Their structures were elucidated by spectroscopic analysis and time-dependent density functional theory (TDDFT)-electronic circular dichroism (ECD) calculations. Saccharothrixones G (3) and H (4) are the first examples of tetracenomycins featuring a novel ring-A-cleaved chromophore. Saccharothrixone I (5) was determined to be a seco-tetracenomycin derivative with ring-B cleavage. The new structural characteristics, highlighted by different oxidations at C-5 and cleavages in rings A and B, enrich the structural diversity of tetracenomycins and provide evidence for tetracenomycin biosynthesis. Analysis of the structure–activity relationship of these compounds confirmed the importance of the planarity of the naphthacenequinone chromophore and the methylation of the polar carboxy groups for tetracenomycin cytotoxicity.Six new tetracenomycin congeners, saccharothrixones E–I ( Streptomyces glaucescens and Streptomyces olivaceus, represent a separate group of aromatic polyketides featuring a tetracyclic naphthacenequinone chromophore with highly hydroxylated cyclohexenone moiety, and exhibit moderate antibacterial and antitumor activities . Th. Th1 and1–6 against the cancer cell lines mentioned above. All the compounds were found to be inactive at 100 µM. The action mechanism of tetracenomycins was assumed to be intercalation with DNA, requiring flat structural moiety to move between the base pairs c 0.31, MeOH); UV (MeOH) λmax (logε 239 (4.30), 266 (4.50), 277 (4.54), 317 (3.80), 328 (3.75), 382 (3.82) nm; ECD  λmax (Δε) 247 (+12.89), 277 (−28.45), 334 (+0.31), 377 (−2.08) nm; IR vmax (cm−1): 3469, 2948, 1730, 1659, 1606, 1374, 1221, 1089, 946, 839; 1H NMR  data, 13C NMR  data, m/z 489.1391 [M + H]+ .Saccharothrixone E (2): Yellow amorphous powder; [α]c 0.15, MeOH); UV (MeOH) λmax (logε) 232 (4.06), 268 (4.28), 277 (4.38), 318 (3.60), 330 (3.53), 382 (3.67) nm; ECD  λmax (Δε) 218 (+4.16), 250 (+5.45), 276 (−15.33), 381 (−1.60) nm; IR vmax (cm−1): 3199, 2921, 1680, 1604, 1205, 1140, 840, 801, 723; 1H NMR  data, 13C NMR  data, m/z 475.1236 [M + H]+ ; 497.1053 [M + Na]+ .Saccharothrixone F (3): Yellow amorphous powder; [α]c 0.17, MeOH); UV (MeOH) λmax (logε) 227 (4.28), 275 (4.51), 315 (3.65), 328 (3.56), 377 (3.75) nm; ECD  λmax (Δε) 217 (−6.40), 274 (+9.45), 315 (+1.74) nm; IR vmax (cm−1): 3367, 2947, 1735, 1622, 1368, 1221, 1087, 1053, 993, 948; 1H NMR  data, 13C NMR  data, m/z 487.1229 [M − H]− .Saccharothrixone G (4): Yellow amorphous powder; [α]c 0.05, MeOH); UV (MeOH) λmax (logε) 226 (4.26), 278 (4.38), 316 (3.68), 328 (3.63), 382 (3.71) nm; ECD  λmax (Δε) 206 (−1.35), 236 (+9.71), 277 (+2.79), 331 (+0.87) nm; IR vmax (cm−1): 3383, 2927, 1736, 1621, 1375, 1218, 1092, 989, 802; 1H NMR  data, 13C NMR  data, m/z 487.1226 [M − H]− .Saccharothrixone H (5): White amorphous powder; [α]c 0.98, MeOH); UV (MeOH) λmax (logε) 258 (4.60), 263 (4.68), 352 (3.71) nm; ECD  λmax (Δε) 206 (−2.01), 244 (+14.00), 262 (−12.01), 299 (−2.54), 350 (−1.51) nm; IR vmax (cm−1): 3346, 2952, 1730, 1677, 1615, 1366, 1203, 1105, 994, 723; 1H NMR  data, 13C NMR  data, m/z 487.1226 [M − H]− .Saccharothrixone I (O-methyltetracenomycin X (6): Yellow amorphous powder; [α]c 1.13, MeOH); UV (MeOH) λmax (logε) 216 (4.47), 240 (4.51), 291 (4.76), 397 (4.19), 414 (4.23) nm; ECD  λmax (Δε) 201 (+13.60), 227 (−4.05), 262 (+28.51), 350 (−5.55), 420 (−1.09) nm; IR vmax (cm−1): 3357, 2934, 1679, 1602, 1369, 1236, 1122, 838, 601; 1H NMR , 13C NMR  data, m/z 473.1101 [M + H]+ ,495.0923 [M + Na]+ .13-De-1 and 2 and the CAM-B3LYP/TZVP and WB97XD/6-311++G levels for 3–5 in MeOH with the PCM model. ECD spectrum of each conformer was simulated by the SpecDis program [S,4sR,5S,12aR-1a, 4S,4aR,5S,12aR-2a, 4S,4aR,5S,12aS-3a, 4R,4aR,5S,12aS-3c, and 4S,4aR,5R,12aR-5a were generated by averaging the calculated data of the lowest energy conformers for each structure according to Boltzmann distribution theory at 298 K based on Gibbs free energies. The corresponding theoretical ECD spectra of 4R,4aS,5R,12aS-1b, 4R,4aS,5R,12aS-2b, 4R,4aS,5R,12aR-3b, 4S,4aS,5R,12aR-3d, and 4R,4aS,5S,12aS-5b were depicted by inverting those of 1a, 2a, 3a, 3c, and 5a, respectively.Conformational analysis was carried out via Monte Carlo searching in the MMFF94 molecular mechanics force field on Molecular Operating Environment (MOE) software . The low program  using a The cytotoxicities of the tested compounds against the human cancer cells HepG2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and K562 (leukemia) were evaluated by the sulforhodamine B (SRB) assay as described previously . The antseco-tetracenomycins were isolated from the rare marine-derived actinomycete Saccharothrix sp. 10-10. Saccharothrixones G (3) and H (4) are the first examples of tetracenomycins featuring a novel ring-A-cleaved chromophore. Saccharothrixone I (5), together with previously identified saccharothrixones A–C from the same cultures, are the only seco-tetracenomycins with a cleaved ring-B skeleton isolated from microbial natural products. This finding not only adds diversity of tetracenomycins, but also provides evidence for tetracenomycin-related polyketides biosynthesis. The structure-activity relationship study indicated that the planarity of the chromophore and methylation of the polar carboxy groups are important for tetracenomycin cytotoxicity.Six new tetracenomycin derivatives including three


ZZ745. The structures of these compounds were determined by means of NMR spectroscopic analysis, HRESIMS data, and optical rotation. Both bagremycins F (1) and G (2) showed antibacterial activity against Escherichia coli, with MIC values of 41.8 and 61.7 μM, respectively.As part of our research to discover novel bioactive natural products from marine microorganisms, five bagremycin analogues, including the previously unreported bagremycins F ( Salinospora strain CNB-392 entered clinical trials against multiple myeloma only three years after its discovery + and 453.1089 [M + Na]+ in the (+)-HRESIMS, corresponding to a molecular formula of C21H22N2O6S. The 13C NMR spectrum , 113.1 (C-2), 140.4 (C-3), 147.7 (C-4), 113.2 (C-5), 119.1 (C-6), 165.7 (C-7), 150.5 (C-8), 122.0 , 127.1 , 134.7 (C-11), 135.8 (C-14), and 114.3 (C-15). The 1H NMR spectrum ; 7.10 ; 7.21 ; 7.55 ; 6.74 ; 5.29 ; and 5.82  (1H and 13C NMR data for the scaffold of 1 were very similar to those of bagremycins C (6) and D (7) + and 306.0734 [M + Na]+, as well as its 13C data. The 13C and 1H NMR spectra , one formamide unit , twelve aromatic carbons and seven aromatic protons, including: δC 118.1 (C-1), 121.9 (C-2), 126.2 (C-3), 152.6 (C-4), 114.8 (C-5), 126.9 (C-6), 150.4 (C-8), 122.1 , 127.1 , 134.7 (C-11), and δH 8.90 ; 6.99 ; 7.74 ; 7.20 ; 7.54 ; and an exocyclic double bond   . These N3) and B (4) was previously proposed, as described in Reference c 0.04, MeOH); UV (MeOH) λmax (log ε) 201 (3.49), 250 (2.92), 331 (2.34) nm; IR (KBr) νmax 3317, 2921, 2853, 1714, 1461, 1254, 1171, 1033 cm−1; 13C and 1H NMR data (in DMSO-d6), see m/z 431.1270 [M + H]+  and 453.1089 [M + Na]+ .Bagremycin G (2): colorless amorphous powder; molecular formula C16H13NO4; UV (MeOH) λmax (log ε) 201 (3.14), 247 (2.97), 321 (1.92) nm; IR (KBr) νmax 3380, 2918, 2853, 1714, 1540, 1464, 1256, 1198, 1050 cm−1; 13C and 1H NMR data (in DMSO-d6), see m/z 284.0929 [M + H]+  and 306.0734 [M + Na]+ .1–5 against MRSA, E. coli, and C. albicans was determined by the micro broth dilution method, as described in the previous study in Reference [The antimicrobial activity of eference .1–5 against human glioma U87-MG and U251 cells was evaluated by SRB assay and DOX was used as a positive control. U87-MG and U251 cells were cultured in MEM  and DMEM , both adding 10% FBS , respectively. All glioma cells were incubated at 37 °C in a humidified incubator with 5% CO2. Cells after the third generation were used for the experiment. The detailed procedure for SRB assay was described in previous publications in References [The activity of ferences ,16.Streptomyces genus, are important sources for the discovery of novel bioactive natural products. In this study, we isolated and identified five bagremycin derivatives (1–5) from the culture of the marine-derived actinomycete Streptomyces sp. ZZ745 in Gause’s liquid medium. Bagremycins F (1) and G (2) are the two new members of bagremycins, which enriched the structural diversity of the bagremycin family. Bagremycins F (1) and G (2) showed antibacterial activity against E. coli.Marine actinomycetes, especially from the


N-ethyl-3-acetylindole with different substituents were investigated: (E)-3-(4-bromophenyl)-1-(1-ethyl-1H-indol-3-yl)prop-2-en-1-one (3a); (E)-3-(3-bromophenyl)-1-(1-ethyl-1H-indol-3-yl)prop-2-en-1-one (3b); (E)-1-(1-ethyl-1H-indol-3-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3c); (E)-1-(1-ethyl-1H-indol-3-yl)-3-mesitylprop-2-en-1-one (3d); and (E)-1-(1-ethyl-1H-indol-3-yl)-3-(furan-2-yl)prop-2-en-1-one (3e). The molecular packing of the studied compounds is controlled mainly by C–H⋅⋅⋅O hydrogen bonds, C–H⋅⋅⋅π interactions, and π···π stacking interactions, which were quantitatively analyzed using Hirshfeld topology analysis. Using density functional theory (DFT) calculations, the order of polarity (3b ˂ 3d ˂ 3e ˂ 3a ˂ 3c) was determined. Several chemical reactivity indices such as the ionization potential (I), electron affinity (A), chemical potential (μ), hardness (η), electrophilicity (ω) and nucleophilicity (N) indices were calculated, and these properties are discussed and compared. In addition, the antiproliferative activity of the five new chalcones was studied.The crystal structures of five new chalcones derived from  Aplidiumcyaneum , found 353.00, C19H16BrNO for 353.04.one (3a) ,23. Yiel(E)-3-(3-Bromophenyl)-1-(1-ethyl-1H-indol-3-yl)prop-2-en-1-one (3b) , found 305.10, C20H19NO2 for 305.38.one (3c) . Yield 1(E)-1-(1-Ethyl-1H-indol-3-yl)-3-mesitylprop-2-en-1-one (3d) , found 317.20; C22H23NO for 317.18.one (3d) . Yield 1(E)-1-(1-Ethyl-1H-indol-3-yl)-3-(furan-2-yl)prop-2-en-1-one (3e) , found 265.10, C17H15NO2 for 265.11.one (3e) . Yield 1X-ray crystal data were collected on a Bruker APEX-II D8 Venture area diffractometer. Crystal data, data collection, and structure refinement details are summarized in The structures of the studied systems were optimized at the B3LYP/6-311G  level of theory using Gaussian 09 . In all The topology analyses were performed using Crystal Explorer 3.1  to deter50 [The anticancer activities for the five compounds were evaluated against five different cancer types . The initial screening was performed at 50 µM, and the inhibitory activity was detected using the crystal violet assay . Subsequ50 . The staN-alkyl-3-acetylindole and five aldehydes. Structures of the obtained chalcones were clarified, and the study of the chemical behavior including (DFT) calculations, ionization potential (I), electron affinity (A), chemical potential (μ), hardness (η), electrophilicity (ω) and nucleophilicity (N) indices and Hirshfeld surface analysis were investigated. The anticancer activity of the synthesized chalcones was carried out and exhibited more sensitivity to the triple negative breast cancer cells (MDA-MB-231) than the ER-positive breast cancer cell line (MCF-7). Further investigation will be carried out in the near future.Five chalcone-based indole scaffolds were successfully synthesized from


