The new compounds were structurally elucidated through spectroscopic analysis including UV (Ultraviolet Spectrum), IR (Infrared Spectrum), 1H-NMR (1Hydrogen-Nuclear Magnetic Resonance), 13C-NMR (13Carbon-Nuclear Magnetic Resonance), DEPT (Distortionless Enhancement by Polarization Transfer), 1H-1H COSY (1H-1H Correlation Spectroscopy), HSQC (Heteronuclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Correlation), NOESY (Nuclear Overhauser Enhancement Spectroscopy) and HR-MS (High Resolution Mass Specttrum) and their absolute configurations were proposed by comparison of NOESY spectra and specific optical rotations with those of known compounds and biosynthesis grounds. Compound 2 is the first sesquiterpene alkaloid isolated from this plant. New compounds 1–4 exhibited some cytotoxic activities against NB4, MKN-45 and MCF-7 cells at 20 μM and of which 4 showed the highest activity against NB4 and MKN-45 cells with inhibition rates of 85.6% and 30.5%, respectively.Four new ( Parnassia wightiana wall (Saxifragaceae), commonly known as Ji-mei-hua-cao, Cang-er-qi, Qiao-mai-ye or Ding-chuang-cao in China, is a perennial herb and mainly distributes in Qinling Mountains in China. Its dried whole plants have been used as Chinese folk medicine for the treatment of leukorrhea, cough, haematemesis, carbuncle, irregular menstruation, hypertension, malaria, kidney stones, gall stones, etc. Recently, we reported eight dihydro-β-agarofuran sesquiterpene polyesters including five new compounds isolated from this plant and their anticancer activity + in HR-ESI-MS  and aromatic groups . The UV spectrum showed the presence of benzoate moieties with maximum absorption peaks at 204, 232, 274 and 282 nm.Compound R-ESI-MS  in conju spectra  afforded1H- and 13C-NMR spectroscopic data of compound 1 , 1.78 , 2.05 ), three tertiary methyl groups , 1.45 , 1.58 ), one secondary methyl group ) and one benzoate group . Additionally, the 13C-NMR and DEPT (Distortionless Enhancement by Polarization Transfer) spectra revealed that 1 also contained one methylene group (δC 43.1 (CH2)), six methine groups with four linked to an oxygen atom (δC 38.1 (CH), 53.3 (CH), 69.0 (CH–O–), 72.6 (CH–O–), 76.0 (CH–O–), 77.2 (CH–O–)), three quaternary sp3 carbons with two bearing an oxygen atom (δC 54.0 (C), 84.0 (C–O–), 88.7(C–O–)), one ketone carbonyl group (δC 204.8) and four ester carbonyl groups . The complete assignments of all carbon and hydrogen signals were carried out by an analysis of 1H-1H COSY (1H-1H Correlation Spectroscopy), HSQC (Heteronuclear Single Quantum Coherence) , 5.35 (s), 5.53  and 5.82 (s) were assigned to H-9, H-6, H-8 and H-1, respectively.The mpound 1  showed therence) , HMBC (Hherence)  and DEPTherence) . The sigH 5.82 /δC 169.5 (CH3CO2–), δH 5.35 /δC 169.8 (CH3CO2–), δH 5.53 /δC 169.3 (CH3CO2–), and δH 5.16 /δC 165.4 (PhCO2–) showed that three acetoxy groups and one benzoyloxy group were at C-1, C-6, C-8 and C-9, respectively. The correlation between δH 5.82  and δC 204.6 (C=O) as well as the downfield chemical shift and singlet of H-1 showed that position 2 was a ketone carbonyl group.The location sites of the ester groups and ketone carbonyl group were determined by an HMBC experiment . The cor1 was determined by a NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectrum  to 3.15 , 1.58  to 7.92 , δH 5.35  to 0.90 , 5.53  to 1.22 , and δH 5.16  to 5.53  and 1.22  showed that three acetoxy groups at C-1, C-6 and C-8 were equatorial and the 9-benzoyloxy group was axial. It is worth noting that 8-OH or 8-AcO in the previously isolated compounds from the same plant are axial D19 = +18.448° , possessed a molecular formula of C34H39NO12, as deduced from HR-ESI-MS data (m/z: 676.2359 [M + Na]+) , hydroxyl (3551 cm−1) and aromatic groups . The UV spectrum showed the presence of benzoate moieties with maximum absorption peaks at 202, 228 and 264 nm. Spraying with potassium heptaiodobismuthate solution gave an orange-red spot on thin layer chromatographic plate, indicating 2 belonged to alkaloids.Compound  + Na]+)  and 13C-1H- and 13C-NMR , six methyl groups , three methylene groups with one linked to an oxygen atom  and 2.22 /41.2, 2.25  and 2.59 /34.3, 4.35  and 4.99 /65.2), five methine groups with four linked to an oxygen atom /79.2, 5.54 /70.9, 5.42 /68.0, 5.41 /69.1, 2.33 /49.1), four quaternary sp3 carbon atoms with three linked to an oxygen atom , two quaternary sp2 carbon atoms  and nine tertiary sp2 carbon atoms (δH = 7.39–9.23 (9H)/δC 124.0–153.0 (nine peaks)). The 1H-NMR, HSQC and 1H-1H COSY spectra revealed the presence of one phenyl group /128.1, 7.54 /133.3, 7.94 /129.8) and one 3-pyridyl group /150.5, 8.68 /153.0, 8.47 /138.0, 7.50 /124.0). The complete assignments of signals of all hydrogen atoms on the dihydro-β-agarofuran rings were carried out by an analysis of the 1H-1H COSY and coupling constants. The signals of H-12, H-13, H-14 and H-15 were assigned on the base of the HMBC analysis , 1.47  and δC = 169.6, between δH = 5.41 , 1.99  and δC = 170.0, and between δH = 4.35 , 4.99 , 2.22  and δC = 170.7 revealed the presence of 1-AcO, 2-AcO and 15-AcO groups while the correlations of both δH = 6.30  and 8.47  to δC = 164.4, and both δH = 5.42  and 7.94  to δC = 165.1 indicated the presence of 6-nicotinoyloxy and 9-benzoyloxy groups. The correlations between δH = 5.41 , 1.90 , 2.22 , 1.41  and δC = 69.7 (C-4) showed that the unique hydroxyl group was located at C-4.In the HMBC spectrum, the correlations between δH = 5.54  toδH = 5.41 , 2.22  and 7.94  showed that 1-AcO and 2-AcO groups were equatorial and axial, respectively. The correlations between δH = 1.41  and 1.90 , 4.35 , 4.99  and 6.30 , and between δH = 6.30  and 4.35 , 4.99  indicated that both 4-OH and 6-nicotinoyloxy groups were located at an equatorial bond. The correlations of δH = 5.42  to 4.35  and 2.59  showed 9-benzoyloxy group was axial. Based on the NOESY analysis above as well as the fact that 1-OH or 1-ester group in all known natural dihydro-β-agarofuran sesquiterpenes were found to be equatorial, the absolute configuration of 2 was determined. Thus, 2 was elucidated as -1,2,15-triacetoxy-9-benzoyloxy-6-nicotinoyloxy dihydro-β-agarofuran. This finding on 2 is of a certain significance because this compound is the first nitrogen-containing dihydro-β-agarofuran sesquiterpene polyester isolated from this plant.In the NOESY spectrum , the cro3, obtained as a pale yellow solid with [α]D19 = +15.364° , possessed a molecular formula of C31H34O9, as deduced from HR-ESI-MS data (m/z: 573.2073 [M + Na]+) (13C-NMR data (−1), hydroxyl (3466 cm−1), and aromatic groups . The UV spectrum showed the presence of benzoate moieties with maximum absorption peaks at 202, 228, 274 and 282 nm.Compound  + Na]+)  and 13C-NMR data . The IR 1H-NMR spectrum of compound 3 , two phenyl groups with two sets of aromatic hydrogen signals  and one hydroxy group  which disappeared after the addition of D2O. The 13C-NMR spectrum showed the presence of one acetic ester  and two benzoic ester moieties . Besides these ester moieties, the 13C-NMR, DEPT and HSQC spectra /31.2, 1.82 /26.8, 1.01/18.4, 1.22/18.9), one methylene group , 2.37 /42.8), six methine groups with four bearing an oxygen atom /53.7, 2.86 /38.4, 5.19 /75.1, 5.44 /72.8, 5.49 /77.3, 5.84 /69.9), three quaternary sp3 carbon atoms  with two linked to an oxygen atom and one ketone group (δC = 211.5), which were very similar to those of the skeleton of -1-acetoxy-6,9-dibenzoyloxy-8-hydroxydihydro-β-agarofuran-2-one isolated from the same plant D21 = +3.152° , possessed a molecular formula of C31H36O8, as deduced from ESI-MS data (m/z: 559.23 [M+Na]+) (13C-NMR data. The IR showed the presence of ester carbonyl (1718 cm−1), hydroxyl (3519 cm−1) and aromatic groups . The UV spectrum showed the presence of benzoate moieties with maximum absorption peaks at 242, 274 and 282 nm.Compound [M+Na]+)  and 13C-1H- and 13C-NMR spectrum data -2-acetoxy-6,9-dibenzoyloxy-1-hydroxydihydro-β-agarofuran) isolated from the same plant + .Compound R,2S,4S,5S,6R,7R,9S,10R)-1,2,15-Triacetoxy-9-benzoyloxy-6-nicotinoyloxydihydro-β-agarofuran (2)(+)-; UV (MeOH): λmax (log ε) 202 (4.14), 228 (4.12), 264 (3.43) nm; IR νmax (KBr) 3551, 3063, 2962, 2929, 1751, 1719, 1593, 1368, 1279, 1096, 1025, 802, 713 cm−1; 13C-NMR and 1H-NMR data, see m/z 676.2359 [M + Na]+ .Compound R,4R,5S,6R,7R,8S,9R,10S)-6-Acetoxy-8,9-dibenzoyloxy-1-hydroxydihydro-β-agarofuran-2-one (3)(+)-; UV (MeOH): λmax (log ε) 202 (4.16), 228 (4.13), 274 (3.10), 282 (3.00) nm; IR νmax (KBr) 3466, 3068, 2963, 2924, 2854, 1730, 1602, 1558, 1283, 1263, 1233, 1105, 1028, 802, 710 cm−1; 13C-NMR and 1H-NMR data, see m/z 573.2073 [M + Na]+ .Compound R,2S,4R,5S,6R,7R,9S,10S)-2-Acetoxy-6,9-dibenzoyloxy-1-hydroxydihydro-β-agarofuran (4)(+)-; UV (MeOH): λmax (log ε) 242 (3.99), 274 (3.16), 282 (3.09) nm; IR νmax (KBr) 3519, 3062, 3010, 2963, 2929, 1718, 1602, 1584, 1452, 1389, 1365, 1276, 1106, 1024, 892, 860, 803, 712 cm−1; 13C-NMR and 1H-NMR data, see m/z 559.23 [M + Na]+.Compound 1–4 was determined according to our reported MTT -2,5-diphenyltetrazolium bromide) method [2. The tested compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted freshly before each experiment to 20 μM concentration in DMEM (high glucose) medium for MKN-45 or MCF-7 cells or RPMI 1640 medium for NB4 cells. The final concentration of DMSO in all experiments was 0.1% (v/v) and had negligible effect on the measured parameters.Cytotoxic activity of new compounds ) method . The hum3 cells/200 μL/well and cultured for 24 h. After removal of the medium, the cells in each well were treated with 200 μL solution containing 20 μM of the tested compounds and vehicle controls which only received an equivalent amount of DMSO in quintuplicate for at 37 °C 48 h. For NB4 cells, the cell suspension at exponential growth phase was treated with the solution of the tested compounds, and seeded into a 96-well plate at 4.2 × 104 cells/200 μL/well in quintuplicate and cultured for 48 h. Thirty microliters of MTT solution (5 mg/mL in PBS) was added to each well with 150 μL fresh medium for an additional 4 h. After removing the supernatant, 150 μL of DMSO was added to completely dissolve the formazan crystals that had formed in viable cells in the wells. Finally, the plates were shaken and the absorbance (A) was determined using microplate reader (Bio-Rad 680) at 570 nm. The wells containing the same media as other test wells but no cells were used as blank controls. All the doses were tested in quintuplicate and the experiments were repeated at least three times. The inhibition rates (IRs) were calculated according to the following formula and expressed as means ± S.D.MKN-45 or MCF-7 cells at exponential growth phase were seeded into a 96-well plate at 4.8 × 10Parnassia wightiana and elucidated the structures of the new compounds through spectroscopic analysis including UV, IR, 1H-NMR, 13C-NMR, 2D-NMR and HR-MS. The first dihydro-β-agarofuran sesquiterpene alkaloid (2) was found in this plant.In this study we isolated four new and six known dihydro-β-agarofuran sesquiterpene polyesters from


