Their in vitro inhibitory activities against HDACs were evaluated. Disulfide and hydroxamic acid derivatives were the most potent ones. Assays with neonatal rat cardiac fibroblasts demonstrated low cytotoxic effects for all compounds. Regarding the parameters associated to cardiac fibrosis development, the compounds showed antiproliferative effects, and triggered a strong decrease on the expression levels of both α-SMA and procollagen I. In conclusion, the new thiazolyl-coumarin derivatives inhibit HDAC activity and decrease profibrotic effects on cardiac fibroblasts.New histone deacetylases (HDAC) inhibitors with low toxicity to non-cancerous cells, are a prevalent issue at present because these enzymes are actively involved in fibrotic diseases. We designed and synthesized a novel series of thiazolyl-coumarins, substituted at position 6 (R = H, Br, OCH Cardiac fibrosis is characterized by the excessive deposition of collagens and extracellular matrix (ECM) proteins that lead to impaired organ function, and is the major factor in the progression of myocardial infarction and heart failure . Cardiac+ dependent -Dithiolan-3-yl-N-[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-pentanamide (5a). Pale yellow solid, m.p.: 91–92 °C. Yield: 80.6%. FTIR (cm−1): 3155 (aromatic); 2931 (-CH-); 1726 ; 1648 (-C= N-); 1540 (-C=C-). 1H-NMR : 1.60 ; 1.94 ; 2.42 ; 2.67 ; 3.65 ; 7.3–7.8 ; 7.5 ; 8.55 . 13C-NMR : 25.5; 27.0; 33.2; 37.6; 39.3; 40.2; 55.1; 107.0; 117.3; 118.1; 122.4; 124.2; 127.5; 133.7; 139.3; 148.2; 154.5; 157.6; 160.4; 175.1. MALDI-MS theoretical m/z: 433.0714; m/z experimental: 433.0694; error (ppm): −4.6. ESI-MS: m/z: 433.0 [M + H]+; 431.0 [M − H]−. 306 [M-H2O + C3H6S2 + H]+; 245 [M-CO-C4H7-C3H5S2 + H]+; 225 [M-H2O + CO + C4H9-C3H5S2 + H]+; 207 n.d. 397 [M-H2S − H]−. Molecular formula: C20H20O3N2S3. Molecular weight (monoisotopic): 432.0636 g/mol5--Dithiolan-3-yl-N-[4-(6-bromo-2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-pentanamide (5b). Pale yellow solid, m.p.: 150–153 °C. Yield: 70.5%. FTIR (cm−1): 3197 (aromatic); 2945 (-CH-); 1697 ; 1625 (-C = N-); 1538 (-C = C-). 1H-NMR :1.56 ; 1.88 ; 2.42 ; 2.67 ; 3.62 ; 7.15 ; 7.20–7.62 ; 7.53 ; 8.49 . 13C-NMR: 25.7; 27.3; 33.0; 36.9; 38.7; 40.8; 56.1; 107.8; 117.6; 119.0; 122.6; 124.7; 128.5; 134.2; 140.2; 149.5; 155.5; 158.4; 160.9; 176.3. MALDI-MS: theoretical m/z: 510.9819; m/zexperimental: 510.9796; error (ppm): -4.5. ESI-MS: m/z: 513.0 [M + H]+; 511.0 [M + H]+; 511 [M − H]−. 397 [M-HBr + H2S + H]+; 325 [M-CO-C4H7-C3H5S2 + H]+; 306 [M-HBr + H2O + C3H6S2 + H]+; 189 [M-Br-C9H4O2-C3HNS-NH2 + H]+. 498 n.d; 396 [M-HBr + H2S + H]+; 323 [M-CO-C4H7-C3H5S2 + H]+; 189 [M-Br-C9H4O2-C3HNS-NH2+H]+. 477 [M-H2S − H]−. Molecular Formula: C20H19O3N2S3Br. Molecular weight (monoisotopic): 509.9741 g/mol.5--Dithiolan-3-yl-N-[4-(6-methoxy-2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-pentanamide (5c). Yellow solid, m.p.: 83–84 °C. Yield: 70.5%. FTIR (cm−1): 3083 (aromatic); 2922 (-CH-); 1702 ; 1640 (-C = N-); 1556 (-C=C-). 1H-NMR : 1.55 ; 1.88 ; 2.41 ; 2.68 ; 3.62 ; 7.15 ; 7.4  7.55 ; 7.7 ; 8.1 ; 8.45 . 13C-NMR : 25.3; 27.15; 31.7; 36.3; 39.5; 40.9; 54.3 55.1; 108.2; 110.5; 117.1; 117.4; 117.7; 119.1; 139.7; 146.3; 146.4; 153.3; 154.5; 158.2; 177.1. MALDI-MS theoretical m/z: 463.0820; m/zexperimental: 463.0797; error (ppm): −5.0. ESI-MS: m/z: 463.0 [M + H]+; n.d [M − H]−. 398 [M-C3OH + H2S2 + H]+; 275 [M-CO-C4H7-C3H5S2 + H]+. Molecular formula: C21H22O4N2S3. Molecular weight (monoisotopic): 462.0742 g/mol.N-[4-(2-Oxo-2 H-chromen-3-yl) -thiazol-2-yl]-3,4-dihydroxyacetyl-benzamide (6a′). Pale yellow solid, m.p.: 175–177 °C. Yield: 75%. FTIR (cm−1): 3064 (aromatic); 2926 (-CH-); 1707 ; 1609 (-C=N-); 1485 (-C=C-). 1H-NMR :3.00 ; 3.17 ; 7.18 ; 7.30–7.98 ; 7.85 ; 8.73 . 13C-NMR : 25.3 (d); 109.2; 113.9; 116.3; 119.7 (d); 121.0; 125.2; 129.2; 131.9; 132.0; 138.5; 139.3; 137.4; 143.7; 144.5; 152.6; 152.8; 157.3; 159.4 (d); 167.9; 173.6. MALDI-MS theoretical m/z: 465.0756; m/z experimental: 465.0737; error (ppm): −4.0. ESI-MS: m/z: 465.0 [M + H]+; n.d [M − H]−. 424 [M-CH3CHO +H]+; 387 [M-H2O + HO-COCH3 + H]+; 258 n.d; 136 [M-H-C9H4O2-C3H-NS-NH-CO + CO-CH2O + H]+. Molecular Formula: C23H16O7N2S. Molecular weight (monoisotopic): 464.0678 g/mol.N-[4-(6-Bromo-2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-3,4-dihydroxyacetyl-benzamide (6b′). Pale yellow solid, m.p.: 214–217 °C. Yield: 70%. FTIR (cm−1): 3068 (aromatic); 2931 (-CH-); 1727 ; 1631 (-C=N-); 1504 (-C=C-). 1H-NMR :3.05 ; 3.19 ; 7.18 ; 7.35-8.00 ; 7.92 ; 8.91 . 13C-NMR :26.4 (d); 109.5; 114.0; 116.5; 119.8; 119.4121.0; 126.6; 130.0; 133.2; 133.4; 139.0; 140.3; 144.1; 144.8; 153.4; 154.1; 157.9; 162.4 (d); 169.8; 177.3. MALDI-MS theoretical m/z: 542.9862; experimental m/z: 542.9839; error (ppm): −4.2. ESI-MS: m/z: n.d. [M + H]+; n.d. [M − H]. Due to the low intensity of the signals, fragmentation characterization at low resolution could not be performed. Molecular Formula: C23H15O7N2SBr. Molecular weight (monoisotopic): 541.9783 g/mol.N-[4-(6-Methoxy-2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-3,4-dihydroxyacetyl-benzamide (6c′). Yellow solid, m.p.: 118–120 °C. Yield: 68.2%.FTIR (cm-1): 3116 (aromatic); 2927 (-CH-); 1714 ; 1611 (-C=N-); 1572 (-C=C-).1H-NMR :3.00 ; 3.16 ; 3.83 ; 7.00–8.10 ; 7.80 ; 8.41 ; 8.72 . 13C-NMR :25.3 (d); 58.8; 108.1; 112.7; 115.3; 118.6; 120.9; 124.5; 128.7; 130.8; 131.7; 137.5; 138.3; 142.5; 144.3; 151.3; 152.0; 156.1; 158.5 (d); 166.6; 172.8. MALDI-MS theoretical m/z: 495.0862; experimental m/z: 495.0840; error (ppm): −4.4. ESI-MS: m/z: 495.0 [M + H]+; 493.0 [M − H]−. 450 [M-CH3 CHO + H]+; 401 [M-H2O + CO2 + CH3OH + H]+; 372 [M-H2O + HO-COCH3 + CH3CHO + H]+; 270 [M-CH2O + C6H4-(O-CO-CH3)2]+; 452 [M-CH2CO− H]−; 479 [M-CH3− H]−. Molecular formula: C24H18O8N2S. Molecular weight (monoisotopic): 494.0784 g/mol.5-[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-pentanoic acid methyl ester (7a′). Pale yellow solid, m.p.: 165–166 °C. Yield: 75%. FTIR (cm−1): 3185 (aromatic); 2981 (-CH-); 1687 ; 1643 (-C= N-); 1539 (-C=C-). 1H-NMR :1.60 ; 2.35 ; 2.49  3.59 ; 7.05–7.47 ; 7.51 ; 8.2 ; 8.48 . 13C-NMR :24.7; 25.7; 32.2; 33.7; 49.9; 109.1; 116.3; 119.5; 120.4; 125.0; 129.1; 132.2; 138.7; 152.7; 154.1; 159.2; 168.2; 169.8; 173.0. MALDI-MS theoretical m/z: 387.1015; experimental m/z: 387.0996; error (ppm): −4.9. ESI-MS: m/z: 387.0 [M + H]+; 385.0 [M − H]−. 355 [M-CH3OH + H]+; 311 [M-H2O + COOCH2 + H]+; 271 [M-C4H9-COOCH3 + H]+; 245 [M-CO-C4H7-COOCH3 + H]+. 353 [M-CH3OH − H]−; 310 [M-H2O + COOCH2− H]−; 243 [M-CO-C4H7-COOCH3− H]−. Molecular formula: C19H18O5N2S. Molecular weight (monoisotopic): 386.0936 g/mol.5-[4-(6-Bromo-2-oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-pentanoic acid methyl ester (7b′). Yellow solid, m.p.: 182–185 °C. Yield: 61.4%. FTIR (cm−1): 3067 (aromatic); 2943 (-CH-); 1704 ; 1639 (-C= N-); 1536 (-C= C-). 1H-NMR :1.70 ; 2.38 ; 2.56 ; 3.61 ; 7.30 ; 7.80 ; 7.83 ; 8.00 ; 8.92 . 13C-NMR :24.9; 25.8; 32.9; 34.5; 51.8; 110.0; 119.7; 120.3; 121.4; 125.5; 129.8; 135.2; 140.6; 153.7; 155.6; 160.0; 170.3; 173.1; 173.9. MALDI-MS theoretical m/z: 465.0120; experimental m/z: 465.0103; error (ppm): −3.7. ESI-MS: m/z: 467.0 [M + H]+; 465.0 [M − H]+; 463.0 [M − H]−. 435 [M-CH3OH + H]+; 325 [M-CO-C4H7-COOCH3 + H]+; 433 [M-CH3OH + H]+; 409 n.d; 323 [M-CO-C4H7-COOCH3 + H]+; 431 [M-CH3OH-H]−; 321 [M-CO-C4H7-COOCH3− H]−. Molecular formula: C19H17O5N2SBr. Molecular weight (monoisotopic): 464. 0042 g/mol.5-[4-(6-Methoxy-2-oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-pentanoic acid methyl ester (7c′). Yellow solid, m.p.: 163–164 °C. Yield: 64%. FTIR (cm−1): 3188 (aromatic); 2935 (-CH-); 1705 ; 1614 (-C = N-); 1540 (-C=C-). 1H-NMR :1.75 ; 2.36 ; 2.50 ; 3.68 ; 6.96 ; 7.06 ; 7.30 ; 7.75 ; 8.23 ; 8.44 . 13C-NMR :24.5; 25.3; 33.1; 35.0; 49.3; 55.1; 109.0; 116.0; 116.4; 117.0; 119.5; 124.0; 138.5; 151.1; 152.5; 158.6; 166.3; 169.8; 173.0. MALDI-MS theoretical m/z: 417.1120; experimental m/z: 417.1102; error (ppm): −4.3. ESI-MS: m/z: 417.0 [M + H]+; 415.0 [M − H]−. 385 [M-CH3OH + H]+; 341 [M-H2O + COOCH2 + H]+; 301 [M-C4H9-COOCH3 + H]+; 275 [M-CO-C4H7-COOCH3 + H]+. 383 [M-CH3OH − H]−; 273 [M-CO-C4H7-COOCH3− H]−. Molecular formula: C20H20O6N2S. Molecular weight (monoisotopic): 416.1042 g/mol.5-[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-N-hydroxypentanamide (8a). Pale yellow solid, m.p.: 150–152 °C (d). Yield: 64%. FTIR (cm−1): 3057 (aromatic); 2925 (-CH-); 1699 ; 1640 (-C=N-); 1539 (-C=C-).1H-NMR : 1.50 ; 2.02 ; 5.50 ; 6.50-7.30 ; 7.97 ; 8.50 ; 8.60 . 13C-NMR :24.7; 25.7; 32.2; 33.7; 49.9; 109.1; 116.3; 119.5; 120.4; 125.0; 129.1; 132.2; 138.7; 152.7; 154.1; 159.2; 168.2; 169.8; 173.0. MALDI-MS theoretical m/z: 388.0967; experimental m/z: 388.0953; error (ppm): −3.6. ESI-MS: m/z: 388.0 [M + H]+; 386.0 [M − H]−. 370 [M-H2O + H]+; 355 [M-NH2OH + H]+; 271 [M-C4H9-CONHOH + H]+; 245 [M-CO-C4H7-CONHOH + H]+. 353 [M-NH2OH − H]−; 259 n.d.; 243 [M- CO-C4H7-CONHOH − H]−; 141 [M-H-C9H4O2-C3HNSNH2− H]−. Molecular Formula: C18H17O5N3S. Molecular weight (monoisotopic): 387.0889 g/mol.5-[4-(6-Bromo-2-oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-N-hydroxypentanamide (8b). Pale yellow solid, m.p.: 106–110 °C (d). Yield: 57%. FTIR (cm−1): 3032 (aromatic); 2951 (-CH-); 1698 ; 1648 (-C=N-); 1537 (-C=C-). 1H-NMR :1.50 ; 2.50 ; 7.24 ; 7.52 ; 7.39 ; 7.40 ; 7.73 ; 8.10 ; 8.50 . 13C-NMR : 31.5; 32.4; 34.4 (d); 110.1; 116.8; 118.5; 121.7; 121.8; 131.3; 134.1; 131.0; 134.1; 137.1; 143.5; 151.5; 158.7; 168.0. Due to the insolubility of the compound it was not possible to perform mass spectrometry analysis. Molecular Formula: C18H16O7N2SBr. 5-[4-(6-Methoxy-2-oxo-2H-chromen-3-yl)-thiazol-2-yl-carbamoyl]-N-hydroxypentanamide (8c). Pale yellow solid, m.p.: 125–127 °C (d). Yield: 68%.FTIR (cm-1): 3136 (aromatic); 2916 (-CH-); 1721 ; 1631 (-C=N-); 1555 (-C=C-).1H-NMR :1.56 ; 1.97 ; 3.82 ; 7.20 ; 7.53 ; 7.34 ; 7.38 ; 7.98 ; 8.50 ; 8.53 . 13C-NMR : 24.8; 25.2; 32.6; 35.2; 56.3; 109.4; 111.1; 111.5; 117.4; 119.7; 120.1; 121.0; 139.0; 147.0; 155.3; 159.2; 168.3; 169.3; 172.1. MALDI-MS theoretical m/z: 418.1073; m/z experimental: 418.1053; error (ppm): −4.8. ESI-MS: m/z: 418.0 [M + H]+; 416.0 [M − H]−. 400 [M-H2O + H]+; 385 [M-CH3OH + H]+; 301 [M-C4H9-CONHOH + H]+; 275 [M-CO-C4H7-CONHOH + H]+. 383 [M-CH3OH − H]−; 288 n.d.; 273 [M-CO-C4H7-CONHOH − H]−; 141 [M-H-CH3O-C9H4O2-C3HNS-NH2− H]−. Molecular Formula: C19H19O6N3S. Molecular weight (monoisotopic): 417.0995 g/mol.