We designed and synthesized carbamates of the clinically-approved HDAC (histone deacetylase) inhibitor vorinostat  in order to validate our previously-proposed hypothesis that these carbamates might serve as prodrugs for hydroxamic acid containing HDAC inhibitors. Biochemical assays proved our new compounds to be potent inhibitors of histone deacetylases in vitro, and they also showed antiproliferative effects in leukemic cells. These results, as well as stability analysis led to the suggestion that the intact carbamates are inhibitors of histone deacetylases themselves, representing a new zinc-binding warhead in HDAC inhibitor design. This suggestion was further supported by the synthesis and evaluation of a carbamate derivative of the HDAC6-selective inhibitor bufexamac. N-hydroxy-N′-phenyloctanediamide, SAHA), belinostat and panobinostat, as well as in many clinical candidates. Hydroxamic acids are known to be problematic with regard to their pharmacokinetic properties, especially the metabolic stability in vivo is limited. Thus, a reasonable prodrug concept for hydroxamic acids might lead to an improvement of stability, metabolism or cellular uptake of such inhibitors.Histone deacetylases (HDACs) are some of the best-studied histone modifying enzymes within the still expanding field of epigenetics. They are validated targets for anticancer therapy with five approved drugs on the market and a growing number of HDAC inhibitors in promising clinical trials ,4,5,6,7.In previous work, we were able to show that carbamates might serve as promising prodrugs for hydroxamic acids . Our hypFor this study, we synthesized carbamates of the FDA-approved HDAC inhibitor SAHA with varying side chains in order to validate our previous findings and to investigate the effect of the different substitution patterns on the stability and inhibition properties. We also synthesized a water-soluble carbamate with a terminal basic moiety, aiming at improved pharmacokinetic properties, which would especially be beneficial for animal studies.1, vorinostat; see N,N-disubstituted carbamoyl chlorides. As carbamoyl chlorides are very unstable and difficult to isolate N-isopropylcarbamate (2a). Remained as a white powder ; 1H NMR : δ (ppm) = 11.24 , 9.84 , 7.61–7.54 , 7.31–7.25 , 7.04–6.99 , 3.64–3.53 2), 2.29 , 2.06 , 1.63–1.48 2), 1.34–1.27 2), 1.08 2); 13C NMR : δ (ppm) = 171.62 (CO), 170.42 (CO), 154.51 (COO), 139.77 (Ar-C1), 129.04 , 123.33 (Ar-C4), 119.42 , 43.46 (CH(CH3)2), 36.80 (Ar-NH-CO-CH2), 32.36 (NH-CO-CH2), 28.80 (CH2), 28.68 (CH2), 25.42 (CH2), 25.09 (CH2), 22.80 ((CH3)2); MS-ESI+: m/z 350 [M + H]+, 372 [M + Na]+.[(8-Anilino-8-oxo-octanoyl)amino]N-benzylcarbamate (2b). Remained as a white solid ; 1H NMR : δ (ppm) = 11.36 , 9.84 , 8.22 , 7.60–7.56 , 7.36–7.22 , 7.04–6.99 , 4.23 , 2.28 , 2.07 , 1.63–1.47 2), 1.36–1.24 2); 13C NMR : δ (ppm) = 171.63 (CO), 170.54 (CO), 155.79 (COO), 139.75 (Ar-C1), 139.49 (Ar-C1), 129.04 , 128.72 , 127.46 , 127.38 (CH2-Ar-C4), 123.32 (NH-Ar-C4), 119.43 , 44.49 (CH2-Ar), 36.78 (Ar-NH-CO-CH2), 32.35 (NH-CO-CH2), 28.81 (CH2), 28.67 (CH2), 25.41 (CH2), 25.09 (CH2); MS-ESI+: m/z 398 [M + H]+, 420 [M + Na]+.[(8-Anilino-8-oxo-octanoyl)amino]N-ethylcarbamate (2c). Remained as a white powder ; 1H NMR : δ (ppm) = 11.27 , 9.84 , 7.65–7.55 , 7.31–7.25 , 7.04–6.99 , 3.09–2.99 , 2.29 , 2.07 , 1.62–1.46 2), 1.35–1.24 2), 1.03 ; 13C NMR : δ (ppm) = 171.69 (CO), 170.42 (CO), 155.20 (COO), 139.82 (Ar-C1), 129.04 , 123.33 (Ar-C4), 119.43 , 36.76 (Ar-NH-CO-CH2), 35.90 (CH2-CH3), 32.35 (NH-CO-CH2), 28.79 (CH2), 28.67 (CH2), 25.42 (CH2), 25.12 (CH2), 14.26 (CH3); MS-ESI+: m/z 336 [M + H]+, 358 [M + Na]+.[(8-Anilino-8-oxo-octanoyl)amino]N-phenylcarbamate (2d). Remained as a white solid ; 1H NMR : δ (ppm) = 11.54 , 10.15 , 9.82 , 7.61–7.55 , 7.48–7.43 , 7.35–7.23  7.08–6.98 , 2.29 , 2.14 , 1.65–1.48 2), 1.39–1.24 2); 13C NMR : δ (ppm) = 171.62 (CO), 170.69 (CO), 152.74 (COO), 139.77 (Ar-C1), 138.68 (Ar-C1′), 129.35 , 129.02 , 123.50 (Ar-C4), 123.31 (Ar-C4′), 119.48 , 118.82 , 36.81 (Ar-NH-CO-CH2), 32.82 (NH-CO-CH2), 28.82 (CH2), 28.57 (CH2), 25.41 (CH2), 25.08 (CH2); MS-ESI+: m/z 384 [M + H]+, 406 [M + Na]+.[[2-(4-Pentoxyphenyl)acetyl]amino]N-isopropylcarbamate (9). Remained as a white solid ; 1H NMR : δ (ppm) = 11.49 , 7.60 , 7.17 , 6.85 , 3.93 , 3.64–3.53 2), 1.73–1.63 , 1.48–1.37 , 1.08 2), 0.93 ; remark: the two benzylic protons overlap with the signal of the solvent, but could be identified and assigned in a two-dimensional NMR experiment; 13C NMR : δ (ppm) = 168.36 (CO), 157.90 (COO), 154.41 (Ar-C4), 130.46 , 127.29 (Ar-C1), 114.61 , 67.52 (CH2-O), 43.42 (CH(CH3)2), 38.34 (Ar-CH2), 31.21 (CH2), 22.81 (CH2), 19.16 (CH3), 14.08 (C(CH3)2); MS-ESI+: m/z 398.0 [M + H]+, 420.0 [M + Na]+.tert-Butyl-piperazine-1carboxylate (3). A solution of 5 mmol di-tert-butyl dicarbonate in dichloromethane (5 mL/mmol) was slowly added to a solution of piperazine (10 mmol) in dichloromethane (25 mL/mmol). The batch was stirred over night before the solvent was removed under reduced pressure. The addition of water led to precipitation of the raw product, which was filtrated and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate and evaporated to result in 0.616 g of white solid (66% yield). 1H NMR : δ (ppm) = 3.40 2), 2.82 2), 1.47 3); 13C NMR : δ (ppm) = 154.88 (CO), 79.41 (C(CH3)3), 45.88 (N-(CH2)2), 44.66 (N-(CH2)2), 28.38 (C(CH3)3).tert-Butyl 4-(imidazole-1-carbonyl)piperazine-1-carboxylate (4). Three-point-three millimoles tert-butyl-piperazine-1carboxylate (3) were dissolved in dichloromethane (5 mL/mmol) and added dropwise to a solution of carbonyldiimidazole  in dichloromethane (5 mL/mmol) under ice cooling. The batch was stirred over night at room temperature before the solvent was removed under reduced pressure, and the raw product was purified via flash chromatography resulting in 0.850 g of a white solid (91% yield). 1H NMR : δ (ppm) = 7.91–7.90 , 7.22–7.21 , 7.13–7.12 , 3.62–3.58 2), 3.56–3.52 2), 1.49 3); 13C NMR : δ (ppm) = 154.25 (CO), 150.92 (CO), 136.81 (imidazole-C2), 130.01 (imidazole-C4), 117.77 (imidazole-C5), 80.72 (C(CH3)3), 46.26 (N-(CH2)2), 43.18 (N-(CH2)2), 28.19 (C(CH3)3).tert-Butyl 4-(3-methylimidazol-3-ium-1-carbonyl)piperazine-1-carboxylate iodide (5). Three millimoles of the carbamoyl imidazole (4) were dissolved in acetonitrile (5 mL/mmol), and 16 mmol methyl iodide were slowly added dropwise under ice cooling. The batch was stirred over night at room temperature. The precipitate was filtrated and washed with diethyl ether resulting in 0.760 g of a light yellow solid (60% yield). 1H NMR : δ (ppm) = 9.56–9.55 , 7.86–7.84 , 7.64–7.63 , 3.86 , 3.50–3.40 2-(CH2)2), 1.37 3); 13C NMR : δ (ppm) = 154.57 (CO), 147.27 (CO), 137.44 (Imidazole-C2), 123.96 (Imidazole-C4), 121.27 (Imidazole-C5), 80.68 (C(CH3)3), 46.11 (N-(CH2)2), 42.62 (N-(CH2)2), 36.76 (N-CH3), 28.35 (C(CH3)3).O4-[(8-Anilino-8-oxo-octanoyl)amino]O1-tert-butyl piperazine-1,4-dicarboxylate (6). One-point-six-three millimoles of the carbamoyl imidazolium salt (5) were dissolved in acetone and added dropwise to a solution of 1.5 mmol SAHA (1) in DMF (1 mL/mmol). After stirring over night at room temperature, the solvent was removed under reduced pressure, and water was added to the remaining solution. The raw product was extracted with dichloromethane, and the combined organic phases were dried over sodium sulfate. After removal of the solvent, the product was purified via flash chromatography (ethyl acetate/cyclohexane 9:1) to result in 0.240 g of a white solid (34% yield). 1H NMR : δ (ppm) = 9.29 , 7.78 , 7.57–7.51 , 7.34–7.29 , 7.13–7.07 , 3.57–3.41 2-(CH2)2), 2.37 , 2.25 , 1.79–1.66 2), 1.48 3), 1.43–1.39 2). 13C NMR : δ (ppm) = 171.98 (CO), 171.72 (CO), 154.42 (COO), 154.11 (CO), 138.08 (Ar-C1), 128.91 , 124.08 (Ar-C4), 119.74 , 80.43 (C-CH3), 44.17 (N-(CH2)2), 43.10 (N-(CH2)2), 37.03 (Ar-NH-CO-CH2), 32.49 (NH-CO-CH2), 28.31 (C(CH3)3), 28.12 (CH2), 27.95 (CH2), 25.04 (CH2), 24.59 (CH2). MS-ESI+: m/z 477.2 [M + H]+, 499.3 [M + Na]+.[(8-Anilino-8-oxo-octanoyl)amino]piperazin-4-ium-1-carboxylate trifluoroacetate (7). Zero-point-one millimoles of the Boc-protected amine (6) were dissolved in dichloromethane (5 mL/mmol) and cooled to 0 °C. Trifluoroacetic acid (3 mL/mmol) was slowly added dropwise. The batch was stirred for 2 h at room temperature, and the reaction progress periodically was detected via thin layer chromatography. After complete elimination of the protecting group was detected, the solvent was removed under reduced pressure. This was done repetitively until the typical scent of trifluoroacetic acid was no longer recognized. After the final evaporation step, 0.043 g of a white solid remained (90% yield). 1H NMR : δ (ppm) = 11.62 , 9.86 , 9.06 , 7.61–7.56 , 7.31–7.24 , 7.04–6.99 , 3.62 2, 3.14 2-NH2), 2.29 , 2.10 , 1.61–1.48 2), 1.33–1.27 2). 13C NMR : δ (ppm) = 171.34 (CO), 170.58 (CO), 158.73 , 153.74 (COO), 139.75 (Ar-C1), 129.02 , 123.34 (Ar-C4), 119.41 , 42.79 (N-(CH2)2), 41.39 (N-(CH2)2), 36.76 (Ar-NH-CO-CH2), 32.18 (NH-CO-CH2), 28.78 (CH2), 28.65 (CH2), 25.38 (CH2), 25.07 (CH2). MS-ESI+: m/z 377 [M + H]+.50 values were determined with GraphPad Prism 4.02 .Recombinant human HDAC1 and 6 were purchased from BPS Biosciences , and recombinant human HDAC8 was produced as described before . InhibitCrystal structures of human HDAC1 (PDB ID 5ICN)  and huma4 cells per well. Cells were immediately incubated with inhibitors. As these cells already differentiate with 1% DMSO content, inhibitors were prepared as 200× stock solutions in DMSO. In all cases, inhibitors were compared to DMSO vehicle only, and three replicates per concentration were used. Growth inhibition was determined using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay according to the manufacturer’s instructions. Briefly, 5 × 104 of HL60 or U937 cells per well were treated with DMSO stocks of the respective inhibitors or with DMSO in 100 µL cell culture medium and incubated for 72 h at 37 °C and 5% CO2 in transparent 96-well microtiter plates (PAA). Subsequently, 20 µL of the detection reagent  was added to each well and incubated for 4 more hours under the same conditions. After this time, absorption was measured at a wavelength of 490 nm . Data were plotted as absorbance units against the logarithm of compound concentration using GraphPad Prism 4.02 . Fifty percent growth inhibition (GI50) was determined as the compound concentration required to reduce the number of metabolically-active cells by 50% compared to DMSO control.HL60 or U937 cells were seeded in 96-well tissue culture plates at a density of 5 × 10g, 5 min). The supernatant was removed, and the cells were washed with PBS, before they were transferred to 1.7-mL reaction tubes. The cells were lysated with Bio-Rad loading buffer, and the cell suspension was heated to 95 °C for 5 min. The proteins then were separated using a 15% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane . Acetylation levels were detected using an anti-acetyl histone H3 antibody  and an anti-acetyl-α-tubulin antibody . An antibody directed against GAPDH (Sigma Aldrich) was used as a loading control.HL60 cells were treated with the inhibitors in the given concentrations. Cells treated with dimethyl sulfoxide as a vehicle were used as controls. After an incubation time of 4 h, cells were transferred into 50-mL Falcon tubes and centrifuged  as a substrate  and IGEPAL CA-630 . After 2 more hours of incubation at 37 °C and 5% CO2, 50 µL stop solution consisting of 0.5 µL TSA (100 µM) and 5 µL trypsin solution (10 mg/mL) in 44.5 µL cell buffer were added. This mixture was incubated for a further 20 min at 37 °C. Fluorescence was measured in a plate reader (BMG Polarstar) with excitation at λ = 390 nm and emission at λ = 460 nm. IC50 values were determined with GraphPad Prism 4.02.To assess cellular inhibition of HDAC activity, 7.5 × 102O containing 0.05% TFA, Eluent B: acetonitrile containing 0.05% TFA, linear gradient conditions ; injection volume: 30 µL, flow rate: 1 mL/min; detection wavelength: λ = 254 nm. For evaluation, the ChemStation software from Agilent was used. To quantify the released amount of SAHA and bufexamac, calibration curves were plotted by incubating DMSO stocks of these compounds under in vitro assay conditions with final compound concentrations ranging from 1 µM–200 µM. HPLC analysis with the conditions described above led to the following equations: y = 13.935x − 27.918 for SAHA and y = 0.6099x + 1.1301 for bufexamac.For stability tests, the carbamates were subjected to the described in vitro assay conditions (see above) without adding the substrate ZMAL and without the second incubation step with trypsin. Compounds were applied in a final concentration of 2.4 mM, and recombinant HDAC1 was used as the enzyme. After 90 min of incubation at 37 °C, the release of SAHA was detected via HPLC on an Agilent 1200 series device. The employed analytical column was a Kinetex 5u XB-C18, 100 Å, 250 × 4.6 mm  with the ReproSil 100 C18 5-µm precolumn . The following conditions were applied: Eluent A: HThe aim of this study was to validate our previously-postulated prodrug concept for hydroxamic acid containing HDAC inhibitors, which would be beneficial, e.g., for increased aqueous solubility or improved cellular uptake. However, the synthesis and evaluation of a subset of carbamates of the FDA-approved HDAC inhibitor SAHA strongly suggest that at least some of the synthesized carbamates are potent inhibitors themselves, representing a completely new warhead for HDAC inhibition. Generally, in light of the similar cellular potency of hydroxamates and some of the carbamates in the cells, we assume a weaker binding of the carbamate function to the zinc and/or steric hindrance for decreased in vitro potency, but an improved cellular uptake and possibly increased cleavage to the hydroxamate in the cellular setting. Thus, this study paves the way toward further analysis of carbamates of hydroxamates as new HDAC inhibitor warheads, but still shows potential for carbamates as prodrugs for hydroxamates.To refine and validate these findings, a larger library of carbamates of different hydroxamic acid containing HDAC inhibitors should be set up and tested for HDAC inhibition, selectivity and stability.