Poly(isosorbide itaconite -co- succinate) 13 as a bio-based unsaturated polyester was cross-linked by a 1,3-dipolar cycloaddition with the received dinitrones 10a/b. The 1,3-dipolar cycloaddition led to a strong change of the mechanical properties which were investigated by rheological measurements. Nitrones derived from methyl acrylate (3a) and methyl crotonate (3b) were used as model systems and reacted with dimethyl itaconate to further characterize the 1,3-dipolaric cycloaddition.Isosorbide was esterified with acryloyl chloride and crotonic acid yielding isosorbide diacrylate ( N-methylnitrone)s and biss , which yielded linear polymers bearing isoxazolidine units with high thermal stability butanoate (3b), were prepared from methyl acrylate (2a) and methyl crotonate (2b) with E-benzaldoxime (1) amino]propanoate (xime (1) , respect3a/b can be easily verified by 1H NMR spectroscopy  and 1147 cm−1 (3b) thereby hinting at the formation of nitrones.The success of the reaction of compounds troscopy . Former m values . The app2/BF3∙OEt2 to reduce several side reactions. For example, it could be determined, that high amounts of catalyst led to the oxidation of the oxime to benzaldehyde or the cleavage of nitrones.In contrast to previous reports , we lowe3a with 4 yields two diastereomers for each 4- and 5-substituted isoxazolidines, resulting in four isomers. The proposed molecular interactions are shown in We expected that the cycloaddition of 3a/b were reacted with an excess of dimethyl itaconate (4) obtaining isoxazolidines 5a/b  was esterified with acryloyl chloride (7) yielding acrylate 9a. Dicrotonate 9b was prepared from isosorbide (6) and crotonic acid , zinc iodide (98%), and boron trifluoride diethyl etherate (46%) were purchased from Sigma-Aldrich and used as received. Isosorbide (98%) and E-benzaldoxime (97%) were purchased from Alfa Aesar, and isosorbide was recrystallized from acetone/ethyl acetate. All used solvents are commercially available and were used without further purification.Acryloyl chloride (97%), crotonic acid (98%), itaconic acid , methyl acrylate (99%), methyl crotonate (98%), succinic acid , 1H NMR and 13C NMR spectroscopic measurements were performed with a Bruker Avance III spectrometer at 300 MHz and at 75 MHz in either DMSO-d6 or CDCl3 as solvents. Chemical shifts were referenced to the solvent value δ = 2.50 ppm for DMSO-d6 and δ = 7.26 ppm for CDCl3. IR measurements were performed by using a FTIR spectrometer (Nicolet 6700 FTIR) equipped with an ATR unit. Molecular weight distributions were measured by Gel Permeation Chromatography (GPC) with a flow rate of 1 mL/min at room temperature and THF  as an eluent. The GPC System compromises a Scharmbeck SFD degasser (Gastor BG12), a FLOW pump (Intelligent PUMP AL-12) and a Scharmbeck SFD (Model S5200) sampler. For detection a Waters 486 Turnable Absorbance Detector and a Schambeck SFD RI 2000 detector were used. A set of columns packed with porous styrene–divinylbenzene–copolymer beads was used for the separation of the analytes . The system was calibrated with polystyrene standards with a molecular range from 575 g/mol to 3,114,000 g/mol. Toluene was added to the analytes as an internal standard. Rheologial measurements were performed by using a Haake Mars II rheometer by ThermoFisher Scientific. For rotation measurements a plate–plate construction  was used. The temperature was measured with an accuracy of ±0.5 ° C in the measuring plate. The control of the thermostat (Haake DC30 or Haake AC 200-G50) was controlled by the software. Differential scanning calorimetry (DSC) measurements were performed by using a Mettler Toledo DSC 822 controller apparatus in a temperature range between 0 and 200 °C with a heating rate of 10 °C·min−1. The glass transition temperature (Tg) values reported are taken from the third heating cycle. EIMS spectra were recorded on a GC–MS integrated system Trace DSG by Thermo Finnigan. The instrument was calibrated in the m/z range of 1050 Da.Synthesis of methyl 3-[benzylidene(oxido)amino]propanoate (3a): Methyl acrylate  and benzaldoxime  were placed in a 100 mL round bottom flask and dissolved in 20 mL toluene. Zinc iodide  and boron triflouride diethyl etherate  were added. The flask was closed with a plug and the reaction mixture was heated to 80 °C for 24 h. The solvent was evaporated and the crude product dissolved in 20 mL of dichloromethane. Filtration and subsequent purification by column chromatography over silica gel (ethyl acetate/petrol ether 1:1) afforded the pure product in 66% yield. 1H NMR  δ (ppm) 8.27–8.21 , 7.91 , 7.46–7.39 , 4.19  3.61 , 2.92 ; 13C NMR  δ (ppm) 171 (C=O), 134 (-C=N-), 131 (Ar-C), 130 (Ar-C), 128 , 61 (-CH2-CH2-), 53 (-O-CH3), 31 (-CH2-CH2-); FTIR (diamond)  (cm−1): 3059, 2996, 2952 , 1731 , 1172 , 1581 , 1149  753, 691 ; EIMS m/z: 221 [M+], 205 [M+ − C12H15NO2], 190 [M+ − C11H13NO2], 132 [M+ −C9H10N], 104 [M+ − C7H6N].Synthesis of methyl 3-[benzylidene(oxido)amino]butanoate (3b): Methyl crotonate  and benzaldoxime  were placed in a 100 mL round bottom flask and dissolved in 40 mL toluene. Zinc iodide  and boron triflouride diethyl etherate  were added. The flask was closed with a plug, and the reaction mixture was heated to 80 °C for 24 h. The solvent was evaporated, and the crude product dissolved in 20 mL of dichloromethane. Filtration and subsequent purification by column chromatography over silica gel (ethyl acetate/petrol ether 1:2) afforded the pure product in 46% yield. 1H NMR  δ (ppm) 8.28–8.22 , 7.95 , 7.44–7.39 , 4.60 , 3.57 , 3.00 , 2.67 , 1.37 ; 13C NMR  δ (ppm) 171 (C=O), 133 (-C=N), 132 (Ar-C), 130 (Ar-C), 129 , 128 , 67 (-CH-CH3), 52 (-O-CH3), 38 (-CH-CH2-), 19 (-CH-CH3); FTIR (diamond)  (cm−1): 3056, 2986, 2951 , 1733 , 1175 , 1148 , 753, 691 ; EIMS m/z: 207 [M+], 192 [M+ − C11H13NO2], 134 [M+ − C9H12N], 132 [M+ − C9H10N], 91 [M+ − C7H7].General procedure for 1,3-dipolaric cycloaddition of nitrons 3a/b with dimethyl itaconate (4): A vial was charged with 2 mmol of nitron 3a or 3b and 20 mmol of dimethyl itaconate (4). The reaction mixture was stirred at 120 °C for 1 h and purified via column chromatography (ethyl acetate/petrol ether 1:4). Yield: 74–75%.Methyl 5-(2-methoxy-2-oxoethyl)-2-(3-methoxy-3-oxopropyl)-3-phenylisoxazolidine-5-carboxylate (5a): cis-isomer: 1H NMR  δ (ppm) 7.39–7.28 , 3.92 , 3.70–3.52 , 3.02–2.92 , 2.95 , 2.89 , 2.48 , 2.42 ; trans-isomer: 1H NMR  δ (ppm) 7.3–7.28 , 3.82 , 3.70–3.52 , 3.02–2.92 , 2.89 , 2.72 , 2.48 ; 13C NMR  δ (ppm) 172–169 (-C=O), 129–127 (Ar-C), 81 (-C-), 69 (Ar-CH-), 53–51 (-O-CH3), 48 (Ar-CH-CH2-), 42 (-C-CH2-C=O), 41 (-N-CH2-CH2-), 32 (-N-CH2-CH2-); FTIR (diamond)  (cm−1): 2953, 2917, 2850 , 1733 , 1195 , 1173 , 756, 701 ; ESIMS m/z: 366 [M+].Methyl 5-(2-methoxy-2-oxoethyl)-2-(4-methoxy-4-oxobut-2-yl)-3-phenylisoxazolidine-5-carboxylate (5b): 1H NMR  δ (ppm) 7.4–7.25 , 4.25–3.80 , 3.72–3.48 , 3.27–2.98 , 3.06–2.84, , 3.02–2.27 , 2.90–2.13 , 1.04–0.90 ; 13C NMR  δ (ppm) 173–169 (-C=O), 129–127 (Ar-C), 81–80 (-C-), 69 (Ar-CH-), 56 (-N-CH-CH2-), 53–51 (-O-CH3), 48–46 (Ar-CH-CH2-), 42 (-C-CH2-C=O), 36 (-N-CH-CH2-), 20–12 (-CH-CH3); FTIR (diamond)  (cm−1): 2953, 2920, 2847 , 1731 , 1197 , 1170 , 757, 701 ; ESIMS m/z: 380 [M+].Synthesis of 2,5-di-O-acryloyl-1,4:3,6-dianhydro-D-glucitol (9a): A 250 mL round bottom flask was charged with isosorbide  dissolved in 100 mL of dichloromethane. Then triethylamine  was added and the reaction mixture was cooled to 0 °C. Acryloyl chloride  was dissolved in 50 mL dichloromethane and added via a dropping funnel under vigorous stirring. The reaction mixture was stirred for additional 24 h after complete addition. Precipitated ammonium chloride was removed by filtration and the obtained solution was washed with 2 × 50 mL of saturated sodium hydrogen carbonate solution. The pure product was received after drying of the organic layer over magnesium sulfate and subsequent removal of the solvent under reduced pressure. Yield: 94%. 1H NMR  δ (ppm) 6.58–6.33 , 6.22–6.01 , 5.89–5.80 , 5.29–5.16 , 4.87 , 4.51 , 4.00–3.80 ; 13C NMR  δ (ppm) 165 , 132 , 128 , 86 , 81 , 78 , 74 , 73 , 70 ; FTIR (diamond)  (cm−1): 2980, 2876 , 1721 , 1634 , 1179 ; EIMS m/z: 255 [M+], 183 [M+ − C9H11O4], 128 [M+ − C6H8O3].Synthesis of 1,4:3,6-dianhydro-2,5-di-O-but-2-enoyl-D-glucitol (9b): A 250 mL round bottom flask was charged with isosorbide , crotonic acid , p-toluenesulfonic acid , phenothiazine  and 80 mL of toluene. The flask was fitted with a Dean–Stark apparatus and the mixture was heated under reflux for 72 h. The solution was washed with 2 × 40 mL of saturated sodium hydrogen carbonate solution and the organic layer was dried over magnesium sulfate. The pure product was obtained by evaporation of the solvent. Yield: 92%. 