C-7a), 181.1 (C-2) and 173.2 (C-9) could be identified. Correlations were observed between the proton signals at δ 3.05 (H-8) and the carbon signals at δ 131.6 (C-4a), 181.1 (C-2) and 173.2 (C-9) (5 could be elucidated as 5-methoxy-2-oxoindolin-3-acetic acid methyl ester. The absolute configuration of the 3-position in compound 5 has not been established yet.The .2 (C-9) . Thus, tO-(β-D-glucuronopyranosyl-6-O-methyl ester)-olean-12-ene-28-olic acid (2)  to afford six fractions  to give 2 (23.4 mg tR = 43.2 min). Fr. III4 was separated by HPLC [MeOH/H2O ] to give 8  and 1 . Fr. III5 was separated by HPLC [MeOH/H2O ] to give 3 . Fraction IV was subjected to reversed-phase silica gel column chromatography  to afford three fractions  to give 4 . Young leaves (6 kg) of 3-O-[(α-L-Rhamnopyranosyl)(1→2)]-[β-D-glucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid (1). White amorphous powder (MeOH). [α]20D +10.5° . IR (KBr): 3408, 2946, 1695, 1387, 1159, 1078, 637 cm−1. HR-ESI-MS m/z 799.4605 [M+Na]+ ; 1H and 13C-NMR (pyridine-5d) data, see 5-Methoxy-2-oxoindolin-3-acetic acid methyl ester (5). Yellow oil (MeOH). [α]20D +4.5° . UV λ MeOH max nm (log ε): 304 (3.11), 257 (3.76), 208 (4.05); IR (KBr): 3376, 2951, 1700, 1615, 1293, 1255, 1091 cm−1. HR-ESI-MS m/z 258.0740 [M+Na]+ ; 1H and 13C-NMR (CD3OD) data, see 1 (3 mg) was hydrolyzed in 1 M HCl (1.0 mL) for 2 h at 85°. The reaction mixture was cooled and partitioned between CHCl3 (2.0 mL) and H2O (2.0 mL). The aqueous layer was washed with CHCl3 (3 × 3.0 mL), neutralized with Ba(OH)2, filtered, and evaporated under reduced pressure. The residue was dissolved in pyridine (1.0 mL) and L-cysteine methyl ester hydrochloride in pyridine  was added. The mixture was heated at 60° for 1 h. An equal volume of Ac2O was added with heating and incubation was continued for 1 h. The acetylated thiazolidine derivatives were analyzed by GC with a DM-5 column  using authentic samples as standards. Temperatures of both injector and detector were 280°. A temperature gradient system was used for the oven, starting at 160° and increasing up to 195° at a rate of 5°/min. The determination of the sugar units in compound 1 was achieved by comparison of retention times of peaks between the hydrolysate and authentic samples of D-glucuronic acid (10.08 min) and L-rhamnose (7.05 min). Compound α-glucosidase (EC.3.2.1.20) enzyme inhibition assay has been performed according to the literature -[β-D-glucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid (1) and 5-methoxy-2-oxoindolin-3-acetic acid methyl ester (5) were isolated from the 30% EtOH fraction of A. senticosus leaves, along with six known compounds. Compounds 1, 22α-hydroxychiisanoside (4) and (+)-afzelechin (6) showed inhibitory activities against α-glucosidase, with IC50 values of 186.0 µM, 908.5 µM and 819.7 µM, respectively.Two new compounds, 3-


Laurencia have been widely studied for their structurally diverse and biologically active secondary metabolites. We report here the natural product investigation of the organic extract of a newly identified South African endemic species, Laurencia alfredensis. A sequence of column chromatography, preparative TLC and normal phase HPLC resulted in the isolation of eleven compounds comprising three labdane-type diterpenes (1–3), four polyether triterpenes (4–7), three cholestane-type ecdysteroids (8–10) and a glycolipid (11). Compounds 1–3, 5–8 and 10 have not previously been reported, while compound 9 is reported here for the first time from a natural source and the known compound 11 isolated for the first time from the genus Laurencia. The structural elucidation and the relative configuration assignments of the compounds were accomplished by extensive use of 1D- and 2D-NMR, HR-ESI-MS, UV and IR spectroscopic techniques, while the absolute configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. All compounds were evaluated against the MDA-MB-231 breast and HeLa cervical cancer cell lines. Compound 2 exhibited low micromolar antiproliferative activity (IC50 = 9.3 µM) against the triple negative breast carcinoma and compound 7 was similarly active (IC50 = 8.8 µM) against the cervical cancer cell line.The marine red algae of the genus  Laurencia  in this family has about 144 identified species +  for the molecular formula C30H50O6 and was identified as another polyether triterpene from its 1H- and 13C-NMR spectra. Six oxygenated methine doublet of doublets including an olefinic proton  and similar olefinic geminal protons  to compound 5  and H3-29  to C-18, along with key COSY correlations between H2-20, H2-21 and H-22, confirmed the presence of an oxane ring in the right hand side chain of 7.Compound mpound 5  were eviine H-18 a. Key HM3-28 was indicative of the trans configuration about the double bond between C-15 and C-16, which was further supported by the ROESY correlation between H-14 and H-16  and δH 2.04 , and five oxymethine protons. Several overlapping multiplets were observed in the methylene envelope between δH 1.11–2.15  and two esters , two aromatic/olefinic carbons , and three signals at δC 68.5 (C-2), 68.3 (C-3), and 73.7 (C-22) suggesting the presence of C-O chemical environments  and its COSY correlation to H-9  and H-14  suggested the ketone carbonyl was adjacent to the C-7-C-8 double bond at C-6 to give the endocyclic α, β-unsaturated ketone, a structural feature of ecdysteroids. HMBC cross-peaks from H2-1 to C-2, C-5, C-9, C-10 and C-19; H2-4 to C-2 and C-3; and H-5 to C-4, C-6, C-9, C-10 and C-19 confirmed the structure of ring A, and that the ketone was at C-6  and H-4β (δH 1.93) respectively, and a further two 1,3-diaxial NOESY correlations from H-5 to H-3 and H-9, where the latter three protons are all in the same α-orientation. The trans-ring-fusion between rings A and B was further corroborated by the absence of a NOESY cross peak between the methine proton H-5 and H3-19. The presence of correlation between H3-18 and H3-19 but not H3-21 established the β-configuration of H3-18 and H3-19 to the steroid backbone suggesting a chair-chair-chair conformation of rings A, B and C. The relative configurations at C-17, C-20, C-21 and C-22 were assigned, by comparison of the 13C-NMR data with that of the structurally related 22R hydroxyl cholesterol +  for the molecular formula C29H46O5. The 1H- and 13C-NMR data for 10 were very similar to both compounds 8 and 9 described above. However, key noticeable differences in the 1H-NMR data of 10 were observed, including the absence of an acetate methyl singlet and the upfield chemical shifts of H-2  and H-3  compared to 8. The 13C-NMR data of 10 showed corresponding changes  consistent with its 1H-NMR. The 1H-1H COSY spin systems were similarly consistent with those observed in compound 8. The position of the single acetate group on ring A was assigned to C-2, on the basis of the chemical shifts of C-2 and C-3 . The structure was elucidated upon analyses of its NMR data and confirmed with reported data as 1, 2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol + .Isoconcinndiol 13-acetate (2): White amorphous solid; c 0.09, CHCl3); IR (cm−1): 3455, 2943, 1732, 1367, 1251, 1095; 1H- and 13C-NMR data (CDCl3), m/z 451.1821 [M + Na]+ .Concinndiol 13-acetate (3): White amorphous solid; c 0.06, CHCl3); IR (cm−1): 3467, 2923, 1732, 1462, 1367, 1241; 1H- and 13C-NMR data (CDCl3), m/z 451.1821 [M + Na]+ .Saiyacenol B (4) + .Alfredensinol B (6): Clear oil; 1H- and 13C-NMR data (CDCl3), m/z 545.3457 [M + Na]+ .Alfredensinol C (7): White amorphous solid; c 0.25, CHCl3); IR (cm−1): 3409, 2940, 1374, 1092; 1H- and 13C-NMR data (CDCl3), m/z 507.3693 [M + H]+ .Alfredensterol (8): White amorphous solid; c 0.13, CHCl3); UV (MeOH) λmax(log ε), nm: 239 (3.43); IR (cm−1): 3425, 2925, 1737, 1643, 1350, 1234, 1035; 1H- and 13C-NMR data (CDCl3), m/z 517.3527 [M + H]+ .14α-Hydroxy Alfredensterol (9): White amorphous solid; c 0.26, CHCl3); UV (MeOH) λmax(log ε), nm: 222 (3.76); IR (cm−1): 3240, 2925, 1738, 1645, 1375, 1234, 1036; 1H- and 13C-NMR data (CDCl3), m/z 533.3488 [M + H]+ .3-Deacetoxy Alfredensterol (10): White amorphous solid; c 0.30, CHCl3); UV (MeOH) λmax(log ε), nm: 231 (3.27); IR (cm−1): 3397, 2948, 1715, 1660, 1325, 1240, 1030; 1H- and 13C-NMR data (CDCl3), m/z 475.3432 [M + H]+ .d-quinovopyranosyl)-glycerol1,2-di-O-Palmitoyl-3-O-(6-sulfo-α- (11) − .rol (11) : White a1 was obtained by recrystallization from methanol and its structure was solved by direct methods from intensity data collected from the crystal specimen at 173(2) K on a Bruker Apex II Duo diffractometer and refined by full-matrix least-squares. The Flack x parameter value of −0.013(6), indicating that the correct absolute configuration had been assigned, was determined using 1652 quotients [(I+) − (I−)]/[(I+) + (I−)]. Salient crystallographic data for 1 are as follows:The crystal for compound 22H37O3Br (M = 429.42 g/mol): monoclinic, space group P21 (no. 4), a = 11.1926(11) Å, b = 7.2138(7) Å, c = 14.8442(13) Å, β = 111.571(2)°, V = 1114.60(18) Å3, Z = 2, T = 173(2) K, μ(MoKα) = 1.861 mm−1, Dcalc = 1.280 g cm−3, 20,619 reflections measured (2.95° ≤ 2θ ≤ 56.02°), 5386 unique  which were used in all calculations. The final R1 was 0.0378 (I > 2σ(I)) and wR2 was 0.0760 .Crystal Data for CFurther technical details of the structure determination and molecular parameters are provided in the CIF file .v/v) heat-inactivated fetal bovine serum (FBS), 1 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 12.5 μg/mL amphotericin (PSA) at 37 °C in a humidified incubator. HeLa cervical cancer cells (ATCC CCL-2) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented as above at 37 °C and 9% CO2 in a humidified incubator.MDA-MB-231 breast cancer cells (ATCC HTB-26) were maintained in culture in phenol-red free L-15 medium supplemented with 10% (v/v DMSO) for 96 h. Thereafter 2.5 µL of WST-1 Cell Proliferation Reagent was added per well and the absorbance at 450 nm after 8 h recorded using a Synergy Mx spectrophotometer (BioTek). The half maximal inhibitory concentration (IC50) for each compound was calculated relative to the vehicle-treated control from a dose response curve (log concentration vs absorbance at 595 nm) using non-linear regression with GraphPad Prism 4 .The antiproliferative effects of the compounds were assessed using the WST-1 assay  as previously described ,36. Brie