The structures of 1−5 were determined by spectroscopic analysis and/or synthesis. Compound 1 inhibited the division of fertilized sea urchin eggs and compound 4 showed mild cytotoxity against HCT116 cells .Pentylphenols  The methyl protons at δH 0.83 were coupled to the methylene protons at δH 1.21. The methylene protons at δH 2.57 were coupled to the methylene protons at δH 1.50, which were in turn coupled to the other methylene protons at δH 1.21  did not show any HMQC correlations, but HMBC correlations to two olefinic carbons, suggesting thepresence of an OH group coupled with the molecular formula of 1. Bromo, n-pentyl and hydroxyl positions on the benzene ring were determined by comparison with calculated 1H and 13C chemical shift values, and by HMBC correlations of OH/C-1, OH/C-2, H-2/C-1, H-2/C-7, H-5/C-3, H-5/C-4, H-5/C-6, H-7/C-2, H-7/C-3, H-7/C-4. Since decomposition of 1 prevented us from characterizing this compound completely, synthesis of 1 was attempted (1a). The Grignard reaction of 1a with pentylmagnesium chloride afforded alcohol 1b. Mesylation of 1b, followed by reduction of the mesylate with NaBH4, gave 4-bromo-3-pentylanisole (1d). Cleavage of the ether 1d with phenyltrimethylsilane/iodine yielded 1 as the sole product. 1H-NMR data of the product were identical with those of the naturally occurring compound. The known 3-pentylphenol (2) was identified by comparison of its NMR data with those of synthetic 2 (2b with H2/Pd-C gave 3-pentylanisole (2e). Cleavage of the ether 2e with phenyltrimethylsilane/iodine yielded 2 as the sole product. 1H-NMR data of the product were in agreement with those of natural compound 2. Compound 1 completely inhibited the first cleavage of fertilized sea urchin eggs at 1 ppm.The  δH 1.21 . Furtherety in 1 . The prottempted . The synthetic 2  12]. Re. Re1H- a13C-NMR and HRFABMS data  for compound 3 provided the molecular formula C17H32O3, which accounted for two degrees of unsaturation. In the 13C-NMR spectrum, 17 carbon signals were observed, including a carbonyl carbon, two methine carbons, 13 methylene carbons and a methyl carbon. The 13C-NMR resonance at δC 176.9 indicated the presence of a carboxyl group, which was substantiated by IR absorption bands at 1700 and 3300 cm−1. Four upfield shifted signals  in the NMR spectra suggested that 3 should contain a cyclopropane ring. These data revealed compound 3 to be a cyclopropane fatty acid. Analysis of COSY, HMQC and HMBC spectra permitted assignment of the protons of the cyclopropane ring . This was confirmed by comparison of the 1H- and 13C-NMR chemical shifts within the cyclopropane ring for 3with those of cis- and trans-1,2-disubstituted cyclopropanes  for compound 4 provided the molecular formula C11H12O4, which indicated six degrees of unsaturation. The IR absorption bands at 1,680 and 3,250 cm−1 indicated the presence of carbonyl and hydroxyl groups. 1H- and 13C-NMR data analysis indicated the presence of a carbonyl carbon (δC 188.0), a cis double bond , a tetrasubstituted double bond , a trisubstituted double bond , an oxygenated methine , two oxygenated methylenes  and a methylene . The NMR data of 4 showed close similarity to those of the known compound 15 for compound 5 provided the molecular formula C11H12O4. Spectroscopic data for 5 showed close similarity to those of 4  of H-9 in 4 was at lower field than that (δH 6.38) in 5 owing to the magnetic anisotropy effect of the carbonyl group, suggesting an E configuration for the C-3,9 double bond of 4 and thus a Z configuration for that of 5. This was confirmed by NOEDS experiments. Irradiation of H-4 of 4 resulted in enhancement of H-9 and H-5b, and H-4 of 5 showed NOEs to H-10 and H-5b. Compound 4 showed weak cytotoxity against HCT116 cells  in a dose dependent manner (IC50: >20 ppm). We could not evaluate the activity of unstable compound 5 because of the loss of 5 with the formation of insoluble material.Analysis of 13C-NMR  and HREIose of 4 . The lar11 compounds 6–21have been isolated from ascidians, sponges and cyanobacteria . Pentylphenols are known to be formed with a loss of CO2 from a C12 parent (six acetates)  to give 12 fractions. An active fifth fraction (240 mg) was subjected to further separation by CC on silica gel using the gradient solvent mixture hexanes-CH2Cl2-MeOH to give 19 fractions. Active fractions were combined and the mixture (100 mg) was subjected to ODS column chromatography (100% MeOH) to give 14 fractions. The second fraction (10.6 mg) was purified by HPTLC on silica gel using hexanes-EtOAc (6:1) to afford 1 (1.0 mg) and 2 (0.5 mg). The fifth fraction (21 mg) was separated by HPLC on silica gel using hexanes-CHCl3-isopropanol (50:10:3) to afford crude 3 (12 mg), which was purified by reversed phased HPLC on ODS using 0.01 M NH4Cl in MeCN-MeOH-H2O (50:48:2) to give 3 (2.0 mg). The sample of second collection  was soaked in 50 mL acetone at room temperature (rt) and left in the dark for 8 h. After filtration, the residue was again extracted with acetone. The acetone extracts were concentrated under reduced pressure to give a residual oil. The oil was quickly partitioned between H2O and EtOAc three times to give 123.2 mg of EtOAc extract. This extract was suspended in MeOH and H2O (1:1) and then successively extracted with hexanes and chloroform (CHCl3), which were evaporated to give hexanes extract and CHCl3 extract. The CHCl3 part was subjected to reversed phased HPLC on ODS using MeOH-H2O (7:3) to furnish compound 4 (20 mg) in pure form, and a mixture of compound 4 and related compound 5 (10 mg). Further purification of the mixture by HPLC on ODS using MeOH-H2O (7:3) to give 4 (6 mg) and 5 (2 mg).The brown tunicate collected from Hateruma Island was kept frozen during transportation. Theascidian 4-Bromo-3-pentylphenol (1). Colorless oil; 1H- and 13C-NMR (CDCl3) data, see m/z 244 , 242 , 109 .cis-3-(2-Undecylcyclopropyl)propionic acid (3). Colorless powder; [α]25D + 11 (c 0.026 CHCl3); FT/IR (film) νmax 2950, 2830, 1700 cm−1; 1H-NMR  δ 2.44 , 1.75 , 1.49 , 1.36 , 1.2–1.3 (14H), 1.15 , 0.86 , 0.71 , 0.71 , 0.60 , −0.27 ; 13C-NMR  δ 176.85, 33.98, 31.87, 30.09, 29.65, 29.60, 29.57, 29.30, 29.18, 28.59, 24.10, 22.63, 15.95, 15.08, 14.05, 10.74; HRAPCIMS m/z (M + H)+ 269.2462 .(E)-5-Hydroxy-7-(3-hydroxypropylidene)-7,7a-dihydrocyclopenta[b]pyran-6(2H)-one (4). Colorless oil; [α]29D + 2.4 (c 0.060 CHCl3); UV (MeOH) λmax 227 (logε 4.0), 330 (logε 3.7) nm; FT/IR (film) νmax 3350, 2920, 2850, 1685, 1420, 1150, 1046 cm−1; 1H-NMR  and 13C-NMR (CDCl3) data, see m/z (M + Na)+ 231.0698 , m/z (M + H − H2O)+ 191.0688 .(Z)-5-Hydroxy-7-(3-hydroxypropylidene)-7,7a-dihydrocyclopenta[b]pyran-6(2H)-one (5). Colorless oil; [α]25D + 5.4 (c 0.058 CHCl3); UV (MeOH) λmax 228 (logε 4.0), 330 (logε 3.6) nm; FT/IR (film) νmax 3350, 2920, 2850, 1684, 1420, 1151, 1058 cm−1; 1H- and 13C-NMR (CDCl3) data, see m/z (M + H − H2O)+ 191.0736 .4-Bromo-3-(1-hydroxybutyl)anisole (1b). To a solution of methyl 2-bromo-5-methoxybenzoate (1a)  in tetrahydrofuran  was added butylmagnesium chloride  via syringe at rt. The mixture was stirred at rt for 6 h and quenched with saturated NH4Cl solution. The products were extracted with CHCl3. The CHCl3 solution was dried (Na2SO4) and concentrated in vacuo. The residual oil (430 mg) was purified by column chromatography on silica gel (20 g) using hexanes-EtOAc (9:1) to afford alcohol 1b . Colorless solid; mp 42−44 °C; UV (MeOH)max 228 (logε 4.0), 281 (logε 3.2) nm; FT IR νmax (KBr) 3300, 3005, 2960, 2920, 2830, 100, 1580, 1460 cm−1; 1H-NMR  δ 7.35 , 7.08 , 6.65 , 4.97 , 3.77 , 1.72 , 1.61 , 1.45 , 1.35 , 0.89 ; 13C-NMR  δ 159.3, 145.0, 133.2, 114.7, 112.5, 112.2, 73.0, 55.5, 37.4, 28.0, 22.5, 14.0; LRESI(+)MS (relative intensity) m/z 295  and 297 .4-Bromo-3-butylanisole (1d). To a solution of alcohol 1b  and triethylamine  via syringe at rt. After being stirred at rt for 2 h, the mixture was quenched with methanol (0.1 mL) and stirred at rt for 1 h. The mixture was diluted with H2O (5 mL) and the products were extracted with CHCl3. The CHCl3 solution was dried (Na2SO4) and concentrated in vacuo. The residual oil (75 mg) was then separated by CC  to give crude mesylate 1c (47 mg). The mesylate (47 mg) was dissolved in dimethyl sulfoxide . To the solution was added a solution of NaBH4  in DMSO (2 mL) via syringe. The mixture was stirred at rt for 24 h and quenched with 8 drops of acetone. After being stirred for 1 h, H2O (10 mL) was added. The products were extracted with hexanes (5 mL × 3). The hexanes solution was dried over Na2SO4 and concentrated in vacuo. The residue (34 mg) was purified by CC on silica gel  to afford 1d . UV (MeOH)max 228 (logε 3.9), 281 (logε 3.3) nm; FT IR νmax (KBr) 3010, 2960, 2930, 2860, 1630, 1560, 1460 cm−1; 1H-NMR  δ 7.37 , 6.74 , 6.59 , 3.76 , 2.65 , 1.59 , 1.33 , 0.89 ; 13C-NMR  δ 160.1, 144.0, 134.1, 117.3, 115.9, 113.7, 55.4, 37.3, 32.4, 30.6, 23.4, 14.8; LRESI(−)MS (relative intensity) m/z 241  and 243. 4-Bromo-3-pentylphenol (1). A solution of ether 1d , phenyltrimethylsilane  and iodine  was heated to 115 °C for 2 h. The mixture was concentrated in vacuo and the residual oil was purified by preparative TLC  to give 4-bromo-3-pentylphenol (1) . Colorless oil; UV (MeOH)max 228 (logε 3.9), 281 (logε 3.3) nm; FT IR νmax (KBr) 3300, 2960, 2920, 2840, 1600, 1575, 1460 cm−1; 1H-NMR  δ 7.33 , 6.69 , 6.52 , 2.62 , 1.55 , 1.32 , 0.89 . 1H NMR  δ 7.20 , 6.32 , 6.06 , 3.98 , 2.58 , 1.52 , 1.21 , 0.83 ; 13C-NMR  δ 156.0, 143.9, 134.0, 117.9, 115.3, 115.2, 36.8, 32.1, 30.2, 23.2, 14.5; LRESI(−)MS (relative intensity) m/z 241  and 243 . 3-Butylanisole (2a). To a solution of alcohol 1b  in methanol (1.0 mL) was added a small amount of 5% palladium on activated carbon. The suspension was stirred at rt under an atmosphere of H2 for 8 h, the mixture was filtered through Celite and concentrated in vacuo. The filtrate was evaporated and the resulting residue (7.5 mg) was then separated by preparative TLC to give ether 2a . Colorless oil; 1H-NMR  δ 7.17 , 6.75 , 6.70 , 3.76 , 2.56 , 1.59 , 1.30 , 0.87 .3-Pentylphenol (2). A solution of ether 2a , phenyltrimethylsilane  and iodine  was heated to 110 °C for 2 h. The mixture was concentrated in vacuo and the residual oil was purified by preparative TLC [hexanes-EtOAc (10:1)] to give 4-pentylphenol (2) . Colorless oil; 1H-NMR  δ 7.12 , 6.73 , 6.73 , 4.70 , 2.53 , 1.56 , 1.29 , 0.87 ;  δ 7.01 , 6.67 , 6.43 , 6.40 , 3.96 , 2.41 , 1.49 , 1.22 , 0.83 ; 13C-NMR  δ 156.8, 145.2, 129.9, 121.2, 116.0, 113.2, 36.5, 32.1, 31.7, 23.2, 14.6.1 and 2, cyclopropane fatty acid 3, and cyclopentenones 4 and 5 from an ascidian, Diplosoma sp. The structures of 1−5 were determined by spectroscopic analysis and/or synthesis. Compounds 1, 3, 4 and 5 were new. Compound 1 inhibited the division of fertilized sea urchin eggs and compound 4 showed mild cytotoxity against HCT116 cells . 1H-NMR spectra of the extract of the separated Prochloron sp. from the body of the ascidian Diplosoma sp. showed the presence of the same peaks as present in those of 4 and 5, suggesting that Prochloron sp. is the actual producers of cyclopentenones.We have isolated pentylphenols