1H NMR  δ (ppm) 7.11–6.94 , 5.96–5.79 , 5.33–5.08 , 4.86 , 4.53 , 4.07–3.79 , 1.93–1.85 ; 13C NMR  δ (ppm) δ 165 , 145 , 122 , 86 , 81 , 78 , 74 , 74 , 70 , 18 ; FTIR (diamond)  (cm−1): 2975, 2876 , 1717 , 1656 , 1172 ; ESIMS m/z: 283 [M+].Synthesis of 1,4:3,6-dianhydro-2,5-bis-O-{3-[benzylidene(oxido)amino]propanoyl}-D-glucitol (10a): Compound 9a  and benzaldoxime  were placed in a 100 mL round bottom flask and dissolved in 20 mL toluene. Zinc iodide  and boron triflouride diethyl etherate  were added. The flask was closed with a plug and the reaction mixture was heated to 80 °C for 24 h. The solvent was evaporated and the crude product washed with 50 mL of isopropanol and 50 mL of diethyl ether. Yield: 95%. 1H NMR  δ (ppm) 8.29–8.17 , 7.90 , 7.48–7.37 , 5.16–5.02 , 4.72 , 4.39 , 4.25–4.13 , 3.85–3.70 , 3.05–2.85 ; 13C NMR  δ (ppm) 170 , 134 , 131 , 130 , 128 , 85 , 80 , 78 , 74 , 72 , 70 , 61 , 31 ; FTIR (diamond)  (cm−1): 2976, 2871 , 1734 , 1173 , 1148 , 756, 691 ; ESIMS m/z: 496 [M+].Synthesis of 1,4:3,6-dianhydro-2,5-bis-O-{3-[benzylidene(oxido)amino]butanoyl}-D-glucitol (10b): Compound 9b  and benzaldoxime  were placed in a 100 mL round bottom flask and dissolved in 20 mL toluene. Zinc iodide  and boron triflouride diethyl etherate  were added. The flask was closed with a plug and the reaction mixture was heated to 80 °C for 24 h. The solvent was evaporated and the crude product washed with 50 mL of isopropanol and 50 mL of diethyl ether. Yield: 60%. 1H NMR  δ (ppm) 8.34–8.16 , 7.98–7.91 , 7.45–7.37 , 5.12–4.93 , 4.69–4.51 , 4.37–4.22 , 3.94–3.59 , 3.10–2.87 , 2.75–2.57 , 1.37 ; 13C NMR  δ (ppm) 170 , 134 , 132 , 131 , 128 , 85 , 82 , 78 , 72 , 71 , 67 , 31 , 19 ; FTIR (diamond)  (cm−1): 2975, 2936, 2876 , 1734 , 1580 , 1176 , 1148 , 755, 692 ; ESIMS m/z: 525 [M+].Synthesis of poly(isosorbide itaconate -co- succinate) 13: A 250 mL round bottom flask was charged with isosorbide , itaconic acid , succinic acid , p-toluenesulfonic acid , phenothiazine  and 80 mL toluene. The flask was fitted with a Dean–Stark apparatus and the solution was heated under reflux for 72 h. The solution was concentrated after cooling to room temperature and the crude product was dissolved in a small amount of dichloromethane and precipitated in 300 mL methanol. The product was again dissolved in dichloromethane and the pure polyester was isolated by removing the solvent under reduced pressure. 1H NMR  δ (ppm) 6.39 , 5.78 , 5.20 , 4.82 , 4.47 , 3.90 , 3.39 , 3.33 , 2.67 ; FTIR (diamond)  (cm−1): 2977, 2935, 2877 , 1729 , 1639 , 1151 ; GPC (THF):  = 3452; D = 1.69; DSC: Tg = 64 °C.


Cnidium monnieri is commercially used as healthcare products for the improvement of impotence and skin diseases. Three new coumarins, 3'-O-methylmurraol (1), rel--1'-O-methylphlojodicarpin (2), and -1'-O-methylvaginol (3), have been isolated from the fruits of C. monnieri, together with 14 known compounds (4–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4–12, and 14–17 exhibited inhibition (IC50 ≤ 7.31 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 7, 9–11, 15, and 17 inhibited fMLP/CB-induced elastase release with IC50 values ≤7.83 µg/mL. This investigation reveals that bioactive isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.The fruit of  Cnidium monnieri (L.) Cusson (Umbelliferae) is an annual herb distributed in China, India, Russia, Korea, Mongolia, Vietnam, Europe, and North America + ion at m/z 297.1105 . The presence of a carbonyl group was revealed by a band at 1727 cm−1 in the IR spectrum, and was confirmed by the resonance at δ 160.9 in the 13C-NMR spectrum. The 1H-NMR spectrum of 1 showed the presence of an (E)-3-methoxy-3-methylbut-1-enyl group , a methoxy group [δ 3.95 ], an AB spin system [δ 6.88  and 7.31 ], and the typical H-3 and H-4 protons of the coumarin nucleus . The 1H-NMR data of 1 was similar to those of murraol D25 = −28.4). Its molecular formula, C16H18O5, was determined on the basis of the positive HRESIMS at m/z 313.1050 [M + Na]+  and supported by the 1H-, 13C-, and DEPT NMR data. The presence of a carbonyl group was revealed by a band at 1733 cm−1 in the IR spectrum, and was confirmed by the resonance at δ 160.2 in the 13C-NMR spectrum. The 1H-NMR data of 2 were similar to phlojodicarpin D25 + 120.4). The molecular formula C15H16O5 was deduced from a sodium adduct ion at m/z 299.0892 [M + Na]+  in the HRESI mass spectrum. The presence of a carbonyl group was revealed by the band at 1727 cm−1 in the IR spectrum. The 1H- and 13C-NMR data of 3 were similar to those of vaginol  of 3 replaced 1'-hydroxy group of vaginol. This was supported by the HMBC correlation between OMe-1' (δ 3.65) and C-1' (δ 79.4) and by the NOESY correlations between OMe-1' (δ 3.65) and both H-1' (δ 5.23) and H-2' (δ 4.53). In addition, the 1'S,2'S-configuration of 3 was established by the following evidences: (a) The small coupling constant (J = 3.0 Hz) between Hβ-1' and Hα-2' of 3 was similar to that (J = 3.6 Hz) of vaginol with 1'S,2'S-configuration D25 = +120.4, which was similar to vaginol ([α]D20 = +119°) with 1'S,2'S-configuration , me+. HR-ESI-MS: m/z = 297.1105 [M + Na]+ .Colorless prisms (MeOH), m.p. 129–131 °C. UV (MeOH): λ2Cl2-MeOH); m.p. 141–143 °C. [α]D25: −28.4 . UV (MeOH): λmax (log ε) = 219 (4.17), 244 (3.95), 253 , 318 (4.15) nm. IR (neat): υmax 1733 (C=O) cm−1. 1H-NMR : δ = 1.08 , 1.27 , 3.48 , 3.81 , 3.94 , 4.84 , 6.27 , 6.91 , 7.44 , 7.64 . 13C-NMR : δ = 19.4 (C-5'), 24.7 (C-4'), 56.8 (C-3'), 56.3 (OMe-7), 57.6 (OMe-1'), 65.6 (C-2'), 75.1 (C-1'), 108.0 (C-6), 113.1 (C-4a), 113.5 (C-3), 114.6 (C-8), 129.2 (C-5), 143.5 (C-4), 153.3 (C-8a), 160.2 (C-2), 160.7 (C-7). ESI-MS: m/z = 313 [M + Na]+. HR-ESI-MS: m/z = 313.1050 [M + Na]+ .Colorless needles (CHmax (log ε) = 214 (4.31), 245 , 256 , 287 , 322 (4.18) nm. IR (KBr): υmax = 3454 (OH), 1727 (C=O) cm−1. 1H-NMR : δ = 1.26 , 1.33 , 1.72 , 3.65 , 4.53 , 5.23 , 6.25 , 6.82 , 7.39 , 7.65 . 13C-NMR : δ = 24.9 (C-4'), 25.3 (C-5'), 57.8 (OMe-1'), 79.4 (C-1'), 96.3 (C-2'), 107.5 (C-6), 112.6 (C-3), 113.2 (C-4a), 114.2 (C-8), 131.2 (C-5), 143.9 (C-4), 152.3 (C-8a), 160.5 (C-2), 164.6 (C-7). ESI-MS: m/z = 299 [M + Na]+. HR-ESI-MS: m/z = 299.0892 [M + Na]+ .Colorless prisms (MeOH), m.p. 166–168 °C. UV (MeOH): λ1H-NMR, and 13C-NMR spectra of three new compounds (1–3) are available as ESI-MS, HR-ESI-MS, The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation and elastase release in fMLP/CB-activated human neutrophils in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS.2+)-free HBSS buffer at pH 7.4 and were maintained at 4 °C prior to use.Human neutrophils from venous blood of healthy, adult volunteers (20–28 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes . Purifiec [c and 1 mM Ca2+, neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with different concentrations (10–0.01 μg/mL) of compounds or DMSO (as control) for 5 min. Cells were incubated with cytochalasin B (1 μg/mL) for 3 min prior to the activation with 100 nM formyl-l-methionyl-l-leucyl-l-phenylalanine for 10 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring . Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).The assay for measurement of superoxide anion generation was based on the SOD-inhibitable reduction of ferricytochrome c ,26. In bp-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with compounds for 5 min. Cells were stimulated with fMLP (100 nM)/CB (0.5 µg/mL), and changes in absorbance at 405 nm were monitored continuously in order to assay elastase release. The results were expressed as the percent of elastase release in the fMLP/CB-activated, drug-free control system.Degranulation of azurophilic granules was determined by measuring elastase release as described previously ,26. Expet-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.Results are expressed as the mean ± SEM, and comparisons were made using Student’s 1–3), were isolated from the fruits of C. monnieri. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) , hydrogen peroxide) and granule proteases  produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of isolates were evaluated by suppressing fMLP/CB-induced O2•− generation and elastase release by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 1, 4–12, and 14–17 can significantly inhibit fMLP-induced O2•− generation and/or elastase release. Osthol (6) and cnidimol A (17) were the most effective among the isolated compounds, with IC50 values of 0.005 ± 0.0002 and 3.20 ± 0.16 µg/mL, respectively, against fMLP-induced O2•− generation and elastase release. Our study suggests C. monnieri and its isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.Seventeen compounds, including three new coumarins (


Their structures were elucidated by spectroscopic analysis, including 1D, 2D NMR, and MS data. The known compounds, (2E)-1--3-(4-methoxyphenyl)-2-propen-1-one (4), (2E)-1--3-phenyl-2-propen-1-one (5), laxichalcone (6), macarangin (7), bonanniol A (8), and bonannione A (9), showed inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) in vitro.Three new C-methylated and isoprenylated chalcone derivatives, dentichalcones A–C (1–3), together with six known compounds, were isolated from the twigs and leaves of Macaranga denticulata. Some compounds showed inhibitory activities against PTP1B in vitro. Three new C-methylated and isoprenylated chalcone derivatives, dentichalcones A–C ( Macaranga Thou. (Euphorbiaceae) comprises about 300 species mainly distributed in the tropical regions of Africa, Asia, Australia, and the Pacific islands + . The IR spectrum showed absorptions for OH (3417 cm−1), carbonyl (1625 cm−1), and aromatic  moieties. The 1H NMR spectrum , two trans-coupled olefinic protons at δH 8.08  and 7.76 , resonances of a 1,4-disubstituted benzene moiety at δH 7.61  and 6.95 , a methyl group at δH 2.05 , and signals of a 2,2-dimethylpyran ring at δH 6.70 , 5.59 , and 1.56 . The 13C NMR spectrum  was located at C-3′ by the HMBC correlations of H3-7′/C-2′, C-3′, C-4′. The 2,2-dimethylpyran group was fused at C-5′ and C-6′, as deduced from the HMBC correlations of H-1″/C-4′, C-5′, C-6′ and H-2″/C-5′, together with the key NOESY correlations of H3-4″, 5″/H-α and H-2, 6. Thus, the structure of 1 was elucidated as (2E)-1--3-(4-hydroxyphenyl)-2- propen-1-one and named dentichalcone A.Dentichalcone A  was assi2) was assigned the molecular formula C21H22O5 by HREIMS . Comparison of its NMR spectroscopic data  and 2.89  and at δC 