® from Invitrogen was purchased to Thermo Fisher . Fetal bovine serum (FBS), trypsin/ethylenediaminetetraacetic acid (EDTA), molecular weight standard and organic and inorganic compounds were purchased from Merck . Reagents for enhanced chemiluminescence (ECL) were purchased from PerkinElmer Life Sciences . Sterile plastic material was obtained from Corning Inc. . Primary anti-histone H4–acetylated antibody and total histone H4 were purchased from R & D Systems . Primary anti–collagen type I and anti–α–SMA were purchased from Sigma-Aldrich . Primary anti–GAPDH antibody and secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained at Santa Cruz Biotechnology . Alamar Blueth, 2011), and experimental protocols were approved by the University of Chile Institutional Ethics Review Committee. CF were isolated from of 2 or 3–day–old Sprague-Dawley rats and cultured as described previously [g. The pellet was resuspended in 10 mL of Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 (DMEM-F12) supplemented with 10% FBS and antibiotics (100 μg/mL streptomycin and 100 units/mL penicillin) and cultured in a humid atmosphere of 5% CO2 and 95% O2 at 37°C until confluence (5 days). The purity of the CFs population was assessed through the expression of several markers. CFs had positive staining against vimentin (Santa Cruz Biotechnology), while being negative against sarcomeric actin and desmin (Sigma-Aldrich). Experiments were performed on cells at passage 1. Cells were cultured in 35-mm well plates and serum starved for 24 h prior to stimulation.Sprague Dawley rats were obtained from the Animal Breeding Facility of the School of Chemical and Pharmaceutical Sciences at the University of Chile. All studies followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , in presence/absence of CTz derivatives for 24 h, at concentrations according to experiment. Cell proliferation was quantified by Alamar Blue®, according to protocol mentioned before. Cell proliferation was expressed as percentage over the control FBS treated cells. To evaluate cytotoxicity, CFs were seeded in 12 microwell plates at a ratio of 2 × 10The inhibitory activity of the synthesized compounds against HDACs Hela cells nuclear extract, HDAC1 (class I) and HDAC6 (class II) were measured utilizing the Fluorometric Drug Discovery Assay Kit BML-AK500-0001 (HeLa cells nuclear extract), BML-AK511 (HDAC1), and BML-AK516 (HDAC6) . The HDACs fluorometric substrate and assay buffer were added to HeLa nuclear extracts and also to HDACs 1 and 2 substrates in a 96–wellplate and incubated at 37 °C for 30 min. TSA at 5 nM served as the positive control. The fluorophore produced was detected in a plate spectrofluorimeter, with an excitation of 360 nm and an emission of 460 nm. The assay was performed according to the manufacturer’s instructions, at various concentrations of the synthesized compounds.6 of CFs seeded in 60 mm plates was used, which were incubated with the inhibitory compounds at a concentration of 10 μM, in the absence/presence of TGF–β1(5ng/mL) for 4 h. Subsequently, total histones were extracted by the technique of saline precipitation . Briefly, CFs were scraped from the plates with 1 mL of cold PBS transferring their contents to Eppendorf tubes to be centrifuged for 60 s at 1500 rpm at 4 °C and re–suspended in 400 μL of cold buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (HEPES-KOH) adjusted to pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). The suspension is left on ice for 10 min and then placed in the vortex for 10 s. The samples were centrifuged for 60 s, subsequently discarding the supernatant fraction. The obtained pellet was re-suspended in 60 μL of cold C buffer  and placed on ice for 20 min for saline precipitation. Cell debris is removed by centrifugation for 120 s at 1500 rpm at 4 °C and the supernatant fraction, which contains the DNA binding proteins, is stored at minus 20 °C. For the analysis of α–SMA and procollagen type I expression levels, 3×105 CFs seeded in 35 mm plates were used, which were incubated together with inhibitor compounds at a 5 μM, in the absence/presence of TGF–β1 (5 ng/mL) for 48 h. To obtain the total proteins, the CFs were washed three times with cold PBS, then scraped and lysed with 50 μL of radio inmuno-precipitation assay (RIPA) lysis buffer  0.1%, deoxycholate 1%, leupeptin 2 μg/mL, aprotinin 10mM, PMSF 1mM and Na3VO4 100μM). The solution was centrifuged for 15 min at 10,000 rpm and at 4 °C. The supernatant fraction containing the total proteins was stored at minus 20 °C. The concentration of proteins obtained was determined by spectrophotometry at 570 nm, using the Bradford method .For the analysis of the protein expression of histone H4–acetylated (ac–H4) and total H4 histone, a number of 1 × 10CFs proteins (25 μg), were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and finally transferred onto a nitrocellulose membrane . The membrane was washed twice with tris–buffered saline solution containing 0.1% Tween 20 (TBST). After being blocked with TBST containing 5% nonfat milk for 40 min, the membrane was incubated with primary antibodies in TBST/1% nonfat milk at 4 °C overnight. The membrane was washed three times with TBST for a total of 15 min and then incubated with primary antibodies . Then the membrane was incubated with goat anti–rabbit or anti-mouse IgG antibodies conjugated with horseradish (diluted 1:1000) for 1 h at room temperature. Signals were developed with ECL reagents and exposure to photographic film.The ADMET properties of the thiazolyl–coumarin derivatives as drug candidate were predicted using the software QikProp .5 and series 8 derivatives are potent HDAC inhibitors. All derivatives inhibit HDAC enzymatic activity (to a greater or lesser degree), suggesting that the thiazolyl–coumarin group is an effective surface recognition CAP. All compounds at lower concentration display diminished cytotoxicity. Regarding the parameters associated to cardiac fibrosis development, compounds 5a and 8a show significant inhibition on CFs proliferation at 1 μM and also showed better decrease of procollagen type I and α–SMA expression levels.Synthesis and evaluation of different ZBGs linked to thiazolyl–coumarin moieties shows that the series 5 and 8 derivatives could be considered as leads to develop drugs with strong antifibrotic activity and are consistent with published reports where HDAC inhibitors could be useful pharmacological tools to prevent cardiac fibrosis development. Finally, the advantage of thiazolyl–coumarin disulfide over hydroxamic acid derivatives is their low cytotoxicity and better ADMET parameters. These results confirm that series


Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25°C for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11β,17β-Dihydroxyandrost-4-en-3-one (2), 11β- hydroxyandrost-4-en-3,17-dione (3), 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (4) and prednisolone (5) were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.A unicellular microalga,  Among of them, Pseudomonas fluorescens showed the greatest bioconversion efficiency D +164° (MeOH) (lit. D +226°(MeOH); (lit. D +91° (MeOH); (lit. D +100° (dioxane); IR νmax  3050, 2980, 1658, 1624, 1600 cm-1; MS (EI) m/z (%) 360 (7) , 300 (19), 122 (100), 91 (14), 148 (38), 124 (40), 74 (35) ,55 (45); 1H-NMR δ 0.9 , 1.3 , 4.42 , 4.60 , 5.96 , 6.20 , 7.20 ; 13C-NMR δ 213 (C20), 185.2 (C3), 170.4 (C5), 156.8 (C1), 127 (C2), 121.8 (C4), 88.6 (C17), 68.8 (C11), 66.4 (C21), 55.4 (C9), 51.2 (C13), 55.4 (C9), 47.2 (C12), 44.2 (C12), 38.8 (C10), 34.2 (C8), 33.6 (C16), 31.8 (C6), 31.2 (C7), 23.9 (C15), 21 (C19), 17.2 (C18); Rf 0.4 .Chlamydomonas reinhardtii and then PCR was applied using two set primers. The sequences were amplified using the primers 5′-GTCAGAGGTGAAATTCTTGGATTTA-3′ as forward and 5′-AGGGCAGGGACGTAATCAACG-3′ as reverse, which amplify a ~600-bp region of the 18S rRNA gene. The applied PCR condition has been described by Nubel et al. [For this purpose, DNA content was first extracted from the l et al. . PCR prol et al.  and the l et al.  software


The latter thiones reacted with the appropriate hydrazonoyl chloride to give the corresponding pyridotriazolopyrimidin-5(1H)-ones. The assigned structures for all the newly synthesized compounds were confirmed on the basis of elemental analyses and spectral data and the mechanisms of their formation were also discussed. Most of the synthesized compounds were tested for in vitro activity against human lung cancer (A-549) and human hepatocellular carcinoma (HepG-2) cell lines compared with the employed standard antitumor drug (cisplatin) and the results revealed that compounds 8, 4e and 7b have promising activities against the A-549 cell line  while compound 7b has promising activity against the HepG-2 cell lines (IC50 = 3.54 ± 1.11 μg/mL). Moreover, computational studies using MOE 2014.09 software supported the biological activity results.A series of new morpholinylchalcones was prepared and then used as building blocks for constructing a series of 7-morpholino-2-thioxo-2,3-dihydropyrido[2,3- In recent years, interest in the chemistry of hydrazonoyl halides has been renewed because of the development of novel synthetic routes and their use as synthons for compounds that find many applications ,7,8,9,102a) and 1-morpholino-1-(2-(p-tolyl)hydrazono)propan-2-one (2b) were prepared by a previously reported method propan-2-ones 2a,b and the appropriate benzaldehyde derivatives 3a–c in glacial acetic acid in the presence of a catalytic amount of concentrated H2SO4, gave the morpholinylchalcone derivatives 4a–f in good yield . The IR spectra of compounds 4a–f revealed in each case two absorption bands in the regions υ 3250–3236, 1683–1676 cm−1 attributed to the NH and C=O groups. The 1H-NMR spectra of compounds 4a–f showed in each case three signals assigned for the CH=CH and NH in addition to the expected signals for the morpholine and aromatic protons . For example, the 1H-NMR spectrum of compound 4a taken as a typical example of the products 4, revealed three signals at δ = 7.45 , 7.79 , 10.58  and morpholine protons ppm in addition to the characteristic signals of the aromatic protons. The 13C-NMR spectrum of compound 4a showed three signals at δ = 25.76, 68.04, 188.22 ppm assignable for the morpholine-C and the carbonyl-C, in addition to twelve aromatic and olifinic carbons. Moreover, the mass spectrum of 4a revealed a molecular ion peak at m/z = 335 which is consistent with its expected molecular weight.The required starting compounds, namely 1-morpholino-1-(2-phenylhydrazono)propan-2-one (d method  34]. Th. Th2a) aod yield . The ass4 were further evidenced via alternative method. Thus, condensation of 2-oxo-N-phenylpropanehydrazonoyl chloride (1) with benzaldehyde 3a in glacial acetic acid in the presence of catalytic amount of concentrated H2SO4, afforded 2-oxo-N,4-diphenylbut-3-enehydrazonoyl chloride (5). Refluxing an equimolar amounts of 5 and morpholine in ethanol for 3 h, gave a product identical in all respects  with compound 4a obtained from the 2a + 3a reaction -one derivatives bearing morpholine moieties. Thus, reaction of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6) and the appropriate chalcones 4a–e in ethanol under reflux for 8–12 h led to the formation of one isolable product as evidenced by TLC analysis of the crude product , in addition to the expected signals due to the morpholine and aryl protons. Also, the 13C-NMR spectra showed the expected number of aliphatic and aromatic signals. The mass spectra of the products 7a–e revealed in each case a molecular ion peak m/z at the expected molecular weight calculated for each compound .The morpholinylchalcone derivatives  product . The str7a–e, a comparison with authentic material prepared from the reaction between thione 8  with morpholine was achieved, the product obtained from this reaction was identical to that from the reaction of 4a and 6  in dioxane in the presence of triethylamine to give 1,5-dihydropyridotriazolopyrimidine derivatives 10a–d, respectively .On the other hand, compound ectively . Spectro10a–d was executed whereby compound 4d was refluxed with 3-acetyl-7-amino-1-phenyl-triazolopyrimidin-5(1H)-one (11) in ethanol afforded the respective authentic sample 10a  and a human hepatocellular carcinoma cell line (HepG-2), in comparison with a well-known anticancer standard drug (cisplatin) under the same conditions using a colorimetric MTT assay. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined. The results are depicted in 8 > 4e > 7b > 7c > 4f > 7a > 5 > 10d > 10c > 4a > 4c > 10a > 10b. The descending order of activity of the newly synthesized compounds towards the human hepatocellular carcinoma cell line (HepG-2) were as follows: 7b > 7c > 10d > 4f > 5 > 7a > 10c > 4e > 10b > 4c > 4a > 10a > 8.The in vitro growth inhibitory activity of the newly synthesized compounds The results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner.The activities of the synthesized compounds depend on the structural skeleton and electronic environment of the molecules.8, 4e and 7b were the most active  against the lung carcinoma cell line (A-549), compared with cisplatin reference drug with IC50 value of 0.95 ± 0.90 μg/mL (50 = 6.79 ± 1.11 − 26.8 ± 0.75 µg/mL).Compounds 90 μg/mL , while t7b, 7c, 10d and 4f were the most active  against the human hepatocellular carcinoma cell line (HepG-2), compared with the reference drug cisplatin with an IC50 value of 1.40 ± 1.1μg/mL (50 = 12.4 ± 0.98 − 29.9 ± 0.93 µg/mL).Compounds 1.1μg/mL . The oth4e is the most active one against the A549 (IC50 = 5.37 ± 0.95 μg/mL) line, while the methylchlorochalcone 4f is the most active one against the HepG-2 cell line (A549) (IC50 = 9.78 ± 0.78 μg/mL).Among the morpholinylchalcone derivatives, the dimethylchalcone d]triazolopyrimidin-5(1H)-ones 10a–d: Compounds 10c and 10d (substituted with COOEt group at position 3) have more in vitro inhibitory activity than compounds 10a and 10b (substituted with a COCH3 group at position 3). Also compound 10d is more active than 10c where the p-substitution with a methyl group increases the activity via its +I effect.For pyridopyrimidin-4(1H)-one (7a), Yellow solid, (70% yield), m.p. 150–152 °C; IR (KBr) υmax 1613 (C=N), 1679 (C=O), 2919, 2983, 3030 (C–H), 3125, 3243, 3447 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 2.30–2.42 , 3.01–308 , 7.01–7.94 , 9.67, 10.65, 11.17 ; 13C-NMR (DMSO-d6): δ 25.80, 78.62 (CH2), 112.96, 115.30, 119.41, 123.23, 123.27, 125.07, 128.83, 129.74, 131.13, 132.11, 132.96, 138.02, 142.96, 148.36, 154.78 (Ar–C and C=N), 162.08 (C=O), 175.01 (C=S); MS m/z (%) 458 , 313 (5), 236 (6), 192 (13), 152 (4), 129 (9), 121 (10), 107 (13), 98 (20), 97 (33), 81 (37), 71 (50), 57 (79), 43 (100). Anal. Calcd. for C24H22N6O2S (458.54): C, 62.87; H, 4.84; N, 18.33. Found: C, 63.17; H, 4.71; N, 18.16%.7-(Morpholino(2-phenylhydrazono)methyl)-2-thioxo-5-(p-tolyl)-2,3-dihydropyridopyrimidin-4(1H)-one (7b), Yellow solid, (70% yield), m.p. 158–160 °C; IR (KBr) υmax 1608 (C=N), 1679 (C=O), 2926, 3057 (C–H), 3175, 3247, 3425 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 2.24 , 2.30 , 3.31 , 7.12–7.33 , 10.57, 11.48, 11.58 ; MS m/z (%) 472 , 368 (9), 313 (7), 237 (7), 178 (6), 143 (54), 115 (16), 98 (22), 83 (24), 69 (44), 43 (100). Anal. Calcd. for C25H24N6O2S (472.57): C, 63.54; H, 5.12; N, 17.78. Found: C, 63.68; H, 4.05; N, 17.59%.5-(4-Chlorophenyl)-7-(morpholino(2-phenylhydrazono)methyl)-2-thioxo-2,3-dihydropyrido pyrimidin-4(1H)-one (7c), Yellow solid, (70% yield), m.p. 149–151 °C; IR (KBr) υmax 1608 (C=N), 1695 (C=O), 2954, 3038 (C–H), 3172, 3397, 3412 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 2.36 , 3.38 , 6.99–7.44 , 10.64, 11.49, 11.59 ; MS m/z (%) 494 , 492 (39), 390 (38), 202 (39), 161 (23), 127 (100), 84 (80), 77 (82). Anal. Calcd. for C24H21ClN6O2 (492.98): C, 58.47; H, 4.29; N, 17.05. Found: C, 58.78; H, 4.01; N, 16.70%.7-(Morpholino(2-(p-tolyl)hydrazono)methyl)-5-phenyl-2-thioxo-2,3-dihydropyridopyrimidin-4(1H)-one (7d), Yellow solid, (70% yield), m.p. 183–185 °C; IR (KBr) υmax 1600 (C=N), 1637 (C=O), 2950, 3058 (C–H), 3246, 3330, 3426 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 2.23 , 2.47 , 3.35 , 6.95–7.42 , 10.64, 11.49, 11.59 ; MS m/z (%) 472 , 368 (9), 313 (7), 237 (7), 178 (6), 143 (54), 115 (16), 98 (22), 83 (24), 69 (44), 55 (45), 43 (100). Anal. Calcd. for C25H24N6O2S (472.56): C, 63.54; H, 5.12; N, 17.78. Found: C, 63.78; H, 4.05; N, 17.19%.5-(4-Chlorophenyl)-7-(morpholino(2-(p-tolyl)hydrazono)methyl)-2-thioxo-2,3-dihydropyrido-pyrimidin-4(1H)-one (7e), Yellow solid, (70% yield), m.p. 160–162 °C; IR (KBr) υmax 1601 (C=N), 1676 (C=O), 2972, 3031 (C–H), 3243, 3326, 3425 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 2.24 , 2.46 , 3.32 , 7.12–7.33 , 10.57, 11.49, 11.59 ; 13C-NMR (DMSO-d6): δ 20.80 (CH3), 25.76, 78.62 (CH2), 111.93, 115.30, 122.76, 125.37, 130.15, 132.25, 135.47, 140.67, 142.01, 145.96, 147.13, 148.22, 152.84, 154.77 (Ar–C and C=N), 162.07 (C=O), 175.02 (C=S); MS m/z (%) 509 , 507 , 409 (11), 304 (38), 272 (24), 240 (42), 208 (20), 195 (17), 174 (39), 163 (23), 131 (28), 79 (100). Anal. Calcd. for C25H23ClN6O2S (507.01): C, 59.22; H, 4.57; N, 16.58. Found: C, 59.25; H, 4.05; N, 16.21%.8  and morpholine  in ethanol (15 mL) was refluxed for 3 h, gave product identical in all respects  with compound 7a which obtained from reaction of 4a + 6.Equimolar amounts of Synthesis of 4-oxo-N,5-diphenyl-2-thioxo-1,2,3,4-tetrahydropyridopyrimidine-7-carbohydrazonoyl chloride (8), A mixture of 5  and 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6)  in ethanol (20 mL) was refluxed for 6 h (monitored by TLC). The formed precipitate after cooling was isolated by filtration, washed with ethanol, dried and recrystallized from ethanol to give product 8 as yellow solid, Yellow solid, (70% yield), m.p. 203–205 °C; IR (KBr) υmax 1588 (C=N), 1641 (C=O), 2973, 3097, 3199 (C–H), 3242, 3316, 3424 (3NH) cm−1; 1H-NMR (DMSO-d6) δ 6.35–7.43 , 10.64, 11.49, 11.59 ; MS m/z (%) 409 , 407 , 392 (25), 370 (10), 344 (26), 332 (15), 310 (100), 283 (33), 268 (74), 108 (10), 92 (39), 77 (24), 56 (16). Anal. Calcd. for C20H14ClN5OS (407.88): C, 58.90; H, 3.46; N, 17.17. Found: C, 59.24; H, 3.15; N, 16.89%.7d  and the appropriate hydrazonoyl halides 1a,b  (1 mmol of each) in dioxane (20 mL) was added triethylamine . The reaction mixture was refluxed till all of the starting materials have disappeared and hydrogen sulfide gas ceased to evolve (6–12 h monitored by TLC). The solvent was evaporated and the residue was treated with methanol. The solid that formed was filtered off and crystallized from the appropriate solvent to give compounds 10a–d.To a mixture of equimolar amounts of thione 3-Acetyl-8-(morpholino(2-(p-tolyl)hydrazono)methyl)-1,6-diphenylpyridotriazolo-pyrimidin-5(1H)-one (10a), Browen solid, (70% yield), m.p. 230–232 °C; IR (KBr) υmax 1594 (C=N), 1654, 1675 (2C=O), 2933, 3042 (C–H), 3429 (NH) cm−1; 1H-NMR (DMSO-d6) δ 2.24 , 2.42 , 2.61 , 3.54 , 6.59–7.93 , 11.22 ; 13C-NMR (DMSO-d6): δ 20.51, 25.58 (CH3), 26.03, 66.80 (CH2), 113.17, 114.84, 115.36, 120.58, 122.46, 124.03, 125.64, 125.77, 129.43, 129.61, 129.81, 129.91, 130.01, 130.30, 130.33, 130.46, 130.78, 132.16, 134.83, 140.43 (Ar–C and C=N), 168.19, 196.22 (2C=O); MS m/z (%) 598 , 353 (12), 247 (20), 196 (15), 121 (15), 108 (27), 105 (24), 92 (49), 77 (47), 69 (21), 65 (25), 43 (100). Anal. Calcd. for C34H30N8O3 (598): C, 68.21; H, 5.05; N, 18.72. Found: C, 68.55; H, 4.85; N, 18.41%.3-Acetyl-8-(morpholino(2-(p-tolyl)hydrazono)methyl)-6-phenyl-1-(p-tolyl)pyridotriazolopyrimidin-5(1H)-one (10b), Brown solid, (70% yield), m.p. 238–240 °C; IR (KBr) υmax 1596 (C=N), 1653, 1682 (2C=O), 2970, 3027 (C–H), 3434 (NH) cm−1; 1H-NMR (DMSO-d6) δ 2.24 , 2.37 , 2.40 , 2.63 , 3.41 , 7.03–7.76 , 11.17 ; MS m/z (%) 612 , 519 (4), 494 (7), 460 (13), 443 (63), 429 (34), 400 (30), 384 (32), 337 (12), 315 (18), 291 (16), 216 (30), 201 (62), 187 (53), 126 (43), 68 (100). Anal. Calcd. for C35H32N8O3 (612.68): C, 68.61; H, 5.26; N, 18.29. Found: C, 68.93; H, 5.01; N, 17.89%.Ethyl 8-(morpholino(2-(p-tolyl)hydrazono)methyl)-5-oxo-1,6-diphenyl-1,5-dihydropyridotriazolopyrimidine-3-carboxylate (10c), Brown solid, (70% yield), m.p. 244–245 °C; IR (KBr) υmax 1597 (C=N), 1658, 1712 (2C=O), 2979, 3061 (C–H), 3427 (NH) cm−1; 1H-NMR (DMSO-d6) δ 1.26 , 2.31–2.43 , 2.59 , 3.54–360 , 4.24 , 6.97–7.97 , 10.52 ; 13C-NMR (DMSO-d6): δ 14.56, 25.61 (2CH3), 27.43, 62.62, 69.48 (3CH2), 115.01, 115.05, 115.36, 120.40, 122.65, 122.87, 126.44, 129.11, 129.37, 129.41, 129.68, 129.75, 129.78, 129.85, 129.94, 130.01, 130.36, 130.41, 138.74, 143.12 (Ar–C and C=N), 162.52, 174.52 (2C=O); MS m/z (%) 628 , 511 (18), 472 (9), 397 (6), 209 (14), 161 (23), 127 (10), 119 (26), 104 (88), 84 (100), 77 (69). Anal. Calcd. for C35H32N8O4 (628.68): C, 66.87; H, 5.13; N, 17.82. Found: C, 67.21; H, 4.80; N, 17.55%.Ethyl 8-(morpholino(2-(p-tolyl)hydrazono)methyl)-5-oxo-6-phenyl-1-(p-tolyl)-1,5-dihydropyrido triazolopyrimidine-3-carboxylate (10d), Brown solid, (70% yield), m.p. 256–258 °C; IR (KBr) υmax 1591 (C=N)1663,1717 (2C=O), 2979, 3028 (C–H), 3436 (NH) cm−1; 1H-NMR (DMSO-d6) δ 1.21 , 2.24 , 2.37 , 2.48 , 4.25 , 3.40 , 6.95–7.98 , 11.18 ; MS m/z (%) 642 , 602 (21), 511 (18), 472 (9), 397 (6), 209 (14), 161 (23), 127 (10), 119 (26), 104 (88), 84 (100), 77 (69). Anal. Calcd. for C36H34N8O4 (642.71): C, 67.28; H, 5.33; N, 17.43. Found: C, 67.59; H, 5.02; N, 17.09%.11  and chalcone 4d  in ethanol (15 mL) was refluxed for 6 h, gave product identical in all respects  with compound 10a which obtained from reaction of 7d + 1a.A mixture of acetyltriazolopyrimidine The cytotoxic evaluation of the synthesized compounds was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt according to the reported method ,37.Docking studies were performed using the MOE 2014.09 software. Regularization and optimization for protein and ligand were performed. Each docked compound was assigned a score according to its fit in the ligand binding pocket (LBP) and its binding mode ,39,40.d]triazolopyrimidin-5(1H)-ones. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their synthesis by alternative methods. The in vitro growth inhibitory activity of the synthesized compounds against hepatocellular carcinoma (HepG-2) and human lung cancer (A-549) cell lines were investigated in comparison with cisplatin as reference drug using MTT assays and the results revealed promising activities of four compounds. Moreover, computational studies using MOE 2014.09 software supported the biological activity results.In our present work, we present an efficient synthesis of novel morpholinylchalcones, which have not been hitherto reported. These chalcones were used as building blocks for constructing a series of pyridopyrimidinethiones and pyrido[2,3-


Their structures were determined on the basis of extensive spectroscopic and spectrometric data . Compounds 2–4; 6; 7; 10; and 11 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophage cells with IC50 values in the range of 25.5–98.4 μM .Four new lignan glycosides; urenalignosides A–D ( Urena lobata, belonging to the family Malvaceae, is an annually shrubby herbage widely distributed around the world, particularly in the tropical and subtropical areas of Asia, South America, and Africa  − ion at m/z 639.2282  in the HRESIMS spectrum (13C-NMR data (1 showed the absorption bands contributing to hydroxy group (3385 cm−1), benzene ring (1615 and 1518 cm−1), and ester carbonyl (1735 cm−1) group. The NMR spectra of 1 , 6.80 . δC 134.1 (C-1), 105.2 , 149.4 , 136.3 (C-4); 133.6 (C-1′), 104.8 , 149.3 , 136.4 (C-4′)], two oxygenated methines , two sp3 methines , two oxygenated methylenes , and four methoxyl groups . Comparison of the above NMR data with those of icariol A2  and a glucopyranosyl moiety were also observed in the NMR spectra of 1. The anomeric proton was presented at δH 4.36 , corresponding to the carbon at δC 104.6 assigned by HSQC experiment, and the relatively large coupling constant (J = 8.0 Hz) of the anomeric proton suggested that the glucopyranosyl moiety was in β configuration. Given that naturally occurring glucose is D-form, and limited by the small amount of 1, we tentatively determined the glucopranosyl moiet in 1 was in D-form. In the HMBC spectra of 1, the correlations between the anomeric proton δH 4.36  and C-9 (δC 69.3) confirmed that the glucopyranosyl moiety was linked at C-9 (δC 172.8). All the protons and carbons were unambiguously assigned  in the HRESIMS spectrum , 6.83 , 6.96 . δC 130.8 (C-1), 113.3 (C-2), 148.7 (C-3), 147.1 (C-4), 115.5 (C-5), 122.1 (C-6)] and a 1,3,4,5-tetrasubstituted benzene moieties , two oxygen-bearing methines , two oxygen-bearing methylenes , two methylenes , and three methoxy groups . Comparison of the above-mentioned NMR data with those of 1-(4′-hydroxy-3′-methoxy-phenyl)-2- propane-1,3-diol, a lignan previously isolated from Bursera tonkinensis  of the glucopyranosyl moiety and the C-3’ of the aglycon demonstrated that the glucopyranosyl moiety was linked at C-3’ in 3 (J = 8.5 Hz) between H-7 and H-8 suggested that the C-7 and C-8 were in threo orientation. The negative Cotton effect at 233 nm in the CD spectrum  in the HRESIMS spectrum , suggesting that the O-glucopyranosyl moiety was linked at C-9′ in 4, but not like that at C-7 in 2. The deduction was confirmed