Dillenia serrata Thunb. showed 64% to 73% inhibition on the production of prostaglandin E2 (PGE2) in lipopolysaccharide-induced human whole blood using a radioimmunoassay technique. Three triterpenoids isolated from the root bark of the plant, koetjapic (1), 3-oxoolean-12-en-30-oic (2), and betulinic (3) acids, exhibited significant concentration-dependent inhibitory effects on PGE2 production with IC50 values of 1.05, 1.54, and 2.59 μM, respectively, as compared with the positive control, indomethacin (IC50 = 0.45 μM). Quantification of compounds 1 and 3 in the methanol extracts and fractions were carried out by using a validated reversed-phase high performance liquid chromatography (RP-HPLC) method. The ethyl acetate fraction of the stem bark showed the highest content of both compound 1 (15.1%) and compound 3 (52.8%). The strong inhibition of the extracts and fractions on cyclooxygenase-2 (COX-2) enzymatic activity was due to the presence of their major constituents, especially koetjapic and betulinic acids.The crude methanol extracts and fractions of the root and stem barks of  NMR spectral data were identical to those given in + 493.3300 ; 1H-NMR  δH (ppm): 0.74 , 0.86 (Hα-16), 0.96 , 0.99 , 0.99 (Hα-15), 1.00 , 1.06 , 1.13 , 1.29 , 1.33 (Hα-5), 1.35 (Hα-7), 1.41 (Hα-1), 1.46 (H2-6), 1.53 (Hβ-7), 1.60 (Hα-19), 1.64 (Hα-9), 1.70 (Hβ-19), 1.73 (Hβ-15), 1.79 (Hβ-21), 1.80 (Hβ-1), 1.88 (H2-23), 1.91 (Hβ-18), 1.98 (Hβ-16), 2.29 (Hα-2), 2.53 (Hβ-2) and 5.19  and 12.10 ; 13C-NMR  δC (ppm): 15.3 (C-25), 16.9 (C-26), 19.6 (C-6), 21.6 (C-24), 23.6 (C-11), 26.0 (C-27), 26.2 (C-15), 26.7 (C-23), 26.8 (C-16), 28.6 (C-28), 28.7 (C-29), 31.1 (C-21), 32.0 (C-17), 32.1 (C-7), 34.2 (C-2), 36.6 (C-10), 38.5 (C-22), 39.0 (C-1), 39.7 (C-8), 41.7 (C-14), 42.8 (C-19), 43.6 (C-20), 46.6 (C-9), 47.2 (C-4), 48.3 (C-18), 54.7 (C-5), 122.2 (C-12), 144.9 (C-13), 178.5 (C-30) and 216.7 (C-3). NMR spectral data were identical to those in − 455.35252 ; 1H-NMR  δH (ppm): 0.64 , 0.75 , 0.84 (Hα-1), 0.85 , 0.86 , 0.92 , 0.97 (Hα-12), 1.09 (Hα-15), 1.15 (Hβ-11), 1.24 (Hα-9), 1.30 , 1.37 , 1.44 , 1.50 , 1.55 (Hβ-1), 1.60 (Hβ-12), 1.64 , 1.79 , 2.11 (Hβ-16), 2.21 , 2.95 , 4.30 , 4.55 , 4.68  and 12.10 ; 13C-NMR  δC (ppm): 14.8 (C-27), 16.2 (C-25), 16.3 (C-24), 16.4 (C-26), 18.4 (C-6), 19.4 (C-30), 20.9 (C-11), 25.5 (C-12), 27.6 (C-2), 28.6 (C-23), 29.7 (C-15), 30.5 (C-21), 32.2 (C-16), 34.4 (C-7), 36.8 (C-22), 37.2 (C-10), 38.0 (C-13), 38.7 (C-1), 38.9 (C-4), 40.7 (C-8), 42.5 (C14), 47.1 (C-19), 48.9 (C-18), 50.4 (C-9), 55.3 (C-5), 55.9 (C-17), 77.2 (C-3), 110.1 (C-29), 150.8 (C-20) and 177.7 (C-28). NMR spectral data were identical with reference -PGE2  and incubated for 18–24 h at 4 °C. After incubation, dextran-charcoal (200 μL) was added to the mixture and incubated for 10 min at 0 °C. The supernatant was then separated by centrifugation at 3000× g for 15 min at 4 °C and pipetted (300 μL) into liquid scintillation cocktail (3 mL). The radioactivity was measured using a liquid scintillation analyzer.Human whole blood was drawn using aseptic vein puncture from the same donors of healthy volunteers when they had not taken any medicine or supplements during the last two weeks and fasted for 8 h prior to blood being withdrawn. The blood sample was prevented from coagulation by adding 10% (2 (pg/0.1 mL) in the blood was calculated using a semi-logarithmic graph of standard PGE2. Previously, standard PGE2 (1 mg/mL) had been serially diluted to concentrations ranging from 2.45 to 400 pg/0.1 mL. The interference of compounds in the crude methanol extracts and fractions towards RIA method was checked by adding crude extracts and fractions to the standards and found not to interfere with the measurements. The average count per min (cpm) values of standards and samples (B) resulting from antibody-antigen (labeled PGE2) binding in the plasma were subtracted from the non-specific binding (Nc) together with the total binding between antibody and antigen (Bo). The normalized percent bound (% B/Bo) was then determined using Equation (1): Concentration of PGEo values were plotted against their respective concentrations of standard PGE2 in picograms (pg) semi-logarithmically. Thus, the interpolation of % B/Bo values for samples and indomethacin using PGE2 standard curve resulted in the determination of PGE2 concentration in the blood. The percentage inhibition (% I) was then calculated using Equation (2).The calculated % B/Bp < 0.05 representing a significant difference as compared to control. GraphPad Prism 5 was used to determine the IC50 value of active extracts and compounds.The HPLC analysis and bioassay were performed in triplicate and the data were expressed as means ± SD. Empower software (Waters) was used to construct a calibration curve of koetjapic and betulinic acids as well as their quantification. The bioactivity data were analyzed using Statistical Package for Social Sciences (SPSS) software Version 17. Data were analyzed using one way ANOVA analysis with a probability D. serrata possesses a promising inhibitory effect on LPS-induced PGE2 production. Three triterpenoids from this plant, koetjapic acid (1), 3-oxoolean-12-en-30-oic acid (2), and betulinic acid (3), were found to inhibit LPS-induced PGE2 production concentration-dependently. Although substantial amounts of koetjapic (1) and betulinic (3) acids in the crude methanol extracts and fractions of D. serrata may affect LPS-induced PGE2 production, the presence of other compounds in the extracts and fractions may also contribute to the observed activity. Further studies need to be carried out to investigate the effect of these compounds on COX-2 especially in vitro prostaglandin biosynthesis catalysed by COX-2 in cell free assays and expression of COX-2 protein.Our study revealed that


The use of spirobifluorene prevents fluorescence quenching by intramolecular energy transfer and/or electron transfer among the chromophores in the excited state. The emission spectra of 4-PySBF exhibited a red shift of 20 nm in comparison to a model compound , but its UV-Vis spectrum remained unchanged.We have synthesized a highly luminescent  pyrene dye based on a spirobifluorene skeleton [2,2′,7,7′-tetrakis(7- Recently, blue emitting dyes with high efficiency   and starSpirobifluorene derivatives are considered the most promising candidates for organic optoelectronics ,9,10,11.In this paper, we report the synthesis and characterization of a new pyrene dye that is based on the spirobifluorene skeleton. Pyrene derivatives are highly absorptive ,24,25,26tert-butyl-1-pyrenyl)-9,9′-spirobi [9H-fluorene] (4-PySBF), and a model compound, 9,9′-dioctyl-2,7-bis(7-tert-butyl-1-pyrenyl)-9H-fluorene (2-PyF), were synthesized in 37% and 24% yields, respectively, by palladium-catalyzed Suzuki-Miyaura coupling reactions (tert-butylpyrene-1-boronic acid pinacol ester (1) and 1-bromo-7-tert-butylpyrene (3), were prepared from pyrene according to a previously reported method ), where kf, kisc, knr, and kq[O2] denote the rate constants for fluorescence radiation, intersystem crossing, nonradiative decay, and fluorescence quenching by oxygen, respectively, and τs denotes the lifetime (s = singlet)  = 0. As knr for aromatic hydrocarbons is known to be negligible [kisc should be the dominant component in kisc+knr [kq[O2] is estimated to be nearly diffusion controlled (up to 3.89 × 1010 m−1 s−1 in hexane) [2] is less than 2.0 × 10−3 M in usual solvents. These data imply that the maximum possible value of kq[O2] is 8.0 × 107 s−1. As can be seen from kf values for 4-PySBF and 2-PyF are much larger than the kq[O2] values. This situation is most probably responsible for the observed large values of Φf for both compounds, even in the presence of oxygen.Fluorescence quantum yield can be expressed as Φsinglet) . Table 1gligible , kisc shkisc+knr ,36,37,38 hexane) ,39, and 4-PySBF) and measured its photophysical properties. In comparison to a model compound, 2-PyF, the emission spectrum of 4-PySBF exhibited a red shift of 20 nm, but its UV-Vis spectrum remained unchanged. It is not easy to find a satisfactory explanation for this observed red-shift at the present stage. However, in our next study, we intend to calculate the electron states of these compounds in the ground and excited states by the molecular orbital (MO) method and examine this conjugation system in greater detail. Further, we intend to apply 4-PySBF in a previously developed cholesteric liquid crystal laser (distributed feedback laser) as the laser dye in the future.In summary, we have synthesized a highly luminescent spirobifluorene dye  with tetrapyrene chromophores (1H- and 13C-NMR spectra were recorded on a 400 MHz JEOL LMN-EX400 instrument with tetramethylsilane (TMS) as the internal standard. FT-IR spectra were recorded on a JASCO FT-IR 469 plus spectrometer. Melting points were obtained by a Stuart Scientific Melting Point Apparatus SMP3. MS spectra (FAB) were obtained by JEOL JMS700 mass spectrometer. UV-Vis spectra were recorded with a Beckman Coulter DU800 UV-Vis Spectrophotometer. Fluorescence spectra were recorded on a JASCO FP-6500 Spectrofluorometer. Quantum Yields were measured by a Hamamatsu Photonics C9920-02 Absolute PL Quantum Yield Measurement system. Fluorescence lifetimes were measured using a Hamamatsu Photonics OB 920 Fluorescence Lifetime Spectrometer. All the 3)4, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane were obtained from TCI. 9,9-Dioctyl-fluorene-2,7-bis(trimethylene-borate) (4) and PdCl2(PPh3)2, were obtained from Aldrich. 2,2′,7,7′-Tetrabromospirobifluorene (2) was a gift of JFE Chemical Coporation (Japan).Unless otherwise noted, all reagents, chemicals and solvents were obtained from commercial sources and used without further purification. Pyrene, 2-chloro-2-methylpropane, Pd-9,9′-spirobi [9H-fluorene] tert-butylpyrene-1-boronic acid pinacol ester  and cesium carbonate  were mixed together with Pd(PPh3)4  and degassed THF (30 mL). The mixture was refluxed for 24 h. After cooling to room temperature, the resulting mixture was extracted with chloroform. The organic extract was washed sequentially with water and brine and then dried over MgSO4. After removal of the solvent, the residue was purified by column chromatography (chloroform:hexane = 1:5) and preparative HPLC (CHCl3). After removal of the solvent, the product was recrystallized from cyclohexane to afford 4-PySBF as a yellow powder in 37% yield. 1H-NMR : δ 8.23–7.36 , 1.53  ppm.; 13C-NMR : δ 149.3, 149.1, 140.9, 140.7, 137.4, 131.3, 130.7, 130.5, 130.3, 128.4, 128.2, 128.1, 127.7, 127.5, 127.3, 127.2, 126.5, 125.0, 124.9, 124.4, 123.1, 122.4, 122.0, 120.2 (aromatic C), 66.3 (spiro-C), 35.2 (Ar-C (CH3)3), 31.9 (Ar-C (CH3)3) ppm; FT-IR (KBr): 3042, 2961, 2901, 2867, 1594, 1457, 1227 cm−1; mp 286–288 °C; HR-MS (FAB+) Calcd. for C105H80 [M] 1341.6294, found [M+] 1341.6290. Under an argon atomosphere, 2,2′,7,7′-tetrabromospirobifluorene , 7-9,9′-Dioctyl-2,7-bis (7-tert-butyl-1-pyrenyl)-9H-fluorene tert-butylpyrene  and 9,9-dioctyl-fluorene-2,7-bis(trimethyleneborate) (0.28 g 0.5 mmol) were mixed together with Pd(PPh3)4 , degassed toluene (20 mL), one drop of Aliquat 336, and 1M aqueous sodium carbonate solution (2 mL). The resulting mixture was refluxed for 24 h. After cooling to room temperature, the mixture was extracted with chloroform. The organic extract was washed sequentially with water and brine and then dried over MgSO4. After removal of the solvent, the residue was purified by column chromatography using chloroform/hexane  following HPLC (CHCl3). After subsequent recrystallization in ethanol, 2-PyF was obtained as a yellow powder in 24% yield. 1H-NMR : δ 8.27–7.97 , 7.69–7.67 , 2.12–2.08 , 1.60 , 1.25–1.19 , 0.96 , 0.82  ppm; 13C-NMR : δ 151.2, 149.1, 140.1, 140.0, 138.1, 131.4, 130.9, 130.4, 129.4, 128.4, 127.6, 127.5, 127.3, 125.4, 125.3, 125.0, 124.5, 123.2, 122.4, 122.0, 119.8 (aromatic C), 55.3 (spiro-C), 40.4 , 35.2 (Ar-C (CH3)3), 31.9 (Ar-C (CH3)3), 30.1, 29.7, 29.4, 29.3, 24.2, 22.7, 14.2  ppm; FT-IR (KBr): 3050, 2953, 2925, 2853, 1608, 1481, 1457, 1310 cm−1; mp 206–208 °C; HR-MS (FAB+) calcd. for C69H74 [M] 902.5791, found [M+] 902.5786. Under an argon atomosphere, 2-bromo-7-