46.6 (C-α), and 30.4 (C-β), and 207.3 (C-γ) and by the HMBC correlations of H2-α/C-γ, C-1 and H2-β/C-α, C-γ, C-1, C-6 -3-(4-hydroxyphenyl)propan-1-one and named dentichalcone B.Dentichalcone B  was assi3) was assigned the molecular formula C21H20O5 by HRESIMS . The 1H and 13C NMR spectra indicated the presence of a chalcone skeleton with a hydrogen-bonded hydroxyl and a methyl group, which was similar to that of 1. Its NMR spectra also displayed signals of a monosubstituted benzene ring  and resonances of a 2-hydroxymethyl-2-methylpyran moiety . The planar structure of 3 was further constructed by the HMBC spectrum -1--3-phenyl-2-propen-1-one and named dentichalcone C.Dentichalcone C (E)-1--3-(4-methoxyphenyl)-2-propen-1-one (4) + (30), 337 (64), 217 (100), 91 (15), 77 (7); HREIMS m/z 352.1308 [M]+ .Red, amorphous powder; UV (MeOH) λmax (log ε) 222 (3.79), 284 (3.82) nm; IR (KBr) νmax 3419, 2975, 2924, 1638, 1606, 1515, 1427, 1218, 1165, 1133, 827, 548 cm−1; 1H NMR and 13C NMR data, see Table m/z 354 [M]+ (49), 339 (100), 233 (35), 219 (33), 191 (36), 107 (47), 91 (16), 77 (21), 65 (8), 43 (8); HREIMS m/z 354.1465 [M]+ .Yellow, amorphous powder; UV (MeOH) α]D25−32.9 ; UV (MeOH) λmax (log ε) 224 (4.33), 240 (4.26), 287 (4.30), 346 (4.41) nm; IR (KBr) νmax 3420, 2975, 2915, 1633, 1595, 1456, 1348, 1171, 1130, 701, 575 cm−1; 1H NMR and 13C NMR data, see Table m/z 351 [M–H]−; HRESIMS m/z 351.1237 [M–H]− .Red, amorphous powder; [, 346 4.4 nm; IR .The bioassay procedure was the same as that reported previously , 23. The


YXf3, an endophytic fungus derived from Ginkgo biloba. Its structure was unambiguously established by analysis of HRMS–ESI and spectroscopic data, and the absolute configuration was determined by low-temperature (100 K) single crystal X-ray diffraction with Cu Kα radiation. This compound is structurally characterized by a new carbon skeleton with an unprecedented 6/5/6 tricyclic ring system bearing an α,β-unsaturated spirolactone moiety in ring B, and represents a new subclass of norditerpenoid, the skeleton of which is named aspergilane. The hypothetical biosynthetic pathway for 1 was also proposed. The cytotoxic, antimicrobial, anti-oxidant and enzyme inhibitory activities of 1 were evaluated.An unusual C Aspergillus sp. YXf3 associated with Ginkgo biloba led to the isolation of new p-terphenyls and novel types of diterpenoids including pimarane-type diterpenoids , a cleistanthane-type diterpenoid (aspergiloid C), and norcleistanthane-type diterpenoids , many of which were reported from this microorganism for the first time + at m/z 329) spectrum that did not match any previously isolated compounds, prompted us to purify it further. The obtained fermented broth (~45 L) was extracted four times with EtOAc  to afford a brown crude extract (9.1 g). Subsequent fractionation by silica gel column chromatography (CC), octadecylsilyl (ODS) CC, Sephadex LH-20 and semi-preparative reversed-phase HPLC yielded 1 (4.3 mg).A large-scale culture broth of 1) was isolated as a colorless lamellar crystal, with the molecular formula C18H26O4  as derived from the ESI high-resolution mass spectrometry  and NMR data (−1 (carbonyl group). The 1H NMR spectrum (acquired in DMSO-d6) displayed signals of a terminal vinyl group at δH 5.72 (H-14), 4.99 (H-15α) and 4.96 (H-15β), two oxygenated methine protons , ten aliphatic protons , three aliphatic methyl groups  and two hydroxy groups . The 13C NMR and DEPT spectra revealed that 1 contained 18 carbons, attributable to three methyl groups , five aliphatic methylenes , one olefinic methylene (δC 113.2), two oxygenated methines , one olefinic methine (δC 142.9), and six non-protonated carbon atoms (one of which was identified as lactone group) . These data show that 1 has two double bonds and one carbonyl which require three degrees of unsaturation, thus, 1 must also contain three rings.Aspergiloid I  attached to C-5 and the other hydroxy group (δH 5.04) attached to C-11 were identified by the 1H,1H couplings (acquired in DMSO-d6) with H-5 (δH 2.81), and H-11 (δH 3.76), respectively. HMBC correlations from two singlet methyl groups’ protons H3-17 and H3-18 to C-3, C-4, and C-5 indicate that C-17 and C-18 were located on the same quaternary carbon C-4, which was connected by C-3 and C-5. HMBC correlations from the hydroxy proton (δH 5.21) to C-5, and C-6 (acquired in DMSO-d6), and from H-5 to C-1, C-6, and C-7 (acquired in CDCl3) assigned the connectivity of the C-6 to C-1, C-5, and C-7. The other singlet methyl group, C-16, and the terminal vinyl group (C-14–C-15), were also located on the same quaternary carbon, C-10, which was flanked by C-9 and C-11 deduced from the HMBC correlations from H2-9 to C-10, C-11, and C-14, from H-11 to C-14, from H2-15 to C-10, and from the methyl protons  to C-10, C-11, and C-14. The HMBC correlations from the hydroxy proton (δH 5.04) to C-12, from H-11 to C-7, C-12, and C-13 indicated that C-12 was linked to C-7, C-11, and C-13 to form an α,β-unsaturated enone fragment. HMBC correlations from H2-8 to C-7 and C-12 secured the connectivity of the C-8 to C-7. The connectivity of C-6 to the ketone carbon C-13 through an ester linkage, which was also supported by the downfield chemical shift of C-6 (δC 88.6) completed the structure of 6/5/6 tricyclic spirolactone. The relative configuration of aspergiloid I (1) could be determined by NOESY correlations. The NOESY spectrum showed correlations of OH-5 with H3-18 and H-8α, of H-9β with H-8α and H3-16, and of H3-16 with OH-11, indicating that OH-5, H3-16, and OH-11 were on the same plane, while the relative configuration of the chiral center C-6 was further confirmed by a NOESY correlation of H-1α with H-8β. Therefore, the structure of compound 1 was elucidated as shown in The gross structure of 1 to C-3 . The COS1 was further confirmed by a low-temperature (100 K) single-crystal X-ray diffraction experiment, which is shown in 1 has a relatively high percentage of oxygen, it shows enough anomalous dispersion of Cu Kα radiation and allows to determinate the absolute stereochemistry with the Hooft parameter 0.17(15) for 992 Bijvoet pairs by single-crystal X-ray diffraction experiment 28D −19.5 ; UV (MeOH) λmax (log ε): 220.0 (3.97) nm; CD (MeOH): Δε = 214 (+5.30), 242 (−8.23), 283 (+0.04), 374 (−0.54), 399 (+0.50), 411 (−0.14), 471 (+1.69), 488 (−0.53), 500 (+1.19) nm; IR (KBr) νmax 3649, 1735, 1700, 1652, 1558, 1540, 1508, 1457, 816 cm−1; 1H and 13C NMR data, see m/z): [M + Na]+ calcd for C18H26O4Na, 329.1723; found, 329.1729.Aspergiloid I  on a Bruker APEX DUO CCD diffractometer, equipped with an Oxford Cryostream 700+ cooler. Structures were solved using the program SHELXS-97 [F2 using SHELXL-97. The absolute configurations were determined by computation of the Hooft parameter [18H26O4, M = 306.39, orthorhombic, a = 6.3349(2) Å, b = 11.1090(3) Å, c = 23.3643(6) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1644.25(8) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.694 mm−1, 7387 reflections measured, 2729 independent reflections (Rint = 0.0603). The final R1 values were 0.0901 (I > 2σ(I)). The final wR(F2) values were 0.2603 (I > 2σ(I)). The final R1 values were 0.1025 . The final wR(F2) values were 0.2816 . The goodness of fit on F2 was 1.126. The Hooft parameter is 0.17(15) for 992 Bijvoet pairs. Crystallographic data for the structure of aspergiloid I (1) have been deposited with the Cambridge Crystallographic Data Centre (deposition no. CCDC 985728). These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: +44-1223-336-033; or desposit@ccdc.cam.ac.uk.Colorless crystal of HELXS-97 , and refarameter , in all Xanthomonas oryzae pv. oryzae Swings, Xanthomonas oryzae pv. oryzicola Swings, Acidovorax avenae subsp. Citrulli, Erwinia amylovora, Pseudomonas syringae pv. Lachrymans, Clavibacter michiganense subsp. Sepedonicus, and Pectobacterium carotovorum subsp. carotovorum) and fungi , and Candida albicans (ATCC 10231), and the antioxidant, acetylcholinesterase (AChE) , α-glucosidase, and topoisomerase IIα inhibitory activities were performed in accordance with the primary literature [Cytotoxic activity against 11 human cancer cell lines, K562 myeloid leukemia, SH-SY5Y beuroblastoma, SGC-7901 gastric adenocarcinoma, HepG2, SMMC-7721 hepatocellular carcinoma, A549 lung cancer, MCF-7, MDA-MB-231 breast cancer, HCT116, SW480 colon cancer, HT29 colorectal cancer, were evaluated with the MTT assay . Antimicterature –20.File 11.1D, 2D NMR spectra, HRMS–ESI, and the X-ray crystallographic structure of


Codium dwarkense. The bioactive fractions were further subjected to chromatographic separations, which resulted in the isolation of a new triterpenic acid; dwarkenoic acid (1) and the known sterols; androst-5-en-3β-ol (2), stigmasta-5,25-dien-3β,7α-diol (3), ergosta-5,25-dien-3β-ol (4), 7-hydroxystigmasta-4,25-dien-3-one-7-O-β-d-fucopyranoside (5), 7-hydroxystigmasta-4,25-dien-3-one (6), and stigmasta-5,25-dien-3β-ol (7). The structure elucidation of the new compound was carried out by combined mass spectrometry and 1D (1H and 13C) and 2D  NMR spectroscopic data. The sub-fractions and pure constituents were assayed for enzymatic inhibition of alpha-glucosidase. Compound 1 showed significant inhibition at all concentrations. Compounds 2, 3, 5, and 7 exhibited a dose-dependent response, whereas compounds 4–6 showed moderate inhibition. Utilizing such marine-derived biological resources could lead to drug discoveries related to anti-diabetics.The marine ecosystem has been a key resource for secondary metabolites with promising biological roles. In the current study, bioassay-guided phytochemical investigations were carried out to assess the presence of enzyme inhibitory chemical constituents from the methanolic extract of marine green alga— Codium are well known for their anticancer activities + in the High Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS), along with the 1H and 13C NMR data, suggested the molecular formula C32H48O5 for compound 1, with eight indices of hydrogen deficiency, thus supporting the pentacyclic system with one olefinic