Onosma bracteatum Wall, a traditional medicinal plant used in the Unani and Ayurvedic systems of medicine. During bioassay-guided isolation, two known benzoquinones, allomicrophyllone (1) and ehretiquinone (2) along with three novel benzoquinones designated as ehretiquinones B–D (3−5) were isolated from O. bracteatum. Their structures were characterized by spectroscopic analysis through 1D and 2D NMR, by MS spectroscopic analysis and comparing with those reported in the literatures. The anti-aging potential of the isolated benzoquinones was evaluated through a yeast lifespan assay, and the results indicated that 1, 2, 4 and 5 significantly extended the replicative lifespan of K6001 yeast, indicating that these benzoquinones obtained from O. brateatum have the ability to be employed as a potential therapeutic agent against age-related diseases.The aim of this study was to investigate anti-aging molecules from  Aging is a natural process marked by progressive deterioration in physiological functions and increases in mortality and is often accompanied by various human pathologies, such as cardiovascular diseases, diabetes, stroke, cancer and neurodegenerative diseases (Parkinson’s and Alzheimer’s disease) . AdvanceNatural products have been an important source of drug discovery since ancient times, and a large number of present-day drugs are derived from natural sources. In our previous studies, we reported many natural compounds with anti-aging properties by using K6001 yeast replicative life span bioassay ,5,6. ThiOnosma bracteatum Wall belongs to family Boraginaceae. It is known as Gaozaban in the Unani system of medicine and as Sedge in the Middle East. O. bracteatum is commonly used as a demulcent, alterative, diuretic, immunity enhancer and spasmolytic and as a major constituent of various Ayurvedic formulations for the treatment of hypertension, leprosy, rheumatism and asthma  and yielded compound (1) .Fr.4 (500 mg) was separated by silica gel open column and eluted with CH3OH/H2O . Ten fractions (Fr.3-1–Fr.3-10) were obtained, and Fr.3-7 was further purified by HPLC  and yielded compound (2) .Fr.3 (113 mg) was separated by using ODS open column and eluted with CH2Cl2:CH3OH . Nine fractions (Fr.2-1–Fr.2-9) were obtained, and Fr.2-4 (20 mg) was subjected to ODS open column chromatography with CH3OH/H2O , and the fifth fraction (6.5 mg) was purified by HPLC  to yield compound (3) . Fr.2-5 (70 mg) was separated by ODS open column using CH3OH/H2O  and the seventh fraction (11 mg) was purified by HPLC  to yield compound (4) . Fr.2-8 (75 mg) was separated by ODS open column using CH3OH/H2O  and the seventh fraction (10 mg) was purified by HPLC  to yield compound (5) .Fr.2 (450 mg) was subjected to silica gel open column and eluted with CHAllomicrophyllone (1). Yellow powder; 3); High-resolution ESI-TOF-MS m/z 389.1359, calculated for C22H22O5Na (M + Na)+ 389.1359; 1H NMR : δH = 6.83 , 6.61 , 6.56 , 6.51 , 6.51 , 5.76 , 5.67 , 5.65 , 3.77 , 2.73 , 2.50 , 1.69 , 1.22 ; 13C NMR : δC = 195.8 (C-1′), 193.1 (C-4′), 150.0 (C-4), 145.0 (C-1), 143.3 (C-8′), 139.4 (C-5′), 138.6 (C-6′), 131.8 (C-9), 127.7 (C-2), 122.5 (C-8), 122.0 (C-7′), 117.6 (C-6), 114.9 (C-5), 114.4 (C-3), 80.7 (C-3′), 71.0 (C-9′), 54.9 (C-2′), 39.1 (C-7), 36.2 (C-10), 29.8 (C-10′), 29.8 (C-11′), 22.6 (C-11). The structure was identified through comparison of MS, 1H NMR and 13C NMR spectra as well as specific rotation with literatures [eratures .Ehretiquinone (2). Red powder; 3OH); high-resolution ESI-TOF-MS m/z 371.1254, calculated for C22H20O4Na (M + Na)+ 371.1252; 1H NMR : δH = 6.83 , 6.62 , 6.58 , 6.53 , 6.52 , 6.25 , 5.65 , 5.50 , 5.04 , 4.97 , 3.83 , 2.78 , 2.52 , 1.73 , 1.70 ; 13C NMR : δC = 195.4 (C-1′), 193.2 (C-4′), 150.0 (C-4), 145.1 (C-1), 140.8 (C-9′), 139.2 (C-5′), 138.8 (C-6′), 137.5 (C-8′), 131.9 (C-9), 127.7 (C-2), 124.1 (C-7′), 122.7 (C-8), 119.3 (C-10′), 117.6 (C-6), 114.9 (C-5), 114.4 (C-3), 80.7 (C-3′), 55.3 (C-2′), 39.4 (C-7), 36.3 (C-10), 22.7 (C-11), 18.3 (C-11′). The structure was identified through comparison of MS, 1H NMR and 13C NMR spectra as well as specific rotation with literature [Ehretiquinone B (3). Red powder; 3OH); high-resolution ESI-TOF-MS m/z 387.1208, calculated for C22H20O5Na (M + Na)+ 387.1203; Data for 1H NMR and 13C NMR are described in Ehretiquinone C (4). Red powder; 3OH); high-resolution ESI-TOF-MS m/z 387.1174, calculated for C22H20O5Na (M + Na)+ 387.1174; Data for 1H NMR and 13C NMR are described in Ehretiquinone D (5). Yellow powder; 3); high-resolution ESI-TOF-MS m/z 405.1300, calculated for C22H22O6Na (M + Na)+ 405.1300; Data for 1H NMR and 13C NMR are described in The bioassay method used for the anti-aging study was previously described . K6001 yp value of < 0.05 was considered statistically significant.The significant differences among groups were determined by one-way ANOVA with Dunnett’s multiple comparison test. GraphPad Prism 5.0 software was used. A O. bracteatum under the guidance of K6001 yeast replicative lifespan assay. The structure of the new benzoquinones (3–5) were elucidated by spectroscopic analysis, and the known ones (1–2) were identified by comparing the spectroscopic data with those in literatures. All the isolated benzoquinones were evaluated for anti-aging activity by yeast replicative lifespan assay and 1, 2, 4 and 5 exhibited significant anti-aging activity. Ehretiquinone C (4) showed the most promising anti-aging activity and can thus be considered a potential lead compound. Its anti-aging mechanism should be further studied.Three new and two known benzoquinones were isolated and characterized from


Eight phenolic compounds in EtOAc extracts were purified through silica gel and Sephadex LH-20 column chromatographies, and then identified by nuclear magnetic resonance and electrospray ionization mass spectra. The results showed that eight phenolic compounds from EtOAc extracts were identified as caffeic acid, cis-p-hydroxycinnamic acid, quercetin, apigenin, albanin A, australone A, moracin M, and 5'-geranyl-5,7,2',4'-tetrahydroxyflavone. The antioxidant and nitrite-scavenging capacities of different solvent extracts correlated positively with their total phenolic (TP) contents. Amongst various extracts, EtOAc extracts possessed the highest TP content and presented the strongest oxygen radical absorbance capacity (ORAC), 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity, 2,2'-azobis-3-ethylbenthiaazoline-6-sulfonic acid (ABTS) radical-scavenging capacity, ferric reducing antioxidant power (FRAP) and nitrite-scavenging capacity. Thus, sugarcane tops could be promoted as a source of natural antioxidant.Sugarcane tops were extracted with 50% ethanol and fractionated by petroleum ether, ethyl acetate (EtOAc), and  Compound 1 was identified as caffeic acid by comparing NMR and MS data with literature +, 147, 138, 121, 119, 93, 65. IR (KBr) νmax: 3400, 1675, 1606, 1511, 1445, 1240, 1211, 988 cm–1. 1H-NMR  δ: 7.66 , 6.70 , 6.62 , 5.68 ; 13C-NMR  δ: 126.1 (C-1), 132.5 , 115.0 , 158.8 (C-4), 140.7 (C-7), 118.4 (C-8), 168.5 (C-9). Compound 2 was confirmed as cis-p-hydroxycinnamic acid by NMR and MS data with literature –. 1H-NMR  δ: 12.49 , 10.76 , 9.58 , 9.35 , 9.30 , 7.68 , 7.55 , 6.89, 6.41, 6.19; 13C-NMR  δ: 146 (C-2), 136 (C-3), 176(C-4), 103.3 (C-4a), 157 (C-5), 98.1 (C-6), 164.4 (C-7), 93.2 (C-8), 163.3 (C-8a), 123.2 (C-1'), 114.9 (C-2'), 145.6 (C-3'), 147.3 (C-4'), 114.3 (C-5'), 121.2 (C-6'). Compound 3 was identified as quercetin by the comparison of NMR and MS data with literature –. IR (KBr) νmax: 3288, 1655, 1606, 1510 cm–1. 1H-NMR  δ: 7.99 , 6.94 , 6.88 , 6.56 , 6.21 ; 13C-NMR  δ: 164.1 (C-2), 103.1 (C-3), 181.6 (C-4), 157.7 (C-4a), 162.0 (C-5), 99.8 (C-6), 164.0 (C-7), 94.3 (C-8), 104.1 (C-8a), 121.1 (C-1'), 128.2 , 116.4 , 161.5 (C-4'). Compound 4 was identified as apigenin by comparing NMR and MS data with literature –. 1H-NMR  δ: 13.13 , 7.18 , 6.56 , 6.51 , 6.31, 6.23 , 5.11 , 3.1 , 1.56 , 1.42 ; 13C-NMR  δ: 161.5 (C-2), 121.6 (C-3), 182.9 (C-4), 105.0 (C-4a), 163.3 (C-5), 99.4 (C-6), 165.3 (C-7), 94.3 (C-8), 159.3 (C-8a), 24.6 (C-9), 122.7 (C-10), 132.0 (C-11), 25.8 (C-12), 17.6 (C-13), 113.0 (C-1'), 157.2 (C-2'), 103.9 (C-3'), 162.3 (C-4'), 108.1 (C-5'), 132.2 (C-6'). Compound 5 was identified as albanin A by comparing NMR and MS data with literature –. 1H-NMR  δ: 7.85 , 6.62 , 6.55, 6.71 , 5.71 , 6.46 , 5.13 , 2.1–2.2 , 1.6-1.8 , 1.64 , 1.57 , 1.45 ; 13C-NMR  δ: 162.6 (C-2), 108.5 (C-3), 183.4 (C-4), 105.6 (C-4a), 156.9 (C-5), 105.7 (C-6), 159.6 (C-7), 95.3 (C-8), 158.0 (C-8a), 116.4 (C-9), 128.0 (C-10), 81.1 (C-11), 27.0 (C-12), 42.2 (C-13), 23.7 (C-14), 124.8 (C-15), 132.2 (C-16), 18.0 (C-17), 25.7 (C-18), 110.6 (C-1'), 160.3 (C-2'), 104.3 (C-3'), 163.0 (C-4'), 109.0 (C-5'), 130.9 (C-6'). Compound 6 was identified as australone A by comparing NMR and MS data with literature –. 