by HMBC correlation between the anomeric proton [δH 4.32 ] and C-9′ (J = 7.0 Hz) between H-7 and H-8 suggested that the C-7 and C-8 were in threo orientation. The absolute configuration of C-8 was assigned as S based on the positive Cotton effect at 233 nm presented in the CD spectrum -threo-4,9,9’-trihydroxy-3,3’,5’-trimethoxy-8-O-4’-neolignan-7-O-β-d-glucopyranoside (5) [6) [R,8R)-threo-guaiacylglycerol-8-O-4’-sinapyl ether-7-O-β-d-glucopyranoside (7) [S,8R)-erythro-4,9,9’-trihydroxy-3,3’-dimethoxy-8-O-4’-neolignan-7-O-β-d-glucopyranoside (8) [S,8S)-threo-4,9,9’-trihydroxy-3,3’-dimethoxy-8-O-4’-neolignan-7-O-β-d-glucopyranoside (9) [R,8S)-4,7,9,3′,9′-pentahydroxy-3-methoxy-8-O-4′-neolignan-9′-O-β-d-glucopyranoside (10) [S,8S)-4,7,9,3’,9’-pentahydroxy-3-methoxyl-8-O-4’-neolignan-4-O-β-d-glucopyranoside (11) [S,7’S,8R,8’R)-icariol A2-9-O-β-d-glucopyranoside (12) [S,7’S,8S,8’S)-icariol A2-4-O-β-d-glucopyranoside (13) [O-β-d-glucopyranoside (14) [O-β-d-glucopyranoside (15) [O-β-d-glucopy ranoside (16) [2–11 and 16 are neolignans which are classified as a subgroup of lignan family [By comparison of their spectroscopic and specific rotation data with those of the known compounds, the remaining 11 compounds were identified as (7side (5) , rourino (5) [6) , side (7) , side (8) , side (9) , (–)-(7Ride (10) , ide (11) ,  ,  , lyonireide (14) , (−)-isoide (15) , and cedide (16) , respectn family .1H-NMR and 125 MHz for 13C-NMR. HRESIMS was recorded on an LCMS-IT-TOF system, fitted with a Prominence UFLC system and an ESI interface . Silica gel , LiChroprep RP-C18 gel , D101 m acroporous adsorption resin  and Sephadex LH-20  were used for open column chromatography (CC). HPLC was performed on a ShimadzuLC-20AT pump system , equipped with an SPD-M20A photodiode array detector monitoring at 254 nm. A semi-preparative HPLC column  was employed for the isolation. TLC was performed using GF254 plates .Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter . IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer  with KBr pellets. UV spectra were obtained using a Shimadzu UV-2450 spectrophotometer . NMR spectra were recorded on a Varian INOVA-500 spectrometer  operating at 500 MHz for Urena lobata L. was collected in Guangxi Province, People’s Republic of China, in September 2013. The plant material was authenticated by one of the authors (P.F. Tu) and a voucher specimen (DTH2013029) was deposited at the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China.U. lobata (13.6 kg) were refluxed with 95% EtOH for three times . After removing the solvent under reduced pressure, the residue (1.35 kg) was suspended in water (6 L), and partitioned with petroleum ether (3 × 6 L), EtOAc (5 × 6 L), and n-BuOH (3 × 6 L), successively. The n-BuOH-soluble fraction (158 g) was subjected to D101 macroporous adsorption resin column and eluted with H2O–EtOH  to yield five fractions (Fr. 1-5). Fr. 2 (20 g) and Fr. 3 (40 g) were combined and subjected to silica gel chromatography and eluted with a stepwise gradient of EtOAc-MeOH-H2O from 30:2:1 to 5:2:1 to give five subfractions (Subfr. A–E). Subfr. B (8 g) was chromatographed on a Sephadex LH-20 column and eluted with MeOH to give six subfractions (Subfr. B1–B6). Subfr. B3 (1 g) was chromatographed on a silica gel column and eluted with gradient of CH2Cl2–MeOH  to give seven subfractions (Subfr. B3a–B3g). Subfr. B3d (0.2 g) was purified by semipreparative HPLC using 27% aqueous MeCN as the mobile phase to afford compound 7 . Subfr. B3g (0.1 g) was applied to semi-preparative HPLC using 25% aqueous MeCN to obtain two compounds 8  and 9 . Subfr. B4 (4 g) was subjected to RP-C18 open column and eluted with a stepwise gradient of MeOH–H2O , to afford five fractions (Subfr. B4a–Subfr. B4e). Subfr. B4a (1.2 g) was applied to semi-preparative HPLC using 25% aqueous MeCN to give compound 1 . Subfr. B4c (1.1 g) was further separated by ODS column chromatography and eluted with MeOH–H2O (1:19→1:3) to obtain six fractions (Subfr. B4c1–B4c6). Subfr. B4c4 was repeatedly separated and purified by semi-preparative HPLC (27% aqueous MeCN) to give two fractions Subfr. B4c4-1 , Subfr. B4c4-2 , and five compounds 3 , 4 , 5 , 12 , and 13 . Subfr. B4c4-2 was purified by semi-preparative HPLC (30% aqueous MeOH) to give compounds 10  and 11 . Subfr. B4c5 was applied to semi-preparative HPLC using 10% aqueous MeOH to give compounds 2 , 6 , 14 , 15 , and 16 .The air-dried Urenalignoside A (1): Colorless powder, c 0.1, MeOH); UV λ (log ε): 208 (4.49), 317 (4.31), 383 (3.91) nm; IR (KBr) νmax: 3385, 2921, 1735, 1615, 1518, 1462, 1428, 1367, 1331, 1217, 1114, 1076, 1036 cm−1; 1H and 13C-NMR data .data see ; negativUrenalignoside B (2): Colorless powder, c 0.1, MeOH); UV λ (log ε): 202 (4.14),226 (4.25), 277 (3.37), 298 (2.63), 317 (2.48), 329 (2.40), 341 (2.43), 348 (2.38) nm; IR (KBr) νmax: 3423, 2926, 1630, 1384, 1253, 1119, 1076, 1037 cm−1; 1H and 13C-NMR data .data see ; negativUrenalignoside C (3): Colorless powder, c 0.1, MeOH); UV λ (log ε): 212 (4.58), 285 (4.00) nm; IR (KBr) νmax: 3389, 2968, 2923, 2852, 1739, 1610, 1456, 1431, 1366, 1259, 1228, 1216, 1174, 1111, 1028 cm−1; 1H and 13C-NMR data .data see ; negativUrenalignoside D (4): Colorless powder, c 0.1, MeOH); UV λ (log ε): 207 (4.62), 263 (4.70), 316 (4.23) nm; IR (KBr) νmax: 3739, 3716, 3660, 3430, 2956, 2924, 2853, 1717, 1592, 1514, 1488, 1455, 1428, 1383, 1367, 1230, 1157, 1125, 1023 cm−1; 1H and 13C-NMR data .data see ; negativ2 at 37 °C. Cell viability was evaluated using MTT assay. The NO concentration was detected by the Griess method. Briefly, RAW264.7 macrophage cells were seeded into 96-well plates at a density of 5 × 104 cells/well and stimulated with 0.5 μg/mL LPS  in the presence or absence of test compounds. After incubation for 24 h at 37 °C, treated RAW264.7 macrophage cells were incubated with 100 μL MTT solution (0.5 mg/mL in medium) for another 4 h at 37 °C, subsequently, the supernatants were removed and residues were dissolved using 150 μL DMSO for each well; 50 μL of cell-free supernatant was mixed with 100 μL of Griess reagent containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.2% (w/v) N-(1-naphthyl) ethylenediamine solution to measure nitrite production. The absorbance was detected at 540 nm using a microplate reader . Compared with a calibration curve prepared using NaNO2 standards. The experiments were performed in triplicate. quercetin was conducted as a positive control. All the compounds were prepared as stock solutions in DMSO .The murine macrophage RAW264.7 cell line was purchased from Peking Union Medical College (PUMC) Cell bank , and was cultured in DMEM supplemented with 10% Fetal Bovine Serum, 100U/mL penicillin G and 100 μg/mL streptomycin, in a humidified 5% CO1–16 were evaluated for their inhibitory effects on the NO production in LPS-stimulated RAW 264.7 macrophage cells. Quercetin was used as a positive control (IC50 = 7.2 ± 0.2 μM). Compounds 2–4, 6, 7, 10, and 11 exhibited weak inhibitory activity against NO production with IC50 values of 90.4 ± 3.2 μM, 74.3 ± 1.8 μM, 88.1 ± 2.2 μM, 98.4 ± 3.6 μM, 97.5 ± 2.6 μM, 97.7 ± 3.5 μM, 25.5 ± 1.2 μM, respectively.Compounds


O-β-d-glucopyranosyl-(1′′→6′)-β-d-glucopyranosyl ester (1), akebonoic acid 28-O-(6′′-O-caffeoyl)-β-d-glucopyranosyl-(1′′→6′)-β-d-glucopyranosyl ester  and 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 3-O-(6′-O-caffeoyl)-β-d-glucopyranoside  were isolated from the stems of Holboellia coriacea Diels, together with five known compounds, eupteleasaponin VIII (4), 3α-akebonoic acid (5), quinatic acid (6), 3β-hydroxy-30-norhederagenin (7) and quinatoside A (8). The structures of these compounds were determined on the basis of spectral and chemical evidence. Compounds 1–5 were evaluated for their inhibitory activity against three human tumors HepG2, HCT116 and SGC-7901 cell lines in vitro.Three new 30-noroleanane triterpenoid saponins, akebonoic acid 28- Holboellia coriacea Diels belonging to family Lardizabalaceae, is an evergreen woody vines mainly distributed in Qinling Mountain region of China at altitudes of 500–2000 m + at m/z 787.4248, which, taken together with the 13C-NMR data analysis, indicated the molecular formula C41H64O13. The 13C-NMR spectrum displayed 41 signals, of which 12 carbons were assignable to sugar moieties and 29 carbons to the aglycone moiety ; 1.02 ; 1.12 ; 1.20 ; 1.22 ), a broad singlet olefinic proton at δH 5.45, and two exo-methylene protons at δH 4.65 (s) and 4.71 (s). These NMR data suggested that the structure of 1 should be a noroleanane triterpenoid saponin + and 961 [M + Cl]− by ESI-MS. The molecule formula of C50H70O16 was confirmed by HR-ESIMS. By comparison of the 1H and 13C-NMR data  and sugar moiety as those in 1, but an additional trans-caffeoyl moiety which was proposed according to the presence of an aromatic ABX system , together with two coupled doublets (J = 15.8 Hz) at δH 6.61 and 7.91 and the signal of an ester carbon C=O at δC 167.1 +; negative ESIMS m/z: 763 [M − H]−, 799 [M + Cl]−; HR-ESIMS m/z: 787.4248 [M + Na]+ .Compound 2: light yellow powder, c = 2.3, MeOH); UV (MeOH) λ max nm (log ε) 345 (4.00), 242 (2.32), 129 (2.94); IR (KBr) υmax 3435, 3420, 3414, 2933, 2854, 1745, 1689, 1620, 1451, 1435, 1380, 1045 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data, see m/z: 949 [M + Na]+; negative ESIMS m/z: 961 [M + Cl]−; HR-ESIMS m/z: 961.4378 [M + Cl]− .Compound 3: light yellow powder, c = 2.0, MeOH); UV (MeOH) λ max nm (log ε) 329 (2.59), 244 (1.78), 217 (2.41); IR (KBr) υmax 3435, 3069, 1719, 1648, 1621, 1298, 1073 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data, see m/z: 821 [M + Na]+; negative ESIMS m/z: 797 [M − H]−, 833 [M + Cl]−; HR-ESIMS m/z: 797.4109 [M − H]− .Compound 1 and 2: A solution of saponins  in 5% KOH (3 mL) was stirred at 85 °C for 4 h. The reaction mixture was acidified to pH 4.0 with 10% HCl and extracted with CHCl3 (3 × 10 mL). The CHCl3 layer was dried (anhydr. MgSO4) and concentrated under reduced pressure. The residue was subjected to Sephadex LH-20 CC using MeOH to afford akebonoic acid (1a) for the aglycone . 1a was identified on the basis of 1H and 13C-NMR spectrum comparisons with that of published data for akebonoic acid [2SO4 (2 drops) was heated in a boiling H2O bath for 1 h. The solution was extracted with n-BuOH (3 × 20 mL). The n-BuOH solution was concentrated in vacuo and desalted  to afford the sugar residue (1.0 mg). The sugar residue was derivatized with Sigma Sil-A for 35 min at 70 °C and analyzed by GC-MS with only one peak detected  [d-glucose.Alkaline and Acid Hydrolysis of oic acid . The aqu M+ 540) , which w3: Compounds 3 (8.0 mg) in 1 N HCl  was heated under reflux for 8 h. After removal of the solvent, the residue was partitioned between CHCl3 and H2O. The CHCl3-soluble portion was evaporated and subjected to ODS column chromatography using 90% MeOH to yield 4 mg of the aglycone (3a) which was identified as 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid [1 and 2, the sugar unit of 3 was identified as d-glucose.Alkaline and Acid Hydrolysis of oic acid . By the 1a: 1H-NMR  δ 5.51 , 4.80/4.76 , 3.25 , 1.25 , 1.03 , 1.01 , 0.88 ; 13C-NMR  δ 39.3 (C-1), 28.4 (C-2), 78.4 (C-3), 39.7 (C-4), 56.1 (C-5), 19.1 (C-6), 33.6 (C-7), 40.1 (C-8), 48.4(C-9), 37.7 (C-10), 24.2 (C-11), 123.3 (C-12), 144.5 (C-13), 42.4 (C-14), 28.6 (C-15), 24.1 (C-16), 47.4 (C-17), 48.3 (C-18), 42.4 (C-19), 149.5 (C-20), 38.7 (C-21), 30.8 (C-22), 29.1 (C-23), 16.9 (C-24), 15.9 (C-25), 17.7 (C-26), 26.5 (C-27), 179.9 (C-28), 107.4 (C-29).Compound 3a: 1H-NMR  δ 5.57 , 4.50 , 3.65/3.64, 3.38 , 1.60 , 1.56 ,1.26 , 0.99 , 0.86 ; 13C-NMR  δ 38.7 (C-1), 28.3 (C-2), 80.2 (C-3), 43.2 (C-4), 56.4 (C-5), 19.1 (C-6), 33.6 (C-7), 39.8 (C-8), 48.1 (C-9), 37.1 (C-10), 23.9 (C-11), 122.6 (C-12), 144.4 (C-13), 42.1 (C-14), 28.4 (C-15), 28.4 (C-16), 46.8 (C-17), 44.4 (C-18), 48.2 (C-19), 69.9 (C-20), 36.3 (C-21), 35.2 (C-22), 23.6 (C-23), 64.5 (C-24), 16.0 (C-25), 17.0 (C-26), 26.0 (C-27), 180.7 (C-28), 25.7 (C-29).Compound 1–5 against three human cancer cell lines, human gastric carcinoma (SGC-7901), human colon carcinoma (HCT 116) and human liver hepatocellular carcinoma (HepG2) were assayed at the National Center for Drug Screening, Shanghai, China. Sulforhodamine B (SRB)  was used to test the effects of the compounds on cell growth and viability [50 values.The cytotoxicity of compounds iability . AdriamyO-β-d-glucopyranosyl-(1′′→6′)-β-d-glucopyranosyl ester (1), Holboelliside A (2) and Holboelliside B (3) isolated from the stem of Holboellia coriacea, along with five known compounds. It is noteworthy that the two caffeoylation of nortriterpenoidal saponins (2 and 3) were isolated from family Lardizabalaceae for the first time. 3α-akebonoic acid (5) showed interesting cytotoxicity against HCT-116, HepG2 and SGC-7901 cell lines, with IC50 values of 9.1, 15.2 and 41.0 μM, respectively. In addition, the saccharide unit attached at C-28 of this type of nortriterpenoids may greatly reduce the in vitro cytotoxicity.The present work reported three new nortriterpenoid saponins, akebonoic acid 28-