and two carbonyl groups. Further fragments appeared at m/z 468 and 453 due to a loss of CO2 and OAc, respectively , ester and acidic carbonyls , and the trisubstituted double bond (1628 and 812 cm−1) by the appearance of characteristic absorption bands for these groups.Compound ectively . The RDA1H NMR spectrum (1 displayed singlets for five tertiary methyl (δ 0.81 (Me-23), 0.93 (Me-24), 1.14 (Me-26), 1.18 (Me-27), and 1.33 (Me-25)) and doublets for two secondary methyl  and 1.23 ) groups. The tertiary methyl group of the acetyl moiety (Me-32) also appeared as a singlet at δ 2.07. The presence of these methyl groups was further confirmed by 13C NMR spectrum, which displayed signals for tertiary methyls at δ 28.9 (Me-23), 21.1 (Me-24), 13.3 (Me-26), 18.3 (Me-27), 20.5 (Me-25), and 21.3 (Me-32), while secondary methyls appeared at δ 17.4 (Me-29) and 23.8 (Me-32). These observations were indicative of the ursane-type skeleton in the molecule. The olefinic proton (H-6) was observed at δ 5.54 as a broad singlet, whereas the proton (H-3) geminal to the acetyl group was observed at δ 5.29 . The 13C NMR spectra (BB) of compound 1 , C-2 (δ 23.5), and C=O (δ 170.2); H-6 proton showed interactions with C-5 (δ 164.9), C-6 (δ 130.5), and C=O (δ 199.4); H-9 proton showed cross peaks with C-8 (δ 45.1) and C-10 (δ 37.3); and H-18 showed cross peaks with C-13 (δ 50.4), C-17 (δ 43.8), and C=O (δ 179.1), thus indicating the relative positions of these groups in the molecule.The HSQC spectrum was helpful for establishing the ns HMBC;  were use1 was assigned by the analysis of NOESY correlations , after the producing organism. The known compounds 2–7 were identified by the comparison of NMR data with those reported in literature +.Amorphous powder; mp: 274–275 °C; [α]et al. [p-nitrophenyl α-d-glucopyranoside (PNP-G) as a substrate and 200 m units/mL enzyme, in 50 mM sodium phosphate buffer containing 100 mM NaCl. Acarbose was used as a positive control. The increment in absorption at 400 nm due to the hydrolysis of PNP-G by α-glucosidase was continuously monitored with ELISA.Enzyme inhibition assay  was performed according to the modified method of Oki et al. . The inh


E-sarcophytoxide (1), (+)-2-epi-12-methoxycarbonyl-11E-sarcophine (2), 3,4-epoxyehrenberoxide A (3), ehrenbergol D (4) and ehrenbergol E (5), were obtained from the soft coral Sarcophyton ehrenbergi. The structures of 1–5 were established on the basis of comprehensive NMR and HR-ESI-MS analyses and by comparison with reported data in the literature. Compounds 4 and 5 showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with EC50 values of 2.0 and 3.0 μM, respectively. Compound 2 exhibited slight antiviral activity against HCMV  with IC50 values of 25.0 μg/mL.Five new polyoxygenated cembranoids, named (+)-1,15-epoxy-2-methoxy-12-methoxycarbonyl-11 Sarcophyton ehrenbergi , ordinarily regarded as the leather coral, belongs to the order Alcyonacea, and is classified as an octocoral because of eight-fold symmetry or eight-branched tentacles in their polyp structure. From a chemical perspective, the alcyonacean soft coral S. ehrenbergi is interesting due to macrocyclic cembranoids with intriguing structural features and various promising biological activities +, corresponding to the molecular formula C22H32O6 with seven indices of hydrogen deficiency. The IR spectrum of 1 showed a strong absorption band at 1712 cm−1, indicating the presence of an α,β-unsaturated methyl ester carbonyl group, as well as from the UV absorption (MeOH) λmax (log ε) at 225 (3.83) nm + for 2 identical to that of (+)-12-methoxycarbonyl-11E-sarcophine +, which coupled with the 13C NMR data  and δC 124.3 (CH) and 136.7 (qC)] in ehrenberoxide A were replaced by those due to a epoxide  in 3. The HMBC correlations , Me-18/H-6a (δH 1.83), H-3/H-5b (δH 1.08), and H-3/H-14a (δH 2.28) -12-carboxy-11E-sarcophytoxide for which X-ray crystallography established the absolute configuration . Additionally, the carbon resonance appearing at δC 173.6  was attributable to an α,β-unsaturated γ-lactone carbon. Correlations deduced from extensive analysis of the 1H–1H COSY correlations of 4 enabled initially the establishment of five partial structures. The connectivity of the above structural fragments was subsequently interconnected by the HMBC correlations (H 2.83), H-2/Me-16, H-2/Me-17, H-3/Me-20, H-5a (δH 2.87)/H-18b (δH 4.03), H-3/H-18a (δH 4.09) and H-3/H-14a (δH 2.68)  further suggested the s-trans geometry of the conjugated double bonds 20D −41.7 , suggesting that the absolute configurations of 7,8-epoxy group in 5 should be 7S and 8R. The full assignments of ehrenbergol E (5) have been disclosed (Ehrenbergol E (pic data . The dec3b δH 2.0 made it pic data  of 5 wermbrene-C . Howeverisclosed  by extenSarcophyton (Alcyoniidae), have attracted much attention from chemists due to their structural complexity and remarkable biological activities such as antitumor 25D +71 ; UV (MeOH) λmax (log ε) 225 (3.83) nm; IR (KBr) vmax 2962, 1712, 1457, 1373, 1263, 1088, 1027 cm−1; 1H NMR and 13C NMR data, see m/z 415 [M + Na]+; HRESIMS m/z 415.2093 [M + Na]+  ((+)-1,15-Epoxy-2-methoxy-12-methoxycarbonyl-1115.2096) .epi-12-Methoxycarbonyl-11E-sarcophine (2): Colorless, viscous oil; [α]25D +56 ; UV (MeOH) λmax (log ε) 224 (3.89) nm; IR (KBr) vmax 2926, 1751, 1712, 1701, 1636, 1576, 1436, 1386, 1244, 1109, 1125, 1099 cm−1; CD  λmax (Δε) 279 (+6.8), 245 (−1.1) and 215 (−2.9) nm; 1H NMR and 13C NMR data, m/z 383 [M + Na]+; HRESIMS m/z 383.1832 [M + Na]+  ((+)-2-83.1834) .3): Colorless, viscous oil; [α]25D +23 ; UV (MeOH) λmax (log ε) 226 (3.85) nm; IR (KBr) vmax 2951, 1715, 1643, 1461, 1376, 1179, 1279, 1240, 1195, 1073 cm−1; 1H NMR and 13C NMR data, see m/z 401 [M + Na]+; HRESIMS m/z 401.2301 [M + Na]+   .4): Colorless, viscous oil; [α]25D +35 ; UV (MeOH) λmax (log ε) 245 (3.67) nm; IR (KBr) vmax 3422, 2959, 1747, 1621, 1438, 1383, 1219, 1041 cm−1; CD  λmax (Δε) 259 (+24.9) and 205 (–9.0) nm; 1H NMR and 13C NMR data, see m/z 339 [M + Na]+; HRESIMS m/z 339.1938 [M + Na]+  (Ehrenbergol D (39.1936) .5): Colorless, viscous oil; [α]25D +40 ; UV (MeOH) λmax (log ε) 249 (3.76) nm; IR (KBr) vmax 3441, 2958, 2932, 1619, 1455, 1376, 1240, 1033 cm−1; 1H NMR and 13C NMR data, see m/z 327 [M + Na]+; HRESIMS m/z 327.2303 [M + Na]+  (Ehrenbergol E (27.2300) .50 values of 0.05, 0.06 and 0.07 μM against P-388, A-549 and HT-29, respectively. The experimental details of this assay were carried out according to a previously described procedure [Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549  tumor cells using a modification of the MTT colorimetric method. The provision of the P-388 cell line was supported by John M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, IL, USA. These HT-29 and A-549 cell lines were purchased from the American Type Culture Collection . The anticancer agent mithramycin was used as the positive control and exhibited ECrocedure ,29.50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [To determine the effects of the natural product upon human cytomegalovirus (HCMV) cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural product. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity is expressed as ICemployed .1–5 were successfully purified from Sarcophyton ehrenbergi. Preliminary cytotoxic screening revealed that compounds 1–3 were not cytotoxic to P-388, A-549, HT-29 and HEL cells. However, compounds 4 and 5 displayed cytotoxicity against P-388 with EC50 values of 2.0 and 3.0 μM, respectively. The result may suggest that the conjugated double bonds at C-1/C-2 and C-3/C-4 are important for the cytotoxicity against P-388. Additionally, at a concentration of 1 μg/mL, compound 2 showed slight antiviral activity against HCMV with an IC50 value of 25.0 μg/mL.Five new polyoxygenated cembranoids


O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant Aspergillus sp. strain F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.Three new asperentin-type compounds, 6- Pale yellow oil; c = 0.44, MeOH), UV (MeOH) λmax 260.0 and 302.0 nm; IR (KBr) νmax 3445 and 1700 cm−1; 1H and 13C NMR, see m/z 439.1975 [M + H]+ .6-O-methylasperentin (3). White needle; c = 0.18, MeOH), m.p. 125.7 °C, UV (MeOH) λmax 268.9 and 333.0 nm; IR (KBr) νmax 3319 and 1657 cm−1; 1H and 13C NMR, see m/z 345.1308 [M + Na]+, .5-Hydroxyl-6-1 (90.0 mg) in 3 N HCl (60 mL) was stirred at 80 °C for 1 h. After the hydrolysis reaction was completed, the reaction mixture was concentrated under vacuum. The resulting residue was suspended in H2O (40 mL) and extracted with CH2Cl2 (3 × 40 mL). Then the aqueous layer was concentrated and dried under vacuum. The resulting residue was acetylated with Ac2O/Py at room temperature to yield an acetate product that was purified by Sephadex LH-20 (80 g) eluting with acetone/MeOH  to afford a tetra-acetate of ribofuranose (4.1 mg). The specific rotation of this tetra-acetate product, c = 0.68, MeOH), is consistent with the reported value of d-ribose [1H-NMR  δ 6.06 , 5.06 , 5.51 , 5.18 , 3.94 , 4.05 , 2.16 , 2.15 , 2.13 , 2.12 . 13C-NMR  δ 90.9 (C-1), 67.3 (C-2), 66.2 (C-3), 66.3 (C-4), 62.7 (C-5), 169.9 (CH3CO), 169.8 (CH3CO), 169.5 (CH3CO), 168.8 (CH3CO), 20.9 (CH3CO), 20.8 (CH3CO), 20.7 (CH3CO), 20.7 (CH3CO).A solution of C. gleosporioides Penz, C. gleosporioides (Penz) Sacc., and B. cinerea Pers was evaluated using a filter-paper disk method. The tested strains were cultivated on PDA plates at 28 °C. Compounds 1–9 were tested at a concentration of 5 mg/mL in MeOH using amphotericin B (5 mg/mL in DMSO) as the positive control. The tested compound solutions (5 μL) were transferred onto filtering paper disks (Φ = 5 mm) placed in the center of assay plates. After 48 h incubation at 28 °C, the diameters of inhibition zones were measured to evaluate the activity, a bigger size of the diameter indicated a stronger inhibition. All experiments were repeated in triplicate.Anti-crop pathogen activity against O-α-d-ribosylasperentin (1), 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with six known asperentin derivatives (4–9), isolated and identified from the fermentation products of a halotolerant fungus Aspergillums sp. strain F00785 from the inner tissue of the marine alga. 1 and 2, were found to be the first members with d-ribofuranose via α-glycosidic linkage. To the best of our knowledge, the α-d-ribofuranosyl moiety is unusual in metabolites from fungi, with only four natural products containing α-d-ribofuranosyl moiety reported up to now [4 exhibited strong inhibitory activity against three crop pathogens. Preliminary study of the structure activity relationships (SARs) of these nine compounds indicated that free hydroxyls at C-6 and C-8 are critical to the antifungal activity, while the hydroxyl substitution at the 4′ or 5′ position of the pyranoid ring is not favorable. In summary, discovery of these new secondary metabolites suggest that chemical investigations of marine microorganisms in relatively uninvestigated environments may provide significant natural chemical diversity for drug discovery.Three new compounds, 6-p to now ,21,23,24