1H-NMR  δ: 13.10 , 7.70 , 7.10 , 6.66 , 6.46 , 6.23 , 5.39 , 5.13 , 3.32 , 2.14 , 2.06 , 1.76 , 1.60 , 1.57 ; 13C-NMR  δ: 163.2 (C-2), 104.2 (C-3), 183.4 (C-4), 105.0 (C-4a), 158.9 (C-5), 99.5 (C-6), 165.1 (C-7), 94.6 (C-8), 160.1 (C-8a), 110.3 (C-1'), 157.5 (C-2'), 108.4 (C-3'), 163.2 (C-4'), 121.4 (C-5'), 130.2 (C-6'), 28.1 (C-1''), 123.6 (C-2''), 136.5 (C-3''), 16.2 (C-4''), 40.5 (C-5''), 27.5 (C-6''), 125.0 (C-7''), 131.8 (C-8''), 25.8 (C-9''), 17.8 (C-10''). Compound 8 was identified as 5'-geranyl-5,7,2',4'-tetrahydroxy-flavone by comparing NMR and MS data with literature [Compound in vitro depends upon the arrangement of functional groups about nuclear structure. Sufficient evidence support that the role of specific structural components are requisites for free radical scavenging, metal chelation and oxidant activity [p < 0.05) ORAC value, followed by water extract, petroleum ether extract, n-BuOH extract, and total extract. EtOAc extract exhibiting the highest TP content and ORAC value  correlated with TP content  correlated with TP content  nitrite-scavenging ratio among five extract, followed by n-BuOH extract, total extract, petroleum ether extract, and water extract. The nitrite-scavenging ratio of EtOAc extract approached to that of vitamin C, indicating that EtOAc extract of sugarcane tops was probably a good nitrite-scavenging additive for the food industry. From p < 0.05), suggesting that phenolic components played an important role in scavenging nitrite. This finding was similar to the previous report by Liu et al. [Maydis stigma had significant scavenging ability on nitrite. Phenolic compounds could inhibit the formation of N-nitroso-dimethylamine due to their hydroxyl groups. At low pH, the nitrite was converted to nitrous acid and subsequently to N2O3, which could be rapidly reduced to NO by phenolic compounds that existed in sugarcane tops. Consequently, the formation of N-nitrosamine was inhibited, and the nitrite was also scavenged.Nitrite-scavenging capacities of different extract from sugarcane tops are shown in u et al. , who fouSugarcane variety of “ROC22” (one of mainly cultivated variety in China) was chosen as plant material. Sugarcane materials were obtained from a cane experimental base in Guangxi Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences on 20 November, 2011. The healthy sugarcane tops were cut, sun-dried and crushed into powder. The dried powdered plant material was stored at room temperature (25 °C) in a desiccator until further analysis.Ascorbic acid, ABTS diammonium salt, DPPH, fluorescein (FL), Folin–Ciocalteu reagent, trolox , and 2,2'-azobis (2-amodinopropane) dihydrochloride (AAPH) were purchased from Sigma Chemical Co. . Other reagents were of analytical grade. n-BuOH). Petroleum ether-soluble fraction, EtOAc-soluble fraction, n-BuOH-soluble fraction, and water-soluble fraction were obtained. All the fractions were concentrated separately under reduced pressure and freeze-dried to get petroleum ether extract, EtOAc extract, n-BuOH extract, and water extract. The crude 50% EtOH extract was expressed as total extract. Five different extracts (1 mg) were precisely weighed, dissolved in 1 mL of 50% EtOH, and then diluted for further analysis.The dry powder of sugarcane tops (4.1 kg) was extracted with 50% ethanol (EtOH) with the solid/liquid rate of 1/10 (m:v) for 90 min at 70 °C. EtOH extract was evaporated to dryness by a RE52AA rotary evaporator  under reduced pressure at 50 °C. The residue was dissolved in water and fractionated by petroleum ether. Petroleum ether-soluble fraction was collected and the residue was extracted by ethyl acetate (EtOAc). EtOAc-soluble fraction was collected and the residue was further extracted n-butyl alcohol  content in sugarcane tops extracts was measured by the method of Dorman et al. . TP cont1 (4 mg) and compound 2 (6 mg). Fraction D (3.4 g) was purified using Sephadex LH-20 column eluting with chloroform-methanol  to give compound 3 (5 mg), compound 4 (7 mg) and compound 5 (5 mg). Fraction E (7.2 g) was subjected to silica gel column chromatography  eluting with chloroform-methanol  to give 7 fractions (E1–E7). Fraction E2 (1.2 g) was further purified using Sephadex LH-20 column eluting with methanol to give compound 6 (8 mg), compound 7 (5 mg), and compound 8 (4 mg).EtOAc extract  from sugarcane tops was purified using silica gel column chromatography , eluting with gradient mixtures of chloroform-methanol  to obtain 10 fractions (A–J). Fraction C (8 g) was separated by silica gel column  using petroleum ether-acetone  as eluents to afford 8 fractions (C1–C8). Fraction C2 and C5 was further subjected to Sephadex LH-20 column chromatography eluting with methanol to afford compound 1H-NMR, 400 MHz; 13C-NMR, 100 MHz). Chemical shift values (δ) were recorded in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. ESI-MS spectra were recorded on a Finnigan LCQ Advantage Max ion trap mass spectrometer . IR spectra were determined on a Jasco FT/IR-480 plus Fourier transform . Melting points (mp) were recorded on a XT-4 micro melting point apparatus  without correction.The chemical structures of isolated phenolic compounds were identified by nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectra (ESI-MS), and infrared (IR) spectroscopy. NMR spectra were measured on a Bruker AV-400 spectrometer   were calculated by applying the following formula:et al. [sample was absorbance of sample, Ablank was absorbance of blank and Acontrol was absorbance of control. DPPH radical scavenging value was determined using a standard curve of Trolox (0.05–0.3 mM) and the results were expressed as micromolar Trolox equivalent per milligram extracts (μM TE/mg).DPPH radical-scavenging capacity was determined according to the modified method of Luo et al.  and expret al. [+) compared with Trolox. ABTS+ stock solution was produced by dissolving ABTS in 2.45 mM potassium persulfate solution and keeping the mixture in the dark for 12–16 h at room temperature. The stock solution was diluted using 10 mM sodium phosphate buffer (pH 7.4) to obtain the working solution with an absorbance of 0.7 at 734 nm. A total of 40 μL of sample was mixed with 4 mL of ABTS·+ working solution for 30 s before measurement at 734 nm. ABTS+ scavenging ratio (%) was calculated as (1 − Asample/0.7) × 100. ABTS+ scavenging value was determined using a standard curve of Trolox (0–1 mM) and the results were expressed as micromolar Trolox equivalent per milligram extracts (μM TE/mg).ABTS radical-scavenging capacity was determined according to the modified method of Roberta et al. . This meet al. [FRAP was conducted according to the modified method of Jayaprakasha et al. . One milsample/Ablank) × 100.Nitrite-scavenging capacity was evaluated by the method of hydrochloric acid naphthalene ethylenediamine coloration. One milliliter of sample or 1 mL of 50% ethanol (blank) was mixed with 1 mL of 5 mg/L nitrite solution and 1 mL of citric acid buffer (pH 3). After reacting for 30 min at 37 °C, 1 mL of 4 g/L amino benzene sulfonic acid sodium (in 20% hydrochloric) was added in the mixture, and then 0.5 mL of 2 g/L hydrochloric acid naphthalene ethylenediamine (in water) was also added after 3 min. The mixture was reacted for 15 min and measured at 538 nm. Ascorbic acid was used as positive control. Nitrite-scavenging ratio (%) = (1 − Ap < 0.05.All experiments were performed in triplicate (n = 3) and the results were expressed as mean ± standard deviation. Statistical analysis was carried out by SPSS 18 statistical software . A difference was considered statistically significant when i.e., total extract, petroleum ether extract, EtOAc extract, n-BuOH extract and water extract) on biological activities (antioxidant and nitrite scavenging capacities) were investigated in vitro. Amongst them, EtAOc extract from sugarcane tops possessed the strongest antioxidant and nitrite scavenging capacities. Further investigation will be performed to study the correlation between antioxidant activity and chemical structure of sugarcane top phenolics. Antioxidant and nitrite scavenging capacities of individual phenolic compounds from sugarcane tops will be determined in following research. The functional products will be also explored from sugarcane tops.Eight phenolic compounds were isolated from EtOAc extract of sugarcane tops. The effects of different extracts (


All compounds were isolated from Illicium plants for the first time. The absolute stereochemistry of all germacranes and m-menthane was established by a combination of NMR and the modified Mosher’s ester method. The biological activity was evaluated on SH-SY5Y neuroblastoma cell line. -1,5-Dihydroxygermacra-4(15),10(14),11(12)-triene (at 62.5 µM) and -1,5-dihydroxygermacra-4(15),10(14),11(12)-triene (at 15.6 µM) promoted the proliferation of SH-SY5Y by 36.2% and 45.8%, respectively, after 48 h incubation, indicating potential neurotrophic activity.Three new germacrane sesquiterpenes and a new  Illicium L. (Illiciaceae) consists of ca. 40 species that form one of the earliest evolutionary branches of the angiosperms + , indicating two degrees of unsaturation. The 1H-, 13C- and DEPT-NMR spectra (1H-1H COSY spectrum displayed correlations between δH 1.72 (H-2) and δH 3.60 , between δH 3.60 (H-3) and δH 1.91  and δH 1.63 , between δH 1.91 (H-4a) and δH 2.23 , between δH 2.23 (H-5) and δH 1.53 (H-6), leading to the connections of between C2-C3-C4-C5-C6  and δC 37.4 (C-5), δC 109.0 (C-9), and δC 149.2 (C-8), as well as correlations between δH 4.70  and δC 21.1 (C-10), δC 37.4 (C-5), and δC 149.2 (C-8), suggested that C-9 and C-10 were connected to C-5 via C-8 (δH 1.53 (H-6)/δC 71.4 (C-1); δH 1.23 /δC 71.4 (C-1) and δC 33.7 (C-2). Two hydroxyl groups were assigned to C-1 and C-3, respectively. In the NOESY spectrum, δH 2.23  displayed correlations with δH 3.60 , and δH 1.23 , indicating the relative stereochemistry of 1β,3β-dihydroxy-(5αH)-m-menth-8-ene. To determine the absolute stereochemistry, modified Mosher’s ester procedure was carried out. Due to the steric hindrance on C-1, only 3-OH was esterified by (R)- and (S)-MTPA chlorides into (S)- and (R)-MTPA derivatives. The absolute configuration of C-3 was finally deduced to be R based on the proton shift difference between (S)- and (R)-MTPA derivatives -1,3-dihydroxy-m-menth-8-ene.Compound  spectra  revealed spectra . The 1H-C4-C5-C6 . The HMB via C-8 . The remtives 5, , indicat6, 7, and 8 were identified to be 3-hydroxyocta-1,5E-dien-7-one + , m/z 201.1639 [M-−2H2O+H]+.-1,5-Dihydroxygermacra-4(15),10(14),11(12)-triene (2). Colorless oil. IR (cm−1): 3359, 2928, 1641, 1439, 1015, 895.  −7° . 13C- and 1H-NMR spectroscopic data (CD3OD): see m/z 219.1735 [M−H2O+H]+ , 201.1630 [M−2H2O+H]+.-1,5-Dihydroxygermacra-4(15),10(14),11(12)-triene (3). White amorphous powder. IR (cm−1): 3260, 2928, 1644, 1434, 1020, 905.  −4° . 13C- and 1H-NMR spectroscopic data (CD3OD): see 1H spectroscopic data : δH 5.24 , 5.03 ; 5.16 , 5.06 ; 4.65 , 4.64 ; 4.16 ; 3.95 ; 2.27 , 1.83 ; 2.13 ; 2.06 ; 1.73 , 1.59 ; 1.58 . HR-ESI-MS m/z 219.1734 [M−H2O+H]+ , 201.1642 [M−2H2O+H]+, 237.1820 [M+H]+. -1,5-Dihydroxygermacra-4(15),10(14),11(12)-triene (4). Colorless oil. IR (cm−1): 3368, 2927, 1642, 1449, 1012, 893.  +11° . 13C- and 1H-NMR spectroscopic data (CD3OD): see m/z 219.1740 [M−H2O+H]+ , 201.1650 [M−2H2O+H]+.-1,3-Dihydroxy-m-menth-8-ene (5). White amorphous powder. 