N-acetyl- hexosaminidases and GH84 β-N-acetylglucosaminidases. Here, we focused on the design, synthesis and inhibition potency of a series of new derivatives of NAG-thiazoline modified at the C-6 position. Dimerization of NAG-thiazoline via C-6 attached triazole linkers prepared by click chemistry was employed to make use of multivalency in the inhibition. Novel compounds were tested as potential inhibitors of β-N-acetylhexosaminidases from Talaromyces flavus, Streptomyces plicatus (both GH20) and β-N-acetylglucosaminidases from Bacteroides thetaiotaomicron and humans (both GH84). From the set of newly prepared NAG-thiazoline derivatives, only C-6-azido-NAG-thiazoline displayed inhibition activity towards these enzymes; C-6 triazole-substituted NAG-thiazolines lacked inhibition activity against the enzymes used. Docking of C-6-azido-NAG-thiazoline into the active site of the tested enzymes was performed. Moreover, a stability study with GlcNAc-thiazoline confirmed its decomposition at pH < 6 yielding 2-acetamido-2-deoxy-1-thio-α/β-D-glucopyranoses, which presumably dimerize oxidatively into S-S linked dimers; decomposition products of NAG-thiazoline are void of inhibitory activity.NAG-thiazoline is a strong competitive inhibitor of GH20 β- Hexyne (0.1 mL) and CuI (30 mg) were added and reaction mixture was stirred overnight at room temperature. The product was isolated after evaporation of solvents by column chromatography . Butyl derivative 3 was isolated as a white solid . 1H-NMR : 0.967 , 1.395 , 1.665 , 2.237 , 2,710 , 3.575 , 4.245 , 4.380 , 4.474 , 4.717 , 6.330 , 7.695 . 13C-NMR : 14.41 (C-6'), 20.82 (CH3), 23.39 (C-5'), 26.18 (C-3'), 32.96 (C-4'), 53.25 (C-6), 73.05 (C-4), 74.17 (C-3), 74.50 (C-5), 80.90 (C-2), 90.66 (C-1), 124.41 (C-1'), 149.36 (C-2'), 170.84 (=C-). HRMS: C14H23O3N4S calcd. 327.14854; m/z [M+H]+ found 327.14848.d-glucopyrano--1,2-Dideoxy-2'-methyl-6-(4-phenyltriazolyl)-α-∆2'-thiazoline (4): Compound 4 was prepared analogously as described for the compound 3 from azide 2 (100 mg) and phenylacetylene (0.1 mL) yielding title compound 4 as a white solid . 1H-NMR : 2.239 , 3.631 , 3.689 , 4.269 , 4.399 , 4.598 , 4.806 , 6.368 , 7.352 , 7.443 , 7.825 , 8.283 . 13C-NMR : 20.92 (CH3), 53.38 (C-6), 72.29 (C-4), 74.18 (C-3), 74.39 (C-5), 80.74 (C-2), 90.63 (C-1), 123.60 (C-1'), 126.95 (C-ortho), 129.59 (C-para), 130.23 (C-meta), 131.92 (C-ipso), 148.94 (C-2'), 171.10 (=C-). HRMS: C16H19O3N4S calcd. 347.11724; m/z [M+H]+ found 347.11704.d-glucopyrano--1,2-Dideoxy-6-(4-hydroxymethyltriazolyl)-2'-methyl-α-∆2'-thiazoline (5): Compound 5 was prepared as described for the compound 3 from azide 2 (100 mg) and propargylalcohol (0.1 mL) yielding hydroxymethyl derivative 5 as a white solid . 1H-NMR : 2.241 , 3.562 , 3.639 , 4.222 , 4.372 , 4.542 , 4.696 , 4.751 , 6.337 , 7.887 . 13C-NMR : 20.86 (CH3), 53.26 (C-6), 56.77 (C-3'), 72.89 (C-4), 74.20 (C-3), 74.39 (C-5), 80.78 (C-2), 90.70 (C-1), 124.41 (C-1'), 149.20 (C-2'), 170.95 (=C-). HRMS: C11H17O4N4S calcd. 301.09650; m/z [M+H]+ found 301.09650.d-glucopyrano--1,2-Dideoxy-2'-methyl-6-(4-trimethylsilyltriazolyl)-α-∆2'-thiazoline (6): Compound 6 was prepared as described for compound 3 from azide 2 (100 mg) and trimethylsilylacetylene (0.1 mL) yielding trimethylsilyl derivative 6 as a white solid . 1H-NMR : 0.334 3), 2.261 , 3.594 , 3.641 , 4.272 , 4.405 , 4.569 , 4.796 , 6.341 , 7.970 . 13C-NMR : −0.78 (Si(CH3)3), 21.10 (CH3), 52.77 (C-6), 72.99 (C-4), 74.27 (C-3), 74.58 (C-5), 80.82 (C-2), 90.61 (C-1), 132.82 (C-1'), 147.47 (C-2'), 171.39 (=C-). HRMS: C13H23O3N4SSi calcd. 343.12546; m/z [M+H]+ found 343.12537.d-glucopyrano--1,2-Dideoxy-6-[4-(hex-5-ynyl)triazolyl]-2'-methyl-α-∆2'-thiazoline (7) d-glucopyrano--and 1,4-Bis[-triazol-4-yl]-butane (9)2  was dissolved in the mixture of acetonitrile (15 mL) and N,N-diisopropylethylamine (0.6 mL). Octa-1,7-diyne (0.08 mL) and CuI (150 mg) were added and the reaction mixture was stirred overnight at room temperature. The products were isolated after evaporation of solvents by column chromatography . Monomeric derivative 7 was isolated as a white solid  and dimeric derivative 9 was then eluted by methanol gradient as a white solid .Compound Compound7: 1H-NMR : 1.571 , 1.802 , 2.233 , 2.242 , 2.738 , 3.567 , 4.249 , 4.382 , 4.474 , 4.723 , 6.331 , 7.718 . 13C-NMR : 19.06 (C-6'), 20.81 (CH3), 25.95 (C-3'), 29.22 (C-5'), 29.78 (C-4'), 53.28 (C-6), 70.01 , 74.14 (C-3), 74.50 (C-5), 80.81 (C-2), 85.12(C-7'), 90.62 (C-1), 124.51 (C-1'), 149.01 (C-2'), 171.10 (=C-). HRMS: C16H22O3N4NaS calcd. 373.13048; m/z [M+Na]+ found 373.13033. Compound9: 1H-NMR : 1.734 , 2.228 , 2.754 , 3.561 , 3.584 , 4.230 , 4.371 , 4.485 , 4.712 , 6.329 , 7.717 . 13C-NMR : 20.86 (CH3), 26.18 (C-3'), 30.00 (C-4'), 53.25 (C-6), 73.01 (C-4), 74.20 (C-3), 74.50 (C-5), 80.90 (C-2), 90.70 (C-1), 124.62 (C-1'), 149.03 (C-2'), 170.82 (=C-). HRMS: C24H35O6N8S2 calcd. 595.21155; m/z [M+H]+ found 595.21133.d-glucopyrano--1,2-Dideoxy-2'-methyl-6-[4-(propargyloxymethyl)-triazolyl]-α-∆2'-thiazoline (8) d-glucopyrano--and bis{[-triazol-4-yl]-methyl} ether (10) 8 and 10 were prepared as described for the compounds 7 and 9, respectively, from the azide 2 (300 mg) and dipropargylether (0.08 mL) yielding monomeric derivative 8 as a white solid  and dimeric derivative 10 as a white solid .Compounds Compound8: 1H-NMR : 2.242 , 2.915 , 3.568 , 3.610 , 4.211 , 4.241 , 4.379 , 4.538 , 4.702 , 4.762 , 6.336 , 7.954 . 13C-NMR : 20.83 (CH3), 53.33 (C-6), 58.28 (C-4'), 63.47 (C-3'), 72.99 (C-4), 74.16 (C-3), 74.35 (C-5), 76.73 (C-6'), 80.49 (C-5'), 80.83 (C-2), 90.65 (C-1), 126.62 (C-1'), 145.41 (C-2'), 170.93 (=C-). HRMS: C14H19O4N4S calcd. 339.11215; m/z [M+H]+ found 339.11203.Compound10: 1H-NMR : 2.236 , 3.567 , 3.615 , 4.238 , 4.378 , 4.549 , 4.667 , 4.763 , 6.341 , 7.967 . 13C-NMR : 20.86 (CH3), 53.32 (C-6), 64.12 (C-3'), 72.95 (C-4), 74.15 (C-3), 74.36 (C-5), 80.81 (C-2), 90.68 (C-1), 126.62 (C-1'), 145.73 (C-2'), 170.91 (=C-). HRMS: C22H31O7N8S2 calcd. 583.17516; m/z [M+H]+ found 583.17503.1; 200 mM) was incubated in 50 mM K2HPO4/KH2PO4 buffer pH 6.5 at 35 °C and 850 rpm. Aliquots (10 μL) were taken at regular time intervals (30–60 min), mixed with 95% CH3CN (40 μL) and analyzed by HPLC. Chromatography was carried out on the Shimadzu Prominence UFLC system  consisting of DGU-20A mobile phase degasser, two LC-20AD solvent delivery units, SIL-20ACHT cooling autosampler, CTO-10AS column oven and SPD-M20A diode array detector. The HILIC column TSKgel Amide-80  was used as a stationary phase. The PDA data were acquired in the 190–320 nm range and the 200 nm signal was extracted. Gradient elution: mobile phase A (CH3CN); mobile phase B (H2O); gradient, 0–2 min, 20% B; 2–15 min, 20%–60% B; 15–16 min, 60% B, 16–18 min, 60%–20% B, 18–21 min, 20% B (column equilibration). Flow rate was 1 mL/min at 25 °C. The experiment was monitored for 24 h. Retention times were found as follows: NAG-thiazoline 1, 6.1 min; α-GlcNAc-SH 11a, 6.8 min; β-GlcNAc-SH 11b, 7.1 min; oxidation products, 9–13 min. In situ reduction of the products of spontaneous oxidation was performed by adding dithiothreitol (1 M) to the reaction mixture in the final concentration of 100 mM and monitored by HPLC and NMR.NAG-thiazoline  was dissolved in 50 mM K2HPO4/KH2PO4 buffer pH 6.5 (4 mL) and incubated at 35 °C and 850 rpm for 5 h. Then, the reaction volume was reduced to 2.5 mL in vacuo and the reaction mixture was loaded on a Biogel P-2 column  in water, flow rate 12 mL/h. A mixture of oxidation products was isolated (20 mg). This mixture was further practically inseparable since the compounds coexisted in equilibrium. Analysis by MS revealed the presence of masses corresponding to C8H15NO5S calcd. 237.07, m/z [M–H]− found 236.0; and to C16H28N2O10S2 (tentative structure GlcNAc-S-(1↔1)-S-GlcNAc) calc. 472.1, m/z [M–H]− found 471.0 (HPLC and MS are presented in the Supplementary Information). After application of dithiothreitol, the oxidation mixture was quickly reduced to the anomeric mixture of 2-acetamido-2-deoxy-1-thio-d-glucopyranose 11a (α) and 11b (β). (11a) 1H-NMR : 1.749 , 3.136 , 3.507 , 3.565 , 3.685 , 3.862 , 5.272 , 13C-NMR : 22.45 (2-Ac), 55.29 (C-2), 60.93 (C-6), 70.73 (C-4), 71.37 (C-5), 71.56 (C-3), 80.37 (C-1), 174.27 (CO). (11b): 1H-NMR : 1.742 , 3.124 , 3.150 , 3.174 , 3.352 , 3.396 , 3.577 , 4.385 , 13C-NMR : 22.57 (2-Ac), 59.72 (C-2), 61.25 (C-6), 70.18 (C-4), 75.78 (C-3), 80.23 (C-5), 80.69 (C-1), 174.63 (C=O). Results of NMR analysis of oxidation product mixture are not in contradiction with the structure GlcNAc-S-(1↔1)-S-GlcNAc as proposed from the MS measurement.NAG-thiazoline  was prepared synthetically as a His6-tagged fusion protein  and cloned into the bacterial expression vector pET-15b via the NdeI and BamHI restriction sites. The gene was intracellularly expressed in E. coli BL21(DE3) pLysS strain  under the IPTG (0.5 mM) induction. After 16 h of cultivation at 25 °C, the cells were harvested by centrifugation  and resuspended in a lysis buffer  with 50 mM NaCl, 4 mM MgCl2, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and lysozyme (1 mg/mL). The cells were then incubated for 30 min at 37 °C and disrupted with ultrasound (6 × 1 min). After centrifugation  the supernatant was loaded onto Ni-NTA agarose column . The column was equilibrated with 50 mM Tris/HCl buffer with 150 mM KCl and 10 mM imidazole, pH 7.4. The fusion protein was eluted from the column using 50 mM Tris/HCl buffer supplemented with 150 mM KCl and 250 mM imidazole, pH 7.4. The purity of the fractions was determined by 10% SDS-PAGE.β-eviously . The genO-GlcNAcase from Bacteroides thetaiotaomicron and human O-GlcNAcase were obtained from Prof. D. Vocadlo . The genes were intracellularly expressed in E. coli BL21(DE3) pLysS strain under the induction by IPTG (0.5 mM). After 4 h of cultivation at 16 °C, the cells were harvested by centrifugation . The procedure for the isolation of both O-GlcNAcases was analogous to the purification procedure for β-N-acetylhexosaminidase from Streptomyces plicatus as described above.The genes of N-Acetylhexosaminidase and O-GlcNAcase activities were assayed in end-point experiments using p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside  as a substrate; starting concentration 2 mM. One unit of enzymatic activity was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per minute in 50 mM citrate-phosphate buffer at pH 7.0 and 35 °C. After incubation of the reaction mixture at 35 °C for 10 min, the liberated p-nitrophenol was determined spectrophotometrically at 420 nm under alkaline conditions (50 μL of the reaction mixture was added to 1 mL of 0.1 M Na2CO3).