Phosphonic diamide derivatives enhance the cellular permeability and in turn their biological activities.Chronic and oral administration of benzylamine improves glucose tolerance. Picolylamine is a selective functional antagonist of the human adenosine AA series of heteroaryl phosphonicdiamide derivatives were designed as therapeutics to control and manage type2 diabetes. Initially defined Lipinski parameters encouraged them as safer drugs. Molecular docking of these compounds against Protein tyrosine phosphatase (PTP), the potential therapeutic target of type 2 diabetes, revealed their potential binding ability explaining their anti-diabetic activity in terms of PTP inhibition. Human intestinal absorption, Caco-2 cell permeability, MDCK cell permeability, BBB penetration, skin permeability and plasma protein binding abilities of the title compounds were calculated by PreADMET server. A convenient method has been developed for the synthesis of title compounds through the formation of 1-ethoxy-N,N’-bis(4-fluorobenzyl/pyridin-3-ylmethyl)phosphinediamine by the reaction of 4-fluorobenzylamine/ 3-picolylamine with ethyldichlorophosphite, subsequently reacted with heteroaryl halides using lanthanum(III) chloride as a catalyst.in vitro anti-oxidant activity and in vivo evaluation in streptozotocin induced diabetic rat models revealed that the normal glycemic levels were observed on 12th day by 9a and 20th day by 5b, 5c, 9e and 9f. The remaining compounds also exhibited normal glycemic levels by 25th day.All the compounds exhibited significant in vitro and in vivo studies are suggesting them as safer and effective therapeutic agents against type2 diabetes.The results from molecular modeling, The online version of this article (doi:10.1186/s40199-014-0076-3) contains supplementary material, which is available to authorized users. The stipulation of anti-diabetic drugs is snowballing hastily, due to millions of people is distressing about diabetes. Several budding essential mechanisms for diabetes are characterized by elevation of blood glucose levels caused by decreased production of the hormone insulin and/or increased resistance to the action of insulin by certain cells. Tyrosine phosphorylation is associated with a group of enzymes which are mainly involved in the negative regulation of insulin signaling and intertwined in the insulin resistance, complementary to type 2 diabetes ,2. Protei-vii reveals that they are ideal for anti-diabetic activity due to the thiazolidine-2,4-dione , pyridinyl , quinolone (iv), urea and amide , Flouro substituted, heteroaryl pyrazine (vi) and benzyl amine (vii). Compound xiii is a α-aminophosphonate with established anti-diabetic property which gave an idea to focus on phosphorus containing drugs.The study of the reported drugs 99mTc(I)-complexs ; Anal. Calcd. for C18H17N4F2OP: C 57.76; H 4.58; N 14.97; Found: C 57.63; H 4.39; N 14.77.Yield: 72%; mp: 162-164°C; IR (KBr): ῡ 3378 (N-H), 1252 (P = O), 1018 (P-CAr) cm−1; 1H NMR : δ 9.06 , 8.32-6.74 , 5.73-5.68 , 5.15 , 3.83-4.08 ; 13C NMR : δ 169.6 (C-2′), 161.6 (C-4), 161.3 (C-4′), 158.2 (C-1′), 155.4 (C-1), 141.9 (C-6′), 122.6-121.8 (C-2 & C-6), 117.8-116.8 (C-3 &C-5), 30.9 (C-7); 31P NMR : δ 27.6; LC MS (%): m/z 407.8 (100%) [MH+•]; Anal. Calcd. for C18H17N4F2O3P: C 53.21; H 4.22; N 13.79; Found: C 53.08; H 4.12; N 13.55.Yield: 70%; mp: 189-191°C; IR (KBr): ῡ 3386 (N-H), 1238 (P = O), 992 (P-CAr) cm−1; 1H NMR : δ 7.65-6.50 , 5.12 , 4.06-3.83 ; 13C NMR : δ 159.1 (C-4), 155.9 (C-1′), 154.8 (C-3′), 154.0 (C-1), 133.6 (C-5′), 133.5 (C-4′), 122.2-121.1 (C-2 & C-6), 119.6 (C-6′), 118.9 (C-7′), 117.7-116.6 (C-3 & C-5), 30.9 (C-7); 31P NMR : δ 28.7; LC MS (%): m/z 442.8 (100%) [MH+•]; Anal. Calcd. for C20H17F5N3OP: C 54.43; H 3.88; N 9.52; Found: 54.15; H 3.51; N 9.22.Yield: 69%; mp: 202-204°C; IR (KBr): ῡ 3354 (N-H), 1261 (P = O), 1010 (P-CAr) cm−1; 1H NMR : δ 8.55-6.79 , 5.14 , 4.08-3.83 ; 13C NMR : δ 162.5 (C-1′), 161.9 (C-4), 155.1 (C-1), 154.3 (C-3′ & C-5′), 124.4 (C-4′), 121.8-121.1 (C-2 & C-6), 117.6-116.4 (C-3 & C-5), 31.4 (C-7); 31P NMR : δ 28.2; LC MS (%): m/z 375.4 (100%) [MH+•]; Anal. Calcd. for C18H17F2N4OP: C 57.76; H 4.58; N 14.97; Found: 57.62; H 4.41; N 14.82.Yield: 75%; mp: 167-169°C; IR (KBr): ῡ 3346 (N-H), 1268 (P = O), 996 (P-CAr) cm−1; 1H NMR : δ 8.65-6.76 , 5.16 , 4.06-3.84 ; 13C NMR : δ 161.6 (C-4), 156.2 (C-4′), 155.2 (C-1), 142.6 (C-2′), 135.9 (C-9′), 129.5 (C-7′), 129.2 (C-10′), 127.5 (C-5′), 127.1 (C-8′), 126.9 (C-6′), 126.2 (C-1′), 122.3-121.5 (C-2 & C-6), 117.7-116.6 (C-3 & C-5), 30.7 (C-7); 31P NMR : δ 27.8; LC MS (%): m/z 424.5 (100%) [MH+•]; Anal. Calcd. for C23H20F2N3OP: C 65.25; H 4.76; N 9.92; Found: C 65.09; H 4.57; N 9.71.Yield: 75%; mp: 221-224°C; IR (KBr): 3364 (N-H), 1274 (P = O), 986 (P-CAr) cm−1; 1H NMR : δ 8.63-6.92 , 5.13 , 4.08-3.83 ; 13C NMR : δ 161.5 (C-4), 157.2 (C-9′), 154.5 (C-1), 148.4 (C-2′), 136.8 (C-3′), 131.7 (C-6′), 128.5 (C-4′), 127.3 (C-7′), 126.8 (C-10′), 126.7 (C-5′), 123.6 (C-8′), 122.1-121.3 (C-2 & C-6), 117.8-116.2 (C-3 & C-5), 30.5 (C-7); 31P NMR : δ 28.9; LC MS (%): m/z 424.6 (100%) [MH+•]; Anal. Calcd. for C23H20F2N3OP: C 65.25; H 4.76; N 9.92; Found: C 65.08; H 4.51; N 9.84.Yield: 72%; mp: 179-181°C; IR (KBr): 3372 (N-H), 1259 (P = O), 1012 (P-C6) (0. 002 mol) in dry tetrahydrofuran (THF) (10 mL), ethyldichlorophosphite (2) (0.001 mol) was added at 0°C in the presence of triethylamine (TEA) (0.002 mol) under N2 atmosphere. After completion of the addition, the reaction mixture was raised to 30°C and stirred for 2 h to form the intermediate 1-ethoxy-N,N’-bis(pyridin-3-ylmethyl)phosphinediamine (7). The reaction progress was monitored by thin layer chromatography (TLC) using ethyl acetate: hexane (1:1) as mobile phase. After completion of the reaction, it was filtered to remove triethylamine hydrochloride. 2-Chloro-4,6-dimethoxy-1,3,5-triazine (8a) (0.001 mol) in dry THF (10 mL) was added to the filtrate under N2 atmosphere at 20°C in the presence of La(III)Cl3.7H2O (20 mol%) and the reaction mixture was refluxed for 3 h. The progress of the reaction was monitored by TLC using ethyl acetate: hexane (1:1). After completion of the reaction, catalyst was removed by filtration and the filtrate was concentrated in rota-evaporator to afford the crude product. It was purified by silica gel column chromatography eluting with ethyl acetate: hexane (1:2) mixture to afford the title compound, 4,6-dimethoxy-1,3,5-triazin-2-yl-N,N’-di(3-pyridylmethyl)phosphonic diamide (9a). The same experimental procedure was adopted for the preparation of the remaining title compounds 9b-f (Scheme To a stirred solution of 3-picolylamine (6) 0. 00 mol in dAr) cm−1; 1H NMR : δ 8.92-7.26 , 5.63 , 4.39-4.35 , 3.72 ; 13C NMR : δ 178.3 (C-3′ & C-5′), 169.2 (C-1′), 149.5 (C-2), 148.1 (C-4), 145.8 (C-1), 135.3 (C-6), 121.5 (C-5), 51.9 (C-OMe), 38.6 (C-7); 31P NMR : δ 21.6; LC MS (%): m/z 402.5 (100%) [MH+•]; Anal. Calcd. for C17H20N7O3P: C 50.87; H 5.02; N 24.43; Found: C 50.66; H 4.81; N 24.23.Yield: 70%; mp: 198-200°C; IR (KBr): ῡ 3371 (N-H), 1242 (P = O), 989 (P-CAr) cm−1; 1H NMR : δ 8.61-7.46 , 5.64 , 4.35-4.31 ; 13C NMR : δ 149.4 (C-2), 148.9 (C-4), 147.5 (C-2′), 145.2 (C-9′), 135.3 (C-6), 134.6 (C-1), 132.3 (C-3′), 131.7 (C-6′), 128.9 (C-4′), 127.5 (C-5′), 127.3 (C-10′), 126.1 (C-7′), 122.5 (C-8′), 121.7 (C-5), 38.8 (C-7); 31P NMR : δ 20.2; LC MS (%): m/z 390.3 (100%) [MH+•]; Anal. Calcd. for C21H20N5OP: C 64.77; H 5.18; N 17.99; Found: C 64.51; H 5.03; N 17.81.Yield: 74%; mp: 206-208°C; IR (KBr): 3376 (N-H), 1247 (P = O), 1005 (P-CAr) cm−1; 1H NMR : δ 8.63-7.45 , 5.62 , 4.37-4.34 ; 13C NMR : δ 152.5 (C-4′), 149.1 (C-2), 148.5 (C-4), 145.2 (C-1), 143.9 (C-2′), 135.1 (C-6), 134.5 (C-9′), 130.2 (C-10′), 129.5 (C-7′), 128.2 (C-6′), 127.4 (C-5′), 127.1 (C-8′), 125.9 (C-1′), 121.3 (C-5), 38.4 (C-7); 31P NMR : δ 19.1; LC MS (%): m/z 390.5 (100%) [MH+•]; Anal. Calcd. for C21H20N5OP: C 64.77; H 5.18; N 17.99; Found: C 64.68; H 5.12; N 17.85.Yield: 73%; mp: 166-169°C; IR (KBr): ῡ 3379 (N-H), 1253 (P = O), 995 (P-CAr) cm−1; 1H NMR : δ 8.48-7.44 , 5.62 , 4.35-4.31 ; 13C NMR : δ 162.2 (C-1′), 155.3 (C-8′), 149.2 (C-2), 148.9 (C-4), 145.5 (C-1), 135.8 (C-6), 134.3 (C-9′), 129.5 (C-4′), 127.1 (C-5′), 125.9 (C-6′), 125.7 (C-3′), 121.2 (C-5), 38.3 (C-7); 31P NMR : δ 21.6; LC MS (%): m/z 396.5 (100%) [MH+•]; Anal. Calcd. for C19H18N5OPS: C 57.71; H 4.59; N 17.71; Found: C 57.62; H 4.41; N 17.58.Yield: 66%; mp: 175-177°C; IR (KBr): ῡ 3383 (N-H), 1245 (P = O), 1013 (P-CAr) cm−1; 1H NMR : δ 8.66-7.41 , 5.63 , 4.32-4.30 ; 13C NMR : δ 157.3 (C-1′), 156.3 (C-3′), 149.3 (C-2), 148.8 (C-4), 145.5 (C-1), 138.4 (C-5′), 135.9 (C-4′), 135.8 (C-6), 121.4 (C-5), 118.3 (C-7′), 113.6 (C-6′), 38.2 (C-7); 31P NMR : δ 22.7; LC MS (%): m/z 365.7 (100%) [MH+•]; Anal. Calcd. for C18H17N6OP: C 59.34; H 4.70; N 23.07; Found: C 59.19; H 4.48; N 22.91.Yield: 68%; mp: 172-174°C; IR (KBr): ῡ 3388 (N-H), 1258 (P = O), 1018 (P-CAr) cm−1; 1H NMR : δ 8.61-7.48 , 5.64 , 4.34-4.31 ; 13C NMR : δ 159.3 (C-1′), 149.1 (C-2), 148.5 (C-4), 145.9 (C-1), 144.8 (C-3′), 135.9 (C-10′), 135.3 (C-6), 129.7 (C-6′), 129.5 (C-9′), 