13C- and 1H-NMR spectroscopic data (CDCl3): see  m/z 193.1202 [M+Na]+ .R)- and (S)-MTPA chloride was used to react with each compound to yield its (S)- and (R)-MTPA derivatives, respectively [2O5 inside. After successive addition of 6 μL of (R)- or (S)-MTPA chloride and 600 μL of pyridine-5d, the NMR tubes were sealed immediately, and shaken vigorously. The tubes were then kept in desiccator overnight until the reaction was complete [1H-NMR spectra of the final (R)- and (S)-MTPA derivatives were recorded, and the chemical shifts were assigned based on the 1H-1H COSY NMR experiments. In case that signals could not be unambiguously assigned, gHSQC and gHMBC experiments were carried out. The ∆SδR- values were calculated [In these experiments,  were incubated with various concentrations of compounds in 24-well culture plates. After 48-h incubation, 20 µL MTT -2.5-diphenyltetrazolium bromide, 5 mg/mL in PBS) were added to the each well. The supernatant were removed after further 4 h incubation. The formazan in each well were dissolved in 300 µL isopropanol with 4 mM HCl and 0.1% Nondet P-40. The absorbance was read at 590 nm with a reference filter of 620 nm by using microplate reader . The cells without treatment were as vehicle control. The cells were treated by corresponding concentration of DMSO as control. The percentage of growth promotion was calculated using the following formula: % cell promotion = 100 × (OD590nm test compound − OD590nm control)/OD590nm control. Results were expressed as the mean of at least three independent experiments.The SH-SY5Y cells were maintained in the Opti-MEM with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. The cells (5 × 101–3, a new m-menthane monoterpene 5, together with a known germacrane sesquiterpene 4, and three other known compounds 6–8 have been identified from I. lanceolatum. All the compounds were isolated from Illicium plants for the first time. Absolute stereochemistry for germacranes and m-menthane were established. Compounds 3 and 4 exhibited proliferative activity on the SH-SY5Y cell lines, indicating potential neurotrophic activity. Further biological evaluation on primary cultures of fetal rat cortical neurons is under planning.Three new germacrane sesquiterpenes


Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HREIMS. Triterpenoids possessing a nitro group were not isolated previously.Three novel multiflorane-type triterpenoids, 3α- Cucurbita pepo is a cultivated plant of the genus Cucurbita that includes varieties of squash, gourd, and pumpkin. Cucurbita pepo L  (Cucurbitaceae) are widely cultivated in America, Europe, and Asia. The zucchini is a hybrid of the cucumber, and has been a commercially important crop in many countries since the 1950–1960s. It is a highly nutritional low caloric food that requires relatively little effort to prepare. It is full of nutrients like vitamin A, vitamin C, potassium, folate and fiber—all of which support a healthy metabolism. Zucchini, grows well in warm climates. This readily available vegetable can also be an important part of weight loss efforts. The species Cucurbita pepo L, Appendino reported 3α-p-aminobenzoyl, 29-benzoylmultiflor-8-en-7β-ol, and 3α-p-aminobenzoylmultiflora-7:9(11)-dien-29-benzoate , three sp3 methine groups including an oxymethine [δH 4.97 ], two trisubstituted olefin , six sp3 quaternary carbons, a benzoyl group  and a p-nitrobenzoyl group . In the HMBC spectrum (C 80.7), C-4, C-5, and C-24; between Me-24 and C-3, C-4, C-5, and C-23; between Me-25 and C-1, C-5, C-9 (δC 144.6), and C-10; between Me-26 and C-8 (δC 142.3), C-13, C-14, and C-15; between Me-27 and C-12, C-13, C-14, and C-18; between Me-28 and C-16, C-17, C-18, and C-22; between Me-30 and C-19, C-20, C-21, and C-29 (δC 73.0); between H2-29 and C-19, C-20, C-21, and C-30; between H-2′ and H-6′ [δH 8.10 (2H)] and C-4′ (δC 150.6), O-C=O (δC 164.4); and between H-2″ and H-6″ [δH 8.08 (2H)] and O-C=O (δC 166.9). In the 1H-1H COSY spectrum  of 1 revealed the partial structures shown in bold face in 4. Furthermore, the same base ion peak was observed in compounds 1 and 4 at m/z 526 . On the basis of the above spectral data, 1 was established to be a novel 3α-p-nitrobenzoylmultiflora-7:9(11)-dien-29-benzoate. Selected NOESY correlations for 1 are shown in p-nitrobenzoyl group at C-3 was established as the α -orientation due to the NOE correlations between H-3 and Me-23 and Me-24, and the coupling constants of H-3 [δH 4.97 ]. The benzoyl group was at C-29 because the NOESY correlation was observed between H2-29 and H-22α and Me-27. Therefore, 1 was determined as 3α-p-nitrobenzoylmultiflora-7:9(11)-dien-29-benzoate. Although, natural products containing nitro groups have been isolated from plants , three sp3 methine groups including an oxymethine [δH 4.67 ], Δ7,9(11)-diene , an acetyl group [δH 2.03 ], and a benzoyl group . In the HMBC spectrum of 2 (H 0.93) and C-9 (δC 144.3); between Me-26 (δH 0.93) and C-8 (δC 141.8); between Me-23 (δH 0.84) and Me-24 (δH 0.98) and C-3 (δC 78.2); and between Me-30 (δH 1.11) and C-29 [δC 72.8 (t)]. The spectral data indicated 2 to be a novel 3α-acetoxymultiflora-7:9(11)-diene-29-benzoate.Compound  spectra  exhibiterum of 2 , long-ra39H52O4  based on HREIMS. The UV spectrum showed a 5(6),7,9(11)-triene moiety . The IR spectrum showed bands assignable to ester groups . The 1H- and 13C-NMR spectra ], three sp3 methine groups including an oxymethine [δH 4.75 ], three trisubstituted olefin , six sp3 quaternary carbons, an acetyl group , and a benzoyl group . In the HMBC spectrum (C 77.4), C-4, C-5 [δC 148.9 (s)], and C-24; between Me-24 (δH 1.22) and C-3, C-4, C-5, and C-23; and between Me-25 (δ 1.17) and C-1, C-5, C-9 [δC 144.6 (s)], and C-10. In the 1H-1H COSY spectrum, H-6 (δH 5.85) correlated with only H-7 (δH 5.61); H-11 (δH 5.34) correlated with H2-12 . EIMS showed a fragment ion peak at m/z 524 [M–AcOH]+ as a base ion peak. Based on the spectral data, the structure of 3 was established as 3α-acetoxymultiflora-5(6):7:9(11)-trien-29-benzoate.The molecular formula of 3 was determined as C spectra  exhibitespectrum , long-ra1–5 were evaluated for cytotoxic activity against HL-60 and P388 cells using MTT methods (2 exhibited weak cytotoxic activity against HL-60 (IC50 25.7 μM) and P388 (IC50 75.1 μM), 1 and 3–5 showed no activity against either cell line. Compound 3 showed melanogenesis inhibitory activity with low cytotoxicity at 100 μM  .Compounds  methods  18]. Al. Al1–5 wy 92.5%) . Compoun1H- and 13C-NMR spectra were obtained on a Varian INOVA 500 spectrometer with standard pulse sequences, operating at 500 and 125 MHz, respectively. CDCl3 was used as the solvent and TMS, as the internal standard. EIMS were recorded on a Hitachi 4000H double-focusing mass spectrometer (70 eV). Column chromatography was carried out over silica gel  and MPLC was carried out with silica gel . HPLC was run on a JASCO PU-1586 instrument equipped with a differential refractometer (RI 1531). Fractions obtained from column chromatography were monitored by TLC . Melting points were determined on a Yanagimoto micro-melting point apparatus and are uncorrected. Optical rotations were measured using a JASCO DIP-1000 digital polarimeter. IR spectra were recorded using a Perkin-Elmer 1720X FTIR spectrophotometer. Cucurbita pepo L. produced in USA , were purchased from JA -Takatsuki in May, 2011.The seeds of 2O and H2O. Evaporation of the Et2O phase gave a yellowish residue (216.1 g) which was subjected to silica gel (3.5 kg) column chromatography. Elution of the column with CHCl3 gave residue A , B  and C . Elution of the column with CHCl3/EtOAc (10:1) afforded residues D  and E  and subsequent column chromatography with CHCl3/EtOAc (2:1) to give residues F . Elution was continued with EtOAc and MeOH to give residues G  and H . Air-dried seeds (10 kg) were ground and extracted × 3 for 3 days each with MeOH (10 L) employing an automatic percolator. Removal of the MeOH under reduced pressure left a greenish residue which was partitioned between Etn-hexane:EtOAc = 20:1~EtOAc to give residues B-1 , B-2 , B-3 . Residue B-1 was separated by HPLC  to give compounds 1 (2.5 mg), 2 (5.1 mg) and 3 (1.8 mg). Residue B was rechromatographed on a silica gel  column using n-hexane:EtOAc = 10:1~EtOAc to give residues C-1 , C-2 , C-3 . Residue C-1–C-3 was separated by HPLC  to give compounds 4 (28.3 mg) and 5 (4.9 mg). Compound 4 was identified as 3α-p-aminobenzoylmultiflora-7:9(11)-dien-29-benzoate on the basis of published data D26 +10.9° ; HREIMS m/z: 693.4024 [M]+ ; UV (EtOH) λmax nm (log ε): 230 (3.85), 237 (3.80), 248 (3.63); IR (KBr) νmax cm−1; 2945, 1713 (O-C=O), 1542 (Ar), 1527 and 1341 (NO2), 1510, 1371, 1287; 1H- and 13C-NMR, see  m/z (rel. int.): 693 (100) [M]+), 526 (41) [M–p-nitrobenzoic acid]+, 389 (26), 253 (71), 227 (37), 211 (37).Colorless crystals; mp 172–174 °C (from MeOH-CHCl3); [α]D26–44.0° ; HREIMS m/z: 586.4016 [M]+ ; UV (EtOH) λmax nm (log ε): 222 (3.93), 237 (3.95); IR (KBr) νmax cm−1; 2974, 1743, 1718 and 1271 (O-C=O), 1559 (Ar), 1521, 1489, 1458, 1271, 1114; 1H- and 13C-NMR, see  m/z (rel. int.): 586 (62) [M]+, 526 (100) [M–HOAc]+, 511 (31), 389 (35), 253 (62).Colorless crystals; mp 93–94 °C ; HREIMS m/z: 584.3864 [M]+ ; UV λmax (EtOH) nm (log ε): 227 (4.19), 304 (3.98), 315 (4.00), 334 (3.72); IR (KBr) νmax cm−1: 2949, 2881, 1725 and 1239 (O-C=O), 1540 (Ar), 1450, 1274, 1105, 992, 773; 1H- and 13C-NMR, see  m/z (rel. int.): 584 (33) [M]+, 524 (100) [M–HOAc]+, 509 (52), 457 (11), 387 (35), 295 (23), 285 (36), 251 (30), 225 (51).Colorless crystals; mp 105–107 °C; [α]4 cells in 100 μL) were treated with test compounds for 72 h, and MTT solution was added to the wells. The grown cells were labeled with 5 mg/mL MTT in phosphate-buffered saline (PBS), and the absorbance of formazan dissolved with 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was measured at 550 nm using a microplate reader .The cytotoxicity assay was determined previously . Briefly4 cells in 500 μL), preincubated for 24 h were treated for 48 h with test samples dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100, 30 or 10 μM, and MTT solution was added. After 3 h of incubation, 2-propanol containing 0.08 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 550 nm using a microplate reader.Cell proliferation was examined according to a method reported previously . BrieflyThe assay of melanin content was performed as described previously . B16 cel1 was established as 3α-p-nitrobenzoylmultiflora-7:9(11)-dien-29-benzoate. This is the first report of a triterpenoid having a nitro group in the molecule. At this time we have no explanation for the presence of a p-nitrobenzoic moiety in a zucchini metabolite, and wish to consider the role of the nitro group in the plant body in the future.The structure of