β-N-acetylglucosaminidase reactions was measured in a kinetic assay at 348 nm (isosbestic point of p-nitrophenol), using pNP-β-GlcNAc as substrate with its concentration in the range of 0.04–0.5 mM ; in the inhibition assays the concentration of the inhibitors ranged from 0.001 to 0.5 mM for O-GlcNAcases and from 0.05 to 2 mM for β-N-acetylhexosaminidases. All data were measured in five parallels; the non-linear regression was calculated using Enzfitter . Lineweaver-Burk plots for the individual experiments are shown in the Supplementary information.Michaelis-Menten kinetics of β-2) was constructed in YASARA by modification of the crystal structure of NAG-thiazoline (1) extracted from the enzyme/inhibitor complex of β-N-acetylhexosaminidase from Streptomyces plicatus with NAG-thiazoline (pdb code: 1hp5) [pNP-β-GlcNAc was constructed in YASARA by modification of di-N-acetyl-d-glucosamine (chitobiose), extracted from the crystal structure of chitobiase from Serratia marcescens (pdb code: 1qbb) [C-6-azido-NAG-thiazoline (e: 1hp5) . Quantume: 1hp5)  at the Be: 1hp5)  and incle: 1qbb) .pNP-β-GlcNAc, inhibitor C-6-azido-NAG-thiazoline (2) and NAG-thiazoline (1) were docked in the active site of selected β-N-acetylhexosaminidases (GH20) and β-N-acetylglucosaminidases (GH84) using rigid docking in AutoDock 4.2.3 [O-GlcNAcase from Bacteroides thetaiotaomicron (pdb code: 2chn) [O-GlcNAcase [N-acetylhexosaminidase from Streptomyces plicatus (pdb code: 1hp5) [N-acetylhexosaminidase from Talaromyces flavus (will be published elsewhere). The obtained complexes with best docking scores were used for molecular dynamics simulation in water with YASARA , counter ions to reach neutrality, YASARA 2 force field [The substrate ck 4.2.3  to accomck 4.2.3 , and 100e: 2chn) ; model oidases GH and β-N-e: 1hp5)  and the ce field , periodice field ). Molecuce field  and YASA2–8) or dimerized at C-6 (compounds 9–10) bearing azido or triazole moieties were designed as potential β-N-acetylhexosaminidase inhibitors and synthesized. New compounds were tested for their inhibitory activities with GH20 β-N-acetylhexosaminidases and GH84 β-N-acetylglucosaminidases. One bacterial and one eukaryotic model enzyme of each glycosidase family were selected for the inhibition study: GH20 β-N-acetylhexosaminidases from Streptomyces plicatus  and Talaromyces flavus  and GH84 β-N-acetylglucosaminidases from Bacteroides thetaiotaomicron  and humans. While all the triazole-substituted NAG-thiazolines 3–10 lacked inhibition activity with the enzymes used, the C-6-azido-NAG-thiazoline (2) inhibited most of the enzymes in a moderate way. Elaborated kinetic study was performed to compare both the original 1 and the new inhibitor 2. Molecular docking of C-6-azido-NAG-thiazoline 2 into the active site of the respective enzymes and consequent molecular dynamics simulations of the resulting complexes were performed to corroborate the conclusions obtained from the “wet” experiments with a molecular explanation. Generally, the affinity of β-N-acetylhexosaminidases to modified NAG-thiazoline 2 is lower than to the substrate having more hydrogen bonding partners. A detailed stability study with unsubstituted GlcNAc-thiazoline 1 has shown its decomposition at pH < 6 resulting in the thiazoline ring opening yielding 2-acetamido-2-deoxy-1-thio-α/β-d-glucopyranoses 11a,b, which presumably dimerize oxidatively into S-S linked dimers. Tentative structures of the products were supported by mass spectrometry and NMR measurements. Decomposition products proved to have no inhibitory activity towards β-N-acetylhexosaminidase (T. flavus).A series of novel derivatives of GlcNAc-thiazolines substituted (compounds


Picrasma quassioides (D. Don) Benn. rich in alkaloids. An innovative technique for quality assessment of KMI was developed using high performance liquid chromatography (HPLC) combined with chemometric methods and qualitative and quantitative analysis of multi-components by single marker (QAMS). Nigakinone , one of the most abundant alkaloids responsible for the major pharmacological activities of Kumu, was used as a reference substance. Six alkaloids in KMI were quantified, including 6-hydroxy-β-carboline-1-carboxylic acid (PQ-1), 4,5-dimethoxycanthin-6-one (PQ-2), β-carboline-1-carboxylic acid (PQ-3), β-carboline-1-propanoic acid (PQ-4), 3-methylcanthin-5,6-dione (PQ-5), and PQ-6. Based on the outcomes of twenty batches of KMI samples, the contents of six alkaloids were used for further chemometric analysis. By hierarchical cluster analysis (HCA), radar plots, and principal component analysis (PCA), all the KMI samples could be categorized into three groups, which were closely related to production date and indicated the crucial influence of herbal raw material on end products of KMI. QAMS combined with chemometric analysis could accurately measure and clearly distinguish the different quality samples of KMI. Hence, QAMS is a feasible and promising method for the quality control of KMI.Kumu injection (KMI) is a common-used traditional Chinese medicine (TCM) preparation made from  It is well known that high performance liquid chromatography (HPLC) is one of the most common techniques used for qualitative and quantitative analysis of organic compounds, especially in a complex multi-component system. There are two main methods for simultaneous quantitation of multiple components by HPLC, the normal external standard method (ESM) using multiple reference standards, and qualitative and quantitative analysis of multi-component by single marker (QAMS). Although it indeed has played an important role in many fields of research and production regulation, ESM for quality control and evaluation of multi-component system has become challenging in some cases due to the high cost of some reference standards, complexity and chemical diversity, the instability of detection, and many other uncertainties ,2,3. In Picrasma quassioides (D. Don) Benn., a perennial plant widely distributed throughout southern China +. 1H-NMR  δ: 7.90 , 8.12 , 7.47 , 9.19 , 7.09 , 7.66 , 11.46 . 13C-NMR  δ: 146.00 (C-1), 133.77 (C-3), 116.96 (C-4), 105.47 (C-5), 151.08 (C-6), 119.03 (C-7), 113.58 (C-8), 135.00 (C-10), 120.40 (C-11), 130.33 (C-12), 135.92 (C-13), 168.04 (C-1′); PQ-2: MS m/z: 281.0921 [MH]+. 1H-NMR  δ: 7.92 , 8.83 , 8.64 , 7.69 , 7.50 , 8.07 , 4.47 , 4.08 . 13C-NMR  δ: 115.64 (C-1), 145.33 (C-2), 152.91 (C-4), 140.01 (C-5), 158.41 (C-6), 117.02 (C-8), 130.82 (C-9), 125.33 (C-10), 122.59 (C-11), 124.76 (C-12), 139.13 (C-13), 128.40 (C-14), 130.06 (C-15), 133.47 (C-16), 61.48 (C-17), 61.40 (C-18); PQ-3: MS m/z: 213.0658 [MH]+. 1H-NMR  δ: 8.45 , 8.48 , 8.32 , 7.63 , 7.32 , 7.84 , 11.84 . 13C-NMR  δ: 141.92 (C-1), 135.35 (C-3), 118.43 (C-4), 122.01 (C-5), 129.32 (C-6), 120.13 (C-7), 113.06 (C-8), 131.50 (C-10), 119.85 (C-11), 130.72 (C-12), 135.45 (C-13), 165.55 (C-1′); PQ-4: MS m/z: 241.0969 [MH]+. 1H-NMR  δ: 8.24 , 7.95 , 8.21 , 7.54 , 7.32 , 7.60 , 11.66 , 2.87 , 3.35 . 13C-NMR  δ: 144.04 (C-1), 137.27 (C-3), 112.84 (C-4), 121.66 (C-5), 127.86 (C-6), 119.22 (C-7), 111.95 (C-8), 134.02 (C-10), 121.01 (C-11), 127.10 (C-12), 140.41 (C-13), 31.32 (C-1′), 28.04 (C-2′), 174.10 (C-3′); PQ-5: MS m/z: 251.0815 [MH]+. 1H-NMR  δ: 7.51 , 8.07 , 6.02 , 8.46 , 7.70 , 7.55 , 8.24 , 3.91 . 13C-NMR  δ: 104.20 (C-1), 136.62 (C-2), 93.34 (C-4), 170.60 (C-5), 157.40 (C-6), 116.14 (C-8), 130.02 (C-9), 125.61 (C-10), 123.07 (C-11), 120.62 (C-12), 136.95 (C-13), 139.42 (C-14), 124.85 (C-15), 140.50 (C-16), 40.96 (C-17); PQ-6: MS m/z: 267.0763 [MH]+. 1H-NMR  δ: 7.51 , 8.07 , 6.02 , 8.46 , 7.70 , 7.55 , 8.24 , 3.91 . 13C-NMR  δ: 114.57 (C-1), 145.88 (C-2), 142.68 (C-4), 136.37 (C-5), 157.98 (C-6), 116.68 (C-8), 130.66 (C-9), 125.73 (C-10), 122.67 (C-11), 125.41 (C-12), 138.63 (C-13), 125.96 (C-14), 130.14 (C-15), 134.22 (C-16), 61.01 (C-17).PQ-1: MS Methanol and acetonitrile were of HPLC grade and obtained from Fisher Scientific Co. . All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. . Ultra-pure water was used in the present study and prepared by a lab purification system .t-test of the results from the QAMS method and those from the ESM. Finally multivariate analyses such as HCA, radar plot analysis, and PCA were conducted for sample classification and quality assessment of KMI based on the contents of six alkaloids  to obtain working solutions of each reference standard and the mixed standard for establishing calibration curves. The concentration ranges were 11.0–41.3 μg·mL−1 for PQ-1, 1.95–9.75 μg·mL−1 for PQ-2, 20.6–62.0 μg·mL−1 for PQ-3, 5.22–31.3 μg·mL−1 for PQ-4, 2.00–14.1 μg·mL−1 for PQ-5, and 2.00–100 μg·mL−1 for PQ-6, respectively. All the standard solutions were stored at 4 °C and brought to room temperature before use.Sample solution was prepared by filtration of the mixture of three aliquots of KMI through a 0.45 μm membrane before it was injected for HPLC analysis. Stock standard solutions of the six analytes were prepared by dissolving approximately 10 mg of each reference standard respectively into a 10 mL volumetric flask with acetonitrile to volume, mixed, and then diluted with acetonitrile/0.2% aqueous phosphoric acid . Samples with a volume of 20 μL were injected into the column for separation at a column temperature of 30 °C. At a flow rate of 1.0 mL·min−1, the mobile phase composed of solvent mixture of acetonitrile (solvent A) and 0.2% aqueous phosphoric acid (solvent B) was programmed with a linear gradient of solvent A as follows, 0–30 min, 7–13%; 30–50 min, 13–50%; 50–55 min, 50%. An additional 80 min chromatographic run with acetonitrile increasing from 50% to 90% during 55–80 min was performed to obtain a full chromatogram for checking whether all the components could be eluted within 55 min , on-line degasser (G1379A), autosampler (G1313A), column temperature controller, and variable wavelength UV detector (G1314A) coupled with an analytical workstation . Analytes were separated on a reverse phase Cn 55 min . The detxf) of the co-existing components were calculated by the ratio of the peak areas and the ratio of the concentration between the analyte and PQ-6 (Equation (1)), and the quantification could be carried out with both their RCFs and peak areas according to the following equation (Equation (2)).The QAMS method is based on the principle of the linear relationship between a detector response and the levels of components within certain concentration ranges . PQ-6 wax and s represent the analyte and the internal referring substance PQ-6, respectively, and xf is the average RCF value of each co-existing component to PQ-6, xA and xC denote the peak area and the concentration of the co-existing component in the single standard solution or in the samples, respectively.HCA and PCA were performed using the software of SPSS  16.0 to further investigate the difference among the KMI samples. Radar plot analysis was manipulated by using Microsoft Excel 2010.In the present study, an innovative technique for quality assessment of Kumu injection, a traditional Chinese medicine preparation, was developed using HPLC combined with chemometric and QAMS methods for the first time. The major bioactive alkaloid nigakinone was applied as reference substance for simultaneous quantitation of six major alkaloids in KMI. The findings provided evidence of the feasibility of the QAMS method using RCFs for simultaneous quantitation of multiple components. Moreover, chemometric combined with QAMS methods could be a powerful and reliable way to provide both qualitative insight and quantitative data for comprehensive quality assessment of complex multi-component systems, especially TCM preparations.