129.1 (C-8′), 128.5 (C-7′), 127.3 (C-5′), 122.6 (C-4′), 121.7 (C-5), 38.4 (C-7); 31P NMR : δ 18.9; LC MS (%): m/z 390.2 (100%) [MH+•]; Anal. Calcd. for C21H20N5OP: C 64.77; H 5.18; N 17.99; Found: C 64.55; H 5.02; N 17.75.Yield: 73%; mp: 185-187°C; IR (KBr): ῡ 3387 (N-H), 1261 (P = O), 1010 (P-Cin silico studies were carried out in the Molecular Operating Environment (MOE) software tool /[Blood], where [Brain] and [Blood] are the steady-state concentration of radio labeled compound in brain and peripheral blood. Predicting BBB penetration helps to know whether the compounds able to pass across the blood-brain barrier or not. This parameter expresses the BBB penetration capacity and absorption rate of compound to CNS. All the compounds were observed to be having moderate absorption to CNS.Km is distribution coefficient between stratum corneum and vehicle, D is average diffusion coefficient (cm2/h) and h is thickness of skin (cm) [The skin permeability is a crucial parameter that can define the transdermal delivery of the compound as the risk assessment during accidental contact with the skin. The skin permeability values are defined as logKp, cm/hr for all the compounds, where Kp = Km*D/h. kin (cm) .The title compounds were assessed for anti-oxidant and anti-hyperglycemic activity. The detailed discussion regarding the assessment method is demonstrated as follows.5a-f and 9a-f were able to reduce the stable radical DPPH to the yellow-colored diphenylpicrylhydrazine. The compounds were evaluated at four different concentrations of 50 μg/mL, 75 μg/mL, 100 μg/mL and 150 μg/mL and the IC50 values were determined from these evaluations. The scavenging effect of title compounds as compared to standard with the DPPH radical is in the following order 5f >5e >9f > Ascorbic acid >5c >9c and the remaining compounds showed less effect than these compounds. The compounds showed almost all same order at all concentrations and the complete results are given in Additional file 50 value of each compound was considered as the concentration (μg/mL) of the compound at which 50% of DPPH reduction was observed. These results are presented in Figure DPPH is usually used as a reagent to evaluate free radical scavenging activity of antioxidants . DPPH is50 values were determined from these evaluations. Amongst the title compounds 5e > BHT >5f >5a >5c >9f have exerted significant inhibitory activity and the remaining compounds exhibited less effect than these compounds on radicals that are generated in vitro and the complete results are given in Additional file 50 value of each compound was considered as the concentration (μg/mL) of the compound at which 50% of NO reduction was observed. These results are presented in Figure In the current investigation, newly synthesized compounds exhibited an excellent NO radicals scavenging activity. The compounds were evaluated at four different concentrations of 50 μg/mL, 75 μg/mL, 100 μg/mL and 150 μg/mL and the ICin vivo and can result in the formation of H2O2via dismutation reaction. Moreover, the conversion of superoxide and H2O2 into more reactive species, for instance, the hydroxyl radical, has been thought to be one of the unfavorable effects caused by superoxide radicals [in vitro and their activity is in comparable to that of Ascorbic acid. The compounds were evaluated for their scavenging effects at four different concentrations of 50 μg/mL, 75 μg/mL, 100 μg/mL and 150 μg/mL and the IC50 values were determined from these evaluations. The scavenging effects of the compounds are in the following order 5e >5f >9f >9c > Ascorbic acid and the remaining compounds exhibited less scavenging effect than these compounds on radicals that are generated in vitro. The compounds exhibited almost all same order at all concentrations and the complete results are presented in Additional file 50 value of each compound was considered as the concentration (μg/mL) of the compound at which 50% of NO reduction was observed. These results are presented in Figure Superoxide radical is known to be a very harmful species to cellular components as a precursor of more reactive oxygen species . The supradicals . The new5e, 5f, 9c, 9f are showing the better antioxidant activity, it may be due to the presence of quinolone group in the structures of the title compounds. These results are supported by the previous reports of Shridhar et al. of the antioxidant activity of eight substituted quinolines [In over view of observation, the compounds inolines .All the title compounds are containing atleast two N-H functional groups in their structure. These N-H groups are playng the main role in the abridging of free radicals, there by oxidative stress decreases. This mechanisim was supported by previous reports. This reduction of oxidative stress is the basis for mechanism for the antihyperglycemic actitivity Figure .Figure 69a) <242 ± 3.42 (9f) <244 ± 2.35 (5f) <245 ± 2.56 (Standard) <245 ± 4.86 (9b) <250 ± 3.24 (5c). On the eighth day the compounds 9a (152 ± 3.46) and 9f (195 ± 3.48) showed the least glycemic levels than the other compounds. On the 12th day, the compound 9a induced rats showed the normal glycemic levels (120 ± 3.15) and compound 9b (146 ± 1.95), 9f (153 ± 2.32), 5c (165 ± 2.76), Glibenclamide (166 ± 2.46) and 9e (168 ± 2.68) induced rats gave moderate glycemic levels. On the 16th day only the compound 9a (103 ± 1.47) gave the good result. The remaining compounds gave the moderate glycemic levels except 5a, 5d, 5e and 9c. On the 20th day all the compounds showed normal glycemic levels but 9d gave moderate and 5a, 5d, 5e and 9c gave the high glycemic levels. Finally on the last day, all the title compounds gave excellent results with glycemic levels in between 82 ± 1.58 (9a) to 109 ± 1.23 (9d) apart from 5a, 5d, 5e and 9c. The detailed observation of results of all title compounds are reproduced in Table All the title compounds showed significant anti-diabetic activity in the diabetes-induced rats when compared with the standard Glibenclamide. All the rats were kept in the observation for 25 days and seasoned the glycemic levels (mg/dL) for every four days after administration of drug. The diabetic rats showing glucose levels ≥350 mg/dL were taken for the experiment on the first day. On the fourth day, the glucose levels were almost all decreased i.e 221 ± 3.25  gave the potential anti-hyperglycemic properties against Diabetic rats. From these results, compound evention .5a-f and 9a-f by two-step reaction. The molecular descriptors of all the compounds from Lipinski parameters explained their drug likeliness and suggesting them as safer drugs. The descriptors also indicate that they are all not harmful to the host system because of their optimal logP values. The molecular docking study revealed their strong ability to interact with the target and inhibits its activity, there by predicting their anti-diabetic activity. PreADMET results demonstrated that the title compounds exhibit good absorption, permeability, penetration abilities in the human body. This prediction is confirmed by in vivo screening of these compounds in the diabetic induced rat models where the test compounds exhibited significant antihyperglycemic activity comparative to the standard Glibenclamide. Almost all the compounds brought the glycemic levels to normal on the 25th day. Especially, 9a was shown normal glycemic levels on 12th day. On the 20th day 5c, 5b, 9a, 9e and 9f were shown normal glycemic levels. All the compounds exhibited good diabetic levels on 25th day except 5a, 5d, 5e and 9c.Lanthanum (III) chloride is stated as an efficient catalyst for the Michaelis-Arbuzov reaction and for the synthesis of the title compounds Supporting Tables of the antioxidant activity and supporting Figures are given in the supporting information as Additional file


The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds  showed high potency against both strains of T. brucei and P. falciparum with the inhibitory concentrations for 50% (IC50) in the nanomolar range (IC50 = 1–96 nM). None of the tested derivatives was significantly active against T. cruzi or L. donovani. Three of the more potent compounds  were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences as possible binding sites for these compounds. Several of the tested compounds are suitable leads for the development of improved antiparasitic agents.A series of 15 alkanediamide-linked bisbenzamidines and related analogs was synthesized and tested  Pneumocystis activity of two key alkanediamide-linked bisbenzamidines when evaluated in an animal model of pneumocystosis , N,N-dimethyl formamide (40 mL) was refluxed for 30–120 min. The resultant precipitate was filtered and washed with the appropriate solvents.A mixture of 4-aminobenzamidine and/or 3-aminobenzamidine (10 mmol), pyridine , appropriate diacid chloride (5 mmol), in N,N′-bis[4-(aminoiminomethyl)phenyl] ethane-1,2-dicarboxamide (1). The product was successively washed with water and acetone to yield 10% solid, m.p. > 300 °C; IR:3130, 1674, 1611, 1514, 1412 cm−1; 1H-NMR: (DMSO-d6) δ11.1 ; 9.2 ; 8.0 , 7.9  ppm. Anal. Calcd. for C16H16N6O2·2HCl·1.5 H2O (424.28): C, 45.29; H, 4.98; N, 19.80. Found: C, 45.55; H, 5.12; N, 19.84.N,N′-bis[4-(aminoiminomethyl)phenyl] butane-1,4-dicarboxamide (2). The product was successively washed with ethanol and ether to yield 40% solid, m.p. > 300 °C; IR:3030, 1669, 1602, 1541, 1415 cm−1; 1H-NMR:(DMSO-d6) δ10.5 ; 9.2 ; 8.8 ; 7.8 ; 2.7  ppm. Anal. Calcd. for C18H20N6O2·2HCl (424.28): C, 50.83; H, 5.21; N, 19.76. Found: C, 50.77; H, 5.44; N, 19.58.N,N′-bis[3-(aminoiminomethyl)phenyl] hexane-1,6-dicarboxamide (7). The product was washed twice with acetone to yield 57% solid, m.p. > 300 °C; IR:3054; 1668; 1564; 1482; 1411; 1314 cm−1; 1H-NMR:(DMSO-d6) δ10.5 ; 9.3 ; 9.1 ; 8.1 ; 7.8 ; 7.5 ; 7.3 ; 2.4 ; 1.7  ppm. Anal. Calcd. for C20H24N6O2·2HCl (453.37): C, 52.98; H, 5.78; N, 18.54. Found: C, 52.74; H, 5.99; N, 18.45.N-[3-(aminoiminomethyl)phenyl] N′-[4-(aminoimino methyl)phenyl] hexane-1,6-dicarboxamide (10). Oily liquid, % yield unknown. IR:3086; 1667; 1601; 1481; 1315; 1264 cm−1; 1H-NMR:(DMSO-d6) δ10.7 ; 10.5 ; 9.4 ; 9.2 ; 9.1 ; 9.0 ; 8.2 ; 7.8 ; 7.5 ; 7.4 ; 2.4 ; 1.6  ppm. HRMS: found 381.2057, calcd. 