Eupatorium lindleyanum DC. The two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water  was selected. From 540 mg of the n-butanol fraction of Eupatorium lindleyanum DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxy-tigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and 1H-NMR.A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from  Eupatorium lindleyanum DC. (family Compositae) is a traditional Chinese herb. Its aerial part, called ‘Yemazhui’, shows antihistaminic, antibacterial and antioxidant activities −; 1H-NMR : 4.93 ; 2.51 ; 2.34 ; 4.34 ; 4.85 ; 5.21 ; 2.92 ; 5.80 ; 2.84 ; 2.30 ; 6.32 ; 5.63 ; 1.52 ; 1.81 ; 6.79 ; 4.38 ; 1.84 . Comparing the above data with the literature data [1 was identified as 3β-Hydroxy-8β-[4′-hydroxytigloyloxy]-costunolide.Peak 1: ESI-MS: ure data , compounm/z 463.3 [M + H]+; 1H-NMR : 5.27 ; 2.43 ; 2.75 ; 5.30 ; 5.23 ; 5.83 ; 2.98 ; 5.49 ; 2.30 ; 3.15 ; 6.38 ; 5.80 ; 4.90 ; 4.71 ; 1.84 ; 6.76 ; 4.30 ; 1.80 ; 2.01 ; 2.19 . Comparing the above data with the literature data ure data , compounEupatorium lindleyanum DC. in high purity by conventional HSCCC. The results of our study demonstrate that HSCCC is an effective method for separating some difficult to separate natural compounds.As is known the separation of natural compounds with similar structures is very difficult. In the present study, three similar sesquiterpenoid lactones were successfully isolated for the first time from


Their structures were elucidated on the basis of spectroscopic analysis and comparison of NMR data with those of known analogues. At a concentration of 50 μM, compounds 1, 2, and 3 showed 26.2%, 23.6%, and 35.7% inhibition towards xanthine oxidase enzyme, respectively. In addition, compound 3 also showed 24.9% inhibition toward angiotensin-converting enzyme (ACE).Three new dimeric abietane-type diterpenoids, abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7α-yl peroxide ( Cryptomeria japonica D. Don (Cupressaceae), belongs to the monotypic genus in the Cupressaceae + and 284 [C20H28O]+  and aromatic  groups. The 1H- and 13C-NMR data of 1 ), an isopropyl group attached to a phenyl group , 1.01 , and 2.85 ), two para aromatic protons  and 6.69 ), a phenolic hydroxyl proton ), and a typical downshifted Hβ-1 signal of a dehydroabietane diterpene ) +  and forterpenoid . The UV ata of 1  showed oata of 1  also exh3.0 Hz)) . Thus, t2 was an isomer of 1 with the same molecular formula C40H56O3, determined by the molecular ion of HR-EI-MS at m/z 584.4237. Its EI-MS also showed the fragmental ions at m/z 300 [C20H28O2]+ and 284 [C20H28O]+, indicating that 2 was also a dimeric diterpenoid. The absorptions for hydroxyl (3423 cm−1) and aromatic  groups were also found in the UV and IR spectra. Comparison of 1H and 13C-NMR data of 2 and 1 ), an isopropyl group attached to a phenyl group , 1.18 , and 3.01 ), two para aromatic protons  and 6.74 ), a phenolic hydroxyl proton ), and a typical downshifted Hβ-1 signal of a dehydroabietane diterpene ) + ; UV (MeOH) λmax (log ε): 220 (4.74), 279 (4.41) nm; IR (KBr) νmax: 3409, 3049, 1593, 1487, 1460, 1407, 1255, 1169, 1043, 1009, 890 cm−l; 1H-NMR and 13C-NMR : See Abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7α-yl peroxide (1). Gum; + ; UV (MeOH) λmax (log ε): 217 (4.80), 276 (4.45) nm; IR (KBr) νmax 3423, 3048, 1590, 1493, 1460, 1407, 1261, 1169, 1062, 1036, 1003, 963, 890, 738 cm−1; 1H-NMR and 13C-NMR : See Abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7β-yl peroxide (2). Gum; +: 598.4018 [M]+ ; UV (MeOH) λmax (log ε): 233 (4.34), 282 (4.08), 310 (3.88) nm; IR (KBr) νmax 3376, 3049, 1639, 1586, 1467, 1407, 1275, 1182, 1036, 990, 897 cm−1; 1H-NMR and 13C-NMR : See 12-Hydroxyabieta-8,11,13-trien-7β-yl 7-oxoabieta-5,8,11,13-tetraen-12-yl peroxide (3). Gum;  × 100, where ∆AInhibitor and ∆ABlank were the peak areas of HA in testing and blank samples, respectively. Captopril, a known inhibitor of ACE, was used as a positive control, whereas a negative control was performed without any inhibitor. ACE assay was performed using the modified spectrophotometric method described by Cushman and Cheung with minor modifications . The ass1), abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7β-yl peroxide (2), and 12-hydroxyabieta-8,11,13-trien-7β-yl 7-oxoabieta-5,8,11,13-tetraen-12-yl peroxide (3), together with four known abietane-type diterpenoids (4–7) were isolated and characterized from the bark of C. japonica. At a concentration of 50 μM, the three new compounds exhibited the xanthine oxidase inhibitory activity. In addition, compounds 3 also showed ACE inhibitory activity.In this study, three new dimeric abietanoid peroxides, abieta-6,8,11,13-tetraen-12-yl 12-hydroxyabieta-8,11,13-trien-7α-yl peroxide (


Reaction conditions for the selective preparation of each of these products were found. The latter was obtained in 90% yield in CHCl3 under a pressure of acetylene of 10–15 atm, whereas the former product was formed in up to 72% yield in CCl4 under a pressure of acetylene of 1–3 atm. Synthesis of the previously unknown E,E-bis(2-chlorovinyl) telluride, E,E-bis(2-chlorovinyl) ditelluride, E-2-chlorovinyl 1,2,2-trichloroethyl telluride and E,E-bis(2-chlorovinyl)-tellurium dibromide is described.The reaction of tellurium tetrachloride with acetylene proceeds in a stereospecific  Selenium was considered a poison for many years, until Schwarz and Foltz identified it as an essential micronutrient for mammals, including human beings . Like se4 to acetylenes was reported in 1962 +, 252 (48) [M-2Cl]+, 226 (71) [ClTeCH=CHCl]+, 200 (12) [TeCl2]+, 191 (28) [TeCH=CHCl]+, 165 (66) [TeCl]+, 130 (52) [Te]+, 122 (82) [C4H4Cl2]+, 87 (86) [C4H4Cl]+, 61 (37) [C2H2Cl]+, 51 (70) [C4H3]+, 36 (52) [HCl]+, 26 (80) [C2H2]+. E-(2-Chlorovinyl)tellurium trichloride (2). Dry CCl4 (50 mL) was saturated with acetylene by bubbling dry acetylene for 1 h at atmospheric pressure. Then TeCl4  was added and dry acetylene was bubbled through the mixture with intensive stirring for 2 h. The solution was decanted from the precipitate and new portion of dry CCl4 (50 mL) was added to the precipitate and stirred for 1 h. The solution was decanted from the precipitate and combined with the first portion of the CCl4 solution. After evaporation of the solvent, the residue was washed with cold hexane and dried to give product 2 —Colorless powder, which upon heating begins to darken at 45–47 °C and then decomposed. Found (%): C, 7.98; H, 0.78; Cl, 47.66. C2H2Cl4Te. Calculated (%): C, 8.13; H, 0.68; Cl, 48.00. 1H-NMR : 7.38 , 7.82 . 13C-NMR : 131.32, 145.67. E-2-Chlorovinyl 2,2,1-trichloroethyl telluride (3). A mixture of TeCl4  and benzene (80 mL) was heated (20–40 °C) in a 1 L rotating autoclave under acetylene pressure (12–14 atm) for 5 h. The solvent was evaporated and the residue was washed with cold hexane and dried to give compound 1  as a colorless powder. The solvent was evaporated from the hexane solution and the residue was subjected to short column chromatography on silica gel (eluent hexane) to give compound 3  as a dark liquid. Found (%): C, 14.58; H, 1.36; Cl, 44.45. C4H4Cl4Te. Calculated (%): C, 14.94; H, 1.25; Cl, 44.11. 1H-NMR : 5.32 ; 6.03 , 6.66 , 6.87 . 13C-NMR : 62.89 (TeCHCl), 74.58 (CHCl2). 116.21 (TeCH=), 127.87 (=CHCl).E,E-Bis(2-chlorovinyl) ditelluride (4). A solution of Na2S2O5  in water (5 mL) was added to a mixture of compound 2  and benzene (2 mL). The resulted mixture was vigorously stirred at room temperature for 24 h under argon. The mixture was extracted with benzene (3 × 5 mL), organic phase was dried, filtered and the solvent was evaporated. The residue was subjected to column chromatography (eluent–hexane) to give ditelluride 4  as a dark red oil. Found (%): C, 13.08; H, 1.20; Cl, 19.06. C4H4Cl2Te2. Calculated (%): C, 12.70; H, 1.07; Cl, 18.75. 1H-NMR : 6.49 , 7.37 . 13C-NMR : 93.28 (TeCH), 125.34 (CHCl). MS (70 eV), m/z : 380 (64) [M]+, 319 (16) [M-C2H2Cl]+, 293 (27) [Te2Cl]+, 258 (18) [Te2]+, 252 (47) [M-Te]+, 191 (100) [TeC2H2Cl]+, 165 (88) [TeC2H]+, 130 (80) [Te]+, 61 (44) [C2H2Cl]+, 51 (33) [C4H3]+.E,E-Bis(2-chlorovinyl) telluride (5). A solution of Na2S2O5  in water (5 mL) was added to a mixture of compound 1  and benzene (2 mL). The resulting mixture was vigorously stirred at room temperature for 24 h under argon. The mixture was extracted with benzene (3 × 5 mL), the organic phase was dried, filtered and the solvent was evaporated. The residue was subjected to column chromatography (eluent–hexane) to give telluride 5  as a dark yellow oil. Found (%): C, 18.94; H, 1.75; Cl, 28.68. C4H4Cl2Te. Calculated (%): C, 19.17; H, 1.61; Cl, 28.30. 1H-NMR : 6.34 , 6.89 . 13C-NMR : 100.02 (TeCH), 128.14 (CHCl).E,E-Bis(2-chlorovinyl)tellurium dibromide (6). A solution of bromine  in hexane (2 mL) was added dropwise to a stirred solution of compound 5  in hexane (5 mL) at 0 °C (an ace bath). The mixture was stirred for 1 h at 0 °C and for 1 h at room temperature. The solvents were decanted and the precipitate was washed with cold hexane and dried under vacuum to give compound 6  as a brown powder, m.p. 265–268 °C. Found (%): C, 11.46; H, 1.08; Cl, 16.97; Br, 39.23. C4H4Cl2Br2Te. Calculated (%): C, 11.71; H, 0.98; Cl, 17.28; Br, 38.94. 1H-NMR : 7.33 , 7.67 . 13C-NMR : 126.12 (TeCH), 133.70 (CHCl).1–6, prospective precursors and synthons for organic synthesis, have been elaborated. The methods are efficient, simple and based on readily available starting materials (acetylene and tellurium tetrachloride). In contrast to the syn-addition of tellurium tetrachloride to substituted acetylenes [viaanti-addition to give the products 1 and 2 of E-stereochemistry (anti-additions of tellurium tetrachloride to acetylenic hydrocarbons. A mechanism involving the formation of the 3-membered intermediates B and C was proposed in order to explain the stereospecific anti-addition (B and C, A (Convenient methods for preparation of the previously unknown compounds etylenes ,13,14,15hemistry . This araddition . In the B and C,  are suppand C, A .