Betula alnoides is a medicinal plant in Thai traditional longevity preparations. The crude extracts of this plant possess various biological activities. However, the isolated compounds from this plant have no reports of anti-HIV-1 integrase (IN) activity. Therefore, the present study aims to investigate the anti-HIV-1 integrase and anti-inflammatory effects of isolated compounds from this plant and predict the interaction of compounds with integrase active sites. From the bioassay-guided fractionation of the ethanol extract of B. alnoides stems using chromatographic techniques, five pentacyclic triterpenoid compounds were obtained. They are betulinic acid (1), betulin (2), lupeol (3), oleanolic acid (4), and ursolic acid (5). Compound 2 exhibited the most potent inhibitory activity against HIV-1 IN, with an IC50 value of 17.7 μM. Potential interactions of compounds with IN active sites were investigated using computational docking. The results indicated that active compounds interacted with Asp64, a residue participating in 3′-processing, and Thr66, His67, and Lys159, residues participating in strand-transfer reactions of the integration process. Regarding anti-inflammatory activity, all compounds exerted significant inhibitory effects on LPS-induced nitric oxide production (IC50 < 68.7 μM). Thus, this research provides additional scientific support for the use of B. alnoides in traditional medicine for the treatment of HIV patients. The catalytic core domain, residues 52–210, contains the catalytic triad characterized by Asp64, Asp116, and Glu152. The C-terminal domain, residues 220–288, contributes to DNA binding [Human immunodeficiency virus (HIV) infection remains a major global public health crisis. In 2017, there were approximately 36.9 million people living with HIV, with 1.8 million people becoming newly infected and 940,000 people died from HIV-related causes globally . The fulBetula alnoides (Betulaceae family) is locally known in Thai as “Khamlang suea khrong.” The stem bark of this plant has traditionally been used for tonic, longevity, and appetite and as a carminative and an aphrodisiac. Methanol and ethanol extracts of this plant possess various biological activities, such as anti-inflammatory [α-glucosidase inhibitory activities [Betula alnoides wood possessed high inhibitory activity against HIV-1 IN with an IC50 of 10.2 and 20.1 μg/mL [ammatory , anti-hytivities , and anttivities . Our pre.1 μg/mL . It is iB. alnoides stems were collected from Chonburi Province, Thailand, in 2015 and were identified by a traditional Thai doctor, Mr. Sarupsin Thongnoppakhun. The voucher specimen (SKP024020101) was deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand.3 on a Varian Unity Inova at 500 MHz for 1H and 125 MHz for 13C . Column chromatography was performed using silica gel , Sephadex LH-20, and Diaion HP-20 . All solvents were analytical reagent grade and purchased from Labscan, Thailand. All reagents were purchased from Sigma, USA.The NMR spectra were recorded in CDClB. alnoides stems (800 g) was extracted three times with 95% ethanol under reflux for 3 h. The filtrate was concentrated at 50°C under reduced pressure to obtain ethanol extract (83.9 g). This extract was subsequently partitioned with various solvents to generate residues of hexane (7.2 g), chloroform (21.5 g), ethyl acetate (15.3 g), water (25.4 g), and water and chloroform emulsion (10.3 g) fractions. These fractions were prepared at concentrations 3–100 μg/mL for screening of their anti-HIV-1 IN activity.The dried coarse powder of 50 values of 20.5 and 25.5 μg/mL, respectively  as white needle crystals.The water and chloroform fractions that exhibited good activity with ICectively , were fu2  as a white powder. Fraction C2 (3.3 g) was chromatographed by VLC using chloroform and increasing polarity with ethyl acetate and methanol as the eluent to give 5 subfractions (C2/1–C2/5). Subfraction C2/2 was rechromatographed on silica gel to afford compound 3  as a white powder. Fractions C3 (2.5 g) and C4 (3.8 g) were purified by the same procedure, successively affording compounds 4  and 5  as white powder, respectively.The chloroform fraction (12.5 g) was chromatographed by VLC using silica gel. Elution was started with hexane and chloroform and followed by ethyl acetate and methanol to give four fractions (C1–C4). Fraction C1 (4.1 g) was chromatographed over silica gel and eluted with chloroform and increasing polarity with ethyl acetate to obtain compound 1–5 were identified by 1H and 13C-NMR analysis as well as by comparison with previously reported data in the literature.The structures of compounds in vitro model using HIV-1 IN enzymes according to the multiplate integration assay (MIA) as previously described [μL) composed of 12 μL of IN buffer (containing 150 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2 (MOPS), 75 mM MnCl2, 5 mM dithiothreitol (DTT), 25% glycerol, and 500 μg/mL bovine serum albumin), 1 μL of 5 pmol/mL digoxigenin-labeled target DNA, and 32 μL of sterilized water was added into each well of a 96-well plate. Subsequently, 6 μL of sample solution in DMSO and 9 μL of a 1/5 dilution of the IN enzyme were added to each well and incubated at 37°C for 80 min. After washing the plate three times with PBS with 0.05% Tween 20 (PBST), 100 μL of 500 mU/mL alkaline phosphatase- (AP-) labeled anti-digoxigenin antibody was added and incubated at 37°C for 1 h. The plates were washed with PBS three times. Then, AP buffer (150 μL) containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2, and 10 mM p-nitrophenyl phosphate was added to each well and incubated at 37°C for 1 h. Finally, the absorbance of p-nitrophenol, the final product of the integration reaction, was measured with a microplate reader  at a wavelength of 405 nm. Suramin, a polyanionic HIV-1 IN inhibitor, was used as a positive control.The anti-HIV IN activity of isolated compounds was determined in an escribed . Briefly5 cells/well) and were maintained to adhere at 37°C for 1 h in a CO2 incubator containing 5% CO2. They were then cultured in RPMI-1640 medium containing lipopolysaccharide  together with the test compounds at various concentrations (3–100 μM). After 24 h of incubation, the nitrite (NO2–) concentration in the culture medium was determined as an indicator of NO production using the Griess reagent to assay the accumulation of NO2–, a stable metabolite of NO. The absorbance was measured using a microplate reader at 570 nm. In this study, NO synthase inhibitor , nuclear translocation of NF-κB inhibitor  and nonsteroidal anti-inflammatory drug, NSAID (indomethacin), were used as positive controls. The percent inhibition was calculated from the following equation, and inhibition concentration at 50% (IC50) values was determined graphically (n = 4):A–C are the NO2– concentration (A = LPS (+), sample (−); B = LPS (+), sample (+); C = LPS (−), sample (−)).To evaluate the anti-inflammatory activity, an inhibitory effect on nitric oxide (NO) production was carried out according to the previous report described by Sudsai et al. . Brieflyμl of MTT solution (5 mg/ml in PBS) was added to each well of 96-well plates and further incubated in a CO2 incubator for 4 h. The formazan products generated by MTT reduction were dissolved in DMSO. At last, the medium was removed, 100 μl of DMSO was then added to each well and thoroughly mixed by gently tapping on the test plate. The absorbance of formazan solution was measured at a wavelength of 570 nm using a microplate reader. The test compounds were considered to be cytotoxic when the viability of the compound-treated group was less than 80% of that in the control (1% DMSO-treated) group.The cytotoxicity of the test compounds after 24 h of incubation was determined by the colorimetric method described by Sudsai et al. . A volumMolecular docking experiments of HIV-1 IN enzyme and pure compound were performed with version 4.2 of the AutoDock program according to the procedure as previously described . Dockingp < 0.05.The results are expressed as the mean value ± S.E.M. of four determinations. Differences between groups were assessed by one-way ANOVA using the post hoc Duncan's test. The significance level was considered at 1 [2 [3 [4 [5 [From bioassay-guided fractionation based on anti-HIV-1 IN activity using the MIA method, the bioactive water and chloroform fractions were purified by chromatographic techniques to afford five known pentacyclic triterpenoid compounds . They we1 , 17; bet1 [2 ; and lup1 [2 [3 , along w[3 [4 [5 .1H-NMR (CDCl3): δ 3.18 , 2.98 , 4.56 , 4.71 , 0.91, , 0.75 , 0.83 , 0.94∗, 0.96∗ , 1.63 . ∗Interchangeable signals.13C-NMR (CDCl3): δ 38.6 (C-1), 27.3 (C-2), 78.8 (C-3), 38.6 (C-4), 55.5 (C-5), 18.3 (C-6), 34.0 (C-7), 40.4 (C-8), 50.5 (C-9), 37.7 (C-10), 20.8 (C-11), 25.5 (C-12), 38.4 (C-13), 42.4 (C-14), 30.5 (C-15), 32.1 (C-16), 56.3 (C-17), 46.9 (C-18), 49.3 (C-19), 150.4 (C-20), 39.7 (C-21), 37.0 (C-22), 28.2 (C-23), 15.3 (C-24), 15.9 (C-25), 16.1 (C-26), 14.5 (C-27), 179.7 (C-28), 109.6 (C-29), 19.4 (C-30).1H NMR (CDCl3): δ 4.62 , 4.54 , 3.78 , 3.31 , 3.16 , 1.66 , 0.96 , 0.99 , 0.95 , 0.80 , 0.74 .13C NMR (CDCl3): δ 150.5 (C20), 109.7 (C-29), 79.0 (C-3), 60.5 (C-28), 55.3 (C-5), 50.4 (C-9), 48.7 (C-18), 47.9 (C-17), 47.8 (C-19), 42.7 (C-14), 40.9 (C-8), 38.8 (C-4), 38.7 (C-1), 37.3 (C-13), 37.1 (C-10), 34.2 (C-7), 34.0 (C-22), 29.7 (C-21), 29.1 (C-16), 28.0 (C-23), 27.4 (C-2), 27.0 (C-15), 25.2 (C-12), 20.9 (C-11), 19.1 (C-30), 18.3 (C-6), 16.1 (C-25), 16.0 (C-26), 15.3 (C-24), 14.7 (C-27).1H NMR (CDCl3): δ 4.68 , 4.55 , 3.20 , 1.66 , 0.92 , 1.01 , 0.95 , 0.85 , 0.79 , 0.74 .13C NMR (CDCl3): δ 151.0 (C-20), 109.3 (C-29), 79.0 (C-3), 55.5 (C-5), 50.5 (C-9), 48.3 (C-18), 48.0 (C-19), 43.0 (C-17), 42.9 (C-14), 40.8 (C-8), 40.1 (C-22), 39.0 (C-13), 38.9 (C-4), 38.6 (C-1), 37.2 (C-10), 35.6 (C-16), 34.3 (C-7), 29.9 (C-21), 28.0 (C-23), 27.4 (C-15), 27.5 (C-12), 24.4 (C-2), 20.9 (C-11), 19.3 (C-30), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.0 (C-26), 15.6 (C-24), 14.5 (C-27).1H NMR (CDCl3): 2.82 , 2.87 , 3.23 , 5.27 , 0.80 , 1.05 , 0.95 , 0.93 , 0.91 , 0.80∗ , 0.79∗ . ∗interchangeable signals.13C NMR (CDCl3): δ 180.1 (C-28), 143.6 (C-13), 123.0 (C-12), 79.0 (C-3), 55.2 (C-5), 48.0 (C-9), 46.6 (C-19), 46.5 (C-17), 42.4 (C-18), 41.8 (C-14), 39.5 (C-8), 39.1 (C-1), 38.9 (C-4), 37.1 (C-10), 33.9 (C-21), 33.5 (C-29), 32.8 (C-7), 33.1 (C-22), 31.1 (C-20), 28.4 (C-23), 28.1 (C-2), 27.8 (C-15), 26.4 (C-27), 23.8 (C-30), 23.8 (C-11), 23.6 (C-16), 18.8 (C-6), 17.2 (C-26), 16.9 (C-24), 15.8 (C-25).1H NMR (CDCl3): δ 5.25 , 3.25 , 1.00 , 1.05 , 0.98 , 1.10 , 0.95 , 0.88 , 0.79 , 0.83 .13C NMR (CDCl3): δ 179.7 (C-28), 138.2 (C-13), 126.0 (C-12), 78.8 (C-3), 55.3 (C-5), 53.9 (C-18), 48.2 (C-17), 47.5 (C-9), 42.1 (C-14), 39.6 (C-8), 39.1 (C-19), 39.0 (C-20), 38.7 (C-1), 38.6 (C-4), 37.6 (C-22), 37.2 (C-10), 33.0 (C-7), 30.2 (C-21), 29.0 (C-23), 28.8 (C-15), 28.0 (C-2), 25.1 (C-16), 23.8 (C-11), 23.6 (C-27), 21.5 (C-30), 18.3 (C-6), 17.2 (C-26), 17.0 (C-29), 15.7 (C-24), 15.5 (C-25).Betula spp.), and exhibited a wide spectrum of biological and pharmacological activities. However, it is important to note that these compounds have not previously been investigated for anti-HIV-1 IN activity. In addition, the anti-inflammatory activity of B. alnoides has been only reported in methanol and ethanol extracts. Therefore, all identified compounds were evaluated for anti-HIV-1 IN effect as well as anti-inflammatory activity.All isolated compounds are known triterpenoids that are found in many plants, especially in birch species (2) is the most potent anti-HIV-1 IN activity with an IC50 value of 17.7 μM. Betulinic acid (1) showed good inhibition of HIV-1 IN with an IC50 value of 24.8 μM. However, oleanolic acid (4) and ursolic acid (5) showed moderate activity with IC50 values of 30.3 and 35.0 μM, respectively, whereas lupeol (3) was inactive against HIV-1 IN  possessed the best binding affinity for the IN enzyme in terms of low binding energy  and lowest inhibiting constants , indicating that it strongly interacted with IN. Betulin exhibited four hydrogen bond interactions with amino acid residues. The hydroxyl group at position C-28 interacted with Asp64, the residue of the catalytic triad, while the hydroxyl group at C-3 formed multiple hydrogen bonds with Thr66, His67, and Lys159. Betulinic acid (1) interacted with Asp64, Thr66, and Lys159. The binding energy of betulinic acid was lower than that of betulin (2). Lupeol (3), which contained a methyl group at position C-17, was an inactive compound against HIV-1 IN. It only interacted with Gln148 and had a weak binding energy . Oleanolic acid (4) and ursolic acid (5) formed two hydrogen bonds with weak interactions that can be observed in terms of binding energy.The interactions of compounds with the amino acid residues of IN are shown in Ki value (5.25 μM) and exhibited lowest binding energy. In addition, it formed six H-bonding with Leu63, Asp64, Asp116, Gln148, and Glu152. In the case of dolutegravir, it formed comparable numbers of H-bonding to elvitegravir but showed weaker interaction with the enzyme than elvitegravir in terms of high binding energy and Ki value (17.62 μM).In this study, the potential interactions of drugs as IN inhibitors  with HIVThe structure of betulin, the compound that possessed the strongest activity, has three remarkable positions, the primary hydroxyl group at position C-28, the secondary hydroxyl group at position C-3, and the alkene moiety at position C-20. In the case of betulinic acid, the structure is substituted with a carboxylic group at C-17. It possessed less activity than betulin, which was confirmed by weaker interactions with the IN active site in terms of the lower number of hydrogen bonds. With respect to lupeol, the structure was substituted with a methyl group in the same position, and lupeol had considerably decreased activity against HIV-1 IN. These results agree with previous reports that found lupeol was poorly active for antiviral activity . InteresKi show that IN inhibitors interact more strongly with HIV-1 IN than isolated compound from B. alnoides. The oxadiazole group of raltegravir is an essential function group to interact with HIV-1 IN active site. In addition, the halobenzyl groups of elvitegravir and dolutegravir display the important role for interaction.These results clearly show the structure-activity relationship that minor structural modifications at C-17 of those pentacyclic triterpenoids lead to significant differences in the inhibitory anti-HIV-I IN effect. In particular, hydroxyl groups are a potential functional group for binding to IN active sites, resulting in the inhibitory action against IN. Asp64, Thr66, and His67 are amino acid residues participating in 3′-processing, and Gln148, Asn155, and Lys159 are residues participating in strand-transfer reactions. Thus, this result underlined that the anti-HIV-1 IN activity of active compounds resulted from interference with the integration process at the IN active site. Docking studies of three IN inhibitors revealed that all inhibitors strongly interacted with amino acid residue of IN enzyme. All inhibitors are found to bind preferably in similar ways close to the catalytic residues, Asp64, Asp116, and Glu152. Their binding energy and α (TNF-α) production [α and interleukin β (ILβ) in LPS-treated macrophages [in vivo which mediated by the downregulation of the expression of NF-κB and TNF-α production [β, IL-6, and TNF-α and to increase the production of IL-10 in macrophages stimulated with LPS.Regarding the anti-inflammatory activity, our study is in accordance with previous studies in which triterpenoid compounds presented anti-inflammatory effects in various models. Betulin, the compound that possessed highest activity in this study, also exhibited an anti-inflammatory effect by the reduction of NO level in the edema paw model . The potoduction . For luprophages  as well rophages . In the oduction . UrsolicBetula pendula Roth, B. pubescens Ehrh, and B. davurica Pall [Dillenia indica which is extensively used as a food additive [Combretum laxum [Euphorbia microsciadia [Syzygium guineense Wild DC [Polypodium vulgare, the common polypody, is a fern widely distributed in Europe [Acacia mellifera [Byrsonima microphylla [Celtis philippinensis [Adenium obesum [Acacia mellifera [Chrysanthemum indicum Linne [Polypodium vulgare [Gentiana veitchiorum [Orthosiphon stamineus [Perilla frutescens var. acuta [Since pentacyclic triterpenes are secondary metabolites widespread in various plants, betulinic acid and betulin are lupane-type triterpenes which can be found in large amount in the outer bark of many species of birch, i.e., ica Pall –30. For additive  and has um laxum . Moreoveosciadia , stem ba Wild DC , and Poln Europe . Betulinellifera , Byrsonirophylla , the twipinensis , and stem obesum . Lupeol ellifera  and Chryum Linne . This co vulgare . In parttchiorum . In additamineus  and the r. acuta .Albizia procera, Areca catechu, Bauhinia strychnifolia, Betula alnoides, Blumea balsamifera, Caesalpinia sappan, Cassia garrettiana, and Stephania venosa possess good activity with IC50 values of <20 μg/ml [Coleus parvifolius have been reported to be potent anti-HIV-1 agents [Albizia procera bark exhibited appreciable activity against HIV-1 IN with an IC50 value of 46.3 μM, whereas protocatechuic acid showed mild activity with 46.0% inhibition at a concentration of 100 μM [Caesalpinia sappan L. was phytochemically investigated by Tewtrakul et al. [50 value of 2.3 μM followed by protosappanin A with an IC50 value of 12.6 μM. For the compounds isolated from the bulbils of Dioscorea bulbifera, myricetin exhibited the most potent activity with an IC50 value of 3.15 μM, followed by 2,4,6,7-tetrahydroxy 9,10-dihydrophenanthrene IC50 value of 14.20 μM [N-methyl-trans-4-hydroxy-L-proline was isolated from Aglaia andamanica leaves. It has been reported to be potent anti-HIV-1 agents with an IC50 value of 11.8 μg/mL [Boesenbergia kingii showed moderate anti-HIV-1 IN with an IC50 value of 47.7 μM [In terms of effect of medicinal plants and constituents against HIV-IN, several medicinal plants have been described as possessing anti-HIV-1 IN activity. Our previous study showed that the crude ethanolic and aqueous extracts from eight plants of Thai medicinal plants in longevity preparations; 20 μg/ml . Regardi1 agents . (+)-Catf 100 μM . The heal et al. , and the14.20 μM . The actRegarding the other biological activities of isolated compounds, betulin has been reported to possess antiviral  and antiB. alnoides, including betulinic acid (1), betulin (2), oleanolic acid (4), and ursolic acid (5). These compounds showed significant anti-HIV activity with IC50 values ranging from 17.7 to 35.0 μM and possessed anti-inflammatory effects. The active compounds against HIV-1 IN interacted with the essential amino acids participating in 3′-processing and strand-transfer reactions, resulting in interference with the integration process. This finding is the first report of the anti-HIV-1 IN activity of compounds from B. alnoides.Pentacyclic triterpenoids were isolated from the stems of


Streptomyces sp. Did-27, was investigated. Three new 2(1H)-pyrazinones derivatives, (S)-6-(sec-butyl)-3-isopropylpyrazin-2(1H)-one (1), (S)-3-(sec-butyl)-6-isopropylpyrazin-2(1H)-one (2) and (S)-6-(sec-butyl)-3-isobutylpyrazin-2(1H)-one (3), together with the known (1H)-pyrazinones analogues deoxymutaaspergillic acid (4), 3,6-diisobutyl-2(1H)-pyrazinone (5) and 3,6-di-sec-butyl-2(1H)-pyrazinone (6), and the diketopiperazine alkaloids cyclo(6-OH-d-Pro-l-Phe) (7), bacillusamide B (8), cyclo(l-Pro-l-Leu) and cyclo(l-Pro-l-Ile) (10) were isolated from this strain. The structures of the compounds were determined by study of their one- and two-dimensional NMR spectra as well as high-resolution mass spectral determinations. Compound 4 was reported previously as a synthetic product, while compound 6 was reported as 2-hydroxy-3,6-di-sec-butylpyrazine. Herein, we report the complete NMR data for compounds 4 and 6. The compounds were evaluated for their cytotoxic activities against three cell lines. Compound 5 showed potent and selective activity against HCT-116 cell line with IC50 of 1.5 μg/mL, while 1–10 showed variable cytotoxic activities against these cancer cell lines. These results provide further understanding about the chemistry and bioactivities of the alkylated 2(1H)-pyrazinone derivatives.As a part of our ongoing effort to allocate marine microbial bioactive leads, a tunicate-derived actinomycete,  Streptomyces was first described by Waksman and Henrici + as a reference mass. For column chromatography, silica gel  and Sephadex LH-20  were used. Precoated silica gel 60 F-254 plates (Merck) were used for TLC. HPLC purifications were performed on a semi-preparative HPLC column .Optical rotations were measured on a JASCO DIP-370 digital polarimeter at 25 °C at the sodium D line (589 nm). UV spectrum were recorded on a Hitachi 300 spectrometer. IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer . 1D and 2D NMR spectra  were recorded on Bruker Avance DRX 600 MHz spectrometers  using CDClDidemnum sp. was collected in November 2013 by hands using SCUBA at depths between 15 and 20 m near Obhur, Saudi Arabia. The tunicate material was identified by Dr. Francoise Monniot at Muséum National d’Histoire Naturelle (MNHN), Paris. A voucher specimen was deposited in the MNHN, Paris, under the Registration Number A2-Did c-476.The marine tunicate Streptomyces on the basis of 16S rRNA gene sequence analysis. Genomic DNA isolation, PCR amplification of 16S rRNA gene and sequence alignment of the strain were performed as described previously [Streptomyces flocculus (DQ442498) and Streptomyces rangoonensis (NR_041110).The actinomycete strain was identified as a member of the genus eviously . Its 16SStreptomyces sp. Did-27 were directly cultured in 2000 mL Erlenmeyer flasks containing 500 mL of ISP-2  [The spores of Project)  fermenta2 VLC eluting with n-hexane/CH2Cl2/MeOH gradients to give six fractions (A–F). Fraction B (390 mg) was subjected to gel filtration on Sephadex LH-20 using MeOH as eluent to give five subfractions (B1–B5). Fraction B3 (139 mg) was further subjected to C18 HPLC separation eluting with 30% ACN to yield 1 (4.5 mg), 2 (1.6 mg), and 3 (3.9 mg). Fraction B4 (180 mg) was subjected to C18 HPLC separation eluting with 35% ACN to yield 7 (6.5 mg), 8 (5.3 mg), 9 (10 mg) and 10 (4.8 mg). Fraction E (320 mg) was purified by gel filtration over Sephadex LH-20 using MeOH giving four subfractions (E1–E4). Fraction E2 (130 mg) was purified by C18 HPLC eluting with 35% ACN to yield 4 (2.9 mg), 5 (4.9 mg) and 6 (4.6 mg).The EtOAc extract (4.3 g) was subjected to SiO1: White solid; c 0.1, CHCl3); UV (MeOH) λmax (log ε): 329 (3.65), 227 (3.50) nm; IR (film) νmax 3430, 1643 cm−1; NMR data: m/z 195.1499 .Compound 2: White solid; c 0.1, CHCl3); UV (MeOH) λmax (log ε): 328 (3.65), 227 (3.52) nm; IR (film) νmax 3435, 1640 cm−1; NMR data: m/z 195.1499 . Compound 3: White solid; c 0.1, CHCl3), UV (MeOH) λmax (log ε): 325 (3.55), 227 (3.50) nm; IR (film) νmax 3440, 1645 cm−1; NMR data: m/z 209.1655 . Compound 4: White solid; UV (MeOH) λmax (log ε): 325 (3.53), 227 (3.50) nm; IR (film) νmax 3440, 1645 cm−1; NMR data: m/z 195.1497 .Compound 1-10 on cell proliferation and cytotoxicity were evaluated using the sulforhodamine B (SRB) assay as described previously [1–10 are displayed in The in vitro antiproliferative and cytotoxic activities of the compounds was evaluated against three human tumor cells including HCT-116 , HepG2  and MCF-7 . The effect of compounds eviously . DoxorubStreptomyces sp. Did-27, afforded three new compounds, namely (S)-6-(sec-butyl)-3-isopropylpyrazin-2(1H)-one (1), (S)-3-(sec-butyl)-6-isopropylpyrazin-2(1H)-one (2) and (S)-6-(sec-butyl)-3-isobutylpyrazin-2(1H)-one (3) and six previously reported ones including deoxymutaaspergillic acid (4), 3,6-diisobutyl-2(1H)-pyrazinone (5), 3,6-di-sec-butyl-2(1H)-pyrazinone (6), cyclo(6-OH-d-Pro-l-Phe) (7), bacillusamide B (8), cyclo(l-Pro-l-Leu) and cyclo(l-Pro-l-Ile) (10). Their structures were assigned by interpretation of their spectral data. In addition, the complete NMR data for compounds 4 and 6 were reported here for the first time. Compound 5 showed selective and potent active agaisnt colorectal carcinoma cell line (HCT-116) with with IC50 of 1.5 µg/mL. All other compounds were moderatly active against MCF-7 and weakly active against HepG2 cell line.In conclusion, investigation of a tunicate-derived actinomycete,


Macleaya (Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde) have been widely reported, as well as more and more concerned from the scientific communities. However, systematic research on the phytochemical information of M. microcarpa is incomplete. The aim of this study was to rapidly and conveniently qualitative analyze alkaloids from M. microcarpa by ultra-performance liquid chromatography/quadrupole-time-of-fight mass spectrometry (UHPLC–Q-TOF-MS) using accurate mass weight and characteristic fragment ions, furthermore separate and identify the main alkaloids, test antitumor activity in vitro and antiangiogenic activity in vivo.Extensive bioactivities of alkaloids from the genus M. microcarpa were identified by UHPLC–Q-TOF-MS, including 5 protopines, 2 benzophenanthridines, 1 dimer, 1 dihydrobenzophenanthridines and 5 unknown structure compounds. Two major alkaloids were isolated by various column chromatographic methods. Their structures were determined by NMR data and related literatures. The two major alkaloids were evaluated for intro cytotoxic activities against HL-60, MCF-7, A-549, and in vivo antiangiogenic activity using transgenic zebrafish.A total of 14 alkaloids from fruits of M. microcarpa in the future.Current qualitative method based on UHPLC–Q-TOF-MS technique provided a scientific basis for isolation, structural identification, and in vitro or in vivo pharmacological further study of alkaloids from  Macleaya contains two species, Macleaya cordata (Willd.) R. Br. and Macleaya microcarpa (Maxim.) Fedde, which mainly distributed in Japan, South and Northwest China, on slopes of grass or thickets at altitudes of 450–1600 m + or [MA–18]+, respectively. The mass spectral cleavage rules of benzophenanthridine alkaloids were as follows  was isolated as colorless square crystal in CH2Cl2: MeOH (1:1). 1H NMR  δ: 6.90 , 6.69 , 6.66 , 6.64 , 5.95 , 5.92 , 3.78 , 3.58 , 2.2–3.2 , 1.91 . 13C NMR  δ: 108.3 (C-1), 146.5 (C-2), 148.1 (C-3), 110.6 (C-4), 132.9 (C-4a), 31.9 (C-5), 57.9 (C-6), 50.9 (C-8), 118.0 (C-8a),146.0 (C-9), 146.1 (C-10), 106.9 (C-11), 125.2 (C-12), 129.1 (C-12a), 46.6 (C-13), 195.1 (C-14), 136.3 (C-14a), 101.3 , 101.0 , 41.6 (N-CH3). The structure was identified by comparison of the NMR data with literature [Protopine (7) was yellow powder. 1H NMR  δ: 9.99 , 8.71 , 8.68 , 8.24 , 8.23 , 8.21 , 7.59 , 6.28 , 4.30 , 4.15 , 5.01 . 13C NMR  δ: 107.1 (C-1), 151.8 (C-2), 150.8 (C-3), 105.1 (C-4), 121.9 (C-4a), 132.6 (C-4b), 52.9 (N-CH3), 152.1 (C-6), 119.9 (C-6a), 147.6 (C-7), 151.8 (C-8), 127.5 (C-9), 121.0 (C-10), 130.2 (C-10a), 127.2 (C-10b), 119.5 (C-11), 132.7 (C-12), 134.4 (C-12a), 104.3 , 62.8 (7-OCH3), 57.6 (8-OCH3). According to related literatures [Chelerythrine  formation in zebrafish embryos were shown in Fig. M. microcarpa by UHPLC–Q-TOF-MS using accurate mass weight and characteristic fragment ions, and combining with their cleavage rules. Some unknown compounds were discovered through this simple and sensitive method. The study on the cleavage rules of these alkaloids is helpful to identify the characteristic alkaloids by LC–MS method, and identify the structural types of alkaloids in M. microcarpa. The two major alkaloids of M. microcarpa, protopine and chelerythrine, exhibited potent cancer cell growth inhibitory activities in vitro, but they showed almost no antiangiogenic activity in transgenic zebrafish vivo model. However, the structure and bioactivity screening of new natural products from M. microcarpa still need further study, which is an integral part of drug discovery progress.In this paper, we rapidly and conveniently qualitative analyze alkaloids from


Penicillium sp. ZZ380 was previously reported to have the ability to produce a series of new pyrrospirone alkaloids. Further investigation on this strain resulted in the isolation and identification of novel penicipyrroether A and pyrrospirone J. Each of them represents the first example of its structural type, with a unique 6/5/6/5 polycyclic fusion that is different from the 6/5/6/6 fused ring system for the reported pyrrospirones. Their structures were elucidated by extensive nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectroscopy (HRESIMS) spectroscopic analyses, electronic circular dichroism (ECD) and 13C NMR calculations and X-ray single crystal diffraction. Penicipyrroether A showed potent antiproliferative activity against human glioma U87MG and U251 cells with half maximal inhibitory concentration (IC50) values of 1.64–5.50 μM and antibacterial inhibitory activity with minimum inhibitory concentration (MIC) values of 1.7 μg/mL against methicillin-resistant Staphylococcus aureus and 3.0 μg/mL against Escherichia coli.The marine-sourced fungus  Analyses of its 1H, 13C, 1H-1H COSY, HSQC and HMBC spectra (9 and pyrrospirones C–I (1–7) are the same. However, the structures of 9 and pyrrospirones have significant differences. The first difference is the ring D with a five-membered ether ring for 9 and a cyclohexanone for pyrrospirones. The second difference is that 9 has no spiro junction for rings D and E. The five-membered ether ring D and its connections with rings C and E were confirmed by 1H-1H COSY correlation  with H-13  and HMBC correlations (δC 48.9), C-9 (δC 87.5), C-11 (δC 126.8), C-12 (δC 48.6), C-14 (δC 102.1), C-16 (δC 139.2) and C-31 (δC 22.0), H-15  with C-11 and C-31 and H-19  with C-14, C-17 (δC 172.7) and C-18 (δC 88.1). The locations of CH3-31, CH3-32 and OH-14 were also established by HMBC correlations of H3-31  with C-11, C-12, C-13 (δC 56.3) and C-15 (δC 79.4) and H3-32  with C-12 and C-15, as well as OH-14  with C-14 and C-16. The planner structure of 9 was further confirmed by X-ray diffraction analysis  and 3J7,8 (13.2 Hz) also confirmed the trans-juncture for A/B and B/C rings + 508.2711 and [M + Na]+ 530.2531 as well as its 13C NMR data, which showed two fewer carbons, when compared to the structure of penicipyrroether A (9). Detailed interpretation of 1H, 13C, 1H-1H COSY, HSQC and HMBC spectra  with the same structural part of rings A, B, F and G but possesses an epoxy moiety at C-10 and C-11 and a different five-membered ether ring D fused with ring E through a spiro carbon of C-15. In addition, the dehydro-pyrrolidinone moiety (ring E) in 9 was replaced by a pyrrolidinone moiety in 10. The presence of the epoxy moiety was confirmed by HMBC correlations  with C-10 (δC 58.8) and C-11 (δC 63.5), H-11  with C-10, C-12 (δC 81.3), C-13 (δC 44.5), C-29 (δC 21.0) and C-30 (δC 26.0) and H3-29  with C-7 (δC 49.9), C-10 and C-11. Similarly, the ether ring D and its connections with rings C and E was indicated by HMBC correlations of H-8  with C-13 and C-14 (δC 180.9), H-11 with C-12 and C-13, H-13  with C-8 (δC 39.2), C-11, C-12, C-14 and C-30, H-18  with C-14, C-15 (δC 79.7), C-16 (δC 172.1), C-17 (δC 86.2) and C-19 (δC 45.0), H3-30  with C-11, C-12 and C-13 and NH-16  with C-15, C-16, C-17 and C-18 (δC 40.9).Compound  spectra  demonstrelations  of H-7  with βH-3 (δH 0.51) and H-9 (δH 4.82), H-7 (δH 1.55) with βH-1 (δH 0.81) and H3-30 (δH 1.59), H-9 with H-13 (δH 3.21) and H-24 (δH 6.94), H-13 with H3-30, NH-16 (δH 8.76) with βH-19 (δH 2.99) and H-25 (δH 6.78) and H-25 with βH-19 and H-24 suggested a β-orientation for these protons; while NOE correlations of H-8 (δH 2.79) with H3-28 (δH 1.05), H3-28 with αH-1 (δH 1.80), H-2 (δH 1.82), H-4 (δH 1.78) and H3-29 (δH 1.20), H3-29 with H-11 (δH 2.46), H-21 (δH 6.92) with αH-19 (δH 2.69) and H-22 (δH 6.82) and H-18 (δH 1.99) with OH-17 (δH 6.24) proved an α-orientation for these protons. The trans-juncture for A/B and B/C rings was also confirmed by the large coupling constants of 3J4,5 (11.6 Hz) and 3J7,8 (14.3 Hz) c 0.10, MeOH); ECD  λmax (Δε) 208 (−57.22), 233 (+64.65), 286 (+34.08) nm; UV (MeOH) λmax (log ε) 202 (4.20), 229 (3.66), 278 (2.67) nm; IR (MeOH) νmax 3375, 2924, 1672, 1437, 1408, 1239, 1098, 1013, 951 cm−1; 13C (125 MHz) and 1H (500 MHz) NMR data, see m/z [M + H]+ 520.3063 , [M + Na]+ 542.2877 .Penicipyrroether A (10): Colorless amorphous powder; molecular formula C30H37NO6; ECD  λmax (Δε) 204 (−55.93), 223 (+59.59), 256 (−11.80), 293 (−17.92) nm; UV (MeOH) λmax (log ε) 211 (4.40), 230 (4.21), 284 (3.36) nm; IR (MeOH) νmax 3347, 2923, 2851, 1748, 1686, 1505, 1460, 1371, 1239, 1169, 1074, 937 cm−1; 13C (125 MHz) and 1H (500 MHz) NMR data, see m/z [M + H]+ 508.2711  and [M + Na]+ 530.2531 .Pyrrospirone J (9): Colorless crystals of penicipyrroether A (9) was obtained from MeOH. X-ray diffraction analysis was performed on an Xcalibur Atlas Gemini Ultra diffractometer  with Cu Kα radiation (λ = 1.54184 Å) at 100 K. Structure was solved by direct method (SHELXL-2018) and refined with full-matrix least-squares on F2 . All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters [9): C32H41NO5 (M = 519.68), orthorhombic crystal (0.13 × 0.12 × 0.09 mm), space group P212121 (no. 19), unit cell dimensions a = 6.32760(10) Å, b = 19.8687(2) Å, c = 25.0617(3) Å, V = 3150.79(7) Å3, α = 90°, β = 90°, γ = 90°; Z = 4; calcedD = 1.231 g/cm3; μ = 0.684 mm−1; 18406 reflection measured (5.676° ≤ 2θ ≤ 147.088°); 6237 unique  which were used in all calculation; the final refinement ( I ≥ 2σ (I)) gave 1R = 0.0418 and 2wR = 0.1116 ; Flack parameter = 0.02 (5). Crystallographic data of penicipyrroether A (9) have been deposited at the Cambridge Crystallographic Data Centre (deposition number: CCDC 1868814). Copies of the crystallographic data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. [fax (+44)1223-336-033; or e-mail: data_request@ccdc.cam.ac.uk).Crystal data of penicipyrroether A  using Merck Molecular Force Field (MMFF) and three conformers were obtained for ECD calculations. The X-ray CIF profile of 9 and the three conformer of 10  were calculated. ECD spectra were generated using the program SpecDis 1.6  and GraphPad Prism 5  from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.2 eV for 9 (or 0.3 eV for 10).Monte Carlo conformational searches of compound er of 10  were ini13C NMR calculation. Briefly, Monte