381.2039 for C20H24N6O2.N,N′-bis[4-(aminoiminomethyl)phenyl] heptane-1,7-dicarboxamide (11). The product was successively washed with ethanol and ether to yield 45% solid, m.p. > 300 °C; IR:3117, 1675, 1616, 1541, 1496 cm−1; 1H-NMR:(DMSO-d6) δ10.5 ; 9.2 ; 8.9 ; 7.8 ; 2.3 ; 1.6 ; 1.3  ppm. Anal. Calcd. for C21H30N6O2·2HCl.0.2H2O) (503.42): C, 50.10; H, 6.40; N, 16.69. Found: C, 50.38; H, 6.03; N, 16.51.N,N′-bis[4-(aminoiminomethyl)phenyl] octane-1,8-dicarboxamide (12). The product was successively washed with water, ethanol and ether to yield 10% solid, m.p. > 300 °C; IR:3063, 1674, 1609, 1541, 1338, 1308 cm−1; 1H-NMR:(DMSO-d6) δ10.5 ; 9.2 ; 8.9 ; 7.8 ; 2.3 ; 1.6 ; 1.3  ppm. Anal. Calcd. for C22H28N6O2·2HCl) (481.42): C, 54.89; H, 6.28; N, 17.46. Found: C, 55.12; H, 6.25; N, 17.28.N,N′-bis[4-(aminoiminomethyl)phenyl] nonane-1,9-dicarboxamide (13). After refluxing for 2 h, the cooled solution was poured into acetone (100 mL). The product was successively washed with acetone and water to yield 50% solid, m.p. > 300 °C; IR:2935, 1696, 1674, 1541, 1330 cm−1; 1H-NMR:(DMSO-d6) δ10.4 ; 9.3 ; 7.8 ; 2.3 ; 1.6 ; 1.3  ppm. Anal. Calcd. for C23H32N6O2·2HCl·0.5C2H5OH) (518.48): C, 55.60; H, 6.80; N, 16.21. Found: C, 55.98; H, 6.61; N, 15.96.N,N′-bis[4-(aminoiminomethyl)phenyl] decane-1,10-dicarboxamide (14). After refluxing for 2 h, thecooled solution was poured into acetone (100 mL). The product was successively washed with acetone and water to yield 50% solid, m.p. > 265 °C decomposed; IR:2931, 1667, 1599, 1559, 1487 cm−1; 1H-NMR:(DMSO-d6) δ 10.4 ; 9.1 ; 7.8 ; 2.4 ; 1.6 ; 1.3  ppm. HRMS: found 437.2672, calcd. 437.2665 for C24H32N6O2.N,N′-bis[4-(aminoiminomethyl)phenyl] dodecane-1,12-dicarboxamide (15). The product was successively washed with water, ethanol and ether to yield 35% solid, m.p. > 300 °C; IR:3093, 1669, 1602, 1541, 1411 cm−1; 1H-NMR:(DMSO-d6) δ 10.4 ; 9.2 ; 8.9 ; 7.8 ; 2.3 ; 1.6 ; 1.3  ppm. HRMS: found 465.2985, calcd. 465.2978 for C26H36N6O2.T. brucei used for the in vitro study were T. b. brucei Lab 110 EATRO (pentamidine-sensitive) and T. b. rhodesiense KETRi 243  [in vitro assays with the two isolates of T. brucei were conducted according to previously described procedures [T. cruzi activity was determined using intracellular amastigote forms of T. cruzi Tulahuen strain C2C4 in rat skeletal myoblasts (L6 cells) as described previously [L. donovani activity was determined using the amastigotes of L. donovani strain MHOM-ET-67/L82 as described previously [P. falciparum NF54 (chloroquine-sensitive) and K1 (chloroquine-resistant) were determined using the erythrocytic stage of P. falciparum as described previously [The two clinical isolates of sistant) . The in ocedures . The antin vivo studies were conducted according to the procedures described previously [The eviously ,14.m value reported in The DNA binding affinity of the tested compounds was determined by measuring the change in mid-point of the thermal denaturation curves for a 1:5 compound to DNA base pair ratio as described previously . Each TmWe used Surflex-Dock to study the DNA binding affinities of 16 bisbenzamidine compounds to the minor groove of AT-rich sites and an AT absent site. Computational details are described in supplementary section.in vitro and in vivo evaluation as potential antiparasitic agents. Potential cytotoxicity of the compounds in a mammalian cell line, as well as their interactions with DNA via thermal denaturation and molecular modeling studies, have been determined. Seven of the compounds  were highly potent (IC50 = 1–96 nM) against drug-sensitive as well as drug-resistant cell lines of T. brucei and P. falciparum. Three compounds  were highly effective in curing mice infected with the drug-sensitive strains (Lab 110 EATRO and KETRI 2002) of T. brucei, whereas 5 and 6 were also efficacious in curing mice infected with the drug-resistant KETRI 2538 clinical isolate. Thermal denaturation studies with DNA showed that these compounds have stronger binding to poly(dA-dT) versus calf thymus DNA and a good correlation was observed between the ΔTm of poly(dA-dT) and the antiparasitic activity of these compounds against T. brucei and P. falciparum. Molecular modeling studies indicated that these compounds exhibited high preference for binding to the minor groove of a single G or GC inserted AT-rich (AAAGTTT or AAAGCTTT) DNA duplexes and they correlated well with the pIC50 values of the compounds against T. brucei and P. falciparum.This study reports on the synthesis of a series of alkanediamide-linked bisbenzamidines  and their


Nitration of β-asarone with AgNO2/I2 in ether yielded 1--2-nitropropene (1) but with NaNO2/I2 in ethylene glycol obtained 1--1-nitropropene (2). Compound 2 was prepared for the first time and characterized using IR, 1H-NMR, 13C-NMR, and GC-MS spectra and it was converted into 1- phenyl-1-propanone (3) using modified Nef reaction. Based on 1D NOESY experiments, compounds 1 and 2 have been assigned E configuration. Compounds 1 and 2 were subjected to cytotoxic activity using five human cancer cell lines, namely, MCF-7, SW-982, HeLa, PC-3, and IMR-32 by MTT assay. Except in breast cancer line (MCF-7) compound 2 exhibited five- to tenfold increase in activity compared to β-asarone and twofold increase over compound 1. Acorus calamus (Acoraceae) also known as sweet flag in Indian traditional medicine is generally used for treatment of cough, fever, bronchitis, inflammation, depression, tumors, haemorrhoids, skin diseases, insomnia, hysteria, epilepsy, and loss of memory . The cell death was calculated as follows:μM at which they inhibit the cell growth by 50% (IC50).Cell lines were maintained in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum  in a COTT assay . Five di Acorus calamus was collected from marshy areas of Kunigal in Karnataka, India in 2012. Melting points were recorded on an Acro melting point apparatus using a calibrated thermometer. Thin layer chromatography (TLC) and column chromatography (CC) were performed with [TLC silica gel 60 F254. Merck] and silica gel , respectively. Chromatograms were developed using hexane-EtOAc . IR spectra were recorded on Thermo-Nicolet instrument in KBr discs. Mass spectra were recorded using GCMS-QP2010S (direct probe). PMR spectra and 13C NMR spectra were recorded in CDCl3 with TMS (tetramethylsilane) as an internal standard on a Bruker AG spectrometer and chemical shifts were recorded in δ units.All chemicals were purchased from Laboratory Reagent (LR) grade. Fresh rhizome of−1.IR (KBr): 3421, 2939, 1589, 1512, 1469, 1211, 1145, 1029, 833 cm1H NMR : δ 1.88 , 3.81 , 3.84 , 3.90 , 5.78 , 6.49 , 6.53 , 6.84 .13C NMR : δ 153.58, 150.58, 143.26, 126.74, 126.42, 119.32, 116.71, 97.28, 56.98 (OCH3), 56.53 (OCH3), 56.38 (OCH3), 14.78 (C-9).m/z) = 208 [M]+ (25), 193 (10), 165 (15), 150 (5), 135 (12), 119 (5), 105 (5), 91 (14).GC-MS  were stirred in anhydrous ether (20 mL) at room temperature under nitrogen for 45 min. β-Asarone  and pyridine  in ether were added and the mixture was stirred at room temperature for 30 h as per the reported procedure  (42), 207 (100), 192 (70), 177 (62), 161 (28), 149 (25), 131 (12), 121 (30), 107 (12).GC-MS  in nitroethane  was added ammonium acetate  and the reaction mixture heated to 75–80°C for 3 hrs. After completion of the reaction, nitroethane was removed under vacuum, and oily orange mass was triturated with hot methanol (3 × 50 mL). Methanol extract was concentrated and allowed to stand at room temperature. Yellow crystals (75–78% yield) were obtained having the melting point 98–100°C. 1--2-nitropropene obtained by nitration of β-asarone  in 150 mL of ethyl acetate containing iodine  at 0°C was added to a solution of sodium nitrite , ethylene glycol , and water 20 mL. The reaction mixture was stirred at room temperature for 48 hrs under nitrogen and the ethyl acetate layer was separated, washed with water and then with 10% thiosulphate, and dried over MgSO4. Ethyl acetate layer was evaporated and recrystallized from methanol to obtain a yellow crystalline compound (70–75% yield).A mixture of MP: 158–160°C.−1.IR (KBr): 2948, 1645, 1346, 1214, 1032, 829, 769 cm1H NMR : δ 1.80 , 3.76 , 3.83 , 3.93 , 6.57 , 6.67 , 7.40 .13C NMR : δ 152.64, 151.44, 149.58, 142.86, 133.74, 115.01, 109.64, 97.28, 56.73 (OCH3), 56.33 (OCH3), 56.05 (OCH3), 14.28 (CH3).m/z) = 253 [M+.] (74), 207 (100), 192 (40), 177 (55), 161 (26), 149 (24), 131 (12), 121 (32).GC-MS  in methanol (25 mL) was added sodium borohydride  and stirred the reaction mixture at 25–30°C for 30 minutes. Completion of the reaction was confirmed by TLC. The reaction mixture was concentrated under vacuum and acidified with dilHCl to pH about 4.0 and extracted with CH2Cl2 (2 × 15 mL) washed with water and dried over Na2SO4. Evaporation of CH2Cl2 layer followed by crystallization from methanol gave colourless crystals.To a solution of compoundMP: 108-109°C.−1.IR (KBr): 2959, 1712, 1649, 1618, 1510, 1215, 1028, 810, 750 cm1H NMR : δ 1.18 , 2.99 , 3.85 , 3.87 , 3.91 , 6.50 , 7.43 .13C NMR : δ 200.73 (C=O), 155.16, 153.58, 143.09, 119.19, 112.80, 96.56, 56.28 (OCH3), 56.18 (OCH3), 56.10 (OCH3), 37.07 (CH2), 8.63 (CH3).m/z) = 224 [M+.] (38), 195 (100), 180 (10), 165 (5), 151 (10), 137 (12), 122 (32), 109 (5).GC-MS -1-propenylbenzene) when subjected to nitration gave 1--2-nitropropene (1) and 1--1-nitropropene (2). Preparation of compound 2 is reported here for the first time. The cytotoxic activities of these nitro derivatives were compared with β-asarone in five human cancer cell lines namely MCF-7, SW-982, HeLa, PC-3 and IMR-32 using MTT assay. Except in breast cancer line (MCF-7) compound 2 exhibited five- to tenfold increase in activity compared to β-asarone and twofold increase over compound 1.1H-NMR, 13C-NMR, 1D-NOESY and GC-MS spectra of 1--2-nitropropene (1), 1--1-nitropropene (2) and 1- phenyl-1-propanone (3) are provided with the supplementary material available online.Copies of