Reacting 1 with [(η6-cymene)RuCl2]2 and RuHCl(CO)(PPh3)3 generated Ru(η6-cymene)[C4H3N-2-(CO-C6H4-2-OMe)]Cl (2) and {RuCl(CO)(PPh3)2[C4H3N-2-(COC6H4-2-OMe)]} (3), respectively, in moderate yields. Successively reacting 2 with sodium cyanate and sodium azide gave {Ru(η6-cymene)[C4H3N-2-(CO-C6H4-2-OMe)]X}  with the elimination of sodium chloride. Compounds 2–5 were all characterized by 1H and 13C-NMR spectra and their structures were also determined by X-ray single crystallography.A series of ruthenium compounds containing a pyrrole-ketone bidentate ligand, 2-(2′-methoxybenzoyl)pyrrole ( Ruthenium compounds ,2,3 cont6-cymene)RuCl2]2 2  and RuHC2]2 [3)3  are two 2]2 2 2 2 2 2 and RuHCl(CO)(PPh3)3 is shown in 6-cymene)RuCl2]2 with two equivalents of 1 in methanol solution at room temperature in the presence of sodium bicarbonate gave a 32.0% yield of Ru(η6-cymene)[C4H3N-2-(CO-C6H4-2-OMe)]Cl (2). The 1H-NMR spectrum of 2 showed four doublets in the range of δ 5.62~5.35 representing the arene CH protons of the cymene fragment. Similarly, while reacting 1 with RuHCl(CO)(PPh3)3 in toluene under refluxing and work-up, the resulting product {RuCl(CO)(PPh3)2[C4H3N-2-(COC6H4-2-OMe)]} (3) was prepared in a 66.9% yield along with the elimination of one equivalent of hydrogen molecules. The 1H-NMR spectrum of 3 showed the pyrrole CH chemical shifts at δ 5.50, 5.59, and 6.65. Due to the weak hydrogen bonding, the isopropyl fragment showed two doublets in the 1H-NMR spectrum representing the slow C-C bond rotation of the isopropyl and the cymene phenyl ring. The 13C-NMR spectra of 3 showed two chemical shifts at δ 205.6 and 183.4, assigned as the carbon of C≡O and C=O, respectively [3 also showed a strong absorption band at 1942 cm−1 representing the stretching frequency of C≡O [A series of ruthenium compounds containing 2-(2′-methoxybenzoyl)pyrrole [C4H3N-2-(CO-C6H4-2-OMe)]X} . Compounds 4 and 5 were both characterized by 1H and 13C-NMR spectra and showed patterns similar to those of Compound 2, indicating that the electronic properties around the ruthenium atom was not affected by the coordinated mono-anionic ligands.Successively replacing the chloride anion of 2–5 for X-ray diffraction analysis were obtained from either methanol or THF saturated solution at −20 °C. The summary of the X-ray crystal data and selected bond lengths and angles are shown in 2–5 are depicted in 2 could be described as a three-legged piano stool with the nitrogen atom of the pyrrole, the oxygen atom of the carbonyl atoms, and the chloride atom forming the three legs and the cymene ring acting as the stool plane. The bond length of the ruthenium atom to the center of the cymene ring is 1.6598(1) Å, relatively close to the bond lengths of published ruthenium-cymene compounds 2 were prepared using a modified procedure 2  in a 50 mL of flask was stirred for 1 h at room temperature. Sodium bicarbonate then was added into the solution and stirred for 3 h. The volatiles were removed under vacuum and the solid was extracted with methylene chloride and then solvent was removed again to give crude product of 2. The product was recrystallized from a methanol solution to generate 0.149 g of red brown crystals in a 32.0% yield. 1H-NMR (CDCl3): 1.21 , 1.23 , 2.30 , 2.81 , 3.77 , 5.35 , 5.40 , 5.55 , 5.62 , 6.37 , 6.79 , 7.36 . 13C-NMR (CDCl3): 18.9 (cymene Me), 22.1 (cymene CHMe2), 22.8 (cymene CHMe2), 31.9 (cymene CHMe2), 55.9 (OMe), 79.3 (cymene CH), 81.8 (cymene CH), 82.0 (cymene CH), 83.5 (cymene CH), 98.4 (cymene ipsoC), 100.5 (cymene ipsoC), 111.8 (pyr CH), 116.1 (pyr CH), 120.4 (phenyl CH), 123.4 (phenyl CH), 125.5 (pyr ipsoC), 131.4 (phenyl CH), 132.1 (phenyl CH), 141.8 (phenyl ipsoC), 143.1 (pyr CH), 157.7 (phenyl ipsoC), 186.7 (C=O). Anal. Calcd for C22H24ClNO2Ru: C, 56.11; H, 5.14; N, 2.97. Found: C, 56.08; H, 5.15; N, 3.36.A methanol solution (10 mL) of 1  and RuHCl(CO)(PPh3)3  in a 50 mL of flask was refluxed for 24 h under nitrogen. The volatiles were removed under vacuum and residue was washed with heptane to remove excess of PPh3. The resulting solid was recrystallized from a saturated THF solution at −20 °C to give 0.24 g of yellow crystals in a 66.9% yield. 1H-NMR (CDCl3): 3.31 , 5.50 , 5.59 , 6.20 , 6.40 , 6.65 , 7.30 . 13C-NMR (CDCl3): 55.0 (OMe), 110.3, 117.3, 119.7, 123.0, 125.4, 126.6, 127.8, 127.9, 128.0, 128.3, 128.6, 128.7, 129.2, 129.5, 130.5, 130.9, 131.0, 131.2, 131.5, 132.2, 134.5, 134.6, 134.7,141.3, 142.6, 156.3, 183.4 (C=O), 205.6 (C≡O). Anal. Calcd for C49H40NO3P2ClRu: C, 66.13; H, 4.53; N, 1.57. Found: C, 66.01; H, 4.86; N, 1.53.A toluene solution (10 mL) of 2 , excess sodium cyanate and methanol (10 mL) was stirred at room temperature for overnight. Methanol was removed under vacuum and residue was extracted with methylene chloride (10 mL × 3). The methylene chloride was removed and residue was recrystallized from a methanol solution at −20 °C to yield 0.045 g of orange crystals of 4 (38.1% yield). 1H-NMR (CDCl3): 1.20 , 1.22 , 2.25 , 2.74 , 3.77 , 5.32 , 5.57 , 5.62 , 6.37 , 6.78 , 7.38 , 7.62 . Anal. Calcd for C23H24N2O3Ru: C, 57.70; H, 5.06; N, 5.86. Found: C, 57.70; H, 5.18; N, 6.04.A 25 mL Schlenk flask charged with 2 , excess sodium azide, and methanol (10 mL) was stirred at room temperature for overnight. Methanol was removed under vacuum and residue was extracted with methylene chloride (10 mL × 3). The methylene chloride was removed and residue was recrystallized from a methanol solution at −20°C to yield 0.026 g of orange crystals of 5 (53.8% yield). 1H-NMR (CDCl3): 1.23 , 1.25 , 2.27 , 2.78 , 3.80 , 5.32 , 5.53 , 5.62 , 6.43 , 6.82 , 7.38 . 13C-NMR (CDCl3): 18.4 (cymene Me), 22.5 (cymene CHMe2), 22.8 (cymene CHMe2), 31.2 (cymene CHMe2), 55.9 (OMe), 78.9 (cymene CH), 81.7 (cymene CH), 82.4 (cymene CH), 84.2 (cymene CH), 98.7 (arene C), 100.5 (ipsoC),111.7 (pyr CH), 116.2 (pyr CH), 120.5 (phenyl CH), 123.7 (phenyl CH), 125.2 (ipsoC), 131.6 (phenyl CH), 132.1 (phenyl CH), 142.1 (ipsoC), 143.2 (pyr CH), 157.7 (ipsoC), 187.2 (C=O). Anal. Calcd for C22H24N4O2Ru: C, 55.34; H, 5.07; N, 11.73. Found: C, 55.32; H, 5.09; N, 11.75.A 25 mL Schlenk flask charged with 2–5 were attached to a fine glass fiber and mounted in goniostat for data collection. Data collections were performed at 150 K under liquid nitrogen vapor for all compounds. Data were collected on a Bruker SMART CCD diffractometer with graphite monochromated Mo-Kα radiation. No significant crystal decay was found. Data were corrected for absorption empirically by means of ψ scans. All non-hydrogen atoms were refined with anisotropic displacement parameters. For all the structures, the hydrogen atom positions were calculated and they were constrained to idealized geometries and treated as rigid where the H atom displacement parameter was calculated from the equivalent isotropic displacement parameter of the bound atom. An absorption correction was performed with the program SADABS [2F’s, in SHELXL [Suitable crystals of Compounds m SADABS  and the m SADABS  and refin SHELXL . All the2–5 and their structures were determined by X-ray single crystallography. A preliminary test of hydrogen transfer reactions of acetophenone and isopropyl alcohol using these ruthenium compounds showed low conversion. We are currently using Compounds 2–5 to investigate the catalytic activities of hydrogen transfer reactions toward varieties of ketones and alcohols and the hydroaminations of inter- and intra-molecular alkenes and alkynes.We have successfully employed a bidentate pyrrole-ketone ligand with ruthenium compounds to form a series of Compounds


All the synthesized derivatives 4(a–f) and 6(a–f) were evaluated for their in vitro antifungal and antibacterial activity, by method recommended by National Committee for Clinical Laboratory Standards (NCCLS). The compound 6c bearing a fluoro group on the chromone ring and oxygen and a hydrazino group (–NHNH2) on the pyrimidine ring, was found to be the most potent antibacterial compound amongst the synthesized derivatives. The compound 6f bearing a methoxy group (–OCH3) on the chromone ring and sulphur group on the pyrimidine ring, was found to exhibit equipotent antifungal activity when compared with the standard drug miconazole. A d-alanine-d-alanine ligase (DdlB) enzyme assay study and an ergosterol extraction and quantitation assay study were performed to predict the mode of action of the synthesized compounds. A molecular docking study was performed to predict the binding interactions with receptors and mode of action of the synthesized derivatives. Further, analysis of the ADMET parameters for the synthesized compounds has shown that these compounds have good oral drug-like properties and can be developed as oral drug candidates. To establish the antimicrobial selectivity and safety, the most active compounds 6c and 6f were further tested for cytotoxicity against the human cancer cell line HeLa and were found to be non-cytotoxic in nature. An in vivo acute oral toxicity study was also performed for the most active compounds 6c and 6f and the results indicated that the compounds are non-toxic in nature.Herein, we report an environmentally friendly, rapid, and convenient ionic liquid ([Et In the past few decades many drug-resistant human pathogenic microbes have been observed  and thisCoumarins are an elite class of naturally occurring compounds with promising therapeutic perspectives ,7. This Nitrogen-containing heterocycles such as pyrimidine are a subject of continuous interest because of their diverse biological activities and occurrence in natural medicinal plants. Pyrimidine and its derivatives are used as pesticides, herbicides and insecticides ,17. MarkMolecular hybridization is a rational drug design approach whereby new chemical entities are obtained by combining two or more pharmacophoric units from different bioactive compounds into a single molecule. Through this approach, medicinal chemists hope that the new hybrid derivatives will present better affinity and efficacy when compared to the individual parent drugs, a modified selectivity profile with improved pharmacokinetic and pharmacodynamic properties, dual or multiple modes of action, reduction of undesirable side effects, decreases in drug-drug interactions, reduced emergence or spread of drug resistance in microorganisms and protozoans and lower cost . Pharmac4(a–f) and 6(a–f) using triethylammonium hydrogen sulphate [Et3NH][HSO4] as an solvent and easily recoverable green catalyst [HSO4] in molten state, which is a low cost, mild, non-volatile and non-corrosive acidic ionic liquid, as an efficient Bronsted acid catalyst under solvent-free conditions for the Biginelli reaction is described, as shown in The utility of triethylammonium hydrogen sulphate [Et3NH][HSO4] was optimized by investigating various parameters such as the mol percent of catalyst at various temperatures. The triethylammonium hydrogen sulphate [Et3NH][HSO4] ionic liquid was screened as catalyst at various loads such as 5, 10, 15, 20 mol % and at various temperatures such as 80, 90, 100, 110 °C as shown in 4a was obtained in 72% yield in 150 min. Furthermore, the effect of the amount of catalyst was examined by examining the effect of various catalyst loads on the yield of product and the time required for completion of reaction for compound 4a. When 10 mol % of catalyst was used at 100 °C, compound 4a was obtained in 76% yield in 105 min. When 15 mol % of catalyst was used at 100 °C, 95% of 4a was obtained in 60 min. The same yield resulted when 20 mol % of catalyst was used at 