Their structures were elucidated by extensive spectroscopic analysis including 1D, 2D NMR , HRMS and IR spectra. This is the first time that tetracyclic triterpenes have been reported in genus Coffea.In present study, four new dammarane-type triterpenoids, namely caffruones A–D (The online version of this article (10.1007/s13659-018-0181-y) contains supplementary material, which is available to authorized users. Coffea arabica L. (Rubiaceae) is the most economic significant species in the coffee trade, occupying 69% of the world’s coffee production + ion peak at m/z 513.3551 , with six degrees of unsaturation. The 1H NMR spectrum , one olefinic methylene , one oxygenated methylene  and two oxygenated methines . The 13C-DEPT NMR spectra , six methines (two oxygenated) and seven quaternary carbons . Apart from a double bond and a carbonyl groups, the remaining elements of unsaturation degrees contributed to four rings, therefore, 1 should be a tetracyclic triterpenoid.Caffruone A  was isol3-18 (δH 1.12) to C-7 (δC 35.4), C-14 (δC 50.4) and C-8 (δC 40.7), from H2-7  to C-8 and C-18 (δC 15.6), from δH 1.43 (H-9) to C-18, and from H3-19 (δH 1.43) to C-9 (δC 50.5), C-10 (δC 36.8) and C-1 (δC 39.6), demonstrated that C-8 and C-10 in 1 were substituted by methyls /δH 0.96 , δH 1.56 (H-7b)/δH 1.12 (H3-18)/δH 1.71 (H-13) and δH 2.25 (H-17)/δH 0.96 (H3-30) confirmed that H3-18, H-13 were β-oriented and H-17 was α-oriented in 1  and an oxygenated methine (δC 73.8) in 1, respectively. The HMBC correlations from the methylene protons  to C-3 (δC 219.0), C-4 (δC 52.4) and C-29 (δC 16.6), together with the ROESY correlation of H2-28/H-5 indicated that C-28 in 1 was connected a hydroxyl group  to the methine carbon  and 1H-1H COSY correlations between δH 1.89, 1.68 (H2-16)/δH 4.28 (H-15) confirmed a hydroxyl group was located at C-15 in 1.Aforementioned information suggested that the structure of en-3-one , except 1 by X-ray crystallography, Snatzke’s method and modified Mosher’s method were failed. However, on the analysis of the literatures available   . Additio2), obtained as white amorphous powder, was assigned the molecular formula C30H50O5 by HRESIMS, as the same as 1. Moreover, its 1H and 13C NMR spectra  showed correlation of β-oriented methyl protons H3-19 (δH 0.90) in 2, rather than H-5α in 1, suggesting that C-29 was the oxygenated methylene in 2, not C-28 in 1. Meanwhile, the ROESY correlation of H3-18 (δH 1.07)/H-15 (δH 4.25) proved the 15-OH to be α-oriented (supporting infomation). Similarly, the absolute configuration of C-24 in 2 was determined as S due to the same chemical shifts of C-24 with 1. These results indicated that 1 and 2 were a pair of epimers. Therefore, 2 was elucidated as (24S)-15α,24,25,29β-tetrahydroxy-dammar-20-en-3-one.Caffruone B , obtaine3) was isolated as white amorphous powder. Its molecular formula, C30H48O5, determined from the [M + Na]+ peak at m/z 511.3396  in the HRESIMS spectrum, suggesting seven degrees of unsaturation. The 1H NMR spectrum ; δH 5.60 ], three oxgenated methylenes , and one oxgenated methine [δH 4.25 ]. The 13C-DEPT NMR spectra , six methines (one oxygenated and one olefinic), and seven quaternary carbons (two olefinic and one ketone carbonyl). Aparting the three degrees ocuppied by two double bonds and a carbonyl, the remaining degrees attributed to a tetracyclic system.Caffruone C  to δC 50.4 (C-9), δC 35.5 (C-7) and δC 40.5 (C-8), from H3-19 (δH 0.90) to δC 36.6 (C-10), 39.7 (C-1), δC 50.4 (C-9) and δC 55.5 (C-5), together with the ROESY correlations of δH 1.59 (H-7a)/δH 0.96 (H3-30), δH 1.48 (H-7b)/δH 1.06 (H3-18)/δH 1.67 (H-13) confirmed that 3 was also a dammarane triterpenoid.Similar with 3 was assigned as a 3-oxo-dammara-20,24-dien-26-ol triterpenoid along with three additonal hydroxy groups + ; 1H and 13C NMR data shown in Tables White amorphous powder, mentclass2pt{minimc = 0.2, MeOH); UV (MeOH) λmax (log ε): 202 (3.77), 268 (2.86), 374 (0.0067) nm; IR (KBr) vmax: 3430, 3083, 2965, 2872, 1680, 1610, 1460, 1380, 1010, 900 cm−1; HRESIMS m/z 513.3551 [M + Na]+ ; 1H and 13C NMR data shown in Tables White amorphous powder, mentclass2pt{minimc = 0.2, MeOH); UV (MeOH) λmax (log ε): 203 (3.36), 278 (2.39) nm; IR (KBr) vmax: 3410, 3080, 2960, 2870, 1724, 1685, 1615, 1460, 1380, 1020, 905 cm−1; HRESIMS m/z 511.3396 [M + Na]+ ; 1H and 13C NMR data shown in Tables White amorphous powder, mentclass2pt{minimc = 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (3.89), 269 (2.83) nm; IR (KBr) vmax: 3420, 3080, 2970, 2880, 1690, 1610, 1460, 1380, 1020, 920 cm−1; HRESIMS m/z 511.3397 [M + Na]+ ; 1H and 13C NMR data shown in Tables White amorphous powder, mentclass2pt{minimSupplementary material 1 (DOCX 3207 kb)Below is the link to the electronic supplementary material.


Based on extensive spectroscopic techniques  and chemical methods, their structures were established as szechenyianine D (1), szechenyianine E (2), szechenyianine F (3), 8-O-methyl-14-benzoylaconine (4), and spicatine A (5). The immunosuppressive effects of compounds 1–5 were studied using a ConA-induced or LPS-induced splenocyte proliferation model. In vitro tests showed that Compounds 2, 4, and 5 suppressed ConA-induced or LPS-induced splenocyte proliferation in a concentration-dependent manner. The CC50/IC50 values of 2, 4, and 5 suggested that these compounds were potential immunosuppressive agents for the treatment of autoimmune diseases characterized by arthritis, such as rheumatoid arthritis. Three new C Aconitum szechenyianum Gay. and A. flavum Hand.-Mazz., which belong to the Aconitum genus of Ranunculaceae, are widely used in folk medicine in Shaanxi province in China +  in the HR-ESI-MS spectrum. The 1H-NMR spectrum , 7.54 , and 7.44 , five OMe protons at δH 3.80, 3.37 , 3.29 (over-lapped), 3.29 (over-lapped), and 3.21 , and two strongly shielded protons at δH 3.32  and δH 4.29 . The 13C-NMR spectrum , and 128.7 (C × 2) were attributed to a benzoyloxy group; δC 62.2, 59.3, 59.0, 55.4, and 50.7 were attributed to five OMe groups; δC 74.7 and 76.4 were attributed to two oxygenated carbons associated with hydroxyl groups. Out of the 10 oxygen atoms in 1, 9 are associated with five methoxy groups, two hydroxyl groups, and one benzoyl group, and the remaining one may be a hydroxyl group or an internal ether. The NMR features of the remaining 19 resonances were characteristic of an aconitine-type alkaloid, where the δC 64.4 and 70.0 resonances were attributed to the two carbons associated with an internal ether. The deduction was confirmed by the chemical shift of C-7 (δC 64.4) and C-17 (δC 70.0) to downfield in 13C-NMR spectra of 1 compared with C-7 (δC 49.6) and C-17 (δC 60.6) of szechenyianine A +  in the HR-ESI-MS spectrum. The 1H-NMR spectrum , 7.56 , 7.45 , four OMe protons at δH 3.75 , 3.30 , 3.19 (3H s), and 3.12 ; one acetoxyl proton at δH 1.35 , and one methylic proton of the hydrocarbon chain at δH 0.86 . The 13C-NMR spectrum , and 128.9 (C × 2) were attributed to a benzoyloxy group; δC 61.5, 59.4, 58.0, and 55.5 were attributed to four OMe groups, δC 172.5 and 21.6 were attributed to one acetoxyl group; δC 173.3 was attributed to C=O, δC 74.3 and 79.0 were attributed to two carbons associated with the hydrocarbon chain, and δC 14.3 was attributed to one CH3 group. The assignments of the NMR signals associated with 2 were derived from HSQC, HMBC, and ROESY experiments. In the HMBC spectrum (H 4.89) to the carbonyl carbon signal of the benzoyl group (δC 166.4) suggested that the benzoyl group was located at C-14. Correlations of OCH3 (δH 3.19) to C-1 (δC 82.4), OCH3 (δH 3.12) to C-6 (δC 83.3), OCH3 (δH 3.75) to C-16 (δC 90.4), and OCH3 (δH 3.30) to C-18 (δC 80.0) suggested that four methoxyl groups were linked at C-1, C-6, C-16, and C-18, respectively. Correlations of CH3 (δH 1.32) to 8-OAc (δC 172.5) suggested that one acetoxyl was linked at C-8, and correlations of H-3 , H-17 (δH 4.11), and H-20  to C-19 (δC 173.3) suggested that C=O was linked at C-19. Correlations of H-20  to C-17 (δC 56.7) and C-21 (δC 33.7), H-21  to C-22 (δC 25), and H-22 , H-25 , and H-26  to C-24 (δC 31.9) suggested the presence of an N-heptyl group. Correlations of H-12 , H-14 (δH 4.89), and H-16 (δH 3.30) to C-13 (δC 74.3), and H-16 (δH 3.30) to C-15 (δC 79.0) suggested that two hydroxyl groups were linked at C-13 and C-15, respectively. This compound differed from the known compound (A-c)-8β-acetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane +  in the HR-ESI-MS spectrum. The 1H-NMR spectrum , one N–CH2CH3 group at δH 1.51 , 4.01 , and 4.42 , and three OMe resonances at δH 3.38 , 3.36 , and 3.16 . The 13C-NMR spectrum  and H-17 (δH 3.79) to C-19 (δC 179.2) suggested that C-19 was involved in the N=CH group. Correlations of OCH3 (δH 3.18) to C-1 (δC 80.6), OCH3 (δH 3.36) to C-16 (δC 81.5) and of OCH3 (δH 3.38) to C-18 (δC 73.4) suggested that three methoxyl groups were linked at C-1, C-16, and C-18, respectively. Correlations of H-6 , H-7 (δH2.22), H-9 (δH 2.47), and H-10 (δH 2.01) to C-8 (δC 72.3) and of H-16 (δH 3.45) to C-14 (δC 75.0) suggested that two hydroxyl groups were linked at C-8 and C-14, respectively. Thus, the planar structure of 3 was deduced as 8,14-dihydroxy-1,16,18-trimethoxy-19-en-aconitane. In the ROESY spectrum , or 4.293 ± 3.20 μm, 3.852 ± 1.57 μm, and 2.283 ± 1.28 μm (LPS-induced), respectively. These three compounds showed low cytotoxic effect . ESI-MS analysis was performed on a Quatro Premier instrument . HR-ESI-MS spectra were recorded on an Agilent Technologies 6550 Q-TOF . 1D- and 2D-NMR spectra were recorded on Bruker-AVANCE 400  and Bruker-AVANCE 600 instrument  using TMS as an internal standard. Analytical HPLC was performed on a Waters e2695 Separations Module system coupled with a 2998 Photodiode Array Detector and an Accurasil C-18 column . Semipreparative HPLC was performed on a system comprising an LC-6AD pump equipped with an SPD-20A UV detector  and an Ultimate XB-C18  or YMS-Pack-ODS-A  column. Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation . A. szechenyianum Gay. were collected from the Xi Mountains in Gansu Province of China in July 2014 and identified by senior experimentalist Jitao Wang. A voucher specimen (herbarium No. 20140728) has been deposited in the Medicinal Plants Herbarium (MPH), Shaanxi University of Chinese Medicine, Xianyang, China. The roots of A. szechenyianum (5.0 kg) were extracted with 80% EtOH at 80 °C . After the removal of EtOH under reduced pressure, the extract (2 L) was dispersed in water (1.5 L), adjusted to pH 0.8 with 9% HCl solution, and extracted with petroleum ether (PE). The acidic water solution was alkalized to pH 10.26 with 25% ammonia solution, extracted with CHCl3 three times, and evaporated under pressure to give crude alkaloids (50 g). The crude alkaloids (47 g) were loaded on a silica gel column and eluted with a gradient solvent system  to yield 12 fractions (Fr.1–Fr.12). Fr.3 (2.5 g) was purified by HPLC  with CH3OH/H2O (83:17) as the mobile phase to obtain 1 . Fr.4 was purified by HPLC with CH3OH/H2O (75:25) as the mobile phase to obtain 4  and 5 . Fr.7 was purified by HPLC with CH3OH/H2O (65:35) as the mobile phase to obtain 2  and 3 . More details of the spectra are provided in the The air-dried and powdered underground parts of (A-c)-14α-benzoyloxy-8β-acetoxyl-13β,15α-dihydroxy-1α,6α,8β,16β,18β-pentamethoxyl-7(17)–oxide-aconitane (szechenyianine D): A white amorphous powder, +  for C31H43NO10.(A-c)-14α-benzoyloxy-8β-acetoxy-N-nonyl-13β,15α-dihydroxy-1α,6α,16β,18β-tetramethoxy-19-oxo-aconitane (szechenyianine E): A white amorphous powder,  +  for C39H55NO11.8β,14α-dihydroxy-1α,16β,18β-trimethoxy-19-en-aconitane (szechenyianine F): A white amorphous powder, +  for C24H38NO5+.5 cells/well) were incubated in triplicate at 37 °C in a humidified incubator with 5% CO2 and 95% air. The assay was performed in a 96-well format, and different concentration of Compounds 1–5 (0.16–100 μm) and CsA（2μm）were added. The cells cultured with media alone were used as controls. Approximately 48 h later, 20 μL of 3--2,5-diphenyltetrazolium bromide  was added to each well. The plates were then incubated for another 5 h. Approximately 150 μL of DMSO (Sigma) was then added to each well. Optical density was measured at 570 nm . The IC50 and CC50 values were calculated according to the dose curves generated by plotting the percentage of viable cells against the test concentration on a logarithmic scale by using SPSS 15.0. CsA  was used as a positive control. Splenocytes (4 × 105 cells/well), different concentration of Compounds 1–5 (0.16–100 μm), and CsA（2 μm）in 96-well plates at 37 °C in a 5% CO2 atmosphere were cultured in triplicate for 48 h using ConA  or LPS . The cells cultured with media alone were used as controls. The cells were pulsed at 0.25 μCi/well of [3H]-thymidine for 8 h before the end of the culture period and then harvested onto glass fiber filters. [3H]-thymidine incorporation was measured using a beta scintillation counter .Splenocytes (4 × 10