Carlo conformational searches were carried out by means of Spartan’s 10 software using MMFF. Four conformers  level in DMSO using the CPCM calculation model. Gauge-independent atomic orbital (GIAO) calculations of 13C NMR chemical shifts were performed by DFT at the mPW1PW91-SCRF (DMSO)/6-311+g level with the CPCM calculation model in Gaussian 09 software . The calculated 13C NMR data of the lowest energy conformers for 10 were averaged based on the Boltzmann distribution theory and their relative Gibbs free energy.The previously described methods ,20,21 wenformers  of compo2 humidified incubator. The cultured cells after the third generation were used for experiment.The activity of the tested compounds in inhibiting the proliferation of human glioma U87-MG and U251 cells was determined by SRB assay . DoxorubE. coli. Vancomycin (an antibiotic against MRSA) and gentamicin (an antibiotic against Gram-positive and Gram-negative bacteria) were used as positive controls. The microorganisms were cultured in MHB medium in 96-well plates at a concentration of 1 × 106 CFU/mL. The MIC was determined after 12 h incubation at 37 °C with tested compounds.The previously described micro broth dilution method ,22 was uPenicillium sp. ZZ380 produced a series of pyrrospirones C–I with a characteristic spiro conjunction for rings D and E in BMPM medium [9), produced by strain ZZ380 in PDB medium and pyrrospirone J (10), a previously unidentified compound isolated from a previous culture of ZZ380 in BMPM medium [9 and 10 possess unique structures with a 6/5/6/5 fused ring system of rings A/B/C/D, rather than the 6/5/6/6 ring fusion for pyrrospirones C–I. Each of the two new alkaloids is the first compound of its structural type. Penicipyrroether A (9) is a cyclo-condensation product of GKK1032 analogue via the addition of a five-membered ether ring and exhibited potent inhibitory activity against the proliferation of glioma U87MG and U251 cells and the growth of MRSA and E. coli.It was reported that the marine-derived M medium . This stM medium . Both co


Siegesbeckia glabrescens (Compositae) showed antibacterial activity against the foodborne pathogen Staphylococcus aureus. Bioactivity-guided separation led to the isolation of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid from nature for the first time.The structure was determined by spectroscopic data analysis . The minimal inhibitory concentration (MIC) of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid against S. aureus was found to be 3.12 μg/mL. In addition, in a further antimicrobial activity assay against Gram-positive , and Gram-negative bacteria (E. coli and P. aeruginosa), and yeast strains , the antimicrobial activity of the compound was found to be specific for Gram-positive bacteria. The MIC values of the compound for Gram-positive bacteria ranged from 3.12 to 25 μg/mL. Furthermore, it was found that the 2-(isobutyryloxy)-4-methylpentanoic acid substituent may operate as a key factor in the antibacterial activity of the compound, together with the laurate group.The crude methanol extract of the dried aerial parts of  Staphylococcus aureus , ten methylenes  and two methines (δC 29.0 and 33.9). These facts were consistent with a molecular formula of C22H40O6, which was supported by HR-EIMS data  and ESIMS . The 1H-1H COSY spectrum of active compound showed proton correlations of δH 5.20 (H-2) with δH 5.11 (H-3); δH 5.11 (H-3) with δH 5.20 (H-2) and δH 2.16 (H-4); δH 2.16 (H-4) with δH 5.11 (H-3), δH 0.93 (H-5) and δH 1.01 (H-6); δH 0.93 (H-5) with δH 2.16 (H-4); δH 1.01 (H-6) with δH 2.16 (H-4); δH 2.63 (H-8) with δH 1.19 (H-9 and H-10). The structure of the active compound was further established according to its HMBC spectrum, in which 1H-13C long-range correlation signals were between δH 5.20 (H-2) and δC 169.8 (C-1), δC 76.5 (C-3), δC176.3 (C-7); δH 5.11 (H-3) and δC 173.5 (C-1'), δC 169.8 (C-1), δC 72.0 (C-2), δC 29.0 (C-4), δC 17.3 (C-5), δC 18.4 (C-6); δH 2.16 (H-4) and δC 72.0 (C-2), δC 76.5 (C-3), δC 17.3 (C-5), δC 18.4 (C-6); δH 0.93 (H-5) and δC 76.5 (C-3), δC 29.0 (C-4), δC 18.4 (C-6); δH 1.01 (H-6) and δC 76.5 (C-3), δC 29.0 (C-4), δC 17.3 (C-5); δH 2.63 (H-8) and δC 176.3 (C-7), δC 18.0 (C-9 and C-10)  .From all the above information, the structure of the active compound was determined to be 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid , isolateS. aureus + ; ESIMS (m/z): 423.8, [M+Na]+ .The air dried Staphylococcus aureus (ATCC 6538P), Bacillus subtilis (ATCC 15245), Enterococcus faecalis (ATCC 11700), Propionibacterium acnes (ATCC 6919), Staphylococcus epidermidis (ATCC 12228), Staphylococcus schleiferi subsp. coagulans (ATCC 49545), Streptococcus agalactiae (ATCC 14364) and Streptococcus pyrogens (ATCC 19615)], Gram-negative bacteria [Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 27853)] and yeast [Candida alibicans (ATCC 10231) and Filobasidiella neoformans (ATCC 34144)] were used in these experiments. The antibacterial activity was determined using both the agar diffusion and broth dilution techniques as described previously by Cheesbrough [et al. [Gram-positive bacteria [esbrough  and Gatsiae ATCC 364 and S5 cfu/mL bacterial cells of the pre-cultured bacteria were inoculated into 3 mL of broth. The samples were then added into approximately 3 mL of broth containing the bacteria and cultured for 24 h at 37 °C. To determine the activity of the samples, we employed a two-fold serial dilution method. The total volume of the mixture was approximately 3 mL, with the test-compound concentrations in the tube ranging from 200 to 0.78 μg/mL and the concentration of standard compound (erythromycin) ranged from 100 to 0.78 μg/mL. After 24 h of incubation at 37 °C, the MIC value was defined as the lowest concentration that inhibited the visible growth of tested microorganism.We pre-cultured the bacterial cells for 24 h at 37 °C in 10 mL broth. Approximately 5 × 10S. glabrescens has antibacterial activities specifically against Gram-positive bacteria. We found that the antibacterial effect of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid was due to both the 2-(isobutyryloxy)-4-methylpentanoic acid group and laurate group. In addition, the antibacterial activity of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid was more potent than lauric acid itself. To the best of our knowledge, this is the first study demonstrating the antibacterial activity of S. glabrescens. In addition, we demonstrated that 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid is an antibacterial compound of S. glabrescens. The compound also has a specific antibacterial activity against Gram-positive bacteria and the strongest activity was observed against S.aureus, which are major foodborne pathogenic microorganisms.It was demonstrated that a new compound, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid isolated from


Aspergillus  and the strain Penicillium chermesinum 113 were described. The strain A. niger KB catalysed carbonyl group reduction, leading to (±)-2,4-cis-7-methoxyflavan-4-ol. Biotransformation with the help of A. ochraceus 456 gave two products: (+)-2,4-trans-7-methoxyflavan-4-ol and 4'-hydroxy-7-methoxyflavone. Transformation by means of P. chermesinum 113 resulted in a dihydrochalcone product, 4,2'-dihydroxy-4'-methoxydihydrochalcone. DPPH scavenging activity test proved that all the biotransformations products have higher antioxidant activity that the substrate.Microbial transformations of racemic 7-methoxyflavanone using strains of the genus  Flavonoids are polyphenolic compounds with diverse chemical structures, which are widely found in plants. Apart from plants, a natural capability to carry out biosynthesis of flavonoid compounds is a feature of some endophytic fungi . Animal O-methylation and hydrolysis 20546= +2.57 , and the enantiomeric excess  ee = 30%. For the unreacted substrate (1) isolated from the reaction mixture the data was as follows: [α]20546 = +6.28  and ee = 24%.For the obtained 2,4-4) was established by means of 1H NMR and 13C-NMR. Hydroxylation at C 4' in ring B is proved by two doublets integrating each for 2 H at δ = 6.95 ppm (J = 8.6 Hz) and δ = 7.81 ppm (J = 8.7 Hz), corresponding to H-3', H-5' and H-2', H-6', respectively. In the 13C-NMR we observe a change in C-4' chemical shift from δ = 128.8 ppm for substrate (1) to δ = 160.5 ppm for product 4. In the 1H-NMR spectrum of 7-methoxyflavone (1) the signal of H-2 appears at δ = 5.47 ppm as a doublet of doublets (J = 13.3 Hz and J = 2.8 Hz), the signal of H-3ax at δ = 3.04 ppm is a doublet of doublets (J = 16.9 Hz and J = 13.3 Hz) and the signal of H-3eq at δ = 2.84 ppm is also a doublet of doublets (J = 16.9 Hz and J = 2.9 Hz). In the 1H-NMR of 4'-hydroxy-7-methoxyflavone (4) a one proton singlet is visible at δ = 5.69 ppm, attributed to H-3, whereas the signal of H-2 disappears, which confirm the presence of a double bond between C-2 and C-3 in ring C. Additionally, in the 13C-NMR the signals of C-2 and C-3 were moved from δ = 80.2 ppm and δ = 44.3 ppm for the substrate (1) to δ = 162.8 ppm and δ = 104.0 ppm for the dehydrogenation product 4.The structure of the independently formed 4'-hydroxy-7-methoxyflavone (1) by the strain P. chermesinum 113 led to formation of product 5 of dihydrochalcone structure (Transformation of (±)-7-methoxyflavanone (tructure .5) was isolated after 10 days of biotransformation in 15% yield (15 mg/100 mg of the substrate). We expected a 4'-hydroxylation product with the C ring of 7-methoxyflavone intact, which would be analogous to the described earlier hydroxylation of flavanone catalysed by the strain P. chermesinum 113 23546 = 0, ; 1H-NMR (CDCl3) δ: 2.13 , 2.52 , 3.79 , 5.05 , 5.17 , 6.45 , 6.59 , 7.36 , and 7.34–7.45 ; 13C-NMR (CDCl3) δ: 40.2 (C-3), 55.3 (OCH3), 65.6 (C-4), 77.1 (C-2), 101.1 (C-8), 108.1 (C-6), 118.2 (C-10), 126.0 , 127.8 (C-4'), 128.1 (C-5), 128.6 , 140.4 (C-1'), 155.5 (C-9), and 160.3 (C-7); IR : 3225 , 1619 , and 1590 ; HRESI-MS [M+H+]  (m/z 257.0990/257.0979).4-ol (2) . C16H16O(+)-2,4-trans-7-Methoxyflavan-4-ol (3). C16H16O3; Melting point 115–117 °C; 4.9% yield; purity 97% (HPLC); [α]20546 = +2.57, , ee = 30%; 1H-NMR (CDCl3) δ: 2.52 , 2.95 , 5.07 , 5.17 , 6.45 , 6.58 , 7.42 , and 7.34–7.47 ; 13C-NMR (CDCl3) δ: 40.0 (C-3), 55.2 (7-OCH3), 65.6 (C-4), 77.2 (C-2), 101.2 (C-8), 108.1 (C-6), 118.0 (C-10), 126.0 , 127.9 (C-4'), 128.2 (C-5), 128.7 , 140.1 (C-1'), 155.0 (C-9), and 160.1 (C-7); IR : 3230 , 1619  and 1595 ; HRESI-MS [M+H+]  (m/z 257.0990/257.0977).4'-Hydroxy-7-methoxyflavone (4)   (m/z 269.0965/269.0960).vone (4) . C16H12O4,2'-Dihydroxy-4'-methoxydihydrochalcone (5)   (m/z 273.1111/273.1105).cone (5) . C16H16O1–5. After 20 min, disappearance of absorbance at 520 nm was measured. The initial concentration of DPPH was determined by means of calibration curve. The IC50 value  was determined graphically—DPPH radical reduction (expressed in %) as a function of concentration of the tested compound. IC50 means concentration of the antioxidant that reduces the initial concentration of DPPH by half. A methanolic solution of DPPH  with an absorbance of about 1.00, was mixed with a proper amount of a tested flavonoid A. niger KB described in this article and in our previous papers prove that this strain is a good catalyst for carbonyl group reduction in flavanone and its monosubstituted derivatives [P. chermesinum 113 performs mainly reactions of hydroxylation in ring B, which are often accompanied with the ether bond cleavage in ring C, leading to dihydrochalcone structure [The study on ivatives ,20. The tructure .trans-7-methoxyflavan-4-ol obtained in the reaction catalysed by A. ochraceus 456 did not exceed 30%.The biotransformations of 7-methoxyflavanone did not afford any enantiomerically pure product. Enantiomeric excess of 2,4-All the products of biotransformations of 7-methoxyflavanone have higher antioxidant properties than the substrate.