100 °C. Therefore, considering 15 mol % as an efficient amount the reaction was carried out at various temperatures like 80 °C, 90 °C, 100 °C and 110 °C. Temperatures both above and below 100 °C gave lower yields, therefore 15 mol % of the [Et3NH][HSO4] ionic liquid as catalyst and solvent at 100 °C was considered to give the best yield (95%) in the shortest reaction time (60 min)  were synthesized according to the abovementioned green synthetic protocol. The time required for the completion of reactions was around 60–85 min time (monitored by TLC). The yields of the synthesized novel compounds were in the 86–95% range. The structures of the synthesized compounds were confirmed by IR, 1H-NMR, 13C-NMR, mass spectral analysis and elemental analysis.A total of six novel ethyl 4-(6-substituted-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 4(a–f) (1 mmol) were next allowed to react with hydrazine hydrate (1.2 mmol) under solvent free condition using [Et3NH][HSO4] as a catalyst and also as a solvent to furnish compounds 6(a–f). The catalyst load and the temperature required for the synthesis of 6(a–f) were also studied. The effect of different [Et3NH][HSO4] ionic liquid catalyst loads on the yield of product and time required for completion of reaction were studied at various catalyst loads such as 5, 10, 15 mol % and at various temperatures such as 70, 80, 90 and 100 °C, as shown in 6a was obtained in 88% yield in 30 min. When 10 mol % catalyst was used at the same temperature, compound 6a was obtained in 87% yield in 30 min. When 15 mol % catalyst was used at 90 °C, compound 6a was obtained in 85% yield in 30 min. Therefore, considering 5 mol % as an efficient amount, the reaction was carried out at various temperatures like 70 °C, 80 °C, 90 °C and 100 °C. At 5 mol % catalyst loading the use of the higher (100 °C) or lower  temperatures than 90 °C gave the compound 6a in reduced yields after longer times, therefore, 5 mol % of the [Et3NH][HSO4] ionic liquid as catalyst and solvent was shown to give the best yield (88%) in a short reaction time (30 min) at 90 °C -6-methyl-2-thioxo/oxo-1,2,3,4-tetra-hydropyrimidine-5-carbohydrazides 6(a–f) were synthesized according to the abovementioned green synthetic protocol. The time required for the completion of reactions was around 20–35 min (monitored by TLC), and the yields of the synthesized novel compounds were in the 80–95% range The structures of the synthesized compounds were confirmed by IR, 1H-NMR, 13C-NMR, mass spectral analysis and elemental analysis.4(a–f) and 6(a–f) were screened for their in vitro antibacterial activity against Staphylococcus aureus (NCIM-2901), Escherichia coli 1411 (standard laboratory strains) and Escherichia coli SM1411 (an acrAB deficient derivative of 1411). Minimum inhibitory concentration (MIC100) values were determined using methods recommended by National Committee for Clinical Laboratory Standards (NCCLS) [HSO4] (15 mol %) was refluxed under solvent-free conditions at 100 °C for the required time while the reactions were monitored by TLC. After completion of the reaction, the reaction mixture was poured into crushed ice and stirred for 5 min. The solid obtained was filtered, washed with cold water and then recrystallized from ethanol to afford the pure product. The ionic liquid present in the filtrate was recovered and was used for other reactions. A mixture of substituted 4-oxo-4Ethyl 6-methyl-2-oxo-4-(4-oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate4a. Yield 95%; M.P.: 270–272 °C; IR (KBr vmax in cm−1): 3238 (N–H stretching), 3005 (C–H stretching), 2900 (–CH3 stretching), 2815 , 1746 (C=O stretching), 1601 (C=C stretching), 1454 (CH bending of CH2), 1356 (C–N stretching), 1002 (–O– stretching); 1H-NMR : 1.14 , 2.28 , 4.05 , 4.72 , 7.41–7.59 , 7.75 , 8.05 , 8.10 , 9.12 ; 13C-NMR : 14.45, 18.51, 45.91, 61.37, 101.94, 118.07, 121.61, 123.87, 126.27, 126.68, 133.85, 149.33, 150.72, 155.65, 155.77, 167.37, 172.17; MS: m/z: 329.21 [M + 1]+; Anal. Calcd. for C17H16N2O5: C, 62.19; H, 4.91; N, 8.53. Found: C, 62.20; H, 4.93; N, 8.50.Ethyl 6-methyl-4-(4-oxo-4H-chromen-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate4b. Yield 87%; M.P.: 210–212 °C; IR (KBr vmax in cm−1): 3235 (N–H stretching), 3005 (C–H stretching), 2905 (–CH3 stretching), 2815 , 1600 (C=C stretching), 1454 (CH bending of CH2), 1360 (C–N stretching), 1140 (C=S stretching), 1002 (–O– stretching); 1H-NMR : 1.17 , 2.29 , 4.74 , 7.47–7.74 , 8.03 , 8.06 , 8.12 , 8.89 ; 13C-NMR : 14.50, 18.44, 50.62, 61.77, 106.24, 118.71, 121.32, 123.99, 126.12, 133.91, 150.11, 155.60, 160.39, 167.33, 171.94, 177.89; MS: m/z: 345.01 [M + 1]+; Anal. Calcd. for C17H16N2O4S: C, 59.29; H, 4.68; N, 8.13. Found: C, 59.31; H, 4.69; N, 8.10.Ethyl 4-(6-fluoro-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate4c. Yield 92%; M.P.: 200–202 °C; IR (KBr vmax in cm−1): 3230 (N–H stretching), 3000 (C–H stretching), 2900 (–CH3 stretching), 2815 , 1745 (C=O stretching), 1600 (C=C stretching), 1454 (CH bending of CH2), 1364 (C–N stretching), 1002 (–O– stretching); 1H-NMR : 1.15 , 2.29 , 4.09 , 4.74 , 6.91–7.11 , 7.43 , 7.74  8.06 , 9.13 ; 13C-NMR : 14.22, 18.11, 45.51, 61.70, 101.99, 111.52, 118.71, 119.23, 122.00, 126.88, 149.14, 150.79, 155.13, 156.11, 164.19, 167.19, 172.10; MS: m/z: 347.11 [M + 1]+; Anal. Calcd. for C17H15FN2O5: C, 58.96; H, 4.37; N, 8.09. Found: C, 58.99; H, 4.39; N, 8.04.Ethyl 4-(6-methoxy-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate4d. Yield 88%; M.P.: 230–232 °C; IR (KBr vmax in cm−1): 3238 (N–H stretching), 3005 (C–H stretching), 2900 (–CH3 stretching), 2845 (–OCH3 stretching), 2815 , 1742 (C=O stretching), 1600 (C=C stretching), 1454 (CH bending of CH2), 1362 (C–N stretching), 1002 (–O– stretching); 1H-NMR : 1.17 , 2.29 , 3.80 , 4.10  4.74 , 7.37 , 7.42 , 7.48–7.52 , 8.06 , 9.13 ; 13C-NMR : 14.45, 18.51, 45.91, 55.08, 61.37, 101.94, 107.44, 117.98, 119.35, 122.42, 149.33, 150.72, 152.63, 155.65, 156.18, 167.37, 171.94; MS: m/z: 359.53 [M + 1]+; Anal. Calcd. for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.35; H, 5.09; N, 7.80.Ethyl 4-(6-methoxy-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate4e. Yield 86%; M.P.: 222–224 °C; IR (KBr vmax in cm−1): 3235 (N–H stretching), 3008 (C–H stretching), 2908 (–CH3 stretching), 2845 (–OCH3 stretching), 2813 , 1740 (C=O stretching), 1600 (C=C stretching), 1454 (CH bending of CH2), 1360 (C–N stretching), 1148 (C=S stretching), 1002 (–O– stretching); 1H-NMR : 1.17 , 2.29 , 3.80 , 4.11 , 4.73 , 7.37 , 7.36 , 7.48–8.08 , 8.89 ; 13C-NMR : 14.45, 18.51, 49.08, 55.80, 61.37, 104.43, 107.44, 117.98, 119.35, 122.42, 126.68, 149.33, 152.63, 154.52, 156.18, 167.37, 171.94, 177.89; MS: m/z: 375.17 [M + 1]+; Anal. Calcd. for C18H18N2O5S: C, 57.74; H, 4.85; N, 7.48. Found: C, 57.77; H, 4.86; N, 7.46.Ethyl 4-(6-fluoro-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate4f. Yield 92%; M.P.: 228–230 °C; IR (KBr vmax in cm−1): 3230 (N–H stretching), 3000 (C–H stretching), 2910 (–CH3 stretching), 2810 , 1742 (C=O stretching), 1605 (C=C stretching), 1450 (CH bending of CH2), 1360 (C–N stretching), 1140 (C=S stretching), 1005 (–O– stretching); 1H-NMR : 1.15 , 2.29 , 4.11 , 4.73 , 6.91–7.74 , 7.76 , 8.08 , 8.89 ; 13C-NMR : 14.44, 18.46, 50.66, 61.79, 106.33, 111.50, 118.73, 119.31, 122.18, 126.89, 149.15, 152.36, 160.19, 162.58, 167.79, 171.96, 177.88; MS: m/z: 363.15 [M + 1]+; Anal. Calcd. for C17H15FN2O4S: C, 56.35; H, 4.17; N, 7.73. Found: C, 56.37; H, 4.19; N, 7.70.H-chromen-3-yl)-6-methyl-2-thioxo/oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivative 4(a–f) (1 mmol), hydrazine hydrate (5) (1.2 mmol) and [Et3NH][HSO4] (5 mol %) was refluxed under solvent-free conditions at 90 °C for the required time, while reaction completion was monitored by TLC. After completion of the reaction, the reaction mixture was poured into crushed ice and stirred for 5 min. The solid obtained was filtered, washed with cold water and then recrystallized from ethanol to afford the pure product. The ionic liquid present in the filtrate was recovered and was used for other reactions.A mixture of ethyl4-(6-substituted-4-oxo-46-Methyl-2-oxo-4-(4-oxo-4H-chromen-3-yl)-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6a. Yield 88%; M.P.: 256–258 °C; IR (KBr vmax in cm−1): 3520 (NH2 stretching), 3450 (N–H stretching), 3000 (C–H stretching), 2900 (–CH3 stretching), 1742 (C=O stretching), 1605 (C=C stretching), 1368 (C–N stretching), 1005 (–O– stretching); 1H-NMR : 2.03 , 3.13 , 4.74 , 7.08 , 7.42 , 7.47–8.13 , 9.39 ; 13C-NMR : 18.49, 46.11, 102.31, 118.71, 121.61, 123.44, 126.23, 126.85, 133.88, 149.45, 150.71, 155.36, 159.17, 168.62, 172.49; MS: m/z: 315.25 [M + 1]+; Anal. Calcd. for C15H14N4O4: C, 57.32; H, 4.49; N, 17.83. Found: C, 57.32; H, 4.49; N, 17.83.6-Methyl-4-(4-oxo-4H-chromen-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6b. Yield 88%; M.P.: 198–200 °C; IR (KBr vmax in cm−1): 3515 (–NH2 stretching), 3450 (N–H stretching), 3000 (C–H stretching), 2908 (–CH3 stretching), 1742 (C=O stretching), 1600 (C=C stretching), 1360 (C–N stretching), 1145 (C=S stretching), 1002 (–O– stretching); 1H-NMR : 2.26 , 3.13 , 4.74 , 7.08 , 7.47 , 7.59–8.13 , 8.89 ; 13C-NMR : 18.97, 51.16, 106.11, 118.23, 121.14, 123.43, 126.28, 126.81, 133.69, 150.11, 155.49, 160.19, 168.66, 172.32, 177.80; MS: m/z: 331.29 [M + 1]+; Anal. Calcd. for C15H14N4O3S: C, 54.53; H, 4.27; N, 16.96. Found: C, 54.55; H, 4.29; N, 16.93.4-(6-Fluoro-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6c. Yield 95%; M.P.: 210–212 °C; IR (KBr vmax in cm−1): 3510 (NH2 stretching), 3455 (N–H stretching), 3000 (C–H stretching), 2910 (–CH3 stretching), 1742 (C=O stretching), 1605 (C=C stretching), 1365 (C–N stretching), 1005 (–O– stretching); 1H-NMR : 2.03 , 3.13 , 4.74 , 6.91 , 7.08 , 7.10–8.06 , 9.39 ; 13C-NMR : 18.26, 46.83, 106.12, 109.11, 118.36, 121.33, 123.39, 126.10, 147.77, 150.18, 155.60, 155.99, 162.45, 168.20, 172.45; MS: m/z: 333.55 [M + 1]+; Anal. Calcd. for C15H13FN4O4: C, 54.22; H, 3.94; N, 16.86. Found: C, 54.25; H, 3.96; N, 16.83.4-(6-Fluoro-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6d. Yield 90%; M.P.: 146–148 °C; IR (KBr vmax in cm−1): 3512 (NH2 stretching), 3458 (N–H stretching), 3000 (C–H stretching), 2912 (–CH3 stretching), 1742 (C=O stretching), 1605 (C=C stretching), 1360 (C–N stretching), 1140 (C=S stretching), 1005 (–O– stretching); 1H-NMR : 2.27 , 3.14 , 4.74 , 6.92 , 7.09 , 7.11–8.09 , 8.90 ; 13C-NMR : 18.10, 52.61, 106.21, 109.14, 118.24, 121.39, 123.41, 126.52, 150.19, 155.62, 160.41, 162.56, 168.21, 171.96, 177.81; MS: m/z: 349.72 [M + 1]+; Anal. Calcd. for C15H13FN4O3S: C, 54.22; H, 3.94; N, 16.86. Found: C, 54.27; H, 3.99; N, 16.80.4-(6-Methoxy-4-oxo-4H-chromen-3-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6e. Yield 82%; M.P.: 208–210 °C; IR (KBr vmax in cm−1): 3520 (NH2 stretching), 3452 (N–H stretching), 3000 (C–H stretching), 2905 (–CH3 stretching), 2845 (–OCH3 stretching), 1742 (C=O stretching), 1605 (C=C stretching), 1362 (C–N stretching), 1005 (–O– stretching); 1H-NMR : 2.03 , 3.13 , 3.80 , 4.74 , 7.08 , 7.37–8.06 , 9.39 ; 13C-NMR : 18.92, 46.48, 56.82, 106.66, 109.11, 118.24, 121.48, 123.49, 125.76, 147.41, 150.62, 155.12, 156.30, 168.61, 172.27; MS: m/z: 345.02 [M + 1]+; Anal. Calcd. for C16H16N4O5: C, 55.81; H, 4.68; N, 16.27. Found: C, 55.84; H, 4.69; N, 16.25.4-(6-Methoxy-4-oxo-4H-chromen-3-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide6f. Yield 80%; M.P.: 158–160 °C; IR (KBr vmax in cm−1): 3522 (NH2 stretching), 3455 (N–H stretching), 3000 (C–H stretching), 2910 (–CH3 