The structures of three new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4–6, 13 and 14 were evaluated for inhibition of nitric oxide production in LPS-stimulated product of nitrite in RAW 264.7 cells with IC50 values of 6.12, 16.43, 18.98, 9.30, 9.30 and 14.90 μg/mL, respectively.Three new benzenoid derivatives, lawsoinermone ( Lawsonia inermis Linn (Lythraceae) is a small tree or tall shrub, native to northern Africa, western and southern Asia, and northern Australasia − ion at m/z 229.0498 . The IR absorption bands implied the presence of an OH (3442 cm−1), a γ-butyrolactone carbonyl group (1772 cm−1), and a conjugated carbonyl group (1682 cm−1). The 1H-NMR spectrum of 1 showed the presence of four mutually coupling aromatic protons , four mutually coupling methylene protons , and an oxymethine proton [δ 6.07 ], and a hydroxyl group [δ 3.88 ]. The 1H-NMR spectrum of 1 was similar to 3,4,5,6-tetrahydro-8-methoxy-2H-benzo[b]furan-2-one (1a) cycloheptafuran-2,6-dione, named lawsoinermone.Lawsoinermone  was obtained as yellow powder. Its molecular formula, C34H30O8, was determined on the basis of the positive HR-ESI-MS at m/z 567.6049 [M + H]+  and supported by the 1H, 13C, and DEPT NMR data. The presence of conjugated carboxyl group was revealed by the bands at 3300~2500 and 1679 cm−1 in the IR spectrum, and was confirmed by the resonance at δ 167.8 in the 13C-NMR spectrum. The 1H-NMR spectrum of 2 displayed the presence of a 4-methoxybenzyloxy moiety , four aromatic protons on para-disubstituted benzene ring [δ 6.95  and 7.49 ], and a conjugated olefinic proton [δ 7.71 ]. Based on the HR-ESI-MS, 1H-, and 13C-NMR data, the number of resonances observed was half that expected, suggesting that 2 had a symmetrical structure. The 1H-NMR data of 2 were similar to 2,3-bis(4-benzyloxy-3-methoxybenzylidene)succinic acid  and the similar chemical shift [δ 7.71] of H-7 and H-7′′ when compared to the analogous -2,3-bis(4-(benzyloxy)benzylidene)succinic acid + ion at m/z 258.0901 . The presence of carboxyl group in 3 was revealed by the bands at 3300~2500 and 1682 cm−1 in the IR spectrum. The 1H-NMR spectrum of 3 showed the presence of a 4-methoxybenzyloxy moiety , four para-substituted aromatic protons [δ 7.01  and 8.05 ], and a carboxyl group [δ 10.68 ]. The 1H-NMR spectrum of 3 was similar to that of 4-(4-methoxybenzyloxy)-benzaldehyde (3a) −; HR-ESI-MS m/z 229.0498 [M − H]− .Lawsoinermone (2): yellow powder; UV (MeOH): λmax (log ε) 291 (4.52), 265 (4.59); IR (KBr) υmax: 3300~2500, 1679, 1607, 1508 cm−1; 1H-NMR : δ 3.74 , 4.98 , 6.92 , 6.95 , 7.33 , 7.49 , 7.71 ; 13C-NMR : δ 55.1 (OMe-4′ and OMe-4′′′), 69.1 (C-7′ and C-7′′′), 113.8 , 115.0 , 125.8 (C-8 and C-8′′), 127.2 (C-1 and C-1′′), 128.5 (C-1′ and C-1′′′), 129.6 , 131.4 , 140.1 (C-7 and C-7′′), 159.1 (C-4′ and C-4′′′), 159.5 (C-4 and C-4′′), 167.8 (COOH); ESI-MS m/z 567 [M + H]+; HR-ESI-MS m/z 567.6049 [M + H]+ .Inermidioic acid (3): amorphous powder; UV (MeOH): λmax (log ε) 321 (4.26), 294 (4.33), 242 (4.31), 220 (4.43); IR (KBr) υmax: 3300~2500, 1682, 1628, 1578, 1530 cm−1; 1H-NMR : δ 3.83 , 5.06 , 6.92 , 7.01 , 7.36 , 8.05 , 10.68 ; 13C-NMR : δ 55.3 (OMe-4′), 70.0 (C-7′), 114.1 (C-3′ and C-5′), 114.6 (C-3 and C-5), 121.5 (C-1), 128.1 (C-1′), 129.3 (C-2′ and C-6′), 132.3 (C-2 and C-6), 159.7 (C-4′), 163.2 (C-4), 169.8 (COOH); EI-MS m/z 258 [M]+; HR-EI-MS m/z 258.0901 [M]+ .Inermic acid (2 incubator (5% CO2 in air) at 37 °C and subcultured every three days at a dilution of 1:5 using 0.05% trypsin-0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).A murine macrophage cell line RAW264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center  of the Food Industry Research and Development Institute . Cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  in a CO5) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for one day to become nearly confluent. Then cells were cultured with compounds 1–14 in the presence of 100 ng/mL LPS (lipopolysaccharide) for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader .Cells . By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured from absorbance at 540 nm.NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentration of samples in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Then, cells were dispensed into 96-well plates, and 100 μL of each supernatant was mixed with the same volume of Griess reagent  and incubated at room temperature for 10 min, the absorbance was measured at 540 nm with a Micro-Reader . Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe’smultiple range tests).The data is expressed as means ± standard errors (SE). The IC


Compounds 3 and 4 are new and their structures were elucidated on the basis of spectroscopic evidence and comparisons with literature data.Two pairs of diastereoisomeric acetylenic norlignan compounds with PhCH(OR Curculigo crassifolia (Bak.) Hook. f. belongs to the Hypoxidaceae family and is found throughout the Western and Southern regions of China. Its rhizomes are used as a tonic and a folk medicine for treating child pneumonitis -; HR-FAB- MS m/z: [M-H]-329.1037 . Fraction 5 (210 g) was refractionated by Sephadex LH-20  to yield 12 crude fractions (5-1 to 5-12). Fraction 5-7 (4.34 g) was purified by Sephadex LH-20  to yield 6 fractions (5-7-1 to 5-7-6). Fraction 5-7-4 (612 mg) was repeatedly purified on Sephadex LH-20 (EtOH) to afford a mixture of -1-O-methylnyasicoside (1) and -1-O-methylnyasicoside (2) (212 mg) and pure 1 (18 mg). White amorphous powder; : +12.37º ; UV (MeOH): λmax (lg ε): 205 (4.34), 255 (3.96), 289 (3.65) nm. IR νmax: 3441, 2926, 2045, 1546, 1473, 1179cm-1; 1H-NMR see 13C-NMR see -MS m/z: 491 [M-H]-; HR-FAB-MS m/z: [M-H]- 491.1565 .The air-dried and powered rhizomes of


This article focuses on the antiradical activity of a number of 2,6-diisobornylphenol-porphyrin conjugates with various spacers between the porphyrin and phenolic fragments in the model reaction of ethylbenzene oxidation initiated by azoisobutyric acid dinitrile. The study has shown that the electronic effects of the groups directly related to the 2,6-diisobornylphenol fragment exert the predominant influence both on the reactivity of the phenolic hydroxyl group in interaction with free radicals and on the antiradical activity of the molecule as a whole. The antiradical activity of the molecule is generally less affected by the nature of the substituents in the porphyrin macrocycle, mainly due to a change in the stoichiometric inhibition coefficient in the presence of relatively easily oxidizable groups. It was found that the length of the spacer between the porphyrin and phenolic fragments does not affect the antiradical activity of the conjugate. A solution of porphyrin 4 , CMPI , acid 2  and DMAP  in 15 mL of toluene was boiled for 1 h under reflux. Then CMPI , acid 2  was added and the mixture was boiled for 1 h. After cooling the reaction mixture was diluted with dichloromethane (100 mL) and washed with a 5% HCl solution to remove excess of DMAP and CMPI conversion products and then with distilled water until neutral washing water, dried with anhydrous Na2SO4, and evaporated under reduced pressure. The residue after evaporation was chromatographed on alumina . The eluate containing the desired product was evaporated under reduced pressure. 8 mg (13%) of compound 5 were obtained as a dark blue-green crystalline powder. UV-Vis (CH2Cl2), λmax, nm(%): 663.0 (33), 607.0 (4), 558.5 (2), 528.0 (3), 501.0 (10), 401.5 (100). m/z (ESI): for [M]+ C65H81N5O8, calculated: 1060.7, found: 1060.6. 1H-NMR : Signals of the terpenylphenol fragment: 0.64, 0.65,0.76, 0.78 , 1.49–1.71, 1.23–1.43, 0.98–0.84 , 1.84–2.19 , 2.84–3.00 , 5.32 , 7.95 . Signals of the porphyrin fragment: −1.73 , −1.53 , 1.72–1.82 , CH3-8(2)), 2.19–2.30 ), 2.43–2.84 ), 3.35 ), 3.53 ), 3.55 ), 3.65 ), 3.81 ), 3.79–3.93 ), 4.32–4.37 and 3.98–4.15 ), 4.41 , 4.51 , 4.70–4.87 ), 5.31 A), 5.60 A), 6.17 ), 6.38 ), 6.80–6.92 ), 8.05–8.18 ), 8.84 , 9.66 , 9.71 . 13C-NMR : Signals of the terpenylphenol fragment: 12.58 ), 21.01 ), 21.15 ), 27.41 ), 34.01 ), 39.98 ), 45.22 ), 46.01 ), 48.19 ), 50.00 ), 120.59 (C(13′)), 127.70 ), 128.80, 129.51 ), 158.69 (C(12′)). Signalsoftheporphyrinfragment: 11.35 (C(7(1))), 11.94 (C(2(1))), 12.17 (C(12(1))), 17.78 (C(8(2))), 19.70 (C(8(1))), 23.06 (C(18(1))), 29.72 (C(17(2))), 31.17 (C(17(1))), 37.94 (C(15(1))), 40.12 (13-CONHCH2CH2), 49.28 (C(18)), 51.62 (C(17(4))), 52.18 (C(15(3))), 53.09 (C(17)), 93.62 (C(20)), 98.82 (C(5)), 101.47 (C(10)), 102.27 (C(15)), 121.63 (C(3(2))), 127.86 (C(13)), 129.69 (C(3(1))), 129.72 (C(12)), 130.22 (C(2)), 134.55 (C(7)), 134.85 (C(4)), 134.93 (C(3)), 136.08 (C(11)), 138.95 (C(1)), 144.76 (C(8)), 149.10 (C(14)), 154.33 (C(9)), 166.66 (C(6)), 167.22 (C(16)), 168.91 (C(19)), 169.71 (13-CON), 173.52 (C(17(3)), (13-CONHCH2CH2OCOAr)), 173.90 (C(15(2))). IR spectrum, (KBr), cm–1: 3314 (NH), 2953, 2876, 2727 , 1734 , 1665 , 1601 («chlorin band»), 1510 («amide–II»).2-{(76) was obtained according to heptan-2-yl}-5-{-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}-N-{4-phenyl}benzamide (7). Thionyl chloride  was added to a solution of acid 2  in dichloromethane (10 mL) and refluxed for 1 h. The solvent and the volatile components were then removed under reduced pressure. Dichloromethane (10 mL) and DMF (0.1 mL) was added to the obtained in situ acid chloride 3, then solution of porphyrin 6  in dichloromethane (15 mL) was added. The reaction mixture was refluxed for 1 h, then cooled, a 5% HCl solution was added and organic phase washed with water until a neutral reaction of the wash water. The resulting solution was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The reaction products were separated by column chromatography on alumina . 12 mg (34%) of compound 7 were obtained as a dark red-violet crystalline powder. UV-Vis (CH2Cl2), λmax, nm (%): 666.5 (1), 594.5 (1), 551.0 (1), 515.5 (3), 420.0 (100). m/z (ESI): for [MH]+ C71H68N5O2, calculated: 1022.5, found: 1022.9. 1H-NMR : Signals of the terpenylphenol fragment: 0.88 , 0.94 , 1.07 , 1.30–2.01 , 2.43–2.55 , 3.14 ,5.34 , 7.90 . Signalsoftheporphyrinfragment: −2.73 , 7.81 , H-10(4),H-10(5),H-15(3),H-15(4),H-15(5),H-20(3),H-20(4),H-20(5)), 8.06 ,H-5(5)), 8.13 , 8.25–8.27 ,H-5(6),H-10(2),H-10(6),H-15(2),H-15(6),H-20(2), H-20(6)), 8.89 , 8.94 . 13C-NMR : Signals of the terpenylphenol fragment: 12.76 ), 20.48 ), 21.44 ), 27.58 ), 34.29 ), 40.18 ), 45.48 ), 46.30 ), 48.49 ), 50.26 ), 124.94 ), 129.25 ), 157.64 (C(12′)), 138.02 (C(15′)). Signalsoftheporphyrinfragment: 118.42 (C(5(3))),(C(5(5))), 119.58, 120.17 ), 126.70 (C(10(3))),(C(10(5))), (C(15(3))),(C(15(5))),(C(20(3))),(C(20(5))), 127.73 (C(10(4))),(C(15(4))),(C(20(4))), 130.06–131.97 (C(1)),(C(2)),(C(3)),(C(4)),(C(60),(C(7)),(C(8)),(C(9)),(C(11)),(C(12)),(C(13)),(C(14)),(C(16)),(C(17)), (C(18)),(C(19))), 134.57, 135.18 (C(5(2))),(C(5(6))),(C(10(2))),(C(10(6))),(C(15(2))),(C(15(6))),(C(20(2))), (C(20(6))), 138.10 (C(5(4))), 142.21 (C(5(1))),(C(10(1))),(C(15(1))),(C(20(1))), 166.90 (–NHCO). IR spectrum, (KBr), cm–1: 3590 (OH), 3316 (NH), 2951, 2876, 2708 , 1641 , 1597(«chlorin band»), 1516 («amide–II»).4-Hydroxy-3-{-{O-heptan-2-yl}-5-{-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}phenyl)methyl]}-4,8,13,18-tetramethyl-20-oxo-9-ethenyl-14-ethylphorbin-3-propanoate (8) was obtained according to heptan-2-yl}-5-{-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}phenyl)methyl]carbamoylethyl}-18-[(N-methyl)carbamoyl] chlorin-20-yl)acetate (9) was obtained according to heptan-2-yl}-5-{-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}phenyl)propyl]carbamoylethyl}-18-[(N-methyl) carbamoyl]chlorin-20-yl)acetate (10) was obtained according to heptan-2-yl}-6-{-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}-4-{10,15,20-tris(4-methoxyphenyl)porphyrin-5-yl}phenol (11) was obtained according to [2-{(1rding to . RSA was estimated in a model reaction of the ethylbenzene initiated oxidation at 333 K in an air atmosphere. Dinitrile of azoisobutyric acid was used as the initiator of free radicals, the initiation rate was Wi = 5 × 10−8 mol/L·s. Ethylbenzene with a dissolved antioxidant was pre-thermostated. After that, an initiator additive was added. The oxygen absorption kinetics was measured with a volumetric unit [ric unit . StatistThus, the estimation of the antiradical activity of the conjugates presented in the present study showed that the electronic effects of groups directly connected with the 2,6-diisobornylphenol moiety generally exert the predominant effect on both the reactivity of phenolic hydroxyl in interaction with free radicals and the antiradical activity of the molecule. The nature of the substituents in the porphyrin macrocycle affects the activity of the compounds to a much lesser extent, due mainly to a change in the stoichiometric inhibition coefficient in the presence of relatively easily oxidisable groups; the length of the spacer between the porphyrin and the terpenylphenol fragment has no effect on the antiradical activity of the conjugates.