stretching), 2845 (–OCH3 stretching), 1742 (C=O stretching), 1605 (C=C stretching), 1360 (C–N stretching), 1146 (C=S stretching), 1005 (–O– stretching); 1H-NMR : 2.26 , 3.13 , 3.80 , 4.74 , 7.08 , 7.37–8.08 , 8.89 ; 13C-NMR : 18.90, 51.50, 105.66, 106.41, 118.90, 121.71, 123.44, 126.47, 150.53, 150.66, 156.30, 160.31, 168.22, 171.96, 177.98; MS: m/z: 361.10 [M + 1]+; Anal. Calcd. for C16H16N4O4S: C, 53.32; H, 4.47; N, 15.55. Found: C, 53.35; H, 4.49; N, 15.51.4(a–f) and 6(a–f) were screened for their in vitro antibacterial activity against Staphylococcus aureus (NCIM-2901) and Escherichia coli 1411 (standard laboratory strains) and Escherichia coli SM1411 (an acrAB deficient derivative of 1411). Minimum inhibitory concentration (MIC100) values were determined using the method recommended by National Committee for Clinical Laboratory Standards (NCCLS). In vitro antibacterial activities of the synthesized compounds 4(a–f) and 6(a–f) were tested in Nutrient Broth (NB) for Bacteria by the two fold serial dilution method. Seeded broth  was prepared in NB from 24 h old bacterial cultures on nutrient agar (Hi-media) at 37 ± 1 °C. The bacterial suspension was adjusted with sterile saline to a concentration of 1 × 10−4–10−5 C.F.U. The synthesized compounds and standard drug d-cycloserine were prepared by two fold serial dilutions to obtain the required concentrations. The tubes were incubated in BOD incubators at 37 ± 1 °C for bacteria. The MICs were recorded by visual observations after 24 h (for bacteria) of incubation [All the synthesized compounds cubation ,29,30,31d-Ala-adding activity of DdlB ligase was monitored by the detection of orthophosphate generated during the reaction based on the colorimetric malachite green method described by Walsh et al. [2, 6.5 mM (NH4)2SO4, 700 µM d-Ala, 500 µM ATP, purified DdlB  and the test compound . All the compounds were soluble in the assay mixture containing 5% DMSO. After 30 min of incubation, 100 µL Biomol reagent was added. After 5 min, absorbance was read at 650 nm. Residual activity was calculated with respect to a similar assay without inhibitor. To exclude possible non-specific (promiscuous) inhibitors, representative compounds 4(a–f) and 6(a–f) were also tested in the presence of Tween (0.003%), Triton X-114 (0.005%), and SDS (420 µM), as described by McGovern et al. [The h et al. . Assays n et al. ,48. No sn et al. .In vitro antifungal activity was determined as per CLSI  guidelines [4(a–f), 6(a–f) and the standard drug miconazole were dissolved in DMSO solvent. The medium yeast nitrogen base was dissolved in phosphate buffer pH 7 and it was autoclaved at 110 °C for 10 min. With each set a growth control without the antifungal agent and solvent control DMSO were included. The fungal strains were freshly sub cultured on to Sabouraud dextrose agar (SDA) and incubated at 25 °C for 72 h. The fungal cells were suspended in sterile distilled water and diluted to get 105 cells/mL. Further, ten microlitres of the standardized suspension was inoculated onto the control plates and the media incorporated with the antifungal agents. The inoculated plates were incubated at 25 °C for 48 h. The readings were taken at the end of 48 h and 72 h. The MIC was the lowest concentration of drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug free control plates [idelines ,30,31. Tl plates .Candida albicans (NCIM-3471) colony from an overnight Sabouraud dextrose agar plate culture was used to inoculate 50 mL of Sabouraud dextrose broth for control and for various concentrations of the molecules. The cultures were incubated for 16 hours and harvested by centrifugation at 2700 rpm (856× g) for five min. The net weight of the cell pellet was determined. Three mL of 25% alcoholic potassium hydroxide solution was added to each pellet and vortex mixed for one min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated in an 85 °C water bath for one hour. Following incubation, the tubes were allowed to cool. Sterols were then extracted by addition of a mixture of 1 mL of sterile distilled water and 3 mL of n-heptane followed by vigorous vortex mixing for 3 min. The heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at −20 °C. Prior to analysis, 0.6 mL aliquot of sterol extract was diluted five fold in 100% ethanol and scanned spectrophotometrically between 240 nm and 300 nm with a spectrophotometer . The presence of ergosterol and the late sterol intermediate 24(28)-dehydroergosterol (DHE) in the extracted sample results in a characteristic four-peaked curve. The absence of detectable ergosterol in extracts was indicated by a flat line. A dose-dependent decrease in the height of the absorbance peaks was evident and corresponded to decreased ergosterol concentration [A single ntration ,50.C. albicans was built using homology modeling [C. albicans and into the E. coli DdlB enzyme (PDB entry: 1IOV [The 3D model structure of cytochrome P450 lanosterol 14-α demethylase of modeling . Moleculry: 1IOV ) were peC. albicans was built using homology modeling. The amino acid sequence of the enzyme was obtained from the Universal Protein Resource (http://www.uniprot.org/) (Accession Code: P10613), and sequence homologous was obtained from Protein Data Bank (PDB) using Blast search. In literature, the structure of cytochrome P450 lanosterol 14α-demethylase was developed homologically using crystal structure of lanosterol 14α-demethylase from C. albicans as template. Based on the result of blast search, we used the crystal structure of human lanosterol 14-α DM (CYP51) with miconazole as a template for homology modeling (PDB ID. 3I3K). A hidden Markov model (HMM) was generated from a sequence alignment for the identification of sequence motifs and query family, which provides the information about which residues are conserved in the consensus sequence. This information was used as constraints in the generation of the protein sequence alignment. In addition, secondary structure prediction algorithms SSpro [C. albicans lanosterol 14α-demethylase to human lanosterol 14α-demethylase (DM) enzyme. The combination of sequence motifs and secondary structure provides accurate picture of each helix. Alignment of C. albicans lanosterol 14α-demethylase with human lanosterol 14α-demethylase enzyme was carried out using the automated alignment program in PRIME v. 2.1 . Following automated alignment, manual inspection was made to ensure the conserved motifs, and loops were correctly aligned. The final model of C. albicans lanosterol 14α-DM enzyme was developed using PRIME v2.1. The binding sites were generated using SITEMAP v2.3 [C. albicans lanosterol 14 α-demethylase model was assessed by using the well-validated programs PROCHECK [The 3D model structure of cytochrome P450 lanosterol 14α-demethylase of -α DM CYP with micMAP v2.3   docking mode for all compounds was performed on generated grid of protein structure [Molecular docking study was performed in Maestro 9.1 using Glide v. 6.8 (Schrodinger LLC). All compounds were built using Maestro build panel and optimized to lower energy conformers using Ligprep v3.5 . The coordinates for tructure . The fintructure .4(a–f) and 6(a–f) was performed for prediction of ADMET properties. The absorption, distribution, metabolism, elimination and toxicity (ADMET) properties of all the synthesized compounds were predicted using Qikprop v. 3.5 (Schrodinger LLC). In the present study, we have calculated the molecular volume (M.V.), molecular weight (M.W.), predicted octanol–water partition coefficient (log P o/w), number of hydrogen bond acceptors (n-ON), number of hydrogen bonds donors (n-OHNH), percentage human oral absorption (% ABS), Van der waals surface area of polar nitrogen and oxygen atoms (Polar Surface Area), Log S (water solubility), BIPCaco-2 (apparent Caco-2 cell permeability), Log Khsa  and Log HERG (toxicity study) [A computational study of the synthesized compounds y study) .in vitro cytotoxicity study of the most active compounds 6c and 6f was performed against HeLa  and PC-3 (human prostate cancer cell line) using Sulforhodamine B (SRB) assay using Adriamycin as positive control [To explore the selective antifungal activity of the synthesized compounds  control . The ima6c and 6f, which are most potent among the synthesized compounds, were further subjected to an in vivo acute oral toxicity study and gross behavioral study as per the OECD guideline No. 425. Each group, consisting of six Swiss albino mice , were kept in colony cage at 25 ± 2 °C with 55% relative humidity and 12 h of light and dark cycle. A specified dose of 100, 250, 500, 750, 1000, 1500 and 2000 mg/kg body weight of mice was administered orally as a single dose. The acute toxic symptoms and the behavioral changes produced by the test compounds were observed continuously for 4 h periods at 8th, 12th and 24th h on set of toxic symptoms and the gross behavioral changes were also recorded. These animals were maintained for further 10 days with observation made daily. The in vivo acute oral toxicity study was carried out at animal house at the Y.B. Chavan College of Pharmacy, Aurangabad IAEC approval number CPCSEA/IAEC/P’col-52/2015-16/115 [Two of the newly synthesized compounds 5-16/115 .4(a–f) and 6(a–f) containing chromone-pyrimidine coupled hybrid derivatives as antifungal and antibacterial agents using a green synthetic protocol. The use of a green method, i.e., use of ionic liquid helped us in the synthesis of the target derivatives in good yield. The eco-friendly reaction conditions, good to excellent yields in a short reaction time and avoidance of cumbersome work-up procedures make this process economically worthwhile for industrial application, with the additional advantage of reusability of the catalyst. The in vitro antibacterial activity results suggest that compounds 4c, 6c and 6d possess potent in vitro antibacterial activity. The in vitro antifungal activity results suggest that compounds 4e, 6e and 6f possess potent in vitro antifungal activity. A d-alanine-d-alanine ligase (DdlB) enzyme assay study indicates that the synthesized derivatives have the ability to inhibit the DdlB enzyme. The synthesized derivative 6f was found to be the most active antifungal agent; its mode of action was studied using an ergosterol extraction and quantitation assay method. The results of this test indicate that the synthesized compound 6f acts by inhibiting ergosterol biosynthesis by inhibiting lanosterol 14α-demethylase enzyme. A molecular docking study was performed to predict the mechanism of action of the synthesized derivatives. Further, analysis of the ADMET parameters for the synthesized compounds performed using Qikprop v3.5 has revealed that these compounds have good oral drug-like properties and which makes them suitable for development as future oral drug candidates. Moreover, the cytotoxicity study revealed that the compounds 6c and 6f did not show any significant cytotoxicity against HeLa and PC-3 cell lines at the maximum evaluated concentration, thus indicating the selectivity of their antimicrobial action. The newly synthesized compounds 6c and 6f were also tested for their in vivo acute oral toxicity study and there was no mortality and no significant behavioral changes observed in Swiss albino mice for the first 24 h. Thus, the synthesized coupled compounds have shown excellent in vitro antimicrobial activity and compounds 4c, 4e, 6c and 6f could be in future further developed as lead antimicrobial molecules.In this study, we have synthesized a suite of novel compounds